Future Path Toward TB Vaccine Development: Boosting BCG or Re-educating by a New Subunit Vaccine

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), kills 5,000 people per day globally. Rapid development and spread of various multi drug-resistant strains of Mtb emphasize that an effective vaccine is still the most cost-effectives and efficient way of controlling and eradicating TB. Bacillus Calmette-Guerin (BCG), the only licensed TB vaccine, still remains the most widely administered human vaccine, but is inefficient in protecting from pulmonary TB in adults. The protective immunity afforded by BCG is thought to wane with time and considered to last only through adolescent years. Heterologous boosting of BCG-primed immune responses using a subunit vaccine represents a promising vaccination approach to promote strong cellular responses against Mtb. In our earlier studies, we discovered lipopeptides of ESAT-6 antigen with strong potential as a subunit vaccine candidate. Here, we have investigated that potential as a booster to BCG vaccine in both a pre-exposure preventive vaccine and a post-exposure therapeutic vaccine setting. Surprisingly, our results demonstrated that boosting BCG with subunit vaccine shortly before Mtb challenge did not improve the BCG-primed immunity, whereas the subunit vaccine boost after Mtb challenge markedly improved the quantity and quality of effector T cell responses and significantly reduced Mtb load in lungs, liver and spleen in mice. These studies suggest that ESAT-6 lipopeptide-based subunit vaccine was ineffective in overcoming the apparent immunomodulation induced by BCG vaccine in Mtb uninfected mice, but upon infection, the subunit vaccine is effective in re-educating the protective immunity against Mtb infection. These important results have significant implications in the design and investigation of effective vaccine strategies and immunotherapeutic approaches for individuals who have been pre-immunized with BCG vaccine but still get infected with Mtb

Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines.

Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV

Heterologous Immunity between Adenoviruses and Hepatitis C Virus (HCV): Recombinant Adenovirus Vaccine Vectors Containing Antigens from Unrelated Pathogens Induce Cross-Reactive Immunity Against HCV Antigens

Host immune responses play an important role in the outcome of infection with hepatitis C virus (HCV). They can lead to viral clearance and a positive outcome, or progression and severity of chronic disease. Extensive research in the past >25 years into understanding the immune responses against HCV have still resulted in many unanswered questions implicating a role for unknown factors and events. In our earlier studies, we made a surprising discovery that peptides derived from structural and non-structural proteins of HCV have substantial amino acid sequence homologies with various proteins of adenoviruses and that immunizing mice with a non-replicating, non-recombinant adenovirus vector leads to induction of a robust cross-reactive cellular and humoral response against various HCV antigens. In this work, we further demonstrate antibody cross-reactivity between Ad and HCV in vivo. We also extend this observation to show that recombinant adenoviruses containing antigens from unrelated pathogens also possess the ability to induce cross-reactive immune responses against HCV antigens along with the induction of transgene antigen-specific immunity. This cross-reactive immunity can (a) accommodate the making of dual-pathogen vaccines, (b) play an important role in the natural course of HCV infection and (c) provide a plausible answer to many unexplained questions regarding immunity to HCV

Harnessing Innate Immunity to Treat <i>Mycobacterium tuberculosis</i> Infections: Heat-Killed <i>Caulobacter crescentus</i> as a Novel Biotherapeutic

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a serious and devastating infectious disease worldwide. Approximately a quarter of the world population harbors latent Mtb infection without pathological consequences. Exposure of immunocompetent healthy individuals with Mtb does not result in active disease in more than 90% individuals, suggesting a defining role of host immunity to prevent and/or clear early infection. However, innate immune stimulation strategies have been relatively underexplored for the treatment of tuberculosis. In this study, we used cell culture and mouse models to examine the role of a heat-killed form of a non-pathogenic microbe, Caulobacter crescentus (HKCC), in inducing innate immunity and limiting Mtb infection. We also examined the added benefits of a distinct chemo-immunotherapeutic strategy that incorporates concurrent treatments with low doses of a first-line drug isoniazid and HKCC. This therapeutic approach resulted in highly significant reductions in disseminated Mtb in the lungs, liver, and spleen of mice compared to either agent alone. Our studies demonstrate the potential of a novel innate immunotherapeutic strategy with or without antimycobacterial drugs in controlling Mtb infection in mice and open new avenues for the treatment of tuberculosis in humans

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi-infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2-DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2-D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI-TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down-regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi

Immunization of mice with Ad vector leads to reduced titer of Vaccinia-HCV chimeric virus.

<p>Mice immunized twice intramuscularly with Ad vector (with or without poly I:C), PBS, or HEK cell lysate, were challenged 8 days after the second immunization with wild-type Vaccinia (WT-Vac) or chimeric Vaccinia-HCV. Ovaries were harvested 5 days after challenge and viral loads in each mouse were determined by plaque-forming assay using TK-1 cells. <b>(A)</b> Challenge with HCV core-NS2-NS3 (Vac-C/NS2/NS3) or wild type vaccinia (WT-Vac). <b>(B)</b> Challenge with Vaccinia-HCV NS3-NS4-NS5 (Vac-NS3/4/5). Data are presented as mean ± standard deviation of % reduction in viral titer compared to corresponding unimmunized control group, and statistical comparison was done by two-tailed <i>t</i>-test (p<0.05 was considered significant).</p

Cross-reactive CD4⁺ and CD8⁺ T cells obtained from Ad vector immunized mice produce cytokines upon <i>ex vivo</i> stimulation with various HCV proteins.

<p>Splenocytes obtained from Ad vector immunized mice were cultured with HCV core, NS3, NS4 or NS5 antigens at 5 μg/ml, and analyzed after 5 days for intracellular IFN-γ and IL-10 expression profile of CD4⁺ and CD8⁺ T cells by flow cytometry. Data are obtained from a pool (n = 5) of spleen cells and are representative of two independent experiments.</p

Cytotoxic killing of target cells loaded with HCV antigens-derived peptides, by splenocytes obtained from Ad vector immunized mice.

<p>Splenocytes harvested from Ad vector immunized mice, were stimulated <i>in vitro</i> with the HCV protein antigens core, NS3, NS4 and NS5 at 5 μg/ml concentrations for 4 days. The target EL4 cells were incubated with corresponding HCV peptides each at 1 μg/ml concentration (core peptides #: 2, 14, 17, 25, 27, 28, 32; NS3 peptides #: 8, 10; NS4 peptides #: 3, 4, 8; NS5a peptides #: 1, 2, 16, 20 and NS5b peptides: 5, 19, 23, 39; or All: a mixture of the above peptides from core, NS3, NS4 and NS5) and peptide-loaded EL4 cells were cultured with effectors at 10:1 (effectors: target) ratio for 4–5 hours. Empty (no peptide loaded) EL4 targets were used as a negative control. CFSE-labeled live targets were quantified by flow cytometry, and % killed targets were calculated using the formula: % Killing = [(Average live cells in PBS control − live cells in immunized group) /Average live cells in PBS control] × 100). Data shown are mean±SD and are representative of three independent experiments.</p

Characterization of Ad vector stock by PCR.

<p>HCV genes core, F, NS3, NS4, NS5a or NS5b are not amplified. First panel shows the DNA ladder, followed by agarose gel electrophoresis of PCR products obtained with HCV core, F, NS3, NS4, NS5a and NS5b specific primers. HEK lysate supernatant and rAd-HCV vectors were used as negative and positive controls. Data are representative of 2–3 repeated experiments.</p

Cross-reactive CD4⁺ and CD8⁺ T cells obtained from Ad vector immunized mice proliferate in HCV antigens-dependent manner.

<p>Splenocytes obtained from Ad vector immunized mice were stimulated <i>ex vivo</i> with various recombinant HCV antigens (core, NS3, NS4 and NS5) or their selected respective peptides (at 5 μg/ml each), and analyzed by flow cytometry. Proliferation of CD4⁺ and CD8⁺ T cells stimulated <i>ex vivo</i> with: <b>(A)</b> HCV proteins; <b>(B)</b> Representative peptides derived from HCV proteins using the CFSE-based assay (loss of CFSE due to cell division represented by shift of peak of CFSE⁺ T cells towards left). Data are obtained from a pool (n = 5) of spleen cells and are representative of two independent experiments.</p