Jagadish Chandrabose Sundaramurthi

In silico identification of potential antigenic proteins and promiscuous CTL epitopes in Mycobacterium tuberculosis.

Cell-mediated immunity is critical for the control of Mycobacterium tuberculosis infection. We hypothesized that those proteins of M. tuberculosis (MTB) that do not have homologs in humans as well as human gut flora, would mount a good antigenic response in man, and employed a bioinformatics approach to identify MTB antigens capable of inducing a robust cell-mediated immune response in humans. In the first step we identified 624 MTB proteins that had no homologs in humans. Comparison of this set of proteins with the proteome of 77 different microbes that comprise the human gut flora narrowed down the list to 180 proteins unique to MTB. Twenty nine of the 180 proteins are known to be associated with dormancy. Since dormancy associated proteins are known to harbor CTL epitopes, we selected four representative unique proteins and subjected them to epitope analysis using ProPred1. Nineteen novel promiscuous epitopes were identified in the four proteins. Population coverage for 7 of the 19 shortlisted epitopes including Rv3852 (58-KPAEAPVSL, 112-VPLIVAVTL, 118-VTLSLLALL and 123-LALLLIRQL), Rv2706c (66-RPLSGVSFL) Rv3466 (8- RIVEVFDAL and 38-RSLERLECL) was >74%. These novel promiscuous epitopes are conserved in other virulent MTB strains, and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

Prevalence and Pattern of Cross-Reacting Antibodies to HIV in Patients with Tuberculosis

In many countries, HIV testing among tuberculosis (TB) patients is recommended so that both infections are appropriately treated. Cross-reacting antibodies to HIV antigens have been reported for several conditions, including TB, leprosy, malaria, and rheumatoid arthritis. To study the pattern and prevalence of cross-reacting antibodies to HIV antigens, we examined sera from 153 HIV-negative TB patients and 40 healthy individuals in Chennai, south India. We also studied the differences in cross-reactivity of various HIV antigens using two different Western blot kits. Of the 153 samples studied, 80 were tested using HIV Western blot and 73 were tested using INNOLIA. Most patients in the study had concordantly negative ELISA and rapid tests, and no subject had a positive Western blot. However, seven TB patients had antibodies that cross-reacted with HIV antigens, giving rise to an indeterminate result. While p51/55 was the most frequently recognized antigen in the Western blot assay, antibodies to sgp120 was most frequently identified in INNOLIA. Sequence similarities between the two organisms could be responsible for eliciting cross-reacting antibodies, since a few related epitopes were identified in HIV and Mycobacterium. These findings could have potential implications for the development of diagnostics and vaccines.

Mycobacterium tuberculosis Whole Genome Sequences From Southern India Suggest Novel Resistance Mechanisms and the Need for Region-Specific Diagnostics

Key points By sequencing 223 M. tuberculosis strains from Southern India, we expanded the studied genetic diversity of lineages 1 and 3. We observed local transmission of strains; unexplained resistance; potential novel resistance mutations; and that isoniazid resistance was gained first.

Informatics resources for tuberculosis – Towards drug discovery

Integration of biological data on gene sequence, genome annotation, gene expression, metabolic pathways, protein structure, drug target prioritization and selection, has resulted in several online bioinformatics databases and tools for Mycobacterium tuberculosis. Alongside there has been a growth in the list of cheminformatics databases for small molecules and tools to facilitate drug discovery. In spite of these efforts there is a noticeable lag in the drug discovery process which is an urgent need in the case of emerging and re-emerging infectious diseases. For example, more than 25 online databases are available freely for tuberculosis and yet these resources have not been exploited optimally. Informatics-centered drug discovery based on the integration and analysis of both bioinformatics and cheminformatics data could fill in the gap and help to accelerate the process of drug discovery. This article aims to review the current standing of developments in tuberculosis-bioinformatics and highlight areas where integration of existing resources could lead to acceleration of drug discovery against tuberculosis. Such an approach could be adapted for other diseases as well.

Immunological and proteomic analysis of preparative isoelectric focusing separated culture filtrate antigens of Mycobacterium tuberculosis.

Isolation of the secreted proteins and studying the immune response they induce is an essential prerequisite for understanding the pathogenesis of M. tuberculosis. In this study, preparative liquid-phase isoelectric focusing was used for the separation of culture filtrate protein (CFP) of M. tuberculosis. This procedure resolved culture filtrate proteins into 20 fractions with a pI range of 2.59 to 12.9. These 20 fractions were subjected to immunological analysis in healthy laboratory volunteers from our endemic area. Eleven fractions (Fractions 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 19) showed increased interferon gamma (IFN-gamma) secretion and 5 fractions induced increased proliferative response, when compared to unfractionated CFP. In the 11 fractions which showed increased IFN-gamma secretion, mass spectrometric analysis identified 19 different proteins. Apart from the already reported immunodominant antigens like FbpB, CFP-10 and ESAT-6, two new T cell antigens (AcpM and PpiA) were also identified in the immunologically active fractions. Immunoinformatic analysis showed that PpiA was predicted to bind more number of class I and class II HLA alleles compared with the immunodominant ESAT-6 and CFP-10. Population coverage calculations also showed that PpiA protein (85%) had a higher population coverage compared with ESAT-6 (79%) and CFP-10 (77%). This result shows that the PpiA protein has a potential to be a novel T cell antigen.

Cellular immune response to Mycobacterium tuberculosis-specific antigen culture filtrate protein-10 in south India

The Mycobacterium tuberculosis (M. tuberculosis)-specific culture filtrate protein-10 (CFP-10) is highly recognized by M. tuberculosis infected subjects. In the present study, the proliferative response and IFN-γ secretion was found for C-terminal peptides of the protein (Cfp651–70, Cfp761–80, Cfp871–90, and Cfp981–100). The alleles HLA DRB1 *04 and HLA DRB1 *10 recognized the C-terminal peptides Cfp7, Cfp8, and Cfp9 in HHC. Cfp6 was predominantly recognized by the alleles HLA DRB1 *03 and HLA DRB1 *15 by PTB. The minimal nonameric epitopes from the C-terminal region were CFP-1056–64 and CFP-1076–84. These two peptides deserve attention for inclusion in a vaccine against tuberculosis in this region.

Resistance-associated epitopes of HIV-1C—highly probable candidates for a multi-epitope vaccine

Earlier studies have identified a large number of immunogenic epitopes in HIV-1. Efforts are required to prioritize these epitopes in order to identify the best candidates for formulating an effective multi-epitope vaccine for HIV. We modeled 155 known cytotoxic T lymphocyte epitopes of HIV-1 subtype C on the 3D structure of HLA-A*0201, HLA-B*2705, and HLA-B*5101 using MODPROPEP, as these alleles are known to be associated with resistance to HIV/slow progression to AIDS. Thirty-six epitopes were identified to bind to all the three HLA alleles with better binding affinity than the control peptides complexed with each HLA allele but not to any of the HLA alleles reported to be associated with susceptibility to HIV infection/rapid progression to disease. As increase in stability of the epitope–HLA complex results in increased immunogenicity, the short-listed epitopes could be suitable candidates for vaccine development. Twenty of the 36 epitopes were polyfunctional in nature adding to their immunological relevance for vaccine design. Further, 9 of the 20 polyfunctional epitopes were found to bind to all three resistance-associated HLA alleles using an additional method, adding worth to their potential as candidates for a vaccine formulation for HIV-1C.

Molecular docking of azole drugs and their analogs on CYP121 of Mycobacterium tuberculosis

The Mycobacterium tuberculosis genome codes for 20 different cytochromes. These cytochromes are involved in the breakdown of recalcitrant pollutants and the synthesis of polyketide antibiotics and other complex macromolecules. It has been demonstrated that CYP121 is essential for viability of the bacterium by gene knock-out and complementation studies. CYP121 could therefore be a probable target for the development of new drugs for TB. It has been widely reported that orthologs of CYP121 in fungi are inhibited by azole drugs. We evaluated whether these azole drugs or their structural analogs could bind to and inhibit CYP121 of M. tuberculosis using molecular docking. Six molecules with known anti-CYP121 activity were selected from literature and PubChem database was searched to identify structural analogs for these inhibitors. Three hundred and fifty seven molecules were identified as structural analogs and used in docking studies. Fifty three molecules were found to be scored better than the azole drugs and five of them were ranked among the top 12 molecules by two different scoring functions. These molecules may be further tested by in vitro experimentation for their activity against CYP121 of M. tuberculosis.

Bioinformatics approach to prioritize known drugs towards repurposing for tuberculosis

New drugs are urgently needed to cure tuberculosis (TB) in a short period of time without causing any adverse effects since currently used drugs for the treatment of multi drug-resistant TB cause several adverse effects with poor success rate. Therefore, we aimed to prioritize known drugs towards repurposing for TB by employing bioinformatics approach in the present study. A total of 1554 FDA approved drugs were obtained from DrugBank. Serine/threonine-protein kinase, pknB (Rv0014c) of Mycobacterium tuberculosis (Mtb) was selected as the drug target since it involves in several vital functions of the Mtb. All of the 1554 drugs were subjected to molecular docking with pknB. Glide and AutoDock Vina were employed using rigid docking followed by induced fit docking protocol for prioritization of drugs. Out of 14 drugs prioritized, six are suggested as high-confident drugs towards repurposing for TB as they were consistently found within top 10 ranks of both methods, and strongly binding in the active site of the pknB. We also found atorvastatin as one of the high-confident drugs, which has already been demonstrated to be active against Mtb under in vitro conditions by other researchers. Therefore, we propose that the prioritized six high-confident drugs as potential candidates for repurposing for TB and suggest for further experimental studies. We also suggest that the bioinformatics procedure we have employed in this study could be effectively applied for prioritization of drugs for other diseases.

Broad and potent cross clade neutralizing antibodies with multiple specificities in the plasma of HIV-1 subtype C infected individuals

Broadly Cross clade Neutralizing (BCN) antibodies are recognized as potential therapeutic tools and leads for the design of a vaccine that can protect human beings against various clades of Human Immunodeficiency Virus (HIV). In the present study, we screened plasma of 88 HIV-1 infected ART naïve individuals for their neutralization potential using a standard panel of 18 pseudoviruses belonging to different subtypes and different levels of neutralization. We identified 12 samples with good breadth of neutralization (neutralized >90% of the viruses). Four of these samples neutralized even the difficult-to-neutralize tier-3 pseudoviruses with great potency (GMT > 600). Analysis of neutralization specificities indicated that four samples had antibodies with multiple epitope binding specificities, viz. CD4-binding site (CD4BS), glycans in the V1/V2 and V3 regions and membrane proximal external region (MPER). Our findings indicate the strong possibility of identifying highly potent bNAbs with known or novel specificities from HIV-1 subtype C infected individuals from India that can be exploited as therapeutic tools or lead molecules for the identification of potential epitopes for design of a protective HIV-1 vaccine.