Marjorie Robert-Guroff

Publications of Marjorie Robert-Guroff

• Replicating Adenovirus-SIV Vectors Efficiently Prime SIV-specific Systemic and Mucosal Immune Responses by Targeting mDCs and Persisting in Rectal Macrophages, Regardless of Immunization Route.

  Authors: L Jean Patterson, Seraphin Kuate, Mara Daltabuit-Test, Qingsheng Li, Peng Xiao, Katherine McKinnon, Janet Dipasquale, Anthony Cristillo, David Venzon, Ashley Haase, Marjorie Robert-Guroff

  Clinical and vaccine immunology : CVI. 03/2012;

  Although priming with replicating Ad5hr-HIV/SIV recombinants followed by HIV/SIV envelope boosting has proven highly immunogenic, resulting in protection from SIV/SHIV challenges, Ad5hr-recombinantAlthough priming with replicating Ad5hr-HIV/SIV recombinants followed by HIV/SIV envelope boosting has proven highly immunogenic, resulting in protection from SIV/SHIV challenges, Ad5hr-recombinant distribution, replication and persistence have not been examined comprehensively in non-human primates. We utilized Ad5hr-GFP and Ad5hr-SIV recombinants to track biodistribution and immunogenicity following mucosal priming of rhesus macaques by intranasal/intratracheal, sublingual, vaginal or rectal routes. Ad-recombinants administered by all routes initially targeted macrophages in BAL and rectal tissue, later extending to mDC in BAL with persistent expression in rectal mucosa, 25 weeks after the last Ad immunization. Comparable SIV-specific immunity was elicited among all groups, including cellular responses, serum binding antibody, and mucosal secretory IgA. The ability of the vector to replicate in multiple mucosal sites irrespective of delivery route, together with the targeting of macrophages and professional APCs which provide potent immunogenicity at localized sites of virus entry warrant continued use of replicating Ad vectors.

• Replicating Adenovirus-Simian Immunodeficiency Virus (SIV) Recombinant Priming and Envelope Protein Boosting Elicits Localized, Mucosal IgA Immunity in Rhesus Macaques Correlated with Delayed Acquisition following a Repeated Low-Dose Rectal SIVmac251 Challenge.

  Authors: Peng Xiao, L Jean Patterson, Seraphin Kuate, Egidio Brocca-Cofano, Michael A Thomas, David Venzon, Jun Zhao, Janet Dipasquale, Claudio Fenizia, Eun Mi Lee, Irene Kalisz, Vaniambadi S Kalyanaraman, Ranajit Pal, David Montefiori, Brandon F Keele, Marjorie Robert-Guroff

  Journal of virology. 02/2012; 86(8):4644-57.

  We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasallyWe have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.

• Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA.

  Authors: Shari N Gordon, Rhonda C Kines, Galyna Kutsyna, Zhong-Min Ma, Anna Hryniewicz, Jeffery N Roberts, Claudio Fenizia, Rachmat Hidajat, Egidio Brocca-Cofano, Nicolas Cuburu, Christopher B Buck, Marcelino L Bernardo, Marjorie Robert-Guroff, Christopher J Miller, Barney S Graham, Douglas R Lowy, John T Schiller, Genoveffa Franchini

  Journal of immunology (Baltimore, Md. : 1950). 12/2011; 188(2):714-23.

  The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV.The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.

• Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques.

  Authors: Izabela Bialuk, Stephen Whitney, Vibeke Andresen, Ruth H Florese, Janos Nacsa, Valentina Cecchinato, Valerio W Valeri, Jean-Michel Heraud, Shari Gordon, Robyn Washington Parks, David C Montefiori, David Venzon, Thorsten Demberg, Marjorie Robert- Guroff, Gary Landucci, Donald N Forthal, Genoveffa Franchini

  Vaccine. 12/2011; 30(1):78-94.

  The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicitThe role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.

• Low-dose penile SIVmac251 exposure of rhesus macaques infected with adenovirus type 5 (Ad5) and then immunized with a replication-defective Ad5-based SIV gag/pol/nef vaccine recapitulates the results of the phase IIb step trial of a similar HIV-1 vaccine.

  Authors: Huma Qureshi, Zhong-Min Ma, Ying Huang, Gregory Hodge, Michael A Thomas, Janet DiPasquale, Veronique DeSilva, Linda Fritts, Andrew J Bett, Danilo R Casimiro, John W Shiver, Marjorie Robert-Guroff, Michael N Robertson, Michael B McChesney, Peter B Gilbert, Christopher J Miller

  Journal of virology. 12/2011; 86(4):2239-50.

  The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestlyThe Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8(+) T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10(3) 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.

• A CD8α(-) subpopulation of macaque circulatory natural killer cells can mediate both antibody-dependent and antibody-independent cytotoxic activities.

  Authors: Diego A Vargas-Inchaustegui, Thorsten Demberg, Marjorie Robert-Guroff

  Immunology. 11/2011; 134(3):326-40.

  Natural killer (NK) cells are important components of the innate immune system that mediate effector and regulatory functions. As effector cells, NK cells help control virus-infected cells throughNatural killer (NK) cells are important components of the innate immune system that mediate effector and regulatory functions. As effector cells, NK cells help control virus-infected cells through cell-mediated antibody-dependent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). Although macaques are an important and reliable animal model for the study of retrovirus-induced human diseases, and despite the crucial role played by NK cells in innate and adaptive immune responses against simian immunodeficiency virus (SIV), only a few studies have attempted to characterize different macaque NK cell subpopulations. In the present study, we identified a subpopulation of circulatory CD8α(-) macaque NK cells that express NK lineage markers and exhibit cytotoxic potential. CD8α(-) NK cells were phenotypically characterized as CD3(-) CD14(-) CD20(-) CD8α(-) cells that express NK cell markers including CD16, CD56, granzyme B, perforin, NKG2D and KIR2D. Based on their CD56/CD16 expression patterns, cells within the CD8α(-) gate can be divided into four subpopulations: CD56(dim) CD16(bright) , CD56(dim) CD16(-) , CD56(bright) CD16(-) , and CD56(-) CD16(-) cells. In contrast, CD8α(+) NK cells are 95% CD56(dim) CD16(bright) , which correlates with their high cytotoxic potential. Upon interleukin-15 activation, CD8α(-) cells up-regulated CD69 expression and produced low levels of interferon-γ and tumour necrosis factor-α. Sorted CD8α(-) NK cells were capable of killing MHC-I-devoid target cells and mediated ADCC responses against SIV gp120-coated target cells in the presence of macaque anti-gp120 antibodies. Taking into account CD8α(-) myeloid dendritic cells, we show that about 35% of macaque CD8α(-) cells represent a novel, functional population of circulatory NK cells that possesses cytotoxic potential and is capable of mediating anti-viral immune responses.

• HIV Envelope-Specific Antibody and Vaccine Efficacy

  Authors: Egidio Brocca-Cofano, Peng Xiao, Marjorie Robert-Guroff

  10/2011;

  ISBN: 978-953-307-665-2

• High-level, lasting antiviral immunity induced by a bimodal AIDS vaccine and boosted by live-virus exposure: prevention of viremia.

  Authors: Robert A Rasmussen, Nagadenahalli B Siddappa, Samir K Lakhashe, Jennifer Watkins, François Villinger, Chris Ibegbu, Ruth H Florese, Marjorie Robert-Guroff, David C Montefiori, Donald N Forthal, David O'Connor, Ruth M Ruprecht

  AIDS (London, England). 09/2011; 26(2):149-55.

  To characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque, RAt-9, which had been challenged sequentially with two related clade C simian/humanTo characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque, RAt-9, which had been challenged sequentially with two related clade C simian/human immunodeficiency viruses (SHIV-Cs) yet remained aviremic for more than 5 years despite indirect evidence of cryptic infection. To measure long-term anti-SHIV-C immunity, host genetics and gene-expression patterns for protective correlates. Long-term immune reactivity was evaluated and identification of virus in RAt-9 was attempted by RT-PCR analysis of concentrated plasma and blood transfer to CD8(+) cell-depleted infant macaques. Full MHC genotyping of RAt-9, TRIM5α and KIR3DL allelic expression analysis of PBMC, and microarray gene expression analysis were performed. All attempts to detect/isolate virus, including blood transfer to CD8(+) cell-depleted infant rhesus macaques, were negative, and the animal maintained normal levels of memory CD4(+) T cells in both peripheral blood and gut tissues. However, RAt-9 maintained high levels of anti-SHIV-C humoral and cellular immunity, including reactivity to nonvaccine neoantigens (Nef and Rev), up to 63 months postinitial challenge, suggesting chronic sub-threshold infection. RAt-9 expressed the Mamu A*001 allele negative for B*008 and B*017, had a B13 serotype, and had increased expression of killer-cell immunoglobulin-like receptors (KIRs) previously linked to favorable outcomes of lentiviral infection. Elements of the gene expression profiling coincided with genotyping results. RAt-9 also displayed CD8 cell noncytotoxic antiviral response (CNAR) activity. Monkey RAt-9 is the first example of a virus-exposed, persistently aviremic animal that has maintained long-term, high-level cellular and humoral antiviral immunity in the absence of an identifiable cryptic reservoir.

• TRIM5α does not affect simian immunodeficiency virus SIV(mac251) replication in vaccinated or unvaccinated Indian rhesus macaques following intrarectal challenge exposure.

  Authors: Claudio Fenizia, Brandon F Keele, David Nichols, Stefano Cornara, Nicolò Binello, Monica Vaccari, Poonam Pegu, Marjorie Robert-Guroff, Zhong-Min Ma, Christopher J Miller, David Venzon, Vanessa Hirsch, Genoveffa Franchini

  Journal of virology. 09/2011; 85(23):12399-409.

  TRIM5α is a natural resistance factor that binds retroviral capsid proteins and restricts virus replication. The B30.2/SPRY domain of TRIM5α is polymorphic in rhesus macaques, and some alleles areTRIM5α is a natural resistance factor that binds retroviral capsid proteins and restricts virus replication. The B30.2/SPRY domain of TRIM5α is polymorphic in rhesus macaques, and some alleles are associated with reduced simian immunodeficiency virus (SIV) SIV(mac251) and SIV(smE543) replication in vivo. We determined the distribution of TRIM5α alleles by PCR and sequence analysis of the B30.2/SPRY domain in a cohort of 82 macaques. Thirty-nine of these macaques were mock vaccinated, 43 were vaccinated with either DNA-SIV/ALVAC-SIV/gp120, ALVAC-SIV/gp120, or gp120 alone, and all were exposed intrarectally to SIV(mac251) at one of three doses. We assessed whether the TRIM5α genotype of the macaques affected the replication of challenge virus by studying the number of SIV variants transmitted, the number of exposures required, the SIV(mac251) viral level in plasma and tissue, and the CD4(+) T-cell counts. Our results demonstrated that TRIM5α alleles, previously identified as restrictive for SIV(mac251) replication in vivo following intravenous exposure, did not affect SIV(mac251) replication following mucosal exposure, regardless of prior vaccination, challenge dose, or the presence of the protective major histocompatibility complex alleles (MamuA01(+), MamuB08(+), or MamuB017(+)). The TRIM5α genotype had no apparent effect on the number of transmitted variants or the number of challenge exposures necessary to infect the animals. DNA sequencing of the SIV(mac251) Gag gene of the two stocks used in our study revealed SIV(mac239)-like sequences that are predicted to be resistant to TRIM5α restriction. Thus, the TRIM5α genotype does not confound results of mucosal infection of rhesus macaques with SIV(mac251).

• Strong viremia control in vaccinated macaques does not prevent gradual Th17 cell loss from central memory.

  Authors: Thorsten Demberg, Amelia C Ettinger, Stanley Aladi, Katherine McKinnon, Thea Kuddo, David Venzon, L Jean Patterson, Terry M Phillips, Marjorie Robert-Guroff

  Vaccine. 06/2011; 29(35):6017-28.

  It has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributedIt has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributed to Th17 and Tc17 cells. Th17 cells can be infected with HIV/SIV, however whether effective vaccination leads to their maintenance following viral challenge has not been addressed. Here we retrospectively investigated if a vaccine regimen that potently reduced viremia post-challenge preserved Th17 and Tc17 cells, thus adding benefit in the absence of sterilizing protection. Rhesus macaques were previously vaccinated with replication-competent Adenovirus recombinants expressing HIVtat and HIVenv followed by Tat and gp140 protein boosting. Upon SHIV(89.6P) challenge, the vaccines exhibited a significant 4 log reduction in chronic viremia compared to sham vaccinated controls which rapidly progressed to AIDS [39]. Plasma and cryopreserved PBMC samples were examined pre-challenge and during acute and chronic infection. Control macaques exhibited a rapid loss of CD4(+) cells, including Th17 cells. Tc17 cells tended to decline over the course of infection although significance was not reached. Immune activation, assessed by Ki-67 expression, was associated with elevated chronic viremia of the controls. Significantly increased plasma IFN-γ levels were also observed. No increase in plasma LPS levels were observed suggesting a lack of microbial translocation. In contrast, vaccinated macaques had no evidence of immune activation within the chronic phase and preserved both CD4(+) T-cells and Tc17 cells in PBMC. Nevertheless, they exhibited a gradual, significant loss of Th17 cells which concomitantly displayed significantly higher CCR6 expression over time. The gradual Th17 cell decline may reflect mucosal homing to inflammatory sites and/or slow depletion due to ongoing low levels of SHIV replication. Our results suggest that potent viremia reduction during chronic SHIV infection will delay but not prevent the loss of Th17 cells.

• Prime-boost vaccination with heterologous live vectors encoding SIV gag and multimeric HIV-1 gp160 protein: efficacy against repeated mucosal R5 clade C SHIV challenges.

  Authors: Samir K Lakhashe, Vijayakumar Velu, Gaia Sciaranghella, Nagadenahalli B Siddappa, Janet M Dipasquale, Girish Hemashettar, John K Yoon, Robert A Rasmussen, Feng Yang, Sandra J Lee [......] Rama Rao Amara, Maria Kahn, Shiu-Lok Hu, Sufen Li, Zhongxia Li, Fred R Frankel, Marjorie Robert-Guroff, Welkin E Johnson, Judy Lieberman, Ruth M Ruprecht

  Vaccine. 06/2011; 29(34):5611-22.

  We sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunizedWe sought to induce primate immunodeficiency virus-specific cellular and neutralizing antibody (nAb) responses in rhesus macaques (RM) through a bimodal vaccine approach. RM were immunized intragastrically (i.g.) with the live-attenuated Listeria monocytogenes (Lm) vector Lmdd-BdopSIVgag encoding SIVmac239 gag. SIV Gag-specific cellular responses were boosted by intranasal and intratracheal administration of replication-competent adenovirus (Ad5hr-SIVgag) encoding the same gag. To broaden antiviral immunity, the RM were immunized with multimeric HIV clade C (HIV-C) gp160 and HIV Tat. SIV Gag-specific cellular immune responses and HIV-1 nAb developed in some RM. The animals were challenged intrarectally with five low doses of R5 SHIV-1157ipEL-p, encoding a heterologous HIV-C Env (22.1% divergent to the Env immunogen). All five controls became viremic. One out of ten vaccinees was completely protected and another had low peak viremia. Sera from the completely and partially protected RM neutralized the challenge virus > 90%; these RM also had strong SIV Gag-specific proliferation of CD8⁺ T cells. Peak and area under the curve of plasma viremia (during acute phase) among vaccinees was lower than for controls, but did not attain significance. The completely protected RM showed persistently low numbers of the α4β7-expressing CD4⁺ T cells; the latter have been implicated as preferential virus targets in vivo. Thus, vaccine-induced immune responses and relatively lower numbers of potential target cells were associated with protection.

• Rapid SIV Env-specific mucosal and serum antibody induction augments cellular immunity in protecting immunized, elite-controller macaques against high dose heterologous SIV challenge.

  Authors: L Jean Patterson, Mara Daltabuit-Test, Peng Xiao, Jun Zhao, William Hu, Ulrike Wille-Reece, Egidio Brocca-Cofano, V S Kalyanaraman, Irene Kalisz, Stephen Whitney, Eun Mi Lee, Ranajit Pal, David C Montefiori, Satya Dandekar, Robert Seder, Mario Roederer, Roger W Wiseman, Vanessa Hirsch, Marjorie Robert-Guroff

  Virology. 03/2011; 411(1):87-102.

  Three Indian rhesus macaques, Ad-SIV primed/protein boosted and exposed twice to high-dose mucosal SIV(mac251) challenges, exhibited elite control of viremia over 6.5 years. They were negative forThree Indian rhesus macaques, Ad-SIV primed/protein boosted and exposed twice to high-dose mucosal SIV(mac251) challenges, exhibited elite control of viremia over 6.5 years. They were negative for host factors associated with control of SIV infection. After a third intrarectal challenge with SIV(smE660), all controlled viremia, with one (macaque #5) maintaining undetectable viremia in blood. Acquisition was not blocked, but virus was contained in the jejunum and draining lymph nodes. Polyfunctional memory T cell responses and high-titered neutralizing and non-neutralizing serum and mucosal antibodies were present before and maintained post-challenge. The level of protection seen for animal #5 was predicted from analyses of gene transcription in jejunum 2 weeks post-challenge. Macaques #7 and #9, exhibiting lower pre-challenge cellular and humoral immunity, partially controlled the SIV(smE660) challenge. Initial vaccine-induced control by macaque #5 extended to the SIV(smE660) challenge due to multiple immune mechanisms that were boosted and augmented by cryptic SIV exposure.

• Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia.

  Authors: Egidio Brocca-Cofano, Katherine McKinnon, Thorsten Demberg, David Venzon, Rachmat Hidajat, Peng Xiao, Mara Daltabuit-Test, L Jean Patterson, Marjorie Robert-Guroff

  Vaccine. 03/2011; 29(17):3310-9.

  An effective HIV vaccine requires strong systemic and mucosal, cellular and humoral immunity. Numerous non-human primate studies have investigated memory T cells, but not memory B cells. HumoralAn effective HIV vaccine requires strong systemic and mucosal, cellular and humoral immunity. Numerous non-human primate studies have investigated memory T cells, but not memory B cells. Humoral immunologic memory is mediated by long-lived antibody-secreting plasma cells and differentiation of memory B cells into short-lived plasma blasts following re-exposure to immunizing antigen. Here we studied memory B cells in vaccinated rhesus macaques. PBMC were stimulated polyclonally using CD40 Ligand, IL-21 and CpG to induce B cell proliferation and differentiation into antibody secreting cells (ASCs). Flow cytometry was used for phenotyping and evaluating proliferation by CFSE dilution. B cell responses were quantified by ELISPOT. Methodology was established using PBMC of vaccinated elite-controller macaques that exhibited strong, multi-functional antibody activities. Subsequently, memory B cells elicited by two replicating Ad-recombinant prime/envelope boost regimens were retrospectively evaluated pre- and post-SIV and SHIV challenges. The vaccine regimens induced SIV and HIV Env-specific IgG and IgA memory B cells. Prior to challenge, IgA memory B cells were more numerous than IgG memory B cells, reflecting the mucosal priming immunizations. Pre- and post-challenge memory B cells were correlated with functional antibody responses including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated viral inhibition (ADCVI) and transcytosis inhibition. Post-challenge, Env-specific IgG and IgA memory B cells were correlated with reduced chronic viremia. We conclude that functional antibody responses elicited by our prime/boost regimen were effectively incorporated into the memory B cell pool where they contributed to control of viremia following re-exposure to the immunizing antigen.

• Early short-term antiretroviral therapy is associated with a reduced prevalence of CD8(+)FoxP3(+) T cells in simian immunodeficiency virus-infected controller rhesus macaques.

  Authors: Jeffy George, Egidio Brocca Cofano, Elizabeth Lybarger, Mark Louder, Bernard A P Lafont, John R Mascola, Marjorie Robert-Guroff, Joseph J Mattapallil

  AIDS research and human retroviruses. 01/2011; 27(7):763-75.

  Regulatory T cells contain a mix of CD4 and CD8 T cell subsets that can suppress immune activation and at the same time suppress immune responses, thereby contributing to disease progression. RecentRegulatory T cells contain a mix of CD4 and CD8 T cell subsets that can suppress immune activation and at the same time suppress immune responses, thereby contributing to disease progression. Recent studies have shown that an increased prevalence of CD8(+)FoxP3(+) T regulatory cells was associated with immune suppression and diminished viral control in simian immunodeficiency virus (SIV)-infected rhesus macaques. Preventing an increase in the prevalence of CD8 T regulatory subsets is likely to lead to a better long-term outcome. Here we show that short-term antiretroviral therapy initiated within 1 week after SIV infection was associated with lower viral set point and immune activation after withdrawal of therapy as compared to untreated animals. Early short-term treated controller animals were found to have better SIV-specific immune responses and a significantly lower prevalence of immunosuppressive CD8(+)FoxP3(+) T cells. Lower levels of CD8(+)FoxP3(+) T cells coincided with preservation of CD4(+)FoxP3(+) T cells at homeostatic levels, and significantly correlated with lower immune activation, suggesting a role for viral infection-driven immune activation in the expansion of CD8(+)FoxP3(+) T cells. Interestingly, initiation of continuous therapy later in infection did not reduce the increased prevalence of CD8(+)FoxP3(+) T cells to homeostatic levels. Taken together, our results suggest that early antiretroviral therapy preserves the integrity of the immune system leading to a lower viral set point in controller animals, and prevents alterations in the homeostatic balance between CD4(+) and CD8(+) T regulatory cells that could aid in better long-term outcome.

• Genomic analysis reveals pre- and postchallenge differences in a rhesus macaque AIDS vaccine trial: insights into mechanisms of vaccine efficacy.

  Authors: Robert E Palermo, L Jean Patterson, Lauri D Aicher, Marcus J Korth, Marjorie Robert-Guroff, Michael G Katze

  Journal of virology. 11/2010; 85(2):1099-116.

  We have employed global transcriptional profiling of whole blood to identify biologically relevant changes in cellular gene expression in response to alternative AIDS vaccine strategies withWe have employed global transcriptional profiling of whole blood to identify biologically relevant changes in cellular gene expression in response to alternative AIDS vaccine strategies with subsequent viral challenge in a rhesus macaque vaccine model. Samples were taken at day 0 (prechallenge), day 14 (peak viremia), and week 12 (set point) from animals immunized with replicating adenovirus type 5 host range (Ad5hr) recombinant viruses expressing human immunodeficiency virus HIV(env)(89.6P), simian immunodeficiency virus SIV(gag)(239), or SIV(nef)(239) alone or in combination with two intramuscular boosts with HIV(89.6P)gp140ΔCFI protein (L. J. Patterson et al., Virology 374:322-337, 2008), and each treatment resulted in significant control of viremia following simian-human immunodeficiency virus SHIV(89.6P) challenge (six animals per group plus six controls). At day 0, 8 weeks after the last treatment, the microarray profiles revealed significant prechallenge differences between treatment groups; data from the best-protected animals led to identification of a network of genes related to B cell development and lymphocyte survival. At peak viremia, expression profiles of the immunized groups were extremely similar, and comparisons to control animals reflected immunological differences other than effector T cell functions. Suggested protective mechanisms for vaccinated animals included upregulation of interleukin-27, a cytokine known to inhibit lentivirus replication, and increased expression of complement components, which may synergize with vaccine-induced antibodies. Divergent expression profiles at set point for the immunized groups implied distinct immunological responses despite phenotypic similarities in viral load and CD4(+) T cell levels. Data for the gp140-boosted group provided evidence for antibody-dependent, cell-mediated viral control, whereas animals immunized with only the replicating Ad5hr recombinants exhibited a different evolution of the B cell compartment even at 3 months postchallenge. This study demonstrates the sensitivity and discrimination of gene expression profiling of whole blood as an analytical tool in AIDS vaccine trials, providing unique insights into in vivo mechanisms and potential correlates of protection.

• Impact of viral dose and major histocompatibility complex class IB haplotype on viral outcome in mauritian cynomolgus monkeys vaccinated with Tat upon challenge with simian/human immunodeficiency virus SHIV89.6P.

  Authors: Aurelio Cafaro, Stefania Bellino, Fausto Titti, Maria Teresa Maggiorella, Leonardo Sernicola, Roger W Wiseman, David Venzon, Julie A Karl, David O'Connor, Paolo Monini, Marjorie Robert-Guroff, Barbara Ensoli

  Journal of virology. 09/2010; 84(17):8953-8.

  The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with TatThe effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P(cy243.) In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID(50)]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID(50) (P < 0.0001), and contained acute CD4 loss at 15 MID(50) (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.

• Multiple vaccine-elicited nonneutralizing antienvelope antibody activities contribute to protective efficacy by reducing both acute and chronic viremia following simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques.

  Authors: Peng Xiao, Jun Zhao, L Jean Patterson, Egidio Brocca-Cofano, David Venzon, Pamela A Kozlowski, Rachmat Hidajat, Thorsten Demberg, Marjorie Robert-Guroff

  Journal of virology. 07/2010; 84(14):7161-73.

  We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting withWe have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)(89.6P) challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.

• Long-lasting humoral and cellular immune responses and mucosal dissemination after intramuscular DNA immunization.

  Authors: Vainav Patel, Antonio Valentin, Viraj Kulkarni, Margherita Rosati, Cristina Bergamaschi, Rashmi Jalah, Candido Alicea, Jacob T Minang, Matthew T Trivett, Claes Ohlen, Jun Zhao, Marjorie Robert-Guroff, Amir S Khan, Ruxandra Draghia-Akli, Barbara K Felber, George N Pavlakis

  Vaccine. 05/2010; 28(30):4827-36.

  Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals wereNaïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat and Vif), long-lasting (up to 90 weeks post third vaccination) and were boosted with each subsequent immunization, even after an extended 90-week rest period. The SIV-specific cellular immune responses were consistently more abundant in bronchoalveolar lavage (BAL) than in blood, and were characterized as predominantly effector memory CD4(+) and CD8(+) T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.

• Construction and immunogenicity of replication-competent adenovirus 5 host range mutant recombinants expressing HIV-1 gp160 of SF162 and TV1 strains.

  Authors: Rachmat Hidajat, Seraphin Kuate, David Venzon, Vaniambadi Kalyanaraman, Irene Kalisz, James Treece, Ying Lian, Susan W Barnett, Marjorie Robert-Guroff

  Vaccine. 04/2010; 28(23):3963-71.

  An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162)An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162) gp160 (subtype B) and HIV(TV1) gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIV(TV1) gp160 induced better cellular immunity than Ad5hr-HIV(SF162) gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIV(TV1) gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector.

• Contribution of nonneutralizing vaccine-elicited antibody activities to improved protective efficacy in rhesus macaques immunized with tat/env compared with multigenic vaccines.

  Authors: Ruth H Florese, Thorsten Demberg, Peng Xiao, LaRene Kuller, Kay Larsen, L Ebonita Summers, David Venzon, Aurelio Cafaro, Barbara Ensoli, Marjorie Robert-Guroff

  Journal of immunology (Baltimore, Md. : 1950). 03/2009; 182(6):3718-27.

  Previously, chronic-phase protection against SHIV(89.6P) challenge was significantly greater in macaques primed with replicating adenovirus type 5 host range mutant (Ad5hr) recombinants encodingPreviously, chronic-phase protection against SHIV(89.6P) challenge was significantly greater in macaques primed with replicating adenovirus type 5 host range mutant (Ad5hr) recombinants encoding HIVtat and env and boosted with Tat and Env protein compared with macaques primed with multigenic adenovirus recombinants (HIVtat, HIVenv, SIVgag, SIVnef) and boosted with Tat, Env, and Nef proteins. The greater protection was correlated with Tat- and Env-binding Abs. Because the macaques lacked SHIV(89.6P)-neutralizing activity prechallenge, we investigated whether Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cell-mediated viral inhibition (ADCVI) might exert a protective effect. We clearly show that Tat can serve as an ADCC target, although the Tat-specific activity elicited did not correlate with better protection. However, Env-specific ADCC activity was consistently higher in the Tat/Env group, with sustained cell killing postchallenge exhibited at higher levels (p < 0.00001) for a longer duration (p = 0.0002) compared with the multigenic group. ADCVI was similarly higher in the Tat/Env group and significantly correlated with reduced acute-phase viremia at wk 2 and 4 postchallenge (p = 0.046 and 0.011, respectively). Viral-specific IgG and IgA Abs in mucosal secretions were elicited but did not influence the outcome of the i.v. SHIV(89.6P) challenge. The higher ADCC and ADCVI activities seen in the Tat/Env group provide a plausible mechanism responsible for the greater chronic-phase protection. Because Tat is known to enhance cell-mediated immunity to coadministered Ags, further studies should explore its impact on Ab induction so that it may be optimally incorporated into HIV vaccine regimens.