Harnessing Innate Immunity to Treat Mycobacterium tuberculosis Infections: Heat-Killed Caulobacter crescentus 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.

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.

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.

Unsolved Puzzles Surrounding HCV Immunity: Heterologous Immunity Adds Another Dimension.

Chronic infection with hepatitis C virus (HCV) afflicts 3% of the world's population and can lead to serious and late-stage liver diseases. Developing a vaccine for HCV is challenging because the correlates of protection are uncertain and traditional vaccine approaches do not work. Studies of natural immunity to HCV in humans have resulted in many enigmas. Human beings are not immunologically naïve because they are continually exposed to various environmental microbes and antigens, creating large populations of memory T cells. Heterologous immunity occurs when this pool of memory T cells cross-react against a new pathogen in an individual. Such heterologous immunity could influence the outcome when an individual is infected by a pathogen. We have recently made an unexpected finding that adenoviruses, a common environmental pathogen and an experimental vaccine vector, can induce robust cross-reactive immune responses against multiple antigens of HCV. Our unique finding of previously uncharacterized heterologous immunity against HCV opens new avenues to understand HCV pathogenesis and develop effective vaccines.

Novel lipopeptides of ESAT-6 induce strong protective immunity against Mycobacterium tuberculosis: Routes of immunization and TLR agonists critically impact vaccine's efficacy.

Mycobacterium tuberculosis (Mtb), the bacterial cause of tuberculosis, is a leading infectious agent worldwide. The development of a new vaccine against Mtb is essential to control global spread of tuberculosis, since the current vaccine BCG is not very effective and antibiotic resistance is a serious, burgeoning problem. ESAT-6 is a secreted protein of Mtb, which is absent in BCG but has been implicated in inducing protective immunity against Mtb. Peptide based subunit vaccines are attractive due to their safety and high specificity in eliciting immune responses, but small synthetic peptides are usually not very immunogenic. We have designed a novel subunit vaccine for Mtb by using simple lipid (palmitic acid) modified derivatives of peptides from ESAT-6 protein corresponding to dominant human T cell epitopes and examined their ability to stimulate protective immunity against Mtb by intranasal and subcutaneous immunization in mice. We also investigated how individual TLR agonists as adjuvants (PolyI:C, MPL and GDQ) contribute to enhancing the induced immune responses and resulting protective efficacy of our vaccine. We observed that single C-terminal palmitoyl-lysine modified lipopeptides derived from ESAT-6 induce significant cellular immune responses on their own upon mucosal and subcutaneous immunizations. Intriguingly, a combination of immunogenic lipopeptides of ESAT-6 antigen exhibited local (pulmonary) and systemic immune responses along with efficient protective efficacy when administered intranasally or subcutaneously. Surprisingly, combination of ESAT-6 derived lipopeptides with a TLR-4 agonist (MPL) enhanced protection, whereas TLR-3 (Poly I:C) and TLR-7/8 agonists (gardiquimod, GDQ) led to reduced protection associated with specific local and systemic immune modulation. Our studies demonstrate the potential of ESAT-6 derived lipopeptides as a promising vaccine candidate against Mtb, and emphasize that selection of adjuvant is critical for the success of vaccines. These findings demonstrate the promise of synthetic lipopeptides as the basis of a subunit vaccine for TB.

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.

Immunization with Recombinant Adenoviral Vectors Expressing HCV Core or F Proteins Leads to T Cells with Reduced Effector Molecules Granzyme B and IFN-γ: A Potential New Strategy for Immune Evasion in HCV Infection.

Multispecific, broad, and potent T cell responses have been correlated with viral clearance in hepatitis C virus (HCV) infection. However, the majority of infected patients develop chronic infection, suggesting that natural infection mostly leads to development of inefficient T cell immunity. Multiple mechanisms of immune modulation and evasion have been shown in HCV infection through various investigations. This study examined the generation and modulation of T cell responses against core and frameshift (F) proteins of HCV. A single immunization of mice with replication incompetent recombinant adenovirus vectors encoding for F or core antigens induces poor T cell responses and leads to generation of CD4+ and CD8+ T cells with low granzyme B (GrB) expression. These T cells have impaired GrB enzyme activity and are unable to kill peptide loaded target cells. The low intracellular expression of GrB is not due to degranulation of cytotoxic granules containing cytotoxic T cells. Addition of exogenous IL-2 in in vitro cultures leads to partial recovery of GrB production, whereas immunization with the Toll-like receptor (TLR) agonist poly I:C leads to complete restoration of GrB expression in both CD4+ and CD8+ T cells. Thus, a possible new strategy of T cell modulation is recognized wherein effector T cells are caused to be dysfunctional by HCV-derived antigens F or core, and strategies are also delineated to overcome this dysfunction. These studies are important in the investigation of prophylactic vaccine and immunotherapy strategies for HCV infection.

Recombinant adenoviral vector expressing HCV NS4 induces protective immune responses in a mouse model of Vaccinia-HCV virus infection: a dose and route conundrum.

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected patients presumably due to failure or inefficiency of the immune responses generated. Both antibody and cellular immune responses have been suggested to be important in viral clearance. Non-replicative adenoviral vectors expressing antigens of interest are considered as attractive vaccine vectors for a number of pathogens. In this study, we sought to evaluate cellular and humoral immune responses against HCV NS4 protein using recombinant adenovirus as a vaccine vector expressing NS4 antigen. We have also measured the effect of antigen doses and routes of immunization on the quality and extent of the immune responses, especially their role in viral load reduction, in a recombinant Vaccinia-HCV (Vac-HCV) infection mouse model. Our results show that an optimum dose of adenovirus vector (2×10(7)pfu/mouse) administered intramuscularly (i.m.) induces high T cell proliferation, granzyme B-expressing CD8(+) T cells, pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-2 and IL-6, and antibody responses that can significantly reduce the Vac-HCV viral load in the ovaries of female C57BL/6 mice. Our results demonstrate that recombinant adenovirus vector can induce both humoral and cellular protective immunity against HCV-NS4 antigen, and that immunity is intricately controlled by route and dose of immunizing vector.

4'-Substituted pyrimidine nucleosides lacking 5'-hydroxyl function as potential anti-HCV agents.

Hepatitis C virus (HCV) infection is one of the major health problems worldwide. If left untreated, it leads to liver cirrhosis, liver cancer and death. Herein, we report synthesis and anti-HCV activity of a new class of pyrimidine nucleosides possessing a 4'-carboxymethyl (9-16, 21 and 23) or 4'-carboxamide function (17-19 and 24). Among these, 10-12 (EC50=33.1-42.4 µM), 14 and 21 (EC50=43.4-59.5 µM) exhibited potent activity in HCV-1a replicon cells without any toxicity to parent Huh-7 cells (CC50=>829-1055 µM). The anti-HCV activities demonstrated by this unusual class of compounds were superior to that of ribavirin (EC50=81.9 µM). Further, the most active analog, 12, was found to interact synergistically with ribavirin to inhibit HCV RNA replication.

A new class of pyrimidine nucleosides: inhibitors of hepatitis B and C viruses.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major health threats worldwide leading to liver cirrhosis, liver cancer and mortality. Herein, we report a new category of dideoxy pyrimidine nucleosides possessing a 4'-carboxyl (or carboxymethyl) function (7-9, 13, 16, 17), which are discovered as potential antiviral agents. For the first time, these nucleosides are recognized to be inhibitors of HBV and/or HCV replication. Among 4'-carboxy compounds, 3',4'-didehydrothymidine (16) was most effective against DHBV, HBV and HCV. Modification of the 4'-position in compound 7 from a carboxyl to carboxymethyl group (17) did not affect the anti-HBV activity but greatly increased the anti-HCV activity. Importantly, 17 yielded synergistic antiviral effect when combined with ribavirin without toxicity. The activity exhibited by a single agent towards both hepatitis viruses and no detectable in vitro cytotoxicity make this new class of compounds of interest.

Improvement in the antifilarial efficacy of doxycycline and rifampicin by combination therapy and drug delivery approach.

The present investigation deals with the evaluation of antifilarial efficacy of liposome entrapped antiwolbachial antibiotics doxycycline and rifampicin (5 doses at 10 mg/kg, subcutaneously for 15 days) alone and/or in combination with standard filaricide diethylcarbamazine (DEC) against human lymphatic filariid Brugia malayi in rodent host Mastomys coucha. The delivery system maintained the sustained release of antibiotics up to 48 h and significantly (P < 0.05) augmented the antifilarial potential of these antibiotics over their free administration. A combination of DEC with each entrapped antibiotics significantly (P<0.05) improved microfilaricidal efficacy, while marginal enhancement was noticed in adulticidal activity. Combination of both antibiotics formulation with DEC demonstrated marginal increase in macrofilaricidal efficacy; however, it was highest ( approximately 75%).

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.

Bacterially expressed recombinant envelope protein domain III of Japanese encephalitis virus (rJEV-DIII) elicits Th1 type of immune response in BALB/c mice.

Japanese encephalitis is a major cause of encephalitis in Asia. Cases occur largely in rural areas of the South and East Asian region resulting in significant morbidity and mortality. Multiple vaccines exist to control Japanese encephalitis, but all suffer from problems. Envelope protein domain III of Japanese encephalitis virus is involved in binding to host receptors and it contains specific epitopes that elicit virus-neutralizing antibodies. Earlier, the protective efficacy of domain III has been evaluated in mice by some researchers, but these studies are lacking in explanation of humoral and cellular immune responses. We have earlier reported cloning, expression, purification and in vitro refolding of Japanese encephalitis virus envelope protein domain III (rJEV-DIII). Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80. In the present study, we have evaluated the immunomodulatory potential of refolded rJEV-DIII protein in BALB/c mice with Freunds complete/incomplete adjuvants. Mice were tested for humoral immune response by ELISA. Cell-mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. The rJEV-DIII generated high IgG antibody and its isotypes (IgG2a and IgG3) and induced significant expression of INF-gamma and IL-2 cytokines. The rJEV-DIII induced significant lymphoproliferation of splenocytes. In conclusion rJEV-DIII induced Th1 type of immune response which plays an important role in protection for intracellular pathogens.

Adjuvanticity and protective immunity of Plasmodium yoelii nigeriensis blood-stage soluble antigens encapsulated in fusogenic liposome.

In our previous studies we established fusogenic properties of lipids isolated from edible yeast Saccharomyces cerevisiae (S. cerevisiae). We demonstrated that liposomes prepared from S. cerevisiae membrane lipid (saccharosome) can deliver encapsulated antigen into cytosol of the antigen presenting cells and elicit antigen specific cell mediated as well as humoral immune responses. In this study, we evaluated immunological behavior of saccharosome encapsulated cytosolic proteins (sAg) of Plasmodium yoelii nigeriensis in BALB/c mice. Immunization with antigen (sAg) encapsulated in saccharosome resulted in enhancement of CD4+ and CD8+ T cell populations and also up-regulated the expression of CD80 and CD86 molecules on the surface of antigen presenting cells. Further, immunization with saccharosome-encapsulated sAg-induced elevated levels of both IFN-gamma and IL-4 cytokines in the immunized mice when compared to egg PC liposome encapsulated sAg or its IFA emulsified form. Saccharosome-mediated immunization resulted in induction of high level of total antibody response with preponderance of IgG2a isotype as well. The data of this study suggest that saccharosome-based vehicle can emerge as an effective vaccine in imparting protection against various intracellular pathogens including Plasmodium yoelii nigeriensis.

Vaccination with 73kDa recombinant heavy chain myosin generates high level of protection against Brugia malayi challenge in jird and mastomys models.

We have earlier reported identification, expression and purification of a 2.0kb cDNA clone coding for Brugia malayi heavy chain myosin which exhibited strong immuno-reactivity with bancroftian sera from endemic normal (EN) human subjects which are considered to be putatively immune. In the present study, immunoprophylactic characterization of B. malayi recombinant myosin was carried out in rodent models and the protective efficacy was evaluated by assessing the microfilarial burden and adult worm counts in vaccinated host after an infective larval challenge. Data indicates that immunization resulted in to a significant reduction in microfilarial burden (approximately 76%) and adult worm establishment (54-58%), accompanied with embryostatic effect (70-75%) in both the animal models. The findings suggest that immune-protection by recombinant myosin was conferred through both humoral and cellular arms of immunity as indicated by an increased antibody titer with predominance of IgG2a and IgG2b isotypes along with elevated level of IgG1 apart from significant proliferation of lymphocytes, increased nitric oxide production and profound adherence of splenocytes causing cytotoxicity to microfilariae and infective larvae. The present study indicates that the recombinant B. malayi myosin is a promising vaccine candidate against human lymphatic filarial infection.

Wuchereria bancrofti: effect of single and multiple larval inoculations on infection dynamics and development of clinical manifestations in non-human primate Presbytis entellus.

We earlier reported the successful experimental transmission of Wuchereria bancrofti from humans to the Indian leaf monkey (Presbytis entellus) [Misra, S., Tyagi, K., Chatterjee, R.K., 1997. Experimental transmission of nocturnally periodic Wuchereria bancrofti to Indian leaf monkey (Presbytis entellus). Experimental Parasitology 86,155-157.; Dube, A., Murthy, P.K., Puri, S.K., Misra-Bhattacharya, S., 2004. Presbytis entellus: a primate model for parasitic disease research. Trends in Parasitology 20(8), 358-360.] using a small number of animals. The present study, involving 27 langur monkeys, found the development of pathological manifestations ranging from filarial fever, lymphangitis, lymphadenitis, hydrocoele, and limb edema to minor histopathological changes in tissues after single, double, triple, or multiple inoculations of infective larvae of W. bancrofti recovered from Culex quinquefasciatus fed on human microfilaraemic blood. Thirty-eight percent of the infected langurs developed detectable microfilaraemia in their blood. Single or double larval exposure resulted in better worm establishment than multiple exposures with small numbers of larvae. All of the langurs receiving a single large inoculum and 85.71% of those receiving two inoculations harbored adult parasites. Worm establishment decreased with increasing number of larval inoculations. In all, 60% of infected langurs developed classical gross-pathological symptoms of lymphatic filariasis. Of these, 29.16% developed thickening of the lymphatics, 25% suffered from periodic rise in rectal temperature, and 16.66% developed scrotal swelling with presence of microfilariae in the hydrocoele fluid. Only one out of 25 langurs (4%) developed acute limb edema. It appeared that a single inoculum of a large number of infective larvae was able to induce maximum pathology. Fifty-six percent of the infected langurs acquired a peculiar sitting posture, retracting both the hind limbs, usually after 4-5 months of larval inoculation and eventually resulting in reduced mobility. Oral administration of diethylcarbamazine (citrate) at 12mg/kg for 12 consecutive days to one langur caused 80% suppression in microfilaraemia on day 8, killing all the adult parasites. Of the two immunosuppressed (cortisone- or prednisolone-treated) male rhesus monkeys included in the study, neither developed any of the above lymphatic symptoms or parasites after receiving larval inoculations on two or three occasions; however, a rise in rectal temperature in one of the animals was noticed. The present study thus reveals that the Indian leaf monkey, P. entellus, may serve as an ideal non-human primate model of human bancroftian filariasis for carrying out longitudinal studies on pathology, host-parasite interactions, and preclinical evaluation of candidate anti-filarial drugs or vaccines.

Use of liposomized tetracycline in elimination of Wolbachia endobacterium of human lymphatic filariid Brugia malayi in a rodent model.

Wolbachia bacteria, being filarial parasite symbiont have been implicated in a variety of roles, including development, fecundity and the pathogenesis of the filarial infections. Among various strategies used in the treatment of experimental filariasis, the elimination of symbiont Wolbachia seem to offer an efficient means of curing the disease. The antiwolbachial property of tetracycline has been well worked out; however, treatment needs to be continued for a prolonged period of time to achieve complete elimination of Wolbachia from the filarial parasites and their subsequent killing. This results in acute toxicity, thus limiting its practical utility for clinical implementation. In order to increase efficacy of the antibiotic with minimal toxic manifestations, we developed liposomized formulation of the tetracycline. The liposomized tetracycline was found to be significantly more effective when compared to the free form of the drug. In contrast to the 90/120 days oral administration of the drug, the treatment schedule using the liposomized form of the drug was reduced to 12 alternate days with better efficacy of the treatment.