International Journal of Drug Development and Research

Key words

MDR- Secretory antigens, culture filterate proteins, Biomarker, Subunit vaccine

Introduction

Tuberculosis, caused by Mycobacterium tuberculosis, remains a major global health problem. TB, AIDS and malaria are the ‘big three’ killer infectious diseases worldwide. TB causes ~2 million deaths annually and latently infects one-third of the world population (estimated ~2 billion). Successful global TB control faces many obstacles including the difficulty of timely diagnosis, lack of effective vaccines, and the fact that TB treatment requires many months of chemotherapy. The situation has been further compounded with the advent of M. tb/HIV co-infection and the emergence of multidrugresistant (MDR) and extensively drug-resistant (XDR) TB. In 2006, the global burden of MDR-TB, defined as resistance to isoniazid and rifampin, was estimated at 500,000 cases. Additionally, the incident of XDR-TB, caused by MDR strains that are also resistant to fluoroquinolone and at least one second-line injectable agent (amikacin, kanamycin, or capreomycin), is increasing in many countries. A deadly association between HIV and TB has been known since the start of the HIV epidemic. Of the 1.7 million people who died from TB in the year 2010, approximately 200,000 were co-infected with HIV. In light of these developments, a new and effective vaccine is urgently needed, which is essential for reducing the estimated 8-10 million new TB infections that occur annually. According to the Global plan to Stop TB (2006-2015), the introduction of effective TB vaccines will be an essential component of any strategy to control TB by 2050

History of BCG Vaccine

In 1908, Camille Guerin and Albert Calmette initiated their attempts to produce an anti-TB vaccine from a virulent bovine strain. In 1921, vaccination with BCG, an attenuated vaccine, was introduced (Sakula, 1983). The efficiency of the BCG vaccine has been questioned since its early use and therefore, a large number of trials have been carried out to determine its efficacy. In these studies it was found that, the BCG vaccine protected efficiently against leprosy (Fine and Rodrigues, 1990) as well as childhood manifestations of TB (disseminated TB) (Rodrigues et al., 1993). However, the protective efficacy against pulmonary TB was limited (Tuberculosis Research Centre [ICMR], Chennai, India, 1999). Fig.:1

Various hypotheses have been suggested to explain the low protective efficacy of BCG against pulmonary TB. These hypotheses include inappropriate treatment and storage of the vaccine, the use of different strains of BCG (Fine, 1995), and lack of an effective stimulation of the optimal blend of T-cell populations and in particular that of the CD8+ T-cells (Hess and Kaufmann, 1999). In addition to these hypotheses, the currently used intradermal route of immunization has been suggested as another factor influencing the capacity of BCG to induce optimal immunity in the lungs. In this regard, intranasal (i. n.) route of immunization has recently been evaluated as a possible route for BCG delivery, in mouse experimental models. Results from this study showed a high degree of protection against challenge with M. tb in BALB/c mice, following BCG vaccination (Falero-Diaz et al., 2000). In a similar model, vaccination with BCG conferred as good, if not better protection than subcutaneous (s. c.) route, against challenge with virulent M. bovis (Lyadova et al., 2001).

Prospects for new vaccines

Given the limitations of BCG in protection against adult pulmonary TB, there is a considerable scope for improved vaccination strategies. Immunological research has a key position in understanding the pathogenesis of TB, and thereby in developing novel designs for effective prophylactic vaccination, immunodiagnostic tools and immunotherapeutic agents. Two approaches have been considered for vaccine development. One involves the replacement of BCG by a more potent vaccination inducing immune responses capable of either complete elimination of the bacilli, or of reliable containment of persistent infection. The second approach involves the post-exposure vaccination to boost immunity in individuals whose natural immunity has already been primed by infection or BCG vaccination (Young and Stewart, 2002). Indeed, over the past decade research efforts have been directed to evaluate potential vaccine candidates as well as alternative routes of vaccine delivery, such as the intranasal (i. n.) route, in order to improve protection.

New vaccine candidates

A wide range of potential vaccine candidates have been generated and subjected to tests for protective efficacy in experimental model of infection. New vaccine candidates include live attenuated vaccines, subunit vaccines and DNA vaccines.

Live attenuated vaccinesM

Advances in the techniques required to genetically modify Mycobacteria, as well as the increase in the knowledge of the pathogenesis of the microorganism have made possible to delete genes encoding for potential virulence factors in M. tb, thereby enabling the generation of attenuated mutants. In addition to attenuated strains of M. tb, the natural attenuated Mycobacteria, such as M. vaccae and M. microti are being studied as possible vaccine candidates (Nor and Musa, 2004). Another approach has been the improvement of the BCG immunogenicity by the addition of genes encoding cytokines, such as IFN-N (Murray et al., 1996) or Mycobacterial proteins, such as the antigen 85 complex (Ag85) (Horwitz et al., 2000). Although encouraging results have been obtained in challenge experiments (Horwitz et al., 2000; Smith et al., 2001), but, still a major consideration for the clinical use of live vaccines is safety, specificity when considering TB vaccination strategies for AIDS patients.

Subunit vaccines

Subunit vaccines are currently the most widely studied. This type of vaccine has been focused in particular on proteins present in filtrates prepared nonsecreted antigens have also been shown to induce protective responses in experimental studies (Coler et al., 2001; Skeiky et al., 2000). The most extensively studied antigens are members of the Ag85 complex, a family of mycolyl transferases enzymes involved in cell wall biosynthesis and present in culture filtrates (Belisle et al., 1997). The Ag85 has been reported to induce strong activation of T-cells in several studies (Andersen et al., 1995; Mustafa et al., 1998). Other antigens being studied are:

(i) Early secreted antigenic target (ESAT-6), which has been reported to be absent from all BCG vaccine strains and to induce very strong T-cell and antibody responses (Brodin et al., 2004).

(ii) Heat-shock proteins (HSP) such as HSP-65 and HSP-70, found to induce a prominent immune response at both, the antibody and the T cell levels (Silva, 1999).

(iii) PstS-1 (38 kDa protein), a glycoprotein exposed on the surface of the Bacillus and reported to be a powerful B and T-cell antigen (Bothamley et al., 1992; Lefevre et al., 1997).

(iv) 19 kDa protein, a lipoprotein found to induce the expression of IL-12 and iNOS inmonocytes and dendritic cells through its binding to TLR2 (Brightbill et al., 1999; Thoma-Uszynski et al., 2000) and to promote neutrophil activation (Neufert et al., 2001).

A major limiting factor of the subunit vaccines is the need of adjuvant for vaccine delivery. Currently research studies are focused on the choice of which adjuvant to use and whether immuno-modulatory, such as cytokines, could be used. Despite this drawback subunit vaccines based on recombinant protein antigens are attractive because the techniques for its production are fully established and this type of vaccines are expected to satisfy the regulatory requirements for use in humans more easily than the live vaccines.

DNA vaccines

Administration of naked DNA has the potential of eliciting both cellular and humoral immunity against encoded antigens. Several Mycobacterial antigens including PstS-1, HSP-65 and Ag85 have been studied and found that they are inducing protection in animal models (Bonato et al., 1998; Fonseca et al., 2001; Huygen et al., 1996). Although the results are promising, various concerns about the safety of DNA vaccination have been raised mainly regarding the possibility of DNA integration into the host genome affecting oncogenes or tumor suppressor genes and thereby inducing the development of cancer. However, the risk of integration has been reported to be low under a variety of experimental conditions (Manam et al., 2000; Martin et al., 1999).

Experimental animal models in TB

Discussions about the value of experimental animal models in TB research have a longstanding history. Experimental animal models are critical for delineating the general mechanisms underlying natural resistance and acquisition of a protective immune response against TB. However, assessment of this information using experimental animals should be conducted carefully since there are differences in the host defense mechanisms between experimental animals and humans.

Many experimental animal species such as mouse, guinea pig and non-human primates have been used for deciphering the mechanisms involved in TB. The mouse, without any doubt it is a very sophisticated and cost-efficient animal model. The immune response of the mouse is very well understood and biological reagents such as monoclonal antibodies against surface antigens and cytokines are available. Moreover, the genetic manipulation of mouse species is highly advanced. Trans-gene expression, gene knockout, gene knock-in have all become standard technologies, and a large variety of mouse mutants with defined immune-deficiencies are available to researchers studying the role of distinct cells and effector molecules in the in-vivo setting of TB. Furthermore, the recent elucidation of the murine genome promises to open a new area of research with enormous impact on our understanding of genetic disorders and also of host mechanisms in TB (Kaufmann, 2003).

Currently, two main vaccination strategies are being pursued. The first strategy uses subunit vaccines in the form of protein-adjuvant formulation, naked DNA, or recombinant bacterial or viral carriers that express defined antigens. Till now, some very promising results have been obtained but so far no vaccine candidate tested in animal models has proven to be better than already available BCG vaccine. The second strategy, comprising viable Mycobacterial vaccines, either attenuated viable M. tb or BCG, or recombinant BCG over expressing certain antigens or immuno-modulator is also being pursued and shows promise.

Recently, a lot of attention has been focused on secretory protein antigens of Mycobacterium tuberculosis, which are synthesized by actively growing M. tb culture or by induction of desired immune response (Anderson and Heron, 1993; Anderson, 1994). These proteins have also been termed as culture filtrate proteins and known to elicit strong immune reaction in humans and animals infected with M. tb/ M. bovis (Anderson et al, 1991 b; Orme et al,1992; Romain et al,1993; Anderson, 1994). As a result of the combined efforts of several laboratories, more than 30 secretory proteins of M. tuberculosis have been characterized (Andersen et al., 1991; Anderson, 1994, Kamath et al., 1999; Sonnenberg and Belisle, 1997; Ingrid et al., 2000; Karin et al., 1998; Gennaro, 2000; Kanaujia et al., 2004; Orme et al. 1992; Romain et al1993; Spencer et al., 2004; Sable et al., 2005; Young et al., 2004).

The secretory proteins have been demonstrated to be strongly recognized by T-cells isolated from human (Tuberculosis) TB patients (Orme, 1997; Spencer et al., 2004) as well as mice and cattle experimentally infected with TB (Anderson and Heron, 1993; Pollock and Anderson, 1997; Lanbo et al., 2004). Experimental work in animal models suggests that both CD4 + and CD4 + T-cells are required for optimal protection against tubercle bacillus (Orme et al., 1992; Bonato et al., 1998; Flynn et al., 1992; Pais et al., 1998; Spencer et al., 2004). These proteins have been the focus of much of the research directed at identifying antigens that induce protective immunity or those that elicit immune responses of diagnostic value (Aub et al., 2002; Lein et al., 1999; Young et al., 2004; Paolo et al., 2004).

The recent identification of novel secreted proteins of M. tb open the way to study their immunological characterization of these protein to define their potential for immonological diagnosis of TB or vaccine design. A few numbers of secretory antigenic protein and peptides from M. tb have already been evaluated as antigens for the immunodiagnosis and vaccines research of TB.

purification by immunological methods, and by screening of expression libraries of M. tb DNA with anti-culture filtrate sera (Wolinsky and Schaeferm, 1973; Ginsberg, 1998; Grange and Laszlo, 1990; Bothamley et al, 1991; Altamirano et al, 1992; Sorensen et al, 1995; Mileler et at, 1994; Bellete et al, 2002). The early secretory antigenic target (ESAT)-6, purified protein and peptides from M. tb have been already under in-depth evaluation as antigens for the immunodiagnosis of TB. Some important antigen molecules (like, 38 KDa, 30/31 KDa, 40 KDa, 42 KDa, SOD, 30 KDa MSP, 85B, ESAT-6, and CFP10 etc,), have been found to be secreted by M. tb. These Mycobacterial antigens are highly effective for vaccine development for tuberculosis.

Biomarkers for TB

Biomarkers have been described as distinct molecular features that indicate a defined status of the host in relation to any process or involvement. For development of TB biomarkers, different conditions are desired. These conditions include protection by vaccination, discrimination of latent and active disease to facilitate quick treatment outcome or assess relapse risk. A number of TB biomarkers have been reported based on liposomes and including a synthetic Mycobacterium glycolipids as immunomodulator, it induces strong and protective T-helper-1 and T-helper-17 adult murine responses to Ag85B-ESAT-6, another DNA-based, extracellular proteins, C-reactive protein (Kamath et al., 2009; Hanekom et al., 2008; Zarate-Blades et al., 2011). Now a days several adjuvants are available in market including AS01, AS02, ISCOMS, Aluminum salts (alum), MF59, IFA (McKee1 et al., 2010), these biomarkers would be/ may be of usefull value to evaluate new candidate subunit vaccines in drug development strategy.

Currently our group is working on novel adjuvant PEGylated liposome (Data not show) for M. tb, which is significant effort in the direction of development of novel, more efficacious vaccine against TB these may/ will be become reliable biomarkers of protective immunity against Mycobacterium tuberculosis.

Challenges from the disease

Though, the TB vaccine research has gained momentum in recent years, there are still major obstacles. A new generation of TB vaccines must offer greater protection than currently used BCG and be safe enough to be used in HIV-endemic countries. Currently, none of the subunit vaccines that are in clinical trials have exhibited greater efficacy than BCG in animal model studies, which is the reason that they are considered as a booster rather than a replacement for BCG. Although recombinant BCG strains (rBCG30, rBCG::SureC-llo+) and the attenuated M. tb phoP mutant consistently reduced the M. tb burden by ~1.0 log compared to BCG alone, but, it is not clear that whether this level of improvement is sufficient. In addition to above, the safety of these recombinant BCG and attenuated M. tb strains remains a question. In 2007, WHO revised its policy to recommend that BCG not be given to children known to be HIV-positive, even if asymptomatic, because of substantial high risk of BCG-induced disseminated disease in HIV-infected individuals. All clinical trials of new TB vaccines have so far excluded HIV positive individuals. While this cautious approach is logical that there is a urgent need to evaluate new TB vaccines in HIV-infected populations because with an annual TB incidence rate of 5-10% they are among the most in need of a new and effective vaccine.

Viewpoint with potential instructions

While TB vaccine development has come a long way in the last decade there are still some major impediments ahead of us. For the leading candidates there is no guarantee that they will progress through phase-III clinical trials and registration. Experiences from HIV and malaria vaccine trials have taught us that it is important to continue pre-clinical research and keep developing new and even better vaccines for the pipeline. Another important point for the development of new vaccines in the future is that the human-adapted members of the M. tb complex are more genetically diverse than generally recognized and has recently been linked to changes in human demography and to both ancient and recent human migrations.

Such diversity is most likely to have functional consequences for the mycobacterial strains and could affect the efficacy of new vaccines. In addition to that, vaccines may show different protective efficacies against different mycobacterial strains. Consequently, the efficacy of new vaccines should ideally be tested against several clinical M. tb isolates and preferably against strains from all 6 major human MTBC lineages. Although, it is clear than ever that designing a vaccine, which can cope with many strategies that M. tb has evolved to escape the host’s immune response will be very complex, there remain reasons to be optimistic. The first new vaccine against M. tb in half a century is progressing through clinical trials at a rapid pace. Phase-II trials are already underway with two vaccines and at least two more expected to reach that stage over the next 1 or 2 year. At the same time vaccines which have shown detectable activity against the latent form of the disease in animal models are already in late preclinical stages and several new adjuvants effective at stimulating cell-mediated responses are apparently safe in humans are also in trials. As we scrutinize the immune response against M. tb and the pathogen’s response, we are becoming capable of designing novel vaccine strategies which could let us tip the balance in the host’s favour.

Conflict of Interest

NIL

Source of Support

NONE

Figures at a glance

Figure 1

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