Neutralising epitopes of simian immunodeficiency virus envelope glycoprotein

Abstract: Monoclonal and polyclonal antibodies with weak SIV neutralising activity bind to the V2 and V4 regions of gp120 or bind to the amino acids DWNND in gp41. Antibodies with the most potent neutralising activity recognise conformation-dependent epitopes involving the V3 and V4 regions of gp120. Monoclonal antibodies that map to the V3 region of SIV_mac failed to neutralise. However, one antibody to SIV AGM neutralised but only in the presence of soluble CD4.     

Protection Afforded by an HIV Vaccine Candidate in Macaques Depends on the Dose of SIVmac251 at Challenge Exposure

We used the simian immunodeficiency virus mac251 (SIV_mac251) macaque model to study the effect of the dose of mucosal exposure on vaccine efficacy. We immunized macaques with a DNA prime followed by SIV gp120 protein immunization with ALVAC-SIV and gp120 in alum, and we challenged them with SIV_mac251 at either a single high dose or at two repeated low-dose exposures to a 10-fold-lower dose. Infection was neither prevented nor modified following a single high-dose challenge of the immunized macaques. However, two exposures to a 10-fold-lower dose resulted in protection from SIV_mac251 acquisition in 3 out of 12 macaques. The remaining animals that were infected had a modulated pathogenesis, significant downregulation of interferon responsive genes, and upregulation of genes involved in B- and T-cell responses. Thus, the choice of the experimental model greatly influences the vaccine efficacy of vaccines for human immunodeficiency virus (HIV).     

A Twin-Cysteine Motif in the V2 Region of gp120 Is Associated with SIV Envelope Trimer Stabilization

The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 (“twin cysteines”) is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV_cpz lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV_cpz as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.     

ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge

Live-attenuated strains of simian immunodeficiency virus (SIV) routinely confer apparent sterilizing immunity against pathogenic SIV challenge in rhesus macaques. Understanding the mechanisms of protection by live-attenuated SIV may provide important insights into the immune responses needed for protection against HIV-1. Here we investigated the development of antibodies that are functional against neutralization-resistant SIV challenge strains, and tested the hypothesis that these antibodies are associated with protection. In the absence of detectable neutralizing antibodies, Env-specific antibody-dependent cell-mediated cytotoxicity (ADCC) emerged by three weeks after inoculation with SIVΔnef, increased progressively over time, and was proportional to SIVΔnef replication. Persistent infection with SIVΔnef elicited significantly higher ADCC titers than immunization with a non-persistent SIV strain that is limited to a single cycle of infection. ADCC titers were higher against viruses matched to the vaccine strain in Env, but were measurable against viruses expressing heterologous Env proteins. In two separate experiments, which took advantage of either the strain-specificity or the time-dependent maturation of immunity to overcome complete protection against SIV_mac251 challenge, measures of ADCC activity were higher among the SIVΔnef-inoculated macaques that remained uninfected than among those that became infected. These observations show that features of the antibody response elicited by SIVΔnef are consistent with hallmarks of protection by live-attenuated SIV, and reveal an association between Env-specific antibodies that direct ADCC and apparent sterilizing protection by SIVΔnef.     

The immunopathogenesis of retroviral diseases: No immunophenotypic alterations in T,B, and NK cell subsets in SIVmac239-challenged rhesus macaques protected by SIVΔnef vaccination

Abstract: Immunophenotype analysis was used to characterize circulating lymphocyte subset levels in both rhesus monkeys that were chronically infected with SIV_mac239 and in those that had resisted SIV_mac239 infection as a result of prior vaccination with an attenuated SIV strain. Alterations in T, NK, and B cell subsets were compared with those previously identified in humans chronically infected with HIV [8–11, 14, 22]. The well-known decrease in CD4⁺ cell levels was observed in the SIV_mac239-infected animals. However, these animals had relatively little activation of circulating CD8⁺ T cells as compared with uninfected monkeys. This contrasts with chronically HIV-infected humans who have substantial activation of circulating CD8⁺ cells as evidenced by elevated HLA-DR and CD38 antigen expression on CD8⁺ cells as well as substantially increased percentages and numbers of total CD8⁺ cells. NK cells of the SIV_mac239-infected animals, on the other hand, demonstrated the same changes recently described in HIV-infected humans, i.e., a decrease in circulating percentages and a decreased amount of FcRIII (CD 16). B cell percentages were markedly increased in the SIV_mac239-infected animals, a finding also noted in some children with HIV infection but not in HIV-infected adults. SIVΔnef-vaccinated/SIV_mac239-challenged animals showed none of the immune alterations found in the SIV_mac239-infected monkeys, providing further confirmation of lack of SIV disease in these vaccinated animals.     

Limited Protection from a Pathogenic Chimeric Simian-Human Immunodeficiency Virus Challenge following Immunization with Attenuated Simian Immunodeficiency Virus

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV_239Δnef and SIV_PBj6.6Δnef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV_239Δnef construct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIV_PBj6.6Δnef was effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV_89.6PD, by intravenous injection. All of the SIV_239Δnef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIV_PBj6.6Δnef totally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.     

Correlation of vaccine-elicited systemic and mucosal nonneutralizing antibody activities with reduced acute viremia following intrarectal simian immunodeficiency virus SIVmac251 challenge of rhesus macaques

Cell-mediated immunity and neutralizing antibodies contribute to control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infection, but the role of nonneutralizing antibodies is not defined. Previously, we reported that sequential oral/oral or intranasal/oral (I/O) priming with replication-competent adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants, followed by intramuscular envelope protein boosting, elicited systemic and mucosal cellular immunity and exhibited equivalent, significant reductions of chronic viremia after rectal SIV_mac251 challenge. However, I/O priming gave significantly better control of acute viremia. Here, systemic and mucosal humoral immunity were investigated for potential correlates with the acute challenge outcome. Strong serum binding but nonneutralizing antibody responses against SIV_mac251 were induced in both groups. Antibody responses appeared earlier and overall were higher in the I/O group. Reduced acute viremia was significantly correlated with higher serum binding titer, stronger antibody-dependent cellular cytotoxicity activity, and peak prechallenge and 2-week-postchallenge antibody-dependent cell-mediated viral inhibition (ADCVI). The I/O group consistently displayed greater anti-envelope immunoglobulin A (IgA) antibody responses in bronchoalveolar lavage and a stronger rectal anti-envelope IgA anamnestic response 2 weeks postchallenge. Pre- and postchallenge rectal secretions inhibited SIV transcytosis across epithelial cells. The inhibition was significantly higher in the I/O group, although a significant correlation with reduced acute viremia was not reached. Overall, the replicating Ad5hr-SIV priming/envelope boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities. The pattern of elevated immune responses in the I/O group is consistent with its better control of acute viremia mediated, at least in part, by ADCVI activity and transcytosis inhibition.     

Induction of immunodeficiency virus-specific immune responses in rhesus monkeys following gene gun-mediated DNA vaccination

Abstract: The Accell® gene delivery system (gene gun) was used to deliver gold particles coated with HIV-1_LAI and SIV_mac239 expression constructs into the epidermis of rhesus macaques, resulting in the elicitation of env- and gag-specific humoral responses. One microgram of vector DNA per dose was sufficient to induce immune responses in monkeys using SIV_mac239 gp160 and gp120 vectors driven by the CMV-intron A promoter. Several parameters, including the identity of the vector, the length of the rest period between immunizations, the number of immunizations, and the amount of DNA per immunization, are all important in designing an optimal DNA immunization regimen. In addition, gene gun-based DNA immunization using low efficiency expression vectors is an effective means of priming for the induction of vigorous antibody responses in macaques following boosting with recombinant subunits.     

Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV_mac239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4⁺ T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV_mac239. However, significantly lower viral loads and higher memory CD4⁺ T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV_mac239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.     

HIV-1 Specific IgA Detected in Vaginal Secretions of HIV Uninfected Women Participating in a Microbicide Trial in Southern Africa Are Primarily Directed Toward gp120 and gp140 Specificities

Background

Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.

Methods and Findings

We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges) for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env) antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035). We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.

Conclusion

Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.     

Dynamics of Memory B-Cell Populations in Blood,Lymph Nodes,and Bone Marrow during Antiretroviral Therapy and Envelope Boosting in Simian Immunodeficiency Virus SIVmac251-Infected Rhesus Macaques

Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection causes B-cell dysregulation and the loss of memory B cells in peripheral blood mononuclear cells (PBMC). These effects are not completely reversed by antiretroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cells/plasmablasts (PC/PB) in blood, bone marrow, and lymph nodes of rhesus macaques during ART and upon release from ART. Macaques previously immunized with SIV recombinants and the gp120 protein were included to assess the effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in numbers of activated memory B cells and increases in numbers of tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27⁺ or CD27⁻ and therefore were defined as CD19⁺ CD38^hi CD138⁺. The numbers of these PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC numbers in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment to allow the generation of virus-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Multiple Vaccine-Elicited Nonneutralizing Antienvelope Antibody Activities Contribute to Protective Efficacy by Reducing both Acute and Chronic Viremia following Simian/Human Immunodeficiency Virus SHIV89.6P Challenge in Rhesus Macaques

We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)_89.6P challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.A major goal of human immunodeficiency virus (HIV) vaccine development is the elicitation of protective antibodies capable of neutralizing the diversity of isolates in the worldwide pandemic (6, 61). Indeed, passively administered neutralizing antibodies have been shown to protect against pathogenic HIV/simian immunodeficiency virus (SIV) challenge in rhesus macaque models (4, 44, 45, 57). However, the extent to which other antibody-mediated protective mechanisms impact HIV/SIV infection is still unclear. Whether these alternate biologic activities would augment vaccine-induced protection has not been definitively established.In HIV-infected individuals, as in SIV- or simian/human immunodeficiency virus (SHIV)-infected rhesus macaques, systemic nonneutralizing antibodies appear early during acute infection, often preceding a neutralizing antibody response (21, 55). Although neutralizing antibody activity is critical for sterilizing immunity, recent studies suggest that antibodies may contribute to protection by other functional activities, such as antibody-dependent cellular cytotoxicity (ADCC) (20, 29), antibody-dependent cell-mediated viral inhibition (ADCVI) (22, 23), and transcytosis inhibition (19, 35, 59). Antibodies in secretions may directly block viral entry into intestinal and endocervical tissues by inhibiting transcytosis across epithelium, whereas local or serum-derived antibodies that mediate ADCC or ADCVI may exert protective effects by eliminating small foci of infected cells during the brief window of time that exists between transmission of virus across an epithelial cell barrier to the lamina propria and subsequent systemic spread (32). In support of this notion, mutation of the Fc portion of the broadly neutralizing monoclonal antibody, IgGb12, thereby preventing interaction with the FcγR on effector cells, rendered the antibody less able to mediate protection upon subsequent passive transfer and challenge of rhesus macaques (34). Thus, neutralizing antibodies themselves may mediate protection by additional functional activities.ADCC bridges innate and adaptive immunity. Mechanistically, it involves FcγR-bearing effector cells, such as NK cells, macrophages, neutrophils, and γδ T cells, and antibodies specific for antigens expressed on the surface of target cells. Upon interaction of these three components, the target cells are killed. Since the effector cells are not major histocompatibility complex restricted, ADCC is broadly applicable to diverse populations. Because the antibody specificity need not be restricted to neutralizing epitopes, ADCC may increase the breadth of antibody reactivity. In fact, we have shown that an HIV clade B immunization regimen elicited antibodies that mediated ADCC across several HIV clades (28). Antibodies that mediate ADCC have been shown to arise early in infection, before neutralizing antibodies (55, 60). They are present in the majority of infected individuals, and they have been associated with slow disease progression following both HIV and SIV infection (5, 8).ADCVI is closely related to ADCC, also requiring antibody that forms a bridge between an infected target cell and an FcγR-bearing effector cell (24). However, ADCVI is a broader activity not restricted solely to target cell lysis but, rather, encompassing several mechanisms by which viral replication following infection of target cells is inhibited. Thus, it may include ADCC activity but also involve noncytotoxic mechanisms of virus control, such as the secretion of inhibitory chemokines or FcγR-mediated phagocytosis of immune complexes (24, 25).Most HIV infections occur via a mucosal route, including cervicovaginal and rectal tissues (39, 52). Several nonmutually exclusive mechanisms for HIV-1 transmission across mucosal epithelia have been proposed (13, 56). Transcytosis of infectious virus across polarized columnar epithelial cells following contact of virally infected cells with apical epithelial cell surfaces is one mechanism for mucosal HIV entry (12). Rather than fusion and infection, interactions between the viral envelope proteins and epithelial surface molecules, such as glycosphingolipid galactosyl-ceramide (GalCer) (13, 47), an important component of endocytotic “raft” membrane microdomains, lead to transcytosis of the virus across the epithelial barrier and its trapping by submucosal dendritic cells which disseminate it to their target CD4⁺ T cells. Studies have shown that mucosal immunoglobulin A (IgA) antibody, a major component of the mucosal immune response, could block mucosal HIV-1 entry via transcytosis in vitro (2, 19). Therefore, mucosal antibodies blocking adherence of virus to epithelial cells and preventing HIV-1 transcytosis across the epithelial barrier and subsequent CD4⁺ T cell infection may afford additional protection against HIV/SIV infection.We have been pursuing a replicating adenovirus (Ad)-HIV/SIV prime/protein subunit boost AIDS vaccine approach (30, 51), which has elicited strong, durable protection against HIV, SIV, and SHIV challenges (11, 18, 41, 42, 50). An underlying goal of these studies has been elucidation of immune responses that correlate with protective efficacy. Recently, we studied the contribution of novel protein boosts to immunogenicity and protective efficacy in a SHIV_89.6P model (49). Immunized rhesus macaques were primed with Ad type 5 host range mutant (Ad5hr)-HIV_89.6Pgp140, -SIV₂₃₉gag, and -SIV₂₃₉nef recombinants. One group was not boosted, one was boosted with HIV_89.6P gp140ΔCFI protein (gp140 envelope with deletions in the cleavage site, fusion peptide, and part of the interspace between the two heptad repeats) (40), and one was boosted with a novel HIV-1 polypeptide “peptomer” representing the CD4 binding site of the envelope (54). The best protection was seen in the gp140-boosted group, with significant reductions in both acute and chronic viremia. Although Env-specific antibody and cellular responses were readily detected, none directly correlated with the better protection. Furthermore, neutralizing antibodies against SHIV_89.6P did not develop until 4 weeks postchallenge. Therefore, we hypothesized that vaccine-elicited nonneutralizing anti-Env antibodies might have contributed to the better control of acute and/or chronic viremia in the gp140 group. Here, we report retrospective evaluations of sera and rectal secretions from macaques in this comparative study for serum binding antibody avidity, an important characteristic of functional antibodies (38, 58), and nonneutralizing activities of systemic and mucosal antibodies, including ADCC, ADCVI, and transcytosis inhibition.     

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated,Low-Dose Vaginal Challenge

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV_mac239 or SIV_mac251_UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV_mac251_UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.Development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) is an urgent public health priority, but remains a formidable scientific challenge. Passive transfer experiments in macaques demonstrate neutralizing antibodies can prevent infection by laboratory-engineered simian-human immunodeficiency virus (SHIV) strains (6, 33, 34, 53, 59). However, no current vaccine approach is capable of eliciting antibodies that neutralize primary isolates with neutralization-resistant envelope glycoproteins. Virus-specific T-cell responses can be elicited by prime-boost strategies utilizing recombinant DNA and/or viral vectors (3, 10, 11, 16, 36, 73, 77, 78), which confer containment of viral loads following challenge with SHIV_89.6P (3, 13, 66, 68). Unfortunately, similar vaccine regimens are much less effective against SIV_mac239 and SIV_mac251 (12, 16, 31, 36, 73), which bear closer resemblance to most transmitted HIV-1 isolates in their inability to utilize CXCR4 as a coreceptor (18, 23, 24, 88) and inherent high degree of resistance to neutralization by antibodies or soluble CD4 (43, 55, 56). Live, attenuated SIV can provide apparent sterile protection against challenge with SIV_mac239 and SIV_mac251 or at least contain viral replication below the limit of detection (20, 22, 80). Due to the potential of the attenuated viruses themselves to cause disease in neonatal rhesus macaques (5, 7, 81) and to revert to a pathogenic phenotype through the accumulation of mutations over prolonged periods of replication in adult animals (2, 35, 76), attenuated HIV-1 is not under consideration for use in humans.As an experimental vaccine approach designed to retain many of the features of live, attenuated SIV, without the risk of reversion to a pathogenic phenotype, we and others devised genetic approaches for producing strains of SIV that are limited to a single cycle of infection (27, 28, 30, 38, 39, 45). In a previous study, immunization of rhesus macaques with single-cycle SIV (scSIV) trans-complemented with vesicular stomatitis virus (VSV) G elicited potent virus-specific T-cell responses (39), which were comparable in magnitude to T-cell responses elicited by optimized prime-boost regimens based on recombinant DNA and viral vectors (3, 16, 36, 68, 73, 78). Antibodies were elicited that neutralized lab-adapted SIV_mac251_LA (39). However, despite the presentation of the native, trimeric SIV envelope glycoprotein (Env) on the surface of infected cells and virions, none of the scSIV-immunized macaques developed antibody responses that neutralized SIV_mac239 (39). Therefore, we have now introduced Env modifications into scSIV that facilitate the development of neutralizing antibodies.Most primate lentiviral envelope glycoproteins are inherently resistant to neutralizing antibodies due to structural and thermodynamic properties that have evolved to enable persistent replication in the face of vigorous antibody responses (17, 46, 47, 64, 71, 75, 79, 83, 85). Among these, extensive N-linked glycosylation renders much of the Env surface inaccessible to antibodies (17, 48, 60, 63, 75). Removal of N-linked glycans from gp120 or gp41 by mutagenesis facilitates the induction of antibodies to epitopes that are occluded by these carbohydrates in the wild-type virus (64, 85). Consequently, antibodies from animals infected with glycan-deficient strains neutralize these strains better than antibodies from animals infected with the fully glycosylated SIV_mac239 parental strain (64, 85). Most importantly with regard to immunogen design, animals infected with the glycan-deficient strains developed higher neutralizing antibody titers against wild-type SIV_mac239 (64, 85). Additionally, the removal of a single N-linked glycan in gp120 enhanced the induction of neutralizing antibodies against SHIV_89.6P and SHIV_SF162 in a prime-boost strategy by 20-fold (50). These observations suggest that potential neutralization determinants accessible in the wild-type Env are poorly immunogenic unless specific N-linked glycans in gp120 and gp41 are eliminated by mutagenesis.The variable loop regions 1 and 2 (V1V2) of HIV-1 and SIV gp120 may also interfere with the development of neutralizing antibodies. Deletion of V1V2 from HIV-1 gp120 permitted neutralizing monoclonal antibodies to CD4-inducible epitopes to bind to gp120 in the absence of CD4, suggesting that V1V2 occludes potential neutralization determinants prior to the engagement of CD4 (82). A deletion in V2 of HIV-1 Env-exposed epitopes was conserved between clades (69), improved the ability of a secreted Env trimer to elicit neutralizing antibodies (9), and was present in a vaccine that conferred complete protection against SHIV_SF162P4 (8). A deletion of 100 amino acids in V1V2 of SIV_mac239 rendered the virus sensitive to monoclonal antibodies with various specificities (41). Furthermore, three of five macaques experimentally infected with SIV_mac239 with V1V2 deleted resisted superinfection with wild-type SIV_mac239 (51). Thus, occlusion of potential neutralization determinants by the V1V2 loop structure may contribute to the poor immunogenicity of the wild-type envelope glycoprotein.Here we tested the hypothesis that antibody responses to scSIV could be improved by immunizing macaques with strains of scSIV engineered to eliminate structural features that interfere with the development of neutralizing antibodies. Antibodies to Env-modified strains were selectively enhanced, but these did not neutralize the wild-type SIV strains. We then tested the hypothesis that immunization might prevent infection in a repeated, low-dose vaginal challenge model of heterosexual HIV-1 transmission. Indeed, while all six naïve control animals became infected, two of eight immunized animals remained uninfected after 20 weeks of repeated vaginal challenge. Relative to the naïve control group, reductions in peak and set point viral loads were statistically significant in the immunized animals that became infected.     

HIV-1 gp120 vaccine induces affinity maturation in both new and persistent antibody clonal lineages

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120_W6.1D). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V_H somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120_W6.1D was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.     

Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1

Background

One of the major functions of Nef is in the enhancement of the infectivity of the human and simian immunodeficiency viruses (HIV and SIV, respectively). However, the detailed mechanism of the enhancement of viral infectivity by Nef remains unclear. Additionally, studies of mechanisms by which Nef enhances the infectivity of SIV are not as intensive as those of HIV-1.

Methods

We generated short-lived Nef constructed by fusing Nef to a proteasome-mediated protein degradation sequence to characterize the Nef role in viral infectivity.

Results

The apparent expression level of the short-lived Nef was found to be extremely lower than that of the wild-type Nef. Moreover, the expression level of the short-lived Nef increased with the treatment with a proteasome inhibitor. The infectivity of HIV-1 with the short-lived Nef was significantly lower than that with the wild-type Nef. On the other hand, the short-lived Nef enhanced the infectivity of SIV_mac239, an ability observed to be interestingly equivalent to that of the wild-type Nef. The short-lived Nef was not detected in SIV_mac239, but the wild-type Nef was, suggesting that the incorporation of Nef into SIV_mac239 is not important for the enhancement of SIV_mac239 infectivity.

Conclusions

Altogether, the findings suggest that the mechanisms of infectivity enhancement by Nef are different between HIV-1 and SIV_mac239. Lastly, we propose the following hypothesis: even when the expression level of a protein is extremely low, the protein may still be sufficiently functional.

     

Maintenance of Intestinal Th17 Cells and Reduced Microbial Translocation in SIV-infected Rhesus Macaques Treated with Interleukin (IL)-21

In pathogenic HIV and SIV infections of humans and rhesus macaques (RMs), preferential depletion of CD4⁺ Th17 cells correlates with mucosal immune dysfunction and disease progression. Interleukin (IL)-21 promotes differentiation of Th17 cells, long-term maintenance of functional CD8⁺ T cells, and differentiation of memory B cells and antibody-secreting plasma cells. We hypothesized that administration of IL-21 will improve mucosal function in the context of pathogenic HIV/SIV infections. To test this hypothesis, we infected 12 RMs with SIV_mac239 and at day 14 post-infection treated six of them with rhesus rIL-21-IgFc. IL-21-treatment was safe and did not increase plasma viral load or systemic immune activation. Compared to untreated animals, IL-21-treated RMs showed (i) higher expression of perforin and granzyme B in total and SIV-specific CD8⁺ T cells and (ii) higher levels of intestinal Th17 cells. Remarkably, increased levels of Th17 cells were associated with reduced levels of intestinal T cell proliferation, microbial translocation and systemic activation/inflammation in the chronic infection. In conclusion, IL-21-treatment in SIV-infected RMs improved mucosal immune function through enhanced preservation of Th17 cells. Further preclinical studies of IL-21 may be warranted to test its potential use during chronic infection in conjunction with antiretroviral therapy.     

Influence of Mismatch of Env Sequences on Vaccine Protection by Live Attenuated Simian Immunodeficiency Virus

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.     

A Tyrosine-Rich Region in the N Terminus of CCR5 Is Important for Human Immunodeficiency Virus Type 1 Entry and Mediates an Association between gp120 and CCR5

Human immunodeficiency virus type 1 (HIV-1) requires the presence of specific chemokine receptors in addition to CD4 to enter target cells. The chemokine receptor CCR5 is used by the macrophage-tropic strains of HIV-1 that predominate during the asymptomatic stages of infection. Here we identify a small tyrosine-rich region of CCR5 proximal to the N-terminal cysteine that is critical for entry of macrophage-tropic and dual-tropic variants of HIV-1. HIV-1 infection of cells expressing CCR5 mutants with changes in this region was substantially reduced compared with the infection of cells bearing wild-type CCR5. Simian immunodeficiency virus (SIV_mac239) entry was also ablated on a subset of these mutants but enhanced on others. These differences in virus entry were correlated with the relative ability of soluble, monomeric HIV-1 and SIV_mac239 gp120 glycoproteins to bind the CCR5 mutants. These results identify a region of CCR5 that is necessary for the physical association of the gp120 envelope glycoprotein with CCR5 and for HIV-1 infection.     

Resistance to Infection,Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251 Viremia,and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIV_mac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIV_mac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8⁺ T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4⁺ central memory and CD4⁺/α4β7⁺ T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4⁺, and CD8⁺ T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.