O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Pediatric Tuberculosis: A Review of Evidence-Based Best Practices for Clinicians and Health Care Providers.

Advances in pediatric TB care are promising, the result of decades of advocacy, operational and clinical trials research, and political will by national and local TB programs in high-burden countries. However, implementation challenges remain in linking policy to practice and scaling up innovations for prevention, diagnosis, and treatment of TB in children, especially in resource-limited settings. There is both need and opportunity to strengthen clinician confidence in making a TB diagnosis and managing the various manifestations of TB in children, which can facilitate the translation of evidence to action and expand access to new tools and strategies to address TB in this population. This review aims to summarize existing guidance and best practices for clinicians and health care providers in low-resource, TB-endemic settings and identify resources with more detailed and actionable information for decision-making along the clinical cascade to prevent, find, and cure TB in children.

PD-1 modulates regulatory T cells and suppresses T-cell responses in HCV-associated lymphoma.

T regulatory (T(R)) cells suppress T-cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also has a pivotal role in regulation of T-cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating T(R)-cell functions that inhibit T-cell responses during virus-associated malignancy, T(R) cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4(+)CD25(+) and CD8(+)CD25(+) T(R) cells, as well as high levels of PD-1 expressions on these T(R) cells were found in the peripheral blood of subjects with HCV-L compared with those from non-HCV-L or HCV alone or HS. T(R) cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of T(R) cells in peripheral blood mononuclear cells from HCV-L improved T-cell proliferation. Additionally, the suppressed T-cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in T(R)-cell number and the ability of T(R) to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating T(R) cells that suppress T-cell functions in the setting of HCV-associated B-cell lymphoma.

Heterologous CD8 T cell immune response to HSV induced by toll like receptor ligands.

A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8(+) T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.

Natural killer cells as novel helpers in anti-herpes simplex virus immune response.

Innate defenses help to eliminate infection, but some of them also play a major role in shaping the magnitude and efficacy of the adaptive immune response. With regard to influencing subsequent adaptive immunity, NK cells aided by dendritic cells may be the most relevant components of the innate reaction to herpes simplex virus (HSV) infection. We confirm that mice lacking or depleted of NK cells are susceptible to HSV-induced lesions. The quantity and quality of CD8(+) cytotoxic T lymphocytes generated in the absence of NK cells were diminished, thereby contributing to susceptibility to HSV-induced encephalitis. We demonstrate a novel helper role for NK cells, in that NK cells compensate for the loss of CD4 helper T cells and NK cell supplementation enhances the function of wild type anti-HSV CD8 T cells. In addition, NK cells were able to partially rescue the dysfunctional CD8(+) T cells generated in the absence of CD4 T helper cells, thereby performing a novel rescue function. Hence, NK cells may well be exploited for enhancing and rescuing the T-cell response in situations where the CD4 helper response is affected.

T regulatory cells: an overview and intervention techniques to modulate allergy outcome.

Dysregulated immune response results in inflammatory symptoms in the respiratory mucosa leading to asthma and allergy in susceptible individuals. The T helper type 2 (Th2) subsets are primarily involved in this disease process. Nevertheless, there is growing evidence in support of T cells with regulatory potential that operates in non-allergic individuals. These regulatory T cells occur naturally are called natural T regulatory cells (nTregs) and express the transcription factor Foxp3. They are selected in the thymus and move to the periphery. The CD4 Th cells in the periphery can be induced to become regulatory T cells and hence called induced or adaptive T regulatory cells. These cells can make IL-10 or TGF-b or both, by which they attain most of their suppressive activity. This review gives an overview of the regulatory T cells, their role in allergic diseases and explores possible interventionist approaches to manipulate Tregs for achieving therapeutic goals.

Seroprevalence of hepatitis delta virus infection among subjects with underlying hepatic diseases in Chennai, southern India.

Four hundred million people are carriers of hepatitis B virus (HBV) worldwide and approximately 5% of these are reportedly positive for hepatitis delta virus (HDV). Several reports indicate a declining trend in the occurrence of HDV infection in the north of tropical India. To our knowledge, no study has been conducted to evaluate whether a similar epidemiological change is occurring in southern India. Therefore we evaluated the seroprevalence of HDV among 153 individuals with HBV-related liver diseases in Chennai, and assessed any change in epidemiological pattern by comparing the results with seroprevalence figures reported previously. Of the 153 patients screened, nine (5.9%) were reactive to anti-delta antibodies, six (3.9%) presented an evidence of past infection (IgG anti-delta positive) and three (2.0%) showed anti-HDV IgM, suggestive of recent HDV infection. Alanine transaminase elevation was not significant in HDV-associated infection compared with HBV alone-infected acute viral hepatitis (AVH) (P=0.82) and chronic liver disease (P=0.77) patients. The anti-HDV positivity in AVH was considerably low (6.6%), compared with previous Indian reports varying from 10.7% to >30%. HDV infection was relatively low and seems to play a minor determining factor of liver diseases in the tropical south Indian population.

The prevalence of hepatitis B virus and hepatitis C virus infection among patients with chronic liver disease in South India.

OBJECTIVE: Determining the identity of hepatitis C virus (HCV) genotypes in liver disease has key implications for ascertaining the duration of antiviral therapy and disease prognosis. We investigated the presence of various genotypes of HCV among 69 chronic liver diseased (CLD) patients with chronic HCV infection. METHODS: Sixty-nine consecutive subjects with underlying chronic hepatitis (n=28), cirrhosis (n=35), and hepatocellular carcinoma (n=6), diagnosed by clinical, biochemical, and histological means, were studied. Hepatitis B virus (HBV) and HCV diagnostic markers were used. HCV-RNA was extracted from sera of HCV-infected subjects and subsequently the HCV genotypes were determined using a commercial line probe assay (Inno-LiPA HCV II). RESULTS: Of the 69 CLD cases screened for possible markers of HBV and HCV infection, 39 (57%) were positive for HBV and 30 (43%) were HCV infected. The overall HCV-RNA positivity was 77% (23/30). Of these, the majority were genotype 1b (13/23, 57%), followed by 1a (6/23, 26%), mixed genotypes 3 and 4(3/23, 13%), and mixed pattern of 1a, 1b, and 4 (1/23, 4.3%). The genotype 1b infected subjects demonstrated significantly elevated transaminase (ALT) levels (p<0.05) as compared with the other non-1b HCV genotypes. CONCLUSIONS: The predominance of HCV genotype 1b among CLD patients could pose a major challenge for the efficient management of HCV disease and the development of effective therapeutic interventions in peninsular India.

Relationship between T-lymphocyte cytokine levels and sero-response to hepatitis B vaccines.

AIM: To investigate the cellular defects by analyzing the (Th1/Th2) cytokine levels in vaccine responders and non-responders. METHODS: Peripheral blood mononuclear cell (PBMC) from responders and non-responders were stimulated with or with out recombinant HBsAg or PHA. Broad spectrum of cytokines viz (Th1) IFN-gamma, IL-2, TNF-alpha, IL-12 and (Th2) IL-10, IL-4 were measured after in vitro stimulation with recombinant HBsAg and were compared with respective antibody titers. RESULTS: A significant decrease (P = 0.001) in Th1 and Th2 cytokines namely, IL-2, INF-gamma, TNF-alpha and IL-10 in non-responders was observed. The level of IL-4 was not significant between the three groups. Furthermore, despite a strong Th1 and Th2 cytokine response, the level of IL-12 was elevated in high-responders compared to other groups (P = 0.001) and demonstrated a positive correlation with anti-HBs titers and Th1 cytokine response. CONCLUSION: Our findings suggest that unresponsiveness to recombinant hepatitis B vaccines (rHB) is multifactorial, including specific failure of antigen presentation or the lack of both T helper Th1 and Th2 response.

Transmission of "a" determinant variants of hepatitis B virus in immunized babies born to HBsAg carrier mothers.

Hepatitis B virus (HBV) surface antigen mutations may lead to immune escape and eventually cause failure of immunization. In this report, we identified immune escape variants in immunized babies born to hepatitis B surface antigen (HBsAg) carrier mothers. A total of 68 babies were followed up for 2 years after the full course of vaccination; 2.9% (2/68) of babies were found to be infected with the variant HBV in spite of preexisting antibody to surface antigen (anti-HBs) at 24 months post immunization. Both infants were positive for HBV-DNA; sequencing results of the "a" determinant region of the surface gene revealed that both babies had point mutations at a different nucleotide position resulting in various amino acid substitutions. In addition, an intriguing variant having an addition-deletion mutation was observed in one of the babies. This is the first report to show the addition-deletion variant of HBV in India. However, the immunological significance of the above HBV variants needs to be further elucidated.

Low frequency of precore mutants in anti-hepatitis B e antigen positive subjects with chronic hepatitis B virus infection in Chennai, Southern India.

The natural course of chronic hepatitis B (CH-B) virus infection is reportedly variable, and the long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B infection are distinct from HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in the south Indian setting remain largely unclear. We prospectively studied 679 consecutive patients for HBsAg, HBeAg, anti-HBeAg, and HBV DNA by qualitative PCR. Randomly selected samples were subjected to bidirectional sequencing to reveal core/precore variants. Of the total 679 chronic HBV cases investigated, 23% (154/679) were replicative HBV carriers. Furthermore, amongst the 560 HBV DNA samples analyzed, 26% (146/560) were viremic. Among the 154 HBeAg positive cases, HBV DNA was positive in 118 cases (77%), significantly (p<0.001) higher than the anti-HBe positive (7%) (28/406) cases. Significant increase in liver disease (p<0.01) with ALT enzyme elevation (p<0.001) was observed in both HBe and anti-HBe viremic cases. Interestingly, low frequencies of mutations were seen in the precore region of the HBV strains studied. HBV precore and core promoter variants were less often detected in subjects with "e" negative chronic HBV infection and, therefore, may not have a prognostic role in determining liver disease sequelae in this part of tropical India.

Comparative efficacy of two dosages of recombinant hepatitis B vaccine in healthy adolescents in India.

BACKGROUND: Inclusion of hepatitis B vaccine in the Universal Programme of Immunization of all Asian and African countries is hampered by the economic burden on the health budget because of the cost of hepatitis B vaccines. Here we evaluated the immunogenicity, safety, efficacy, and the persistence of antibody to hepatitis B surface antigen (anti-HBs) titers of a new and a low cost recombinant hepatitis B vaccine GeneVac B, with 2 different dosages in healthy adolescents in India. METHODS: GeneVac-B, a recombinant hepatitis B vaccine (Serum Institute of India, Pune, India), was administered in 10 or 20 microg dose intramuscularly to 2 groups of 100 healthy school-going adolescents at 0-, 1-, and 6-month intervals, who were followed up for 1 year. Group I received 20 mug doses whereas Group II received 10 mug doses. Blood samples were collected 1 month after each dose and 1 year after the third dose. The anti-HBs titers were assayed using commercially available kits to assess the immunogenicity of the 2 dosage schedules. Safety studies were also carried out. RESULTS: The geometric mean titer value of the anti-HBs titer 1 month after the third dose was 2629 (mlU/mL) in Group I and 1373 mlU/mL for Group II subjects. One year after the third dose, the persistence of anti-HBs in those who had received 20 mug was 2262 mlU/mL whereas it was 1039 mlU/mL in the group receiving 10 microg doses. All the subjects in both the groups were seroprotected at 1 year after vaccination. None of the vaccinees exhibited serious adverse reactions throughout the study period. CONCLUSIONS: The study demonstrated the immunogenicity of the recombinant hepatitis B vaccine, and confirms that the 0.5 mL (10 microg) dose of GeneVac B can be administered with satisfactory safety and immunogenicity to adolescents up to 19 years of age, reducing the cost to less than U.S. $1.00 per dose making it acceptable for the Universal Programme of Immunization of developing and under developed countries.

Coinfection of hepatitis B and hepatitis C virus in HIV-infected patients in south India.

AIM: To screen for the co-infection of hepatitis B (HBV) and hepatitis C virus (HCV) in human immunodeficiency virus (HIV) infected patients in southern India. METHODS: Five hundred consecutive HIV infected patients were screened for Hepatitis B Virus (HBsAg and HBV-DNA) and Hepatitis C virus (anti-HCV and HCV-RNA) using commercially available ELISA kits; HBsAg, HBeAg/anti-HBe (Biorad laboratories, USA) and anti-HCV (Murex Diagnostics, UK). The HBV-DNA PCR was performed to detect the surface antigen region (pre S-S). HCV-RNA was detected by RT-PCR for the detection of the constant 5' putative non-coding region of HCV. RESULTS: HBV co-infection was detected in 45/500 (9%) patients and HCV co-infection in 11/500 (2.2%) subjects. Among the 45 co-infected patients only 40 patients could be studied, where the detection rates of HBe was 55% (22/40), antiHBe was 45% (18/40) and HBV-DNA was 56% (23/40). Among 11 HCV co-infected subjects, 6 (54.5%) were anti-HCV and HCV RNA positive, while 3 (27.2%) were positive for anti-HCV alone and 2 (18%) were positive for HCV RNA alone. CONCLUSION: Since the principal routes for HIV transmission are similar to that followed by the hepatotropic viruses, as a consequence, infections with HBV and HCV are expected in HIV infected patients. Therefore, it would be advisable to screen for these viruses in all the HIV infected individuals and their sexual partners at the earliest.

Comparison of three different recombinant hepatitis B vaccines: GeneVac-B, Engerix B and Shanvac B in high risk infants born to HBsAg positive mothers in India.

AIM: To evaluate a low cost Indian recombinant hepatitis B vaccine GeneVac-B for its immunogenicity and safety in comparison to Engerix B and Shanvac B vaccine in high risk newborn infants born to hepatitis B surface antigen (HBsAg) positive mothers. METHODS: A total of 158 infants were enrolled in the study. Fifty eight infants were enrolled in the GeneVac-B group while 50 each were included for Engerix B and Shanvac B groups. A three-dose regimen of vaccination; at birth (within 24 h of birth), 1st mo and 6 mo. were adopted with 10 mug dosage administered uniformly in all the three groups. Clinical and immunological parameters were assessed for safety and immunogenicity of the vaccines, in all the enrolled infants. RESULTS: Successful follow up until seven months of age was achieved in 83% (48/58) for GeneVac-B, 76% (38/50) and 64% (32/50) for Engerix B and Shanvac B groups respectively. 100% seroconversion and seroprotection was achieved in all the three groups of infants. The geometric mean titers of anti-HBs one month after the completion of three dose of vaccination were 90.5, 80.9 and 72.5 mIU/mL in GeneVac-B, Engerix B and Shanvac B vaccine group respectively. Furthermore the level of anti-HBs increases with age of babies who were born to HBsAg positive mothers. The GMT values of anti-HBs were 226.7, 193.9 and 173.6 mIU/mL respectively in GeneVac-B, Engerix B and Shanvac B groups one year after the completion of the three doses of vaccine. No systemic reactions were reported in infants during the entire vaccination process of GeneVac-B and the other two vaccines. Clinical safety parameters remained within the normal limits throughout the study period. CONCLUSION: The study concludes that there is no significant difference between the three recombinant hepatitis B vaccines. Administration of these vaccines within 24 h of birth to babies, born to HBsAg positive mothers will reduce the incidence of HBV infection.

Boosting BCG-primed responses with a subunit Apa vaccine during the waning phase improves immunity and imparts protection against Mycobacterium tuberculosis.

Heterologous prime-boosting has emerged as a powerful vaccination approach against tuberculosis. However, optimal timing to boost BCG-immunity using subunit vaccines remains unclear in clinical trials. Here, we followed the adhesin Apa-specific T-cell responses in BCG-primed mice and investigated its BCG-booster potential. The Apa-specific T-cell response peaked 32-52 weeks after parenteral or mucosal BCG-priming but waned significantly by 78 weeks. A subunit-Apa-boost during the contraction-phase of BCG-response had a greater effect on the magnitude and functional quality of specific cellular and humoral responses compared to a boost at the peak of BCG-response. The cellular response increased following mucosal BCG-prime-Apa-subunit-boost strategy compared to Apa-subunit-prime-BCG-boost approach. However, parenteral BCG-prime-Apa-subunit-boost by a homologous route was the most effective strategy in-terms of enhancing specific T-cell responses during waning in the lung and spleen. Two Apa-boosters markedly improved waning BCG-immunity and significantly reduced Mycobacterium tuberculosis burdens post-challenge. Our results highlight the challenges of optimization of prime-boost regimens in mice where BCG drives persistent immune-activation and suggest that boosting with a heterologous vaccine may be ideal once the specific persisting effector responses are contracted. Our results have important implications for design of prime-boost regimens against tuberculosis in humans.

The effect of methanolic extract of Tamarindus indica Linn. on the growth of clinical isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei (Pseudomonas pseudomallei) causes melioidosis, a life-threatening infection common among paddy cultivators in Southeast Asian countries. No plant materials have been investigated for its activity against B. pseudomallei. Therefore, a preliminary study was carried out using disc diffusion and minimum inhibitory concentration (MIC) methods to evaluate the anti-B. pseudomallei activity of five Indian medicinal plants documented to have been used for several ailments in the ancient Indian scriptures. The leaf extracts of Tamarindus indica, Lawsonia inermis, and Hibiscus rosa-sinensis, the rhizome extracts of Curcuma longa and the seeds of Vigna radiata were prepared using methanol as solvent. The disc diffusion and MIC methods were used to assess the anti-B. pseudomallei activity of the plants tested. Only methanol leaf extracts of Tamarindus indica exhibited anti-B. pseudomallei activity starting from disc concentrations of 150 mug by the disc diffusion method. The other plants failed to show any zone of inhibition. MIC assay revealed that the MIC and minimum bactericidal concentration (MBC) for B. pseudomallei were 125 mug/ml. Our preliminary finding showed that methanolic extracts of Tamarindus indica has anti-B. pseudomallei inhibitory potentials under in vitro conditions. Extensive animal studies may be required before investigating the role of Tamarindus indica for treating melioidosis.

Attrition of T-cell functions and simultaneous upregulation of inhibitory markers correspond with the waning of BCG-induced protection against tuberculosis in mice.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live attenuated vaccine. However, the correlates of protection and waning of its immunity against tuberculosis is poorly understood. In this study, we correlated the longitudinal changes in the magnitude and functional quality of CD4(+) and CD8(+) T-cell response over a period of two years after mucosal or parenteral BCG vaccination with the strength of protection against Mycobacterium tuberculosis in mice. The BCG vaccination-induced CD4(+) and CD8(+) T cells exhibited comparable response kinetics but distinct functional attributes in-terms of IFN-γ, IL-2 and TNF-α co-production and CD62L memory marker expression. Despite a near life-long BCG persistence and the induction of enduring CD4(+) T-cell responses characterized by IFN-γ and/or TNF-α production with comparable protection, the protective efficacy waned regardless of the route of vaccination. The progressive decline in the multifactorial functional abilities of CD4(+) and CD8(+) T cells in-terms of type-1 cytokine production, proliferation and cytolytic potential corresponded with the waning of protection against M. tuberculosis infection. In addition, simultaneous increase in the dysfunctional and terminally-differentiated T cells expressing CTLA-4, KLRG-1 and IL-10 during the contraction phase of BCG-induced response coincided with the loss of protection. Our results question the empirical development of BCG-booster vaccines and emphasize the pursuit of strategies that maintain superior T-cell functional capacity. Furthermore, our results underscore the importance of understanding the comprehensive functional dynamics of antigen-specific T-cell responses in addition to cytokine polyfunctionality in BCG-vaccinated hosts while optimizing novel vaccination strategies against tuberculosis.

Bacillus Calmette-Guérin vaccination using a microneedle patch.

Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be a leading cause of mortality among bacterial diseases, and the bacillus Calmette-Guérin (BCG) is the only licensed vaccine for human use against this disease. TB prevention and control would benefit from an improved method of BCG vaccination that simplifies logistics and eliminates dangers posed by hypodermic needles without compromising immunogenicity. Here, we report the design and engineering of a BCG-coated microneedle vaccine patch for a simple and improved intradermal delivery of the vaccine. The microneedle vaccine patch induced a robust cell-mediated immune response in both the lungs and the spleen of guinea pigs. The response was comparable to the traditional hypodermic needle based intradermal BCG vaccination and was characterized by a strong antigen specific lymphocyte proliferation and IFN-γ levels with high frequencies of CD4(+)IFN-γ(+), CD4(+)TNF-α(+) and CD4(+)IFN-γ(+)TNF-α(+) T cells. The BCG-coated microneedle vaccine patch was highly immunogenic in guinea pigs and supports further exploration of this new technology as a simpler, safer, and compliant vaccination that could facilitate increased coverage, especially in developing countries that lack adequate healthcare infrastructure.

Mycobacterium tuberculosis components stimulate production of the antimicrobial peptide hepcidin.

We investigated the in vitro production of the antimicrobial peptide hepcidin by cells of the innate immune system that harbor Mycobacterium tuberculosis. Stimulation of mouse lung macrophages with M. tuberculosis or IFN-γ + M. tuberculosis induced hepcidin mRNA. In human alveolar A549 epithelial cells, lipoglycans of M. tuberculosis, in particular mannose-capped lipoarabinomannan and phosphatidyl-myo-inositol mannosides, were strong inducers of hepcidin mRNA. In mouse dendritic cells, hepcidin mRNA was increased by subcellular fractions and culture filtrate proteins of M. tuberculosis and by TLR2 and TLR4 agonists, but not by TLR9 agonists, IL-1α, IL-6 or TNF-α. Flow cytometry evaluation of human peripheral blood mononuclear cells demonstrated that CD11c(+) myeloid dendritic cells stimulated with killed M. tuberculosis or live M. bovis BCG produced hepcidin. The production of the antimicrobial peptide hepcidin by cells that interact with M. tuberculosis suggests a host defense mechanism against mycobacteria.