Protection in macaques immunized with HIV-1 candidate vaccines can be predicted using the kinetics of their neutralizing antibodies

Background

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.

Methods and Findings

We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV_SF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.

Conclusions

Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses.     

Induction of simian AIDS in infant rhesus macaques infected with CCR5- or CXCR4-utilizing simian-human immunodeficiency viruses is associated with distinct lesions of the thymus

Newborn rhesus macaques were infected with two chimeric simian-human immunodeficiency virus (SHIV) strains which contain unique human immunodeficiency virus type 1 (HIV-1) env genes and exhibit distinct phenotypes. Infection with either the CCR5-specific SHIV(SF162P3) or the CXCR4-utilizing SHIV(SF33A) resulted in clinical manifestations consistent with simian AIDS. Most prominent in this study was the detection of severe thymic involution in all SHIV(SF33A)-infected infants, which is very similar to HIV-1-induced thymic dysfunction in children who exhibit a rapid pattern of disease progression. In contrast, SHIV(SF162P3) induced only a minor disruption in thymic morphology. Consistent with the distribution of the coreceptors CXCR4 and CCR5 within the thymus, the expression of SHIV(SF162P3) was restricted to the thymic medulla, whereas SHIV(SF33A) was preferentially detected in the cortex. This dichotomy of tissue tropism is similar to the differential tropism of HIV-1 isolates observed in the reconstituted human thymus in SCID-hu mice. Accordingly, our results show that the SHIV-monkey model can be used for the molecular dissection of cell and tissue tropisms controlled by the HIV-1 env gene and for the analysis of mechanisms of viral immunopathogenesis in AIDS. Furthermore, these findings could help explain the rapid progression of disease observed in some HIV-1-infected children.     

Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.     

Improved delivery of broadly neutralizing antibodies by nanocapsules suppresses SHIV infection in the CNS of infant rhesus macaques

Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIV_SF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.     

An Envelope Modification That Renders a Primary, Neutralization-Resistant Clade B Human Immunodeficiency Virus Type 1 Isolate Highly Susceptible to Neutralization by Sera from Other Clades

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162ΔV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162ΔV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162ΔV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.     

Quaternary Epitope Specificities of Anti-HIV-1 Neutralizing Antibodies Generated in Rhesus Macaques Infected by the Simian/Human Immunodeficiency Virus SHIVSF162P4

Monoclonal antibodies (MAbs) that neutralize human immunodeficiency virus type 1 (HIV-1) have been isolated from HIV-1-infected individuals or animals immunized with recombinant HIV-1 envelope (Env) glycoprotein constructs. The epitopes of these neutralizing antibodies (NAbs) were shown to be located on either the variable or conserved regions of the HIV-1 Env and to be linear or conformational. However, one neutralizing MAb, 2909, which was isolated from an HIV-1-infected subject, recognizes a more complex, quaternary epitope that is present on the virion-associated functional trimeric Env spike of the SF162 HIV-1 isolate. Here, we discuss the isolation of 11 anti-HIV NAbs that were isolated from three rhesus macaques infected with the simian/human immunodeficiency virus SHIV_SF162P4 and that also recognize quaternary epitopes. A detailed epitope mapping analysis of three of these rhesus antibodies revealed that their epitopes overlap that of the human MAb 2909. Despite this overall similarity in binding, however, differences in specific amino acid and glycosylation pattern requirements for MAb 2909 and the rhesus MAbs were identified. These results highlight similarities in the B-cell responses of humans and macaques to structurally complex neutralization epitopes on related viruses, HIV-1 and SHIV.HIV-1 infection typically elicits high levels of antibodies directed against the viral surface envelope (Env) glycoprotein, gp160. The initial anti-Env antibody response is nonneutralizing (28), but within 1 or 2 months after infection, neutralizing antibodies (NAbs) emerge which tend to be highly strain specific for the autologous virus and exhibit little or no neutralizing activity against heterologous HIV-1 strains (10, 22). However, several recent reports have indicated that approximately 25% of HIV-1-infected, antiretroviral-naïve patients develop broad cross-neutralizing antibody responses (5, 23, 26). In some cases, these broad neutralizing antibody responses can be mapped to the CD4-binding site of Env while in most cases a single epitope specificity cannot be identified to recapitulate the neutralizing breadth of the corresponding plasma (1, 4, 14, 15, 23, 25). Detailed analyses of the epitope specificities of broad plasma neutralizing antibody responses performed by several groups revealed the presence in HIV-positive (HIV⁺) plasmas of NAbs with as yet undefined epitope specificities (1, 15, 18, 23). It is possible that these undefined specificities include quaternary neutralizing epitopes (QNEs) and/or sugar molecules which coat the HIV Env spike expressed on the surface of viral particles.The human monoclonal antibody (MAb) 2909 recognizes a QNE present on the oligomeric Env spike present on the surface of HIV-1 SF162 virions (8). MAb 2909 can bind and neutralize SF162 virions but does not bind to the corresponding soluble SF162 Env. The binding of MAb 2909 to its QNE depends on the presence of the second and third variable regions of gp120 (the V2 and V3 loops, respectively). One particular amino acid at the amino terminal side of the V2 loop (K at position 158, based on the SF162 numbering, or position 160, based on the strain HxB2 numbering) appears to be critical for its binding (11). MAb 2909 was isolated from a person who was not infected with SF162, but a virus isolated from the donor of MAb 2909 bears a V2 loop with similarities to that of SF162 and, in particular, possesses the same K158 residue (M. K. Gorny, unpublished data). More recently, two additional human MAbs, PG9 and PG16, were isolated from a subject infected with clade A HIV-1 and were shown to bind to a QNE that also includes the V2 and V3 loops (30). In contrast, however, to the narrow neutralizing potential of MAb 2909, MAbs PG9 and PG16 display far broader neutralizing abilities.Similar to the infection of humans by HIV-1, chronic infection of rhesus macaques by simian/human immunodeficiency viruses (SHIVs) or chimpanzees by HIV-1 also results in the elicitation of potent NAbs against the autologous virus and, to a much lesser extent, against heterologous SHIV isolates or HIV-1 viruses (3, 6, 12, 17). Here, we describe a panel of MAbs from SHIV_SF162P4-infected rhesus macaques that demonstrates extremely potent neutralization against the homologous virus (that expresses the same Env as HIV-1 SF162) and that recognizes QNEs present on the surface of intact virions. Similar to the human MAbs 2909, PG9, and PG16, these rhesus macaque monoclonal antibodies (RhMAbs) recognize QNEs that include the V2 and V3 loops. Also, similar to MAb 2909, the RhMAbs neutralize only viruses expressing the SF162 Env. Consequently, we compared the fine epitope specificities of these RhMAbs to the epitope specificity of the human MAb 2909. Our detailed epitope mapping analysis reveals that although the human MAb 2909 and the RhMAbs recognize that same overall Env complex region, their specific requirements for binding differ. Thus, these studies of human and rhesus MAbs indicate that infection of humans and rhesus macaques with viruses expressing distinct Envs can result in the elicitation of antibodies that bind to overlapping conserved quaternary epitopes.     

Neutralizing Polyclonal IgG Present during Acute Infection Prevents Rapid Disease Onset in Simian-Human Immunodeficiency Virus SHIVSF162P3-Infected Infant Rhesus Macaques

Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIV_SF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression.     

In Vivo Evaluation of Safety and Toxicity of a Lactobacillus jensenii Producing Modified Cyanovirin-N in a Rhesus Macaque Vaginal Challenge Model

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across the cervicovaginal mucosa in women is influenced by many factors including the microbiota and the presence of underlying inflammation. It is important that potential HIV preventative agents do not alter the mucosal environment in a way that enhances HIV acquisition. We examined the impact of a “live” microbicide on the vaginal mucosal environment in a rhesus macaque repeated vaginal simian-HIV (SHIV_SF162P3) challenge model. The microbicide contained a human vaginal Lactobacillus jensenii expressing the HIV-1 entry inhibitor, modified Cyanovirin-N (mCV-N), and henceforth called LB-mCV-N. Macaques were colonized vaginally each week with LB-mCV-N and sampled six days after colonization for culturable bacteria, pH and cervical-vaginal cytokines during the duration of the six-week study. We show that macaques that retained the engineered LB-mCV-N strain in their vaginal microbiota, during SHIV challenge, had lower pH, when colonization levels were higher, and had no evidence of inflammatory cytokines. Indeed, Interleukin-13, a mediator of inflammation, was detected less often in LB-mCV-N colonized macaques than in controls and we found higher levels of Interleukin 1 receptor antagonist (IL-1RA) in LB-mCV-N colonized macaques during the SHIV challenge period. We noted an inverse correlation between levels of mucosal IL-1RA and peak plasma viral load, thus higher IL-1RA correlated with lower viral load in LB-mCV-N treated macaques. These data support the use of LB-mCV-N as a safe “live” microbicide and suggest that lactobacilli themselves may positively impact the mucosal environment.     

Addition of a single gp120 glycan confers increased binding to dendritic cell-specific ICAM-3-grabbing nonintegrin and neutralization escape to human immunodeficiency virus type 1

The potential role of dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) binding in human immunodeficiency virus transmission across the mucosal barrier was investigated by assessing the ability of simian-human immunodeficiency chimeric viruses (SHIVs) showing varying degrees of mucosal transmissibility to bind the DC-SIGN expressed on the surface of transfected cells. We found that gp120 of the highly transmissible, pathogenic CCR5-tropic SHIV(SF162P3) bound human and rhesus DC-SIGN with an efficiency threefold or greater than that of gp120 of the nonpathogenic, poorly transmissible parental SHIV(SF162), and this increase in binding to the DC-SIGN of the SHIV(SF162P3) envelope gp120 translated into an enhancement of T-cell infection in trans. The presence of an additional glycan at the N-terminal base of the V2 loop of SHIV(SF162P3) gp120 compared to that of the parental virus was shown to be responsible for the increase in binding to DC-SIGN. Interestingly, this glycan also conferred escape from autologous neutralization, raising the possibility that the modification occurred as a result of immune selection. Our data suggest that more-efficient binding of envelope gp120 to DC-SIGN could be relevant to the enhanced mucosal transmissibility of SHIV(SF162P3) compared to that of parental SHIV(SF162).     

Different Tempo and Anatomic Location of Dual-Tropic and X4 Virus Emergence in a Model of R5 Simian-Human Immunodeficiency Virus Infection

We previously reported coreceptor switch in rhesus macaques inoculated intravenously with R5 simian-human immunodeficiency virus SF162P3N (SHIV_SF162P3N). 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 SHIV_SF162P3N. 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 SHIV_SF162P3N infection of rhesus macaques as a model to study the mechanistic basis, dynamics, and sites of HIV-1 coreceptor switch.The human immunodeficiency virus (HIV) enters target cells via binding of the viral envelope glycoprotein to the CD4 receptor, triggering envelope conformational changes that allow for interaction with either the CCR5 or CXCR4 chemokine receptor (1, 3, 8, 15, 16, 18). Most HIV type 1 (HIV-1) transmissions are initiated with CCR5-using (R5) viruses (58, 68). With time, CXCR4-tropic (X4) viruses emerge and coexist with R5 viruses in close to 50% of subtype B-infected individuals, and this is accompanied by a rise in viremia, rapid CD4⁺ T-cell loss, and progression to disease (4, 7, 11, 34, 57, 65). The mechanistic basis and reasons for HIV-1 coreceptor switch, however, are still not well understood. Several factors including high viral load, low CD4⁺ T-cell numbers, reduced availability of CCR5⁺ cells, and progressive immune dysfunction have been proposed as playing important roles (48, 54). Since X4 virus emergence is associated with a faster rate of disease progression, insights into the determinants of HIV-1 coreceptor switch are of interest in understanding viral pathogenesis. Furthermore, with the introduction of CCR5 entry inhibitors as anti-HIV therapeutics (19, 23, 24, 38), there is a need not only to identify the presence of X4 variants in patients when treatment options are considered but also to understand the factors that influence X4 virus evolution. Although the majority of individuals failing on short-term CCR5 antagonist monotherapy harbor preexisting minor X4 variants (71), it is conceivable that given the right conditions and selective forces, inhibiting HIV-1 entry via CCR5 may drive the virus to evolve to CXCR4 usage and exacerbate disease. An animal model that faithfully recapitulates the process of coreceptor switch will be highly useful to study and identify the determinants and conditions that facilitate the change in coreceptor preference. In addition, an animal model provides the opportunity to track the kinetics of coreceptor switching at different anatomical sites, which may inform on the mechanisms of X4 virus emergence.In this regard, we recently reported coreceptor switch in two of nine rhesus macaques (RM) inoculated intravenously with simian-human immunodeficiency virus SF162P3N (SHIV_SF162P3N) that bears an HIV-1 CCR5-tropic Env (28, 29). In order to establish a reproducible model for coreceptor switch, however, it was crucial to document additional switching events. Furthermore, since the majority of HIV transmission occurs via mucosal surfaces, it was important to demonstrate coreceptor switch in macaques infected with R5 SHIV_SF162P3N by the mucosal route to validate this animal model in studying the in vivo evolution of HIV-1 coreceptor usage. Additionally, the tissue compartment(s) where CXCR4-using viruses evolve and expand is not well characterized. A recent study indicates that the thymus may play an important role in the evolution and/or amplification of coreceptor variants in pediatric HIV infection (56). Since the thymus is the primary source of T lymphopoiesis during early life (45) and since CXCR4 is the predominant coreceptor expressed on thymocytes (33, 64), this organ would seem to provide the ideal milieu for X4 amplification in infants and children. Indeed, we previously showed that whereas X4 SHIV infection of newborn RM resulted in severe thymic involution, R5 SHIV infection induced only a minor disruption in thymic morphology (55), lending support to the idea that the thymus is a preferred site for X4 replication in pediatric HIV infections. Nevertheless, thymopoietic function declines with age (17, 42, 60), and naïve T cells that express high levels of CXCR4 are also enriched in peripheral lymph nodes (5, 27, 36, 66). Thus, the role of the thymus and other lymphoid tissues in HIV-1 coreceptor switch in older individuals remains to be determined. To address these issues, we inoculated adult RM intrarectally (i.r.) with R5 SHIV_SF162P3N and performed frequent longitudinal blood and tissue samplings. Our goal was to document changes in coreceptor preference in mucosally infected macaques, as well as to obtain a more detailed picture of the kinetics and site of X4 virus evolution and amplification in vivo.     

Increased mucosal transmission but not enhanced pathogenicity of the CCR5-tropic,simian AIDS-inducing simian/human immunodeficiency virus SHIV(SF162P3) maps to envelope gp120

Through rapid serial transfer in vivo, the chimeric CCR5-tropic simian/human immunodeficiency virus SHIV(SF162) evolved from a virus that is nonpathogenic and poorly transmissible across the vaginal mucosa to a variant that still maintains CCR5 usage but which is now pathogenic and establishes intravaginal infection efficiently. To determine whether envelope glycoprotein gp120 is responsible for increased pathogenesis and transmissibility of the variant SHIV(SF162P3), we cloned and sequenced the dominant envelope gene (encoding P3 gp120) and characterized its functions in vitro. Chimeric SHIV(SF162) virus expressing P3 gp120 of the pathogenic variant, designated SHIV(SF162PC), was also constructed and assessed for its pathogenicity and mucosal transmissibility in vivo. We found that, compared to wild-type SHIV(SF162) gp120, P3 gp120 conferred in vitro neutralization resistance and increased entry efficiency of the virus but was compromised in its fusion-inducing capacity. In vivo, SHIV(SF162PC) infected two of two and two of three rhesus macaques by the intravenous and intravaginal routes, respectively. Nevertheless, although peak viremia reached 10(6) to 10(7) RNA copies per ml of plasma in some infected animals and was associated with depletion of gut-associated CD4(+) lymphocytes, none of the animals maintained a viral set point that would be predictive of progression to disease. Together, the data from this study suggest a lack of correlation between entry efficiency and cytopathic properties of envelope glycoproteins with viral pathogenicity. Furthermore, whereas env gp120 contains the determinant for enhanced mucosal transmissibility of SHIV(SF162P3), the determinant(s) of its increased virulence may require additional sequence changes in env gp41 and/or maps to other viral genes.     

Development of a pigtail macaque model of sexually transmitted infection/HIV coinfection using Chlamydia trachomatis, Trichomonas vaginalis, and SHIV(SF162P3)

Background Sexually transmitted infections (STIs) are associated with an increased risk of HIV infection. To model the interaction between STIs and HIV infection, we evaluated the capacity of the pigtail macaque model to sustain triple infection with Trichomonas vaginalis, Chlamydia trachomatis, and SHIV_SF162P3. Methods Seven SHIV_SF162P3‐infected pigtail macaques were inoculated with T. vaginalis only (n = 2), C. trachomatis only (n = 1), both T. vaginalis and C. trachomatis (n = 2), or control media (no STI; n = 2). Infections were confirmed by culture and/or nucleic acid testing. Genital mucosa was visualized by colposcopy. Results Characteristic gynecologic signs were observed for both STIs, but not in control animals. Manifestations were most prominent at days 7–10 post‐infection. STIs persisted between 4 and 6 weeks and were cleared with antibiotics. Conclusions These pilot studies demonstrate the first successful STI‐SHIV triple infection of pigtail macaques, with clinical presentation of genital STI symptoms similar to those observed in humans.     

Characterization of primary isolate-like variants of simian-human immunodeficiency virus

Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.     

Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques

Maternal HIV-1-specific antibodies are efficiently transferred to newborns, but their role in disease control is unknown. We administered neutralizing IgG, including the human neutralizing monoclonal IgG1b12, at levels insufficient to block infection, to six newborn macaques before oral challenge with simian-HIV strain SF162P3 (SHIV(SF162P3)). All of the macaques rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for six months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings.     

The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region

Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162DeltaV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162DeltaV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162DeltaV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162DeltaV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.     

“Resistance” to PSC-RANTES Revisited: Two Mutations in Human Immunodeficiency Virus Type 1 HIV-1SF162 or Simian-Human Immunodeficiency Virus SHIVSF162-p3 Do Not Confer Resistance

Resistance of human immunodeficiency virus type 1 (HIV-1) to small-molecule CCR5 inhibitors is well demonstrated, but resistance to macromolecular CCR5 inhibitors (e.g., PSC-RANTES) that act by both CCR5 internalization and receptor blockade had not been reported until recently (3). The report of a single simian-human immunodeficiency virus SHIV_SF162-p3 variant with one V3 and one gp41 sequence change in gp160 that conferred both altered replicative fitness and resistance to PSC-RANTES was therefore surprising. We introduced the same two mutations into both the parental HIV-1_SF162 and the macaque-adapted SHIV_SF162-p3 and found minor differences in entry fitness but no changes in sensitivity to inhibition by either PSC-RANTES or the small-molecule allosteric inhibitor TAK-779. We attribute the earlier finding to confounding fitness effects with inhibitor sensitivity.A recent study by Dudley et al. (3) claimed to be “the first to describe the immediate selection and infection of a drug-resistant SHIV [simian-human immunodeficiency virus] variant in the face of a protective vaginal microbicide, PSC-RANTES.” The article further concluded, “This rhesus CCR5-specific/PSC-RANTES resistance selection is particularly alarming given the relative homogeneity of the SHIV_SF162-p3 stock compared to the potential exposure to a heterogeneous HIV-1 [human immunodeficiency virus type 1] population in human transmission.” The study described a SHIV_SF162-p3 variant with two amino acid substitutions, K315R in the V3 loop region (present as a minor component of the p3 challenge stock) and N640D in HR2 of gp41, that conferred greater replicative fitness and greater relative resistance to both the CCR5 inhibitor PSC-RANTES (to which the single macaque harboring this variant had been exposed prior to infection) and the small-molecule allosteric CCR5 inhibitor TAK-779 (1).While the development of HIV-1 strains resistant to small-molecule CCR5 inhibitors has been observed (11, 14), this result was surprising for several reasons. First, the inhibitory mechanism of PSC-RANTES is different from those of the small-molecule allosteric inhibitors; the ability of the macromolecule to induce profound and prolonged intracellular coreceptor sequestration, together with its ability to sterically block coreceptor use, should provide additional barriers to the development of resistant viruses that retain use of CCR5 (10). Second, this interpretation is supported by the failure to generate PSC-RANTES-resistant strains in multiple long-term in vitro selection studies (R.N. and D.E.M., unpublished results). Finally, the development of escape mutants in an in vivo setting would be expected to require sustained inhibitory concentrations of the drug at sites of replication. The Dudley et al. study was based on a single-dose experiment under conditions in which even at the highest dose used, no detectable systemic exposure occurred (6).The determination of resistance can be confounded by the fitness of a virus isolate (7), and the claim of resistance to PSC-RANTES was surprising given that infection with the parental HIV-1_SF162 isolate with the consensus GPGR₃₁₅ sequence is highly susceptible to PSC-RANTES inhibition (D.E.M., unpublished data) (Fig. ​(Fig.11 and Table ​Table1).1). These concerns prompted us to determine the impact of the K315R and N640D sequence variants on the entry fitness and sensitivity of both HIV-1_SF162 and SHIV_SF162-p3 to PSC-RANTES or TAK-779 in a single-round infection assay using either human or rhesus CCR5-expressing U87.CD4 target cells. We felt that it was important to extend the experiments of Dudley et al. (3) to HIV-1 since it is inhibition of HIV-1 infection of humans that is the intended application of a microbicide containing PSC-RANTES or related recombinant molecules (4, 12). We used site-directed mutagenesis to create three variants of the wild-type SF162 env sequence (R315, N640): K315, N640; R315, D640 (equivalent to the “resistant” SHIV_SF162-p3 variant from macaque 584), and K315, D640. These four env genes were used to complement a luciferase reporter HIV-1 construct in a standard single-round infection assay that has the advantage of a dynamic range of up to 8 logs (9, 15). We found that D640 conferred a small but significant entry advantage over N640 in the single-cycle assay (Table ​(Table1),1), in agreement with the results reported by Dudley et al. (3). However, none of the SF162 mutations conferred any significant resistance to either PSC-RANTES or TAK-779 (Fig. ​(Fig.11 and Table ​Table1),1), whether or not we corrected for the modest difference in entry efficiency. We repeated these experiments using the SHIV_SF162-p3 env clone that has the same sequence as that used in the experiments of Dudley et al. (3, 5) to determine if the finding of resistance was related to the other sequence differences between the macaque-adapted SHIV and SF162. Neither R315, D640, nor the combination of the two “resistance” mutations conferred resistance to either PSC-RANTES or TAK-779 on target cells expressing either human or rhesus CCR5 (Table ​(Table11 and Fig. 1C and D). The D640 substitution again conveyed a small entry advantage over N640 (data not shown). We thus conclude that the two mutations in SHIV_SF162-p3 that were claimed to confer resistance to PSC-RANTES using either human or rhesus CCR5 for entry were selected by replicative fitness in macaque 584 and not by drug resistance. We find no evidence that the two mutations have any impact on the PSC-RANTES sensitivity of either HIV-1_SF162 or SHIV_SF162-p3 (Fig. ​(Fig.1),1), and we were unable to confirm the 5.5-fold increase in resistance to PSC-RANTES on target cells expressing human CCR5 or the 7-fold increase on target cells expressing rhesus CCR5 reported by Dudley et al. (3). We therefore attribute the conclusions of Dudley et al. (3) to confounding fitness effects with inhibitor sensitivity. Multiple rounds of replication in the assays employed by Dudley et al. (3) likely amplified the relatively minor differences in entry fitness that we (and they) observed and made the precise assessment of 50% inhibitory concentration (IC₅₀) values more difficult, particularly given that 10-fold dilutions of inhibitors were used in their experiments.Open in a separate windowFIG. 1.Inhibition of HIV-1_SF162 env mutants by PSC-RANTES. (A) Single-round infection assay performed with U87.CD4.human CCR5 target cells using the four SF162 sequence variants with half-log dilutions of PSC-RANTES added 30 min prior to infection. Data are relative light units (RLU) and are summarized in a different format in the first row of data in Table ​Table1.1. (B) Means ± standard errors (SE) of the 50% inhibitory concentration of PSC-RANTES on each SF162 variant from three replicate experiments plotted as the reciprocal of the log IC₅₀ (pIC₅₀) in moles. Higher pIC₅₀ values indicate greater sensitivity to inhibition, but the differences depicted are not statistically significant. (C) Means ± SE of the 50% inhibitory concentrations of PSC-RANTES from three replicate experiments using the four sequence variants of SHIV_SF162-p3 with target cells expressing human CCR5. Note the different order of the columns for the SHIV_SF162-p3 variants; the “wild-type” SHIV has a different V3 sequence than the “wild-type” HIV-1_SF162, as well as 31 amino acid substitutions in other regions of envelope (5). (D) Means ± SE of the 50% inhibitory concentration of PSC-RANTES from three replicate experiments using the four sequence variants of SHIV_SF162-p3 with rhesus CCR5-expressing U87.CD4 target cells. WT, wild type; M, moles.

TABLE 1.

HIV-1 SF162 or SHIV_SF162-p3 V3 and/or HR2 mutations do not confer resistance to CCR5 inhibitors for entry via either human or rhesus CCR5

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.     

Pathogenic Consequences of Vaginal Infection with CCR5-Tropic Simian-Human Immunodeficiency Virus SHIVSF162P3N

We previously reported efficient transmission of the pathogenic R5 simian-human immunodeficiency virus SHIV(SF162P3N) isolate in Indian rhesus macaques by intravenous and intrarectal inoculations, with a switch to CXCR4 coreceptor usage in ～50% of infected animals that progressed rapidly to disease. Since women continue to be disproportionately affected by HIV, we developed an animal model based on the intravaginal challenge of female rhesus monkeys with SHIV(SF162P3N) and sought to validate the utility of this model to study relevant aspects of HIV transmission and pathogenesis. The effect of viral dose on infection outcome was evaluated to determine the optimal conditions for the evaluation of HIV-1 preventive and therapeutic strategies. We found that the virus can successfully cross the vaginal mucosal surface to establish infection and induce disease with coreceptor switch, but with lower efficiencies compared to intravenous and rectal transmissions. In contrast to intrarectal infection, peak and cumulative viral load over a 1 year-infection period were significantly greater in macaques exposed intravaginally to lower rather than higher inoculum doses. Moreover, low and transient viremia was observed only in macaques that were challenged intravaginally twice within the same day with a high dose of virus, which can be seen as doubling the dose. Taken together, these results show that SHIV(SF162P3N) can successfully transmit across the genital mucosa, undergo coreceptor switch, and induce disease. However, the administered dose appears to impact SHIV(SF162P3N) vaginal infection outcome in an unexpected manner.     

Rectal Application of a Highly Osmolar Personal Lubricant in a Macaque Model Induces Acute Cytotoxicity but Does Not Increase Risk of SHIV Infection

Background

Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication in vitro. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although in vivo evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15.

Methods

Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIV_SF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure.

Results

Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID₅₀ (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models).

Conclusions

Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the in vivo evaluation of lubricants with regards to HIV transmission.