New generation vaccines  

A

Seminar

on

DIVISION OF BACTERIOLOGY AND MYCOLOGY, IVRI

Mamta singh

Phd Scholar

Roll no. 177

Approaches to veterinary bacterial vaccine designVaccine generation

Type Development process

Vaccine examples

I Inactivated whole bacteria (bacterins) or culturesupernatants

Chemical or physical inactivation to eliminate infectivity but retainimmunogenicity

Leptospira Hardjo,Pasteurella multocida

I Live attenuated whole bacteria

Application of in vitro passage orrandom chemical mutagenesis toattenuate the strain and lose infectivity

Brucella abortus S19,Salmonella Pullorum

I and II Subunit or extract vaccines

1.Purified proteins that are chemicallyInactivated2. Purified capsular polysaccharide thatare conjugated with protein

Clostridium tetani toxoid,Clostridium perfringens typeD epsilon toxoidActinobacillus pleuropneumoniaeserotype 5b capsularpolysaccharide-tetanus toxoid conjugate (Andresen et al., 1997)

II Gene deletion vaccines

Rational attenuation through mutagenesisor gene knockout procedures

Chlamydophila abortustemperature sensitive livevaccine (Chalmers et al., 1997)

II Recombinant component vaccines

Cloning of appropriate genes andexpression of their products

Subunit vaccine candidatesfor Streptococcus equi(Timoney et al., 2007;Wallera et al., 2007)

III Reverse vaccinology

In silico analysis of the genome toselect vaccine targets

Subunit vaccine candidates forDichelobacter nodosus(Myers et al., 2007)

Approaches to veterinary bacterial vaccine design

New generation vaccine-IIIrd generation

Changing in antigen type

Changing in delivery methods

Changing in adjuvants type

New generation vaccines

Category –I Antigens Generated by Gene Cloning

Recombinant vaccines

• Recombinant vaccines commercially

available for Mannheimia haemolytica

and Actinobacillus pleuropneumoniae

based upon the leukotoxins produced

by these organisms, as well as

transferrin-binding proteins

• Recombinant cocktail vaccine comparising rOmpA and rOmp C provide 83.33% protection against salmonella challenge (IVRI, annual report,2013-14)

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• Transposon technology• Precise genomic excision of

genes• Used as marker vaccine

Category –II Genetically Attenuated Organisms(Gene deleted vaccines)

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Gene deleted vaccines• Gene-deleted Salmonella enterica serovar typhimurium and

serovar enteritidis vaccines have been licensed for use in poultry (Babu et al., 2004; Meesun et al., 2007)

• AroA gene-deleted Streptococcus equi vaccine Equilis StrepE vaccine from the S. equi TW928 deletion mutant lacking bp 46 to 978 of the aroA gene has been licensed for use in horses (Meesun et al., 2007)

• A double gene (gE and TK) deleted Pseudorabies virus marker vaccine licensed for use in pigs( Meesun et al., 2007)

• gE deleted a Bovine herpesvirus-1 marker vaccine has been licensed for use in cattle (Meesun et al., 2007)

• A deletion mutant of Brucella abortus S19 (IVRI, annual report,2013-14)

Category –III Recombinant vectored vaccine

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Category –III Recombinant vectored vaccine

Viral Vectored Vaccines– Pox viruses, Adenoviruses and Herpesvirus-as

delivery systems for foreign antigens

– Virus – simply as a vector• E.g. Vaccinia-rabies recombinant vaccine

– Virus – both as vector & vaccine against the infection by the wild vector itself• E.g. recombinant capripox virus expressing PPR

virus Ag

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Advantages and disadvantages of recombinant vectored vaccines

Advantages – Rapid generation– No need for protein expression and purification – Potentially generic and low-cost manufacturing

processes– Thermostability– Leading technology for T cell induction

Disadvantages – Affected by maternal antibody

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Category –IV DNA Vaccines/Polynucleotide immunization

Gene for an antigenic determinant is inserted into a plasmid

Genetically engineered plasmid injected into the host

Within the host cells, the foreign gene can be expressed from the plasmid DNA

If sufficient amounts of the foreign protein are produced, they will elicit an immune response

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Production of DNA vaccines

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Delivary of DNA Vaccines

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Advantage of DNA vaccine

Only protective antigen is included like subunit vaccine

Avoid the problem of incorrect folding and glycosylation of antigenic protein

Easy inclusion of regulatory cytokines

Stable, less variation and cheaper to prepare

Safe and long lived immunity(HMI & CMI)

Inexpensive & Multivalency could be achieved

Can induce immune responses in the presence of maternal antibodies

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DNA Vaccines-Disadvantages

• Limited to protein immunogen only

• Potential integration of plasmid into host genome

leading to insertional mutagenesis

• Chromosomal integration – cell transformation

• Induction of autoimmune responses (e.g. pathogenic

anti-DNA antibodies)

• Induction of immunologic tolerance (e.g. where the

expression of the antigen in the host may lead to

specific non-responsiveness to that antigen)

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DNA vaccine – to further improve

• CpG motifs inclusion

• Cytokines inclusion

• Co-stimulatory molecules inclusion

• Conventional adjuvants

• Prime boost approach –Priming with DNA vaccine but

boost with protein or some other recombinant virus

vector expressing the protein

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Vaccines against Johne’s disease

Vaccine Vector SpeciesVaccinationages

Dosage Immunity

DNA vaccine (Kathaperumal et al. 2008)

Four rAgs (85A, 85B, 85Cand superoxide dismutase)with two adjuvants(monophosphoryl lipid Aand bovine IL-12)

Cattle5–10 days

100 μg ofeach antigenand 100 μgof IL-12 im

Antibodies within 3 weeks;significant IFN-g productionwithin 11 weeks Significant increases in CD4+ and CD8+ T cells against all four rAgs;rAg-specific expression of IL-2,IL-12 and TNF-α. 4/8 animals did not show bacteria in tissue

DNA vaccine(Sechi et al.2005)

Three rAgs (Mycobacteriumavium 85A, BCG 85Aand 65K)

Sheep5 months

Three dosesof 1 mg ofeach antigenim. 20 daysapart

Increased IFN-g and IL-10expression, increased CD4+ T cells,Absence of lesions and bacteria in tissues

New generation vaccine-IIIrd generation

• Sequencing of whole bacterial genomes has led to new

approaches to vaccine design (Scarselli et al., 2005), and a

‘‘third generation’’ of vaccines

• New methods of antigen discovery and design including

reverse vaccinology, structural biology, and systems biology

(Rinaudo et al. 2009)

• First example of the use of a genome sequence to produce

vaccine antigens was Neisseria meningitidis (Pizza et al.

2000)

Reverse vaccinology

Application of Reverse vaccinology in vaccine design

Genome-based approaches: strategies in selecting protective antigens– In silico analysis

– for detection of virulence factors– for detection of secreted or surface-associated

proteins– for prediction of T cell and B cell epitopes

– Functional genomics in vaccine design– Proteomics– DNA microarray analysis– Other technologies

– Pan-genomic approach in vaccine design

In silico analysis for detection of virulencefactors

• Comparison of the predicted coding sequence with the

known genes in a database using BlastP or BlastN

homology search tools is a convenient way to identify a

putative virulence gene

• Blast sequence comparison cannot be used for prediction

of new families of virulence factors (Grandi, 2001)

Programme Location and/or function Reference

PSORTb (http://www.psort.org/psortb/)

For predicting the location of proteins in Gram-negative bacteria (cytoplasm, cytoplasmic membrane,periplasm, and outer membrane or extracellular space)

Nakai (2000)

SignalP (http://www.cbs.dtu.dk/services/SignalP/)

For predicting the presence and location of signal peptidase I (SPaseI) cleavagesites within the N-terminal 70 amino acids of secreted proteins

Bendtsen et al. (2004)

TMpred (http://www.ch.embnet.org/software/TMPRED_form.html)

For detecting N-terminaltrans-membrane helices

Hoffman and Stoffel (1993)

In silico analysis for detection of secreted orsurface-associated proteins

Functional genomics in vaccine design

• Techniques- Proteomics DNA microarray analysis Other technologies

• Aim of functional genomics is to reveal the links between

a specific genotype and its corresponding phenotype

• Phenotype results from the expression of genes through

conversion into systemic, catalytic and regulatory

products, and is a complex function of genotype and

environment (Dharmadi and Gonzalez, 2004)

Proteomics

Proteomics can be divided into three main areas:

• ‘‘Protein micro-characterization’’, which deals with large-scale

identification of proteins and their post-translational modifications

• ‘‘Differential display’’ proteomics, a way to compare protein

quantities, and which can be used to investigate the microbial

pathogenicity in regards to protein expression levels

• ‘‘Protein–protein interactions’’ using techniques such as mass

spectrometry (Pandey and Mann, 2000)

These three applications in conjunction with the characterization of

membrane and surface-associated proteins are important for vaccine

development (Serruto et al., 2004)

Proteomics approach to bacterial vaccine development

• Either all the bacterial proteins, or preferably only the

surface proteins (the ‘‘surfaceome’’Cullen et al., 2005), are

first resolved into their individual components using 2DE

• Each separated protein is digested into its discrete peptide

fragments using a suitable enzyme, and the molecular

mass of each proteolytic digested fragment is then

accurately measured using matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry

(MALDI-TOF MS)

Proteomics approach to bacterial vaccine development

• SERological Proteome Analysis (SERPA)- Combining proteomics

with serological analysis is another useful refinement for

identifying potential vaccine candidates, used by Vytvytska et al.

(2002) for identification of vaccine candidates against S. aureus

• In SERPA the bacterial surface proteins were first resolved by 2-DE

and then electro-transferred onto a membrane that was blotted

with different serum pools of S. aureus infections

• Identified a set of highly immunoreactive staphylococcal proteins

Proteomics approach to bacterial vaccine development

• Proteome microarrays

– Used with serum from immunized or infected animals to help

identify immunodominant antigens of bacteria

– Method has been applied to identify antigens of Francisella

tularensis that are of relevance for vaccine development

(Eyles et al., 2007)

Disadvantages-limitation of the proteomic approach is the large

number of proteins expressed by the pathogen under in vitro

growth conditions (Etz et al., 2002a)

DNA microarray analysis

• Preparation of DNA microarrays from whole genome sequence

data that represent the whole bacterial genome

• Microarrays can be hybridized with cDNA that is prepared from

extracted RNA of a microorganism grown under different growth

conditions (e.g. in vivo versus in vitro growth)

• In this way analysis of the transcriptome allows identification of

genes that are up regulated in vivo (Rappuoli, 2000a)

– N. meningitidis - (Grifantini et al., 2002)

– P. multocida - to identify a core set of 13 upregulated and 16 down-

regulated genes in isolated from the liver and bloodstream of infected

chickens (Boyce et al.,2004)

Disadvntages of DNA microarray

(1) Use of DNA microarrays is a semiquantitative method

due to the lack of linearity and proportionality of

mRNA/ cDNA concentration to signal intensity at high

concentration

(2) An inherent limitation of DNA microarrays is that the

resulting transcriptome does not account for post-

transcriptional events

(3) Processing and analysing transcriptome is difficult

Reverse vaccinology approach used with bacterial pathogens include

– Porphyromonas gingivalis (Ross et al., 2002)

– Streptococcus pneumoniae (Wizemann et al., 2001)

– B. anthracis (Ariel et al., 2002)

– Chlamydia pneumoniae (Montigiani et al., 2002)

– Mycobacterium tuberculosis (Betts, 2002)

– Staphylococcus aureus (Vytvytska et al., 2002)

– Edwardsiella tarda (Srinivasa Rao et al., 2003)

– Leptospira interrogans (Gamberini et al., 2005)

Other technologies for functional genomics

• ‘‘In vivo induce antigen technology’’ (IVAT)-convalescent sera from several

naturally infected individuals are pooled and pre-adsorbed with surface expressed

antigens of the target pathogen grown in vitro, to remove antibodies recognizing

antigens expressed in vitro

• ‘‘Expression library immunization’’ (ELI)-

– a genomic DNA expression library from the pathogen of interest is prepared

and divided into pools

– Plasmid DNA from each pool of clones is used to immunize a model

subsequently used as protein subunits or as components of DNA vaccines

(Barry et al., 2004)

Protective antigens in Mycobacterium avium subspecies paratuberculosis, the

agent of Johne’s disease (Huntley et al., 2005)

Pan-genomic approach in vaccine design• Genomic sequence of a single strain provides a huge potential

resource to determine relationships between genotype and phenotypes within the species, but it fails to differentiate genetic variation between strains (e.g. avirulent and virulent strains)

• All available genome sequences of different strains of pathogen were divide into two subgenomes:– A core genome containing genes present in all strains -

responsible for the basic bacterial metabolisms – A dispensable genome composed of genes that are unique to

each strain –responsible for genetic diversity, pathogenicity, colonization,and antibiotic resistance

• Vaccine candidates against Streptococcus agalactiae (Medini et al., 2005)

• Recombinant subunit vaccine against rickettsial septicaemia in fish caused by Piscirickettsia salmonis (Wilhelma et al., 2006)

Adjuvants-Immunomodulators

Conclusion• Vaccines are valuable and specialized products, of great diversity

have already achieved great success in controlling many diseases of economics importance in farm and companion animals

• While present they do not cover all infections, access to modern techniques are used for designing to new vaccine ,not only prolongation of immunity, but also to better practical aspects, such as product stability and less dependence on cold-storage

• With improvements in vaccines and reduction in “cold-chain” requirements will contribute to better standards of animal health and farming prosperity, which in turn benefit human health and the cost of producing safe food

THANK

S