Alterations in potential sites for glycosylation predominate during evolution of the simian immunodeficiency virus envelope gene in macaques.

Genetic diversity is a hallmark of the human immunodeficiency virus (HIV) genome, but the role of distinct HIV variants in the development of AIDS is unclear. Envelope (env) is the most highly variable gene in HIV as well as in other retroviruses. We have previously demonstrated that variation in simian immunodeficiency virus (SIV) env is primarily localized in two regions (V1 and V4) during progression to simian AIDS. To determine whether there is a common genotype that evolves as AIDS develops, a total of 160 SIV env genes isolated directly from the tissue DNAs of four macaques infected with cloned virus were compared. Common amino acid sequence changes were identified within V1, V4, and, in the late stages of disease, near V3. At several positions, the same amino acid change was seen frequently in the variant genomes from all four animals. As AIDS developed, the majority of viruses evolved an extended sequence in V1 that was rich in serine and threonine residues and shared similarity with proteins modified by O-linked glycosylation. Several of the predominant common sequence changes in V1 and V4 created new sites for N-linked glycosylation. Thus, common features of the SIV variants that evolve during progression to AIDS are motifs that potentially allow for structural and functional changes in the env protein as a result of carbohydrate addition.     

Human immunodeficiency virus evolution towards reduced replicative fitness in vivo and the development of AIDS

Human immunodeficiency virus (HIV) infection progresses to AIDS following an asymptomatic period during which the virus is thought to evolve towards increased fitness and pathogenicity. We show mathematically that progression to the strongest HIV-induced pathology requires evolution of the virus towards reduced replicative fitness in vivo. This counter-intuitive outcome can happen if multiple viruses co-infect the same cell frequently, which has been shown to occur in recent experiments. According to our model, in the absence of frequent co-infection, the less fit AIDS-inducing strains might never emerge. The frequency of co-infection can correlate with virus load, which in turn is determined by immune responses. Thus, at the beginning of infection when immunity is strong and virus load is low, co-infection is rare and pathogenic virus variants with reduced replicative fitness go extinct. At later stages of infection when immunity is less efficient and virus load is higher, co-infection occurs more frequently and pathogenic virus variants with reduced replicative fitness can emerge, resulting in T-cell depletion. In support of these notions, recent data indicate that pathogenic simian immunodeficiency virus (SIV) strains occurring late in the infection are less fit in specific in vitro experiments than those isolated at earlier stages. If co-infection is blocked, the model predicts the absence of any disease even if virus loads are high. We hypothesize that non-pathogenic SIV infection within its natural hosts, which is characterized by the absence of disease even in the presence of high virus loads, could be explained by a reduced occurrence of co-infection in this system.     

Dynamics of simian immunodeficiency virus SIVmac239 infection in pigtail macaques

Pigtail macaques (PTM) are an excellent model for HIV research; however, the dynamics of simian immunodeficiency virus (SIV) SIVmac239 infection in PTM have not been fully evaluated. We studied nine PTM prior to infection, during acute and chronic SIVmac239 infections, until progression to AIDS. We found PTM manifest clinical AIDS more rapidly than rhesus macaques (RM), as AIDS-defining events occurred at an average of 42.17 weeks after infection in PTM compared to 69.56 weeks in RM (P = 0.0018). However, increased SIV progression was not associated with increased viremia, as both peak and set-point plasma viremias were similar between PTM and RM (P = 0.7953 and P = 0.1006, respectively). Moreover, this increased disease progression was not associated with rapid CD4(+) T cell depletion, as CD4(+) T cell decline resembled other SIV/human immunodeficiency virus (HIV) models. Since immune activation is the best predictor of disease progression during HIV infection, we analyzed immune activation by turnover of T cells by BrdU decay and Ki67 expression. We found increased levels of turnover prior to SIV infection of PTM compared to that observed with RM, which may contribute to their increased disease progression rate. These data evaluate the kinetics of SIVmac239-induced disease progression and highlight PTM as a model for HIV infection and the importance of immune activation in SIV disease progression.     

Persistence of gut mucosal innate immune defenses by enteric α-defensin expression in the simian immunodeficiency virus model of AIDS

Gastrointestinal mucosa is an early target of HIV and a site of viral replication and severe CD4(+) T cell depletion. However, effects of HIV infection on gut mucosal innate immune defense have not been fully investigated. Intestinal Paneth cell-derived α-defensins constitute an integral part of the gut mucosal innate defense against microbial pathogens. Using the SIV-infected rhesus macaque model of AIDS, we examined the level of expression of rhesus enteric α-defensins (REDs) in the jejunal mucosa of rhesus macaques during all stages of SIV infection using real-time PCR, in situ hybridization, and immunohistochemistry. An increased expression of RED mRNAs was found in PC at the base of the crypts in jejunum at all stages of SIV infection as compared with uninfected controls. This increase correlated with active viral replication in gut-associated lymphoid tissue. Loss of RED protein accumulation in PC was seen in animals with simian AIDS. This was associated with the loss of secretory granules in PC, suggesting an increase in degranulation during advanced SIV disease. The α-defensin-mediated innate mucosal immunity was maintained in PC throughout the course of SIV infection despite the mucosal CD4(+) T cell depletion. The loss of RED protein accumulation and secretion was associated with an increased incidence of opportunistic enteric infections and disease progression. Our findings suggest that local innate immune defense exerted by PC-derived defensins contributes to the protection of gut mucosa from opportunistic infections during the course of SIV infection.     

Variation in simian immunodeficiency virus env is confined to V1 and V4 during progression to simian AIDS.

We have monitored changes in the simian immunodeficiency virus (SIV) envelope (env) gene in two macaques which developed AIDS after inoculation with a molecular clone of SIV. As the animals progressed to AIDS, selection occurred for viruses with variation in two discrete regions (V1 and V4) but not for viruses with changes in the region of SIV env that corresponds to the immunodominant, V3 loop of human immunodeficiency virus. Within the highly variable domains, the vast majority of nucleotide changes encoded an amino acid change (98%), suggesting that these envelope variants had evolved as a result of phenotypic selection. Analysis of the biological properties of these variants, which have been selected for in the host, may be useful in defining the mechanisms underlying viral persistence and progression to simian AIDS.     

Major histocompatibility complex class I alleles associated with slow simian immunodeficiency virus disease progression bind epitopes recognized by dominant acute-phase cytotoxic-T-lymphocyte responses

Certain major histocompatibility complex class I (MHC-I) alleles are associated with delayed disease progression in individuals infected with human immunodeficiency virus (HIV) and in macaques infected with simian immunodeficiency virus (SIV). However, little is known about the influence of these MHC alleles on acute-phase cellular immune responses. Here we follow 51 animals infected with SIV(mac)239 and demonstrate a dramatic association between Mamu-A*01 and -B*17 expression and slowed disease progression. We show that the dominant acute-phase cytotoxic T lymphocyte (CTL) responses in animals expressing these alleles are largely directed against two epitopes restricted by Mamu-A*01 and one epitope restricted by Mamu-B*17. One Mamu-A*01-restricted response (Tat(28-35)SL8) and the Mamu-B*17-restricted response (Nef(165-173)IW9) typically select for viral escape variants in early SIV(mac)239 infection. Interestingly, animals expressing Mamu-A*1 and -B*17 have less variation in the Tat(28-35)SL8 epitope during chronic infection than animals that express only Mamu-A*01. Our results show that MHC-I alleles that are associated with slow progression to AIDS bind epitopes recognized by dominant CTL responses during acute infection and underscore the importance of understanding CTL responses during primary HIV infection.     

Virally induced CD4+ T cell depletion is not sufficient to induce AIDS in a natural host

Peripheral blood CD4+ T cell counts are a key measure for assessing disease progression and need for antiretroviral therapy in HIV-infected patients. More recently, studies have demonstrated a dramatic depletion of mucosal CD4+ T cells during acute infection that is maintained during chronic pathogenic HIV as well as SIV infection. A different clinical disease course is observed during the infection of natural hosts of SIV infection, such as sooty mangabeys (Cercocebus atys), which typically do not progress to AIDS. Previous studies have determined that SIV+ mangabeys generally maintain healthy levels of CD4+ T cells despite having viral replication comparable to HIV-infected patients. In this study, we identify the emergence of a multitropic (R5/X4/R8-using) SIV infection after 43 or 71 wk postinfection in two mangabeys that is associated with an extreme, persistent (>5.5 years), and generalized loss of CD4+ T cells (5-80 cells/microl of blood) in the absence of clinical signs of AIDS. This study demonstrates that generalized CD4+ T cell depletion from the blood and mucosal tissues is not sufficient to induce AIDS in this natural host species. Rather, AIDS pathogenesis appears to be the cumulative result of multiple aberrant immunologic parameters that include CD4+ T cell depletion, generalized immune activation, and depletion/dysfunction of non-CD4+ T cells. Therefore, these data provide a rationale for investigating multifaceted therapeutic strategies to prevent progression to AIDS, even following dramatic CD4 depletion, such that HIV+ humans can survive normal life spans analogous to what occurs naturally in SIV+ mangabeys.     

Acute phase cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus infection

Cytotoxic T-lymphocyte (CTL) responses peak coincident with the decline in acute HIV viremia. Despite two reports of CTL-resistant HIV variants emerging during acute infection, the contribution of acute CTL escape to HIV pathogenesis remains unclear. Difficulties inherent in studying acute HIV infection can be overcome by modeling virus-host interactions in SIV-infected rhesus macaques. We sequenced 21 complete simian immunodeficiency virus (SIV)mac239 genomes at four weeks post-infection to determine the extent of acute CTL escape. Here we show that viruses from 19 of 21 macaques escaped from CTLs during acute infection and that these escape-selecting CTLs were responsive to lower concentrations of peptide than other SIV-specific CTLs. Interestingly, CTLs that require low peptide concentrations for stimulation (high 'functional avidity') are particularly effective at controlling other viral infections. Our results suggest that acute viral escape from CTLs is a hallmark of SIV infection and that CTLs with high functional avidity can rapidly select for escape variants.     

Suppression of simian immunodeficiency virus replication in vitro by CD8+ lymphocytes

The AIDS-like disease in rhesus monkeys induced by the simian immunodeficiency virus (SIV) has been used as a model to explore the nature of the T lymphocyte response after infection with viruses of the human immunodeficiency virus family. Activated CD8+ lymphocytes are present in increased numbers in the paracortex of lymph nodes of SIV-infected rhesus monkeys with a lymphadenopathy syndrome. We demonstrate that SIV is more readily isolated from CD8+ lymphocyte-depleted PBL of SIV-infected animals than from their unfractionated PBL. Rather than reflecting the fact that the CD8+ lymphocyte-depleted cell populations are simply enriched for CD4+ lymphocytes, this indicates that CD8+ cells themselves are critical in this regulatory interaction. In fact, CD8+ lymphocytes from SIV-infected but not uninfected rhesus monkeys can block SIV replication in vitro in PBL populations. A T lymphocyte population that blocks replication of viruses of the HIV family may contribute to containing the progression of AIDS.     

Persistence of simian immunodeficiency virus Mne variants upon transmission.

In macaques infected with a clone of simian immunodeficiency virus (SIV) Mne, viral variants consistently evolve multiple new potential glycosylation sites in the first variable region (V1) prior to the development of AIDS. In the present study, we asked whether viruses with these glycosylation sites persist when they are transmitted to a naive macaque. Variants that evolved after transmission to a recipient macaque were compared with virus that evolved in the donor, which had been infected by cloned SIV Mne. Upon transmission, the specific serine/threonine-rich motifs potentially encoding novel O-linked glycosylation site(s) in V1 were conserved in virus isolated from lymph node, spleen, and liver tissue from the recipient. There was some accumulation of changes in V3 of envelope in virus from the recipient, whereas changes in this region were not observed in virus from the donor macaque. Some variants detected in the tissue of the recipient at necropsy were most closely related to viruses present in the donor inoculum even though these particular variants were not detected early after infection in the recipient's peripheral blood mononuclear cells. Overall, virus with the predominant V1 sequences associated with progression to disease are transmitted to and persist in the recipient animal.     

Cytotoxic T-lymphocyte escape monitoring in simian immunodeficiency virus vaccine challenge studies

Several vaccine studies have ameliorated disease progression in simian-human immunodeficiency virus (SHIV) infections. The successes of these vaccines have been largely attributed to protective effects of cytotoxic T-lymphocyte (CTL) responses, although the precise correlates of immune protection remain poorly defined. It is now well established that vigorous CTL and antibody responses can rapidly select for viral escape variants after HIV and SIV infection. Here we suggest that viral variation analyses should be performed on viruses derived from vaccinated, SIV-, or SHIV-challenged animals as a routine component of vaccine evaluation to determine the contribution of immune responses to the success (or failure) of the vaccine regimen. To illustrate the importance of escape analysis, we show that rapid emergence of escape variants postchallenge contributed to the failure of a DNA prime/MVA boost vaccine regimen encoding SIV Tat.     

Replication and persistence of simian immunodeficiency virus variants after passage in macaque lymphocytes and established human cell lines.

In lentivirus infections, there are typically few cells in the host that harbor the provirus. For this reason, molecular clones of human and simian immunodeficiency viruses (HIV and SIV) are generally derived after passage and amplification of the virus in cell culture. To determine whether SIV variants that persist in culture are similar to the variants that predominate in the host, we examined the proviral sequence of the SIV envelope (env) gene before and after cocultivation of lymphocytes from a macaque with AIDS with naive macaque lymphocytes or human cell lines. Many of the predominant variants in the monkey replicated and persisted in macaque lymphocytes and CEMx174 cells in culture, but a more limited population of variants replicated in C8166 cells. Passage of virus, harvested after 4 weeks of coculture, onto naive cells further demonstrated that the majority of proviruses detected by polymerase chain reaction were also viral variants that were expressed and packaged into infectious virions.     

HIV/AIDS: in search of an animal model

AIDS is among the most devastating diseases of our time, claiming the lives of approximately 3 million people per year. The primary cause of AIDS, human immunodeficiency virus type 1 (HIV-1), is a pathogen that is highly specific for humans and generally does not infect or cause disease in other species. This property complicates the generation of animal models that are urgently needed to test new antiretroviral therapies and vaccines. The most practical animal models developed to date consist of infection of rhesus macaques with a simian immunodeficiency virus (SIV) or chimeric HIV/SIV viruses. Although these models are useful for particular applications, the fact that SIV is a distinct virus compared with HIV-1 represents a significant limitation to their use. Here, we discuss the uses and limitations of existing models and recent advances that might lead to better animal models for HIV/AIDS.     

Divergent TLR7 and TLR9 signaling and type I interferon production distinguish pathogenic and nonpathogenic AIDS virus infections

Pathogenic HIV infections of humans and simian immunodeficiency virus (SIV) infections of rhesus macaques are characterized by generalized immune activation and progressive CD4(+) T cell depletion. In contrast, natural reservoir hosts for SIV, such as sooty mangabeys, do not progress to AIDS and show a lack of aberrant immune activation and preserved CD4(+) T cell populations, despite high levels of SIV replication. Here we show that sooty mangabeys have substantially reduced levels of innate immune system activation in vivo during acute and chronic SIV infection and that sooty mangabey plasmacytoid dendritic cells (pDCs) produce markedly less interferon-alpha in response to SIV and other Toll-like receptor 7 and 9 ligands ex vivo. We propose that chronic stimulation of pDCs by SIV and HIV in non-natural hosts may drive the unrelenting immune system activation and dysfunction underlying AIDS progression. Such a vicious cycle of continuous virus replication and immunopathology is absent in natural sooty mangabey hosts.     

Apoptosis in SIV infection

Pathogenic human immunodeficiency virus (HIV)/Simian immunodeficiency virus (SIV) infection is associated with increased T-cell apoptosis. In marked contrast to HIV infection in humans and SIV infection in macaques, the SIV infection of natural host species is typically nonpathogenic despite high levels of viral replication. In these nonpathogenic primate models, no observation of T-cell apoptosis was observed, suggesting that either SIV is less capable of directly inducing apoptosis in natural hosts (likely as a result of coevolution/coadaptation with the host) or, alternatively, that the indirect T-cell apoptosis plays the key role in determining the HIV-associated T-cell depletion and progression to acquired immune deficiency syndrome (AIDS). Understanding the molecular and cellular mechanisms responsible for the disease-free equilibrium in natural hosts for SIV infection, including those determining the absence of high levels of T-cell apoptosis, is likely to provide important clues regarding the mechanisms of AIDS pathogenesis in humans.     

Primate models for human immunodeficiency virus infection. Evolution of receptor use during pathogenesis

Animal models greatly facilitate understanding of transmission, pathogenesis and immune responses in HIV and SIV infection and provide models for studies on the effect of candidate drugs or vaccines. However, there are several aspects that one should consider when drawing conclusions from results obtained from animal models. First, the genetic relationship of primate lentiviruses cannot be disregarded because it is known that HIV-1 is more closely related to SIV of chimpanzee origin (SIVcpz) than to SIV from sooty mangabey (SIVsm) origin. Nevertheless, SIVsm and SIVmac are the ones most often used as model systems. Second, there are differences in the biological properties, like CXCR4 use and CD4-independent coreceptor use, of HIV and SIV. These differences might be relevant in virus transmission, pathogenesis and in evoking immune responses. Third, in vivo and in vitro selection may influence the results. Neutralizing antibodies may play a role in selection of variant viruses since neutralization sensitive, CD4-independent SIVsm variants seemed to be suppressed in animals that mounted a neutralizing antibody response. It is tempting to speculate that neutralizing antibodies shape the SIV/HIV infection by selecting variants with a more "closed" envelope conformation with consequences for both receptor binding and neutralization sensitivity. The SIV/monkey model, although it has important advantages, may not answer all questions asked about HIV-1 infection in human.     

Highly pathogenic simian immunodeficiency virus mne variants that emerge during the course of infection evolve enhanced infectivity and the ability to downregulate CD4 but not class I major histocompatibility complex antigens

The replicative, cytopathic, and antigenic properties of simian immunodeficiency virus (SIV) variants influence its replication efficiency in vivo. To further define the viral properties and determinants that may be important for high-level replication in vivo and progression to AIDS, we compared a minimally pathogenic SIVmne molecular clone with two highly pathogenic variants cloned from late stages of infection. Both variants had evolved greater infectivity than the parental clone due to mutations in nef. Interestingly, a pol determinant in one of the highly pathogenic variants also contributed to its increased infectivity. Furthermore, because replication in vivo may also be influenced by the ability of a virus to evade the cellular immune response of the host, we examined whether the variants were more capable of downregulating surface expression of class I major histocompatibility complex (MHC). Decreased MHC class I expression was not observed in cells infected with any of the viruses. Furthermore, the Nef proteins of the highly pathogenic variants only slightly reduced surface MHC class I expression in transfected cells, although they efficiently downregulated CD4. Together, these data demonstrate that mutations which can enhance viral infectivity, as well as CD4 downregulation, may be important for efficient replication of SIV in the host. However, Nef-mediated reduction of MHC class I expression does not appear to be critical for the increased in vivo replicative ability of highly pathogenic late variants.     

Macaque long-term nonprogressors resist superinfection with multiple CD8+ T cell escape variants of simian immunodeficiency virus

Human immunodeficiency virus (HIV)-positive individuals can be superinfected with different virus strains. Individuals who control an initial HIV infection are therefore still at risk for subsequent infection with divergent viruses, but the barriers to such superinfection remain unclear. Here we tested long-term nonprogressors' (LTNPs') susceptibility to superinfection using Indian rhesus macaques that express the major histocompatibility complex class I (MHC-I) allele Mamu-B 17, which is associated with control of the pathogenic AIDS virus SIVmac239. The Mamu-B 17-restricted CD8(+) T cell repertoire is focused almost entirely on 5 epitopes. We engineered a series of SIVmac239 variants bearing mutations in 3, 4, or all 5 of these epitopes and used them to serially challenge 2 Mamu-B 17-positive LTNPs. None of the escape variants caused breakthrough replication in LTNPs, although they readily infected Mamu-B 17-negative naive macaques. In vitro competing coculture assays and examination of viral evolution in hosts lacking Mamu-B 17 suggested that the mutant viruses had negligible defects in replicative fitness. Both LTNPs maintained robust immune responses, including simian immunodeficiency virus (SIV)-specific CD8(+) and CD4(+) T cells and neutralizing antibodies. Our results suggest that escape mutations in epitopes bound by "protective" MHC-I molecules may not be sufficient to establish superinfection in LTNPs.     

The cytokine network of acute HIV infection: a promising target for vaccines and therapy to reduce viral set-point?

Cytokines play a central role in the pathogenesis of many diseases, including HIV infection. However, the role of the cytokine network in early HIV infection is only now starting to be elucidated. A number of studies conducted in recent years have indicated that cytokines of the acute/early stages of HIV and SIV infection can impact viral set-point months later, and this is of critical importance since viral set-point during chronic HIV infection affects virus transmission and disease progression. This raises the question whether modulating the cytokine environment during acute/early HIV infection can be a target for novel approaches to develop a vaccine and therapeutics. In this review we focus on the kinetics and function of cytokines during acute HIV and SIV infection and how these may impact viral set-point. We also discuss unresolved questions that are essential for our understanding of the role of acute infection cytokines in HIV infection and that, if answered, may suggest novel therapeutic and vaccine strategies to control the worldwide HIV pandemic.     

Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359))

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.