Shilpa M. Velhal

Identification of CD4-independent HIV Receptors on Spermatozoa

Problem: Human immunodeficiency virus (HIV) has been demonstrated to bind and enter into the spermatozoa facilitating the transmission into urogenital cells. However, spermatozoa has been reported to be devoid of the conventional CD4 receptors for HIV. This suggests that there exists an alternate modality of HIV entry into spermatozoa using receptors other than CD4. Present communication describes the identification of HIV receptors on the spermatozoa.

CD4 Independent Binding of HIV gp120 to Mannose Receptor on Human Spermatozoa

Objective:To characterize the CD4-independent HIV-binding protein of 160kDa on human spermatozoa. Methods:The N-terminal amino acid sequence of the 160kDa protein and its peptide obtained by tryptic digestion were determined. Polymerase chain reaction amplification of human testicular cDNA was performed using degenerate primers corresponding to peptide sequences of the 160kDa protein. Localization of 160kDa protein on sperm was performed using fluorescently labeled gp120, followed by inhibition experiments using antagonists to determine the specificity. Results:The partial cDNA sequence of the 160kDa protein demonstrated 99% identity with human macrophage mannose receptor. Sequence of testicular mannose receptor was obtained and exhibited 99% identity with that of macrophage mannose receptor. Furthermore, mannose receptor protein from sperm extract was found to have a molecular weight of 160kDa, congruent with that of 160kDa HIV-binding protein. gp120 binding and mannose receptor expression were localized to the equatorial segment in 10% of ejaculated sperm, which increased after capacitation. Mannan at molar excess concentrations completely inhibited gp120 binding to sperm. Conclusions:The 160kDa, CD4-independent HIV-binding sperm protein has been identified as the human mannose receptor protein. The role of mannose receptor in HIV transmission and association with risk of sexual transmission merit further investigation.

Evaluation of cystatin C activities against HIV

Background & objectives: Several host defense proteins known to possess antimicrobial activities are present on mucosal surfaces and are consequently found in body fluids of vertebrates. Naturally occurring protease inhibitors like cystatins, especially cystatin C (cys C), are abundantly present in human seminal plasma. Although its antiviral activity against herpes simplex virus (HSV) has been demonstrated, the role of this protein against HIV is not well studied. Therefore, the aim of the present study was to evaluate the anti-HIV activities of cys C, which is present innately in the male reproductive tract. Methods: Protein-protein interaction of cys C with various HIV proteins was studied using a commercially available HIV blot and specific interaction with HIV protease was studied by dot-blot technique using commercially available cys C. To purify biologically active cys C from human seminal plasma to be used for subsequent experiments, gel-permeation chromatography followed by affinity chromatography was used. The HIV infectivity inhibition activity of the purified cystatin C was tested in TZM-bl cells. To study its activity on HIV protease, time-course enzyme kinetics studies were performed using spectrometric assay. Results: Cystatin C reacted with some HIV proteins including HIV protease. Biologically active cys C was purified using gel permeation chromatography followed by affinity chromatography. When tested in TZM-bl cells, purified cystatin C demonstrated HIV-infectivity inhibitory activity (IC50: 0.28 μM). Enzyme kinetic studies demonstrated that it abrogated the action of HIV protease on its substrate. Interpretation & conclusions: The present data demonstrate that cystatin C possesses anti-HIV activities. Molecular models need to be designed with this protein which would assist towards prevention/therapeutics against HIV.

Characterization of human immunodeficiency virus (HIV1C) variants in peripheral blood mononuclear cells and spermatozoa

The presence of distinct viral variants in different cells and secretions of the same person influences the transmission of HIV as well as the response to the host defense and to therapy. Sperm‐associated virus is also a risk factor for sexual transmission of HIV. Characterization of the C2–V3 region of HIV1C env gene by the Heteroduplex Mobility Assay (HMA) and sequencing demonstrated the presence of distinct variants in the peripheral blood mononuclear cells (PBMCs) and the sperm of the same individual (n = 6). The translated amino acid sequences of HIV variants in the PBMCs of all the study participants (n = 12) and spermatozoa of the six participants characterized showed the presence of distinct variants with different numbers of N‐linked glycosylation (NLG) sites. Infectivity of PBMCs of these persons by co‐culture with PBMCs from healthy individuals as detected by the p24 levels in the culture supernatant did not show a correlation with the blood plasma viral load. Interestingly, the infectivity of the sperm samples from four of the five individuals showed positive correlation with the viral load in seminal plasma. The study suggests the presence of distinct viral variants in the sperm and PBMCs of the same person with differential infectivity, and the NLG sites may be associated with the affinity of HIV to receptor/co‐receptor usages as well as affinity toward neutralizing antibodies which may influence the risk of sperm associated virus in sexual transmission of HIV and transmit the virus further to distal cells. J. Med. Virol. 83:760–767, 2011.

Nevirapine Loaded Core Shell Gold Nanoparticles by Double Emulsion Solvent Evaporation: In vitro and In vivo Evaluation

BACKGROUND HIV/AIDS is a macrophage resident infection localized in the reticuloendothelial system and remote locations of brain and bone marrow. We present core shell nanoparticles of gold(AuNPs) and nevirapine(NVP) for targeted delivery to the multiple HIV reservoirs. The aim of the study was to design core shell NVP loaded AuNPs with high drug loading and to evaluate biodistribution of the nanoparticles in possible HIV reservoirs in vivo. A specific objective was to assess the possible synergy of AuNPs with NVP on anti-HIV activity in vitro. METHOD Core shell nanoparticles were prepared by double emulsion solvent evaporation method and characterized. RESULTS Glyceryl monostearate-nevirapine-gold nanoparticles(GMS-NVP-AuNPs) revealed high entrapment efficiency (>70%), high loading (~40%), particle size <250 nm and zeta potential -35.9± 1.41mv and exhibited sustained release with good stability. Surface plasmon resonance indicated shell formation while SEM coupled EDAX confirmed the presence of Au. TEM confirmed formation of spherical core shell nanoparticles. GMS-NVP-AuNPs revealed low hemolysis (<10 %) and serum stability upto 6 h. GMS-NVP-AuNPs exhibited rapid, high and sustained accumulation in the possible HIV reservoir organs, including the major organs of liver, spleen, lymph nodes, thymus and also remote locations of brain, ovary and bone marrow. High cell viability and enhanced uptake in PBMCs and TZM-bl cells were observed. While uptake in PBMCs proposed monocytes/macrophages enabled brain delivery. GMS-NVP-AuNPs demonstrated synergistic anti-HIV activity. CONCLUSION The superior anti-HIV activity in vitro coupled with extensive localization of the nanoparticles in multiple HIV reservoirs suggests great promise of the core shell GMS-NVP-AuNPs for improved therapy of HIV.

Immunogenicity of Semliki Forest virus based self-amplifying RNA expressing Indian HIV-1C genes in mice.

Development of recombinant vaccines is considered as a promising approach to prevent transmission and eradication of HIV/AIDS. Candidate vaccines tested so far have shown poor to modest efficacy. Self-amplifying RNAs of positive strand alphaviruses are reported to be promising vectors for development of recombinant vaccines. This study describes the construction, in vitro expression and in vivo immunogenicity of recombinant RNA vaccines developed by individually cloning gag, env and polRT genes of primary HIV-1C Indian isolates using Semliki Forest virus (SFV) vector. HIV-1C specific T cell responses were detected in mice immunized with rSFV2gen/gag RNA by IFN-γ ELISPOT assay. Furthermore, using flow cytometry based intracellular cytokine staining (ICCS) assay HIV-1C specific IL-2 responses were detected in immunized mice that were mediated by both CD4(+) and CD8(+) T cells. Mice immunized with rSFV2gen/env RNA elicited HIV-1C Env-specific antibodies as detected by gp120 ELISA. The Env, Gag and Pol (RT) RNA constructs in combination elicited better HIV-1C Env-specific humoral responses compared to mice immunized with Env RNA alone. In conclusion, rSFV2gen RNA constructs encoding HIV-1C antigens elicited clear cell mediated and humoral immune responses in mice, thus demonstrating the potential of self-amplifying rSFV2gen RNA as a promising candidate for anti-HIV vaccine development.

Immunogenicity of virus-like Semliki Forest virus replicon particles expressing Indian HIV-1C gag, env and polRT genes

Development of a vaccine targeting human immunodeficiency virus-1 subtype C (HIV-1C) is an important public health priority in regions with a high prevalence of the clade C virus. The present study demonstrates the immunogenicity of recombinant Semliki Forest virus (SFV)-based virus-like replicon particles (VRPs) expressing Indian HIV-1C env/gag/polRT genes. Immunization of mice with recombinant VRPs in a homologous prime-boost protocol, either individually or in combination, elicited significant antigen-specific IFN-γ T cell responses as detected by the ELISPOT assay. Additionally, Gag-specific TNF-α secreting CD8+ and CD4+ T cells and Env-specific IL-2 secreting T cells were also elicited by mice immunized with Gag and Env constructs, respectively, as estimated by intracellular cytokine staining assay. Moreover, an HIV Pol-specific TNF-α response was elicited in mice immunized with a combination of the three VRP constructs. Furthermore, HIV-1C Gag and Env-specific binding antibodies were elicited as verified by gp120 ELISA and p24 Gag ELISA, respectively. The immunogenicity of VRPs was found to be higher as compared to that of RNA replicons and VRPs may therefore be promising preventive and therapeutic candidate vaccines for the control and management of HIV/AIDS.

Surface functionalized nanoparticles of NVP an improved strategy to tackle deadly HIV infection

Results GMS nVP auNPs with > 75% entrapment efficiency & 85% binding to the ligand. In cytotoxicity study nanoformulations showed higher cell viability at all time points as compared to NVP. Cell uptake study revealed higher uptake of nanoparticles as compared to NVP only. In vitro anti HIV assay of surface modified revealed 10 fold increased activity as compared to NVP.

Genotypic characterization of HIV variants from PBMCs and spermatozoa.

Background Several studies have shown that detectable levels of HIV are observed in semen in spite of undetectable viral load in blood following antiretroviral therapy (ART). Also, different HIV variants have been detected in blood and semen of the same individual, suggesting that drugs may not be uniformly effective to control the viral load and infectivity in different tissues and secretions, which in turn may affect sexual transmission of HIV. Hence, genotypic characterization of HIV variants in PBMCs and sperm of the same individual may provide information about the association of these variants with sexual transmission of HIV.

Analysis of HIV variants in blood and semen in serodiscordants by heteroduplex mobility assay

Purpose of the study The resistance of antiretroviral drugs is mainly attributed by the generation of HIV variants in different secretions of the same individual; distinct viral variants have been detected in different cells and body fluids of the same individual. Genotypic and phenotypic characterization of viral variants in blood and urogenital secretions may provide strategies for designing modalities in prevention of sexual transmission of HIV and to explore the mechanism of sexual transmission of human immunodeficiency virus type 1(HIV-1).