The nonnucleoside reverse transcriptase inhibitor MIV-150 in carrageenan gel prevents rectal transmission of simian/human immunodeficiency virus infection in macaques.

Development of a microbicide that prevents rectal transmission of human immunodeficiency virus (HIV) is a vital component in reducing HIV spread. We recently demonstrated that a formulation of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan reduced vaginal infection of macaques with simian immunodeficiency virus SIVmac239 with HIV-1(HxB2) reverse transcriptase (SHIV-RT). Herein, we performed the first testing of MIV-150-carrageenan against rectal infection. Rhesus macaques were treated rectally with MIV-150-carrageenan or methyl cellulose (MC) placebo gel up to 4 h prior to rectal challenge with 10³ or 10(4) 50% tissue culture infective doses (TCID50) of SHIV-RT. Infection was assessed by measuring plasma virus RNA as well as T and B cell responses. MIV-150-carrageenan protected all animals challenged with 10³ TCID(50 when gel was applied either 30 min or 4 h prior to challenge, while 100% of the MC-treated animals became infected (n = 4 each; P < 0.03). Partial protection (2 of 4 animals) by MIV-150-carrageenan was observed for rectal challenge with 10-fold more virus applied 4 h after the gel. Sequencing of the RT gene from plasma virus RNA isolated at peak viremia confirmed that both of these animals (like infected MC controls) were infected with wild-type virus. Infection correlated with the development of SIV-specific T and B cell responses. MIV-150 was detected in the rectal fluids and tissues 4 h after gel application but was not detected in the blood at any time (0.5 to 24 h). These data are promising for the development of NNRTI-containing gels to prevent rectal HIV transmission.

Progesterone implants enhance SIV vaginal transmission and early virus load.

Simian immunodeficiency virus (SIV) can cross the intact vaginal epithelium to establish a systemic infection in macaques (mac). Using this SIVmac model, we found that subcutaneous progesterone implants, which could mimic hormonally based contraceptives, thinned the vaginal epithelium and enhanced SIV vaginal transmission 7.7-fold over that observed in macaques treated with placebo implants and exposed to SIV in the follicular phase of the menstrual cycle. Progesterone treatment also increased the number of SIV DNA-positive cells in the vaginal lamina propria as detected by in situ polymerase chain reaction analysis. Moreover, plasma viral RNA was elevated for the first three months in macaques with progesterone implants, and three of the progesterone-treated macaques developed relatively rapid disease courses. This study shows that SIV genital infection and disease course are enhanced by subcutaneous implants containing progesterone when compared with the rate of vaginal transmission in the follicular phase.

Effects of in vivo CD8(+) T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine.

The role of CD8(+) T lymphocytes in controlling replication of live, attenuated simian immunodeficiency virus (SIV) was investigated as part of a vaccine study to examine the correlates of protection in the SIV/rhesus macaque model. Rhesus macaques immunized for >2 yr with nef-deleted SIV (SIVmac239Deltanef) and protected from challenge with pathogenic SIVmac251 were treated with anti-CD8 antibody (OKT8F) to deplete CD8(+) T cells in vivo. The effects of CD8 depletion on viral load were measured using a novel quantitative assay based on real-time polymerase chain reaction using molecular beacons. This assay allows simultaneous detection of both the vaccine strain and the challenge virus in the same sample, enabling direct quantification of changes in each viral population. Our results show that CD8(+) T cells were depleted within 1 h after administration of OKT8F, and were reduced by as much as 99% in the peripheral blood. CD8(+) T cell depletion was associated with a 1-2 log increase in SIVmac239Deltanef plasma viremia. Control of SIVmac239Deltanef replication was temporally associated with the recovery of CD8(+) T cells between days 8 and 10. The challenge virus, SIVmac251, was not detectable in either the plasma or lymph nodes after depletion of CD8(+) T cells. Overall, our results indicate that CD8(+) T cells play an important role in controlling replication of live, attenuated SIV in vivo.

Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques.

To determine the role of CD8(+) T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8(+) T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo.

A fusion inhibitor prevents spread of immunodeficiency viruses, but not activation of virus-specific T cells, by dendritic cells.

Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4(+) T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4(+), not CD8(+), T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4(+) T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4(+) and CD8(+) T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4(+) T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.

Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs.

Infection of macaques with chimeric simian-human immunodeficiency virus (SHIV) provides an excellent in vivo model for examining the influence of envelope on HIV-1 pathogenesis. Infection with a pathogenic CCR5 (R5)-specific enveloped virus, SHIVSF162P, was compared with infection with the CXCR4 (X4)-specific SHIVSF33A.2. Despite comparable levels of viral replication, animals infected with the R5 and X4 SHIV had distinct pathogenic outcomes. SHIVSF162P caused a dramatic loss of CD4+ intestinal T cells followed by a gradual depletion in peripheral CD4+ T cells, whereas infection with SHIVSF33A.2 caused a profound loss in peripheral T cells that was not paralleled in the intestine. These results suggest a critical role of co-receptor utilization in viral pathogenesis and provide a reliable in vivo model for preclinical examination of HIV-1 vaccines and therapeutic agents in the context of the HIV-1 envelope protein.

Protection against vaginal SIV transmission with microencapsulated vaccine.

Although protection in animal models against intravenous challenges with simian immunodeficiency virus (SIV) has been reported, no previous vaccines have protected against a heterosexual route of infection. In this study, five of six macaques were protected against vaginal challenge when immunized with formalin-treated SIV in biodegradable microspheres by the intramuscular plus oral or plus intratracheal route. Oral immunization alone did not protect. After a second vaginal challenge, three of four intramuscularly primed and mucosally boosted macaques remained protected. The data suggest that protection against human immunodeficiency virus vaginal transmission could be provided by microsphere-based booster vaccines when used to immunize women who are systemically primed.

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques.

Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20(+) B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3-7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-alpha] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1alpha/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

DNA-immunization with a V2 deleted HIV-1 envelope elicits protective antibodies in macaques.

Rhesus macaques immunized with the HIV-1 SF162DeltaV2 gp140 envelope using the DNA-prime plus protein-boost vaccination methodology, developed HIV envelope-specific T-cell lymphoproliferative responses and potent neutralizing antibodies. To evaluate the protective potential of these antibodies during acute infection, the animals were depleted of their CD8+ T lymphocytes using specific monoclonal antibodies and subsequently challenged intravenously with the pathogenic SHIV(SF162P4) isolate. As compared to non-vaccinated animals (one of which died from AIDS 16 weeks post-exposure) the vaccinated macaques had lower levels of peak viremia, rapidly cleared virus from the periphery and developed delayed seroconversion to SIV core antigens.

Comparison of restimulation methods to elicit SIV specific cytotoxic T-lymphocytes (CTL) in vitro: Staphylococcal enterotoxin B (SEB) provides a novel method for the quantification of SIV specific CTL precursors.

Most investigators believe that an effective HIV-1 vaccine will have to induce high levels of HIV-1 specific cytotoxic T-cells (CTL). The macaque SIV challenge/protection model system has been used to test candidate vaccines, but quantitative immunogenicity measurements are difficult due to technical limitations of the assays available. The quantification of SIV specific CTLp is crucial to understanding correlates of immunity for these vaccines, but are difficult to measure. We have compared various methods to quantify SIV specific CTLp, and describe a novel method of SIV specific CTL in vitro stimulation using the superantigen Staphylococcal enterotoxin B (SEB). SEB can stimulate high levels of CTLp in vitro, and provides an alternative method to induce SIV specific CTL.

Vaginal transmission of SIV: assessing infectivity and hormonal influences in macaques inoculated with cell-free and cell-associated viral stocks.

Cell associated and cell-free simian immunodeficiency virus (SIV) were used to investigate transmission of SIV across the vaginal mucosa of rhesus macaques. The intact vaginal epithelium was found to be a strong but penetrable barrier to cell-free SIV infection. We found that 10,000-fold more cell-free SIV was needed to infect 100% of the macaques by the vaginal route when compared to the dose needed to infect 100% by the intravenous (i.v.) route. Like cell-free SIV, cell-associated SIV was an efficient means of transmission if given by the i.v. route; as few as 2 SIV-infected peripheral blood mononuclear cells (PBMC) were infectious inoculum. However, macaques were resistant to cell-associated SIV when exposed by the vaginal route; 10,000 SIV-infected PBMC failed to infect vaginally inoculated macaques. It was also found that vaginal transmission of cell-free SIV to macaques increased during the luteal phase of the menstrual cycle compared to the follicular phase. Results with this animal model predict that cell-free human immunodeficiency virus (HIV) is likely to be the more efficient mode of HIV vaginal transmission and that susceptibility may vary during the menstrual cycle.

The immunodeficiency virus coreceptor, Bonzo/STRL33/TYMSTR, is expressed by macaque and human skin- and blood-derived dendritic cells.

Dendritic cells (DCs) have been shown to be important in the replication of human and simian immunodeficiency viruses (HIV and SIV, respectively) in vivo and in vitro. DCs express CD4 and several chemokine receptors, such as CCR5 and CXCR4, which are important for viral entry. In vivo, DCs are abundant at body surfaces, where they might be one of the first cells that encounter naturally transmitted virus. Furthermore, DCs pulsed with HIV or SIV in vitro can efficiently transmit virus to T cells, thereby propagating vigorous viral replication. Reports have implicated Bonzo/STRL33/TYMSTR to be an additional alternative coreceptor for HIV and especially SIV infection. However, at present there are no reports regarding the expression of Bonzo/STRL33/TYMSTR by human or macaque DCs. Here we demonstrate the presence of Bonzo/STRL33/TYMSTR transcripts in rhesus macaque and human skin-derived DCs, in immature and mature blood monocyte-derived DCs, and in T cells from both skin and blood. Therefore, Bonzo/STRL33/TYMSTR is expressed in DCs and T cells that can play a role in the transmission of immunodeficiency viruses.

Combined systemic and mucosal immunization with microsphere-encapsulated inactivated simian immunodeficiency virus elicits serum, vaginal, and tracheal antibody responses in female rhesus macaques.

We determined the efficacy of immunization with microsphere-encapsulated whole inactivated simian immunodeficiency virus (SIV) by combined systemic and mucosal administration to protect female rhesus macaques against vaginal challenge with homologous rhesus PBMC-grown SIVmac251. Animals in one group were primed and boosted intramuscularly. Two groups were primed intramuscularly and boosted either intratracheally or orally. A final group was primed by vaccinia/rgp140 scarification and subdivided for either intratracheal or oral boosting. Strong ELISA titers of circulating SIV-specific IgG and modest IgA responses were elicited in the animals primed intramuscularly. Intratracheal boosting in the intramuscularly primed macaques resulted in high bronchial alveolar wash (BAW) IgG and less pronounced IgA. SIV-specific vaginal wash (VW) IgG was also present in the intramuscular/intramuscular and intramuscular/intratracheal groups. Vaccinia/rgp140 priming gave low ELISA titers to whole SIV, and failed to elicit mucosal antibody regardless of the booster route. No animal in any group developed serum neutralizing antibody to homologous SIVmac251. On vaginal challenge none of the immunized groups was infected at a lesser frequency than the unimmunized controls. These data suggest that the use of microspheres in a combined parenteral and mucosal regimen is an effective method of eliciting IgG and IgA antibody at mucosal surfaces.

The effect of contraceptives containing nonoxynol-9 on the genital transmission of simian immunodeficiency virus in rhesus macaques.

The ability of two nonoxynol-9 spermicide preparations to prevent the genital transmission of SIV in rhesus macaques was compared. Administration of one mL of contraceptive foam before the intravaginal inoculation of cell-free SIV prevented the genital transmission of SIV to three of six animals, and using one mL of contraceptive gel prevented the genital transmission of SIV to two of six animals. Thus, both contraceptive foams and gels containing nonoxynol-9 provided protection against the genital transmission of SIV.

A method of video-assisted thoracoscopic surgery for collection of thymic biopsies in rhesus monkeys (Macaca mulatta).

To support an infectious disease study, we developed a surgical procedure to collect serial thymic biopsies in rhesus monkeys (Macaca mulatta). In many instances in which thymic tissue is required from living animals, open surgical approaches (thoracotomies) are used, which result in greater postoperative pain and longer recovery periods than those associated with thoracoscopic procedures. Our intent was to develop a surgical procedure that allowed serial biopsy of the thymus with minimal surgical morbidity. We modified a previously published experimental method of thoracoscopic total thymectomy in the dog to collect thymic biopsies in M. mulatta. Of the 15 animals evaluated, 8 underwent two biopsy procedures separated by a 5- to 6-week interoperative interval. The other seven animals underwent a single biopsy procedure. Thymic tissue was collected successfully during all procedures, with an average surgical time of 15 min. No significant intra- or postoperative complications were noted, and the animals recoveries from the surgical procedures were uneventful. Minimally invasive thoracoscopic surgical techniques can be used successfully to collect thymic tissue from adult and juvenile rhesus monkeys with minimal surgical morbidity.

Primary SIVsm isolates use the CCR5 coreceptor from sooty mangabeys naturally infected in west Africa: a comparison of coreceptor usage of primary SIVsm, HIV-2, and SIVmac.

Genetically divergent strains of simian immunodeficiency virus (SIV) from macaques (mac), chimpanzees, and sooty mangabeys (SM) efficiently used rhesus and human CCR5 (R5), but not CXCR4 (xR4), for cell entry. Thus far, however, no studies have characterized primary SIVsm strains for their use of coreceptors derived from their own natural host. Coreceptor usage of two primary, blood-derived SIVsm isolates, SIVsmSL92b and SIVsmFNS from naturally infected sooty mangabeys, was determined. Primary SIVsm efficiently used SM-CCR5 expressed on HOS.CD4 and U87.CD4 cells. Sequence polymorphisms in CCR5 found in four sooty mangabeys did not alter viral entry. Unlike primary rhesus blood-derived R5-tropic SIVmac251, primary SM blood-derived R5-tropic SIVsm was strongly CD4 dependent. The SM-CXCR4 gene was fully functional for xR4-tropic primate lentiviruses, but was not used by primary SIVsm. Therefore, the lack of xR4 tropism among naturally occurring SIVsm strains was not due to CxCR4 gene defects in the natural host. SIVmac derived from four macaques with AIDS also did not use macaque- or SM-derived CXCR4, showing that xR4 tropism did not develop during progression to disease as for humans infected with HIV-1. Three of four primary HIV-2 strains used CCR5 from human, sooty mangabey, and macaque. The fourth, HIV-27924A, obtained from a patient with AIDS, was xR4-tropic. Because SIVmac is most closely related to HIV-2, SIVmac might be expected to rnimic tropisms of HIV-2 infections. However, the correlation between xR4 tropism and AIDS may be a species-specific phenomenon limited to humans. The R5-tropic primary SIVsm and HIV-2 strains grew in CCR5-negative human PBMC, consistent with their use of non-CCR5 coreceptors. However, primary SIVsmSL92b did not use non-CCR5 coreceptors efficiently. The two primary SIVsm isolates replicated poorly in CEMx174 cells, which do not express CCR5, compared to CCR5-positive PM1 cells. SIVmac grew equally well in both cell lines. The findings show that SM-chemokine receptors are fully functional for virus entry and that multicoreceptor tropism is a common property of primary lentiviruses within the SIVsm/HIV-2 subfamily.

SIVrcm infection of macaques.

In a prior report, we described the isolation and characterization of SIVrcm, a distinct primate lentivirus found in a household pet Red-Capped Mangabey (RCM) in Gabon. SIVrcm is divergent from HIV-1 and HIV-2/SIV families of primate lentiviruses. In this report, additional in vitro replication studies and the results of SIVrcm infection in macaques are presented. SIVrcm causes little cytopathic effedct in Molt 4 Clone 8 cells and in rhesus and human PBMCs. In vivo, SIVrcm is non-pathogenic after 200 days in rhesus macaques and after one year in cynomolgous macaques, but does cause a chronic infection in both macaques.

In vivo adaptation of SHIV(SF162): chimeric virus expressing a NSI, CCR5-specific envelope protein.

The chemokine receptor CCR5 is known to be a critical determinant of human immunodeficiency virus (HIV) transmission and pathogenesis in the human host. Towards the development of a macaque model to evaluate the efficacy of vaccines and therapeutics against infection with CCR5-specific viruses, and to delineate the pathogenic properties of such viruses, we constructed a chimeric simian human immunodeficiency virus, SHIV(SF162), containing the env, tat, rev, and vpu genes from HIV-1(SF162) (R5, MT/NSI) in the context of the molecular clone simian immunodeficiency virus, SIV(mac239). Virus generated from this molecular clone was used to intravenously infect two juvenile macaques, followed by three consecutive serial blood/bone marrow transfusions. Animals infected with parental SHIV(SF162) (P1) had detectable levels of viral replication (as determined by p27(gag) production) within days of infection; however, viral set-points fell below detection by Week 3. Late passage animals (P3 and P4) had a two-log increase in the level of plasma p27(gag) antigen. These results demonstrate that in vivo serial passage of the R5-specific SHIV(SF162) enhanced its replicative capacity.

Enhanced infectivity of an R5-tropic simian/human immunodeficiency virus carrying human immunodeficiency virus type 1 subtype C envelope after serial passages in pig-tailed macaques (Macaca nemestrina).

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 strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.

Antigenic variations in the CD4 induced sites of the CCR5-tropic, pathogenic SHIVsf162p3 gp120 variants.

In vivo passage of non-pathogenic, CCR5-tropic simian/human immunodeficiency virus (SHIV) - SHIVsf162 resulted in a pathogenic isolate, SHIVsf162p3. In an attempt to characterize envelope (Env)-mediated properties that may contribute to its pathogenicity, major (P3 major) and minor (P3 minor) Env gp120 variants were cloned from the plasma of a SHIVsf162p3-infected animal, and expressed in the context of luciferase reporter viruses. Entry mediated by these envelopes and susceptibility to neutralization by CD4 induced-site (CD4i) antibodies (MAbs) was analyzed in comparison to parental SF162. Sequence analysis revealed that the P3 major and minor variant Envs contained 14 and 17 amino acid changes, respectively, compared with SF162. The rank order of entry mediated by the three envelopes was P3 major > SF162 > P3 minor, whereas the reverse order was observed for susceptibility to neutralization by CD4i MAbs. Since CD4i epitopes overlap the coreceptor (CoR) binding site, these findings suggest that the amino acid changes accumulated upon in vivo passage of SHIVsf162 result in Env gp120 structural rearrangements that modulate the exposure and/or conformation of the CoR binding site. This, in turn, led to increased entry and infectivity of the P3 major variant and may be responsible, in part, for the enhanced pathogenicity of SHIVsf162p3.