www.elsevierhealth.com/journals/tube

Tuberculosis

Approaches towards the development ofa vaccine against tuberculosis: recombinantBCG and DNA vaccine

Norazmi Mohd Nora,*, Mustaffa Musab

aSchool of Health, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, MalaysiabMedical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan 16150, Malaysia

Summary The last few years have witnessed intense research on vaccine develop-ment against tuberculosis. This has been driven by the upsurge of tuberculosis casesglobally, especially those caused by multi-drug-resistant Mycobacterium tuberculosisstrains. Various vaccine strategies are currently being developed which can bebroadly divided into the so-called living and non-living vaccines. Examples areattenuated members of the M. tuberculosis complex, recombinant mycobacteria,subunit proteins and DNA vaccines. Given current developments, we anticipate thatrecombinant BCG and DNA vaccines are the most promising. Multiple epitopes of M.tuberculosis may need to be cloned in a vaccine construct for the desired efficacy tobe achieved. The technique of assembly polymerase chain reaction could facilitatesuch a cloning procedure.& 2003 Elsevier Ltd. All rights reserved.

KEYWORDS

Assembly PCR;

DNA vaccine;

Recombinant BCG;

TB vaccine

Introduction

A third of the world’s population is exposed to therisk of tuberculosis (TB). Mycobacterium tubercu-losis, the main causative agent of TB, infectsapproximately eight million individuals and causestwo million deaths annually.1 This problem iscompounded by the global emergence of M.tuberculosis strains which are resistant to themajor antibiotics used to treat TB and the suscept-ibility of HIV-infected individuals to TB,2 thusmaintaining a reservoir of TB-infected individuals.Currently, the only available vaccine against TB, M.bovis bacille Calmette–Guerin (BCG), has failed tocontrol the increase of new cases worldwide andthe disease therefore remains a global healthproblem.3 BCG vaccination seems to be highly

variable and poorly protective in certain countries4

perhaps due to differences in the efficacy of thevarious BCG vaccine strains used,5 environmentalfactors6 as well as host genetic factors.7 Therefore,there is an urgent need to develop better orimproved TB vaccines as an alternative to BCG.There are a number of living and non-livingcandidate vaccines being developed. Live vaccinesinclude genetically modified forms of the M.tuberculosis complex, recombinant or re-engi-neered BCG, and the use of vectors such as vacciniavirus and Salmonella strains. Non-living candidatevaccines include subunit and DNA vaccines. Thisreview will expound on some of the recentadvances in TB vaccine development with specialreference to recombinant BCG and DNA vaccines. Inaddition, we describe the use of assembly poly-merase chain reaction (PCR) for the construction ofpotential candidate vaccines containing multi-epitopes of TB and other organisms.

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*Corresponding author.E-mail address: norazmi@kb.usm.my (N.M. Nor).

1472-9792/$ - see front matter & 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.tube.2003.08.011

Tuberculosis (2004) 84, 102–109

Short history of BCG

BCG, an attenuated strain of M. bovis initiallydeveloped at the Pasteur Institute, Paris, at thebeginning of the last century by Albert Calmetteand Camille Guerin, is the current vaccine usedagainst TB. BCG has been administered worldwideto approximately three billion individuals.8,9 How-ever, doubts about its efficacy are increasing,reflected by its highly variable protective efficacyin controlled clinical trials.4 This discrepancy maybe due to the number of mutations and deletionsthat have occurred since its initial isolation in1908,5 although no direct evidence has been putforward to show that these mutations have indeedcompromised the efficacy of BCG strains.

The history of BCG strain development has beenreported by Brewer and Colditz.9 From the originalstrain, three different lineages of BCG, wereobtained on three different media which latercontributed to many daughter strains of BCG buttwo of the lineages were abandoned after 1932.10

At present, a number of BCG vaccine strains, suchas Pasteur, Bilthoven, Japan, Dakar, Sweden,Copenhagen, Chicago, Brazil, Russia and Glaxo,have been produced by different manufacturersand named according to where they are manufac-tured. These BCG vaccine strains are different withregard to their bacteriological, biochemical andimmunological characteristics.11–13 The efficacy ofthe various BCG vaccine strains has been reportedto be around 80% down to 0%.4 A number ofsuggested reasons for the variation of BCG efficacyinclude differences in strains, dosage and vaccina-tion schedule, previous exposure to environmentalmycobacteria, and genetic variability of the popu-lation.4–8 In addition, BCG vaccination may provideprotection against only primary infection but has alimited role on already infected individuals or incases of reactivation TB, suggesting that childhoodvaccination may play a minimal role in preventingadult re-infection.14 Thus, better or improvedvaccines for the control of TB are urgently neededas an alternative to BCG. An ideal TB vaccine shouldbe one that is safe, stable and inexpensive to massproduce. It should require only a single injection toprovide lifelong and worldwide protection as wellas being effective in preventing reactivation.

Various approaches for the developmentof novel TB vaccines

For the past several years, progress has been madein the search for a potential candidate TB vaccine.

A number of alternative living and non-livingputative TB vaccines are being studied and dis-cussed by many authors (see for example15–17). Livevaccines include attenuated mycobacteria such asauxotrophic M. tuberculosis18,19 or less virulentmycobacteria such as M. microti, M. habana, M.vaccae, or M. smegmatis that overproduce immu-nogenic antigens of M. tuberculosis15,16 and re-combinant BCG.20,21 Vehicles such as vacciniavirus22 and attenuated Salmonella strains23 havealso been used. Non-living vaccines include subunitvaccines consisting of secreted and non-secretedproteins, and/or lipid or carbohydrate componentsof M. tuberculosis24–26 and DNA-based vaccinescontaining genes coding for candidate TB epi-topes.27,28

Live vaccines

Auxotrophic mutants of M. tuberculosis complexEfforts to generate auxotrophic mutants of M.tuberculosis or BCG have been actively pursued forthe production of improved live vaccine forTB.18,29–32 In principle, auxotrophic mutants weregenerated by knocking out certain genes or byinduction of one or more mutations in an essentialmetabolic pathway. Other progress in generatingauxotrophs with different levels of attenuationusing signature-tagged mutagenesis technique hasalso been reported.33,34 However, none of theauxotrophic mutants tested thus far has providedbetter protection than the standard BCG in animalstudies, and, furthermore, their stability and safetyfor human use is still in doubt.

Re-engineered BCGAnother approach is to modify or re-engineer BCG.This strategy relies on the basic premise that BCGwhich already has an impressive safety record andis cheap and easy to mass produce, could be re-engineered to enhance its efficacy through insert-ing genes encoding immunodominant antigens orimmunostimulatory genes. For example, recombi-nant BCG expressing various cytokines such asinterleukin-2, interferon gamma, or granulocyte-macrophage colony-stimulating factor have beenshown to improve the response against M. tubercu-losis antigens20,35 and recombinant BCG expressinglisteriolysin of Listeria monocytogenes showed anenhanced capacity to stimulate CD8þ T cells.36 Inaddition, some of the genes in BCG that have beendeleted during the attenuation process may bereintroduced and re-expressed in the recombinantBCG vaccines. Others have produced deletedmutants of BCG, but this approach is mainly aimed

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to make BCG safer to be used in immunocompro-mised individuals rather than to enhance theefficacy of BCG.37 Recently, a recombinant BCGthat overexpresses the 30 kDa Ag85 protein hasbeen reported to provide better protection thanBCG21 (see below).

Other attenuated strains of mycobacteriaNaturally attenuated strains of mycobacteria havethe potential to be used as vaccines against TB.These include the enviromental Mycobacteriumspecies such as M. vaccae, M. microti and M.habana. Skinner et al.38 reported that heat-killedM. vaccae was able to stimulate CD8þT cellsspecific for macrophage-infected M. tuberculosis.Recombinant M. vaccae39 and M. smegmatis37

which express M. tuberculosis epitopes have alsobeen reported. However, these strains have notshown better protective efficacy than BCG itself.15

Non-mycobacterial live vectorsSeveral recombinant non-mycobacterial living vac-cine vehicles such as vaccinia virus22 and attenu-ated Salmonella strains23,40 containing candidategenes of M. tuberculosis have been constructed.For example, a recombinant Salmonella expressionsystem was able to express the immunodominantAg85B M. tuberculosis antigen via the MHC Class Ipresentation pathway for appropriate immunerecognition.41 However, the safety of these vectorsfor human use has not been thoroughly ascertained.

Non-living vaccines

Subunit proteinsWhole organism-based vaccines which containmany epitopes may induce irrelevant immuneresponse. Subunit vaccines therefore have theadvantage of safety and they are generally easyto produce. A number of subunit vaccines which arebased on various secreted and non-secreted pro-teins, and/or lipid or carbohydrate components ofM. tuberculosis have been studied.24–26 Potentialimmunodominant antigens of M. tuberculosis whichreact to antisera and T cells from TB patients orinfected animals have been identified.42 In addi-tion, potential non-peptide antigens such as my-colic acids and glycolipids have been reported toactivate T cells via the CD1 family of antigen-presenting molecules.43 However, these non-livingsubunit proteins have still failed to providebetter protection than BCG. Furthermore, themain drawbacks of subunit proteins are thatthey are expensive to produce and requiremultiple boosters.

DNA vaccinesThe development of DNA vaccines has opened anew era in vaccinology. In fact, this innovativeapproach has overcome many problems associatedwith conventional protein-based subunit vaccinesand has become the current trend in vaccinedevelopment. In principle, this approach involvesdirect injection of plasmid DNA encoding specificantigens or epitopes that can lead to protectiveimmunity.44–46

The development of DNA vaccination againsttuberculosis has been reported by many research-ers.27,28,47–49 Intramuscular administration of DNAcoding for the hsp65 or Ag85A into mice resulted insimilar protection against M. tuberculosis as com-pared to BCG.48,47 Thus far, as exemplified by theabove study, DNA vaccines have provided protec-tion at levels only close to that provided by thestandard BCG vaccine. However, these may beovercome by the cloning of multiple epitopes into asingle DNA vaccine construct (see below).

Potential vaccine strategies againsttuberculosis

The remainder of this review will focus on two ofthe most promising approaches to be exploredfor the development of an effective vaccineagainst TB.

Recombinant BCG

It is important to realize that the use of recombi-nant BCG would most likely retain most of theadvantages and disadvantages of the conventionalBCG vaccine. The major advantage would be thatthe vaccine would share the remarkable safety andlow side effects of BCG. Furthermore, it should beinexpensive and easy to mass produce becausecostly purification and synthesis are not required. Itis also noteworthy that BCG vaccination couldprotect against leprosy,50 and M. avium-intracellu-lare,51 a major opportunistic pathogen in immuno-compromised individuals. Another advantage ofusing re-engineered BCG is that it has beensuggested to protect against TB meningitis andmilitary TB4 and to minimize the dissemination ofthe bacteria from the primary lesion to other partsof the lungs.15

One disadvantage of a recombinant BCG vaccinewould be the possibility (albeit very rare) ofdisseminated disease in vaccinees, especially inimmunocompromised individuals such as those whoare HIV-infected.52 However, others have reported

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104 N.M. Nor, M. Musa

that BCG vaccination to infants who are HIV-positive is safe and probably protective.53 Inaddition, BCG could still confer modest protectionto healthy HIV-positive individuals against variousforms of TB.54 In fact, BCG-vectored HIV vaccine iscurrently being developed55 as one of the vaccinestrategies against HIV. Another major drawbackthat would be shared by a recombinant BCG vaccineis the interference with the tuberculin skin test,which is still the gold standard for detecting M.tuberculosis infection. However, we believe that inthe near future, this disadvantage may be alle-viated by diagnostic tests capable of distinguishingBCG vaccination and TB. In fact, our group iscurrently in the initial stages of negotiations for thecommercialization of such a test.

The recent findings reported by Horwitz andcoworkers21 on the better protective efficacy of arecombinant BCG expressing an epitope of M.tuberculosis compared to the parent strain havestrengthened the justification for using such astrategy. The authors showed that guinea pigsimmunized with the recombinant BCG and chal-lenged with virulent M. tuberculosis via aerosols,had fewer bacilli in their lungs and spleen andsmaller and fewer lesions in these organs ascompared to those immunized with the parentBCG strains. Interestingly, their choice of using re-engineered BCG was somewhat a ‘last resort’having been unsuccessful in inducing better protec-tion over BCG following their attempts at usingsubunit vaccines administered together with var-ious experimental adjuvants and immunostimula-tory molecules such as interleukin-12 and CpGmotifs. To date, this recombinant BCG vaccine isthe first vaccine reported to induce greaterprotective immunity against TB than the standardBCG vaccine in an animal model.

We are currently involved in the construction ofrecombinant BCG containing two T cell epitopes ofthe ESAT-6 antigen of M. tuberculosis.56 This waspart of our overall plan to construct a multi-epitoperecombinant BCG containing genes encoding se-lected epitopes from M. tuberculosis and Plasmo-dium falciparum, the causative agent for malaria.In a previous study, we attempted to clone the Cterminus of merozoite surface protein-1, into BCG.This malaria epitope was chosen because it wasreported to induce inhibitory antibodies that blockmerozoite invasion of erythrocytes,57 a processthat may arrest the pathology of malaria. However,our initial attempts to clone this malaria epitopeinto BCG have been disappointing. We hypothesizedthat the unstable and low expression of the malariaepitope may probably be due to the codon usageand base composition of the epitope: plasmodia is

A:T rich whereas BCG, like other mycobacteria, isG:C rich. Subsequently, we were able to overcomethis caveat by constructing a synthetic version ofthe epitope using a polymerase chain reaction(PCR)-based technique known as ‘DNA shuffling’ orassembly PCR,58 a modification to the methodinitially described by Stemmer and coworkers59

Recombinant BCG containing the synthetic versionof the malaria epitope showed a greatly enhancedexpression of the epitope as compared to recombi-nant BCG containing the ‘native’ version of theepitope.

The assembly PCR technique involves the designand construction of a series of short oligonucleo-tides (about 40 bp each) which are then assembledvia two rounds of PCR, into a complete DNAfragment that codes for the desired epitope (Fig.1). Based on the success of cloning the malariaepitope, we subsequently cloned a composite DNAfragment which included the hsp65 mycobacteriapromoter,60 the MTP63 signal peptide,61 two T-cellepitopes from ESAT-656 and a fragment of theerythrocyte binding antigen of P. falciparum.62

Western-blot analyses showed that the compositemalaria and TB epitopes were expressed in thesecreted form suggesting that the signal peptidewas functional. In addition, the recombinant BCGwas found to be immunogenic in mice wherebyantibody response was observed against the ma-laria epitope while cell-mediated immune responsewas observed against the TB epitope (unpublishedresults).

DNA vaccine

The use of a DNA vaccine offers many advantages: itcan be easily manipulated, safe, and easily storedand transported. Most importantly, as shownin animal studies, DNA vaccines are able toinduce both humoral and cellular immune re-sponses.28,48,63 The other advantage of a DNAvaccine is that it can be constructed together withimmunostimulatory molecules such as cytokinegenes and immunostimulatory DNA sequences, likethe CpG sequences, to enhance its immunogeni-city.64–66 The ability of certain immunostimulatorymolecules to polarize the immune response toTh167,68 may be of advantage with respect to theprotective immunity against TB. Furthermore, oneDNA plasmid can yield many copies of the antigenover prolonged period and that the antigens wouldbe endogenously expressed and thus can lead tomore efficient presentation on the MHC class Imolecules.69 This would efficiently activate cyto-toxic T lymphocytes (CTL) hence enhancing the

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Recombinant BCG and DNA vaccine 105

immune response against an intracellular pathogenlike M. tuberculosis.

However, uncertainty regarding the safety of DNAvaccine still existsFwhether the DNA wouldintegrate into the host genome or whether it cancause autoimmune disease. These questions awaitsclarification. Nevertheless, several clinical trialshave been initiated to test the efficacy of DNAvaccines against hepatitis B,70 malaria71 and HIVinfection.72

It is likely that an improvement to the efficacy ofa DNA vaccine can be achieved by incorporatingmultiple epitopes of M. tuberculosis into a singleconstruct. The application of multi-epitope DNAvaccines in some viral infection such as Epstein-Barr virus and HIV has been reviewed by Suhrbier.73

Thomson et al.74 have constructed a plasmidencoding multiple CTL epitopes and vaccination ofmice with this construct stimulated MHC-restrictedCTL responses to each of the epitopes. Morerecently, Fonseca et al.75 have also shown that

DNA vaccination with multiple epitopes of the 38-kDa protein resulted in a broader Th1 response thanthat of the single-epitope control plasmids. Basedon the study using single and combination DNAvaccine constructs, it has been suggested that theeffective DNA vaccination against TB may requiremulti-subunit vaccination or combination of differ-ent genes.28,76 Recently, Andersen17 has reviewed anumber of potential candidate antigens such asESAT-6, antigen 85A and B, MPT64 and hsp60 for thedevelopment of DNA vaccines against TB.

By adopting the technique of assembly PCR asdescribed above, we have also successfully con-structed a composite of M. tuberculosis epitopesfrom ESAT-6,56 MTP40,77 MTP6478 and 38 kDa49

antigens cloned into an appropriate plasmid vector.The immunogenicity of this construct is beingstudied in C57/b mice. Our preliminary dataindicated that stimulation of mice splenocytesimmunized with the recombinant plasmid with therelevant peptides in vitro resulted in lymphocyteproliferation and IFNg release. Further experimentsare in progress to confirm this.

A combination vaccination protocol using therecombinant BCG and DNA vaccines expressing aseries of M. tuberculosis candidate epitopes will bestudied in a prime-boost approach. In fact, primingwith DNA vaccine expressing the M. tuberculosisAg85B antigen followed by BCG was more effectivein protecting against M. tuberculosis infection inmice as compared to BCG alone.79 Furthermore,boosting of a previously recombinant BCG-vacci-nated child with a DNA vaccine at adolescence mayboost a waning memory immune response as hasbeen speculated previously.80

Conclusions and future directions

The failure of BCG vaccination to control the globalTB epidemic and the spread of multi-drug-resistantMycobacterium tuberculosis strains underline theneed for a better vaccine against TB. Despite theintense efforts towards this end, no experimentalTB vaccine has entered clinical trials and thus avaccine against TB is still anxiously awaited.However, the recent completion of the M. tuber-culosis genome81 would certainly propel TB vaccineresearch forward. The current available technolo-gies of vaccine production using living and non-living approaches are fairly well established and itis probable that an effective TB vaccine would bedeveloped via one or a combination of theseapproaches.

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16, 40-meroligonucleotides

gene assembly55-cycle PCR

23-cycle PCR95°C, 30 sec60°C, 30 sec72°C, 30 sec

95°C, 30 sec55°C, 30 sec72°C, 30 sec

add primers withdesired restrictionenzyme site

cut with relevant enzyme and clone into appropriate vector

5' 3'

5'3'

Figure 1 The assembly PCR procedure used to constructa 300 bp synthetic malaria epitope as describedpreviously [58].

106 N.M. Nor, M. Musa

However, it should be realized that improve-ments to vaccine design would be a continuousprocess. Antigens, such as the 19 kDa antigen of M.tuberculosis, initially thought to be a potentialvaccine candidate was eventually found to have adetrimental rather than a protective effect.82 Thedevelopment of an effective vaccine against TBwould require us to continually improve our under-standing of the host response to mycobacterialinfection as well as to unravel the mechanisms ofhow the hosts curb or eliminate the infection.Therefore, despite recent advances, it is generallyaccepted that the development of putative vac-cines against TB would largely be empirical. One ora combination of several such constructs that couldprovide the desirable level of protection in agenerally accepted animal model would then bechosen for further trials in humans. Progressivemodifications of the putative vaccines would thenhave to be made following the increase in knowl-edge regarding the disease pathogenesis and hostresponses before an ideal TB vaccine becomes areality.

Based on current developments, our strategy isto concentrate on two of the most promisingapproachesFrecombinant BCG and DNA vaccine.Despite the controversies surrounding its use, BCGhas several advantages that may not be easilyreplaced by other candidate vaccines. The reportby Horwitz and coworkers21 further strengthen theargument in favor of BCG. It is believed thatoverexpression and perhaps continuous expressionof selected candidate epitopes as is the case of alive vaccine like BCG may be pivotal in creating aneffective vaccine. DNA vaccine, although non-livingwould still fulfill these necessary features since it iscapable of replication and endogenously expressingthe antigens over a prolonged period, thus inducingimmunity efficiently.69,75 We believe that a combi-nation of recombinant BCG and DNA vaccinesexpressing various epitopes of M. tuberculosis andadministered in a prime-boost manner would beone of the best approaches to be explored.

The possibility of constructing a multi-epitope TBvaccine has been made fairly simple by the use ofassembly PCR. This technique would also provide uswith an elegant platform to incorporate regulatorygenes as well as genes from different chromosomesand from different organisms. The flexibility of thistechnique would allow us to easily constructvarious versions of the putative TB vaccines asmore information on the best candidate antigensemerged from studies on comparative and func-tional genomics as well as structural biology ofcandidate antigens and immunobiology of M.tuberculosis.

Acknowledgements

We thank Zainul F. Zainuddin, Rapeah Suppian,Salwana Ahmad and Jamaruddin Mat Asan for thediscussions and technical assistance afforded.The work described herein is partly funded by TheMinistry of Science, Technology and the Environ-ment, Malaysia (IRPA Grants: No. 06-02-05-8012,06-02-05-0022 and 06-02-05-9015)

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