Ruth M Ruprecht

Publications of Ruth M Ruprecht

• Dynamics of Envelope Evolution in Clade C SHIV-Infected Pig-Tailed Macaques during Disease Progression Analyzed by Ultra-Deep Pyrosequencing.

  Authors: For Yue Tso, Damien C Tully, Sandra Gonzalez, Christopher Quince, On Ho, Patricia Polacino, Ruth M Ruprecht, Shiu-Lok Hu, Charles Wood

  PloS one. 01/2012; 7(3):e32827.

  Understanding the evolution of the human immunodeficiency virus type 1 (HIV-1) envelope during disease progression can provide tremendous insights for vaccine development, and simian-humanUnderstanding the evolution of the human immunodeficiency virus type 1 (HIV-1) envelope during disease progression can provide tremendous insights for vaccine development, and simian-human immunodeficiency virus (SHIV) infection of non-human primate provides an ideal platform for such studies. A newly developed clade C SHIV, SHIV-1157ipd3N4, which was able to infect rhesus macaques, closely resembled primary HIV-1 in transmission and pathogenesis, was used to infect several pig-tailed macaques. One of the infected animals subsequently progressed to AIDS, whereas one remained a non-progressor. The viral envelope evolution in the infected animals during disease progression was analyzed by a bioinformatics approach using ultra-deep pyrosequencing. Our results showed substantial envelope variations emerging in the progressor animal after the onset of AIDS. These envelope variations impacted the length of the variable loops and charges of different envelope regions. Additionally, multiple mutations were located at the CD4 and CCR5 binding sites, potentially affecting receptor binding affinity, viral fitness and they might be selected at late stages of disease. More importantly, these envelope mutations are not random since they had repeatedly been observed in a rhesus macaque and a human infant infected by either SHIV or HIV-1, respectively, carrying the parental envelope of the infectious molecular clone SHIV-1157ipd3N4. Moreover, similar mutations were also observed from other studies on different clades of envelopes regardless of the host species. These recurring mutations in different envelopes suggest that there may be a common evolutionary pattern and selection pathway for the HIV-1 envelope during disease progression.

• No Acquisition: a New Ambition for HIV Vaccine Development?

  Authors: Samir K Lakhashe, Guido Silvestri, Ruth M Ruprecht

  Current opinion in virology. 10/2011; 1(4):246-253.

  Development of a safe and effective prophylactic HIV-1 vaccine presents unique challenges. The pessimism following the failure of two HIV-1 vaccine concepts in clinical trials, HIV-1 gp120 and anDevelopment of a safe and effective prophylactic HIV-1 vaccine presents unique challenges. The pessimism following the failure of two HIV-1 vaccine concepts in clinical trials, HIV-1 gp120 and an adenovirus-based approach to induce only cellular immune responses, has been replaced by cautious optimism engendered by the RV144 trial outcome, the isolation of several new broadly reactive neutralizing monoclonal antibodies, and recent primate model data indicating prevention of viral acquisition by active or passive immunization. Intense efforts are underway to optimize immunogen design, adjuvants, and the tools for preclinical evaluation of candidate vaccines in primates, where correlates of protection can be examined in detail - as proof-of-concept for clinical trials.

• Efficiency of neutralizing antibodies targeting the CD4-binding site: influence of conformational masking by the V2 loop in R5-tropic clade C simian-human immunodeficiency virus.

  Authors: Jennifer D Watkins, Juan Diaz-Rodriguez, Nagadenahalli B Siddappa, Davide Corti, Ruth M Ruprecht

  Journal of virology. 09/2011; 85(23):12811-4.

  In R5-tropic clade C simian-human immunodeficiency viruses (SHIV-Cs), we identified a 3-asparagine (3N) deletion mutation in the V2 loop stem of gp120 as the major determinant of neutralizationIn R5-tropic clade C simian-human immunodeficiency viruses (SHIV-Cs), we identified a 3-asparagine (3N) deletion mutation in the V2 loop stem of gp120 as the major determinant of neutralization escape of the anti-CD4-binding site (anti-CD4-bs) neutralizing monoclonal antibody (nMAb) b12. However, the more potent anti-CD4-bs nMAbs VRC01 and VRC03 were not sensitive to this mutation. Using isogenic tier 1 or tier 2 proviruses differing only in the 3N mutation, we showed that this mutation might result in selective conformational b12 epitope masking. Therefore, human immunodeficiency virus (HIV) Env immunogens targeting the CD4-bs and designed to neutralize tier 2 viruses should take conformational masking by the V2 loop into account.

• 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.

• Molecularly cloned SHIV-CN97001: a replication-competent, R5 simian/human immunodeficiency virus containing env of a primary Chinese HIV-1 clade C isolate.

  Authors: Qiang Liu, Yue Li, GuiBo Yang, JieJie Dai, Ruth M Ruprecht, Yiming Shao

  Journal of medical primatology. 09/2011; 40(6):427-36.

  The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating AIDS candidate vaccines. InThe increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating AIDS candidate vaccines. In China, the prevalent HIV strains comprise a circulating recombinant form, BC (CRF07_BC), in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in non-human primate models, we constructed a simian/human immunodeficiency virus (SHIV) carrying most of the envelope sequence of a primary HIV-1 clade C strain isolated from an HIV-positive intravenous drug user from YunNan province in China. Furthermore, to determine whether in vivo adaptation would enhance the infectivity of SHIV-CN97001, the parental infectious strain was serially passaged through eight Chinese rhesus macaques. Infection of six Chinese rhesus macaques with SHIV-CN97001 resulted in a low level of viremia and no significant alteration in CD4+ T-lymphocyte counts. However, the hallmarks of SHIV infectivity developed gradually, as shown by the increasingly elevated peak viremia with each passage. These findings establish that the R5-tropic SHIV-CN97001/Chinese rhesus macaque model should be very useful for the evaluation of HIV-1 subtype C vaccines in China.

• Schistosoma mansoni enhances host susceptibility to mucosal but not intravenous challenge by R5 Clade C SHIV.

  Authors: Nagadenahalli B Siddappa, Girish Hemashettar, Vivekanandan Shanmuganathan, Amma A Semenya, Elizabeth D Sweeney, Katherine S Paul, Sandra J Lee, W Evan Secor, Ruth M Ruprecht

  PLoS neglected tropical diseases. 08/2011; 5(8):e1270.

  The high prevalence of HIV-1/AIDS in areas endemic for schistosomiasis and other helminthic infections has led to the hypothesis that parasites increase host susceptibility to immunodeficiency virusThe high prevalence of HIV-1/AIDS in areas endemic for schistosomiasis and other helminthic infections has led to the hypothesis that parasites increase host susceptibility to immunodeficiency virus infection. We previously showed that rhesus macaques (RM) with active schistosomiasis were significantly more likely to become systemically infected after intrarectal (i.r.) exposure to an R5-tropic clade C simian-human immunodeficiency virus (SHIV-C) than were parasite-free controls. However, we could not address whether this was due to systemic or mucosal effects. If systemic immunoactivation resulted in increased susceptibility to SHIV-C acquisition, a similarly large difference in host susceptibility would be seen after intravenous (i.v.) SHIV-C challenge. Conversely, if increased host susceptibility was due to parasite-induced immunoactivation at the mucosal level, i.v. SHIV-C challenge would not result in significant differences between parasitized and parasite-free monkeys. We enrolled two groups of RM and infected one group with Schistosoma mansoni; the other group was left parasite-free. Both groups were challenged i.v. with decreasing doses of SHIV-C. No statistically significant differences in 50% animal infectious doses (AID(50)) or peak viremia were seen between the two groups. These data strongly contrast the earlier i.r. SHIV-C challenge (using the same virus stock) in the presence/absence of parasites, where we noted a 17-fold difference in AID(50) and one log higher peak viremia in parasitized monkeys (P<0.001 for both). The lack of significant differences after the i.v. challenge implies that the increased host susceptibility is predominantly due to parasite-mediated mucosal upregulation of virus replication and spread, rather than systemic effects. The major impact of schistosome-induced increased host susceptibility is at the mucosal level. Given that >90% of all new HIV-1 infections worldwide are acquired through mucosal contact, parasitic infections that inflame mucosae may play an important role in the spread of HIV-1.

• 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.

• R5-SHIV induces multiple defects in T cell function during early infection of rhesus macaques including accumulation of T reg cells in lymph nodes.

  Authors: Michael Santosuosso, Elda Righi, E David Hill, Pierre R Leblanc, Brett Kodish, Hari N Mylvaganam, Nagadenahalli B Siddappa, Liljana Stevceva, Shiu-Lok Hu, Musie Ghebremichael, Agnes-L Chenine, Avi-Hai Hovav, Ruth M Ruprecht, Mark C Poznansky

  PloS one. 01/2011; 6(4):e18465.

  HIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated inHIV-1 is a pathogen that T cell responses fail to control. HIV-1gp120 is the surface viral envelope glycoprotein that interacts with CD4 T cells and mediates entry. HIV-1gp120 has been implicated in immune dysregulatory functions that may limit anti-HIV antigen-specific T cell responses. We hypothesized that in the context of early SHIV infection, immune dysregulation of antigen-specific T-effector cell and regulatory functions would be detectable and that these would be associated or correlated with measurable concentrations of HIV-1gp120 in lymphoid tissues. Rhesus macaques were intravaginally inoculated with a Clade C CCR5-tropic simian-human immunodeficiency virus, SHIV-1157ipd3N4. HIV-1gp120 levels, antigen-specificity, levels of apoptosis/anergy and frequency and function of Tregs were examined in lymph node and blood derived T cells at 5 and 12 weeks post inoculation. We observed reduced responses to Gag in CD4 and gp120 in CD8 lymph node-derived T cells compared to the peripheral blood at 5 weeks post-inoculation. Reduced antigen-specific responses were associated with higher levels of PD-1 on lymph node-derived CD4 T cells as compared to peripheral blood and uninfected lymph node-derived CD4 T cells. Lymph nodes contained increased numbers of Tregs as compared to peripheral blood, which positively correlated with gp120 levels; T regulatory cell depletion restored CD8 T cell responses to Gag but not to gp120. HIV gp120 was also able to induce T regulatory cell chemotaxis in a dose-dependent, CCR5-mediated manner. These studies contribute to our broader understanding of the ways in which HIV-1 dysregulates T cell function and localization during early infection.

• Vaccination against heterologous R5 clade C SHIV: prevention of infection and correlates of protection.

  Authors: Samir K Lakhashe, Wendy Wang, Nagadenahalli B Siddappa, Girish Hemashettar, Patricia Polacino, Shiu-Lok Hu, François Villinger, James G Else, Francis J Novembre, John K Yoon, Sandra J Lee, David C Montefiori, Ruth M Ruprecht, Robert A Rasmussen

  PloS one. 01/2011; 6(7):e22010.

  A safe, efficacious vaccine is required to stop the AIDS pandemic. Disappointing results from the STEP trial implied a need to include humoral anti-HIV-1 responses, a notion supported by RV144 trialA safe, efficacious vaccine is required to stop the AIDS pandemic. Disappointing results from the STEP trial implied a need to include humoral anti-HIV-1 responses, a notion supported by RV144 trial data even though correlates of protection are unknown. We vaccinated rhesus macaques with recombinant simian immunodeficiency virus (SIV) Gag-Pol particles, HIV-1 Tat and trimeric clade C (HIV-C) gp160, which induced cross-neutralizing antibodies (nAbs) and robust cellular immune responses. After five low-dose mucosal challenges with a simian-human immunodeficiency virus (SHIV) that encoded a heterologous R5 HIV-C envelope (22.1% divergence from the gp160 immunogen), 94% of controls became viremic, whereas one third of vaccinees remained virus-free. Upon high-dose SHIV rechallenge, all controls became infected, whereas some vaccinees remained aviremic. Peak viremia was inversely correlated with both cellular immunity (p<0.001) and cross-nAb titers (p<0.001). These data simultaneously linked cellular as well as humoral immune responses with the degree of protection for the first time.

• Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV.

  Authors: James B Whitney, Saied Mirshahidi, So-Yon Lim, Lauren Goins, Chris C Ibegbu, Daniel C Anderson, Richard B Raybourne, Fred R Frankel, Judy Lieberman, Ruth M Ruprecht

  Journal of immune based therapies and vaccines. 01/2011; 9:2.

  We have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine theWe have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine the impact of prior Lm vector exposure on the development of new immune responses against HIV antigens. Two groups of rhesus macaques one Lm naive, the other having documented prior Lm vector exposures, were evaluated in response to oral inoculations of the same vector expressing recombinant HIV-1 Gag protein. The efficacy of the Lm vector was determined by ELISA to assess the generation of anti-Listerial antibodies; cellular responses were measured by HIV-Gag specific ELISpot assay. Our results show that prior Lm exposures did not diminish the generation of de novo cellular responses against HIV, as compared to Listeria-naïve monkeys. Moreover, empty vector exposures did not elicit potent antibody responses, consistent with the intracellular nature of Lm. The present study demonstrates in a pre-clinical vaccine model, that prior oral immunization with an empty Lm vector does not diminish immunogenicity to Lm-expressed HIV genes. This work underscores the need for the continued development of attenuated Lm as an orally deliverable vaccine.

• An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV.

  Authors: Jennifer D Watkins, Nagadenahalli B Siddappa, Samir K Lakhashe, Michael Humbert, Anton Sholukh, Girish Hemashettar, Yin Ling Wong, John K Yoon, Wendy Wang, Francis J Novembre [......] Chris Ibegbu, Kalpana Patel, Davide Corti, Gloria Agatic, Fabrizia Vanzetta, Siro Bianchi, Jonathan L Heeney, Federica Sallusto, Antonio Lanzavecchia, Ruth M Ruprecht

  PloS one. 01/2011; 6(3):e18207.

  Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizingNeutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient "Hit and Run" infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target.

• Development of a tier 1 R5 clade C simian-human immunodeficiency virus as a tool to test neutralizing antibody-based immunoprophylaxis.

  Authors: Nagadenahalli B Siddappa, Girish Hemashettar, Yin Ling Wong, Samir Lakhashe, Robert A Rasmussen, Jennifer D Watkins, Francis J Novembre, François Villinger, James G Else, David C Montefiori, Ruth M Ruprecht

  Journal of medical primatology. 11/2010; 40(2):120-8.

  While some recently transmitted HIV clade C (HIV-C) strains exhibited tier 1 neutralization phenotypes, most were tier 2 strains (J Virol 2010; 84:1439). Because induction of neutralizing antibodiesWhile some recently transmitted HIV clade C (HIV-C) strains exhibited tier 1 neutralization phenotypes, most were tier 2 strains (J Virol 2010; 84:1439). Because induction of neutralizing antibodies (nAbs) through vaccination against tier 2 viruses has proven difficult, we have generated a tier 1, clade C simian-human immunodeficiency virus (SHIV-C) to permit efficacy testing of candidate AIDS vaccines against tier 1 viruses. SHIV-1157ipEL was created by swapping env of a late-stage virus with that of a tier 1, early form. After adaptation to rhesus macaques (RM), passaged SHIV-1157ipEL-p replicated vigorously in vitro and in vivo while maintaining R5 tropism. The virus was reproducibly transmissible intrarectally. Phylogenetically, SHIV-1157ipEL-p Env clustered with HIV-C sequences. All RM chronically infected with SHIV-1157ipEL-p developed high nAb titers against autologous as well as heterologous tier 1 strains. SHIV-1157ipEL-p was reproducibly transmitted in RM, induced cross-clade nAbs, and represents a tool to evaluate anti-HIV-C nAb responses in primates.

• A live attenuated Listeria monocytogenes vaccine vector expressing SIV Gag is safe and immunogenic in macaques and can be administered repeatedly.

  Authors: Gaia Sciaranghella, Samir K Lakhashe, Mila Ayash-Rashkovsky, Saied Mirshahidi, Nagadenahalli B Siddappa, Francis J Novembre, Vijayakumar Velu, Rama Rao Amara, Chenghui Zhou, Sufen Li, Zhongxia Li, Fred R Frankel, Ruth M Ruprecht

  Vaccine. 11/2010; 29(3):476-86.

  Listeria monocytogenes (Lm) is known to induce strong cellular immune responses. We constructed a live-attenuated Lm vector, Lmdd-BdopSIVgag, which encodes SIVmac239 gag. Intragastric (i.g.)Listeria monocytogenes (Lm) is known to induce strong cellular immune responses. We constructed a live-attenuated Lm vector, Lmdd-BdopSIVgag, which encodes SIVmac239 gag. Intragastric (i.g.) administration of 3 × 10(12) bacteria to rhesus macaques was safe and induced anti-Gag cellular but no humoral immune responses. Boosting of Gag-specific cellular responses was observed after i.g. administration of Lmdd-BdopSIVgag to previously vaccinated RM despite preexisting anti-Lm immunity shown by lymphoproliferative responses. Surprisingly, anti-Lm cellular responses were also detected in non-vaccinated controls, which may reflect the fact that Lm is a ubiquitous bacterium. The novel, live-attenuated Lmdd-BdopSIVgag may be an attractive platform for oral vaccine delivery.

• AIDS and optic neuritis in a rhesus monkey infected with the R5 clade C SHIV-1157ipd3N4.

  Authors: Anapatricia García, Nagadenahalli B Siddappa, Qingsheng Li, Ashley T Haase, Katherine Paul, Fawn Stroud, Xiaodong Zhang, Jack A Fountain, François Villinger, Francis J Novembre, James G Else, W Evan Secor, Ruth M Ruprecht

  Journal of medical primatology. 10/2010; 39(5):356-60.

  A Chinese rhesus macaque infected with the pathogenic CCR5-tropic clade C simian-human immunodeficiency virus, SHIV-1157ipd3N4, had persistent viremia, depletion of CD4(+) T cells to <200 cells/μl,A Chinese rhesus macaque infected with the pathogenic CCR5-tropic clade C simian-human immunodeficiency virus, SHIV-1157ipd3N4, had persistent viremia, depletion of CD4(+) T cells to <200 cells/μl, opportunistic infections, coagulopathy, and gradual development of bilateral blindness. MRI revealed marked thickening of both optic nerves. Histopathological evaluation showed diffuse cellular infiltration at necropsy and a focus of SHIV-infected cells. This is the first report of CNS pathology following chronic infection with an obligate R5 SHIV.

• Bimodal AIDS vaccine approach: induction of cellular as well as humoral immunity can protect from systemic infection.

  Authors: Robert A Rasmussen, Samir K Lakhashe, Ruth M Ruprecht

  Vaccine. 05/2010; 28 Suppl 2:B25-31.

  HIV clade C (HIV-C) strains comprise approximately 56% of all HIV infections worldwide, and AIDS vaccines intended for global use must protect against this subtype. Our vaccine strategy has been toHIV clade C (HIV-C) strains comprise approximately 56% of all HIV infections worldwide, and AIDS vaccines intended for global use must protect against this subtype. Our vaccine strategy has been to induce balanced antiviral immunity consisting of both neutralizing antibody and cell-mediated immune responses, an approach we tested in primates. As reported earlier, after isolating recently transmitted HIV-C strains from Zambian infants, we used env from one such virus, HIV1084i, to generate a multimeric gp160 immunogen. From another virus, isolated from a different child of the same mother-infant cohort, we cloned env to generate a recombinant simian-human immunodeficiency virus (SHIV), which was adapted to rhesus monkeys to yield SHIV-1157ip. Infant macaques were immunized with recombinant viral proteins, including multimeric HIV-C Env 1084i. To test whether cross-protection could be achieved, we mismatched HIV-C Env immunogens and challenge virus env. All vaccinated and control monkeys were exposed orally to low-dose SHIV-1157ip. Animals with no or only transient infection were rechallenged intrarectally with a high dose of R5 SHIV-1157ipd3N4, a "late", animal-evolved variant of SHIV-1157ip. Compared to controls, the vaccinees had significantly lower peak viral RNA loads, and one vaccinee remained completely virus-free, even in lymphoid tissues. Data from our novel heterologous mucosal challenge model and our protein-only immunogens imply that significant protection against heterologous viruses circulating in the local community may be achievable with a strategy that seeks to simultaneously induce cellular immunity as well as neutralizing antibody responses.

• Relative transmissibility of an R5 clade C simian-human immunodeficiency virus across different mucosae in macaques parallels the relative risks of sexual HIV-1 transmission in humans via different routes.

  Authors: Agnès L Chenine, Nagadenahalli B Siddappa, Victor G Kramer, Gaia Sciaranghella, Robert A Rasmussen, Sandra J Lee, Michael Santosuosso, Mark C Poznansky, Vijayakumar Velu, Rama R Amara, Chris Souder, Daniel C Anderson, François Villinger, James G Else, Francis J Novembre, Elizabeth Strobert, Shawn P O'Neil, W Evan Secor, Ruth M Ruprecht

  The Journal of infectious diseases. 03/2010; 201(8):1155-63.

  Worldwide, approximately 90% of all human immunodeficiency virus (HIV) transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, thenWorldwide, approximately 90% of all human immunodeficiency virus (HIV) transmissions occur mucosally; almost all involve R5 strains. Risks of sexual HIV acquisition are highest for rectal, then vaginal, and finally oral exposures. Mucosal lacerations may affect the rank order of susceptibility to HIV but cannot be assessed in humans. We measured relative virus transmissibility across intact mucosae in macaques using a single stock of SHIV-1157ipd3N4, a simian-human immunodeficiency virus encoding a primary R5 HIV clade C env (SHIV-C). The penetrability of rhesus macaque mucosae differed significantly, with rectal challenge requiring the least virus, followed by vaginal and then oral routes (P = .031, oral vs vaginal; P < .001 rectal vs vaginal). These findings imply that intrinsic mucosal properties are responsible for the differential mucosal permeability. The latter paralleled the rank order reported for humans, with relative risk estimates within the range of epidemiological human studies. To test whether inflammation facilitates virus transmission--as predicted from human studies--we established a macaque model of localized buccal inflammation. Systemic infection occurred across inflamed but not normal buccal mucosa. Our primate data recapitulate virus transmission risks observed in humans, thus establishing R5 SHIV-1157ipd3N4 in macaques as a robust model system to study cofactors involved in human mucosal HIV transmission and its prevention.

• R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

  Authors: Nagadenahalli B Siddappa, Jennifer D Watkins, Klemens J Wassermann, Ruijiang Song, Wendy Wang, Victor G Kramer, Samir Lakhashe, Michael Santosuosso, Mark C Poznansky, Francis J Novembre, François Villinger, James G Else, David C Montefiori, Robert A Rasmussen, Ruth M Ruprecht

  PloS one. 01/2010; 5(7):e11689.

  HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. AlthoughHIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

• Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection.

  Authors: Sergei Popov, Saied Mirshahidi, Sosthène Essono, Ruijiang Song, Xiaowei Wang, Ruth M Ruprecht

  DNA and cell biology. 02/2009;

  We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interestWe developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase-deficient (Tk(-)) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk(-) rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins.

• Overlapping synthetic peptides encoding TPD52 as breast cancer vaccine in mice: Prolonged survival.

  Authors: Saied Mirshahidi, Victor G Kramer, James B Whitney, Sosthène Essono, Sandra Lee, Glenn Dranoff, Karen S Anderson, Ruth M Ruprecht

  Vaccine. 02/2009;

  Peptide-based vaccines, one of several anti-tumor immunization strategies currently under investigation, can elicit both MHC Class I-restricted (CD8(+)) and Class II-restricted (CD4(+)) responses.Peptide-based vaccines, one of several anti-tumor immunization strategies currently under investigation, can elicit both MHC Class I-restricted (CD8(+)) and Class II-restricted (CD4(+)) responses. However, the need to identify specific T-cell epitopes in the context of MHC alleles has hampered the application of this approach. We have tested overlapping synthetic peptides (OSP) representing a tumor antigen as a novel approach that bypasses the need for epitope mapping, since OSP contain all possible epitopes for both CD8(+) and CD4(+) T cells. Here we report that vaccination of inbred and outbred mice with OSP representing tumor protein D52 (TPD52-OSP), a potential tumor antigen target for immunotherapy against breast, prostate, and ovarian cancer, was safe and induced specific CD8(+) and CD4(+) T-cell responses, as demonstrated by development of specific cytotoxic T cell (CTL) activity, proliferative responses, interferon (IFN)-gamma production and CD107a/b expression in all mice tested. In addition, TPD52-OSP-vaccinated BALB/c mice were challenged with TS/A breast carcinoma cells expressing endogenous TPD52; significant survival benefits were noted in vaccine recipients compared to unvaccinated controls (p<0.001). Our proof-of-concept data demonstrate the safety and efficacy of peptide library-based cancer vaccines that obviates the need to identify epitopes or MHC backgrounds of the vaccinees. We show that an OSP vaccination approach can assist in the disruption of self-tolerance and conclude that our approach may hold promise for immunoprevention of early-stage cancers in a general population.

• Passive Immunization with Human Neutralizing Monoclonal Antibodies Against HIV-1 in Macaque Models: Experimental Approaches.

  Authors: Ruth M Ruprecht

  Methods in molecular biology (Clifton, N.J.). 02/2009; 525:1-8.

  After more than 20 years of intense research, a safe and effective vaccine against HIV-1/AIDS has not been developed. Passive immunization has been used as a tool to demonstrate the role ofAfter more than 20 years of intense research, a safe and effective vaccine against HIV-1/AIDS has not been developed. Passive immunization has been used as a tool to demonstrate the role of neutralizing antibodies in conferring protection against HIV-1 challenge in chimpanzees. Because these animals are endangered and studies are difficult to conduct with this species, chimeric viruses, termed simian-human immunodeficiency viruses (SHIVs), have been generated that encode the HIV-1 envelope gene in the backbone of the simian immunodeficiency virus (SIV). SHIVs replicate in several macaque species and can induce AIDS in these animals.Passive immunization with human neutralizing monoclonal antibodies (nmAbs) against HIV-1 has protected rhesus macaques from SHIV infection and provided proof-of-concept of the protective effects of neutralizing antibodies. At the same time, human nmAbs can be evaluated for safety and efficacy in the SHIV/macaque model as therapeutic modalities in their own right for prevention, post-exposure prophylaxis, or possibly therapeutic use. Experimental details are provided for testing human nmAbs in infant rhesus monkeys, which allows testing without the need to generate large amounts of nmAbs.