Figure 4.1: Gel picture showing Generation of HIV-1subtype C codon optimized env expressing recombinant plasmid pvax-1:

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2 Full-fledged work is in progress towards construction and cloning of codon optimized envelope with subsequent aims towards immunization of mice to study immune responses Figure 4.1: Gel picture showing Generation of HIV-1subtype C codon optimized env expressing recombinant plasmid pvax-1: 1-1kb DNA ladder 2-Codon optimized gp150 in commercial vector - Codon optimized gp150 in commercial vector- EcoR1&HindIII digest 4-linear pvax 5-codon optimized gp150 cloned in pvax 6-codon optimized gp150 in pvax- EcoR1&HindIII digest B. Study on Mother to Infant Transmission (MIT) of HIV [Principal Investigator:Dr R S Paranjape] This study has been initiated at the Obstetrics and Gynecology Department of the Sassoon General Hospital in collaboration with Johns Hopkins University, USA. NARI has been involved in providing laboratory support by testing enrolled mothers and infants for HIV-1 PCR, HIV-1 plasma viral load and determining absolute CD4 counts. Automated DNA/RNA Extractor ABI 9800 Thermal Cycler We hypothesize that codon-optimized envelope with native env and gag constructs along with immuno modulatory genes will have immunological advantage in terms of CTL and antibody diversity. 7

3 C. Alterations in immune responses induced by selective modification in env gene of HIV-1 subtype C from India. [Principal Investigator: Dr. S. P. Tripathy] Objectives: To characterize Indian subtype C HIV-1 envelope protein in eliciting immune response and to study the effect of envelope protein with selective modifications in mouse model towards eliciting antibody response Significant progress has been made in the following areas: Envelope genes were amplified and cloned into Adenovirus expression vector Modifications in glycosylation sites done by site directed mutagenesis Immunization of experimental animals are in process Figure 4.2.Functional assay for expression of HIV-1 env in adenovirus using Flow Cytometer. Cell surface and Intracellular staining of 29T cells infected for 48hrs with 240DMab,showed expression of gp160 inside the cells. Mock 29T KDA 77 KDA80 KDA81 8

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5 Table 4.1: Details of age and gender distribution and exposure to different antiretroviral drug combinations Age range (in years) Drug naive Gender distribution Male Female Drug Class combination NRTIs + PIs + NRTIs PIs, NRTIs, NRTIs only Treatment experienced A. Drug Resistance Laboratory OUT IN B. Inside the Laboratory C. Thermal Cycler E. In vitro anti-retroviral testing. [Principle Investigator: Dr. S S Kulkarni] Background: In view of the magnitude of the AIDS pandemic, absence of protective vaccine, lack of non-toxic therapy and resistance developed by the virus to different therapeutic drugs, there is an urgent need for development of new, specific and non-toxic drugs. NARI is contributing by testing activity of indigenously developed drugs in the laboratory. Anti-HIV activity of thiazolidinones: The anti-hiv activity of initially synthesized nine thiazolidinones was tested at NARI. The activity was tested against CXCR4 tropic HIV-IIIB using MT2 cell line and CCR5 tropic Indian isolate using activated PBMCs. Four preparations, exhibited complete reduction (100%) against CXCR4 tropic as well as CCR5 tropic viruses. In addition to this, four herbal (includes three spermicides) and one synthetic preparation were tested for antihiv activity. None of the preparations demonstrated anti-hiv activity. 40

6 ABI 100 Sequencer 1.Viral load testing (Service Component): NARI is participating in various NIH funded anti-retroviral trials, for which viral load testing is considered as an end point. The break up of samples processed for viral load is given against the respective projects Table 4.2: Samples processed for plasma viral load (April 05-March 06) Name of Project No. Tested Mother to child transmission (MIT) study HIV Prevention Trial Network (HPTN) 04 and HPTN 052 studies Intramural NARI projects and samples referred by physicians AIDS Clinical Trial Group (ACTG) , Diagnostic gag gene PCR of Non Project samples collected from in and around Maharastra (Service Activity): National AIDS Research Institute being a service centre for diagnosis of HIV infection from various risk categories like mother to child transmission, accidental exposure, and medico legal issues, whole blood samples are collected from in and around Maharashtra to perform gag gene PCR for diagnosis of HIV-1 infection. In this year, from April 2005 till March 2006, we have collected and tested about 187 samples. We have found 165 samples negative, 25 samples positive and 17 samples are in process for HIV-1 gag gene PCR. Simultaneously with the increasing evidence of HIV-1 infection, we have come across with few cases of HIV-2 infected individuals. We have tested such samples for HIV-2 specific protease and envelope gene PCR to confirm HIV-2 infection. The break up of samples processed for viral load is given against the respective projects are given in Table 4. F. Support Services: NARI is participating in various NIH funded anti-retroviral trials, for which viral load testing is considered as an end point. The break up of samples processed for viral load is given against the respective projects. Table 4.: Samples processed for DNA PCR (2006) Name of Project Mother to child transmission (MIT) study Intramural NARI projects and samples referred by physicians No. Tested