When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides.
Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.
Most human immunodeficiency virus (HIV) transmissions are initiated with CCR5 (R5)-using viruses across mucosal surfaces, with the majority in regions where HIV type 1 (HIV-1) clade C predominates. Mucosally transmissible, highly replication competent, pathogenic R5 simian-human immunodeficiency viruses (SHIVs) encoding biologically relevant clade C envelopes are therefore needed as challenge viruses in vaccine efficacy studies with nonhuman primates. Here we describe the generation of three lineage-related subtype C SHIVs through four successive rapid transfers in rhesus macaques of SHIVC109F.PB4, a molecular clone expressing the soluble-CD4 (sCD4)-sensitive CCR5-tropic clade C envelope of a recently infected subject in Zambia. The viruses differed in their monkey passage histories and neutralization sensitivities but remained R5 tropic. SHIVC109P3 and SHIVC109P3N were recovered from a passage-3 rapid-progressor animal during chronic infection (24 weeks postinfection [wpi]) and at end-stage disease (34 wpi), respectively, and are classified as tier 1B strains, whereas SHIVC109P4 was recovered from a passage-4 normal-progressor macaque at 22 wpi and is a tier 2 virus, more difficult to neutralize. All three viruses were transmitted efficiently via intrarectal inoculation, reaching peak viral loads of 107 to 109 RNA copies/ml plasma and establishing viremia at various set points. Notably, one of seven (GC98) and two of six (CL31, FI08) SHIVC109P3- and SHIVC109P3N-infected macaques, respectively, progressed to AIDS, with neuropathologies observed in GC98 and FI08, as well as coreceptor switching in the latter. These findings support the use of these new SHIVC109F.PB4-derived viruses to study the immunopathology of HIV-1 clade C infection and to evaluate envelope-based AIDS vaccines in nonhuman primates.
A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer), enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP) model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA), consists of two non-replicating recombinant adenoviral (Ad) vectors, one expressing the circumsporozoite protein (CSP) and another expressing the apical membrane antigen-1 (AMA1) of Plasmodium falciparum. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM) immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold) in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.
We previously showed that a prototype gel comprising zinc acetate (ZA) in carrageenan (CG) protected mice against vaginal and rectal herpes simplex virus 2 (HSV-2) challenge as well as macaques against vaginal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) challenge. In this work, we modified buffers and cosolvents to obtain a stable, nearly iso-osmolal formulation and evaluated its safety and efficacy against SHIV-RT and HSV-2. In vitro toxicity to lactobacilli and Candida albicans was determined. Macaques were given daily doses of ZA and CG (ZA/CG) or CG alone vaginally for 14 days and challenged with SHIV-RT 24 h later. Mice were challenged vaginally or rectally with HSV-2 immediately after a single gel treatment to measure efficacy or vaginally 12 h after daily gel treatment for 7 days to evaluate the gel's impact on susceptibility to HSV-2 infection. The modified ZA/CG neither affected the viability of lactobacilli or C. albicans nor enhanced vaginal HSV-2 infection after daily ZA/CG treatment. Vaginal SHIV-RT infection of macaques was reduced by 66% (P = 0.006) when macaques were challenged 24 h after the last dose of gel. We observed 60% to 80% uninfected mice after vaginal (P < 0.0001) and rectal (P = 0.008) high-dose HSV-2 challenge. The modified ZA/CG gel is safe and effective in animal models and represents a potential candidate to limit the transmission of HIV and HSV-2.
Mucosally transmissible and pathogenic CCR5 (R5)-tropic simian-human immunodeficiency virus (SHIV) molecular clones are useful reagents to identity neutralization escape in HIV-1 vaccine experiments and to study the envelope evolutionary process and mechanistic basis for coreceptor switch during the course of natural infection.
We observed progression to AIDS in rhesus macaques infected intrarectally with molecular clones of the pathogenic R5 SHIVSF162P3N isolate. Expansion to CXCR4 usage was documented in one diseased macaque that mounted a neutralizing antibody response and in another that failed to do so, with the latter displaying a rapid progressor phenotype. V3 loop envelop glycoprotein gp120 sequence changes that are predictive of a CXCR4 (X4)-using phenotype in HIV-1 subtype B primary isolates, specifically basic amino acid substations at positions 11 (S11R), 24 (G24R) and 25 (D25K) of the loop were detected in the two infected macaques. Functional assays showed that envelopes with V3 S11R or D25K mutation were dual-tropic, infecting CD4+ target cells that expressed either the CCR5 or CXCR4 coreceptor. And, consistent with findings of coreceptor switching in macaques infected with the pathogenic isolate, CXCR4-using variant was first detected in the lymph node of the chronically infected rhesus monkey several weeks prior to its presence in peripheral blood. Moreover, X4 emergence in this macaque coincided with persistent peripheral CD4+ T cell loss and a decline in neutralizing antibody titer that are suggestive of immune deterioration, with macrophages as the major virus-producing cells at the end-stage of disease.
The data showed that molecular clones derived from the R5 SHIVSF162P3N isolate are mucosally transmissible and induced disease in a manner similar to that observed in HIV-1 infected individuals, providing a relevant and useful animal infection model for in-depth analyses of host selection pressures and the env evolutionary changes that influence disease outcome, coreceptor switching and vaccine escape.
R5 SHIV molecular clone; Coreceptor switch; Antiviral antibody response; Macrophage infection
Development of an effective vaccine or topical compound to prevent HIV transmission remains a major goal for control of the AIDS pandemic. Using a nonhuman primate model of heterosexual HIV-1 transmission, we tested whether a topical microbicide that reduces viral infectivity can potentiate the efficacy of a T-cell-based HIV vaccine.
A DNA prime and rAd5 virus boost vaccination strategy was employed, and a topical microbicide against the HIV nucleocapsid protein was used. To rigorously test the combination hypothesis, the vaccine constructs contained only two transgenes and the topical microbicide inhibitor was used at a sub-optimal dose. Vaccinees were exposed in the absence and presence of the topical microbicide to repeated vaginal R5 SHIVSF162P3 challenge at an escalating dose to more closely mimic high-risk exposure of women to HIV.
Infection status was determined by PCR. Antiviral immune responses were evaluated by gp120 ELISA and intracellular cytokine staining.
A significant delay in SHIV acquisition (Log-rank test; p=0.0416) was seen only in vaccinated macaques that were repeatedly challenged in the presence of the topical microbicide. Peak acute viremia was lower (Mann-Whitney test; p=0.0387) and viral burden was also reduced (Mann-Whitney test; p=0.0252) in the combination-treated animals.
The combined use of a topical microbicide to lower the initial viral seeding/spread and a T-cell-based vaccine to immunologically contain the early virological events of mucosal transmission holds promise as a preventive approach to control the spread of the AIDS epidemic.
HIV; vaccine; topical microbicide; nucleocapsid inhibitor; prevention
Previously we showed that repeated vaginal application of a MIV-150/zinc acetate carrageenan (MIV-150/ZA/CG) gel and a zinc acetate carrageenan (ZA/CG) gel significantly protected macaques from vaginal simian human immunodeficiency virus reverse transcriptase (SHIV-RT) infection. Gels were applied either daily for 2 weeks or every other day for 4 weeks, and the animals were challenged 4–24 h later. Herein, we examined the effects of a single vaginal dose administered either before or after virus challenge. Encouraged by the vaginal protection seen with MIV-150/ZA/CG, we also tested it rectally. Vaginal applications of MIV-150/ZA/CG, ZA/CG, and CG gel were performed once 8–24 h before, 1 h after, or 24 h before and 1 h after vaginal challenge. Rectal applications of MIV-150/ZA/CG and CG gel were performed once 8 or 24 h before rectal challenge. While vaginal pre-challenge and pre/post-challenge application of MIV-150/ZA/CG gel offered significant protection (88%, p<0.002), post-challenge application alone did not significantly protect. ZA/CG gel reduced infection prechallenge, but not significantly, and the effect was completely lost post-challenge. Rectal application of MIV-150/ZA/CG gel afforded limited protection against rectal challenge when applied 8–24 h before challenge. Thus, MIV-150/ZA/CG gel is a highly effective vaginal microbicide that demonstrates 24 h of protection from vaginal infection and may demonstrate efficacy against rectal infection when given close to the time of HIV exposure.
We previously showed that a carrageenan (CG) gel containing 50 μM MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8 h before challenge. Additionally, when 100 mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24 h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC50, greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo.
We recently described a coreceptor switch in rapid progressor (RP) R5 simian-human immunodeficiency virus SF162P3N (SHIVSF162P3N)-infected rhesus macaques that had high virus replication and undetectable or weak and transient antiviral antibody response (S. H. Ho et al., J. Virol. 81:8621-8633, 2007; S. H. Ho, N. Trunova, A. Gettie, J. Blanchard, and C. Cheng-Mayer, J. Virol. 82:5653-5656, 2008; and W. Ren et al., J. Virol. 84:340-351, 2010). The lack of antibody selective pressure, together with the observation that the emerging X4 variants were neutralization sensitive, suggested that the absence or weakening of the virus-specific humoral immune response could be an environmental factor fostering coreceptor switching in vivo. To test this possibility, we treated four macaques with 50 mg/kg of body weight of the anti-CD20 antibody rituximab every 2 to 3 weeks starting from the week prior to intravenous infection with SHIVSF162P3N for a total of six infusions. Rituximab treatment successfully depleted peripheral and lymphoid CD20+ cells for up to 25 weeks according to flow cytometry and immunohistochemical staining, with partial to full recovery in two of the four treated monkeys thereafter. Three of the four treated macaques failed to mount a detectable anti-SHIV antibody response, while the response was delayed in the remaining animal. The three seronegative macaques progressed to disease, but in none of them could the presence of X4 variants be demonstrated by V3 sequence and tropism analyses. Furthermore, viruses did not evolve early in these diseased macaques to be more soluble CD4 sensitive. These results demonstrate that the absence or diminution of humoral immune responses by itself is insufficient to drive the R5-to-X4 switch and the neutralization susceptibility of the evolving viruses.
Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α4β7 on lymphocytes. α4β7 can be engaged by HIV-1 on the cell-surface and CD4+ T cells expressing high levels of this integrin (α4β7high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α4β7high CD4+ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c+ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α4β7 expression on CD4+ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α4β7highCD4+ T cells. These factors may play a role in increasing the susceptibility to HIV-1.
The vast majority of HIV-1 infections occur through genital and rectal mucosa. A better understanding of the characteristics of the mucosal microenvironment that help HIV-1 replication is critical to developing strategies for prevention of HIV-1 transmission. HSV-2 infects genital and rectal mucosa and infected individuals carry an increased risk for HIV-1 infection. Clarifying the mechanisms involved in the increased susceptibility of HSV-2 positive individuals to HIV-1 infection may help understating the characteristics of mucosal microenvironment that facilitate HIV-1 transmission. We previously described a specific interaction between HIV-1 and integrin α4β7, a signature molecule that allows lymphocytes to gain access to the gut tissue, a major site of HIV-1 replication. Vitamin A and its metabolite, retinoic acid, have an important role in balancing the immune response in the gut and in the expression of integrin α4β7. Here we describe that HSV-2 rectal infection in monkeys increases the frequency of α4β7+ CD4+ T cells in blood and rectal tissue and that this could be at least partially explained by the ability of HSV-2 infected DCs to secrete retinoic acid and up-regulate α4β7 on CD4+ T cells. These phenomena could be responsible for increasing HIV-1 replication in DC-T cell co-cultures.
Repeated use, coitus-independent microbicide gels that do not contain antiretroviral agents also used as first line HIV therapy are urgently needed to curb HIV spread. Current formulations require high doses (millimolar range) of antiretroviral drugs and typically only provide short-term protection in macaques. We used the macaque model to test the efficacy of a novel combination microbicide gel containing zinc acetate and micromolar doses of the novel non-nucleoside reverse transcriptase inhibitor MIV-150 for up to 24 h after repeated gel application.
Methods and Findings
Rhesus macaques were vaginally challenged with SHIV-RT up to 24 h after repeated administration of microbicide versus placebo gels. Infection status was determined by measuring virologic and immunologic parameters. Combination microbicide gels containing 14 mM zinc acetate dihydrate and 50 µM MIV-150 afforded full protection (21 of 21 animals) for up to 24 h after 2 weeks of daily application. Partial protection was achieved with the MIV-150 gel (56% of control at 8 h after last application, 11% at 24 h), while the zinc acetate gel afforded more pronounced protection (67% at 8–24 h). Marked protection persisted when the zinc acetate or MIV-150/zinc acetate gels were applied every other day for 4 weeks prior to challenge 24 h after the last gel was administered (11 of 14 protected). More MIV-150 was associated with cervical tissue 8 h after daily dosing of MIV-150/zinc acetate versus MIV-150, while comparable MIV-150 levels were associated with vaginal tissues and at 24 h.
A combination MIV-150/zinc acetate gel and a zinc acetate gel provide significant protection against SHIV-RT infection for up to 24 h. This represents a novel advancement, identifying microbicides that do not contain anti-viral agents used to treat HIV infection and which can be used repeatedly and independently of coitus, and underscores the need for future clinical testing of their safety and ability to prevent HIV transmission in humans.
While mucosal responses are important for preventing infections with HIV, the optimal strategies for inducing them remain unclear. To evaluate vaccine strategies targeting the oral mucosal lymphoid tissue inductive sites as an approach to provide immunity at distal sites, we vaccinated healthy macaques via the palatine/lingual tonsils with aldrithiol 2 (AT-2) inactivated SIVmac239, combined with CpG-C immunostimulatory oligonucleotide (CpG-C ISS-ODN , C274) as the adjuvant. Macaques received 5 doses of C274 or control ODN C661 and AT-2 SIV on the tonsillar tissues every 6 weeks before being challenged rectally with SIVmac239, 8 weeks after the last immunization. Although no T or B cell responses were detected in the blood prior to challenge, Ab responses were detected in the rectum . Immunization with AT-2 SIV significantly reduced the frequency of infection compared to non-immunized controls, irrespective of adjuvant. In the vaccinated animals that became infected, peak viremias were somewhat reduced. SIV-specific responses were detected in the blood once animals became infected with no detectable differences between the differently immunized groups and the controls. This work provides evidence that vaccine immunogens applied to the oral mucosal-associated lymphoid tissues can provide benefit against rectal challenge, a finding with important implications for mucosal vaccination strategies.
SIV; mucosal; vaccine; CpG ISS-ODN
HIV-infected individuals rely on antiretroviral therapy (ART) to control viral replication. Despite abundant demonstrable benefits, the multiple limitations of ART point to the potential advantages of therapeutic vaccination approaches that could provide sustained host control of viral replication after discontinuation of ART. We provide evidence from a non-human primate model that a therapeutic vaccine applied to the tonsils can maintain low viral loads after cessation of ART.
Animals received 40 weeks of ART initiated 9 weeks after rectal SIVmac239 infection. During ART, animals were vaccinated (or not) with AT-2 inactivated SIVmac239 using CpG-C ISS-ODN (C274) or polyICLC as adjuvants. PolyICLC/AT-2 SIV vaccinated animals maintained viral loads <3×103 copies/ml for up to 16 weeks post-ART, whereas the C274/AT-2 SIV vaccinated and non-vaccinated animals' viremia ranged between 1×104–4×105 copies/ml (p<0.03). Neutralizing Ab activity in plasma was increased by polyICLC/AT-2 tonsillar vaccination under ART, compared to controls (p<0.03). Subsequent vaccination of all animals with polyICLC/AT-2 SIV in the absence of ART did not alter viral loads. Other immune parameters measured in blood and tissues were comparable between groups.
These results provide support for the potential benefit of mucosally delivered vaccines in therapeutic immunization strategies for control of AIDS virus infection.
The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost.
To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4+ cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4+ cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4+ cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4+ cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation.
We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology.
We previously reported coreceptor switch in rhesus macaques inoculated intravenously with R5 simian-human immunodeficiency virus SF162P3N (SHIVSF162P3N). Whether R5-to-X4 virus evolution occurs in mucosally infected animals and in which anatomic site the switch occurs, however, were not addressed. We herein report a change in coreceptor preference in macaques infected intrarectally with SHIVSF162P3N. The switch occurred in infected animals with high levels of virus replication and undetectable antiviral antibody response and required sequence changes in the V3 loop of the gp120 envelope protein. X4 virus emergence was associated with an accelerated drop in peripheral CD4+ T-cell count but followed rather than preceded the onset of CD4+ T-cell loss. The conditions, genotypic requirements, and patterns of coreceptor switch in intrarectally infected animals were thus remarkably consistent with those found in macaques infected intravenously. They also overlapped with those reported for humans, suggestive of a common mechanism for coreceptor switch in the two hosts. Furthermore, two independent R5-to-X4 evolutionary pathways were identified in one infected animal, giving rise to dual-tropic and X4 viruses which differed in switch kinetics and tissue localization. The dual-tropic switch event predominated early, and the virus established infection in multiple tissues sites. In contrast, the switch to X4 virus occurred later, initiating and expanding mainly in peripheral lymph nodes. These findings help define R5 SHIVSF162P3N infection of rhesus macaques as a model to study the mechanistic basis, dynamics, and sites of HIV-1 coreceptor switch.
Herpes simplex virus type-2 (HSV-2) infection enhances the transmission and acquisition of human immunodeficiency virus (HIV). This occurs in symptomatic and asymptomatic stages of HSV-2 infection, suggesting that obvious herpetic lesions are not required to increase HIV spread. An animal model to investigate the underlying causes of the synergistic action of the two viruses and where preventative strategies can be tested under such complex physiological conditions is currently unavailable.
We set out to establish a rhesus macaque model in which HSV-2 infection increases the susceptibility to vaginal infection with a model immunodeficiency virus (simian-human immunodeficiency virus, SHIV-RT), and to more stringently test promising microbicides. HSV-2 exposure significantly increased the frequency of vaginal SHIV-RT infection (n = 6). Although cervical lesions were detected in only ∼10% of the animals, long term HSV-2 DNA shedding was detected (in 50% of animals followed for 2 years). Vaginal HSV-2 exposure elicited local cytokine/chemokine (n = 12) and systemic low-level HSV-2-specific adaptive responses in all animals (n = 8), involving CD4+ and CD8+ HSV-specific T cells (n = 5). Local cytokine/chemokine responses were lower in co-infected animals, while simian immunodeficiency virus (SIV)-specific adaptive responses were comparable in naïve and HSV-2-infected animals (n = 6). Despite the increased frequency of SHIV-RT infection, a new generation microbicide gel, comprised of Carraguard® and a non-nucleoside reverse transcriptase inhibitor MIV-150 (PC-817), blocked vaginal SHIV-RT infection in HSV-2-exposed animals (n = 8), just as in naïve animals.
We established a unique HSV-2 macaque model that will likely facilitate research to define how HSV-2 increases HIV transmission, and enable more rigorous evaluation of candidate anti-viral approaches in vivo.
Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIVSF33A isolate revealed a threshold inoculum for establishment of systemic virus infection, and a dose dependency in overall viral burden and CD4+ T cell depletion. While exposure to inoculum size of 1000 or greater 50% tissue infectious dose (TCID50) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID50 of the same virus. The latter was characterized by intermittent detection of low level virus with no evidence of seroconversion or CD4+ T cell decline, but with signs of an ongoing antiviral T cell immune response. Upon vaginal re-challenge with the same limiting dose 11-12 weeks after the first, classic pathogenic X4 SHIVSF33A infection was established in four of the seven previously exposed seronegative macaques, implying enhanced susceptibility to systemic infection with prior exposure. Pre-existing peripheral SIV gag-specific CD4+ T cells were more readily demonstrable in macaques that became systemically infected following re-exposure than those that were not. In contrast, early presence of circulating polyfunctional cytokine secreting CD8+ T cells, or strong virus-specific proliferative responses in draining lymph nodes and in the gut associated lymphoid tissue (GALT) following the first exposure was associated with protection from systemic re-infection. These studies identify the gut and lymphoid tissues proximal to the genital tract as sites of robust CD8 T lymphocyte responses that contribute to containment of virus spread following vaginal transmission.
We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N (SHIVSF162P3N)-infected macaque CA28, supporting the use of this experimental system to examine factors that drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIVCA28NP) used both CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIVCA28NL) used CXCR4 almost exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4 usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent mutational pathways for change in the coreceptor preference of the R5 SHIVSF162P3N isolate in vivo.
Anti-HIV microbicides are being investigated in clinical trials and understanding how promising strategies work, coincident with demonstrating efficacy in vivo, is central to advancing new generation microbicides. We evaluated Carraguard® and a new generation Carraguard-based formulation containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (PC-817). Since dendritic cells (DCs) are believed to be important in HIV transmission, the formulations were tested for the ability to limit DC-driven infection in vitro versus vaginal infection of macaques with RT-SHIV (SIVmac239 bearing HIV reverse transcriptase). Carraguard showed limited activity against cell-free and mature DC-driven RT-SHIV infections and, surprisingly, low doses of Carraguard enhanced infection. However, nanomolar amounts of MIV-150 overcame enhancement and blocked DC-transmitted infection. In contrast, Carraguard impeded infection of immature DCs coincident with DC maturation. Despite this variable activity in vitro, Carraguard and PC-817 prevented vaginal transmission of RT-SHIV when applied 30 min prior to challenge. PC-817 appeared no more effective than Carraguard in vivo, due to the limited activity of a single dose of MIV-150 and the dominant barrier effect of Carraguard. However, 3 doses of MIV-150 in placebo gel at and around challenge limited vaginal infection, demonstrating the potential activity of a topically applied NNRTI. These data demonstrate discordant observations when comparing in vitro and in vivo efficacy of Carraguard-based microbicides, highlighting the difficulties in testing putative anti-viral strategies in vitro to predict in vivo activity. This work also underscores the potential of Carraguard-based formulations for the delivery of anti-viral drugs to prevent vaginal HIV infection.
The basis for the switch from CCR5 to CXCR4 coreceptor usage seen in ∼50% of human immunodeficiency virus type 1 (HIV-1) subtype B-infected individuals as disease advances is not well understood. Among the reasons proposed are target cell limitation and better immune recognition of the CXCR4 (X4)-tropic compared to the CCR5 (R5)-tropic virus. We document here X4 virus emergence in a rhesus macaque (RM) infected with R5-tropic simian/human immunodeficiency virus, demonstrating that coreceptor switch can happen in a nonhuman primate model of HIV/AIDS. The switch to CXCR4 usage in RM requires envelope sequence changes in the V3 loop that are similar to those found in humans, suggesting that the R5-to-X4 evolution pathways in the two hosts overlap. Interestingly, compared to the inoculating R5 virus, the emerging CXCR4-using virus is highly neutralization sensitive. This finding, coupled with the observation of X4 evolution and appearance in an animal with undetectable circulating virus-specific antibody and low cellular immune responses, lends further support to an inhibitory role of antiviral immunity in HIV-1 coreceptor switch.
We examined the effect of inoculum dose on SHIV transmission and infection. We found that repeated low-dose intravaginal exposure with either R5-SHIVSF162P3 or X4-SHIVSF33A results in infections that are blunted and rapidly controlled. Interestingly, although the transmission rate after all repeated exposures is comparable for the two viruses, the probability of low-dose vaginal transmission is greater for the X4 than R5 virus. Furthermore, X4-SHIVSF33A replication predominates in low-dose dually-exposed macaques, suggesting that it is better at establishing a systemic infection following transmission. However, X4-SHIVSF33A advantage in transmission and infection is not observed in macaques inoculated intravenously with low-dose mixed inoculum. The finding that although matched in tissue culture infectious dose, the X4 inoculum is more complex leads us to hypothesize that the greater genetic heterogeneity of the X4 virus population may have rendered it less susceptible to the severe bottleneck effects imposed by IVAG inoculation with small doses, allowing for greater probability of transmission and establishment of a generalized infection. These data have implications for HIV-1 transmission and infection in humans.
SHIV; vaginal transmission; dose effect
Emergence of drug-resistant strains of human immunodeficiency virus type 1 (HIV-1) is a major obstacle to successful antiretroviral therapy (ART) in HIV-infected patients. Whether antiviral immunity can augment ART by suppressing replication of drug-resistant HIV-1 in humans is not well understood, but can be explored in non-human primates infected with simian immunodeficiency virus (SIV). Rhesus macaques infected with live, attenuated SIV develop robust SIV-specific immune responses but remain viremic, often at low levels, for periods of months to years, thus providing a model in which to evaluate the contribution of antiviral immunity to drug efficacy. To investigate the extent to which SIV-specific immune responses augment suppression of drug-resistant SIV, rhesus macaques infected with live, attenuated SIVmac239Δnef were treated with the reverse transcriptase (RT) inhibitor tenofovir, and then challenged with pathogenic SIVmac055, which has a five-fold reduced sensitivity to tenofovir.
Replication of SIVmac055 was detected in untreated macaques infected with SIVmac239Δnef, and in tenofovir-treated, naïve control macaques. The majority of macaques infected with SIVmac055 experienced high levels of plasma viremia, rapid CD4+ T cell loss and clinical disease progression. By comparison, macaques infected with SIVmac239Δnef and treated with tenofovir showed no evidence of replicating SIVmac055 in plasma using allele-specific real-time PCR assays with a limit of sensitivity of 50 SIV RNA copies/ml plasma. These animals remained clinically healthy with stable CD4+ T cell counts during three years of follow-up. Both the tenofovir-treated and untreated macaques infected with SIVmac239Δnef had antibody responses to SIV gp130 and p27 antigens and SIV-specific CD8+ T cell responses prior to SIVmac055 challenge, but only those animals receiving concurrent treatment with tenofovir resisted infection with SIVmac055.
These results support the concept that anti-viral immunity acts synergistically with ART to augment drug efficacy by suppressing replication of viral variants with reduced drug sensitivity. Treatment strategies that seek to combine immunotherapeutic intervention as an adjunct to antiretroviral drugs may therefore confer added benefit by controlling replication of HIV-1, and reducing the likelihood of treatment failure due to the emergence of drug-resistant virus, thereby preserving treatment options.
We used experimental infection of rhesus macaques with envelope gp120 V3 loop isogenic simian-human immunodeficiency virus (SHIV) molecular clones to more clearly define the impact of human immunodeficiency virus type 1 coreceptor usage in target cell selectivity and the rates of CD4+-T-cell depletion. Functional assays demonstrate that substitution of the V3 loop of the pathogenic CXCR4-tropic (X4) SHIVSF33A2 molecular clone with the corresponding sequences from the CCR5-tropic (R5) SHIVSF162P3 isolate resulted in a switch of coreceptor usage from CXCR4 to CCR5. The resultant R5 clone, designated SHIVSF33A2(V3), is replication competent in vivo, infecting two of two macaques by intravenous inoculation with peak viremia that is comparable to that seen in monkeys infected with X4-SHIVSF33A2. But while primary infection with the X4 clone was accompanied by rapid and significant loss of peripheral and secondary lymphoid CD4+ T lymphocytes, infection with R5-SHIVSF33A2(V3) led to only a modest and transient loss. However, substantial depletion of intestinal CD4+ T cells was observed in R5-SHIVSF33A2(V3)-infected macaques. Moreover, naïve T cells that expressed high levels of CXCR4 were rapidly depleted in X4-SHIVSF33A2-infected macaques, whereas R5-SHIVSF33A2(V3) infection mainly affected memory T cells that expressed CCR5. These findings in a unique isogenic system illustrate that coreceptor usage is the principal determinant of tissue and target cell specificity of the virus in vivo and dictates the dynamics of CD4+-T-cell depletion during SHIV infection.
Immunization with a killed or inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic coronaviruses (CoVs). However, the promise of this approach in humans is hampered by serious concerns over the risk of leaking live severe acute respiratory syndrome (SARS) viruses. In this study, we generated a SARS vaccine candidate by using the live-attenuated modified vaccinia virus Ankara (MVA) as a vector. The full-length SARS-CoV envelope Spike (S) glycoprotein gene was introduced into the deletion III region of the MVA genome. The newly generated recombinant MVA, ADS-MVA, is replication incompetent in mammalian cells and highly immunogenic in terms of inducing potent neutralizing antibodies in mice, rabbits, and monkeys. After two intramuscular vaccinations with ADS-MVA alone, the 50% inhibitory concentration in serum was achieved with reciprocal sera dilutions of more than 1,000- to 10,000-fold in these animals. Using fragmented S genes as immunogens, we also mapped a neutralizing epitope in the region of N-terminal 400 to 600 amino acids of the S glycoprotein (S400-600), which overlaps with the angiotensin-converting enzyme 2 (ACE2) receptor-binding region (RBR; S318-510). Moreover, using a recombinant soluble RBR-Fc protein, we were able to absorb and remove the majority of the neutralizing antibodies despite observing that the full S protein tends to induce a broader spectrum of neutralizing activities in comparison with fragmented S proteins. Our data suggest that a major mechanism for neutralizing SARS-CoV likely occurs through blocking the interaction between virus and the cellular receptor ACE2. In addition, ADS-MVA induced potent immune responses which very likely protected Chinese rhesus monkeys from pathogenic SARS-CoV challenge.