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Host and Pathogenic Variation Remains the Challenges in Management

This study explores the risk of HIV transmission through different routes and the factors influencing the transmission in the male and female genital tracts. It also examines the role of CD4-independent HIV binding proteins in the transmission process. The study concludes that the variability in host and pathogenic factors poses challenges in the management and control of HIV/AIDS.

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Host and Pathogenic Variation Remains the Challenges in Management

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  1. Host and Pathogenic Variation Remains the Challenges in Management and Control of HIV/AIDS A H Bandivdekar National Institute for Research in Reproductive Health J M Street, Parel, Mumbai 400 012 INDIA

  2. Risk of HIV Transmission through Different Routes Sexual Intercourse : a. Female to Male 1 in 700 to 3000 b. Male to Female 1 in 200 to 2000 c. Male to Male 1 in 10 to 1600 Needle Stick : 1 in 200 Needle Sharing : 1 in 150 Mother to infant : a. Without ART 1 in 4 b. With ART Less than 1 in 10 Infected Blood : 95 in 100 Cooggins and Segal, J Repr Imm 1998

  3. HIV IN MALE • - Free Virus and Infected Cells may Enter through Prostate, • Seminal Vesicles and Urethra • - Seminal Leukocytes have been Initially Thought to be the Major • and Sole Source of HIV in Semen • - HIV Binds CD4 Independently to Sperm and Further Transmit the • Virus in to Distal Cells • - Free Virus does not Bind to Oocyte • But HIV Infected Sperm can Fertilize Oocyte • - HIV Infected Sperm Showed Normal Acrosomal Reaction, Egg • Penetration, Induce Segmentation and Vehicle HIV Particle into Egg • Concomitant STDs such as CMV and HSV Induces HIV Viral Load • in Seminal Fluid and Circulation

  4. HIV Transmission in Female Genital Tract HIV Infect Vaginal, Ectocervical and Endocervical Mucosa Vagina and Ectocervix is Multilayered Endocervix is Single Layer Columnar Epithelium HIV/SIV Establish Initial Infection in Vagina Vaginal Epithelial Cells Devoid of CD4 Receptor and CXCR4 and CCR5 Coreceptors Few Endo and Ectocervical Cells are CD4 positive Precise Mechanism of HIV Entry is not known

  5. Precise Mechanism of Sexual Transmission of HIV Not Fully Known due to Host Factors - Immune Responses - CD4 Dependent and Independent Receptors for HIV Binding and Entry into Host Cells Variation in Pathogenic Factors - Viral Variation - Presence of Distinct Variants in Different Cells and Secretions of the Same Individual

  6. Dendritic Cell-Specific ICAM-3 Grabbing Nonintegrin • (DC-SIGN) • High Affinity for HIV-1 Env Gene • Expression in Human Cervix, Rectum and Uterus • No Expression on Plasmacytoid DCs • No Expression on Langerhans Cellsin Vaginal Epithelium • Moderate Expression in Subepithelial DCs in the Lamina Propria • High Levels of Expression in Cells of Rectal Mucosa • Localization of CCR5 CD4 DC-SIGN • DCs of Rectal Tissue Beneath the Luminal Epithelium • Accessibility to DC-SIGN is Limited in an Intact Vaginal Mucosa • Galactosyl Ceramide (GalCer) • Neural and Intestinal Epithelial Cells • HIV Env gp120 V3 loop Binds to GalCer

  7. Syndecan-3 HIV-1 Attachment Receptor on DCs DC-specific Heparan Sulfate (HS) Proteoglycan Interaction with HIV-1 Envelope Glycoprotein gp120 Stabilizes Captured Virus, Enhances DC Infection in cis, Promotes Transmission to T Cells Heparan Sulfate Proteoglycan (HS) Surface of Stratified Squamous Epithelium Removal of the HSs from the Cell Surface by Heparinase III or by Silencing Syndecan-3 by siRNA Partially Inhibited HIV-1 Transmission by Immature DCs, Neutralizing of Syndecan-3 and DC-SIGN Completely Abrogated Lot de Witte et al PNAS 2007

  8. CD4 Independent HIV Binding Proteins • HIV binds to conventional CD4 receptor • DC-SIGN is CD4 independent HIV receptor on • Dendritic cells • Sperm and vaginal epithelial cells are devoid of CD4 • receptor • 160 kDa human Mannose Receptor (hMR) CD4 independent HIV binding protein on sperm • (Bandivdekar et al 2003, Fanibunda et al 2008) • Astrocytes are also CD4 negative cells in brain • Large nos. of astrocytes in the brain, • HIV1 neuropathogenesis via hMR • Immature monocyte-derived dendritic cells (MDDC), • dermal dendritic cells, macrophages • hepatic endothelial cells

  9. a b c d e hMR Localization on Human spermatozoa uncapacitated capacitated

  10. Expression of hMR in Vaginal Epithelial Cells L S N 201bp hMR product Sample Negative Control (40X) Expression of hMR by RT PCR L : 100 – 1000bp • Human Vaginal Epithelial Cells are CD4-ve • Few Human Cervical Cells CD4 Positive

  11. Expression of hMR and HIV gp 120 Binding to Vaginal Epithelial Cells hMR expression in Vaginal Epithelial Cell Line using hMR FITC labelled IgG HIV gp120 binding to Vaginal Epithelial Cell Line 1 1 2 3 2 3 (40X) 1 1 - Green Channel 2 - Red Channel 3 - Merged Negative Control IgG FITC in Vaginal epithelial Cell Line 2 3

  12. hMR Expression on Vaginal Epithelial Cells of Healthy Indiviual by Flow Cytometry CD206 hMR CCR5 CD4

  13. Peri-ovulatory * Pre-ovulatory Post-ovulatory * * n= 10 n= 5 n= 6 hMR expression Binding of HIV-gp120 n = 3 n = 3 n = 5 Pre- Peri- Post Ovulatory

  14. HIV gp120 Binding Induces MMP-9 but not MMP-2 Production in Vaginal Epithelial Cells MMP-9 MMP-2 Control 0.08nM 0.83nM 83nM MMP9 Induces Breakdown of Extracellular Matrix

  15. Expression of hMR in Vaginal Epithelial Cells of Serodiscordant Females (n=40) % Cells Stained No. Subjects % Subjects +ve hMR 0 14 35 1-2 10 25 3-5 5 12.5 6-8 8 20 9-11 2 5 78 1 General Population 90-95 52

  16. Screening of CCR5 ∆32 Deletionand CXCR4 gene Mutation in Serodiscordants Female Partner (HIV Negative)Male Partner

  17. CD4 Receptor and CCR5 and CXCR4 are the HIV Receptors CD4 Independent HIV Receptor Human Mannose Receptor DC SIGN Gal Cer Heparin Sulfate Syndecan-3 HIV-1 Attachment Receptor on DCs Role of CD4 independent HIV Receptors Need to be investigated

  18. Global HIV Diversity(Mutation,Superinfection and Recombination) • High error rate of Reverse transcriptase • Circulating recombinants forms (CRF) • Unique recombinant forms (URF) • Superinfection and quasispecies formation • HIV rapidly evolves in vivo to escape from cellular and humoral immune responses An obstacle to the development of a“global HIV vaccine”. Verani P et al.,1993

  19. Participant Recruitment Total No : 81 Males : 81 Females : 18 Males Sequences : 26 PBMCs : 11 Sperm Serodiscordants 5 Blood and Semen Females : Total 18 Sequences : 18 PBMCs : 8 Vaginal epithelial cells : 8 Cervical Cells

  20. Viral Load and CD4 Count in Infected Individuals

  21. HIV1 Seropositive Males : Viral Load and NLG Sites ALN: ZDV+3TC+NVP ALE: ZDV+3TC+EFV SLN: D4T+3TC+NVP SLE: D4T+3TC+EFV TND: Target not detected QNS:Quantity not sufficient NA :Sample not available NLG: N-linked glycosylation

  22. Undetectable Viral Load in Blood Plasma but Detectable in Seminal Plasma CD4 Seminal Viral load ART Regimen Copies/ml 298 Detected D4T+3TC+NVP 301 Detected D4T+3TC+NVP 198 Detected D4T+3TC+NVP 178 Detected D4T+3TC+NVP 383 Detected D4T+3TC+NVP Positive for proviral DNA in spermatozoaby PCR D4T - Stavudine 3TC - Lamividine NVP - Navirapin ZDV - Zidovudine EFV - Efavirine

  23. HIV1 Seropositive Females : Viral Load and NLG Sites ALN: ZDV+3TC+NVP ALE: ZDV+3TC+EFV SLN: D4T+3TC+NVP SLE: D4T+3TC+EFV TND:Target not detected QNS: Quantity not sufficient NA : Sample not available NLG : N-linked glycosylation

  24. Primers for PCR Amplification of C2-V3 Region of env Gene of HIV1 First round PCR using primers corresponding to C2-V5 region of HIV-1C env gene Forward Primer ED5 5' ATGGGATCAAAGCCTAAAGCCATGTG 3' Reverse Primer ED 12 5' AGTGCTTCCTGCTGCTCCCAACCCAA 3' Second round PCR using primers corresponding to C2-V3 region of HIV-1C env gene Forward Primer ED31 5' CCTCGCCATTACACAGGCCTGTCCAAAG 3' Reverse Primer ED33 5' TTACAGTAGAAAAATTCCCCTC 3'

  25. C2 –V3 Region of gp120 env Gene of HIV1 C in Male PBMCs C2221 231 241 251 261 271PB1:AGYAILKCNN* KTFS GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB2: AGYVILRCNN* KTFS GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB3: AGYAILKCNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB4:AGYAILKCNN*KTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEIIIRSEN*LTDN PB5: AGFAILKCNN*KTFN*GTGPCNN*VSTVQCTHG IKPVVTTQLL LN*GSLAEEDI IIRSEN*LTNN PB6: AGYAILKCND KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEDI IIRSEN ITNN PB7: AGCAILKCNN* ETFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLVKGEE IIIRSEN*LTD PB8: AGYAIVSVIIRIKR RKDHGRG LYSTLYKMK IKPVVSTQLL LN*GSLAEGDI IVTSEN*LTNN PB9:AGYAILKCNN* KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB10: AGYAILKCNN* KTFN*GTGPCRN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB11: AGYAILKCNN* KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIIPKN*LTNN PB12: AGYAIIKCNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB13: AGYAILKCNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI IIRSEN*LTNN PB14: AGYAILKCNN* KTFN*GTGPCYN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB15: AGYAILKSNN*ETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAVKEIIISSEN*LTNN PB16: AGYAILKSNN*ETFN*GTGPCN N*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN PB17: AGYAILKSNN*ETFN*GSGPCNN*VSTVQCTHG IKPVVSTQLLLN*GSLAEKEI IISSEN*LTNN PB18: AGYAILKCNN*KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI IIRSEN*LTNN PB19:AGYAILKSNN*ETFN*GSGPCN N*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN PB20: AGYAILKCNN*KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI IIRSEN*LTNN PB21: AGYAILKSNN*ETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN PB22: AGYSLLKCNDKTFN GIGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEREI VIRSED LKSN PB23: AGYAILKSNDETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN GRLAEKEI IISSEN*LTNN PB24: AGYAILKSNN*ETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN PB25: AGYAILKSNN*ETFN*GSGPCCN*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN PB26: AGYAILKSNN*ETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN

  26. C2 –V3 Region of gp120 env Gene of HIV1 C in Male PBMCs C2 V3 281 291 296 301 311 321NLG PB1:AKTIIVQLK E SVQIK CTRPND NTRQSIRIG PGQTF FATGE VIGDIRQAHC4 X4 PB2:AKTIIVHLK E SVQIN*CTRPSN*NTRQSIRIG PGQTF YATEE VIGNIRQAYC6 PB3:AKTIIVQLK E SVQIM CTRPSN*NTRQSIRIG PGQTF FATGE VIGDIRQPHC6 PB4: VKTIIVQLN*ESVEIV CTRPNN*NTRKSIRIG PGQTF YAMGD IIGDIRQAHC 7 PB5: AKTIIVHLN E PVEIM CTRPNN*NTRKSIRIG PGQTF YATGE IIGDIRQAYC 6 PB6: AKTIIVHLN*ESVEIN*CTRPNN*NTRKSIRIG PGQAF YATGA IIGNIRQAHC 6 PB7: NAKTIINKF L NFVLK VGQRPG HNTRKSIRMGARAN*LSMQQET--ET-DKH5 X4 PB8: AKTIIVHLN*QSIEIV CTRPNN*NTRKSIRIG PGQTF YATGS IIGDIRQAHC 4 PB9:AKTIIVHLN*QSVEIV CTRPNN*NTRKSIRIG PGQTF YATGD IIGDIRQAHC 7 PB10: AKTIIVHLN*ESVEIV CTRPDN*NTRKSIRIG PGQTF YATGE VIGDIRQAHC 7 PB11: AKIIIVHLN*ESVQIK CTRPNN*NTRKSIRIG PGQTF LCNRRHNRRHKTSTL7 PB12: AKTIIVQLK E SVQIM CTRPS D NTRQSIRIG PGQTF FATGE VIGDIRQAHC 5 X4 PB13: AKTIIVHLN*ESVEIE CIRPNN*NTRKSIRIG PGQTF YATGK IIGDIRQAHC 7 PB14: AKTIIVHLN*ESVEIT CIRPGN*NTRKSIRIG PGQTF YATGA IIGDIRQAHC 7 PB15: AKIIIVRLN*Q SVENV CTRPHN*NTRKSIRIG PGQTF YATGD IIGDIRQAHC7 PB16: AKIIIVRLN*QSVEIV CTRLNN*NTRKSIRIG PGQTF YATGD IIGDIRQAHC7 PB17: AKIIIVRLN*QSVEIV CTRPHY NTRKSIRIG PGQTF YATGD IIGDIRQAHC6 X4 PB18: VKTIIVHLN*ESVEIV CTRPNN*NTRRSIRIG PGQVF YATSD IIGDIRQAHC 7 PB19: AKIIIVRLN*Q SVEIV CTRPNY NTRKSIRRG PGQTF YATGD IIGDIRQAHC 6 X4 PB20: VKTIIVHLN*ESVEIV CTRPNN*NTRRSIRIG PGQVF YATSD IIGDIRQAHC 7 PB21: GKIIIVRLN*QSVEIV CTRPLN*NTRKSIRIG PGQTF YATGD IIGDIRQAHC 7 PB22: VKTIIVHLN*GSVEIE CTRPSN*NTRKSIRIG PGQTF YATGA IIGDIRQAHC 4 PB23: AKIIIVRLN*QSVEIV CTRPHN*NTRKSIRIG PGQTF YATGD IIGDIRQAHC 6 PB24: AKTIIIRLN*QSVEIV CTRPLN*NTRKSIRIG PGQTF YATGD IIGEIRQAHC 7 PB25: AKIIIIRLN*QSVEIV CTRPHY NTRKSIRIG PGQTF YATGH IIGDIRQAHC 6 X4 PB26: AKIIIVRLN*QSVERV CTRPHN*NTRKSIRIG PGQTF YATGD IIGEIRQAHC 7

  27. C2 –V3 Region of gp120 env Gene of HIV1 C in Sperm C2221 231 241 251 261 271 SP1: AGYAIIKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLSEEEI IIRFEN*LTNN SP2:AGYAIIKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN SP3:AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN SP4:AGYAILKCNN* KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN SP5:AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI MIRSEN*LTNN SP15: AGYAILKSNDKTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLLLN*GSLAERVIIISSEN*LTNN SP16: AGYAILKSND KTFN*GTGPCNN*VSTVQCTHGIKPVVSTQLLLN*GSLAEREIIISSEN*LTNN SP17: AGYAILKSNDKTFN*GSGPCNN*VSTVQCTHGIKPVVSTQLLLN*GSLAEREIIISSEN*LTNN SP18: AGYAILKSNDKTFN*GSGPCYN*VSTVQCTHGIKPVVSTQLL LN*GSLAEREIIISSEN*LTNN SP19: LDYAILKCNN* KTFN*GTGPCH N*VSTVQCTHGIKPVVSTQLLLN*GSLAEGEIIIRSEN*LTNN SP20: SWICDSKCNN* KTFN*GTGPCH N*VSTVQCTHGIKPVVSTQLLLN*GSLAEGEIIIRSEN*LTNN C2 V3 281 291 296 301 311 321 NLG SP1:AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGPTFFATGE VIGDIRQAHC5 X4 SP2: AKTIIVQLK E SVQIK CTRPND NTRQSIRIG PGQTFFATGE VIGDIRQAHC5X4 SP3:AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGQTFFATGE VIGDIRQAHC5 X4 SP4: AKTIIVHLN*ESVEIV CIRPSN*NTRKSIRIG PGQAFYATGGIIGNIRQAHC 7 SP5:AKIIIVQLN*ESVQIM CTRPNN*NTRQSIRIG PGQTFYATGE VIGNIRQAHC 7 SP15:AKIIIVRLN*Q SVENV CTRPHN*NTRKSIRIGPGQTFYATGDIIGDIRQAHC6 SP16: AKIIIVRLN*QSVEIV CTRPH N*NTRKSIRIG PGQTFYATGDIIGDIRQAHC 6 SP17: AKIIIVRLN*QSVEIV CTRPH Y NTRKSIRIGPGQTFYATGDIIGDIRQAHC5 X4 SP18: AKIIIVRLN*QSVECV CTRPHY NTRKSIRIGPGQTFYATGDIIGDIRQTHC 5 X4 SP19: VKTIIVHLN*ESVEIV CIRPNN*NTRRSIRIGPGQVFYATSDIIGDIRQAHC 7 SP20: VKTIIVHLN*E SVEIV CTRPNN*NTRKSIRIGPGQVFYANSDIIGNIRQAHC 7

  28. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs and Sperm C2221 231 241 251 261 271 PB1:AGYAILKCNN* KTFS GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN SP1: AGYAIIKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLSEEEI IIRFEN*LTNN PB2: AGYVILRCNN* KTFS GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN SP2:AGYAIIKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB3: AGYAILKCNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN SP3:AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB4:AGYAILKCNN*KTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEIIIRSEN*LTDN SP4:AGYAILKCNN* KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB5: AGFAILKCNN*KTFN*GTGPCNN*VSTVQCTHG IKPVVTTQLL LN*GSLAEEDI IIRSEN*LTNN SP5:AGYAILKCNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI MIRSEN*LTNN 281 291 296 301 311 321NLG PB1:AKTIIVQLK E SVQIK CTRPND NTRQSIRIG PGQTFFATGE VIGDIRQAHC4 R4 SP1:AKTIIVQLK E SVQIM CTRPS D NTRQSIRIG PGPTFFATGE VIGDIRQAHC5 R4 PB2:AKTIIVHLK E SVQIN*CTRPSN*NTRQSIRIG PGQTFYATEE VIGNIRQAYC6 R5 SP2: AKTIIVQLK E SVQIK CTRPN D NTRQSIRIG PGQTFFATGE VIGDIRQAHC5 R4 PB3:AKTIIVQLK E SVQIM CTRPS N*NTRQSIRIG PGQTFFATGE VIGDIRQPHC6 R5 SP3:AKTIIVQLK E SVQIM CTRPS D NTRQSIRIG PGQTFFATGE VIGDIRQAHC5 R4 PB4: VKTIIVQLN*ESVEIV CTRPNN*NTRKSIRIG PGQTFYAMGDIIGDIRQAHC 7 SP4: AKTIIVHLN*ESVEIV CIRPS N*NTRKSIRIG PGQAFYATGGIIGNIRQAHC 7 PB5: AKTIIVHLN E PVEIM CTRPN N*NTRKSIRIG PGQTFYATGE IIGDIRQAYC 6 SP5:AKIIIVQLN*ESVQIM CTRPN N*NTRQSIRIG PGQTFYATGE VIGNIRQAHC 7

  29. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs and Sperm C2221 231 241 251 261 271 PB15: AGYAILKSNN*ETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAVKEIIISSEN*LTNN SP15: AGYAILKSNDKTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLLLN*GSLAERVIIISSEN*LTNN PB16: AGYAILKSNN*ETFN*GTGPCN N*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN SP16: AGYAILKSND KTFN*GTGPCNN*VSTVQCTHGIKPVVSTQLLLN*GSLAEREIIISSEN*LTNN PB17: AGYAILKSNN*ETFN*GSGPCNN*VSTVQCTHG IKPVVSTQLLLN*GSLAEKEI IISSEN*LTNN SP17: AGYAILKSNDKTFN*GSGPCNN*VSTVQCTHGIKPVVSTQLLLN*GSLAEREIIISSEN*LTNN PB18: AGYAILKCNN*KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI IIRSEN*LTNN SP18: AGYAILKSNDKTFN*GSGPCYN*VSTVQCTHGIKPVVSTQLL LN*GSLAEREIIISSEN*LTNN PB19: AGYAILKSNN*ETFN*GSGPCN N*VSTVQCTHG IKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN SP19: LDYAILKCNN* KTFN*GTGPCH N*VSTVQCTHGIKPVVSTQLLLN*GSLAEGEIIIRSEN*LTNN PB20: AGYAILKCNN*KTFN*GTGPCHN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI IIRSEN*LTNN SP20: SWICDSKCNN* KTFN*GTGPCH N*VSTVQCTHGIKPVVSTQLLLN*GSLAEGEIIIRSEN*LTNN

  30. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs and Sperm C2 V3 281 291 296 301 311 321 NLG PB15: AKIIIVRLN*Q SVENV CTRPHN*NTRKSIRIG PGQTFYATGD IIGDIRQAHC7 SP15:AKIIIVRLN*Q SVENV CTRPHN*NTRKSIRIGPGQTFYATGDIIGDIRQAHC6 PB16: AKIIIVRLN*QSVEIV CTRLNN*NTRKSIRIG PGQTFYATGD IIGDIRQAHC7 SP16: AKIIIVRLN*QSVEIV CTRPH N*NTRKSIRIG PGQTFYATGDIIGDIRQAHC 6 PB17: AKIIIVRLN*QSVEIV CTRPHYNTRKSIRIG PGQTFYATGD IIGDIRQAHC6 R4 SP17: AKIIIVRLN*QSVEIV CTRPH YNTRKSIRIGPGQTFYATGDIIGDIRQAHC5 R4 PB18: VKTIIVHLN*ESVEIV CTRPNN*NTRRSIRIG PGQVFYATSD IIGDIRQAHC 7 R5 SP18: AKIIIVRLN*QSVECV CTRPHY NTRKSIRIGPGQTFYATGDIIGDIRQTHC 5 R4 PB19: AKIIIVRLN*Q SVEIV CTRPNY NTRKSIRRG PGQTFYATGD IIGDIRQAHC 6 R4 SP19: VKTIIVHLN*ESVEIV CIRPNN*NTRRSIRIGPGQVFYATSDIIGDIRQAHC 7 R5 PB20: VKTIIVHLN*ESVEIV CTRPNN*NTRRSIRIG PGQVFYATSD IIGDIRQAHC 7 SP20: VKTIIVHLN*E SVEIV CTRPNN*NTRKSIRIGPGQVFYANSDIIGNIRQAHC 7

  31. C2 –V3 Region of gp120 env Gene of HIV1 C in Female PBMCs C2221 231 241 251 261 271 PB1:AGYAILKCNSKTFN*GTGPCNN*VSTVQC THGIKPVVSTQLL LN*GSLAEEKI IIRSEN*LTNN PB2:AGYAILKCND ETFN*GTGPCRN*VSTVQC THG IKPVVSTQLL LN*GSLAKGDI IIRSEN*LTNN PB3:AGYAILKCNN* KTFN*GTGPCQN*VSTVQC THG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB4: AGNAILKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB5: VVR---CSNK* TTFN*GTGPCQ N*VSTVQC THG IKPVVSTQLL LN*GSLAEEDI IIRSEN*LTDN PB6:SQNPIVLRWF CDSK SEIIRL H GTDLHN*VST VQCHMELASG IN*STTVKW-P SKKK-- LARK PB7:AGYAILKCNN* KTFN*GTGPCNN*VSTVQC THG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN PB8:AGYAILKCND KTFN*GTGPCTN*VSTVQC THG IKPVVSTQLL LN*GSLAETEI VIRSEN*ITDN PB9: AGYAILKCND KTFN*GTGPCHN*VSTVQC THG IKPVVSTQLL LN*GSLAEGDIIIKSEN*LTDN PB10: AGYAILKCNN N*KTFDGKGPCN*VSTVQC THG IRPVLSTQLL LN*GSLAEGEIMIRSEN*LTDG PB11:SWLCDSKCLDK TFNGTGPCN N*VSPVQC THG IKPVVSTQLL LN*GSLAKGEIIIRSEN*LTDN PB12:LVMRF-SVII R SSMEQDPAK M SAQYNV HME LSQ-YPLNYC–MAA-QKKR-- -FRSEN*LTNN PB13: AGYAILKCLV R SSMEQDPVQN*VSTVQC THG IKPVVN-QLL LN*GSLAEEDIVIRSEN*FTNN PB14:AGYAILKCLV R SSMEQDPVQ N*VSTVQC THG IKPVVN QLL LN*GSLAEEDIVIRSEN*FTNN PB15:WLCDSQKRKKK TFNGAGPCSN*VSTVQC THG IKPVVSTQLL LN*GSLAAEEIIIRSEN*LTNN PB16: AGFAILKCND E DFSGKGPCKN*VSTVQC THG IRPVVSTQLL LN*GSLAKTGV KVRSEN*ITGN PB17:KATLSWLCDSK VWKNWDRTLQ KCQHST MQG IKPVVSTQLL LN*GSLAEEEIIIRFEN*LTNN PB18:ANGMRF-SVMV RS-RNRTLYK CQHSTM -HG IKPVVSTQLL LN*GSLAEEDIVIRSEN*LTNN

  32. Translated Amino Acid Sequence of C2 –V3 Region of gp120 env Gene of HIV1 C in Female PBMCs C2 V3 281 291 296 301 311 321NLG PB1:AKTIIVHLN*Q SVEIE CTRPGN*NTRKSIRIGPGQTFYATGDIIGNIRQAHC6 PB2:AKTIIVHLN*E SVEIV CTRPNN*NTRKSIRIGPGQSFHATGEIIGKIRHAHC 6 PB3:AKTIIVQLN*E SVEIK CTRPNN*NTRQSIRIGPGQTFYATGEVIGDIRQAHC7 PB4: AKTIIVQLK E SVQIM CTRPN N*NTRQSIRIG PGQTFFATGE VIGDIRQAHC 6 PB5: AKIIIVHLN*E SVKIM CTRPGN*NTRKSIRIG PGQTFFATGD IIGDIRLAHC7 PB6:LKKIPKQYS TS—S-V KLFYK T HIIPEKYKK GPGQHFMQRR HNRNIKQAHC 2 PB7: AKTIIVHLN*E SVKIE CTRPN N*NTRKIIRIG PGQTFYATGD IIGNIRQAYC 7 PB8: VKTIIVQLN*D SVEIV CIRPN N*NTRKSVRIG PGQTFYATGD IIGDIRKAYC 6 PB9: AKTILVHFN*QSVEIV CVRPNN*NTRKSVRIG PGQTFYATGD IIGNIRKAYC 6 PB10:VKTIIVHLN*Q SVKIG WMTPS N*NTRKSVRIG PGQFFYATGE VIGDIKQPRC 6 PB11: AKTVIVQLN*QSVSIT CTRPG N*NTRKSIRIG PGQTFYATGD IIGDIRQAHC 5 PB12:VKTIIVHLK D SVQIN*CTRPN N*NTRQSVRIG PGRTFMQRRD NRRHKTSTL-3 PB13: AKTIIVQLK D SVDIK CTRDP N*NTRKSFFFKRAGRAFYATGDIIGDIRQAH4 PB14:LKPCSRTL- --VNFN*CTRPN N*NTRKSIHIG PGESILCNRRHNRKYKTSTL4 PB15: AKTIIVHLN*ESVEIV CTRPN N*NTRKSFFDERQDNHSMQQEK—-ET-DKHI5 PB16:VKIIIVHLAT PVRIN*CTSPN N*NTRRSIRIG PGQVFFTSEI IGQVRQAHC4 PB17: AKTIIVHLK DPVEIM CTRPN N*NTRKSIRIG PGRAFYAPGD IIGDIRQAYC 3 PB18: AKTIIVQLK DPVDIN*CTRPN N*NTRKSIHIG PGRAFYATGD IIGDIRQAHC 4

  33. Translated Amino Acid Sequence of C2 –V3 Region of gp120 env Gene of HIV1 C Vaginal Epithelial Cells C2 221 231 241 251 261 271 VG1: AGYAILKCNN* KTFS GTGPCQN*VSTVQCTHGIKPVVSTQLLLN*GSLAEEEIMIRSEN*LTNN VG2: AGYEIIKCNN* KTFN*GTGPCQN*VSTVQCTHGIKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN VG3:AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHGIKPVVSTQLL LN*GSLAEEEIIIRSEN*LTNN VG4:AGYAILKCNN* KTLK GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI MIRSEN*LTNN VG5: AGYAIIKSNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSNQLL LN*GSLAEEEI IIRSEN*LTNN VG6:AGYAILKCNN* KTFN*GTGPCQ N*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN VG7: AGYAILKYNN* KTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN VG8: AGYAI-V--- EGGG QDHAIM LAQYKEHKEN -ASIINSITV KW -PSKRDNN YISLKI TNN C2 V3 NLG 281 291 296 301 311 321 VG1:ARIIIVQLN*K SVEIN*CTRPNN*NTRQSIRIGPGQTFYATGEVIGDIRQAHC7 VG2:AKTIIVQLK E SVQIM CTRPND NTRQSIRIG PGQTFFATGE VIGDIRQAHF4 X4 VG3:AKTIIVQLK E SVQIM CTRPS D NTRQSIRIG PGQTFFATGE VIGDIRQAHC5 X4 VG4: AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGQTFFCNRRSNRRHKTSTL4 X4 VG5: AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGPTFFATGE VIGDIRPAPC 5 X4 VG6: AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGQTFFATGE VIGDIRQAHC 5 X4 VG7: AKTIIVHLN*E SVEIV CTRPGN*NTRKSIRRG PGQTFYATGDIIGDIRRAHC 7 VG8: VKTIIVHLN*E SVNIE CVRPN N*NTRQSVRIG PGQTFYAAGE IIGDIIQAYC 2

  34. Translated Amino Acid Sequence of C2 –V3 Region of gp120 env Gene of HIV1 C in Cervical Cell 221 231 241 251 261 271CX1:AGYAILKCNN* KTFS GTGPCQN*VSTVQCTHGIKPVVSTQLLLN*GSLAEEEIIIRSEN*LTNN CX2: AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHGIKPVVSTQLLLN*GSLAEEEIIIRSEN*LTNN CX3:AGYAIIKCNN* KTFS GTGPCQN*VSTVQCTHGIKPVVSTQLLLN*GSLAEEEIIIRSEN*LTNN CX4: AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN CX5: AGYAILKCNN* KTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN CX6: AGYAILKCNN* KTFN*GTGPCQN*VSTVQCTHG IKPVVSTQLL LNG*SLAEEEI MIRSEN*LTNN CX7: LVMRL-RNNN* KTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN CX8: LVMRF-SVID EMFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAKE-I IIRSEN*ITNN C2 V3 NLG 281 291 296 301 311 321CX1:AKTIIVQLK E SVQIM CTRPNN*NTRQSIRIGPGQTFFATGE VIGDIRQPHC5 CX2:AKTIIVQLN*E SVQIK CTRPNN*NTRQSIRIGPGQTFFATGE VIGDIRQAHC7 CX3:AKTIIVQLK E SVQIR CTRPNN*NTRQSIRIGPGQTFFATGE VIGDIRQPHL5 CX4:AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGQTFFATGE VIGDIRQAHC 4 X4 CX5: AKTIIVHLN*E SVKIE CTRPN N*NTRKIIRIG PGQTFYATGDIIGNIRQAYC 7 CX6: AKTIIVQLN*E SVQIM CTRPN N*NTRQSIRIG PGQTFFATGE VIGDIRQAHC 7 CX7: AKTIIVHLN*E SVEIE CTRPN N*NTRKSIRIG PGQTFYATGD IIGDIRQAYC 7 CX8: VKTIIVHLN*E SVNIE CVRPN N*NTRQSVRIG PGQTFYAAGE IIGDIRQAYC 6

  35. C2-V3 Region of gp120 Env Gene of HIV1C in Vaginal and Cervical Cells VG9 LVYAILKCNN KTFNGTGPCH NVSTVQCTHG IKPVVSTQLL LNGSLAEGEI IIRSENLTNN VG10SGYFLLNLLF HLSCGGCPGDNVSTVQCTHG IKPVVSTQLL LNGSLAEGEI MIRSENLTDN VG11TIVAPAGICDSKVDIQWDRTMPCQHSTMYTRNQASGINST TVKW-PSRRRDNNVKSRQNN VG13 AGFAIIKRNNKTFNGTGPCS NVSTVQCTHG IKPVVSTQLL LNGSLAEEGI IIRSENLTNN VG14 AGCAIIKCNNKTFNGTGPCSNVSTVQCTHGIKPVVSTQLL LNGSLAEEGI IIRSENLTNN VG9VKTIIVHLNE SVEIV CTRPN NNTRKSIRIG PGQVFYATGG IIGDIRQAHC5 VG10VKTIIVQLNQSVEIVCTRPNNNTRKSIRIG PGQTFYATGE VIGNIRQAHC 5 VG11AKTIIVHLYESVEIV CTRPS NNTRKSIRIG PGQAFYATGG IIGNIRQAHC 1 VG13 AKIIIVHLDQ PVEIVRTRPN NNTRKSIRIE PGQTFYATGD IIGDIRRAYC5 VG14 AKTIIVHLDQPVEIVRTRPN NNTRKSIRIGPGQPFYATGDIIGDIRRAHR 4 CX9 AGYAILKCND KTFNGTGPCQNVSTVQCTHG IKPVVSTQLL LNGSLAEGDI IIKSQNLTDN CX10 CX11SWLCDSKCNDKTFNGTGPCNNVSTVQCTHG IKPVVSTQLL LNGSLAEEEI IIRSENLTDN CX12AGYAILKCNSKKFNGTGPCTNVSTVQCTHGIKPVVSTQLLLNGSLAEEDIVIRSENLTNN CX13WLCDSKE-MV RKFNGTGPCT NVSTVQCTHG IKPVVSTQLL LNGSLAEEDI VIRSENLTNN CX14AGYAILKCNGKKFKGTGPCTNVSTVQ-THG IKPVVSTQLL LNGSLAEEDI VIRSENLTNN CX9AKTILVHLNQ SVEIV CVRPN NNTRKSVRIG PGQTFYATGD IIGNIRKAYC 5 VX10 SVKIVCTRPNNNTRRSIRIG PGQLFYENNDIIGDIRQAHW 1 CV11GKTVIVQLNKPVNIT CTRPG NNTRKSIRI 4 CX12AKTIIVQLKD PVDINCTRPN NNTRKSIHIG PGRAFYATGD IIGDIRQAHC 6 CX13 AKTIIVQLKD PVDIN CTRPN NNTRKSIHIG PGRAFYATGD IIGDIRQAHC5 CX14AKTIIVQLKD PVDINCTRPN NNTRKSIHIG PGRAFYATGD IIGDIRQAHC 6

  36. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs, Vaginal and Cervical Cells C2221 231 241 251 261 271 PB1:AGYAILKCNSKTFN*GTGPCNN*VSTVQC THGIKPVVSTQLL LN*GSLAEEKI IIRSEN*LTNN VG1: AGYAILKCNN* KTFS GTGPCQN*VSTVQC THGIKPVVSTQLLLN*GSLAEEEIMIRSEN*LTNN CX1:AGYAILKCNN* KTFS GTGPCQN*VSTVQC THGIKPVVSTQLLLN*GSLAEEEIIIRSEN*LTNN PB2:AGYAILKCNDETFN*GTGPCRN*VSTVQC THG IKPVVSTQLL LN*GSLAKGDI IIRSEN*LTNN VG2: AGYEIIKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN CX2: AGYAILKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLLLN*GSLAEEEIIIRSEN*LTNN PB3:AGYAILKCNN* KTFN*GTGPCQN*VSTVQC THG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN VG3:AGYAILKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLL LN*GSLAEEEIIIRSEN*LTNN CX3:AGYAIIKCNN* KTFS GTGPCQN*VSTVQC THGIKPVVSTQLLLN*GSLAEEEIIIRSEN*LTNN PB4: AGNAILKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN VG4:AGYAILKCNN* KTLK GTGPCQ N*VSTVQC THG IKPVVSTQLL LN*GSLAEEEI MIRSEN*LTNN CX4: AGYAILKCNN* KTFN*GTGPCQ N*VSTVQC THG IKPVVSTQLL LN*GSLAEEEI IIRSEN*LTNN PB5: VVR---CSNK* TTFN*GTGPCQN*VSTVQC THG IKPVVSTQLL LN*GSLAEEDI IIRSEN*LTDN VG5: AGYAIIKSNN* KTFN*GTGPCQN*VSTVQC THG IKPVVSNQLL LN*GSLAEEEI IIRSEN*LTNN CX5: AGYAILKCNN* KTFN*GTGPCNN*VSTVQC THG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN PB6:SQNPIVLRWF CDSK SEIIRL H GTDLHN*VST VQCHMELASG IN*STTVKW-P SKKK—- LARK VG6:AGYAILKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLLLN*GSLAEEEI IIRSEN*LTNN CX6: AGYAILKCNN* KTFN*GTGPCQN*VSTVQC THGIKPVVSTQLLLNG*SLAEEEI MIRSEN*LTNN PB7:AGYAILKCNN* KTFN*GTGPCNN*VSTVQC THG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN VG7: AGYAILKYNN* KTFN*GTGPCNN*VSTVQC THG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN CX7: LVMRL-RNNN* KTFN*GTGPCN N*VSTVQC THG IKPVVSTQLL LN*GSLAEGEI ILRSEN*LTNN PB8:AGYAILKCNDKTFN*GTGPCTN*VSTVQC THG IKPVVSTQLL LN*GSLAETEI VIRSEN*ITDN VG8: AGYAI-V--- EGGG QDHAIM L AQYKEH KEN -ASIINSITV KW -PSKRDNN YISLKI TNN CX8: LVMRF-SVID EMFN*GTGPCNN*VSTVQC THG IKPVVSTQLL LN*GSLAKE-I IIRSEN*ITNN

  37. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs, Vaginal and Cervical Cells 281 291 296 301 311 321NLG PB1:AKTIIVHLN*Q SVEIE CTRPGN*NTRKSIRIGPGQTFYATGDIIGNIRQAHC6 VG1:ARIIIVQLN*K SVEIN*CTRPNN*NTRQSIRIGPGQTFYATGEVIGDIRQAHC7 CX1:AKTIIVQLKE SVQIM CTRPNN*NTRQSIRIGPGQTFFATGEVIGDIRQPHC5 PB2:AKTIIVHLN*E SVEIV CTRPNN*NTRKSIRIGPGQSFHATGEIIGKIRHAHC6 R5 VG2:AKTIIVQLKE SVQIM CTRPN D NTRQSIRIG PGQTFFATGE VIGDIRQAHF4 R4 CX2:AKTIIVQLN*E SVQIK CTRPNN*NTRQSIRIGPGQTFFATGE VIGDIRQAHC7 R5 PB3:AKTIIVQLN*E SVEIK CTRPNN*NTRQSIRIGPGQTFYATGEVIGDIRQAHC7 R5 VG3:AKTIIVQLK E SVQIM CTRPS D NTRQSIRIG PGQTFFATGE VIGDIRQAHC5 R4 CX3:AKTIIVQLK E SVQIR CTRPNN*NTRQSIRIGPGQTFFATGE VIGDIRQPHL5 R5 PB4: AKTIIVQLKE SVQIM CTRPN N*NTRQSIRIG PGQTFFATGEVIGDIRQAHC 6 R5 VG4: AKTIIVQLK E SVQIM CTRPS D NTRQSIRIG PGQTFFCNRRSNRRHKTSTL4 R4 CX4:AKTIIVQLK E SVQIM CTRPSD NTRQSIRIG PGQTFFATGEVIGDIRQAHC 4 R4 PB5: AKIIIVHLN*E SVKIM CTRPGN*NTRKSIRIG PGQTFFATGDIIGDIRLAHC7 R5 VG5: AKTIIVQLKE SVQIM CTRPS D NTRQSIRIG PGPTFFATGEVIGDIRPAPC 5 R4 CX5: AKTIIVHLN*E SVKIE CTRPN N*NTRKIIRIG PGQTFYATGDIIGNIRQAYC 7 R5 PB6:LKKIPKQYS TS—S-V KLFYK T HIIPEKYKK GPGQHFMQRR HNRNIKQAHC 2 R4 VG6: AKTIIVQLKE SVQIM CTRPSD NTRQSIRIG PGQTFFATGE VIGDIRQAHC 5 R4 CX6: AKTIIVQLN*E SVQIM CTRPNN*NTRQSIRIG PGQTFFATGE VIGDIRQAHC 7 R5 PB7: AKTIIVHLN*E SVKIE CTRPNN*NTRKIIRIG PGQTFYATGD IIGNIRQAYC 7 VG7: AKTIIVHLN*E SVEIV CTRPGN*NTRKSIRRG PGQTFYATGDIIGDIRRAHC 7 CX7: AKTIIVHLN*E SVEIE CTRPNN*NTRKSIRIG PGQTFYATGD IIGDIRQAYC 7 PB8: VKTIIVQLN*D SVEIV CIRPN N*NTRKSVRIG PGQTFYATGD IIGDIRKAYC 6 VG8: VKTIIVHLN*E SVNIE CVRPN N*NTRQSVRIG PGQTFYAAGE IIGDIIQAYC 2 CX8: VKTIIVHLN*E SVNIE CVRPN N*NTRQSVRIG PGQTFYAAGE IIGDIRQAYC 6

  38. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs, Vaginal and Cervical Cells P9 221 231 241 251 261 271 PBAGYAILKCND KTFNGTGPCHNVSTVQCTHG IKPVVSTQLL LNGSLAEGDI IIKSENLTDN VGLVYAILKCNN KTFNGTGPCH NVSTVQCTHG IKPVVSTQLL LNGSLAEGEI IIRSENLTNN CVAGYAILKCND KTFNGTGPCQNVSTVQCTHG IKPVVSTQLL LNGSLAEGDI IIKSQNLTDN 281 291 301 311 321 NLG PBAKTILVHFNQ SVEIV CVRPN NNTRKSVRIG PGQTFYATGD IIGNIRKAYC 5 VGVKTIIVHLNE SVEIV CTRPN NNTRKSIRIG PGQVFYATGG IIGDIRQAHC5 CVAKTILVHLNQ SVEIV CVRPN NNTRKSVRIG PGQTFYATGD IIGNIRKAYC 5 P10 221 231 241 251 261 271 PBAGYAILKCNNNKTFDGKGPCNVSTVQCTHG IRPVLSTQLL LNGSLAEGEI MIRSENLTDG VGSGYFLLNLLF HLSCGGCPGDNVSTVQCTHG IKPVVSTQLL LNGSLAEGEI MIRSENLTDN 281 291 301 311 321 NLG PB VKTIIVHLNQSVKIGWMTPSNNTRKSVRIG PGQFFYATGEVIGDIKQPRC6 VG VKTIIVQLNQSVEIVCTRPNNNTRKSIRIG PGQTFYATGE VIGNIRQAHC 5 VC SVKIVCTRPNNNTRRSIRIG PGQLFYENNDIIGDIRQAHW 1 P11 221 231 241 251 261 271 PBSWLCDSKCLDKTFNGTGPCNNVSPVQCTHG IKPVVSTQLL LNGSLAKGEI IIRSENLTDN VGTIVAPAGICDSKVDIQWDRTMPCQHSTMYTRNQASGINST TVKW-PSRRRDNNVKSRQNN CVSWLCDSKCNDKTFNGTGPCNNVSTVQCTHG IKPVVSTQLL LNGSLAEEEI IIRSENLTDN 281 291 301 311 321 NLG PBAKTVIVQLNQSVSIT CTRPG NNTRKSIRIG PGQTFYATGD IIGDIRQAHC 5 VGAKTIIVHLYESVEIV CTRPS NNTRKSIRIG PGQAFYATGG IIGNIRQAHC 1 CV GKTVIVQLNKPVNIT CTRPG NNTRKSIRI 4

  39. C2-V3 Region of gp120 Env Gene of HIV1C in PBMCs, Vaginal and Cervical Cells P12221 231 241 251 261 271 PB LVMRF-SVIIRSSMEQDPAKMSAQYNVHMELSQ-YPLNYC–MAA-QKKR--FRSENLTNN CV AGYAILKCNSKKFNGTGPCTNVSTVQCTHGIKPVVSTQLLLNGSLAEEDIVIRSENLTNN 281 291 301 311 321 NLG PB VKTIIVHLKD SVQINCTRPN NNTRQSVRIG PGRTFMQRRD NRRHKTSTLP3 CV AKTIIVQLKD PVDINCTRPN NNTRKSIHIG PGRAFYATGD IIGDIRQAHC 6 P13221 231 241 251 261 271 PB AGFAIIKCNNKTFNGTGPCS NVSTVQCTHG IKPVVSTQLL LNGSLAEEGI IIRSENLTNN VG AGFAIIKRNNKTFNGTGPCS NVSTVQCTHG IKPVVSTQLL LNGSLAEEGI IIRSENLTNN CV WLCDSKE-MV RKFNGTGPCT NVSTVQCTHG IKPVVSTQLL LNGSLAEEDI VIRSENLTNN 281 291 301 311 321 NLG PB AKIIIVHLDQ PVEIVRTRPN NNTRKSIRIG PGQTFYATGD IIGDIRRAYC5 VG AKIIIVHLDQ PVEIVRTRPN NNTRKSIRIE PGQTFYATGD IIGDIRRAYC5 VC AKTIIVQLKD PVDIN CTRPN NNTRKSIHIG PGRAFYATGD IIGDIRQAHC5 P14221 231 241 251 261 271 PB PAGYAILKCL VRSSMEQDPV QNVSTVQCTH GIKPVVNQLL LNGSLAEEDI VIRSENFTNN VG AGCAIIKCNNKTFNGTGPCSNVSTVQCTHGIKPVVSTQLL LNGSLAEEGI IIRSENLTNN CV AGYAILKCNGKKFKGTGPCTNVSTVQ-THG IKPVVSTQLL LNGSLAEEDI VIRSENLTNN 281 291 301 311 321 NLG PB AKTIIVQLKDSVDIKCTRPN NNTRKSFFFKRAGRAFYATG DIIGDIRQAH3 VG AKTIIVHLDQPVEIVRTRPN NNTRKSIRIGPGQPFYATGDIIGDIRRAHR 4 CV AKTIIVQLKDPVDINCTRPN NNTRKSIHIGPGRAFYATGDIIGDIRQAHC4

  40. C2-V3 Region of gp120 Env Gene of HIV 1 C in PBMCs and Spermatozoa of Serodiscordants 221 231 241 251 261 271 PB1:AGYAILKSNN* ETFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAVKEIIISSEN*LTNN SP1:AGYAILKSNN*KTFN*GTGPCN N*VSTVQCTHGIKPVVSTQLL LN*GSLAERVIIISSEN*LTNN 281 291 301 311 321 PB1:AKIIIVRLN*QSVENV CTRPHN*NTRKSIRIGPGQTFYATGD IIGDIRQAHCNLG 7 SP1:AKIIIVRLN*QSVENV CTRPHN*NTRKSIRIGPGQTFYATGD IIGDIRQAHCNLG 7 221 231 241 251 261 271 PB2:AGYAILKSNN* ETFN*GTGPCN N*VSTVQCTHGIKPVVSTQLL LN*GSLAEKEIIISSEN*LTNN SP2:AGYAILKSNDKTFN*GTGPCNN*VSTVQCTHG IKPVVSTQLL LN*GSLAEREIIISSEN*LTNN 281 291 300 PB2:AKIIIVRLN*QSVEIV CTRLNN*NTRKSIRIGPGQTFYATGDIIGDIRQAHCNLG 7 SP2:AKIIIVRLN*QSVEIV CTRPHN*NTRKSIRIGPGQTFYATGDIIGDIRQAHCNLG 6 221 231 241 251 261 PB3:AGYAILKSNN* ETFN*GSGPCNN*VSTVQCTHGIKPVVSTQLL LN*GSLAEKEIIISSEN*LTNN SP3:AGYAILKSNN* KTFN*GTGPCNN*VSTVQCTHGIKPVVSTQLL LN*GSLAEREIIISSEN*LTNN 281 291 301 311 321 PB3:AKIIIVRLN*QSVEIV CTRPH N*NTRKSIRIGPGQTFYATGD IIGDIRQAHCNLG 7 SP3:AKIIIVRLN*QSVEIV CTRPHN*NTRKSIRIGPGQTFYATGD IIGDIRQAHCNLG 7

  41. C2-V3 Region of gp120 Env Gene of HIV 1 in PBMCs and Spermatozoa of Serodiscordants 221 231 241 251 261 271 PB4:AGYAILKSNN* ETFN*GSGPCNN*VSTVQCTHGIKPVVSTQLL LN*GSLAEKEI IISSEN*LTNN SP4:AGYAILKSNN* KTFN*GSGPCYN*VSTVQCTHGIKPVVSTQLL LN*GSLAEREI IISSEN*LTNN 281 291 301 311 321 PB4:AKIIIVRLN*QSVEIV CTRPN N*NTRKSIRRGPGQTFYATGDIIGDIRQAHCNLG 7 SP4:AKIIIVRLN*QSVECV CTRPH N*NTRKSIRIGPGQTFYATGDIIGDIRQTHCNLG 6 221 231 241 251 261 271 PB5:AGYAILKCNN*KTFN*GTGPCHN*VSTVQCTHGIKPVVSTQLL LN*GSLAEGEIIIRSEN*LTNN SP5:LDYAILKCNN*KTFN*GTGPCHN*VSTVQCTHGIKPVVSTQLLLN*GSLAEGEIIIRSEN*LTNN 281 291 301 311 321 PB5:VKTIIVHLN*ESVEIV CTRPN N*NTRRSIRIG PGQVFYATSDIIGDIRQAHCNLG 7 SP5:VKTIIVHLN*ESVEIV CIRPNN*NTRRSIRIG PGQVFYATSDIIGDIRQAHCNLG 7

  42. Major Conserved Regions in C2-V3 Region of HIV 1 C in PBMCs C2 Region 240 to 263 PBMCs of Twelve Males C2 Region 244 to 263 Four out of Ten Females V3 Region 306 to 313 PBMCs of Twelve Males Five out of Ten Females V3 Region 315 to 330 PBMCs of Twelve Males Three out of Ten Females

  43. Genotypic Characterization of HIV1 C gp41 by Next Generation Sequencing (NGS) Whole Blood PBMCs Plasma Pro Viral DNA Viral RNA cDNA gp41 MPER region nested PCR Next generation sequencing Quasi species reconstruction QuRe Sample specific Quasi species

  44. Characterization of HIV1 gp41 by NGS (QuRe) QuRe

  45. Mutations at Different Positions

  46. Variants at Different Positions

  47. Gp 36 and Gp120 of HIV -2 Sequence Homology gp36 gp120 Sr no. Participants 1 X1HIV-2 isolate CRIK 147 from India 87% HIV1 isolate 63% 2 X2 HIV-2 isolate India env gene, 91% HIV-1 isolate 77% 3 X3 HIV-2 isolate CRIK 147 from India, 87% HIV-1 isolate 78% 4 X4 HIV-2 isolate India env gene, 89% HIV-1 isolate 72% 5 X5 HIV-2 isolate CRIK 147 from India, 88% HIV-1 isolate 83% 6 X6 HIV-2 isolate CRIK 147 from India, 92% HIV-1 isolate 92% 7 X7 HIV-2 isolate India env gene, 88% HIV-1 isolate 75% 8 X8 HIV-2 isolate India env gene, 90% HIV-1 isolate 76% 9 X9 HIV-2 isolate CRIK 147 from India, 92% HIV-1 isolate 84% 10 X10 HIV-2 isolate CRIK 147 from India, 88% HIV-1 isolate 75% 11 X11 HIV-2 isolate CRIK 147 from India, 90% HIV-1 isolate 63%

  48. Long Term Non Progressors (LTNPs) Sr. nos. Age ( Sex) Duration of CD4 count Viral load P24 assay HIV infection 1] 34 (F) 7yrs 568 13197 20.08 2] 34 (F) 9yrs 715 668998 20.40 3] 53 (F) 8yrs 762 32694 18.65 4] 46 (M) 5yrs 651 98036 21.25 5] 29 (F) 11yrs 441 67441 19.09 6] 34 (F) 10yrs 717 74765 18.41 7] 33 (F) 6yrs 448 546103 19.90 8] 28 (M) 5yrs 443 1179 18.75 9] 17 (F) 6yrs 451 250699 25.86 10] 44 (F) 6yrs 574 411977 19.67

  49. Summary • Presence of distinct viral variants in PBMCs and Urogenital cells • and Secretions of the same individual play important role in • transmission and pathogenesis of HIV • Presence of virus in the sperm, vaginal epithelial and cervical cells • suggests its involvement in sexual transmission of HIV • ART may reduce viral load in blood but may not in urogenital secretions • of the same individual therefore may not always reduce the risk of sexual • transmission of HIV • Binding of HIV to hMR on vaginal epithelial cells increases expression of • Matrix Metallo-proteinases-9 (MMP9) which may further induce the cellular • permeability • Lower number of hMR expressing vaginal epithelial cells of the female • partners prevented male to female sexual transmission of HIV • hMR: CD4 independent HIV receptor responsible for sexual transmission • of HIV • HIV variants may have different affinity for CD4 receptor and hMR and • other receptors which may influence HIV transmission, response to ART • and progression to AIDS

  50. Thank You

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