who recommendations to assure the quality, safety, and ......105 assure the quality, safety, and...

ENGLISH ONLY

FINAL

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 19 to 23 October 2009

WHO recommendations to assure the quality, safety, and efficacy

of influenza vaccines (human, live attenuated) for intranasal

administration

NOTE:

Any questions on this document should be addressed to the World Health Organization, 1211

Geneva 27, Switzerland, attention: Quality Safety and Standards (QSS). Questions may also

be submitted electronically to the Responsible Officer: Dr Claudia Alfonso at e-mail:

[email protected].

This document will be published in the WHO Technical Report Series after professional

editing to be in conformity with the "WHO style guide" (WHO/IMD/PUB/04.1).

© World Health Organization 2009

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for the views expressed in this publication.

Adopted by the 60th

meeting of the WHO Expert Committee on Biological Standardization, 19-23 October 2009. A

definitive version of this document, which will differ from this version in editorial but not scientific details, will be

published in the WHO Technical Report Series.

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RECOMMENDATIONS TO ASSURE THE QUALITY, 1

SAFETY, AND EFFICACY OF INFLUENZA VACCINES (HUMAN, 2

LIVE ATTENUATED) FOR INTRANASAL ADMINISTRATION 3

4

Recommendations published by WHO are intended to be scientific and advisory in nature. The 5

parts of each section printed in type of normal size have been written in a form, such that, should 6

a national regulatory authority so desire, they may be adopted as they stand as definitive national 7

requirements or used as the basis of such requirements. Those parts of each section printed in 8

small type are comments and recommendations for guidance for those manufacturers and 9

national regulatory authorities which may benefit from additional information. 10

11

It is recommended that modifications be made only on condition that the modifications ensure 12

that the vaccine is at least as safe and efficacious as that prepared in accordance with the 13

recommendations set out below. In order to facilitate the international distribution of vaccine 14

made in accordance with these recommendations, a summary protocol for the recording of 15

results of tests is given in Appendix 1. 16

17

The terms “national regulatory authority” and “national control laboratory” as used in these 18

recommendations, always refer to the country in which the vaccine is manufactured. 19

20

Table of Contents 21 22

Introduction................................................................................................................................ 4 23

General considerations ............................................................................................................... 4 24

Part A. Manufacturing recommendations.................................................................................... 7 25

A.1. Definitions .................................................................................................................. 7 26

A.1.1 International name and proper name .................................................................. 7 27

A.1.2 Descriptive definition ........................................................................................ 7 28

A.1.3 International standards....................................................................................... 7 29

A.1.4 Terminology...................................................................................................... 7 30

A.2 Background on influenza vaccine (human, live attenuated) production....................... 11 31

A.3 General manufacturing recommendations................................................................... 15 32

A.3.1 Procedures and facilities .................................................................................. 15 33

A.3.2 Eggs and cell cultures ...................................................................................... 16 34

A.4 Control of source materials......................................................................................... 18 35

A.4.1 Choice of vaccine strain................................................................................... 18 36

A.4.2 Substrate for virus propagation ........................................................................ 19 37

A.4.3 Master cell bank and manufacturer’s working cell bank................................... 20 38

A.4.5 Virus strains .................................................................................................... 21 39

A.5 Control of vaccine production .................................................................................... 25 40

A.5.1 Production precautions .................................................................................... 25 41

A.5.2 Production of monovalent virus pool ............................................................... 25 42

A.5.3 Control of monovalent virus pools................................................................... 28 43

A.5.4 Control of final bulk ........................................................................................ 31 44

A.6 Filling and containers ................................................................................................. 31 45

A.7 Control tests on final lot ............................................................................................. 32 46

A.7.1 Identity............................................................................................................ 32 47

A.7.2 Sterility............................................................................................................ 32 48

A.7.3 Infectivity (potency) ........................................................................................ 32 49

A.7.4 Endotoxin........................................................................................................ 33 50

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A.7.5 Residual moisture of lyophilized vaccines ....................................................... 33 51

A.7.6 Inspection of final containers ........................................................................... 33 52

A.8 Records...................................................................................................................... 33 53

A.9 Retained samples ....................................................................................................... 33 54

A.10 Labeling................................................................................................................... 33 55

A.11 Distribution and transport......................................................................................... 34 56

A.12 Stability.................................................................................................................... 34 57

A.12.1 Stability testing.............................................................................................. 34 58

A.12.2 Storage conditions ......................................................................................... 34 59

A.12.3 Expiry date .................................................................................................... 35 60

Part B. Nonclinical evaluation of new influenza vaccines (human, live attenuated)................... 35 61

B.1 General remarks ......................................................................................................... 35 62

B.2 Process development and product characterization ..................................................... 37 63

B.3 Nonclinical toxicity and safety testing ........................................................................ 38 64

B.3.1 Preclinical toxicity........................................................................................... 38 65

B.3.2 Preclinical safety testing .................................................................................. 40 66

B.4 Nonclinical immunogenicity and efficacy................................................................... 40 67

B.4.1 Nonclinical immunogenicity ............................................................................ 40 68

B.4.2 Nonclinical efficacy......................................................................................... 41 69

Part C. Clinical evaluation of new influenza vaccines (human, live attenuated)......................... 41 70

C.1 General remarks ......................................................................................................... 41 71

C.2 Clinical evaluation strategy ........................................................................................ 42 72

C.2.1 Clinical studies for licensure ............................................................................ 43 73

C.2.2 Post marketing studies ..................................................................................... 44 74

C.3 Clinical safety ............................................................................................................ 44 75

C.3.1 Initial safety assessment................................................................................... 44 76

C.3.2 Expanded safety assessment............................................................................. 45 77

C.4 Clinical efficacy and immunogenicity ........................................................................ 46 78

C.4.1 Clinical efficacy studies ................................................................................... 46 79

C.4.2 Clinical correlates of protection ....................................................................... 46 80

C.4.3 Clinical serological parameters ........................................................................ 46 81

C.4.4 Other clinical measures of immunity................................................................ 47 82

C.5 Evaluation in special populations................................................................................ 47 83

C.5.1 Pediatric .......................................................................................................... 47 84

C.5.2 Geriatric .......................................................................................................... 47 85

C.5.3 Risk groups...................................................................................................... 48 86

C.6 Strain change considerations for seasonal vaccines..................................................... 48 87

C.7 Vaccines intended for pandemic influenza.................................................................. 48 88

Part D. Recommendations for national regulatory authorities.................................................... 49 89

D.1 General remarks ......................................................................................................... 49 90

D.2 Release and certification ............................................................................................ 49 91

D.3 Manufacturing changes .............................................................................................. 50 92

Acknowledgements .................................................................................................................. 52 93

References................................................................................................................................ 53 94

Appendix 1: Summary protocol for influenza vaccines (human, live attenuated) for intranasal 95

administration (master/working seed lot Type A or Type B) ..................................................... 60 96

Appendix 3. Model certificate for the release of influenza vaccine (human, live attenuated) for 97

intranasal administration .......................................................................................................... 67 98

99

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Introduction 100

101 The WHO requirements for influenza vaccine (LIVE) date from 1979 ( 2). The purpose of these 102

updated recommendations is to provide vaccine manufacturers and national regulatory 103

authorities with applicable considerations and guidance in developing specific processes to 104

assure the quality, safety, and efficacy of influenza vaccines (human, live attenuated) for 105

intranasal administration including their non-clinical and clinical evaluation. 106

107

These recommendations apply to influenza vaccines (human, live attenuated) for intranasal 108

administration using embryonated hen's eggs as substrates. The production of such vaccines 109

using cell cultures as substrates is anticipated and guidance is also provided for this eventuality. 110

These recommendations, which are directed to vaccines intended for intranasal administration, 111

are not specific for a particular form of influenza vaccine virus attenuation used to prepare the 112

final influenza virus vaccine product. These recommendations should apply to the production 113

and quality control of viruses intended for use in the manufacturing of influenza vaccine (human, 114

live attenuated) for intranasal administration including reassortant viruses prepared through 115

either classical reassortment methods or reverse genetics techniques. 116

117

The recommendations, with possible modifications, are meant to apply to influenza vaccines 118

(human, live attenuated) for intranasal administration produced with seasonal vaccine strains for 119

use during the inter-pandemic period as well as vaccines produced with strains for use during 120

pandemics. However, these recommendations cannot anticipate every arising situation and 121

alternative considerations may be needed for specific public health circumstances. 122

123

The first draft of the document being updated was based on the WHO requirements for influenza 124

vaccines (LIVE) from 1979 ( 2) and the WHO recommendations for the production and control 125

of influenza vaccines (inactivated) ( 3). Sections on the pre-clinical, non-clinical, and clinical 126

evaluation were added to the updated recommendations to assure the quality, safety and efficacy 127

of influenza vaccines (human, live attenuated) for intranasal administration. The section on 128

testing animals for adventitious agents from the WHO 1979 requirements was removed from 129

these proposed recommendations as animal testing for that purpose is not generally 130

recommended anymore. 131

132

The recommendations in this document do not apply to the potential vector systems (i.e. other 133

viral or bacterial hosts) that could be used to deliver the antigenic components of influenza 134

viruses. Further recommendations may be developed in the future as additional strategies 135

emerge for immunologic control of influenza virus infections. 136

137

General considerations 138

139

Influenza virus is a significant cause of morbidity and mortality and each year results in major 140

social and economic impact throughout the world. Influenza viruses undergo continuous 141

evolutionary change, which makes control of influenza challenging. In order to assist national 142

regulatory authorities and manufacturers in control efforts, WHO provides annual reviews of 143

epidemiologic information and recommendations on the influenza viruses to use in vaccines in 144

countries of the Northern and Southern hemispheres ( 4). 145

146

Many people require medical treatment and/or hospitalisation, and excess mortality often 147

accompanies viral influenza epidemics with the vast majority of those affected being elderly. 148

Because the elderly constitute the most rapidly increasing sector of the population in many 149

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countries, the epidemiology of viral influenza can be expected to change accordingly, especially 150

in high income countries. Although the elderly are typically the most impacted by mortality, 151

viral influenza epidemics originate in settings that bring together immunologically susceptible 152

individuals who are capable of rapidly spreading influenza throughout a community, such as 153

school-aged children. Infants are particularly susceptible to the severe consequences of viral 154

influenza infections, but children of all ages may experience complications of influenza 155

including pneumonia and death ( 7). 156

157

At present the primary means of influenza prophylaxis generally available is vaccination. 158

Potential means of prevention, apart from personal and societal hygienic measures, include 159

antiviral medications. The indefinite nature of exposure to influenza virus, which could 160

necessitate long duration of compliance with an antiviral medication regimen, as well as issues 161

of potential or real emergence of drug resistant virus strains, have prompted strategies to reserve 162

chemoprophylaxis for specific circumstances ( 5, 5 6). 163

164

Inactivated influenza vaccines, which function mainly by inducing IgG antibodies specific for 165

influenza virus haemagglutinins, have a long and solid record of use. The interest in live 166

influenza virus vaccines stems from their potential to simplify administration by intranasal drops 167

or spray ( 8). In addition, they stimulate not only systemic humoral immunity but also local and 168

systemic immune protective mechanisms, including mucosal IgA antibodies and cellular 169

immunity. Thus, the possibility of controlling influenza virus infection and illness by the use of 170

live attenuated virus vaccines given by the intranasal route has been investigated extensively 171

during the latter half of the 20th

century. Live influenza virus vaccines arising from studies of 172

“cold adapted” donor strains have been used as an effective public health tool in industrialized 173

countries including the Russian Federation ( 9) and the United States of America ( 10). Although 174

current “cold adapted” vaccines are manufactured in embryonated hen's eggs, there is ongoing 175

research to develop influenza vaccines (human, live attenuated) using other methods of virus 176

attenuation and produced in cell culture. 177

178

The principle of using a live vaccine to control a viral infection has a sound basis, having been 179

employed in preventing other viral infectious diseases, such as polio, measles, mumps and 180

rubella. Attenuated polio virus vaccines are given orally to infect the cells of the intestinal tract, 181

which stimulates protective immune responses mimicking those occurring after natural polio 182

virus infections. By analogy, it may be possible to initiate a benign influenza virus infection in 183

the nasopharynx with an attenuated influenza virus strain to give protection against the prevalent 184

wild type influenza strains. 185

186

The successful deployment of live attenuated virus donor strains depends on ensuring an 187

appropriate balance between attenuation on the one hand, and immunogenicity on the other. The 188

aim is to produce an attenuated virus incorporating the key immunizing antigens and antigenic 189

determinants of circulating wild influenza viruses, simultaneously retaining stable genetic and 190

phenotypic characteristics of the attenuated donor strain when given to susceptible individuals on 191

a wide scale. The ideal candidate would provide strong strain-specific protection, broad cross-192

reactivity, stimulate all categories of protective immunity, produce few or no symptoms in the 193

most susceptible hosts, and be able to infect all hosts in whom specific protective immunity is 194

lacking. Pragmatically, compromise on one or more of these features may be required. 195

196

The continuing commercial development and public health use of influenza vaccines made from 197

live attenuated influenza virus strains make it appropriate to review and update the WHO 198

recommendations for such vaccines. Since the WHO requirements for influenza vaccine (LIVE) 199

were published in 1979 ( 1 2), significant advances in influenza virus vaccine production have 200

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occurred. For example, current reverse genetics techniques allow the selection of a 201

homogeneous pre-defined viral composition by using a system that reconstitutes influenza 202

viruses from genetic codes specific for each of the eight influenza viral gene segments ( 11, 12, 203

13). There is also increased knowledge of the genetic markers associated with virus attenuation, 204

which allows more stringent control of the vaccine. Additionally, there has been considerable 205

effort directed to pandemic planning to ensure that safe and effective vaccines can be produced 206

quickly in response to a pandemic emergency ( 14, 15). Consequently, it is necessary to revise 207

the previous guidance on live attenuated influenza vaccines in order to reflect new developments 208

and current practices in the field. In accordance with current WHO policy, the revised document 209

is renamed as “Recommendations”. 210

211

212

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Part A. Manufacturing recommendations 213

214

A.1. Definitions 215

216

A.1.1 International name and proper name 217

218

The international name is "influenza vaccine (human, live attenuated)". The proper name is 219

"influenza vaccine (human, live attenuated)" translated into the language of the country of 220

origin. 221

222 The use of the proper name should be limited to vaccines that satisfy the recommendations formulated 223 below. 224

225

A.1.2 Descriptive definition 226

227

Influenza vaccine (human, live attenuated) is a preparation of live attenuated influenza virus 228

originating from human or other species. Influenza vaccine (human, live attenuated) is an 229

aqueous suspension, which may be lyophilised, and is intended for intranasal administration. 230

Influenza vaccines (human, live attenuated) contain a strain or strains of influenza virus types A 231

or B or a mixture of these two types, which have been grown individually in embryonated hen’s 232

eggs or in cell cultures. The influenza vaccines (human, live attenuated) shall be named "human" 233

as they are to be administered to human subjects. 234

235

A.1.3 International standards 236

237

No international reference preparations are currently available for quality control or release 238

testing of influenza vaccine (human, live attenuated) for intranasal administration. In addition, 239

these influenza vaccines may differ between manufacturers such that infectivity tests and /or 240

potency assays may not be standardized for universal application. Therefore, no 241

recommendations based on the use of international reference preparations can be formulated at 242

present for influenza vaccine (human, live attenuated) for intranasal administration. 243

244

Each manufacturer of influenza vaccines (human, live attenuated) for intranasal administration 245

should provide preparations of reference live attenuated influenza viruses and specific antisera 246

for use in tests of virus infectivity and/or potency specific to the live attenuated vaccine (see Part 247

A, sections 5.3.1, 5.4.1, and 7.3). The manufacturer should cooperate with national regulatory 248

authorities to determine the acceptability of the proposed reference reagents. 249

250

The infectivity tests and /or potency assays should be established and validated during vaccine 251

development and approved by the national regulatory authority. Strains used for quality control 252

or release testing of influenza vaccines (human, live attenuated) for intranasal administration 253

should be preparations antigenically representative of viruses with surface antigens 254

(haemagglutinin and neuraminidase) identical or closely related to the WHO-recommended 255

vaccine strains ( 4). 256

257

A.1.4 Terminology 258

259

Adventitious agents: Contaminating microorganisms of the cell culture or line including 260

bacteria, fungi, mycoplasmas and viruses that have been unintentionally introduced ( 16). 261

WHO is developing further guidance on adventitious agents. 262

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263

Attenuated donor virus strain: An attenuated influenza virus that provides the genes of 264

attenuation for vaccine strains that can be shown to be safe during clinical trials in human 265

beings ( 24). 266

267

Candidate influenza vaccine viruses for seasonal or non-highly pathogenic influenza A 268

subtype viruses with pandemic potential: These influenza viruses, approved by WHO as 269

suitable for making influenza vaccine , are typically prepared in vaccine virus 270

reassortment laboratories by "classical" reassortment, but reverse genetics may also be 271

considered for preparing candidate influenza vaccine viruses for seasonal and non-highly 272

pathogenic viruses ( 17). 273

274

Candidate influenza vaccine viruses for H5N1and other highly pathogenic influenza A 275

subtype viruses: These influenza viruses, approved by WHO as suitable for making 276

influenza vaccine, are prepared in vaccine virus reassortment laboratories by reverse 277

genetics ( 17). 278

279

Cell bank: A collection of ampoules containing material of uniform composition stored 280

under defined conditions, each ampoule containing an aliquot of a single pool of cells 281

( 16). 282

283

Cell seed: A quantity of well characterized cells of human, animal or other origin stored 284

frozen at -100oC or below in aliquots of uniform composition derived from a single tissue 285

or cell, one or more of which would be used for the production of a master cell bank ( 16). 286

287

Classical genetics (classical reassortment): Process of finding and assembling a set of 288

genes that affect a biological property of interest. In this process, mutants (reassortants) 289

are generated by employing mutagens that accelerate the normal mutation rate or by 290

growing the organism allowing spontaneous mutations to occur. Mutants are selected for 291

a particular biological property (phenotype) differentiating them from the wild type. The 292

location of the mutations responsible for the mutant phenotype is identified and analysed 293

to determine the role of the altered DNA on the studied biological property ( 18). 294

295

Clinical evaluation of vaccines: Include all of the clinical trials and other clinical studies 296

conducted in human beings pre- and post- licensure to determine the safe and effective 297

use of vaccines intended for the control of specific diseases. Clinical evaluation is done in 298

phases in order to gather information in a coherent manner that respects the rights and 299

dignity of all study participants, reduces the risks to the participants of the studies, and 300

provides an understanding of the potential benefit of the vaccine under study ( 19). 301

302

Egg infectivity dose 50% (EID50): The quantity of a virus suspension that will infect 50% 303

of embryonated hen's eggs inoculated with it ( 1 2). 304

305

Final bulk: The finished vaccine prepared from one or more monovalent pools present in 306

the container from which the final containers are filled. It may contain one or more virus 307

strains ( 3). 308

309

Final lot: A collection of sealed final containers which are homogeneous with respect to 310

the risk of contamination during filling procedures (including lyophilisation). A filling lot 311

must, therefore, have undergone the filling procedures (including lyophilisation) in one 312

working session from a single final bulk ( 3). 313

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314

Genetic reassortment:In this process genes from two or more influenza viruses are mixed 315

in different combinations, resulting in hybrid viruses with genetic characteristics of each 316

parent virus. This process occurs in nature but can also be done in a laboratory using 317

"classical" reassortment or reverse genetics ( 18, 17). 318

319

High-growth reassortant viruses: These are influenza viruses that have been genetically 320

modified to grow better in embryonated hen's eggs for optimal vaccine production ( 17). 321

322

Highly pathogenic influenza viruses: These are influenza viruses (typically from an avian 323

host) that cause at least 75% mortality when inoculated intravenously into 4 to 8-week-324

old chickens ( 20). 325

326

Influenza reference viruses: These are wild-type influenza viruses that WHO has 327

selected as representative of important groups of influenza viruses on the basis of 328

extensive antigenic and genetic studies and comparisons with viruses from many 329

countries. As the influenza viruses evolve in nature, new reference viruses are selected 330

( 17). 331

332

Influenza virus subtype(s): Type A influenza viruses are further classified according to 333

their combinations of haemagglutinin (H) and neuraminidase (N) antigens (i.e. specific 334

proteins on the virus surface), e.g. H5N1; 16 H subtypes and nine N subtypes have been 335

distinguished ( 17). 336

337

Master cell bank: A quantity of fully characterized cells of human or other animal origin 338

derived from the cell seed stored frozen at -100oC or below in aliquots of uniform 339

composition derived from the cell seed. The master cell bank is itself an aliquot of a 340

single pool of cells generally prepared from a selected cell clone under defined conditions, 341

dispensed into multiple containers and stored under defined conditions. The master cell 342

bank is used to derive all working cell banks. The testing performed on a replacement 343

master cell bank (derived from the same cell clone or from an existing master or working 344

cell bank) is the same as for the initial master cell bank, unless a justified exception is 345

made ( 3). 346

347

Master seed lot: The virus used to prepare the master seed lot is an attenuated influenza 348

virus that combines the attenuating features of attenuated virus donor strain and the 349

immunizing features of the wild type virus reference strain. The master seed lot is a virus 350

preparation which is antigenically representative of a WHO-recommended strain that has 351

been processed at one time to assure a uniform composition, is fully characterized, and 352

may be used for the preparation of working seed lots or for production of vaccine. The 353

national regulatory authority approves the master seed lot and its passage level. 354

355

Monovalent virus pool: A pool of a number of single harvests of a single virus strain 356

processed at the same time ( 3). 357

358

Nonclinical evaluation of vaccines: All in vivo and in vitro testing performed before and 359

during the clinical development of vaccines. The potential toxicity of a vaccine should be 360

assessed not only prior to initiation of human trials, but throughout clinical development 361

( 21). 362

363

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Non-highly pathogenic influenza viruses: These are influenza viruses (sometimes also 364

termed low-pathogenic) that cause less that 75% mortality when inoculated intravenously 365

into 4 to 8-week-old chickens ( 20) 366

367

Novel (new) subtype of human influenza A virus: This term refers to human influenza 368

viruses that have haemagglutinin and/or neuraminidase antigens that are distinct from 369

seasonal influenza viruses and have the potential to cause a pandemic ( 17). 370

371

Plaque forming units (pfu): The smallest quantity of a virus suspension that will produce 372

a plaque in monolayer cell cultures ( 22). 373

374

Preclinical evaluation of vaccines. All in vivo and in vitro testing carried out prior to the 375

first testing of vaccines in humans. This is a prerequisite to the initiation of clinical trials 376

and includes product characterization, proof of concept/immunogenicity studies and 377

animal safety testing ( 21). 378

379

Production cell cultures: A collection of cell cultures used for biological production that 380

have been prepared together from one or more containers from the working cell bank or, 381

in case of primary cell cultures, from the tissues of one or more animals ( 16). 382

383

Reverse genetics: The technique of determining a gene's function by first sequencing it, 384

then mutating it, and identifying the nature of the change in the phenotype ( 18). 385

386

Single harvest. A quantity of virus suspension harvested from the growth substrate 387

inoculated with the same virus strain and incubated and harvested together in one session 388

( 16). 389

390

Specific pathogen free (SPF): animals that have been shown by the use of appropriate 391

tests to be free from specified pathogenic microorganisms, and also refers to eggs derived 392

from SPF birds ( 23). 393

394

Specific antibody negative (SAN): animals that have been shown by the use of 395

appropriate tests to be free from antibodies to specified avian pathogenic microorganisms, 396

and also refer to eggs derived from SAN birds ( 23). 397

398

Tissue culture infectivity dose 50 % (TCID50): The quantity of a virus suspension that 399

will infect 50% of tissue culture inoculated with it. 400

401

Wild type influenza viruses: These are influenza viruses that have been cultured either in 402

eggs or cells (i.e. isolated) directly from clinical specimens and have not been modified 403

( 17). 404

405

Wild type reference virus strain: An influenza virus that has been selected to 406

antigenically represent the circulating viruses against which the vaccine should protect 407

recipients ( 24). 408

409

WHO recommended viruses for vaccine use: These are wild-type influenza viruses that 410

are recommended by WHO as the basis for an influenza vaccine ( 17). 411

412

Working seed lot: A quantity of fully characterized virus of uniform composition that is 413

derived from a master seed lot by a number of passages that does not exceed the 414

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maximum approved by the national regulatory authority. The working seed lot can be 415

used for production of vaccines ( 3). 416

417

Working cell bank: A quantity of cells of uniform composition derived from the master 418

cell bank at a finite passage level, dispensed in aliquots into individual containers 419

appropriately stored, usually frozen at -100oC or below, one or more of which would be 420

used for production purposes. All containers are treated identically and, once removed 421

from storage, are not returned to the stock ( 16). 422

423

A.2 Background on influenza vaccine (human, live attenuated) production 424

425

Even before inactivated influenza vaccines entered commercial use, interest was placed into 426

influenza vaccines (human, live attenuated). The motivating principles in the development of 427

influenza vaccines (human, live attenuated) include mimicking the immune responses to natural 428

influenza virus infection. 429

430

A number of candidate attenuated virus donor strains have been examined over the years 431

including A/Puerto Rico/8/34 (H1N1), A/Okuda/57 (H2N2) (attenuated by simple serial passage), 432

avian-human reassortant viruses (attenuated by host range characteristics of the avian donor 433

strains), and temperature sensitive (ts) mutants (attenuated by chance recovery of viruses, serial 434

passage, or introduction of mutations by directed mutagenesis) ( 36, 37). However, many 435

candidates have been withdrawn from consideration during clinical development. For example, 436

reassortants prepared with A/Puerto Rico/8/34 are not always well attenuated and introduction of 437

a temperature sensitive mutation without other stabilizing mutations resulted in a virus strain 438

prone to reversion to a non-attenuated form, which may occur after replication in a vaccine 439

recipient. 440

441

The most successful strategy to prepare attenuated virus donor strains so far has been the 442

development of cold adapted attenuated influenza viruses by serial passage at sequentially lower 443

temperature, which produced mutations in multiple gene segments. ( 25, 26, 27, 28, 29). The 444

presence of multiple mutations involving several influenza virus gene segments appears to 445

contribute to the stability of the live attenuated virus genomes and to lower the probability of a 446

reversion to virulence. Methods other than cold adaptation are also being explored and are in 447

early stages of evaluation. ( 30, 31, 32). If new live influenza vaccines are approved for human 448

use, WHO recommendations to assure the quality, safety, and efficacy of influenza vaccine 449

(human, live attenuated) for intranasal administration may be revised. 450

451

Influenza vaccines contain the antigens of one or more influenza A and B viruses that represent 452

the wild type influenza viruses prevalent in human populations. Influenza A viruses are 453

separated into subtypes based on structurally and antigenically distinct haemagglutinins and 454

neuraminidases. Influenza B viruses are not separated into subtypes, but they do have genetic 455

lineages of haemagglutinins and neuraminidases, which may be antigenically distinguishable. 456

Influenza A subtypes and influenza B lineages undergo progressive evolutionary changes of 457

haemagglutinin and neuraminidase antigens (antigenic drift), which may reduce the efficacy of 458

vaccines when the vaccines are inadequately matched to the prevalent viruses. For influenza A 459

virus subtypes not previously circulating in human populations (antigenic shift), it is expected 460

that vaccines will be effective only if they incorporate the antigens of the novel influenza A 461

subtype. 462

463

The composition of influenza vaccines (human, live attenuated), like that of other influenza virus 464

vaccines, is constantly under review to facilitate optimum protective efficacy against prevalent 465

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epidemic strains. Accordingly, twice annually WHO publishes recommendations concerning the 466

strains to be included so that virus strains antigenically matched to circulating strains are 467

included in vaccines manufactured for distribution in the Northern and Southern hemispheres ( 4). 468

469

Antigenic modifications in the haemagglutinin and neuraminidase molecules typically involve 470

variation in surface amino acid residues in the region of the molecule furthest from the viral 471

envelope. Future antigenic variations cannot be predicted because the mechanism of selection of 472

antigenic variants (antigenic drift) is not known and several evolutionary pathways appear 473

possible. Antigenic shifts (the appearance of a new influenza A haemagglutinin subtypes) are 474

also unpredictable. 475

476

In addition to antigenic drift and shift, there is another type of variation among influenza viruses 477

caused by the preferential growth of virus subpopulations in different host cells in which the 478

virus is cultivated. Influenza viruses grown in embryonated hen's eggs often exhibit genetic, 479

antigenic and biological differences from those isolated and maintained in mammalian cells. 480

Sequence analyses of the haemagglutinin genes of egg-adapted variants show that human 481

influenza viruses grown in eggs are less likely to maintain fidelity to the original sequence than 482

the same viruses grown in mammalian cells. An important consideration in vaccine preparation 483

is, therefore, ensuring that antigenic changes in the haemagglutinin molecule do not impair the 484

protective effects of the vaccine. 485

486

There is a long history of safety for egg-grown influenza virus vaccines ( 33, 34). However, it is 487

known that influenza viruses grown in embryonated hen's eggs can be contaminated with other 488

viral agents. Although the use of eggs from flocks husbanded to meet agricultural health criteria 489

( 38) for freedom from specific pathogens may reduce the chances of introduction of a microbial 490

agent, adventitious agents can be introduced from any egg source. The recommendations in this 491

document have been revised in view of the findings with egg-grown viruses, the increasing use 492

of mammalian cells for virus isolation and vaccine production, and the improved methods of 493

detecting adventitious agents ( 16, 35). WHO is developing further guidance on adventitious 494

agents testing. 495

496

Influenza A virus pandemic alerts have occurred on multiple occasions since 1997 (H5N1 497

subtype in 1997, 2003, 2005 and later years; H7N7 subtype in 2003; and H9N2 subtype in 1999) 498

when avian influenza A viruses caused illness often serious enough to require hospitalization and 499

to cause death in infected humans. In addition, the experience with pandemic influenza A 500

(H1N1) 2009 demonstrates the potential for non avian influenza viruses to cause significant 501

morbidity and mortality. Experience gained from these events illustrates that different strategies 502

for the production and clinical use of a vaccine may be needed in response to a pandemic. For 503

example: 504

505

• it may be necessary to generate a vaccine virus from a highly pathogenic virus by 506

reverse genetics, 507

• monovalent vaccines may be preferred, and 508

• two vaccine doses may be needed by all vaccine recipients. 509

510

Reflecting the special needs of an influenza pandemic, WHO has developed recommendations 511

to assure the quality, safety, and efficacy of the vaccine for pandemic situations ( 14) 512

513

The use of reverse genetics for vaccine virus development is relevant to both interpandemic and 514

pandemic vaccines ( 24). Reverse genetics technology has already been introduced as a method 515

WHO/BS/09.2111

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for generating reassortants for manufacturing commercial influenza vaccines (human, live 516

attenuated). Since reverse genetics allows necessary genetic modifications such as removal of 517

virulence motifs, it is also used to produce reassortants for use in vaccines in the event of a 518

pandemic. Reverse genetics technology involves transfecting mammalian cells with plasmids 519

coding for influenza virus genes in order to produce a virus reassortant. Production of 520

reassortants by reverse genetics is similar in concept to classical reassorting methods, but there 521

are some quality issues important to reverse genetic techniques: 522

523

• The source of the influenza virus haemagglutinin and neuraminidase genes for 524

reverse genetics is reduced in importance since the process of extraction of 525

nucleic acid eliminates concerns about adventitious agents: an egg isolate, an 526

isolate in cells not validated for human vaccine production, or a clinical specimen 527

may all be adequate to provide the nucleic acid needed for the start of reverse 528

genetics. 529

• The reverse genetic reassortant virus is generated in mammalian cells acceptable 530

to national regulatory authorities. 531

• In some countries, a reassortant produced using reverse genetics is classified as a 532

“genetically modified organism” and vaccine should comply with national 533

regulations or as indicated in the "WHO biosafety risk assessment and guidelines 534

for the production and quality control of human influenza pandemic vaccines" 535

( 15). 536

537

In developing a new candidate attenuated donor virus, knowledge gained in developing safe and 538

immunogenic vaccines should be applied. Significant information has accumulated to indicate 539

that not all attenuation methods result in donor vaccine virus strains of acceptable stability. For 540

example, it is known that an attenuated donor virus strain based on a mutation conferring 541

temperature sensitivity alone can be expected to revert to a virulent form after a single passage in 542

human recipients ( 26). Attenuation due to multiple mutations appears to be more stable and 543

probably more useful for long-term implementation of influenza vaccines (human, live 544

attenuated). Genetic stability and retention of key phenotypic features of the attenuated donor 545

strain are extremely important quality characteristics. Assurance that reversion to virulence is 546

unlikely to occur in humans should be determined in preclinical and clinical studies, and 547

monitored carefully during the post-marketing period. 548

549

Multiple strategies to derive candidate master seed and working seed viruses may be considered 550

for seasonal vaccines and vaccines against influenza A subtype viruses not classified as highly 551

pathogenic (e.g. pandemic influenza A (H1N1) 2009 virus). Only reverse genetics is appropriate 552

for candidate vaccine viruses derived from highly pathogenic avian influenza viruses. However, 553

both classical reassorting methods and reverse genetics techniques represent controlled methods 554

to be considered for developing seed viruses once a satisfactory attenuated virus donor strain is 555

obtained. 556

557

Classical reassortment between the attenuated virus donor strain and the new wild virus requires 558

selection steps that may not always be readily successful in providing the construct of choice. 559

However, classical reassortment can be an effective and relatively rapid method of producing 560

attenuated seed viruses with wild parent surface antigens. General experience with live virus 561

vaccines shows that candidate seed viruses derived by classical reassortment should be cloned at 562

least three times by limiting dilution passage in SPF-SAN embryonated eggs or plaque purified 563

in qualified cells to assure purity of the desired attenuated seed virus. Reverse genetics methods 564

permit a directed and more defined preparation of the desired reassortant virus. These methods 565

also allow elimination of the highly pathogenic phenotype of avian influenza viruses, since the 566

WHO/BS/09.2111

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major molecular determinants of pathogenicity (multiple basic amino acids at the haemagglutinin 567

cleavage site) can be removed during the preparation of the plasmids used to produce the reverse 568

genetics reassortant. Neither reverse genetics nor classical reassortment eliminates the 569

requirement for careful genetic and phenotypic assessment of the potential master seed and 570

working seed viruses to ensure retention of attenuation. 571

572

Influenza viruses present a challenge for vaccine preparation as they exhibit continuous antigenic 573

change in surface antigens of circulating influenza virus strains. Both inactivated and live 574

influenza virus vaccines can confer a degree of cross-protection against related virus strains 575

within a common haemagglutinin or neuraminidase subtype. For either type of vaccine, the use 576

of a vaccine strain with haemagglutinin and neuraminidase antigenically identical to the 577

naturally prevalent virus strain is expected to provide optimum protective efficacy. However, in 578

practice, it may not always be possible for the haemagglutinin and neuraminidase to be identical 579

with influenza viruses, since further evolution may occur during the several month period needed 580

for vaccine preparation. 581

582

The interval between the appearance of an influenza virus variant and its spread throughout the 583

world may take only months. Thus, the development of an appropriate vaccine virus strain must 584

be rapid. The time available for the yearly preparation and testing of new influenza vaccine 585

(human, live attenuated) lots must be carefully estimated and planned. Clinical studies, if 586

undertaken, must be focused but are likely to be severely limited in information they can provide 587

about potential vaccine performance. 588

589

Where appropriate, the technologies and experience acquired over several decades in the 590

production and control of all live virus vaccines should be applied to influenza vaccines (human, 591

live attenuated) for intranasal administration. As for all live vaccines, the substrate on which the 592

virus is propagated is critically important to maintaining the consistency and safety of the 593

vaccine product. Influenza vaccines (human, live attenuated) for intranasal administration are 594

usually produced in 9 to 11-day old vaccine-quality embryonated eggs. For influenza vaccines 595

(human, live attenuated) for intranasal administration a large measure of assurance of safety can 596

be provided by sourcing the eggs from closed layer flocks that are continuously monitored for 597

known specific pathogenic agents and antibodies against them. These specific pathogen free 598

(SPF) specific antibody negative (SAN) layer flocks are now available in several countries, and 599

the eggs - or cell cultures derived from them - have been widely used in the production of a 600

number of vaccines including measles and mumps vaccines ( 39). 601

602

The production of influenza vaccines (human. Live attenuated) using cell culture is currently 603

under active research. When considering the use of animal cells as in vitro substrates for the 604

production of biologicals, specific WHO guidelines should be taking into account ( 16, 35). 605

Since live influenza vaccines depend on the viability of the virus in filled containers, the storage 606

conditions as well as the short and long term stability of liquid and lyophilised products should 607

be established by rigorous studies similar to those undertaken in the preparation of other live 608

virus vaccines 63). 609

610

Apart from addressing the technical challenges associated with the rapid development of suitable 611

attenuated vaccine seed viruses, manufacturers and national regulatory authorities should 612

cooperate to define the need for special administrative arrangements for registration and 613

licensing, and establish the nature of data needed for review of the vaccine product preparation 614

and use. Close collaboration between manufacturers and the national regulatory authorities is 615

required, particularly during the phases of development, production, and testing of the initial 616

batches of live vaccines. The time for consideration of risks and benefits of the vaccines and for 617

WHO/BS/09.2111

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completion of licensing procedures should be as short as practically possible if appropriate 618

influenza vaccines (human, live attenuated) for intranasal administration are to be available soon 619

after the emergence of a variant. 620

621

In a public health emergency, i.e. influenza pandemic, the abnormal vaccine demand may create 622

difficulties if all tests recommended in the WHO guidelines on regulatory preparedness for 623

human pandemic influenza vaccines ( 14) are to be carried out. Decisions to modify these 624

requirements in the interest of public health during a public health emergency are the 625

responsibility of national regulatory authorities. Since progress in the development and 626

implementation of live attenuated influenza vaccines may result in improvements or, conversely, 627

additional concerns to be addressed, the WHO recommendations to assure the quality, safety, 628

and efficacy of influenza vaccines (human, live attenuated) for intranasal administration are 629

expected to need updating from time to time. 630

631

A.3 General manufacturing recommendations 632

633

The general requirements for manufacturing establishments contained in the WHO Good 634

Manufacturing Practices for biological products ( 40) should apply to establishments 635

manufacturing influenza vaccine (human, live attenuated) for intranasal administration, with the 636

addition of the following: 637

638

A.3.1 Procedures and facilities 639

640

Details of standard operating procedures for the production and testing of influenza vaccines 641

adopted by a manufacturer together with evidence of appropriate validation of the production 642

process should be submitted to the national regulatory authority for approval. Proposals for 643

modification of the manufacturing/control methods should also be submitted to the national 644

regulatory authority for approval. 645

646

Production areas should be cleaned, disinfected and/or decontaminated by validated procedures 647

before their use for the manufacture of influenza vaccines (human, live attenuated) for intranasal 648

administration. The areas where processing of live attenuated influenza vaccines takes place and 649

the procedures used for manufacturing should be designed to ensure that it is not possible to 650

contaminate the influenza vaccine (human, live attenuated) for intranasal administration with 651

another product. It is considered that filling of influenza vaccine (human, live attenuated) for 652

intranasal administration could occur on a campaign basis in the same facility used for filling 653

other vaccines provided that the manufacturer develops and performs a risk analysis and 654

evaluation and puts in place validated procedures for risk control ( 15). 655

656

Facilities for vaccine production should be constructed with adequate containment features to 657

accommodate the candidate influenza vaccines that are derived from the wild type influenza 658

viruses. The WHO biosafety risk assessment and guidelines for the production and quality 659

control of human influenza pandemic vaccines ( 15) should be followed. Standard operating 660

procedures need to be developed for dealing with emergencies involving accidental spillage, 661

leakage, or other dissemination of virus. High levels of bio-containment are required for work 662

with highly pathogenic wild type influenza viruses, which may be used in generating master and 663

working seed viruses. 664

665

The production of influenza vaccines (human, live attenuated) for intranasal administration 666

should be conducted by staff who have not handled other infectious microorganisms or animals 667

WHO/BS/09.2111

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on the same working day. The staff must practice good sanitation and health habits. Staff with 668

respiratory or any other apparent infectious illness should be excluded. Particular attention 669

should be paid to the recommendations given in the WHO Good Manufacturing Practices for 670

biological products ( 40) regarding the training and experience of personnel in charge of 671

production and testing and of those assigned various responsibilities in the manufacturing 672

establishment. Personnel employed in the production and control facilities should be adequately 673

trained and protected against accidental infection with influenza virus according to guidance in 674

the WHO Biosafety Manual ( 41) and the WHO biosafety guidelines for personnel engaged in the 675

production of vaccines and biological products ( 42). Personnel protection and containment 676

measures should also follow the WHO biosafety risk assessment and guidelines for the 677

production and quality control of human influenza pandemic vaccines ( 15). 678

679

Manufacturers and national regulatory authorities should consider whether the influenza vaccine 680

(human, live attenuated) for intranasal administration presents any significant environmental, 681

agricultural, or human risks. The WHO biosafety risk assessment and guidelines for the 682

production and quality control of human influenza pandemic vaccines ( 15) provides a detailed 683

strategy to minimize the risks of introducing influenza virus strains into the community. 684

685

A.3.2 Eggs and cell cultures 686

687

Fertile eggs are currently the preferred substrate for vaccine production with an estimated 600 688

million eggs used annually for this purpose worldwide. In general, two kinds of vaccine-quality 689

embryonated eggs are available for the production of influenza vaccines (human, live attenuated) 690

for intranasal administration: Specific Pathogen Free-Specific Antibody Negative (SPF-SAN) 691

and non- Specific Pathogen Free (non-SPF eggs) ( 43). 692

693

Only vaccine-quality embryonated hen’s eggs obtained from layer flocks meeting the health 694

surveillance requirements of the relevant national animal health authority and the national 695

regulatory authority for the production of influenza vaccine (human, live attenuated) for 696

intranasal administration should be introduced into or handled in the production area. 697

698 General requirements for animal health surveillance have been established by the World Organisation for 699 Animal Health ( 38). Internationally accepted requirements on hygiene and disease security procedures in 700 poultry breeding flocks and hatcheries have also been established ( 44). The relevant national animal health 701 authority and the national regulatory authority in Member Countries and Territories of the World 702 Organisation for Animal Health are bound to follow the OIE Terrestrial Animal Health Code ( 38, 44). The 703 relevant national animal health authority and the national regulatory authority should work together to 704 establish national animal health requirements for layer flocks from which vaccine-quality embryonated 705 eggs are obtained for production of influenza vaccines (human, live attenuated) for intranasal 706 administration. 707

708

Use of vaccine-quality SPF-SAN embryonated eggs: 709

710

The use of vaccine-quality SPF-SAN embryonated eggs is encouraged for the manufacture of 711

influenza vaccines (human, live attenuated) for intranasal administration. The use of vaccine-712

quality embryonated eggs from SPF-SAN layer flocks does not eliminate the need for 713

adventitious agent (as defined in section A.1.4.) testing. 714

715 The animal health requirements for SPF-SAN layer flocks are similar across regions and countries ( 45, 46, 716 38, 44). Hens and roosters in SPF-SAN layer flocks are kept under strictly isolated conditions to guarantee 717 freedom from the avian pathogens (SPF layer flocks) and antibodies (SAN layer flocks) against the avian 718 pathogens which are laid down in the national animal health and regulatory requirements. These flocks are 719

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not vaccinated against avian pathogens and must be kept in filtered-air positive-pressure poultry housing in 720 isolation from commercial poultry ( 39, 52). 721 722 A health surveillance program in SPF-SAN layer flocks is strictly followed and tests are performed 723 regularly to assure the SPF and SAN status. In some countries, SPF-SAN layer flocks are monitored 724 weekly for quality control. All birds are bled when an SPF-SAN layer flock is established, and thereafter a 725 percentage of the birds are bled at specified time intervals. The sera are screened for antibodies to the 726 relevant avian pathogens. These pathogens may also be detected in the flocks by culture or other detection 727 methods including PCR. Any death in an SPF-SAN layer flock is investigated to determine causality. 728 Permanent records of mortality and results of layer flock testing are kept for several (usually five) years. 729 Egg users should be notified immediately when any test results indicate infection with a specified pathogen 730 and when any deterioration in egg production or hatchability is observed in the source layer flock ( 47). 731

732 Layer flocks providing vaccine-quality SPF-SAN embryonated eggs for production of influenza vaccines 733 (human, live attenuated) for intranasal administration should be evaluated on a frequent basis to detect 734 exposure of the flock to avian pathogens, which have the potential to cause quality failure in assessments 735 for adventitious agents (as defined in section A.1.4.). The quality of SPF-SAN embryonated eggs varies 736 according to the extent of avian pathogen testing performed in the layer flocks ( 48). Avian pathogens of 737 interest in SPF-SAN layer flocks may vary by geographic region ( 45, 46, 39, 47, 44) and include as a 738 minimum: avian adenoviruses, avian encephalomyelitis virus, avian infectious bronchitis viruses, avian 739 infectious laryngotracheitis virus, avian leukosis viruses, avian nephritis virus, avian orthoreoviruses, avian 740 reticuloendotheliosis virus, chicken anemia virus, egg drop syndrome virus, fowlpox virus, infectious 741 bursal disease viruses, influenza A viruses, Marek's disease virus, Newcastle disease virus, Mycobacterium 742 avium, Mycoplasma gallisepticum, Mycoplasma synoviae, Salmonella gallinarum, Salmonella pullorum, 743 Salmonella species, and Haemophilus paragallinarum. 744 745 If vaccine-quality SPF-SAN embryonated eggs are used for production of influenza vaccine (human, live 746 attenuated) for intranasal administration, the manufacturer should ensure that layer flock health 747 surveillance is consistent with the requirements of the relevant national animal health authority and the 748 national regulatory authority. The SPF-SAN egg supplier should provide the manufacturer with a quality 749 control certificate showing the testing methods used and the test results performed in accordance with the 750 requirements of the relevant national animal health authority and the national regulatory authority. 751 752 The use of vaccine-quality embryonated eggs from SPF-SAN layer flocks for production of influenza 753 vaccine (human, live attenuated) for intranasal administration is not a regulatory requirement in any 754 country where such vaccine is currently manufactured. 755 756

Use of vaccine-quality non-SPF embryonated eggs: 757

758

As a large number of embryonated eggs are needed for human influenza vaccine production, it 759

may not always be feasible to use vaccine-quality embryonated eggs from SPF-SAN layer flocks. 760

Nowadays vaccine-quality non-SPF eggs represent the largest volume of embryonated eggs used 761

for human influenza vaccine production worldwide ( 43, 49). 762 763 If vaccine-quality non-SPF embryonated eggs are used for production of influenza vaccines (human, live 764 attenuated) for intranasal administration, the manufacturer should ensure that the layer flock health 765 surveillance is consistent with the requirements of the relevant national animal health authority and the 766 national regulatory authority ( 38, 43). The manufacturer should ensure that the non-SPF layer flocks are 767 managed with strict attention to environmental cleanliness and control of access to the flock. The 768 manufacturer should ensure that the vaccine-quality non-SPF embryonated eggs used for vaccine 769 production are highly consistent in their physical and biological qualities thereby meeting specified 770 requirements of cleanliness and viability. 771 772 Hens and roosters in non-SPF flocks are kept under conditions similar to the parent flock for the production 773 of day-old commercial layer chicks. The vaccination program against common avian pathogens in non-774 SPF layer flocks has similarities by region and country and generally includes: Marek's disease virus, 775 Salmonella species, avian coccidia, Newcastle disease viruses, avian infectious bronchitis viruses, 776 Gumboro, avian infectious laryngotracheitis virus, avian encephalomyelitis virus, Escherichia coli, and 777 chicken anemia virus (if necessary). 778 779

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Sera are collected throughout the life of the non-SPF layer flock and tested for antibodies against 780 Mycoplasma gallisepticum, Mycoplasma synoviae, Salmonella species, Newcastle disease viruses, avian 781 infectious bronchitis viruses, avian reoviruses, avian adenoviruses, avian infectious laryngotracheitis virus, 782 avian influenza viruses, avian encephalomyelitis virus, and chicken anemia virus. Vaccine-quality non-783 SPF embryonated eggs can be produced free of antibodies for specific diseases by adjusting the vaccination 784 program of the source layer flock ( 38, 44). 785 786 If vaccine-quality non-SPF embryonated eggs are used for production of influenza vaccine (human, live 787 attenuated) for intranasal administration, the manufacturer should ensure that the flock health surveillance 788 program is consistent with the requirements of the relevant national animal health authority and the 789 national regulatory authority. The supplier of vaccine-quality non-SPF embryonated eggs should provide 790 the manufacturer with a quality control certificate showing the vaccination program and the tests carried 791 out under their animal health surveillance program. The control certificate should be in accordance with 792 the requirements of the relevant national animal health authority and the national regulatory authority. 793

794 Much greater scrutiny should be placed on adventitious agents (as defined in section A.1.4.) testing when 795 vaccine-quality non-SPF embryonated eggs are used throughout the vaccine development and production 796 process. If the vaccine strain is produced in vaccine-quality non-SPF embryonated eggs, the national 797 regulatory authority and national control laboratory should specify the additional tests for the detection of 798 adventitious agents that could be derived from the substrates used in preparation of the donor virus, seed 799 virus strains, and vaccine virus. Knowledge of local, regional, and national zoonotic avian diseases would 800 aid in decision making on adventitious agents testing for influenza vaccines (human, live attenuated) for 801 intranasal administration ( 50, 51). Furthermore, knowledge of the vaccination and antibody testing 802 programs of non-SPF layer flocks required by the relevant national animal health authority and the national 803 regulatory authority should provide a foundation to make sound decisions on what adventitious agents to 804 test when vaccine-quality non-SPF embryonated eggs are used. 805 806

Only cell cultures meeting the requirements of the national regulatory authority should be used 807

in production of influenza vaccine (human, live attenuated) for intranasal administration. Cell 808

cultures should also conform to the principles established in the WHO Requirements for use of 809

animal cells in vitro substrates for the production of biologicals ( 16, 35). 810

811

A.4 Control of source materials 812

813

A.4.1 Choice of vaccine strain 814

815

The World Health Organization, with participation of the WHO Collaborating Centres and WHO 816

Essential Regulatory Laboratories, twice annually reviews the global influenza epidemiological 817

situation and recommends influenza reference viruses for the composition of seasonal influenza 818

vaccines for the Northern and Southern hemispheres in accordance with available evidence ( 4). 819

WHO, with participation of the WHO Collaborating Centres, WHO Essential Regulatory 820

Laboratories and WHO H5 Reference Laboratories, also reviews and reports the circulation of 821

wild type influenza viruses of pandemic potential as well as the status of development of 822

candidate influenza vaccine viruses to be used for pandemic preparedness ( 4). 823

824

Preparation of the live attenuated vaccine master seed virus is typically the responsibility of the 825

laboratories engaged in production of a specific influenza vaccine (human, live attenuated) for 826

intranasal administration. The preparing laboratory uses an attenuated master donor virus (which 827

contributes the essential attenuating genes) and a wild type influenza virus reference strain to 828

derive a reassortant virus. The reassortant virus is characterized fully to determine that it has the 829

proper genetic and phenotypic properties, and if satisfactory, becomes a master seed virus, which 830

may be used to produce a working seed virus and vaccine. 831

832 Although master and working seed viruses for an influenza vaccine (human, live attenuated) for intranasal 833 administration are typically used exclusively by the preparer, it is possible under some circumstances that a 834

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seed virus could be provided to other manufacturers. In this case, it is expected that validated quality 835 control procedures at the receiving manufacturer would be in place to identify the genetic characteristics as 836 well as the antigenic and attenuating properties of the seed virus adequately before its use in vaccine 837 production. 838

839

Influenza vaccines (human, live attenuated) for intranasal administration should include the 840

surface glycoproteins (haemagglutinin and neuraminidase) of the influenza virus reference 841

strains recommended by WHO for inclusion in vaccines ( 4), or from strains antigenically closely 842

related to them, as approved by the national regulatory authority. 843

844 Influenza reference viruses for antigenic analysis and for preparation of reassortant viruses may be obtained 845 from the WHO Collaborating Centres for Reference and Research on Influenza, or other custodian 846 laboratory (see Appendix 2). 847

In some years the specific influenza reference viruses used for the preparation of influenza vaccines 848 (human, live attenuated) and influenza vaccines (inactivated) differ, but the vaccine viruses in any case are 849 antigenically representative of the WHO recommended influenza reference viruses 4). 850

851

The passage history of the parental and reassortant viruses along with full documentation of the 852

characterization of the genetic and phenotypic properties of the master seed virus should be 853

submitted to the national regulatory authority for approval. 854

855

A.4.2 Substrate for virus propagation 856

857

A.4.2.1 Eggs used for seed virus growth 858 859

Vaccine seed virus is produced in vaccine-quality embryonated eggs from healthy layer flocks 860

which are monitored by methods approved by the relevant national animal health authority and 861

the national regulatory authority (see section A.3.2.). 862

863 In some countries, vaccine-quality 9 to 11-day old embryonated eggs are used. 864

865

In production of vaccine virus seed, the egg source layer flock should not have been vaccinated 866

with live Newcastle disease virus vaccine. In addition, layer flocks should not be receiving any 867

chemotherapeutic agents (e.g. antimicrobial agents or coccidiostats). It is also recommended that 868

vaccine-quality embryonated eggs be obtained from young hens. 869

870 In countries where use of live Newcastle disease vaccine or any other live vaccine is mandatory, 871 vaccination should take place during the first few weeks of the hen's life and well before the use of flocks 872 for egg supply. 873

874

Hence, the use of vaccine-quality SPF-SAN embryonated eggs is required for growth of the seed 875

virus for influenza vaccine (human, live attenuated) for intranasal administration (see section 876

A.3.2.). 877

878

A.4.2.2 Eggs used for vaccine production 879 880

Vaccine is produced in vaccine-quality embryonated eggs from healthy layer flocks which are 881

monitored by methods approved by the relevant animal health authority and the national 882

regulatory authority (see section A.3.2.). 883

884 In some countries, vaccine-quality 9 to 11-day old embryonated eggs are used. 885

886

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In production of vaccine, the eggs source layer flock must not have been vaccinated with live 887

Newcastle disease virus vaccine. In addition, layer flocks should not be receiving any 888

chemotherapeutic agents (e.g. antimicrobial agents or coccidiostats). It is also recommended that 889

vaccine-quality embryonated eggs be obtained from young hens. 890

891 In countries where use of live Newcastle disease vaccine or any other live vaccine is mandatory, 892 vaccination should take place during the first few weeks of the hen's life and well before the use of flocks 893 for egg supply. 894

895

Hence, the use of vaccine-quality SPF-SAN embryonated eggs is encouraged for the 896

manufacture of influenza vaccine (human, live attenuated). See section A.3.2. 897

898

A.4.3 Master cell bank and manufacturer’s working cell bank 899

900

A.4.3.1 Cell bank system 901 902

If a cell line is used for the manufacture of influenza vaccine (human, live attenuated) for 903

intranasal administration, it should be based on a cell bank system. The national regulatory 904

authority should approve the master cell bank and should establish the maximum number of 905

passages (or population doublings) by which the manufacturer’s working cell bank is derived 906

from the master cell bank and the maximum number of passages (or population doublings) of the 907

production cultures. 908

909 WHO has established a reference cell bank of Vero cells characterized in accordance with WHO 910 requirements for the use of animal cells as in vitro substrates for the production of biologicals ( 16, 35). 911 The requirements for use of animal cells may be revised during the lifespan of this document on influenza 912 vaccines (human, live attenuated) for intranasal administration. Manufacturers and national regulatory 913 authorities are encouraged to monitor WHO publications for corresponding updates 914 (http://www.who.int/immunization/en/) 915 916 The WHO 10-87 cell bank of Vero cells is stored at the European Collection of Animal Cell Cultures 917 (ECACC), Porton Down, England and at the American Type Culture Collection (ATCC), Rockville, 918 Maryland, USA. This cell bank should not be considered as the master cell bank for direct use in vaccine 919 production, but may be used to establish master cell banks for thorough requalification. Producers of 920 biologicals and national regulatory authorities can obtain culture of these Vero cells (free of charge), as 921 well as additional background information, from Quality, Safety and Standards of Biologicals, World 922 Health Organization, 1211 Geneva 27, Switzerland. 923

924

A.4.3.2 Identity test 925 926

The master cell bank should be characterized according to the WHO requirements for the use of 927

animal cells as in vitro substrates for the production of biologicals as they relate to continuous 928

cell lines, or to human diploid cells, as appropriate ( 16, 35). 929

930

The manufacturer’s working cell bank should be identified by means, inter alia, of biochemical 931

(e.g. isoenzyme analysis), immunological and cytogenetic marker tests, and DNA fingerprinting 932

approved by the national regulatory authority and the national control laboratory. 933

934

A.4.4 Cell culture medium 935

936

At every stage of preparation of donor viruses and vaccines, the sera used for propagation of 937

cells (including the donor strains, master seed lot, working seed lot, master cell bank, working 938

cell bank and production cells cultures) should be tested to demonstrate freedom from bacteria, 939

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fungi and mycoplasma according to the WHO general requirements for sterility of biological 940

substances ( 54). 941

942

The sera used in propagation of cells should also be tested to demonstrate freedom from 943

adventitious agents according to the WHO requirements ( 16, 35) Manufacturers are encouraged 944

to explore possibilities of using serum-free media for the production of influenza vaccines 945

(human, live attenuated) for intranasal administration. 946

947

Suitable tests for detecting bovine viruses in serum using either primary bovine testis cells or 948

continuous bovine kidney-cell lines known to be sensitive to bovine viruses are given in 949

Appendix 1 of the WHO recommendations for the production and control of poliomyelitis 950

vaccine (oral) ( 55, 56) 951 952 Where approved by the national regulatory authority and the national control laboratory, alternative tests 953 for bovine viruses may be used. As an additional monitor of quality, sera may be examined for freedom 954 from phage and endotoxin. 955

956

The source(s) of serum of bovine origin should be approved by the national regulatory authority. 957

The serum should comply with the WHO guidelines on transmissible spongiform 958

encephalopathies in relation to biological and pharmaceutical products ( 57). 959

960

Human serum should not be used. If human serum albumin is used, it should meet the WHO 961

requirements for the collection, processing and quality control of blood, blood components and 962

plasma derivatives ( 58) as well as the WHO guidelines on transmissible spongiform 963

encephalopathies in relation to biological and pharmaceutical products ( 57). 964

965

Penicillin and other beta-lactam antimicrobial agents should not be used at any stage of 966

manufacture. 967

968 Other antimicrobial agents may be used at any stage of manufacture, provided the quantity present in the 969 final lot is acceptable to the national regulatory authority. Non-toxic pH indicators may be added, e.g. 970 phenol red in a concentration of 0.002%. Only substances that have been approved by the national 971 regulatory authority may be added. 972

973

Trypsin used for preparing the master cell bank, working cell bank, and production cell cultures 974

should be tested and found free of cultivable bacteria, fungi, mycoplasmas, and infectious 975

viruses, especially parvoviruses appropriate to the species of animal used. The methods used to 976

ensure this should be approved by the national regulatory authority and the national control 977

laboratory. 978

979

The source of trypsin of bovine origin, if used, should be approved by the national regulatory 980

authority. Bovine trypsin, if used, should comply with current WHO guidelines on transmissible 981

spongiform encephalopathies in relation to biological and pharmaceutical products ( 57). 982

983

A.4.5 Virus strains 984

985

A.4.5.1 Origin and preparation of virus strains 986 987

Strains of influenza virus used in the production of influenza vaccines (human, live attenuated) 988

for intranasal administration should be identified by historical records, which should include 989

information on the origin of the donor virus strains, both the attenuated virus donor strain and the 990

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wild type virus reference strain as well as on the process used for preparing the attenuated master 991

seed lot and working seed lot. 992

993

Only strains that have been isolated in vaccine-quality embryonated eggs (see section A.3.2.), in 994

cells derived from eggs or in mammalian cells approved by the national regulatory authority for 995

human vaccine production should be used (unless reverse genetics technology is being used for 996

preparation of the seed lot as described later in this section). The national regulatory authority 997

should also approve the attenuated virus donor strain and wild type virus reference strain used 998

for preparing the master seed lot. The vaccine seed viruses should have the attenuation 999

phenotype of the attenuated donor virus strain and the surface antigens (haemagglutinin and 1000

neuraminidase) should correspond to the influenza reference viruses recommended by WHO for 1001

vaccine preparation ( 4). 1002

1003

Experience shows that candidate seed viruses derived by classical reassortment should be cloned 1004

at least three times by limiting dilution passage in vaccine-quality SPF-SAN embryonated eggs 1005

or plaque purified in qualified cells to assure purity of the desired attenuated seed virus. Since 1006

antigenic changes are possible during the development of reassortant viruses, the absence of 1007

antigenic changes in the master seed lot and working seed lot should be demonstrated at least by 1008

haemagglutination inhibition tests using antibodies to the haemagglutinins of the candidate 1009

influenza vaccine virus and of the wild type reference virus strain. 1010

1011

Where reassortant viruses are used, the method for producing the reassortant should be approved 1012

by the national regulatory authority. Preparation of the live attenuated vaccine master seed 1013

viruses are typically the responsibility of the laboratories engaged in production of specific 1014

influenza vaccines (human, live attenuated) for intranasal administration. 1015

1016 Master and working seed viruses for an influenza vaccine (human, live attenuated) for intranasal 1017 administration are typically used exclusively by the preparer. If a master or working seed virus is provided 1018 to another manufacturer, the receiving manufacturer should use validated methods to identify the genetic 1019 characteristics and the antigenic and attenuating properties of the seed virus before it is used in vaccine 1020 production. The receiving manufacturer should also have cGMP in place to handle the master seed. 1021

1022

Where reverse genetics is used to generate the reassortant vaccine virus, the influenza 1023

haemagglutinin and neuraminidase genes may be derived from a variety of sources (egg isolate, 1024

mammalian cell isolate or virus in clinical specimen). With reverse genetics the source of viral 1025

genes is not as critical as for classical reassortment because the haemagglutinin and 1026

neuraminidase genes are expected to be free of adventitious agents associated with wild type 1027

virus by virtue of the recombinant DNA technology employed ( 24). 1028

1029

The cell substrate used for transfection to generate the reassortant virus by reverse genetic 1030

techniques should be appropriate for human vaccine production and approved by the national 1031

regulatory authority. The overall process for derivation of the reassortant virus prepared by 1032

reverse genetics should be approved by the national regulatory authority. WHO Guidance on 1033

development of influenza vaccine reference viruses by reverse genetics ( 24) and WHO 1034

Guidelines for assuring the quality of pharmaceutical and biological products prepared by 1035

recombinant DNA technology ( 59) should be considered. 1036

1037

For work with highly pathogenic wild type influenza viruses and newly emerging pandemic 1038

viruses, higher levels of bio-containment are required as described in the WHO biosafety risk 1039

assessment guidelines for the production and quality control of human influenza pandemic 1040

vaccines should be followed ( 15). Facilities for vaccine production should provide containment 1041

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features to accommodate the candidate influenza vaccines that are derived from the wild type 1042

influenza viruses. 1043

1044

If any materials of animal (non-avian) origin are used in vaccine production, they should comply 1045

with the WHO guidelines on transmissible spongiform encephalopathies in relation to biological 1046

and pharmaceutical products ( 57) and should be approved by the national regulatory authority. 1047

1048

Reference strains for antigenic analysis may be obtained from the WHO collaborating centres for 1049

reference and research on influenza (see Annex 2), or other custodian laboratory. 1050

1051

A.4.5.2 Seed lot system 1052 1053

The production of vaccine should be based on a virus seed lot system. The influenza virus 1054

contained in each seed lot should be identified as the appropriate strain by methods acceptable to 1055

the national regulatory authority. The maximum number of passages between a master seed lot 1056

and a working seed lot should be approved by the national regulatory authority. The virus in the 1057

final vaccine should be not more than one passage removed from the working seed lot. The 1058

haemagglutinin and neuraminidase of the seed lot viruses should be identified by suitable tests. 1059

1060 The master seed lot may be considered for use as a working seed lot with the approval of the national 1061 regulatory authority. If the master seed lot is used as the working seed lot, the final vaccine should be not 1062 more than one passage removed from the master seed lot. 1063

1064

A.4.5.3 Tests on seed lots 1065 1066

A.4.5.3.1 Adventitious agents 1067 1068

The master seed lot and working seed lot should be shown to be free from relevant adventitious 1069

agents (as defined in section A.1.4.) by tests or procedures approved by the national regulatory 1070

authority and the national control laboratory in accordance with the WHO general requirements 1071

for the sterility of biological substances ( 53, 54 Part A, sections 5.2 and 5.3). 1072

1073 Strategies to ensure freedom from adventitious agents (as defined in section A.1.4.) in the final vaccine 1074 involve a combination of testing the seed virus and validation of the production process which depends on 1075 the substrate used for production and on the process developed for vaccine manufacture. 1076 1077 Validation of processes 1078

1079 Since the manufacturing of influenza vaccine (human, live attenuated) for intranasal administration is 1080 unlikely to include processes that are effective in inactivating potential contaminating agents, reliance is 1081 placed primarily on general precautions against microbial contamination in manufacture, on the quality of 1082 materials used for manufacture, and on testing for adventitious agents ( 53, 54). In some instances, 1083 removal of contaminating agents may be possible. For example, filtration steps may be used to remove 1084 bacteria or fungi derived from eggs. The production process should be validated, including steps designed 1085 for removal of potential contaminating microbial agents. Process validation may be performed with 1086 appropriate model agents. If removal of a potential contaminant cannot be demonstrated and validated, a 1087 testing strategy should be implemented to document the ability of individual steps and the overall process 1088 to prevent the introduction of potential microbial contaminating agents ( 53, 54). 1089 1090 Cells used to prepare vaccine 1091 1092 The susceptibility of cell cultures to various human pathogens should be taken into account and used in 1093 considering a list of potential human pathogens to be included in testing for adventitious agents in master 1094 seed lots and working seed lots passaged in the cells. Pathogens to be considered include adenovirus, 1095

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parainfluenza virus, respiratory syncytial virus, coronavirus, rhinovirus, enterovirus, human herpesvirus 4 1096 (Epstein-Barr virus), herpes simplex virus, cytomegalovirus and mycoplasmas ( 16, 35). 1097 1098 It is recognized that when a vaccine strain changes, there may be time constraints that make testing master 1099 seed lots and working seed lots for adventitious agents problematic, and the full results of such testing may 1100 not always be available before further processing. Therefore, the development and use of properly validated 1101 rapid assays such as immunoassays or PCR is encouraged. 1102 1103 If an adventitious agent is detected in a master seed lot and/or working seed lot prepared in cell culture, 1104 these lots should not be used for vaccine production. 1105 1106 Embryonated eggs used to prepare vaccine 1107 1108 Whether vaccine-quality SPF-SAN or non-SPF embryonated eggs are used for vaccine preparation, a 1109 judicious testing strategy for specific potential adventitious agents is needed (see section A.3.2.). The use 1110 of properly validated rapid assays such as immunoassays or PCR is encouraged. 1111 1112 The use of vaccine quality SPF-SAN embryonated eggs does not eliminate the need for adventitious agent 1113 testing. However, much greater scrutiny should be placed on adventitious agents testing when vaccine-1114 quality non-SPF embryonated eggs are used for passage of the attenuated virus donor strain, wild type virus 1115 reference strain, master seed lot, working seed lot or vaccine. In this case, the national regulatory authority 1116 should specify any additional tests for the detection of the adventitious agents that could be derived from 1117 the substrates used in preparation of the master seed and working seed lots and in preparation of the 1118 vaccine (see section A.3.2.). 1119 1120 If a potential contaminating agent is detected, the virus master seed lot or virus working seed lot should not 1121 be used for vaccine production. 1122

1123

A.4.5.3.2 Attenuation 1124 1125

Retention of key genetic and phenotypic characteristics related to attenuation may be 1126

demonstrated for either the master seed lot or working seed lot of a virus strain to be introduced 1127

into clinical use. 1128

1129

The methods used to demonstrate attenuation may vary for each vaccine virus but should include 1130

at least one of the following: 1) the full genetic sequence of the virus master seed lot or virus 1131

working seed lot, 2) in vitro tests for the key phenotypic markers of the attenuated virus donor 1132

strain, and 3) studies in an appropriate animal model to assess the in vivo aspects of vaccine 1133

virus attenuation. Specific tests used to assess virus attenuation should be approved by the 1134

national regulatory authority and the national control laboratory. 1135

1136

The development of influenza vaccine (human, live attenuated) for intranasal administration 1137

should include an early assessment of the biosafety level required to work with the attenuated 1138

donor virus and the master seed viruses derived from the donor. Direct handling of an 1139

established attenuated donor virus strains for which much experience exists (such as the cold 1140

adapted H2N2 strains) and master seed viruses prepared for seasonal influenza vaccine 1141

viruses can generally be done using established BSL-2 practices ( 15, 41, 64). 1142

1143

Work with highly pathogenic wild type influenza viruses and newly emerging pandemic viruses, 1144

require higher levels of bio-containment and the WHO biosafety risk assessment and guidelines 1145

for the production and quality control of human influenza pandemic vaccines should be followed 1146

( 15). Facilities for vaccine production should provide adequate containment features to 1147

accommodate the candidate influenza vaccines that are derived from the wild type influenza 1148

viruses. Guidance for safe handling of influenza viruses is subject to revision based on evolving 1149

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experience, and the level of biosafety to be used should be decided in consultation with the 1150

national regulatory authorities. 1151

1152

For candidate influenza vaccine viruses prepared using highly pathogenic avian influenza viruses 1153

as the starting material, additional tests should be performed. Optimally, the strategy to 1154

demonstrate elimination of highly pathogenic characteristics includes assessment of the ability of 1155

the candidate influenza vaccine virus to produce plaques in cell culture with and without trypsin, 1156

the ability to cause chick embryo death, pathogenicity in chickens (as defined in section A.1.4.) , 1157

and attenuation in ferrets ( 15). Specific tests used to assess the removal of highly pathogenic 1158

features should be approved by the national regulatory authority and the national control 1159

laboratory. 1160

1161

For candidate influenza vaccine viruses prepared using non-highly pathogenic influenza viruses 1162

of animal-origin (avian, swine, equine, canine, others), additional safety tests may also be needed 1163

as described in WHO published guidance ( 15). Specific tests used to assess the candidate 1164

influenza vaccine viruses should be approved by the national regulatory authority and the 1165

national control laboratory. 1166

1167

The virus seed lot should be stored at a temperature lower than -60°C; unless it is in the 1168

lyophilized form, in which case it should be stored at a temperature lower than -20°C. 1169

1170

1171

A.5 Control of vaccine production 1172

1173

A.5.1 Production precautions 1174

1175

The general production precautions formulated in the WHO good manufacturing practices and 1176

quality assurance for biological products should be followed ( 40, 60, 59). Although the WHO 1177

recommendations for the production and control of influenza vaccine (inactivated) ( 3) are similar 1178

in many ways, the manufacture of influenza vaccines (human, live attenuated) for intranasal 1179

administration has important differences. The following should be observed: 1180

1181

• for embryonated egg-derived vaccines, only allantoic and amniotic fluids may be 1182

harvested, 1183

1184

• beta-lactam antimicrobial agents should not be used at any stage in the manufacture 1185

of the vaccine, and should not be permitted to come in contact with any part of the 1186

production equipment. 1187

1188

Minimal concentrations of other suitable antimicrobial agents may be used. 1189 1190

Small quantities of antimicrobial agents other than beta-lactam agents may be added with the approval of 1191 the national regulatory authority. However, if antimicrobial agents are added, samples for sterility testing 1192 should be taken before the antibiotic is added. 1193

1194

A.5.2 Production of monovalent virus pool 1195

1196

A.5.2.1 Single harvests 1197 1198

For egg-derived vaccines, each strain of virus should be grown in the allantoic cavity of vaccine-1199

quality embryonated eggs derived from healthy layer flocks (see section A.3.2.). After 1200

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incubation at a controlled temperature, both the allantoic and amniotic fluids may be harvested to 1201

prepare a single harvest. 1202

1203 In some countries, vaccine-quality 9 to 11 day old embryonated eggs are used. 1204 1205 It is recognized that adventitious agents (as defined in section A.1.4.) can be introduced at any point in the 1206 process. It is a wise precaution to pool the allantoic fluids from a limited number of eggs (e.g. 30-50) and 1207 to test these small pools for sterility and virus titre before blending into the Single Harvest. The pools 1208 should be stored at a temperature of 2-8°C. In case any contamination is detected in downstream processes, 1209 the manufacturer can test this small allantoic fluid pool for adventitious agents and virus titre to trace back 1210 where contamination or deterioration of virus titre occurred. 1211 1212 The use of vaccine quality SPF-SAN embryonated eggs does not eliminate the need for adventitious agent 1213 testing. If the vaccine is prepared in vaccine-quality SPF-SAN embryonated eggs, it may possible to obtain 1214 single harvests that are free of adventitious agents. However, in the event it is not achievable, a bioburden 1215 limit on single harvests may be considered with approval of the national regulatory authority. 1216

1217 Much greater scrutiny should be placed on adventitious agents testing when vaccine-quality non-SPF 1218 embryonated eggs are used to produce single harvests. If vaccine-quality non-SPF embryonated eggs are 1219 used, the national regulatory authority should specify any additional tests for the detection of the 1220 adventitious agents that could be derived from the egg substrates (see section A.3.2.) used in preparation of 1221 attenuated virus donor strains, wild type virus reference strains, virus master seed lot and virus working 1222 seed lots and in preparation of the vaccine. 1223

1224 For influenza vaccines (human, live attenuated) for intranasal administration prepared in cell 1225

cultures, each virus strain should be grown in cells approved by the national regulatory authority. 1226

1227

For both egg-derived and cell-derived vaccines, a number of single harvests of the same virus 1228

strain may be combined to give a monovalent virus pool. Cell-derived monovalent virus pools 1229

should not be mixed with egg-derived monovalent virus pools. 1230

1231

A.5.2.2 Tests of control eggs or cell cultures 1232 1233

The national regulatory authority should determine the need for control samples, the sample size 1234

to be examined, the time at which the control samples should be taken during the production 1235

process, and how the control samples are to be maintained. 1236

1237

When using vaccine-quality embryonated eggs, a portion (2% or at least 20 eggs, whichever of 1238

the quantities is greater) of each batch of the eggs used for vaccine virus propagation is held as 1239

un-inoculated control eggs. These control eggs are incubated for the same time, same 1240

temperature, and same humidity as the inoculated embryonated eggs. At the time of harvesting 1241

the virus from the inoculated embryonated eggs, allantoic fluids are also taken from the un-1242

inoculated control embryonated eggs and examined for the existence of haemagglutinating 1243

agents (see Part A, section 5.2.2.1). 1244

1245 To ensure freedom from adventitious agents (as defined in section A.1.4.), the greatest reliance is placed on 1246 the continuous health monitoring of the layer flock from which the vaccine-quality embryonated eggs are 1247 obtained (see section A.3.2.). 1248 1249 The use of vaccine quality SPF-SAN embryonated eggs does not eliminate the need for adventitious agent 1250 testing. Much greater scrutiny should be placed on adventitious agents testing when vaccine-quality non-1251 SPF embryonated eggs are used. 1252 1253 The national regulatory authority and the national control laboratory should approve alternative strategies 1254 and methods for ensuring freedom from adventitious agents. 1255 1256

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The national regulatory authority and the national control laboratory may include additional tests for 1257 microorganisms if necessary. Tests for avian leukosis virus in embryonated eggs are considered essential. 1258

1259 When testing is performed on control cell cultures, a sample equivalent to at least 500 ml of the 1260

cell culture suspension, kept at the same cell concentration employed for vaccine production, is 1261

tested. Control cell cultures are incubated for at least two weeks and are examined during this 1262

observation period for evidence of cytopathic changes. For the test to be valid, not more than 1263

20% of the control cell cultures may have been discarded for non-specific reasons. 1264

1265

If any test described in this section shows evidence of the presence of an adventitious agent in a 1266

control cell culture or a control embryonated egg, the influenza virus grown in the corresponding 1267

inoculated cultures or eggs should not be used for vaccine production. 1268

1269

Samples not tested immediately should be stored at –60oC or below. 1270

1271

A.5.2.2.1 Tests for haemagglutinating and haemadsorbing agents 1272 1273

For influenza vaccine (human, live attenuated) for intranasal administration prepared in vaccine-1274

quality embryonated eggs, a sample of 0.25 ml of allantoic fluid taken from each control egg 1275

should be tested for haemagglutinating agents by the addition of chick erythrocytes both directly 1276

and after one passage of the control allantoic fluid through vaccine-quality SPF-SAN eggs. The 1277

details of the test should be approved by the national regulatory authority and the national 1278

control laboratory. 1279

1280

For influenza vaccine (human, live attenuated) for intranasal administration prepared in cell 1281

culture, testing for the presence of haemadsorbing viruses at the end of the observation period or 1282

at the time the virus is harvested from the production substrate, whichever comes later, should 1283

include at least 25% of control cells. The control cells should be tested using guinea pig red 1284

blood cells. If the guinea pig red blood cells have been stored, the duration of storage should not 1285

have exceeded seven days, and the storage temperature should have been in the range of 2-8oC. 1286

In testing for haemadsorbing viruses, calcium and magnesium ion should be absent from the 1287

medium. 1288 1289 This test is usually done using a suspension of 1% guinea pig red blood cells. However, in some countries, 1290 the national regulatory authority requires that additional tests for haemadsorbing viruses should be made in 1291 other types of red blood cells, including those from humans (blood group O), monkeys and chickens (or 1292 other avian species). 1293 1294 The results of all tests should be read after incubation for 30 min at 0-4oC and again after a further 1295 incubation for 30 min at 20-25

oC. A further reading for test with monkey red blood cells should also be 1296

taken after incubation for 30 min at 34-37 o

C. 1297 1298 In some countries the sensitivity of each new batch of red blood cells is demonstrated by titration against a 1299 haemagglutinin antigen before use in the haemadsorption test. 1300

1301

A.5.2.2.2 Tests on supernatant fluids from cell cultures 1302 1303

Samples of at least 10 ml of the pooled supernatant fluid from the control cell cultures collected 1304

at the end of the observation period should be tested for the presence of adventitious agents (as 1305

defined in section A.1.4.) in monolayers of three indicator cell lines: 1306

1307

• Cultures of cells of the same species and tissue type as those used for production, 1308

• Cultures of a human diploid cell line, 1309

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• Cultures of another cell line from a non-human species. 1310

1311

The samples should be inoculated into containers of these indicator cell cultures, in such a way 1312

that the dilution of the supernatant fluid in the nutrient medium does not exceed one in four. The 1313

area of the indicator cell sheet should be at least 3 cm2/ml of supernatant fluid. At least one 1314

container of each of the cell cultures should remain un-inoculated and serve as a control. 1315

1316

The cultures should be incubated at a temperature of 35-37 o

C and should be observed for 1317

cytopathic effect (and other evidence of a replicating adventitious agent) in the indicator cells for 1318

a period of at least two weeks. 1319 1320 The use of properly validated rapid assays such as immunoassays or PCR that could be conducted within 1321 the time constraints of the procedure is encouraged. 1322 1323

A.5.2.2.3 Identity test for continuous cell culture 1324 1325

For monovalent virus pools produced in continuous cell culture, the control cells should be 1326

identified by means approved by the national regulatory authority and the national control 1327

laboratory. Biochemical (e.g. isoenzyme analysis), immunological and/or cytogenetic marker 1328

tests may be considered in confirming identity. 1329

1330

A.5.2.3 Clarification and purification 1331 1332

The monovalent virus pool should be clarified and purified by centrifugation and/or other 1333

suitable methods approved by the national regulatory authority. 1334

1335 The aim of clarification and purification is to remove cell debris. For vaccine prepared in vaccine-quality 1336 embryonated eggs, a sterile filtration step may be considered to reduce bioburden. It is advisable to clarify 1337 and purify the influenza virus under conditions optimized to preserve its infectivity and antigenic properties. 1338 1339 Sterile filtration should be validated. 1340

1341

A.5.3 Control of monovalent virus pools 1342

1343

At the time the allantoic or tissue culture fluids are pooled to prepare the monovalent virus pool 1344

and before clarification and purification, samples should be set aside for examination for 1345

adventitious agents (as defined in section A.1.4.). If the samples are not tested immediately, they 1346

should be stored at -60°C or below. 1347

1348

For the purposes of the tests recommended in this section to verify neutralization of virus harvest, 1349

hyper-immune antibody preparations should be of an origin that will not cross react with the 1350

antigens of cells or eggs used in production of the monovalent virus pool . The virus used for the 1351

production of the hyper-immune antibody preparations should be grown either in non-avian cell 1352

cultures or in vaccine-quality SPF-SAN embryonated eggs. If vaccine quality embryonated eggs 1353

are used, they should be obtained from a layer flock different from that used to supply the 1354

vaccine production embryonated eggs. 1355

1356 For vaccines intended for use in a pandemic situation, hyperimmune sera for tests described in this section 1357 may not be readily available when they are needed for release and distribution of vaccines. As an 1358 alternative for both pandemic and non-pandemic situations, a strategy to identify potential contaminating 1359 agents by PCR methods may be considered. Alternative strategies should be approved by the national 1360 regulatory authority and the national control laboratory. 1361

1362

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A.5.3.1 Infectivity (potency) 1363 1364

The influenza virus content in the clarified monovalent virus pool should be determined by 1365

titration in vaccine-quality embryonated eggs or cell cultures. The number of infectious doses of 1366

vaccine should be expressed in EID50, TCID50 or pfu per unit volume (see section A.7.3). 1367

1368 Additional equivalent methods dependent on the replication of virus in embryonated eggs or cell cultures 1369 (e.g. fluorescent focus assay expressed as fluorescent focus units) may be considered for determining 1370 potency. 1371

1372

A.5.3.2 Attenuation 1373 1374

Retention of key genetic and phenotypic characteristics related to attenuation of the candidate 1375

influenza vaccine virus should be demonstrated for the monovalent virus pool of a strain to be 1376

introduced into clinical use. The methods used to demonstrate attenuation of the monovalent 1377

virus pool may vary ( 61) and may include at least one of the following: 1) the full genetic 1378

sequence of the virus of the monovalent virus pool, 2) in vitro tests of the monovalent virus pool 1379

for the key phenotypic markers of the attenuated virus donor strain, and 3) studies in an 1380

appropriate animal model (e.g. ferrets) to assess the in vivo aspects of attenuation of the vaccine 1381

preparation ( 15). Attenuating features of the vaccine virus in the monovalent pool are expected 1382

to be identical with those of the seed virus (see section A.4.5.3.2). 1383

1384

A.5.3.3 Identity 1385 1386

Each monovalent virus pool lot should be identified as containing live attenuated influenza virus 1387

of the appropriate strain by methods acceptable to the national regulatory authority. 1388

1389

A.5.3.4 Adventitious agent tests 1390 1391

The tests described in this section form the traditional basis for identifying adventitious agents 1392

(as defined in section A.1.4.). For vaccines intended for use in a pandemic situation, 1393

hyperimmune sera needed for tests in vaccine-quality embryonated eggs or cell cultures 1394

described in this section may not be readily available when they are needed for release and 1395

distribution of vaccines. Therefore, more recent developments involving PCR methods need to 1396

be considered both to improve the specificity and sensitivity of adventitious agent testing, as well 1397

as to anticipate situations in which delays in vaccine availability would be detrimental. The 1398

national regulatory authority and the national control laboratory should approve the specific 1399

methods used to fulfil adventitious agent testing. 1400

1401 The use of vaccine quality SPF-SAN embryonated eggs does not eliminate the need for adventitious agent 1402 testing. Much greater scrutiny should be placed on adventitious agents testing when vaccine-quality non-1403 SPF embryonated eggs are used. 1404

1405

The monovalent virus pool passes if there is no evidence of any adventitious agent attributable to 1406

the virus pool. 1407

1408

A.5.3.4.1 Tests in vaccine-quality embryonated eggs 1409 1410

For vaccines produced in vaccine-quality embryonated eggs, a sample of at least 10 ml of each 1411

monovalent virus pool should be tested for the presence of adventitious agents (as defined in 1412

section A.1.4.) by inoculation of embryonated eggs. After neutralization of the influenza virus by 1413

hyper-immune antibody preparation, the monovalent virus pool should be inoculated in vaccine-1414

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quality embryonated eggs. At least 0.25 ml of the virus/antibody mixture per egg should be used 1415

for inoculation of one group of eggs by the allantoic route and a separate group of eggs by the 1416

yolk sac route. The national regulatory authority should approve the method of incubation of the 1417

embryonated eggs and the observation time. None of the embryonated eggs should show 1418

evidence of the presence of any adventitious agents. 1419

1420

A.5.3.4.2 Tests in cell cultures 1421 1422

For vaccines produced in embryonated eggs or cell cultures, a sample of at least 5 ml of the 1423

monovalent virus pool should be tested for freedom from adventitious agents (as defined in 1424

section A.1.4.) using cell cultures as described below. After neutralization of the influenza virus 1425

by hyper-immune antibody preparation, the monovalent virus pool should be inoculated on cell 1426

cultures of human cells, simian cells, chicken cells, or cells of the species used for vaccine 1427

production. The national regulatory authority should approve the cell cultures, the method of 1428

incubation and the period of observation. None of the cell cultures should show evidence of any 1429

adventitious agents. When chicken cells are used for vaccine production, the absence of avian 1430

leukosis viruses should be ascertained by testing. 1431

1432

A.5.3.4.3 Tests for bacteria, fungi, and mycoplasma 1433 1434

The monovalent virus pool should be tested to demonstrate freedom from bacteria, fungi and 1435

mycoplasma according to the WHO general requirements for sterility of biological substances 1436

( 53, 54). 1437

1438

A.5.3.4.4 Test for mycobacteria 1439 1440

Each monovalent virus pool should be tested for the presence of mycobacteria by methods 1441

appropriate for the detection of the organisms most likely to be found in the embryonated eggs or 1442

cell cultures used. 1443

1444 Its is common practice to concentrate the virus harvest by centrifugation and to inoculate the pellet into 1445 guinea pigs or on to solid media shown to be suitable for the detection of mycobacteria. 1446

1447 1448

A.5.3.5 Residual cell substrate DNA in cell-derived vaccines 1449 1450

For viruses grown in continuous cell culture, the purified monovalent virus pool should be tested 1451

for residual cellular DNA. The purification process should be shown to consistently reduce the 1452

level and molecular size of cellular DNA ( 16). This test should be appropriate for the cell culture 1453

used and should be approved by the national regulatory authority. This test may be omitted with 1454

the agreement of the national regulatory authority, if the manufacturing process is validated for 1455

residual DNA. 1456

1457

A.5.3.6 Tests for chemicals used in production 1458 1459

The concentration of each chemical added during production should be determined in the 1460

monovalent virus pool vaccine using methods approved by the national regulatory authority. The 1461

concentrations should not exceed the limits specific by the national regulatory authority. For 1462

preservatives, the national regulatory authority should approve the method of testing and the 1463

concentration. 1464

1465

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Alternatively, tests for chemicals may be performed on the final bulk. 1466

1467

A.5.4 Control of final bulk 1468

1469

Final bulks are prepared by mixing and diluting monovalent virus pools of the relevant strains. 1470

In the preparation of the final bulk, only preservatives, stabilizers or other substances, including 1471

diluents, approved by the national regulatory authority should be added. Such substances should 1472

have been shown by appropriate tests not to impair the safety or effectiveness of the product in 1473

the concentrations used, and should not be added before samples have been taken for any tests 1474

that would be affected by their presence. The operations necessary for preparing the final bulk 1475

should be conducted in such a manner as to avoid contamination of the product. 1476

1477

A.5.4.1 Infectivity (potency) 1478 1479

The influenza virus content of the clarified final bulk suspension should be determined by 1480

infectivity titration in vaccine-quality embryonated eggs or cell cultures. The number of 1481

infectious doses of vaccine should be determined (total and for each component virus), and the 1482

results expressed as EID50, TCID50 or pfu per human dose (see Section A.7.3). 1483

1484 Vaccines for use during pandemics are likely to contain only one strain. Additional methods dependent 1485 on the replication of virus in vaccine-quality embryonated eggs or cell cultures (e.g. fluorescent focus 1486 assay expressed as fluorescent focus units) may be considered for determining potency. 1487 1488 This test may be omitted if such a test is performed on each final lot. 1489

1490

A.5.4.2 Sterility 1491 1492

Each final bulk should be tested for sterility by a method approved by the national regulatory 1493

authority and national control laboratory. 1494

1495

Many countries have regulations governing sterility testing. Where these do not exist, the WHO 1496

general requirements for the sterility of biological substances ( 53, 54) should be satisfied. 1497

1498

If a preservative, stabilizer or other substance has been added to the vaccine, appropriate 1499

measures should be taken to prevent it from interfering with the sterility test. 1500

1501

A.5.4.3 Protein and residual DNA content 1502 1503

Measurements of protein and residual DNA may be specified based on the nature of the vaccine 1504

being produced ( 16, 62). For example, a vaccine could be prepared from allantoic fluid and not 1505

be further purified before preparation of the final vaccine; this preparation would have an 1506

extremely high protein and ovalbumin content. Alternatively, a vaccine could be prepared from 1507

the harvested medium used to propagate cell cultures and purified to remove residual DNA and 1508

protein. The aim should be to establish parameters suitable for the production and use intended 1509

for a vaccine to ensure consistency of the vaccine’s composition and its clinical performance. 1510

The parameters specified should be approved by the national regulatory authority. 1511

1512

A.6 Filling and containers 1513

1514

The requirements concerning filling and containers given in WHO Good Manufacturing 1515

Practices for biological products ( 40) should apply. Single dose containers may be preferred. If 1516

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multi-dose containers are used, a suitable method to protect the integrity of product before 1517

administration should be approved by the national regulatory authority. 1518

1519

The WHO guidelines on nonclinical evaluation of vaccines: regulatory expectations ( 21) provide 1520

guidance on vaccine formulation and delivery devices for alternative routes of administration. 1521

The consistent performance of the intranasal vaccine delivery device should be demonstrated for 1522

single dose dispensing of influenza vaccines (human, live attenuated) for intranasal 1523

administration. 1524

1525

Care should be taken to ensure that the materials of which the components of the container 1526

system are made do not adversely affect the virus content of the vaccine under the recommended 1527

conditions of storage. 1528

1529

A.7 Control tests on final lot 1530

1531

Samples should be taken from each filled final lot for the tests mentioned in this section and its 1532

sub-sections. 1533

1534

A.7.1 Identity 1535

1536

An identity test should be performed by a method approved by the national regulatory authority 1537

and national control laboratory on at least one container from each final lot. 1538

1539

The virus strains in the final containers should be identified by appropriate methods to identify 1540

the haemagglutinin and neuraminidase antigens. Phenotypic and genetic information may be 1541

useful in full specific identification of the vaccine viruses. 1542

1543

A.7.2 Sterility 1544

1545

Vaccine (reconstituted if lyophilised) should be tested for sterility as described in section A.5.4.2. 1546

1547

A.7.3 Infectivity (potency) 1548

1549

The virus content of each of at least three containers selected at random from each filled final lot 1550

should be determined individually. The national regulatory authority and national control 1551

laboratory should approve the details of the test. 1552

1553

The requirement for virus content per single human dose should be based on the inoculation of at 1554

least five vaccine-quality embryonated eggs or cultures per dilution, using ten fold dilutions. 1555

1556

The number of infectious doses per single human dose of vaccine should be determined (total 1557

and for each component virus), and the results should be expressed in EID50, TCID50 or pfu per 1558

dose. 1559

1560 Additional methods dependent on the replication of virus in vaccine-quality embryonated eggs or cell 1561 cultures (e.g. fluorescent focus assay expressed as fluorescent focus units) may be considered for 1562 determining potency. 1563 1564

The requirements for virus content per single human dose should be based on clinical trials in 1565

humans that demonstrate the dose range needed to insure vaccine safety and effectiveness of the 1566

influenza vaccines (human, live attenuated) for intranasal administration as approved by the 1567

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national regulatory authority. The need for re-characterizing the relation of infectious doses per 1568

human dose may develop, which should be undertaken with approval of the national regulatory 1569

authority. 1570

1571

A.7.4 Endotoxin 1572

1573

A test for endotoxin should be included (e.g. the Limulus Amoebocyte Lysate (LAL) test) as a 1574

test of safety and manufacturing consistency of the final lot. 1575

1576

A.7.5 Residual moisture of lyophilized vaccines 1577

1578

The residual moisture in a representative sample of each lyophilised lot should comply with 1579

cGMP in manufacture of vaccines and should be determined by a method approved by the 1580

national regulatory authority and the national control laboratory. 1581

1582

A.7.6 Inspection of final containers 1583

1584

Each container in each final lot should be inspected visually, and those showing abnormalities 1585

such as lack of integrity should be discarded. 1586

1587

A.8 Records 1588

1589

The requirements given in section 8 of WHO Good Manufacturing Practices for biological 1590

products ( 40) should apply. 1591

1592

A.9 Retained samples 1593

1594

The requirements given in section 9 of the WHO Good Manufacturing Practices for biological 1595


1597

A.10 Labeling 1598

1599

The requirements given in section 7 of the WHO Good Manufacturing Practices for biological 1600

products ( 40) should apply. With the addition of the following information, the label on the 1601

carton, the container or the leaflet accompanying the container should state: 1602

1603

• that the vaccine has been prepared from virus propagated in vaccine-quality 1604

embryonated eggs (as per description in section A.3.2.) or in cell cultures 1605

• the type of cell line i.e. monkey, dog, others (if appropriate) 1606

• the strain or strains of influenza virus present in the preparation 1607

• the infectivity titre per human dose of the vaccine expressed as EID50, TCID50 or 1608

pfu (or other similar means of determining infectivity e.g. fluorescent focus assay 1609

expressed as fluorescent focus units) 1610

• the volume per dose and the nominal volume of vaccine in the container available 1611

for recovery and administration 1612

• the seasonal influenza vaccine composition for which the vaccine is intended ( 4) 1613

• the name and quantity of any antimicrobial agent in the vaccine 1614

• the name and concentration of any preservative added 1615

• the temperature recommended during storage and transport 1616

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• the expiry date 1617

• any special dosing schedules (e.g. two doses for a pandemic vaccine) 1618

1619

A.11 Distribution and transport 1620

1621

The requirements given in section 8 of WHO Good Manufacturing Practices for biological 1622


1624

A.12 Stability 1625

1626

A.12.1 Stability testing 1627

1628

Adequate stability studies form an essential part of the development of influenza vaccines 1629

(human, live attenuated) for intranasal administration. Current guidance on evaluation of 1630

vaccine stability is provided in the WHO guidelines on stability evaluation of vaccines ( 63). 1631

1632

The stability of the influenza vaccine (human, live attenuated) for intranasal administration, 1633

including each of the active components in the vaccine in final form and at the recommended 1634

storage temperatures for the vaccine in final form, should be demonstrated to the satisfaction of 1635

the national regulatory authority on final containers from at least three final lots. 1636 1637

In some countries, vaccine infectivity titre should comply with the final lot specifications at the expiry date 1638 (see section A.7). 1639 1640 Since vaccine may be stored in monovalent form for significant duration prior to preparation of the final 1641 bulk vaccine, stability studies may be performed on the single harvests or the monovalent pools as well as 1642 the final vaccine. 1643

1644

Formulation of the influenza vaccine (human, live attenuated) for intranasal administration must 1645

be stable throughout its shelf life. Acceptable limits for stability should be agreed with the 1646

national regulatory authority. 1647

1648

Following licensure, ongoing monitoring of vaccine stability is recommended to support shelf 1649

life specifications and to refine the stability profile. Data should be provided to the national 1650

regulatory authority on an annual basis. 1651

1652

For influenza vaccines (human, live attenuated) for intranasal administration, thermal stability 1653

for lot release should be explored to determine whether the testing provides any value in the 1654

overall understanding of vaccine quality (safety and efficacy) and the effect of production 1655

variables. If there is no added value, thermal stability should not be required as a lot release 1656

assay ( 63). 1657

1658

When any changes are made in the production process that may affect stability of the product, 1659

the influenza vaccine (human, live attenuated) for intranasal administration produced by the new 1660

method should be shown to be stable. The national regulatory authority should be informed of 1661

and approve any changes that may affect stability of the product ( 63). 1662

1663

A.12.2 Storage conditions 1664

1665

Storage conditions for influenza vaccines (human, live attenuated) for intranasal administration 1666

should be fully validated and approved by the national regulatory authority ( 63). 1667

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1668

A.12.3 Expiry date 1669

1670

The expiry date should be fixed with the approval of the national regulatory authority and should 1671

take into account experimental and clinical data on the stability of the influenza vaccine (human, 1672

live attenuated) for intranasal administration ( 63). 1673 1674

In general, the expiry date should not exceed one year from the date of issue by the manufacturer 1675 because the strains used in one year’s vaccine may not be appropriate the next year ( 4). The national 1676 regulatory authority may approve an expiry date for a pandemic vaccine that differs from that of a 1677 seasonal influenza vaccine. 1678 1679

1680

Part B. Nonclinical evaluation of new influenza vaccines (human, 1681

live attenuated) 1682

1683

B.1 General remarks 1684

1685

Nonclinical evaluation refers to all in vivo and in vitro testing performed before and during the 1686

clinical development of vaccines ( 21). Preclinical testing, as a subcategory of nonclinical testing, 1687

is a prerequisite to move a candidate vaccine from the laboratory to the clinic and includes all 1688

aspects of product characterization, proof of concept/immunogenicity studies and safety testing 1689

in animals conducted prior to introducing the product into humans. 1690

1691

A non-exhaustive summary of typical preclinical evaluations used to prepare a new influenza 1692

vaccine (human, live attenuated) for entry into clinical studies is provided in Table B1. Some of 1693

the in vitro assessments will also form the basis for and be incorporated into the ongoing 1694

nonclinical activities of the quality control and quality assurance oversight of product release. 1695

1696

The yearly changes made to keep vaccines current with influenza epidemiology are not expected 1697

to require repeated nonclinical studies. Some in vitro and in vivo studies, however, may be 1698

useful to assess significant changes in manufacturing processes or alterations of critical product 1699

characteristics. Furthermore, the continuation of some nonclinical activities would be expected to 1700

be appropriate for maintaining current good manufacturing practices for influenza vaccine 1701

(human, live attenuated) for intranasal administration. Current WHO guidelines on nonclinical 1702

evaluation of vaccines ( 21) describe broadly applicable principles of preclinical and nonclinical 1703

assessments in greater detail. Guidance that may apply to the preclinical and nonclinical 1704

assessments of influenza vaccines (human, live attenuated) for intranasal administration to be 1705

used as pandemic vaccines is also provided in the WHO guidelines on regulatory preparedness 1706

for human pandemic influenza vaccines ( 14). 1707

1708

Each candidate attenuated virus donor strain to be developed for the preparation of candidate 1709

influenza vaccines (human, live attenuated) for intranasal administration should be characterized 1710

as completely as possible to identify critical genetic and phenotypic markers, to assess viral 1711

attenuation, to assess potential virus toxicity, and to determine whether the genetic basis of 1712

attenuation prevents reversion to partial or total virulence. Early laboratory studies should 1713

determine the genetic elements responsible for the virus attenuation, singly and in the 1714

combination to be present in the distributed vaccine. It is advisable to select attenuated virus 1715

donor strains that possess stable markers that are not dependent on retention of the markers of 1716

attenuation. The presence of additional stable markers can be used to differentiate vaccine strains 1717

from wild type virus reference strains or other vaccine viruses in epidemiological surveillance. 1718

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1719

The delivery device may have an impact on the uptake of a vaccine, its safety, and effectiveness 1720

( 21). Influenza vaccines (human, live attenuated) may be administered intranasally using 1721

proprietary or non-proprietary delivery devices including syringes, sprayers, and other liquid 1722

delivery devices. The preclinical evaluation of influenza vaccines (human, live attenuated) 1723

should include a rigorous assessment to establish suitability of the intranasal delivery device to 1724

support the safety and effectiveness of the vaccine. The WHO guidelines on nonclinical 1725

evaluation of vaccines: regulatory expectations ( 21) provide guidance on vaccine formulation 1726

and delivery devices for alternative routes of administration. The consistent performance of the 1727

intranasal vaccine delivery device should be demonstrated for single dose dispensing of 1728


1730 The evaluation of single dose dispensing may include parameters of liquid particle size, volume and/or 1731 virus quantity delivered. 1732 1733

1734

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1735

1736

B.2 Process development and product characterization 1737

1738

Before clinical trials are initiated, it is generally advisable for production processes and the 1739

product itself to be in the anticipated final form. However, the information needed to proceed 1740

into clinical trials may vary depending on the nature of the vaccine. Therefore, the preclinical 1741

Table B1. Nonclinical evaluation of new influenza vaccines (human, live attenuated)

for intranasal administration

Area of nonclinical

evaluation

Primary concern Scope of nonclinical

evaluation

In vitro assessments

Process development,

quality control and quality

assurance

Process is expected to meet

cGMP standards

Process control and

laboratory studies related to

all steps in process to

prepare vaccine

Product characterization Product quality appropriate

for use in preclinical and

clinical studies

Genetic, biochemical, and

biological characteristics of

vaccine including features of

attenuation

In vivo assessment

Toxicity and safety testing Product risks are appropriate

for anticipated uses

Safety indicators such as:

range of safe dose; single

and repeated safe doses;

safety parameters for clinical

monitoring; potential for

reversion to virulence

Immunogenicity and

efficacy

Product effects are

appropriate for anticipated

uses

Effect indicators such as:

type of immune responses

for clinical evaluation;

frequency and duration of

immune responses; priming

and boosting parameters;

protective effects in animal

challenge studies

Sources of guidance on nonclinical evaluation of vaccines:

World Health Organization (WHO)

International Conference on Harmonization (ICH) European Medicines Agency (EMA)

United States Food and Drug Administration (FDA)

United Kingdom Medical Research Council (MRC)

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and nonclinical development plans should be discussed with and approved by the national 1742

regulatory authority and the national control laboratory. 1743

1744

Although all preclinical and most nonclinical studies will be performed pre-licensure for an 1745

influenza vaccine (human, live attenuated) for intranasal administration, the suitability of an 1746

attenuated virus donor strain needs continuous careful review (see section A.4.5.3.2.). This 1747

review is done to ensure that the candidate influenza vaccine viruses containing prevalent 1748

antigens from new wild type virus reference strains do not substantially alter the established 1749

parameters of safety and effectiveness confirmed by clinical studies. Therefore, the development 1750

and use of influenza vaccines (human, live attenuated) for intranasal administration require long-1751

term commitment by manufacturers and national regulatory authorities to conduct laboratory 1752

studies as part of the nonclinical evaluation of the vaccine. 1753

1754

Attenuation of influenza vaccines (human, live attenuated) for intranasal administration is based 1755

on the attenuated virus donor strain used for vaccine preparation (see section A.4.5.3.2.). A 1756

complete genetic sequence of the attenuated virus donor strain should be obtained in order to 1757

map as carefully as possible where attenuating mutations have been introduced. Since each 1758

passage of an influenza virus may introduce new mutations, the possibility exists for reversion to 1759

a less or fully virulent form of virus. Therefore, studies should be pursued to determine whether 1760

reverting mutations appear at any point in the planned production and use of vaccine strains. 1761

These studies should also determine, to the extent possible, whether the haemagglutinins and 1762

neuraminidases of the wild type influenza viruses affect the stability of attenuating mutations. 1763

For example, when a highly pathogenic avian influenza virus is used, close attention should be 1764

given to ensure that the haemagglutinin retains the modifications that eliminate the highly 1765

pathogenic phenotype. 1766

1767

Phenotypic markers should be mapped genetically to the extent possible with in vitro systems. 1768

Key phenotypic features and adaptations of the attenuated virus donor strain should be 1769

demonstrated. For example, the ability to replicate efficiently at relatively low temperature 1770

should be demonstrated consistently for a cold adapted attenuated virus donor strain. For 1771

temperature sensitive attenuated virus donor strains, it should be possible to demonstrate 1772

reproducibly the temperature at which viral replication is inhibited. In vitro studies should be 1773

confirmed for the candidate influenza vaccine (human, live attenuated) seed viruses in order to 1774

ensure that attenuating features are fully retained throughout the vaccine process steps. 1775

1776

B.3 Nonclinical toxicity and safety testing 1777

1778

B.3.1 Preclinical toxicity 1779

1780

Preclinical toxicity studies are designed with the primary purpose of demonstrating the safety 1781

and tolerability of a candidate influenza vaccine (human, live attenuated) for intranasal 1782

administration. The design of a preclinical toxicity study should meet the criteria outlined in the 1783

protocol in order to support an intended clinical trial. The protocol should state the background, 1784

rationale, and objectives of the nonclinical studies and describe the design, methodology and 1785

organization, including statistical considerations, and the conditions under which studies are to 1786

be performed and managed. WHO guidelines on nonclinical evaluation of vaccines are 1787

published ( 21). 1788

1789

Toxicity tests for influenza vaccines (human, live attenuated) for intranasal administration 1790

should include: 1791

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1792

• an evaluation of the initial safe dose and of subsequent dose escalation schemes 1793

relevant to the clinical dose; 1794

• an evaluation of single and repeated doses as appropriate; 1795

• a determination of a set of relevant safety parameters for clinical monitoring; 1796

• a demonstration of potential reversibility of virulence of attenuated vaccines 1797

strains; and 1798

• local tolerability studies, which are typically included as part of the general 1799

toxicity evaluation. 1800

1801

The toxicity assessment of influenza vaccines (human, live attenuated) for intranasal 1802

administration formulations can be done either in stand-alone animal toxicity studies or in 1803

combination with studies of safety and activity that have toxicity end-points into the design ( 21). 1804

The parameters to be considered in designing animal toxicology studies with influenza vaccines 1805

(human, live attenuated) for intranasal administration include: 1806

1807

• relevant animal species e.g. ferret, mice, non-human primate, others; 1808

• ability to infect the animal including sero-susceptibility status; 1809

• virus strain i.e. seasonal influenza strains, novel human influenza viruses, 1810

pandemic viruses; 1811

• dosing schedule i.e. one/two/three doses; 1812

• method of vaccine administration e.g. nasal spray or nasal drops; 1813

• timing of evaluation of endpoints; and 1814

• clinical chemistry, antibody responses, histologic examination of target organs, 1815

and necropsy. 1816

1817

Despite efforts to maximize the predictive value of nonclinical toxicity studies, animal models 1818

have limitations in applicability to human experiences and it is possible that the animal studies 1819

will not accurately reflect the risks of using influenza vaccines (human, live attenuated) for 1820

intranasal administration in humans. 1821

1822

The design and value of repeated-dose toxicity tests should be considered on a case-by-case 1823

basis. If an influenza vaccine (human, live attenuated) for intranasal administration is intended 1824

to be clinically tested in women of childbearing age, the need for reproductive toxicity studies 1825

and studies of embryo-fetal and perinatal toxicity should be considered on a case-by-case basis. 1826

Reproductive toxicity studies, where appropriate, will need to be undertaken before licensing 1827

( 21). 1828

1829

Any changes in the composition of the human influenza vaccine (live, attenuated) for intranasal 1830

administration should be carefully considered both by the manufacturer and the national 1831

regulatory authority. In general, there should be no need to repeat the toxicity assessments for an 1832

annual strain change as long as attenuation has been documented adequately. However, it may 1833

be necessary to repeat some or all of the toxicity studies when a novel human influenza virus 1834

subtype emerges (e.g.. influenza A virus H5N1) and a new influenza vaccine (human, live 1835

attenuated) for intranasal administration is prepared. Special considerations on the preclinical 1836

and nonclinical evaluation of influenza vaccines (human, live attenuated) for intranasal 1837

administration for pandemic use are provided in the WHO guidelines on regulatory preparedness 1838

for pandemic influenza vaccines ( 14). 1839

1840

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Any changes in formulation, ingredients and excipients in the human influenza vaccine (live, 1841

attenuated) for intranasal administration may require repeating some or all of the toxicity studies. 1842

The manufacturer should timely discuss these changes with and have the approval of the national 1843

regulatory authority. 1844

1845

B.3.2 Preclinical safety testing 1846

1847

Every effort should be made in the preclinical studies to identify markers of attenuation (genetic 1848

sequences) which can be used to monitor the results during clinical evaluation phases. Primary 1849

investigations of attenuation in animals may be done in a number of influenza responsive species 1850

such as ferrets, mice, and non-human primates to establish the relationship of specific genetic 1851

features on the attenuation phenotype. Studies in animals may be designed to confirm the 1852

location and degree of replication of candidate influenza vaccine (human, live attenuated) in the 1853

host's respiratory tract. 1854

1855

Results from animals inoculated with the attenuated virus donor strain (or its derivative 1856

candidate vaccine virus) compared with results from animals inoculated with the wild type 1857

reference virus strain should indicate whether the attenuated virus donor strain (or the candidate 1858

vaccine virus) is adequately safe to conduct limited clinical trials. 1859

1860

The potential of influenza vaccines (human, live attenuated) for intranasal administration for 1861

neuro-invasiveness and neuro-virulence should be considered if the wild type virus used for seed 1862

virus preparation demonstrates neuro-invasiveness. The national regulatory authority should be 1863

consulted on the need for preclinical evaluation of the potential for neurological effects in 1864

suitable animal models. 1865

1866

Animal studies cannot completely predict what will happen in humans because of differences in 1867

anatomy, host temperature, and other variables. Features that appear to be attenuating in animals 1868

may not fully predict the genotype or phenotype of attenuation in humans. In addition, the 1869

vaccine dose tolerated by animals may greatly differ from dose tolerated by humans. Therefore, 1870

results from studies in animals do not eliminate the need for confirmation in clinical trials in 1871

humans. 1872

1873

B.4 Nonclinical immunogenicity and efficacy 1874

1875

B.4.1 Nonclinical immunogenicity 1876

1877

Assessment of immune responses in animals can provide some assurance that an influenza 1878

vaccine (human, live attenuated) has replicated in the host. Influenza virus infection triggers a 1879

multitude of immune responses in the innate and adaptive immune systems, which can be 1880

assessed in an animal model. Although there is not a specific correlate of protection defined for 1881

influenza vaccine (human, live attenuated) for intranasal administration, antibodies directed 1882

against viral haemagglutinins in the blood and mucosal secretions have been shown to have 1883

protective effects in animal models and in humans. Antibody responses to neuraminidase and 1884

other viral proteins as well as cellular responses involving T helper and cytotoxic cells have also 1885

been identified during recovery from influenza infection. Despite the lack of specific correlates 1886

of protection, immune responses in animals should be evaluated for their potential to provide an 1887

in vivo correlation with prevention of infection and/or the reduction of disease signs and 1888

pathogenicity. 1889

1890

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The specific immune parameters to assess in animals depend, in part, on the nature of the 1891

influenza vaccine (human, live attenuated) for intranasal administration. Parameters should at 1892

least include antibodies directed against haemagglutinins (e.g. serum neutralizing, 1893

haemagglutination inhibition, or single radial haemolysis antibodies). 1894

1895

While immune responses in animals do not necessarily predict immune responses in humans, it 1896

is nevertheless possible to derive useful information from preclinical immunogenicity studies. 1897

1898

B.4.2 Nonclinical efficacy 1899

1900

Protective efficacy can be assessed directly after immunization in animals by infectious 1901

challenge with wild type or selected laboratory strains of influenza viruses. Preclinical studies in 1902

animals also enable the assessment of the protective effects of a candidate influenza vaccine 1903

(human, live attenuated) for intranasal administration against potentially lethal influenza virus 1904

infections, which may become important in preparing vaccines for highly pathogenic influenza 1905

viruses of pandemic potential. Furthermore, animal studies can be designed to assess the breadth 1906

of immune and clinical protection of a given influenza vaccine (human, live attenuated) for 1907

intranasal administration against different influenza A subtypes and drift variants which may 1908

indicate the need for new antigens in the vaccine. 1909

1910

While efficacy identified in an animal model does not necessarily predict the protective effect in 1911

humans, it is nevertheless possible to derive useful information from preclinical efficacy studies 1912

in animals. 1913

1914

Part C. Clinical evaluation of new influenza vaccines (human, live 1915

attenuated) 1916

1917

C.1 General remarks 1918

1919

The clinical evaluation of new influenza vaccines (human, live attenuated) for intranasal 1920

administration includes studies undertaken as part of the developmental process, the licensure 1921

procedure, and/or studies performed in the post marketing period. Extensive guidance for 1922

manufacturers and regulatory authorities on the clinical development of vaccines is found in the 1923

WHO guidelines on clinical evaluation of vaccines: regulatory expectations ( 19) and the WHO 1924

guidelines for good clinical practices (GCP) for trials on pharmaceutical products ( 65). Guidance 1925

that may apply to the clinical evaluation of influenza vaccines (human, live attenuated) for 1926

intranasal administration to be used as pandemic vaccines is provided in the WHO guidelines on 1927

regulatory preparedness for human pandemic influenza vaccines ( 14). 1928

1929

There are a number of issues to be considered in the clinical development and use of safe and 1930

effective influenza vaccines (human, live attenuated) for intranasal administration. Since 1931

influenza vaccines generally have been multivalent to provide protection against influenza A and 1932

influenza B viruses, each component of influenza vaccines (human, live attenuated) for 1933

intranasal administration must be proven safe and protective in the context of a formulated 1934

multivalent vaccine ( 67). Since wild type influenza viruses constantly mutate, influenza 1935

vaccines (human, live attenuated) for intranasal administration will require frequent re-1936

evaluations of composition and change as recommended by WHO on the influenza viruses to use 1937

in vaccines in countries of the Northern and Southern hemispheres ( 4). 1938

1939

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There is not an established immune correlate of protection for influenza vaccines (human, live 1940

attenuated) for intranasal administration and the possibility exists that immune correlates may be 1941

specific for individual influenza vaccines (human, live attenuated) ( 66). Despite potential 1942

difficulties, influenza vaccines (human, live attenuated) for intranasal administration have 1943

demonstrated in large field trials and in routine immunization campaigns that satisfactory 1944

products can be prepared, distributed and administered ( 9 10, 27, 67). 1945

1946

Due to possible variation in the attenuating features of each influenza vaccine (human, live 1947

attenuated) for intranasal administration, it is important to carefully characterize the vaccine 1948

viruses derived from a new attenuated donor virus before licensure. Although animal studies 1949

may not perfectly predict human clinical experiences, nonclinical and preclinical studies with an 1950

influenza vaccine (human, live attenuated) for intranasal administration should assist in 1951

determining the genetic elements of attenuation in humans, in evaluating the possibility of 1952

reversion to partial or full virulence post vaccine administration to people, and in determining 1953

potential targets for safety data collection as the clinical trials begin. 1954

1955

The delivery device may have an impact on the uptake of a vaccine, its safety, and effectiveness 1956

( 21). Influenza vaccines (human, live attenuated) may be administered intranasally using 1957

proprietary or non-proprietary delivery devices including syringes, sprayers, and other liquid 1958

delivery devices. The clinical evaluation of influenza vaccines (human, live attenuated) should 1959

include a rigorous assessment to establish suitability of the intranasal delivery device to support 1960

the safety and effectiveness of the vaccine in humans. 1961

1962

C.2 Clinical evaluation strategy 1963

1964

The clinical development of a new influenza vaccine (human, live attenuated) for intranasal 1965

administration will resemble other vaccine product development; a typical strategy is provided in 1966

table C1. 1967

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1968

Table C.1. Clinical evaluation of new influenza vaccines (human, live attenuated) for

intranasal administration

Phases of clinical

evaluation

Primary concern Scope of clinical

evaluation

Phase I Safety (for common events) Small scale trials to

determine whether

significant risks exist

Phase II Preliminary effect

(immunogenicity) and

general safety (for typical

and less common events)

Larger scale trials to further

define risks and understand

potential benefits in

populations expected to use

the vaccine

Phase III Protective efficacy (pivotal)

and safety (for uncommon

events)

Trial size defined to provide

statistical certainty for

specific endpoints related to

safety and efficacy

Post marketing clinical

trials

Safety (for rare events) and

effectiveness (to expand on

original observations)

Trials purposely designed to

further refine information

on safety and efficacy in

larger populations and/or in

new populations for vaccine

use

Post marketing

surveillance

Safety (for unexpected and

rare events and/or signals)

Potentially includes all

vaccine recipients to

identify safety signals

arising from routine use and

expanded population studies

Sources of guidance on clinical evaluation of vaccines:

World Health Organization (WHO)

International Conference on Harmonization (ICH)

European Medicines Agency (EMA)

United States Food and Drug Administration (FDA)

1969

1970

C.2.1 Clinical studies for licensure 1971

1972

Early (Phase I) studies focus on safety aspects including replication, shedding and transmission 1973

potential of the vaccine virus, relevant pharmacokinetics, and neurotoxic and immunological 1974

effects identified in the nonclinical evaluation of the vaccine virus. Early clinical studies (Phase I 1975

and Phase II) should be designed to confirm the dose range that is well tolerated. These studies 1976

are recommended because replication of influenza vaccine (human, live attenuated) may be 1977

affected by previous host exposure to influenza viruses or vaccines as well as by underlying host 1978

factors. 1979

1980

WHO/BS/09.2111

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As studies progress (Phase II), more information is acquired on immunological and protective 1981

effects, and trials to confirm efficacy (usually Phase III) are also pursued. Later clinical studies 1982

(Phase II and Phase III) should expand the number of subjects to be evaluated by groups targeted 1983

for vaccine use and to define the types and frequencies of already identified adverse events. 1984

Later clinical studies should also provide the possibility of identifying rarer events that can only 1985

be detected when a sufficiently large population has been examined. 1986

1987

Later clinical studies also assess the potential for variation among people of different ages. 1988

People with pulmonary, cardiac, or immune dysfunction should be addressed as part of 1989

expanding vaccine studies. It is also desirable to establish immune correlates of protection in 1990

human populations, since this may aid future improvements in the production and evaluation of 1991

influenza vaccine (human, live attenuated) for intranasal administration. 1992

1993

The information needed for the successful completion of clinical trials may vary somewhat 1994

depending on the nature of the vaccine; therefore, clinical development plans should be 1995

discussed with and approved by the national regulatory authority and the national control 1996

laboratory. Current WHO guidelines on clinical evaluation of vaccines describe broadly 1997

applicable principles in greater detail ( 19). Guidance that may apply to the clinical assessments 1998

of influenza vaccines (human, live attenuated) for intranasal administration to be used as 1999

pandemic vaccines is provided in the WHO guidelines on regulatory preparedness for human 2000

pandemic influenza vaccines ( 19, 14). 2001

2002

C.2.2 Post marketing studies 2003

2004

Although most of the clinical development will precede licensure of influenza vaccines (human, 2005

live attenuated) for intranasal administration, postmarketing trials and surveillance will provide 2006

significant understanding about safety and effectiveness of vaccines that cannot be predicted or 2007

detected in the prelicensing period, particularly for rare adverse events. Therefore, manufacturers 2008

and national regulatory authorities should anticipate a substantial commitment to 2009

pharmacovigilance and to the conduct of clinical studies as part of a continuing, post marketing 2010

review of influenza vaccines (human, live attenuated) for intranasal administration. In the case 2011

of a significant change in the manufacturing process, the national regulatory authority may also 2012

require clinical studies. 2013

2014

While clinical studies with novel influenza virus subtypes or lineages may be considered, once 2015

adequate experience with multiple candidate influenza vaccine viruses within a subtype or 2016

lineage is accumulated, the conduct of postmarketing clinical trials may no longer be justified for 2017

changes recommended by WHO for the influenza viruses to be used in vaccines in countries of 2018

the Northern and Southern hemispheres ( 4). 2019

2020 Some national regulatory authorities may require a limited clinical evaluation to assess safety 2021 parameters for licensing purposes whenever a new candidate influenza vaccine virus is introduced. 2022

2023

C.3 Clinical safety 2024

2025

C.3.1 Initial safety assessment 2026

2027

An influenza vaccine (human, live attenuated) is usually given intranasally so that virus 2028

replicates in the respiratory tract tissues, predominantly the nasopharyngeal mucosa. As a result 2029

of replication of influenza vaccines (human, live attenuated) viruses in the respiratory tract, mild 2030

upper respiratory symptoms in some individuals and occasional benign systemic reactions have 2031

WHO/BS/09.2111

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been observed, but in much lower frequency and severity as compared to wild type influenza 2032

viruses. Initial vaccine safety trials should include a careful assessment of symptoms related to 2033

influenza virus infection. 2034

2035

Since vaccinated individuals may shed influenza vaccine (human, live attenuated) viruses via 2036

respiratory secretions, virus excretion should be assessed for quantity and duration, and, if 2037

possible, compared to shedding during infection with relevant virulent wild type influenza 2038

viruses. It is desirable that the excreted attenuated virus strains maintain the characteristic 2039

phenotypes of influenza vaccine (human, live attenuated) viruses, which may be partially 2040

predicted during the nonclinical and preclinical vaccine evaluation. 2041

2042

Initial clinical studies (Phase I), particularly with newly developed influenza A subtypes may be 2043

best performed in an environment offering the maximum guarantee of isolation and discipline. 2044

The WHO biosafety risk assessment and guidelines for the production and quality control of 2045

human influenza pandemic vaccines ( 15) offer some guidance on this matter. These clinical 2046

trials are to make sure that the virus is not excreted at an unacceptably high titre or for an 2047

excessively long interval. Clinical trials should show that the frequency of virus transmission is 2048

consistent with public health goals. 2049

2050

In addition to detailed assessment of safety, early clinical trials should attempt to show that the 2051

benefit–risk ratio of vaccine use is acceptable. 2052

2053

C.3.2 Expanded safety assessment 2054

2055

When determining acceptability of an influenza vaccine (human, live attenuated) for intranasal 2056

administration in larger scale clinical trials, limited studies administering the vaccine to 2057

immunologically primed individuals from different age groups may be undertaken before 2058

administering the vaccine to immunologically naïve individuals. The follow up period for the 2059

assessment of safety should take into account effects that may occur many days or weeks post 2060

vaccine administration. It should also include purposeful review of events for at least six months 2061

after final vaccine administration for a trial protocol for both early and later trials examining 2062

vaccine safety. Guidance on age progression (from adult to younger age groups) for an influenza 2063

vaccine (human, live attenuated) for intranasal administration in children is provided in WHO 2064

documents ( 14, 65). 2065

2066

Clinical trials (Phase II and Phase III) should delineate the type, frequency, and severity of local 2067

and systemic respiratory infection-related events such as sneezing, nasal discharge, cough, sore 2068

throat, fever, myalgia, as well as other respiratory and non-respiratory events that have been 2069

observed in preclinical studies and Phase I clinical trials. As numbers expand during Phases II 2070

and III studies and during post marketing studies, rarer adverse events may be identified for 2071

further attention. 2072

2073

Each influenza virus passage may introduce new mutations. Therefore, studies should be 2074

pursued to determine whether reverting mutations appear at any point in the planned production 2075

and use of new attenuated donor viruses. 2076

2077

The possibility of influenza vaccine (human, live attenuated) virus transmission by contact of 2078

susceptible individuals with virus-containing secretions exists. However, the probability of 2079

transmission of influenza vaccine (human, live attenuated) viruses should be small compared to 2080

that of wild type influenza viruses, even in immunologically naïve people. 2081

2082

WHO/BS/09.2111

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When novel wild type reference virus strains (e.g. in the case of a pandemic vaccine) are used in 2083

the preparation of influenza vaccines (human, live attenuated), clinical studies may be 2084

considered as a part of the assessment to determine that the newly introduced haemagglutinins 2085

and neuraminidases have not altered the safety profile of the influenza vaccine (human, live 2086

attenuated) for intranasal administration in any adverse manner. 2087

2088

C.4 Clinical efficacy and immunogenicity 2089

2090

With current methods and capabilities, clinical endpoint studies provide the definitive 2091

assessment of efficacy of influenza vaccines (human, live attenuated) for intranasal 2092

administration ( 67). Several antibody responses, including neutralizing antibodies in blood and 2093

respiratory secretions, are associated with prevention of influenza virus infection as well as 2094

reduction of symptomatology and overall pathologic effects in humans. However, broadly 2095

applicable immunological correlates of protection have not been identified and relevant immune 2096

parameters may vary for influenza vaccines (human, live attenuated) for intranasal 2097

administration ( 66). Nonetheless, opportunities to correlate specific immune parameters with 2098

prevention of infection or illness should not be missed during clinical studies. 2099

2100

C.4.1 Clinical efficacy studies 2101

2102

Vaccine efficacy is the reduction in the odds of developing clinical disease post-vaccination 2103

relative to the odds when unvaccinated. Efficacy of influenza vaccines (human, live attenuated) 2104

for intranasal administration in a specified population should be demonstrated in adequately 2105

powered well-controlled clinical trials (Phase III). Since this undertaking is usually complex, 2106

expensive, and dependent on an infected population to study, the design and feasibility (e.g. 2107

pandemic vaccines) of efficacy clinical trials should be discussed thoroughly with the competent 2108

authority ( 19, 14). 2109

2110

In general, efficacy studies should demonstrate that influenza vaccines (human, live attenuated) 2111

for intranasal administration prevent laboratory (e.g. culture) confirmed influenza illnesses. The 2112

ability to document vaccine efficacy depends upon an adequate attack rate of influenza 2113

(proportion of population exposed to influenza viruses who become clinically ill) in relation to 2114

the ability to prevent infection in a study with an appropriate sample size. Since attack rates for 2115

influenza vary from year to year and different influenza types or subtypes predominate in 2116

different years, efficacy studies will generally require a large enrolment and sometimes a multi 2117

year plan for thorough evaluation of each component strain, type or subtype contained in 2118


2120

C.4.2 Clinical correlates of protection 2121

2122

If possible, clinical trials should establish a correlate of protection that can be used to guide 2123

development of influenza vaccines (human, live attenuated) for intranasal administration. Since 2124

influenza vaccines (human, live attenuated) are administered by the respiratory route, it should 2125

be anticipated that immune parameters other than antibodies in blood could serve as correlates of 2126

protection ( 66). Novel methods to measure antibody or cellular responses should be validated. 2127

2128

C.4.3 Clinical serological parameters 2129

2130

Anti- haemagglutinin antibodies in blood are the most widely studied immune mechanisms in 2131

humans. However, antibody responses to neuraminidase and other influenza viral proteins may 2132

WHO/BS/09.2111

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also contribute to protection. The specific serological parameters to assess in clinical trials 2133

(Phases I-III) depend in part of the nature of the influenza vaccine (human, live attenuated) for 2134

intranasal administration but a reasonable starting point is determination of neutralizing, 2135

haemagglutination inhibition, or single radial haemolysis antibodies ( 66). 2136

2137

C.4.4 Other clinical measures of immunity 2138

2139

Assessment of innate, mucosal and cellular immune mechanisms may be helpful in further 2140

developing a rationale for protection resulting from influenza vaccines (human, live attenuated) 2141

for intranasal administration. 2142

2143

Influenza vaccines (human, live attenuated) for intranasal administration are expected to induce 2144

local innate immune responses including interferon and other cytokines as well as adaptive 2145

immune responses such as secretory mucosal antibodies i.e. IgA ( 9 10, 66). 2146

2147

Cellular immune mechanisms are less well understood but it is expected that influenza vaccines 2148

(human, live attenuated) for intranasal administration may also mimic the T cell responses 2149

identified during naturally occurring infections in humans. 2150

2151

2152

C.5 Evaluation in special populations 2153

2154

In general, clinical trials should be performed first in the least vulnerable population (i.e. healthy 2155

adults). Placebo groups are essential to evaluating the incidence and intensity of adverse events 2156

following immunization. 2157

2158

If the initial clinical trials in healthy adults demonstrate vaccine safety at specific doses, 2159

additional studies may be undertaken to delineate the uses of influenza vaccines (human, live 2160

attenuated) for intranasal administration in different age and risk groups. Because it may be 2161

inappropriate to expose some vulnerable populations to an influenza vaccine (human, live 2162

attenuated) for intranasal administration, early clinical studies should also provide an 2163

understanding of the frequency of transmission and the potential for adverse consequences of 2164

transmission to non-vaccinated individuals. 2165

2166

C.5.1 Pediatric 2167

2168

If clinical trials in adults demonstrate a safety profile that is suitably benign, studies in children 2169

may be undertaken to evaluate safety and efficacy of influenza vaccines (human, live attenuated) 2170

for intranasal administration ( 14, 65). A strategy to study older children first and progressively 2171

younger children may be prudent, since younger children would be expected to be more prone to 2172

develop local and systemic adverse events which may not be apparent in older people who have 2173

had exposure to wild type influenza viruses and vaccines. 2174

2175

C.5.2 Geriatric 2176

2177

If clinical trials in healthy adults demonstrate a safety profile that is suitably benign, studies in 2178

elderly populations, in particular those with chronic diseases may be undertaken to evaluate the 2179

safety and efficacy of influenza vaccines (human, live attenuated) for intranasal administration. 2180

Although there may be similarities between healthy adults and the elderly, it might be anticipated 2181

that different responses to influenza vaccines (human, live attenuated) for intranasal 2182

WHO/BS/09.2111

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administration may result from lifetime exposure to influenza and the accumulation of chronic 2183

illnesses, particularly cardiac and pulmonary, in the elderly. 2184

2185

C.5.3 Risk groups 2186 2187 A solid understanding of the typical post vaccination clinical course in the healthy host is 2188

imperative before any study is initiated in a population with special risk status. All studies should 2189

be conducted with strict ethical considerations for the welfare of participants and no one should 2190

be exposed to unreasonable risk ( 65, 19). 2191

2192

Special studies my be considered for populations for whom influenza vaccination is especially 2193

important (such as subjects with chronic bronchitis and asthma) but for whom the potential 2194

adverse events related to a live vaccine might be more severe. 2195

2196

People with immune defects that do not contraindicate exposure to a live virus vaccine should be 2197

evaluated cautiously to determine whether an influenza vaccine (human, live attenuated) for 2198

intranasal administration is beneficial without any unusual consequences such as prolonged 2199

shedding of virus. 2200

2201

C.6 Strain change considerations for seasonal vaccines 2202

2203

After an initial licensure, the vaccine is reformulated according to the strains predicted to 2204

circulate in the upcoming season ( 4). For inactivated influenza vaccines, small scale 2205

immunogenicity studies are requested in some parts of the world. However, without 2206

immunological correlates of protection, such studies with influenza vaccines (human, live 2207

attenuated) for intranasal administration are of limited value. Furthermore, a clinical efficacy 2208

trial is generally not feasible annually because of methodological constraints and timing. 2209

For influenza vaccines (human, live attenuated) for intranasal administration, some national 2210

regulatory authorities may recommend a small scale safety study. The value of such studies to 2211

re-assess the benefit-risk balance is unknown. In general, when the vaccine production process 2212

remains unchanged, it is likely that the benefit-risk balance as assessed at the time of licensure 2213

will not change. The need for additional clinical evaluations prior to annual re-licensure may 2214

depend upon factors including major changes in sub types (i.e. antigenic drifts or shifts), 2215

previously identified risks and/or reported safety signals, and impaired efficacy during vaccine 2216

use in the previous season. In those cases, a clinical study to reconfirm the benefit-risk balance 2217

may be deemed necessary. 2218

2219

It follows that a quality assessment of vaccine formulation for the upcoming year may be 2220

recommended and be supplemented with a structured annual post marketing surveillance 2221

program in countries where vaccine is used in wide scale vaccination programs. Such post 2222

marketing surveillance programs may change over time depending on experience. In the initial 2223

years following licensure, post marketing surveillance studies may be requested and later be 2224

replaced by a continuous surveillance program. In any case marketing authorisation holders are 2225

recommended to consult with national regulatory authorities where vaccine is to be marketed and 2226

discuss details concerning the post marketing surveillance program. 2227

2228

C.7 Vaccines intended for pandemic influenza 2229

2230

It is anticipated that attenuated virus donor strains developed for seasonal influenza vaccines 2231

(human, live attenuated) for intranasal administration may be evaluated for use in pandemic 2232

WHO/BS/09.2111

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situations. Newly attenuated virus donor strains may be prepared specifically for pandemic use. 2233

If a new attenuated virus donor strain is planned for pandemic use, the quality, safety and 2234

efficacy of pandemic influenza vaccines (human, live attenuated) for intranasal administration 2235

should be assured by meeting the recommendations stated in the present document. Furthermore 2236

special regulatory considerations for pandemic influenza vaccines should be followed ( 14). 2237

2238

For an attenuated donor specifically prepared for pandemic use, studies should demonstrate an 2239

attenuated phenotype and stability of the attenuated donor strain genome. Clinical trials of 2240

influenza vaccines (human, live attenuated) for intranasal administration containing the 2241

haemagglutinin and neuraminidase of novel influenza viruses should be conducted in isolation 2242

units, while the novel virus with pandemic potential is not spreading efficiently from human to 2243

human (i.e. WHO pandemic phases 1 through 5). During WHO pandemic phases 1 through 5, 2244

isolation procedures should be maintained for recipients of vaccines against novel influenza 2245

viruses for the duration of vaccine virus shedding. It is expected that advice will be provided by 2246

WHO and by national health authorities on declaration of pandemic phases. Assessment of 2247

efficacy in the absence of widespread disease is especially challenging. Given the potential 2248

efficacy of influenza vaccines (human, live attenuated) for intranasal administration in paediatric 2249

populations, consideration and approval for the conduct of studies in children will be needed 2250

from the national regulatory authorities. 2251

2252

Comprehensive guidance that applies to influenza vaccines (human, live attenuated) for 2253

intranasal administration to be used as pandemic vaccines is provided in the WHO guidelines on 2254

regulatory preparedness for human pandemic influenza vaccines ( 14). 2255

2256

Part D. Recommendations for national regulatory authorities 2257

2258

D.1 General remarks 2259

2260

The general recommendations for national regulatory authorities contained in the WHO Good 2261

Manufacturing Practices for biological products ( 40) should apply. Extensive list of WHO 2262

guidelines with specific recommendations for the quality assurance of biological products to be 2263

followed by national regulatory authorities is provided in the reference section of this document. 2264

2265

The national regulatory authority should give directions to manufacturers concerning the 2266

influenza virus strains to be used, and the recommended human dose. 2267

2268 The national regulatory authority should take into consideration all information available on strains before 2269 deciding on those permitted for vaccine production ( 4). 2270

2271 In addition, the national regulatory authority should provide a reference preparation of influenza 2272

vaccines (human, live attenuated) for intranasal administration to check the normal susceptibility 2273

of the titration system. The national regulatory authority should also specify the requirements 2274

for virus content to be fulfilled in order to achieve adequate immunization of human beings with 2275

the recommended human dose. 2276 2277

As a practical matter, the national regulatory authority will need to collaborate with the manufacturer to 2278 develop an acceptable reference preparation. 2279

2280

D.2 Release and certification 2281

2282

WHO/BS/09.2111

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A vaccine lot should be released only if it fulfils national requirements and/or Part A of these 2283

recommendations. A protocol based on the model in Appendix 1, signed by the responsible 2284

official of the manufacturing establishment, should be prepared and submitted to the national 2285

regulatory authority in support of the request for release of vaccine for use. A statement signed 2286

by the authorized official of the national regulatory authority should be provided at the request of 2287

the manufacturing establishment and should certify that the lot of vaccine in question meets all 2288

national requirements as well as the manufacturing recommendations provided in Part A of these 2289

recommendations. 2290

2291 Regulations in some countries may require performing general safety testing as part of lot release. 2292

2293

The release certificate should state the number under which the lot was released by the national 2294

regulatory authority and the number appearing on the labels of the containers. Importers of 2295

influenza vaccines (human, live attenuated) for intranasal administration should be given a copy 2296

of the official national release document of country of production. The purpose of certificate is to 2297

facilitate the exchange between countries of influenza vaccines (human, live attenuated) for 2298

intranasal administration. An example of a suitable certificate is given in Appendix 3. 2299

2300

D.3 Manufacturing changes 2301

2302

In the case of a new vaccine, the national regulatory authority should assess vaccine safety and 2303

efficacy by arranging for studies in human volunteers of one or more of the lots of influenza 2304

vaccine (human, live attenuated) for intranasal administration that have satisfied the above-2305

mentioned recommendations. Such studies should include the assessment of the immune 2306

responses and adverse reactions in various age groups. 2307

2308 In the case of significant change in the manufacturing process, preclinical, nonclinical and clinical studies 2309 may also be required by the national regulatory authority. 2310 2311 Some national regulatory authorities require a limited clinical evaluation for licensing purposes whenever a 2312 new vaccine strain is introduced. 2313

2314

Authors 2315 2316

The first draft of these revised WHO recommendations for the production and control of 2317

influenza vaccine (human, live attenuated) was prepared by Dr. Roland Levandowski and WHO 2318

Headquarters Responsible Officer Dr Claudia P Alfonso in May 2008. 2319

2320

Comments to this draft were provided by Dr. Elena Govorkova, Department of Infectious 2321

Diseases, St. Jude Children's Research Hospital, Memphis, U.S.A.; Dr. Michael Pfleiderer, Paul-2322

Ehrlich-Institut, Germany; and Dr. Jerry Weir, Center for Biologics Evaluation and Research, 2323

Food and Drug Administration, U.S.A. 2324

2325

A draft incorporating these comments was produced in December 2008 and further revised to 2326

produce a document that was reviewed at the informal consultation on WHO recommendations 2327

for the production and control of influenza vaccine (human, live attenuated), 26-27 February 2328

2009, Geneva, Switzerland. This meeting was attended by stakeholders from national 2329

immunization programs, basic research and public health sciences, regulatory authorities, and 2330

vaccine manufacturers: Dr Kathryn Edwards, Pediatric Clinical Research Office, Vanderbilt 2331

University School of Medicine, Nashville, TN, USA; Dr Roland Levandowski, Bethesda MD, 2332

USA; Mrs Therese Marengo, Nesles-la-Vallee, France; Professor Albert Osterhaus, Head of the 2333

Department of Virology, Erasmus MC, Rotterdam, The Netherlands; Dr Michael Pfleiderer, 2334

WHO/BS/09.2111

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Head of section Viral Vaccines, Paul-Ehrlich-Institut, Langen, Germany; Dr Huib Van de Donk, 2335

Den Haag, The Netherlands; Dr Bettie Voordouw, Clinical Assessor for Vaccines, Pijnacker, 2336

The Netherlands; Dr Jerry Weir, Director, Division of Viral Products, Center for Biologics 2337

Evaluation and Research, Food and Drug Administration, Rockville, MD, USA; Professor Le 2338

van Phung, Director, National Institute for Control of Vaccine and Biologicals, Hanoi City, Viet 2339

Nam; Mrs Tasanee Lorchaivej, Head of Biological Products Section, Drug Control Division, 2340

Food and Drug Administration, Ministry of Public Health, Nonthaburi, Thailand; Professor 2341

Larissa Georgievna Rudenko, Head, Department of Virology, Russian Academy of Medical 2342

Science, Institute for Experimental Medicine, St Petersburg, Russian Federation; Mrs Kusmiaty, 2343

Head of Biological Products Division, National Quality Control Laboratory of Drug and Food 2344

Institution, National Agency of Drug and Food Control, Jakarta Pusat, Indonesia, Mrs 2345

Prapassorn Savaitnisagon Thanaphollert, Senior Pharmacist, Biological Products Section, Drug 2346

Control Division, Food and Drug Administration, Ministry of Public Health, 2347

Nonthaburi,Thailand; Dr John Wood, Division of Virology, National Institute for Biological 2348

Standards & Control, Herts, United Kingdom; Dr François Cano, Unité Contrôle des Vaccins 2349

Viraux et Sécurité Virale, AFSSAPS Direction des Laboratoires et des Contrôles, Lyon, France; 2350

Dr Nurma Hidayati, Head of Sub Directorate of New Drug Evaluation, Directorate of Drug and 2351

Biological Product Evaluation, National Agency of Drug and Food Control, Jakarta Pusat, 2352

Indonesia; Dr Rick A. Bright, Scientific Director, Influenza Vaccine Project Vaccine 2353

Development Global Program, Program for Appropriate Technology in Health (PATH), 2354

Washington, DC, USA; Dr Belinda Breedveld, Senior Regulatory Affairs Scientist, Global 2355

Regulatory Affairs - Schering-Plough, Oss, The Netherlands; Dr Kathleen Coelingh, Senior 2356

Director, Scientific Affairs, MedImmune, Inc., Seattle WA, USA; Dr Tony Colegate, Scientific 2357

Coordinator, IFPMA B&V IVS ITF, Influenza Technical Affairs Manager, Novartis Vaccines, 2358

Liverpool, United Kingdom of Great Britain & Northern Ireland; Dr Han van den Bosch, R&D 2359

Director, Nobilon, part of Schering–Plough, Boxmeer, The Netherlands; Dr Robert Walker, Vice 2360

President, Medical and Scientific Affairs, MedImmune, Inc., Gaithersburg MD, USA; Dr Rajeev 2361

Dhere, Director, Vaccine Production, Serum Institute of India Ltd., Pune, India; Dr Igor 2362

Krasilnikov, Head of Research and Development, "Microgen" State Company, Moscow, Russian 2363

Federation; Dr Alexander Mironov, Head of Clinical Trials Department, “Microgen” State 2364

Company, Moscow, Russian Federation; Dr Jean-Marie Okwo-Bele, Director, Immunization, 2365

Vaccines and Biologicals (IVB), WHO, Geneva, Switzerland; Dr David Wood, Coordinator, 2366

Quality, Safety and standards (QSS), WHO, Geneva, Switzerland; Dr Claudia P. Alfonso, 2367

Quality, Safety and standards (QSS), WHO, Geneva, Switzerland; Prakash Ghimire, Global 2368

Influenza Programme (GIP), WHO, Geneva, Switzerland; Dr Laszlo Palkonyay, Initiative for 2369

Vaccine Research (IVR), WHO, Geneva, Switzerland; Dr Dina Pfeifer, Quality, Safety and 2370

standards (QSS), WHO, Geneva, Switzerland; Dr Jinho Shin, Quality, Safety and standards 2371

(QSS), WHO, Geneva, Switzerland 2372

2373

In June 2009, a revised draft was produced in light of line by line comments and input received 2374

from stakeholders from national immunization programs, basic research and public health 2375

sciences, regulatory authorities, and vaccine manufacturers during the informal consultation of 2376

February 2009. This draft was send to these stakeholders for review and submission of 2377

additional comments by the 15 July 2009. 2378

2379

A revised draft was produced in light of comments received. This draft was proposed for 2380

endorsement by the Expert Committee on Biologicals Standardization of October 2009 convened 2381

in Geneva, Switzerland. 2382

2383

WHO/BS/09.2111

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The present revised document reflects the comments and discussions with the Expert Committee 2384

on Biologicals Standardization of October 2009 convened in Geneva, Switzerland. The 2385

Committee endorsed the document with request for modifications. 2386

2387

WHO Headquarters Responsible Officer: Dr Claudia P. Alfonso 2388

2389

Acknowledgements 2390

2391 Acknowledgments are due to Dr Roland Levandowski for his expert advice and having laid the 2392

foundation of these recommendations. 2393

2394

Acknowledgements are due to Mrs. Christine Jolly and Mrs. Diana de Toth for their secretarial 2395

assistance and program support at WHO Headquarters. 2396

WHO/BS/09.2111

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Organization, 1998, Annex 3 (WHO Technical Report Series, No. 872), 2637



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(Addendum 2000) Annex 1, WHO Technical Report Series, No. 910, 2002 2647

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and pharmaceutical products, Blood Safety and Clinical Technology Department, Health 2652

Technology and Pharmaceutical Cluster, WHO Geneva, 2003, 2653

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components and plasma derivatives (Requirements for Biological Substances No. 27, 2657

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prepared by recombinant DNA technology. In: WHO Expert Committee on Biological 2663

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World Health Organization, 1992, Annex 2 (WHO Technical Report Series, No. 822), 2671



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MtgRep.IK.26_Sep_07.pdf, accessed 19 January 2009 2680

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Expert Committee on Biological Standardization, Fifty-Seventh report, 2006 (WHO 2683

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%20BS%20204909_Nov_06.pdf, accessed 26 January 2009 2686

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and Human Services, Public Health Service, Centers for Disease Control and Prevention 2689

and National Institutes of Health, Fifth Edition, 2007 2690

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In: World Health Organization Expert Committee on the use of essential drugs, Forty-2693

Fifth report, 1995 Annex 3 (WHO Technical Report Series, No. 850), 2694

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vaccine development, FDA/NIH/WHO Public Workshop, 2698

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2700

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67. Belshe, R, Gruber, W, Mendelman, P, Mehta, H, Mahmood, K, Reisinger, K, Treanor, J, 2701

Zangwill, K, Hayden, F, Bernstein, D, Kotloff, K, King, J, Piedra, P, Block, S, Yan, L, 2702

Wolf, M. Correlates of immune protection induced by live, attenuated, cold-adapted, 2703

trivalent, intranasal influenza virus vaccine. J Infect Dis. 2000 Mar;181(3):1133-7, 2704

http://www.jstor.org/pss/30111429, accessed 24 February 2010 2705

2706

2707

2708

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Appendix 1: Summary protocol for influenza vaccines (human, live 2709

attenuated) for intranasal administration (master/working seed lot Type A or 2710

Type B) 2711

2712 The model summary protocol that follows is provided as general guidance to manufacturers. It is not 2713 intended to constrain them in the presentation of data relevant to the complete review of the quality 2714 control tests performed on the vaccine. It is important to note that satisfactory test results do not 2715 necessarily imply that the vaccine is safe or effective, since many other factors must be taken into account, 2716 including the characteristics of the manufacturing facilities. 2717

2718 Name and address of manufacturer_________________________________________ 2719 2720 Laboratory reference No. of lot 2721 2722 No. of lyophilised lot (if applicable) 2723 2724 Date when the processing was completed 2725 2726 Information on manufacture 2727 2728 Substrate for manufacture: 2729 2730

a) Vaccine-quality embryonated eggs or cell culture 2731 b) For vaccine-quality embryonated eggs, qualification of eggs 2732

a. Layer flock status e.g. specific pathogen free - specific antibody negative 2733 b. Supplier name and address 2734 c. Remarks 2735

c) For cell culture, qualification of cells 2736 a. Cell bank reference no. 2737 b. Species and tissue origin 2738 c. Passage history 2739 d. Remarks 2740

2741 Virus used to inoculate vaccine-quality embryonated eggs or cells for the manufacture of the lot: 2742 2743

a) Strain and sub-strain 2744 b) Passage level 2745 c) Source and reference no. 2746 d) Remarks 2747 2748 Results of sterility test 2749

Method: 2750 Media and temperature of incubation: 2751 Volume inoculated: 2752 Date of start of test: 2753 Date of end of test: 2754 Result: 2755

2756 Results of tests for adventitious agents 2757 2758 Results for tests of attenuation 2759 2760 Conditions of storage 2761

2762

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Monovalent virus pool Type A or Type B 2763 2764 Name and address of manufacturer 2765 2766

Laboratory reference No. of the virus pool 2767 2768

Virus used to inoculate vaccine-quality embryonated eggs or cells 2769 2770

a) Master seed strain and source 2771 b) Passage level of master seed 2772 c) Working seed lot, reference No. and source 2773 2774 Date of inoculation 2775 2776 Date of harvesting allantoic or amniotic fluid or cell culture fluids 2777 2778 Storage conditions prior to use in final bulk 2779 2780 Clarification/Purification procedure 2781 2782 Antibiotics used during preparation, if any 2783 2784 Identification of preservatives/stabilizers, if any 2785

2786 Tests on monovalent pool 2787 2788 Determination of infectivity (potency) 2789 Method 2790 No. of vaccine-quality embryonated eggs or cell cultures inoculated 2791 Date of test 2792 Results 2793 2794 Determination of attenuation 2795 Method 2796 Date of test 2797 Results 2798 2799 Determination of identity of virus 2800 Method 2801 Date of test 2802 Results 2803 2804 Test for adventitious virus agents 2805 Method 2806 Date of test 2807 Results 2808 2809 Test in vaccine-quality embryonated eggs (for egg based vaccine) 2810 Method 2811 Date of test 2812 Results 2813 2814 Test in cell cultures 2815 Method 2816 Date of test 2817

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Results 2818 2819 Test for adventitious bacterial agents 2820 Method 2821 Date of test 2822 Results 2823 2824 Test for adventitious fungal agents 2825 Method 2826 Date of test 2827 Results 2828 2829 Test for adventitious mycoplasma agents 2830 Method 2831 Date of test 2832 Results 2833 2834 Test for adventitious mycobacterial agents 2835 Method 2836 Date of test 2837 Results 2838 2839 Test for chemicals used in production (may be performed on final bulk) 2840 Method 2841 Date of test 2842 Results 2843 2844 Test for residual DNA (for cell culture vaccine) 2845

Method 2846 Date of test 2847 Results 2848 2849 Final bulk 2850 2851 Name and address of manufacturer 2852 2853 Identification of final bulk 2854 2855 Identification of monovalent virus pool(s) used to prepare final bulk 2856 2857 Date of manufacture 2858 2859 Control of final bulk 2860 2861 Test for infectivity (potency) (may be performed on final lot) 2862 Method 2863 Date of test 2864 Results 2865 2866 Test for sterility 2867 Method 2868 Date of test 2869 Results 2870 2871

Test for total protein 2872

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Method 2873 Date of test 2874 Results 2875 2876 Test for ovalbumin (for egg based vaccine) 2877 Method 2878 Date of test 2879 Results 2880 2881 Test for residual DNA (for cell culture vaccine) 2882 Method 2883 Date of test 2884 Results 2885 2886

Test for chemicals used in production 2887 Method 2888 Date of test 2889 Results 2890 2891 Control of Final Lot 2892 2893

Test for infectivity (potency) 2894 Method 2895 Date of test 2896 Results 2897 2898 Test for identity 2899 Method 2900 Date of test 2901 Results 2902 2903 Test for sterility 2904 Method 2905 Date of test 2906 Results 2907 2908

Test for endotoxin 2909 Method 2910 Date of test 2911 Results 2912 2913 Test for residual moisture (if applicable) 2914 Method 2915 Date of test 2916 Results 2917 2918 Inspection of final containers 2919 Results 2920 2921 Other tests 2922 Additional comments (if any) 2923 2924 A sample of a completed final container label and package insert should be attached. 2925 2926 Certification by producer 2927

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2928

Name of head of production of the final lot 2929

2930

Certification by head of the quality assurance department taking overall responsibility for 2931

production and control of the final lot: 2932

2933

I certify that lot No.___________________ of influenza vaccine (human, live attenuated) for 2934

intranasal administration, whose number appears on the label of the final container, meets all 2935

national requirements1 and satisfies Part A of the Requirements for Biological Substances No. 17, 2936

revised 1990. 2937

2938

Signature ___________________________________________ 2939

2940

Name typed ___________________________________________ 2941 2942 Date: ___________________________________________ 2943

2944

2945

Certification by the national regulatory authority 2946

2947

If the vaccine is to be exported, provide a copy of the certificate from the national regulatory 2948

authority as described in section D.2, a label of a final container, and a leaflet of instructions to 2949

users. 2950

2951

1 If any national requirement(s) is (are) not met, specify which one(s) and indicate why release

of the lot has nevertheless been authorized.

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Appendix 2: Reference Laboratories 2952 2953

WHO Collaborating Centres for Reference and Research on 2954

Influenza 2955

2956

• WHO collaborating centre for reference and research on influenza, Melbourne, 2957

Australia 2958

2959

• WHO collaborating centre for reference and research on influenza, National Institute 2960

of Infectious Disease, Tokyo, Japan 2961

2962


for Medical Research, London, UK 2964

2965

• WHO collaborating centre for the surveillance, epidemiology and control of influenza, 2966

Center for Disease Control and Prevention, Atlanta, USA 2967

2968

• WHO collaborating centre for studies on the ecology of influenza in animals, St. Jude 2969

Children's Research Hospital, Memphis, TN, USA 2970

2971

Essential Regulatory Laboratories: 2972 2973

• Therapeutic Goods Administration, Immunology and Vaccines, Canberra, Australia 2974

2975

• National Institute of Infectious Disease, Tokyo, Japan 2976

2977

• National Institute for Biological Standards and Control, South Mimms, UK 2978

2979

• Centre for Biologics Evaluation and Research, Division of Viral Products, Food and 2980

Drug Administration, Rockville, Maryland, USA 2981

2982

WHO reference laboratories for diagnosis of influenza A/H5 2983

infection 2984

2985

• WHO collaborating centre for reference and research on influenza, Melbourne, 2986

Australia 2987

• Department of Microbiology, Faculty of Medicine, University of Hong Kong, Hong 2988

Kong SAR China 2989

• National Influenza Centre, Centre for Health Protection, Hong Kong SAR China 2990

• Virology and Zoonotic Disease Research Program, US Naval Medical research Unit 2991

3 (NAMRU-3), Cairo, Egypt 2992


for Medical Research, London, UK 2994

• Institute Pasteur, Unité de Génétique Moléculaire des Virus Respiratoires, Paris, 2995

France 2996

• National Institute of Virology, Pune, India 2997

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of Infectious Disease, Tokyo, Japan 2999

• WHO collaborating centre for the surveillance, epidemiology and control of influenza, 3000

Center for Disease Control and Prevention, Atlanta, USA 3001

• WHO collaborating centre for studies on the ecology of influenza in animals, St. Jude 3002

Children's Research Hospital, Memphis, TN, USA 3003

• Federal State Research Institution, State Research Centre for Virology and 3004

Biotechnology VECTOR Novosibirsk Region, Russian Federation 3005

3006

3007

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Appendix 3. Model certificate for the release of influenza vaccine (human, live 3008

attenuated) for intranasal administration 1

3009

3010

The following lots of influenza vaccine (human, live attenuated) for intranasal 3011

administration 3012

Called [Trade name and/or common name of the product] 3013

Produced by 3014

[Name and address of manufacturer]______________________________2 3015

in [List of manufacturing sites]____________________________3 3016

whose marketing authorization number is [insert marketing authorization number] and 3017

whose numbers appear on the labels of the final containers, meet all national requirements4, 3018

the manufacturing recommendations in part A of the WHO recommendations to assure the 3019

quality, safety, and efficacy of influenza vaccines (human, live attenuated) for intranasal 3020

administration (revised 2009)5

and the WHO recommendations for good manufacturing 3021

practice and quality assurance for biological products6, and has been approved for release.

3022

Lot Number [including

sub-lot number and

packing lot numbers if

relevant]

Container type and

number of doses per

container

Storage conditions Number of

containers/batch

size

Date of start of period

of validity (e.g.

manufacturing date)

and/or

expiry date

3023

1 To be provided by the national regulatory authorities of the country where the vaccines have been manufactured 2 Name and address of manufacturer 3 Country 4 If any national requirement(s) is (are) not met, specify which one(s) and indicate why release of the lot(s) has nevertheless been authorized by

the national regulatory authority 5 To be published in WHO Technical Report Series, No. (to be announced), 2009, Annex (to be announced); with the exception of the previsions

on shipping, which the national regulatory authority may not be in a position to control. 6 WHO Technical Report Series, No. 822, 1992, Annexes 1 and 2

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As a minimum, this certificate is based on examination of the manufacturing protocol. 3024

3025 The number of this certificate is: _____________________________________________ 3026 The date of issue of this certificate is: _____________________________________________ 3027 3028 Title of authorizing official (typed):_________________________________________7

3029 3030 Name of official (typed): __________________________________ 3031

3032

3033

Signature of official: ______________________________________ 3034

3035

Date: _______________________________ 3036

3037

3038

3039

3040

7 Or her or his representative