Ranajit Pal

Advanced BioScience Laboratories Inc., Роквилл, Maryland, United States

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Publications (100)431.15 Total impact

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    ABSTRACT: The FLSC vaccine is fusion protein consisting of a modified full length gp120 protein, derived from HIVBaL, and the first 2 domains (D1D2) of human CD4, genetically fused via a 20 amino acid linker. Experiments in Rhesus macaques demonstrated protection using HEK 293 cell produced rhesus FLSC. A master cell bank expressing human FLSC was prepared from G293H, a derivative of HEK-293 cell that grows in suspension, using the GPEx retrovector transduction system. Tumorgenicity studies performed in athymic nude mice demonstrated that the FLSC MCB generated tumors at the same rate as the HEK-293 cell line available from ATCC. Bioreactor production rates were consistent from the 2 L to the 200 L scale, yielding approximately 1 g/L after downstream purification. Drug substance was predominately monomeric, and expressed the expected CD4 induced structure. A drug product formulation with aluminum phosphate was developed. Potency studies demonstrated a significant relationship between the dose of the FLSC/Alum (IHV01) and the induction of the desired CD4i directed immune response. Immunogenicity studies performed in rhesus macaques showed that the FLSC/Alum formulation induced antibodies directed to CD4i epitopes and mediated ADCC activity while T cell responses were modest. A repeat-dose (N+1) toxicity study in rabbits demonstrated that the majority of the effects observed were attributed to general inflammation occurring with intramuscular vaccine administration and/or an active immune response towards the antigen. An immunotoxicity study in cynomolgus monkeys showed no deleterious autoimmune effects directed to CD4. A phase 1 clinical trial with IHV01 is anticipated to start in September, 2015. Copyright
    No preview · Article · Jan 2016 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    ABSTRACT: An effective vaccine for HIV still remains elusive. Our previous studies showed that a protein-based FLSC (a genetic fusion of gp120 and CD4) protected macaques against repeat rectal challenges with SHIV162P3 but efficacy was low. We hypothesized that DNA would provide a superior prime to a subunit boost than subunit alone and that the adjuvants IL-12 and/or LTA1 would improve priming. FLSC delivery regimens were evaluated in a challenge model using SIVsmE543 antigens and a cross clade challenge with SIVmac251. Eight macaques/group were immunized with DNA expressing a gag/pol fusion and FLSC with/without LTA1, IL-12 or LTA1+IL-12 by electroporation on weeks 0, 4 and 8. Booster immunizations of 300 [mu]g of FLSCsmCG7V/alum were given at week 42. Two weeks later, animals were weekly challenged rectally 10 times with SIVmac251. Immune measures were compared using T tests (parametric) or Mann-Whitney Rank Sum Tests (non-parametric). Infection rates were compared using Log Rank tests. The DNA/IL-12 prime regimen provided 75% efficacy compared to the same regimen without IL-12. Efficacy did not correlate with antibody binding or neutralizing titers to Tier 1 SIVmac251 whereas ADCC titers and antibody binding ratios of FcgR3/FcgR1 correlated with protection, provided T cell responses were low. The DNA/LTA1/IL-12 regimen generated substantially superior CMI responses, but showed no protection. These results suggest that vaccines generating ADCC capable antibodies with high FcgR3/FcgR1 ratios that evoke minimal T cell responses may be the most protective. Copyright
    No preview · Article · Jan 2016 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    ABSTRACT: Full length single chain (FLSC) is a novel HIV vaccine that presents conserved CD4i epitopes on HIV envelope. The constrained structure is achieved with single chain complexes of gp120 and CD4 fragments. FLSC elicits cross-reactive antibodies and heterologous protection against SHIV/SIV in three independent low-dose rectal challenge studies in rhesus macaques and is being developed as a subunit vaccine for clinical evaluation. We performed preclinical immunotoxicology studies to assess potential safety issues specifically derived from a deleterious autoimmune response to CD4. Two studies were performed in cynomolgus macaques. The presence of deleterious antibody responses to CD4 were assessed by ELISA, CD4+ cell staining, as well as impact to a mixed lymphocyte reaction (MLR). CD4+ T cell loss and impact to the immune response to KLH were also assessed. In study 1, we verified that depletion of CD4+ cells impacted the induction of primary and secondary KLH-specific IgG and IgM antibody responses, justifying the use of the antibody response to KLH as an indicator of an autoimmune response to CD4. In study 2, immunization with multiple high doses of FLSC did not induce an autoimmune response to CD4 that had any deleterious effects in any assays that were employed. Little to no impact was seen on CD4 binding/function by flow cytometry and MLRs. Therefore, FLSC did not induce any deleterious autoimmune responses to CD4 and continues to be a promising vaccine candidate for evaluation in a Phase I clinical trial. Supported by: BMGF OPP1017606. Copyright
    No preview · Article · Jan 2016 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    ABSTRACT: Objective: Few studies have examined the eclipse time of SIV/HIV infection via the anal route. We aimed to measure the eclipse time after SIVmac251 intra-rectal inoculation, and to investigate the factor(s) associated with early dissemination. Design: 40 macaques were intra-rectally challenged with SIVmac251 three times at 2 -week intervals. Methods: Plasma viral RNA was monitored at 4, 7, 11, 14, 21 and 28 days after infection. Rectal/vaginal tissues were obtained and tissue viral loads (VLs) were measured at day 14 post-infection. Results: 26 out of 40 macaques (65%) had first detectable viral RNAs in the plasma at day 7 after the challenge that led to productive infection. Strikingly, 6 animals (15%) had detectable viral RNA in the plasma as early as at day 4. The Ki67 viral target CD4 T cells in the colorectal tissues were significantly higher in the early or middle-transmitter groups than those in the late-transmitter group. The rectal VL did not correlate with plasma VL at 14-day post-inoculation, but did positively correlate with plasma VLs at days 21 and 28 post-infection. Conclusions: The median eclipse time after intrarectal challenge was 7 days, with a few early transmitters at 4 days. More rapid viral dissemination was associated with a high frequency of colorectal Ki67CCR5CD4T cells, which fuel the local viral replication. Furthermore, local viral replication in the colorectal tissue during the early stage might affect the plasma VL in a delayed manner. Therefore, to reduce/limit these target cells at the portal of viral entry is essential.
    No preview · Article · Nov 2015 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    ABSTRACT: Background Insights into the host factors that contribute to an effective antiviral immune response may be obtained by examining global gene expression in simian-human immunodeficiency virus (SHIV)-infected non-human primates that exhibit different virological outcomes.Methods Six chronically SHIV-infected macaques were rectally challenged with SIVmac251. Viral RNA and proviral DNA load in blood were measured. Gene expression profiles in CD4+ T cells were examined and compared between animals with different levels of infection following challenge.Results and Conclusions Viral RNA was markedly controlled in four challenged animals, whereas two animals had persistent high viremia. Analysis of the gene expression profiles at early infection revealed gene expression signatures between protectors and non-protectors and identified potential protective biomarkers. Pathway analyses revealed that IFN pathway genes are down-regulated in protectors compared to unprotectors. This study suggests that high levels of expression of type 1 IFN-related genes may paradoxically promote virus replication.
    No preview · Article · Aug 2015 · Journal of Medical Primatology
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    ABSTRACT: Many viral infections, including HIV, exhibit sex-based pathogenic differences. However, few studies have examined vaccine-related sex differences. We compared immunogenicity and protective efficacy of monomeric SIV gp120 with oligomeric SIV gp140 in a pre-clinical rhesus macaque study and explored a subsequent sex bias in vaccine outcome. Each immunization group (16 females, 8 males) was primed twice mucosally with replication-competent Ad-recombinants encoding SIVsmH4env/rev, SIV239gag and SIV239nefΔ1-13 and boosted twice intramuscularly with SIVmac239 monomeric gp120 or oligomeric gp140 in MF59 adjuvant. Controls (7 females, 5 males) received empty Ad and MF59. Up to 9 weekly intrarectal challenges with low-dose SIVmac251 were administered until macaques became infected. We assessed vaccine-induced binding, neutralizing, and non-neutralizing antibodies, Env-specific memory B cells and plasmablasts/plasma cells (PB/PC) in bone marrow and rectal tissue, mucosal Env-specific antibodies, and Env-specific T-cells. Post-challenge, only one macaque (gp140-immunized) remained uninfected. However, SIV acquisition was significantly delayed in vaccinated females but not males, correlated with Env-specific IgA in rectal secretions, rectal Env-specific memory B cells, and PC in rectal tissue. These results extend previous correlations of mucosal antibodies and memory B cells with protective efficacy. The gp140 regimen was more immunogenic, stimulating elevated gp140 and cyclic V2 binding antibodies, ADCC and ADCP activities, bone marrow Env-specific PB/PC, and rectal gp140-specific IgG. However, immunization with gp120, the form of envelope immunogen used in RV144, the only vaccine trial to show some efficacy, provided more significant acquisition delay. Further over 40 weeks of follow-up, no gp120 immunized macaques met euthanasia criteria in contrast to 7 gp140-immunized and 2 control animals. Although males had higher binding antibodies than females, ADCC and ADCP activities were similar. The complex challenge outcomes may reflect differences in IgG subtypes, Fc glycosylation, Fc-R polymorphisms, and/or the microbiome, key areas for future studies. This first demonstration of a sex-difference in SIV vaccine-induced protection emphasizes the need for sex-balancing in vaccine trials. Our results highlight the importance of mucosal immunity and memory B cells at the SIV exposure site for protection.
    Full-text · Article · Aug 2015 · PLoS Pathogens
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    ABSTRACT: Recent preclinical studies have demonstrated the use of properly folded trimeric HIV-1 envelope proteins as immunogen for eliciting protecting immune response in macaques. Trimeric gp145 protein of Indian clade C HIV-1 (93IN101) was characterized for antigenicity by evaluating its binding to sCD4, and several monoclonal antibodies to HIV-1 by bio-layer interferometry. Ten macaques were immunized four times with purified gp145 in adjuplex adjuvant, and serum antibodies were characterized for binding to gp145 and neutralization. Immunized macaques were subjected to weekly low-dose vaginal challenge with SHIV1157-ipEL-p for 8 weeks. Env protein elicited strong antibody response in macaques. Following challenge, seven of ten immunized macaques resisted challenge, while six of eight control animals were infected. Env proteins from a clade C Indian isolate can elicit protective immune response and therefore may be a candidate for inclusion in a multiclade-based HIV-1 vaccine. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
    No preview · Article · Jun 2015 · Journal of Medical Primatology
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    ABSTRACT: To evaluate antibody specificities induced by SIV versus HIV-1 envelope antigens in non-human primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that overall HIV-1 Env IgG responses were dominated by V3, notably with the exception of responses to the RV144 vaccine strain immunogen (A244 Env) that was dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of vaccine regimens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    No preview · Article · May 2015 · Journal of Virology
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    ABSTRACT: The Thai RV144 trial, using the canarypox ALVAC-HIV prime and AIDSVax B/E gp120 boost, has been the only trial so far to demonstrate a modest, yet significant 31.2% protection from HIV-1 infection in a cohort of 16,402 patients. However, given the limited efficacy of the vaccination approach, other poxviral vectors with higher immunogenicity profiles have emerged as candidate HIV-1 vaccines for humans. Among them, the vaccinia-derived NYVAC vector has shown safety and elevated immunogenicity in phase 1 and 2 human trials. To directly compare the two vectors, we assessed the relative efficacy and immunogenicity of ALVAC- or NYVAC- based SIVmac251 vaccine approaches versus placebo controls in a cohort of 65 Rhesus macaques (Macaca mulatta) of Chinese origins. Both strategies induced high titer serum anti−gp120 antibodies that had similar epitope specificity in both groups including the V1/V2 region, as tested by binding assays on overlapping gp120 peptides. In contrast, the NYVAC-based approach elicited significantly higher T-cell responses than the ALVAC-based as evaluated by IFN-γ ELISpot against SIV/gp120. In flow-cytometry assays we found that NYVAC, but not ALVAC, incremented the overall percentage of circulating blood plasmablasts after vaccination, with a decrease in the percentage of α4β7+ and an increment of CXCR3+ plasmablasts in the same group, indicating that the NYVAC-based vaccination directed the plasmablasts to the sites of inflammation rather than the mucosal sites. However, none of the variations observed in the NYVAC-vaccinated group was found to correlate to acquisition or viral load. In contrast, we found that the overall plasmablasts levels before vaccination correlated negatively to protection in the ALVAC-based approach. Thus, despite the high T-cell responses elicited by the NYVAC-based vaccine, only the ALVAC-based approach was able to significantly decrease the risk of infection following repeated low-dose intravaginal SIVmac251 challenge. Our results suggest that substituting the ALVAC vector with NYVAC does not increase the efficacy of an RV144-based vaccine approach.
    No preview · Conference Paper · May 2015
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    ABSTRACT: Off-therapy control of viremia by HIV-infected individuals has been associated with two likely players: a restricted viral reservoir and an efficient cell-mediated immune response. We previously showed that a combination of highly suppressive antiretroviral therapy and two experimental drugs, i.e., auranofin and buthionine sulfoximine, was able to reduce the viral reservoir, elicit efficient cell-mediated antiviral responses, and induce intermittent posttherapy viral load control in chronically SIVmac251- infected macaques. We here show that the macaques that had received this drug combination and then stopped antiretroviral therapy were also able to maintain low numbers of activated CD4+ T cells at viral rebound. Moreover, these macaques consistently displayed low-level simian immunodeficiency virus (SIV) diversity, which was in line with the strong and broadly reactive cell-mediated immune responses against conserved Gag antigens. Extended follow-up showed that the two macaques that had received the complete drug combination remained healthy and did not develop AIDS in 2 years of follow-up after therapy suspension. This disease-free survival is longer than twice the average time of progression to AIDS in SIVmac251-infected rhesus macaques. These results suggest that limited numbers of activated T cells at viral rebound and subsequent development of broadly reactive cell-mediated responses may be interrelated in reducing the viral reservoir.
    No preview · Article · May 2015 · Journal of Virology
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    ABSTRACT: A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.
    Full-text · Article · Mar 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Simian-human immunodeficiency viruses (SHIVs) that mirror natural transmitted/founder (T/F) viruses in man are needed for evaluation of HIV-1 vaccine candidates in nonhuman primates. Currently available SHIVs contain HIV-1 env genes from chronically-infected individuals and do not reflect the characteristics of biologically relevant HIV-1 strains that mediate human transmission. We chose to develop clade C SHIVs, as clade C is the major infecting subtype of HIV-1 in the world. We constructed 10 clade C SHIVs expressing Env proteins from T/F viruses. Three of these ten clade C SHIVs (SHIV KB9 C3, SHIV KB9 C4 and SHIV KB9 C5) replicated in naïve rhesus monkeys. These three SHIVs are mucosally transmissible and are neutralized by sCD4 and several HIV-1 broadly neutralizing antibodies. However, like natural T/F viruses, they exhibit low Env reactivity and a Tier 2 neutralization sensitivity. Of note, none of the clade C T/F SHIVs elicited detectable autologous neutralizing antibodies in the infected monkeys, even though antibodies that neutralized a heterologous Tier 1 HIV-1 were generated. Challenge with these three new clade C SHIVs will provide biologically relevant tests for vaccine protection in rhesus macaques.
    Full-text · Article · Feb 2015 · Virology
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    ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype and sensitivity to known human broadly neutralizing monoclonal antibodies. Since many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope specific antibody PG9 and to CD4 induced epitope specific antibody 17b. Sera from rabbits immunized with the gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3 specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates, in future cocktail of HIV-1 vaccines. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Feb 2015 · Journal of Biological Chemistry
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    ABSTRACT: NK cells are essential components of the immune system, and due to their rapid response potential, can have a great impact during early anti-viral immune responses. We have previously shown that IL-2-dependent NK and CD4+ T cell co-operative immune responses exist in long-term SIV-infected controlling macaques and can be rescued in SIV-infected non-controlling macaques by a short course of antiretroviral therapy (ART). Given that co-operative responses may play an important role in disease prevention and therapeutic treatment, in the present study we sought to determine if these responses can be enhanced in chronically SIV-infected macaques by vaccination with a single-dose of envelope protein given during ART. To this end, we treated 14 chronically SIV-infected macaques with ART for 11 weeks and gave 10 of these macaques a single intramuscular dose of SIV gp120 at week 9 of treatment. ART significantly decreased plasma and mucosal viral loads, increased the numbers of circulating CD4+ T cells in all macaques, and increased T cell-dependent envelope- and gag-specific IFN-gamma and TNF-alpha production by circulatory CD56+ NK cells. The therapeutic envelope immunization resulted in higher envelope-specific responses compared to those in macaques that received ART only. Functional T cell responses restored by ART and therapeutic Env immunization were correlated with transiently reduced plasma viremia levels following ART release. Collectively our results indicate that SIV-specific T cell-dependent NK cell responses can be efficiently rescued by ART in chronically SIV-infected macaques and that therapeutic immunization may be beneficial in previously vaccinated individuals.This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2015 · Immunology
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    ABSTRACT: Following the RV144 clinical trial, the vaccine field has refocused on gp120 as a booster immunogen. To address if oligomeric gp140 would contribute similarly to vaccine efficacy we compared monomeric SIV gp120 vs oligomeric SIV gp140 boosting. Rhesus macaques were primed mucosally (wks 0 and 12) with replication-competent Ad5hr-SIVsmH4env/rev, Ad5hr-SIV239gag, and Ad5hr-SIV239nefdelta1-13, and boosted (wks 39 and 51) with SIV gp120 or gp140 in MF59. A repeated low-dose rectal SIVmac251 challenge (begun at wk 59) infected all but 1 macaque in the gp140 group. Acute viremia was reduced significantly in both immunization groups. Env-specific mucosal IgA correlated with reduced acute viremia in the gp140 group (P = 0.014) and with chronic viremia in both groups combined (P = 0.016). Unexpectedly, female but not male macaques exhibited significantly delayed SIV acquisition (P = 0.0071). The gp140 regimen was more immunogenic overall than gp120, but no differences in antibody activities including neutralization, ADCC and ADCP or in bone marrow levels of memory B cells, plasma blasts (PB) or plasma cells (PC) secreting Env-specific IgA were seen between sexes. Among differences, males exhibited higher binding antibody titers and Env-specific IgG memory B cell and PB/PC levels. Delayed SIV acquisition in vaccinated females correlated significantly with Env-specific memory B cell and PC levels in rectal tissue, 2 wks post-infection (P = 0.0011 and P < 0.0001, respectively). We have previously correlated vaccine-induced Env-specific secretory IgA with delayed SIV acquisition. Together, the data illustrate the importance of local B cell maturation for vaccine efficacy, and suggest that mucosal IgA influences the challenge outcome.
    No preview · Article · Nov 2014 · JAIDS Journal of Acquired Immune Deficiency Syndromes
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    ABSTRACT: High-level T cell expression of PD-1 during SIV infection is correlated with impaired proliferation and function. We evaluated the phenotype and distribution of T cells and Tregs during antiretroviral therapy plus PD-1 modulation (using a B7-DC-Ig fusion protein) and post-ART. Chronically SIV-infected rhesus macaques received: 11 weeks of ART (Group A); 11 weeks of ART plus B7-DC-Ig (Group B); 11 weeks of ART plus B7-DC-Ig, then 12 weeks of B7-DC-Ig alone (Group C). Continuous B7-DC-Ig treatment (Group C) decreased rebound viremia post-ART compared to pre-ART levels, associated with decreased PD-1(hi) expressing T cells and Tregs in PBMCs, and PD-1(hi) Tregs in lymph nodes. It transiently decreased expression of Ki67 and α4β7 in PBMC CD4(+) and CD8(+) Tregs for up to 8 weeks post-ART and maintained Ag-specific T-cell responses at low levels. Continued immune modulation targeting PD-1(hi) cells during and post-ART helps maintain lower viremia, keeps a favorable T cell/Treg repertoire and modulates antigen-specific responses.
    Full-text · Article · Dec 2013 · Virology
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    Full-text · Conference Paper · Nov 2013
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    ABSTRACT: Previously, priming rhesus macaques with Adenovirus type 5 host range mutant-recombinants encoding Tat and Env and boosting with Tat and Env protein in MPL-SE controlled chronic viremia by 4 logs following homologous intravenous SHIV89.6P challenge. Here we evaluated Tat, Env, and Tat/Env regimens for immunogenicity and protective efficacy using clade C Env, alum adjuvant, and a heterologous intrarectal SHIV1157ipd3N4 challenge. Despite induction of strong cellular and humoral immunity, Tat/Env group T and B-cell memory responses were not significantly enhanced over Tat- or Env-only groups. Lack of viremia control post-challenge was attributed to lower avidity Env antibodies and no anamnestic ADCC response or SHIV1157ipd3N4 neutralizing antibody development post-challenge. Poor biologic activity of the Tat immunogen may have impaired Tat immunity. In the absence of sterilizing immunity, strong anamnestic responses to heterologous virus can help control viremia. Both antibody breadth and optimal adjuvanticity are needed to elicit high-quality antibody for protective efficacy.
    Full-text · Article · Mar 2013 · Virology
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    ABSTRACT: SIV stocks for in vivo non-human primate models of AIDS are typically generated by transfection of 293T cells with molecularly-cloned viral genomes or by expansion in productively infected T cells. Although stocks are titered for infectivity in vitro prior to in vivo inoculation, virus production methods may differentially affect stock features that are not routinely analyzed but may impact in vivo infectivity, mucosal transmissibility, and early infection events. We performed a detailed analysis of nine SIV stocks, comprising five infection-derived SIVmac251 viral swarm stocks and paired infection- and transfection/293T-derived stocks of both SIVmac239 and SIVmac766. Representative stocks were evaluated for: 1) virus content, 2) infectious titer, 3) sequence diversity/polymorphism frequency by single genome amplification and 454 pyrosequencing, 4) virion-associated Env content, and 5) cytokine/chemokine content by 36-plex Luminex analysis. Regardless of production method, all stocks had comparable particle-to-infectivity ratios, with the transfection/293T stocks possessing the highest overall virus content and infectivity titers despite containing markedly lower levels of virion-associated Env than infection-derived viruses. Transfection/293T stocks also contained fewer and lower levels of cytokines/chemokines than infection-derived stocks, which had elevated levels of multiple analytes, with substantial variability among stocks. Sequencing of the infection-derived SIVmac251 stocks revealed variable levels of viral diversity between stocks, with evidence of stock-specific selection and expansion of unique viral lineages. These analyses suggest that there may be underappreciated features of SIV in vivo challenge stocks with the potential to impact early infection events, which may merit consideration when selecting virus stocks for in vivo studies.
    Full-text · Article · Feb 2013 · Journal of Virology
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    ABSTRACT: The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8+ T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIVmac251 that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4+ and CD8+ T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIVmac251 acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIVmac251-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIVmac251 infectivity in cells that express high levels of α4β7 integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.
    Full-text · Article · Nov 2012 · Journal of Virology

Publication Stats

3k Citations
431.15 Total Impact Points

Institutions

  • 1991-2015
    • Advanced BioScience Laboratories Inc.
      Роквилл, Maryland, United States
    • Columbia University
      • College of Physicians and Surgeons
      New York, New York, United States
  • 2002-2006
    • National Cancer Institute (USA)
      • Basic Research Laboratory
      베서스다, Maryland, United States
    • Beth Israel Deaconess Medical Center
      • Department of Medicine
      Boston, Massachusetts, United States
  • 1995
    • Università degli studi di Cagliari
      Cagliari, Sardinia, Italy