TB Vaccine Development Research Articles

M.tuberculosis Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to M. bovis BCG Vaccine in an Experimental Mouse Model

The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4+ and particularly CD8+ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG.

Mycobacterium tuberculosisRpfB drives Th1-type T cell immunity via a TLR4-dependent activation of dendritic cells

The failure of Mycobacterium bovis BCG as a TB vaccine against TB reactivation suggests that latency-associated proteins should be included in alternative TB vaccine development. Further, antigens known to generate protective immunity against the strong Th1 stimulatory response to reactivated TB should be included in novel vaccine design. Recent studies have emphasized the importance of Rpfs from Mycobacterium tuberculosis in the reactivation process and cellular immunity. However, little is known about how RpfB mediates protective immunity against M. tuberculosis. Here, we investigated the functional roles and signaling mechanisms of RpfB in DCs and its implications in the development of T cell immunity. DCs treated with RpfB displayed features of mature and functional status, with elevated expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF-α, IL-1β, IL-6, and IL-12p70). Activation of DCs was mediated by direct binding of RpfB to TLR4, followed by MyD88/TRIF-dependent signaling to MAPKs and NF-κB signaling pathways. Specifically, we found that the RpfB G5 domain is the most important part in RpfB binding to TLR4. RpfB-treated DCs effectively polarized naïve CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2. Importantly, RpfB induced the expansion of memory CD4(+)/CD8(+)CD44(high)CD62L(low) T cells in the spleen of M. tuberculosis-infected mice. Our data suggest that RpfB regulates innate immunity and activates adaptive immunity through TLR4, a finding that may help in the design of more effective vaccines.

Advances in TB vaccinology

Development of a vaccine with enhanced efficacy compared to that of the Bacillus Calmette Guerin (BCG) vaccine is essential to reduce the global TB burden. This is challenging since an effective vaccine will need to induce protection that surpasses that induced by natural infection with M. tuberculosis and currently there are no identified biomarkers of protection. Nonetheless progress has been made over the last 20 years in the development and clinical evaluation of novel TB vaccines, evidenced by 15 candidate vaccines being tested in phase I clinical trials and 4 reaching phase IIb and III trials. In order to make further progress there is a need to further understanding of the interactions between M. tuberculosis and its host and for the development of ongoing partnerships to facilitate the design, harmonisation and funding of clinical trials. Keywords: BCG, biomarker, correlates of protection, TB vaccine.

Global progress in TB vaccine development

Global progress in TB vaccine development

Identification of Two New Virulence Factors of Mycobacterium Tuberculosis that Induce Multifunctional CD4 T Cell Responses

In our previous studies, we had successfully completed biopanning of three new phage fusion proteins cpsA (Rv3484), LpqA (Rv3016) and PapA2 (Rv3820c) of Mycobacterium Tuberculosis (Mtb). In the current study, the effects of anti-Tuberculosis (TB) chemotherapy on antigen-specific interferon-γ (IFN-γ) release in response to three proteins were analyzed. Our results suggest that cpsA and PapA2 may be the virulent factors that are actively secreted by Mtb at the active stage of TB. New subunit vaccines of Mtb were obtained by combining Ag85B (Rv1886c)+ESAT-6 (Rv3875) with cpsA and PapA2 phage display protein. A comprehensive analysis of the immune response, including humoral immune response, cytokine expression levels and the level of multiple T cell subset expression, was carried out for each combination group. This helped in the systematic identification of useful anti-TB host T cell subsets and our findings provide an effective platform and evaluation standard for TB vaccine development.

Identification of Growth Promoting Effect of <i>r</i>BCG/BCG Culture Supernatant and Its Potential Applications

Background: Although BCG is the most widely administered vaccine in the world, there have never been as many cases of TB as there are now. Globally, more than 8.8 million people developed active TB and 1.4 million—many of them—died in 2010. It is estimated that half of pulmonary TB cases arise from latent Mtb infection, making the study of latency and reactivation of utmost importance. Methods: Widely administered BCG vaccines and a gene modified recombinant BCG (rBCG) strain, AERAS-422, were used as models to investigate the growth promoting function of resuscitation-promoting factors (Rpfs) in different bacilli culture phases. Different supernatant fractions were prepared by ultrafiltration, and the promoting function of each fraction containing secreted Rpf(s) was evaluated by growth curve monitoring and colony counting on 7H10 agar plates. Results: The promoting effect of culture supernatants was mainly associated with the high molecular weight fraction (>30 kDa), which stimulated bacterial growth, but did not extend the exponential phase of stimulated culture. Anti-RpfB antibody showed significant growth restriction of the tested cultures. When comparing rBCG cultures containing 7H9 medium, the 10 - 30 kDa fraction, or the >30 kDa fraction, only the >30 kDa fraction was displayed with down-regulation of the secretion of RpfC, D and E. In colony counting tests, the plates containing the >30 kDa fraction had total countable colony numbers 2 to 3 fold higher than the plates with the 10 - 30 kDa fraction, and colonies appeared one to two weeks earlier than on the regular plates. The potential applications of the prepared supernatant fractions containing RpfA and RpfB are discussed, which may include accelerating diagnosis of Mtb infection and future TB vaccine development.

Lymph node targeting of BCG vaccines amplifies CD4 and CD8 T-cell responses and protection against Mycobacterium tuberculosis

Vaccination with Mycobacterium bovis BCG provides limited protection against pulmonary tuberculosis and a risk of dissemination in immune-compromised vaccinees. For the development of new TB vaccines that stimulate strong T-cell responses a variety of strategies is being followed, especially recombinant BCG and attenuated M. tuberculosis. The objective of the current study was to test potential benefits of vaccination through direct lymph-node targeting of wildtype BCG; the recommended route of vaccination with BCG is intradermal. C57BL/6 mice were immunised with BCG by intradermal, subcutaneous or intralymphatic injections. Cellular immune responses and protection against M. tuberculosis were determined. Intralymphatic vaccination was 100–1000 times more effective in stimulating BCG-specific immune responses than intradermal or subcutaneous immunisation. Intralymphatic administration stimulated high frequencies of mycobacterium-specific lymphocytes with strong proliferating capacity and production of TNF-α, IL-2, IL-17 and, especially, IFN-γ secretion by. CD4 and CD8 T cells. Most importantly, intralymphatic vaccination with 2×103CFU BCG induced sustained protection against M. tuberculosis in intratracheally challenged C57BL/6 mice, whereas subcutaneous vaccination with 2×105CFU BCG conferred only a transient protection. Hence, direct administration of M. bovis BCG to lymph nodes demonstrates that efficient targeting to lymph nodes may help to overcome the efficacy problems of vaccination with BCG.

Comparing the safety and immunogenicity of a candidate TB vaccine MVA85A administered by intramuscular and intradermal delivery

BackgroundNew vaccines to prevent tuberculosis are urgently needed. MVA85A is a novel viral vector TB vaccine candidate designed to boost BCG-induced immunity when delivered intradermally. To date, intramuscular delivery has not been evaluated. Skin and muscle have distinct anatomical and immunological properties which could impact upon vaccine-mediated cellular immunity. MethodsWe conducted a randomised phase I trial comparing the safety and immunogenicity of 1×108pfu MVA85A delivered intramuscularly or intradermally to 24 healthy BCG-vaccinated adults. ResultsIntramuscular and intradermal MVA85A were well tolerated. Intradermally-vaccinated subjects experienced significantly more local adverse events than intramuscularly-vaccinated subjects, with no difference in systemic adverse events. Both routes generated strong and sustained Ag85A-specific IFNγ T cell responses and induced multifunctional CD4+ T cells. The frequencies of CD4+ T cells expressing chemokine receptors CCR4, CCR6, CCR7 and CXCR3 induced by vaccination was similar between routes. ConclusionsIn this phase I trial the intramuscular delivery of MVA85A was well tolerated and induced strong, durable cellular immune responses in healthy BCG vaccinated adults, comparable to intradermal delivery. These findings are important for TB vaccine development and are of relevance to HIV, malaria, influenza and other intracellular pathogens for which T cell-inducing MVA-based vaccine platforms are being evaluated.

Key issues in the clinical development and implementation of TB vaccines in South Africa

Significant progress has been made in advancing the development pipeline for a new and more effective TB vaccine with some candidate vaccines now in late stage clinical evaluation. However, progress has been hampered by an incomplete understanding of the components of a protective immune response and limited animal models, rendering the field unable to reliably predict vaccine efficacy earlier in preclinical development, including by evaluation in animal models, and limiting the predictive utility of comparing immunogenic effects across vaccine candidates in phase I/II studies. Consequently, new candidate vaccines have to be evaluated for efficacy in large-scale phase II/III trials using clinical endpoints. Apart from the technical challenges of characterising TB incidence in target populations at high risk of acquiring TB disease and standardising case definitions in order to improve both the sensitivity and more importantly the specificity of trial endpoints, there is an urgency in expanding and supporting the considerable trial infrastructure that will be required to evaluate and ultimately license a new TB vaccine. In the longer term, implementation strategies are dependent on what policy makers most value. Economic analyses will be essential to guide policy and implementation. This paper outlines the gaps and challenges and identifies solutions for effectively developing and efficiently introducing a new TB vaccine.

Driving HIV-specific vaccine responses in immune suppressed recipients (113.20)

Abstract Malaria, TB and HIV-1 remain tremendous disease burdens in much of the world’s population. Vaccines for these diseases are desperately needed. However, vaccine efficacy is likely to be compromised due to the systemic Th2 biasing and immune suppression caused by infection with parasitic helminthes. Our lab and others have shown that helminth infection suppresses Th1-type vaccine-specific responses. Although Sub-Saharan populations will benefit most from these vaccines, a large percentage of these populations live in helminth endemic regions. A goal of our research is to find vaccine vectors that drive significant HIV-specific immune responses in helminth infected recipients, despite the immune status of the individual. In the current study, we demonstrate that administration of an HIV vaccine (Listeria expressing HIV-1 gag protein) drives significant HIV-specific CTL and Th1 responses in mice chronically infected with the helminth parasite Schistosoma mansoni. These results suggest that Listeria vector vaccines likely will drive HIV-specific responses in helminth-infected human populations. Kinetic studies show that the HIV-vaccine-specific responses generated in schistosome infected mice are durable and capable of inducing CD4+ and CD8+ T cell memory. Further, Listeria vectors should be considered in the development of new generation HIV-1, malaria or TB vaccines to be administered to populations in sub-Saharan Africa where helminth infection is endemic.

SATVI - after 10 years closing in on a new and better vaccine to prevent tuberculosis

The vision of the South African Tuberculosis Vaccine Initiative (SATVI) (www.satvi.uct.ac.za) is 'A World Without TB' and our mission is 'Innovative and high-quality TB vaccine research in Africa, to impact the global epidemic'. Over the last 10 years, our focus has been twofold: first, clinical trials of BCG and of new candidate vaccines, and second, complementary research that addresses critical questions in TB vaccine development. SATVI is now widely regarded as the leading TB vaccine clinical research site in the world.

Bridging the gap: Engaging researchers and advocates to build support for TB vaccine research and development

The success of global efforts to develop new TB vaccines will rely on both addressing the scientific challenges identified throughout this Blueprint and mobilizing sufficient support and resources to sustain and advance the TB vaccine pipeline. As outlined in the TB Vaccine Blueprint, activities over the next decade will include expanding financing to provide sufficient resources, raising awareness of the need for new TB vaccines, and broadening the base of advocates, allies and champions for TB vaccine R&D. These activities will only be successful if advocates and researchers – including scientists, clinicians and product developers – work together. Researchers and advocates play an essential role in promoting the advancement of TB vaccine research and development (R&D), but have too often operated independently of each other, with researchers focusing on the science and advocates and civil society focused on advocacy, communications and resource mobilization. As we look toward the next decade of TB vaccine development, it will be critical for the research and advocacy communities to work more closely together to support the common goal of developing new, more effective TB vaccines.

Variability between countries in cytokine responses to BCG vaccination: what impact might this have on protection?

*All authors contributed equally to this manuscript. In 2010, there were an estimated 8.8 million incident cases of TB and 1.4 million deaths globally despite the widespread use of BCG [101]. With the aim of elimination of TB by 2050, there is a concerted drive to combine interventions in order to improve diagnosis, prevent infection, treat active disease and reduce the risk factors for TB [102]. With the rising burden of drug-resistant strains of TB and with TB susceptibility in those with HIV/AIDS, there is an urgent need for new effective vaccines that work for all forms of TB in preand post-exposed individuals of all ages in all parts of the world. Whilst there are promising signs in TB vaccine development [1], there are still many unanswered questions, in particular as to what constitutes a protective immune response. Geographic variations in protective immunity induced by the current vaccine, BCG [2], are still poorly understood, and without biomarkers of protection, there are many challenges ahead to identify and test potential vaccines for TB globally. Smaller immunological studies have shown variability in cytokine responses to BCG vaccination between countries, including between BCG-vaccinated adolescents and infants in Malawi and the UK [3–5]. Whereas UK adolescents made strong IFN-γ responses to Mycobacterium tuberculosis (Mtb) purified protein derivative (PPD) post-vaccination, Malawian adolescents and young adults who were presensitized to PPD failed to show a significant increase [3]. Malawian infants also produced weaker IFN-γ responses following BCG vaccination than infants in the UK [4,5]. Three questions arise from these observations: are the differences real, are they important and what implications might they have on protection induced by BCG and future vaccines?

A novel genetically engineered Mycobacterium smegmatis-based vaccine promotes anti-TB immunity

Evaluation of: Sweeney KA, Dao DN, Goldberg MF et al. A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis. Nat. Med. 17, 1261–1268 (2011).Pulmonary TB remains a global health threat. Prophylactic immunization with Mycobacterium bovis BCG is the only key strategy to control TB. Ineffectiveness of BCG immunization in TB-endemic areas and BCG-related safety issues in HIV-positive infants have prompted the development of new TB vaccines. As enhanced understanding of the immune evasion mechanism of Mycobacterium tuberculosis will help develop new vaccines, Sweeney et al. studied the role of the esx-3 locus in mycobacterial pathogenesis. They have identified a previously unappreciated function of the esx-3 locus in innate immune evasion. They further discovered that Mycobacterium smegmatis with the esx-3 genes deleted could function as a novel vaccine vector with an enhanced innate immune-activating property. This vector, when engineered to express M. tuberculosis esx-3, was found to be a potent TB vaccine capable of a level of protection superior to that of BCG when administered via the intravenous route.

MTB-PCDB: Mycobacterium tuberculosis proteome comparison database.

The Mycobacterium tuberculosis Proteome Comparison Database (MTB-PCDB) is an online database providing integrated access to proteome sequence comparison data for five strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC 1551, F11 and KZN 1435) sequenced completely so far. MTB-PCDB currently hosts 40252 protein sequence comparison data obtained through inter-strain proteome comparison of five different strains of MTB. 2373 proteins were found to be identical in all 5 strains using MTB H(37)Rv as reference strain. To enable wide use of this data, MTB-PCDB provides a set of tools for searching, browsing, analyzing and downloading the data. By bringing together, M. tuberculosis proteome comparison among virulent & avirulent strains and also drug susceptible & drug resistance strains MTB-PCDB provides a unique discovery platform for comparative proteomics among these strains which may give insights into the discovery & development of TB drugs, vaccines and biomarkers. The database is available for free at http://www.bicjbtdrc-mgims.in/MTB-PCDB/

Driving HIV-1 specific responses in immune suppressed recipients: Listeria as a vaccine vector (53.20)

Abstract Malaria, TB and HIV-1 remain tremendous disease burdens in much of the world’s population and functional vaccines are desperately needed. Although Sub-Saharan populations are those that will benefit most from these vaccines, they are also coincident with areas of endemic helminth infection. Infection with one or more species of parasitic helminths suppresses the immune system and has been shown, by our lab and others, to suppress vaccine-specific responses. One goal of our research is to find vaccines that drive significant vaccine-specific immune responses in helminth infected recipients, without the need to eliminate helminth infection prior to vaccination. In the current study, we demonstrate that administration of a Listeria vector HIV-1 gag vaccine to mice chronically infected with the helminth parasite Schistosoma mansoni drives significant immune responses to HIV-1 gag CTL and helper epitopes. This observation suggests that Listeria vector vaccines are capable of driving vaccine-specific responses in helminth-infected populations. Kinetic studies show the antigen-specific responses are both durable and capable of inducing CD8+ central memory. In addition to HIV-1, Listeria vectors should be considered in the development of new generation malaria and TB vaccines to be administered to sub-Saharan Africa populations where helminth infection is endemic. Studies are underway to determine if other vectors are also capable of overcoming helminth-induced immune suppression.

Development of cattle TB vaccines based on heterologous prime-boosting strategies

No Abstract. Ethiopian Journal of Health Development Vol. 22 (special issue) 2008: pp. 100-104

Pattern Recognition in Pulmonary Tuberculosis Defined by High Content Peptide Microarray Chip Analysis Representing 61 Proteins from M. tuberculosis

BackgroundSerum antibody-based target identification has been used to identify tumor-associated antigens (TAAs) for development of anti-cancer vaccines. A similar approach can be helpful to identify biologically relevant and clinically meaningful targets in M.tuberculosis (MTB) infection for diagnosis or TB vaccine development in clinically well defined populations.MethodWe constructed a high-content peptide microarray with 61 M.tuberculosis proteins as linear 15 aa peptide stretches with 12 aa overlaps resulting in 7446 individual peptide epitopes. Antibody profiling was carried with serum from 34 individuals with active pulmonary TB and 35 healthy individuals in order to obtain an unbiased view of the MTB epitope pattern recognition pattern. Quality data extraction was performed, data sets were analyzed for significant differences and patterns predictive of TB+/−.FindingsThree distinct patterns of IgG reactivity were identified: 89/7446 peptides were differentially recognized (in 34/34 TB+ patients and in 35/35 healthy individuals) and are highly predictive of the division into TB+ and TB−, other targets were exclusively recognized in all patients with TB (e.g. sigmaF) but not in any of the healthy individuals, and a third peptide set was recognized exclusively in healthy individuals (35/35) but no in TB+ patients. The segregation between TB+ and TB− does not cluster into specific recognition of distinct MTB proteins, but into specific peptide epitope ‘hotspots’ at different locations within the same protein. Antigen recognition pattern profiles in serum from TB+ patients from Armenia vs. patients recruited in Sweden showed that IgG-defined MTB epitopes are very similar in individuals with different genetic background.ConclusionsA uniform target MTB IgG-epitope recognition pattern exists in pulmonary tuberculosis. Unbiased, high-content peptide microarray chip-based testing of clinically well-defined populations allows to visualize biologically relevant targets useful for development of novel TB diagnostics and vaccines.

Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of Mycobacterium tuberculosis in a mouse model

BALB/c mice were vaccinated three times (2-week intervals) with plasmid DNA separately encoding antigen Ag85B, ESAT-6 or Ag85A from Mycobacterium tuberculosis. The protective efficacy of these DNA vaccines against intravenous M. tuberculosis H37Rv challenge infection was measured by counting bacterial loads in spleen and lung and recording changes in lung pathology. The splenocyte proliferative response to the corresponding antigens and antigen-specific interferon (IFN)-gamma secreted by splenocytes of the vaccinated mice were also detected. We found a clear hierarchy of protective efficacies among the three DNA vaccines tested in this study. Plasmid DNA encoding Ag85A provided the strongest protection and showed the least change in lung pathology, followed by plasmid DNAs encoding Ag85B and ESAT-6. However, DNA-85B reduced comparative bacterial load in lung tissue, as did DNA-85A. Compared to the control group, protective efficacies conferred by different DNA vaccines were consistent with the lymphoproliferative responses to the corresponding antigens as well as the secretions of antigen-specific IFN-gamma. Our study demonstrates that both Ag85A and Ag85B are the most promising of the candidate antigens tested for future TB vaccine development.

Translational Mini-Review Series on Vaccines:
Development and evaluation of improved vaccines against tuberculosis

Tuberculosis (TB) continues to be a major global health disaster, despite the widespread use of BCG and effective drug therapies. The development of an efficacious new TB vaccine would be an important component of disease control in the future. Many approaches are being utilised to enhance understanding of the requirements of a successful vaccine. Numerous vaccines are being designed and assessed in a series of animal models, with a few progressing to clinical trials. Here, the steps involved in the development and evaluation of TB vaccines will be discussed, including description of the most frequently used animal models and the processes involved in advancing vaccines to phase III trials.