Neutralization Epitope Mapping in HIV-1 Envelope of Indian Origin and Construction of Envelope-Based Peptide Immunogen for Generation of Broadly Cross-Reactive Neutralizing Antibodies (DBT-ICMR)
	Comprehensive molecular analysis of HIV incidence, genetic diversity and anti-retroviral drug-resistance mutations in diverse risk groups across wide geographic locales of India (DBT-ICMR)
	Construction and Characterization of genetically engineered chimeric HIV-1 envelope protein modulating virus neutralization (DBT)
	Studies on the role of gag and vpu on HIV-1 envelope assembly in T cells and macrophages (Intramural)
	Collaboration of AIDS Vaccine Discovery and Vaccine Immune Monitoring Consortium (CAVD-VIMC)
	Full-Length Genome Analysis of HIV-1 isolates from India
	Studies on cellular tropism of HIV-1 of India clade and characterization of their biological properties in T cells and monocyte-derived macrophages (Intramural)
	Interaction of HIV-1 Rev with host factors in central nervous system (CNS).
	In vitro testing of indigenously developed active principles for identification and characterization of candidate anti-retroviral Microbicides (SSK)

1. Neutralization Epitope Mapping in HIV-1 Envelope of Indian Origin and Construction of Envelope-Based Peptide Immunogen for Generation of Broadly Cross-Reactive Neutralizing Antibodies

Brief Description:

The envelope (env) gene is the most diverse of the HIV-1 genes and can differ in more than 30% of the amino acids. Clade C is the major subtype circulating in India where the HIV/AIDS pathogenesis is predominantly driven by sexual transmission. CD4 and either CCR5 or CXCR4 expressed on cell surface are essential for virus entry; however relative usages of either CCR5 or CXCR4 is known to play vital role in preferential transmission of HIV-1 in vivo. The envelope glycoprotein of HIV-1 is organized as trimers on virus particles, with gp120 surface unit (SU) attached to gp41 transmembrane subunits in a non-covalent manner. The entry of HIV-1 into target cells is initiated by binding of gp120 to cell surface CD4 resulting in induction of conformational alteration within gp120 that in turn exposes site for binding with one or more coreceptor/s. This interaction between gp120 with CD4 and coreceptor then triggers gp41 transmembrane to enable fusion of virus and cellular membrane thereby facilitate delivering virion core into cellular cytoplasm. The flexible domains on envelope trimer of HIV-1 possess critical motifs that modulate affinity to CD4, coreceptor binding and sensitivity to neutralization. Our current research is focused on the motifs that play modulatory role in determining cell type that are infected in vivo besides CD4+T cells as well as critical domains and motifs in env that modulate sensitivity to neutralizing antibodies and entry inhibitors. Using molecularly cloned gene products and constructing pseudovirus or virus like particles (VLPs) as well as chimera, we use engineered cell lines to address our objectives and test it in primary cells such as CD4+T cells as well as macrophages to mimic the process that perhaps is taken place in vivo. Recently, we have cloned and characterized HIV-1 envelopes (gp160) that uses a range of co-receptors, a unique phenotype that has been identified for the first time from patients in India. In addition, we are also working on envelope clones obtained from asymptomatic ART (anti-retroviral therapy) naïve patients and studying to map motifs in variable domains (variable loops) that modulate virus sensitivity to neutralizing antibodies. Our research is significant in understanding mechanism of HIV-1 entry and has relevance in vaccine and drug discovery that target entry.

Current status:

Active and ongoing

2. Comprehensive molecular analysis of HIV incidence, genetic diversity and anti-retroviral drug-resistance mutations in diverse risk groups across wide geographic locales of India

Brief Description:

In this study, we are investigating recombinant viral strains using Real Time PCR based MHAbce technology.

3. Construction and Characterization of genetically engineered chimeric HIV-1 envelope protein modulating virus neutralization

Brief Description:

In this study, we are investigating domain in HIV-1 clade C envelope modulating neutralization resistance and escape.

4. Studies on the role of gag and vpu on HIV-1 envelope assembly in T cells and macrophages

Brief Description:

In this study, we are investigating how viral proteins such as Gag and Vpu regulates HIV-1 envelope trafficking and assembly onto virions.

5. Collaboration of AIDS Vaccine Discovery and Vaccine Immune Monitoring Consortium (CAVD-VIMC)

Brief Description:

Our role in this multi-centric project is support with ancillary research support on HIV-1 clade C neutralization.

6. Full-Length Genome Analysis of HIV-1 isolates from India

Summary:

This study is funded by DBT, Government of India. In this study, we have documented and published novel forms of B/C recombinants in India. In continuation of the same project, we have screened additional HIV-1 strains for disseminating their near full length genetic properties. Virus samples obtained from Viral Bank and infected PBMCs from other projects of NARI have been infected and co-cultured with donor PBMCs respectively that were isolated from donor blood by Density Grade centrifugation method. Genomic DNA was isolated by using Qia Amp DNA Blood Mini kit (Qiagen, Inc.) and near full length genomes of HIV (9 KB) were amplified using Expand Long Template Polymerase (Roche, Inc.). Purified amplicons were cloned in pCR 2.1 vector (Invitrogen, Inc.). Plasmid DNA was isolated and sequenced by using ABI 3100 genetic analyzer using Big Dye Terminator method. We have cloned near full length genome of 37 viruses (at least 2 clones for each virus) and sequencing has been completed for 26 viruses. Interestingly, though all the clones have been found as subtype C, we have found one clone exhibiting novel form of A1/C recombinant and one subtype A virus. Further characterization is being carried out.

7. Studies on cellular tropism of HIV-1 of India clade and characterization of their biological properties in T cells and monocyte-derived macrophages.

The principal aim of the study is of two fold; to study the co-receptor usage and tropism of HIV-1 envelope of Indian clade C in different disease stages and to identify and characterize HIV-1 R5 variants that modulate broader and narrow tropism resulting in modulation in virus entry, transmission and pathogenesis. Our objectives are:

In the proposed study, we would like to ask following questions,

1. To study the receptor biology and tropism of HIV-1 envelope of India clade C in different disease stages.
2. To identify and characterize HIV-1 R5 variants to see if there is any association of broad and narrow tropism with preferential transmission and pathogenesis in CD4+ T cells and monocyte derived macrophages.
3. To correlate envelope-receptor/coreceptor interaction with envelope sensitivity to entry inhibitors and reagents that block env-CD4 interaction.

In this study, we have so far collected a total of 10 primary isolates of HIV-1 from NARI virus bank. These viruses were obtained from patients who were mostly found symptomatic at the time of collection of blood. The viruses were co-cultivated in HIV-1 seronegative donor peripheral blood mononuclear cells (PBMC) to prepare HIV+ PBL and stock viruses. Full length gp160 genes were amplified using high fidelity proof reading polymerase by PCR and subsequently cloned into mammalian expression vectors either by blunt end ligation in pcDNA TOPO 3.1 or by staggered end ligation using conserved KpnI sites in pSVIIIenv (Figure 1). A total of 24 functional gp160 clones were obtained. These plasmid DNA clones were co-transfected with a full length infectious HIV-1 molecular clone (SG?3) with defective env in 293T cells to produce pseudoviruses. These viruses were capable of single cycle replication.

Figure 1. CMV-based (pcDNA3.1) and LTR-based (pSVIIIenv) vectors expressingHIV-1 envelope

We tested the envelope clones for their capacity to use co-receptors other than CCR5 as it has been documented that India clade C are strictly CCR5 tropic irrespective of disease stages. Interestingly, we found that envelope clones obtained from two patients could utilize multiple co-receptors in addition to CCR5. While gp160 clones from one virus (NARI-VB105) could infect CCR5, CXCR4, CCR2b, CCR3, BOB, BONZO and V28, clones from another virus (NARI-VB52) could use CCR5, CCR8 and CXCR6 (Figure 1). To test whether these clones indeed were using multiple co-receptors in vivo, donor PBMCs and primary CD4+T cells were infected with primary viruses of NARI-VB52 and NARI-VB105 and their corresponding clones in presence of TAK-779, a CCR5 inhibitor and AMD-3100, a CXCR4 inhibitor. Infectivity data (Table 1) confirmed that indeed they use multiple co-receptors in vivo. This is the first report to date of sexually acquired HIV-1 India clade C using multiple co-receptors in vivo.

8. Interaction of HIV-1 Rev with host factors in central nervous system (CNS).

In this project, we are working to understand the mechanism by which HIV-1 Rev modulates RNA splicing in macrophages with particular reference to virus replication in Central Nervous System (CNS). HIV-1 rev has both nuclear localization signal (NLS) and nuclear export signal (NES) that plays vital role in HIV RNA splicing and export in cellular cytoplasm towards constituting mature viral particles. This mechanism differs between compartments such as systemic circulation and CNS. We aim to use CNS model to delineate the mechanism of retroviral splicing in macrophages and host factor/s that assist in such function. Incidentally, rev NLS recognizes a conserved RRE (rev response element) in viral envelope that is important for target cell entry and delivery of viral nucleic acid. While rev function in CNS is poorly known, our research would provide insights that could well serve as a target for intervention.

9. In vitro testing of indigenously developed active principles for identification and characterization of candidate anti-retroviral Microbicides

Research Fellows (Ph.D.) Registered under Pune University and their Area of Thesis Study:

Dr. Smita Kulkarni, Scientist D, NARI, Pune

Co-Investigators: 

Dr J Bhattacharya, Scientist D, NARI, Pune.

Dr A R Risbud, Scientist F, NARI, Pune

Dr Kamini Walia, Scientist C, ICMR, New Delhi

Co-PIs:

Dr S P Joshi, Scientist, National Chemical Laboratory, Pune

Dr KVR Reddy, Deputy Director, National Institute of Research on Reproductive Health, Mumbai.

Dr Kamalinder Singh, Professor of Pharmaceutics, C.U. Shah College of Pharmacy, Mumbai.

Duration:

2007- 2009.

Objectives:

1. Establishing a screening programme at national level to select appropriate anti-HIV candidate leads.
2. Development, selection and identification of candidate leads.
3. Screening of leads for anti-HIV activity using high throughput assay.
4. Confirmation of anti-HIV activity of leads using additional in-vitro models.
5. Evaluation of potential leads against other STIs.

Summary:

Mucosal exposure through heterosexual transmission is the major mode of HIV transmission. Women account for more than half of HIV infections worldwide, signifying urgent need for women controlled barriers like microbicides. Many microbicide candidates are undergoing clinical trials, however failure of candidates at advanced stages of the trials indicate the need of rigorous search for new microbicide products.

Medicinal plants have a long history of use and their use is widespread in both developing and developed countries. Extracts of plants and phytochemicals have been shown to possess activity against sexually transmitted pathogens including HIV. Such extracts may serve as a good source of new anti-HIV agents. We have initiated an exploratory approach for testing anti-HIV activity of indigenously developed products under this project, jointly funded by the Department of Biotechnology and the Indian Council of Medical Research, Govt of India.

For selection of appropriate candidate leads, the Product Selection Committee (PSC) has been formed by involving experts in the fields of pharmacology, toxicology and medicinal chemistry. The committee has come up with stringent criteria which will be of national use, making product selection very discriminatory. Following the guidelines, a number of candidate leads developed by four collaborating institutes have been selected. The anti-HIV testing of these candidate leads using a fixed algorithm under the GLP compliance, following strict quality control and quality assurance guidelines is in progress at NARI.

A high throughput assay using TZM-bl cell line has been standardized and is being used for testing anti-HIV activity of the candidate leads. The lead compounds are being assessed to inactivate HIV, to block HIV entry and to inhibit replication, using additional model systems. Test preparations showing reduction of HIV growth in the these assays will be tested for their ability to inhibit HIV-1 using primary target cells (cervico-vaginal and rectal explant cultures) and transwell Epithelial-PBMC infection model system. The candidate preparations showing reduction of HIV growth will be further tested for activity against other STI pathogens. Lead preparations showing anti-HIV and anti-STI activity will be tested in combination to check whether they act additively or synergistically. Thus the study will create complete microbicide profile of the lead candidates. The planned exploratory approach if successful, may lead in identifying novel, effective anti-HIV/STI agents of indigenous nature that would help in preventing HIV infection.

Research Fellows (Ph.D.):

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