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1.  Prevention of SIV Rectal Transmission and Priming of T Cell Responses in Macaques after Local Pre-exposure Application of Tenofovir Gel 
PLoS Medicine  2008;5(8):e157.
Background
The rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model.
Methods and Findings
A total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-γ)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 spot forming cells (SFC)/106 PBMC.
Conclusions
These results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.
Martin Cranage and colleagues find that topical tenofovir gel can protect against rectal challenge with SIV in a macaque model, and can permit the induction of SIV-specific T cell responses.
Editors' Summary
Background.
About 33 million people are now infected with the human immunodeficiency virus (HIV), which causes AIDS by killing immune system cells. As yet, there is no cure for AIDS, although HIV infections can be held in check with antiretroviral drugs. Also, despite years of research, there is no vaccine available that effectively protects people against HIV infection. So, to halt the AIDS epidemic, other ways of preventing the spread of HIV are being sought. For example, pre-exposure treatment (prophylaxis) with antiretroviral drugs is being investigated as a way to prevent HIV transmission. In addition, because HIV is often spread through heterosexual penile-to-vaginal sex with an infected partner, several vaginal microbicides (compounds that protect against HIV when applied inside the vagina) are being developed, some of which contain antiretroviral drugs.
Why Was This Study Done?
Because HIV can cross the membranes that line the mouth and the rectum (the lower end of the large intestine that connects to the anus) in addition to the membrane that lines the vagina, HIV transmission can also occur during oral and anal sex. The lining of the rectum in particular is extremely thin and overlies tissues rich in activated T cells (the immune system cells that HIV targets), so unprotected anal intercourse carries a high risk of HIV infection. Anal intercourse is common among men who have sex with men but is also more common in heterosexual populations than is generally thought. Tenofovir (an antiretroviral drug that counteracts HIV after it has entered human cells) given by mouth partly protects macaques against rectal infection with simian immunodeficiency virus (SIV; a virus that induces AIDS in monkeys and apes) so the researchers wanted to know whether this drug might be effective against rectal SIV infection if applied at the site where the virus enters the body.
What Did the Researchers Do and Find?
To answer this question, the researchers rectally infected several macaques with SIV up to 2 h after rectal application of a gel containing tenofovir, after rectal application of a gel not containing the drug, or after no treatment. In addition, a few animals were treated with the tenofovir gel after the viral challenge. Most of the animals given the tenofovir gel before the viral challenge were partly or totally protected from SIV infection, whereas all the untreated animals and most of those treated with the placebo gel or with the drug-containing gel after the viral challenge became infected with SIV. High blood levels of tenofovir 15 min after its rectal application correlated with protection from viral infection. The researchers also collected rectal and small intestine samples from tenofovir-treated macaques that had not been exposed to SIV and asked which samples were resistant to SIV infection in laboratory dishes. They found that only the rectal samples were resistant to infection and only rectal cells contained tenofovir. Finally, activated T cells that recognized an SIV protein were present in the blood of some of the animals that were protected from SIV infection by the tenofovir gel.
What Do These Findings Mean?
These findings, although based on experiments in only a few animals, suggest that rectal treatment with antiretroviral drugs before rectal exposure to HIV might prevent rectal HIV transmission in people. However, results from animal experiments do not always reflect what happens in people. Indeed, clinical trials of a potential vaginal microbicide that worked well in macaques were halted recently because women using the microbicide had higher rates of HIV infection than those using a control preparation. The finding that immune-system activation can occur in the absence of overt infection in animals treated with the tenofovir gel additionally suggests that a combination of a local antiretroviral/microbicide and vaccination might be a particularly effective way to prevent HIV transmission. However, because HIV targets activated T cells, viral rechallenge experiments must be done to check that the activated T cells induced by the virus in the presence of tenofovir do not increase the likelihood of infection upon re-exposure to HIV before this potential microbicide is tried in people.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050157.
Read the accompanying PLoS Medicine Perspective by Florian Hladik
An overview of HIV infection and AIDS is available from the US National Institute of Allergy and Infectious Diseases
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including an article on safer sex, which includes information on the risks associated with specific types of sex and on microbicides and other methods to prevent the sexual transmission of HIV
Information on all aspects of HIV/AIDS is available from Avert, an international AIDS charity, including information on HIV prevention and on microbicides
The World Health Organization has a fact sheet on microbicides
The UK charity NAM also provides detailed information on microbicides
PrEP Watch is a comprehensive information source on pre-exposure prophylaxis for HIV prevention
Global Campaign for Microbicides is an international coalition of organisations dedicated to accelerating access to new HIV prevention options
doi:10.1371/journal.pmed.0050157
PMCID: PMC2494562  PMID: 18684007
2.  Limited Impact of Passive Non-Neutralizing Antibody Immunization in Acute SIV Infection on Viremia Control in Rhesus Macaques 
PLoS ONE  2013;8(9):e73453.
Background
Antiviral antibodies, especially those with neutralizing activity against the incoming strain, are potentially important immunological effectors to control human immunodeficiency virus (HIV) infection. While neutralizing activity appears to be central in sterile protection against HIV infection, the entity of inhibitory mechanisms via HIV and simian immunodeficiency virus (SIV)-specific antibodies remains elusive. The recent HIV vaccine trial RV144 and studies in nonhuman primate models have indicated controversial protective efficacy of HIV/SIV-specific non-neutralizing binding antibodies (non-NAbs). While reports on HIV-specific non-NAbs have demonstrated virus inhibitory activity in vitro, whether non-NAbs could also alter the pathogenic course of established SIV replication in vivo, likewise via neutralizing antibody (NAb) administration, has been unclear. Here, we performed post-infection passive immunization of SIV-infected rhesus macaques with polyclonal SIV-specific, antibody-dependent cell-mediated viral inhibition (ADCVI)-competent non-NAbs.
Methods and Findings
Ten lots of polyclonal immunoglobulin G (IgG) were prepared from plasma of ten chronically SIVmac239-infected, NAb-negative rhesus macaques, respectively. Their binding capacity to whole SIVmac239 virions showed a propensity similar to ADCVI activity. A cocktail of three non-NAb lots showing high virion-binding capacity and ADCVI activity was administered to rhesus macaques at day 7 post-SIVmac239 challenge. This resulted in an infection course comparable with control animals, with no significant difference in set point plasma viral loads or immune parameters.
Conclusions
Despite virus-specific suppressive activity of the non-NAbs having been observed in vitro, their passive immunization post-infection did not result in SIV control in vivo. Virion binding and ADCVI activity with lack of virus neutralizing activity were indicated to be insufficient for antibody-triggered non-sterile SIV control. More diverse effector functions or sophisticated localization may be required for non-NAbs to impact HIV/SIV replication in vivo.
doi:10.1371/journal.pone.0073453
PMCID: PMC3767751  PMID: 24039947
3.  Cytotoxic Capacity of SIV-Specific CD8+ T Cells against Primary Autologous Targets Correlates with Immune Control in SIV-Infected Rhesus Macaques 
PLoS Pathogens  2013;9(2):e1003195.
Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8+ T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4+ T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4+ T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0–91.7%] vs. 23.7% [0.0–58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = −0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8+ T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8+ T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.
Author Summary
Clues regarding the features of effective immunity against lentiviruses have come from the study of non-human primates. We evaluated rhesus macaques infected with Simian Immunodeficiency Virus (SIV), a lentivirus closely related to Human Immunodeficiency Virus (HIV). In contrast to most SIV-infected rhesus macaques that develop progressive disease, a small proportion are able to control SIV replication and remain healthy for prolonged durations. In this study, we found that these long-term nonprogressor/elite controller (LTNP/EC) macaques have CD8+ T cells that are extremely effective at killing SIV-infected cells. It seems that this control is mediated by the efficient delivery of active granzyme B, a key molecule involved in the elimination of virus-infected cells. Furthermore, we correctly predicted the presence or absence of control of SIV infection in the majority of animals through measurements of the killing capacity of their CD8+ T cells. These findings indicate that measuring these functions could be used in the evaluation of vaccines against SIV in non-human primates.
doi:10.1371/journal.ppat.1003195
PMCID: PMC3585127  PMID: 23468632
4.  Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection 
PLoS Pathogens  2012;8(9):e1002928.
Chronic immune activation is a key determinant of AIDS progression in HIV-infected humans and simian immunodeficiency virus (SIV)-infected macaques but is singularly absent in SIV-infected natural hosts. To investigate whether natural killer T (NKT) lymphocytes contribute to the differential modulation of immune activation in AIDS-susceptible and AIDS-resistant hosts, we compared NKT function in macaques and sooty mangabeys in the absence and presence of SIV infection. Cynomolgus macaques had significantly higher frequencies of circulating invariant NKT lymphocytes compared to both rhesus macaques and AIDS-resistant sooty mangabeys. Despite this difference, mangabey NKT lymphocytes were functionally distinct from both macaque species in their ability to secrete significantly more IFN-γ, IL-13, and IL-17 in response to CD1d/α-galactosylceramide stimulation. While NKT number and function remained intact in SIV-infected mangabeys, there was a profound reduction in NKT activation-induced, but not mitogen-induced, secretion of IFN-γ, IL-2, IL-10, and TGF-β in SIV-infected macaques. SIV-infected macaques also showed a selective decline in CD4+ NKT lymphocytes which correlated significantly with an increase in circulating activated memory CD4+ T lymphocytes. Macaques with lower pre-infection NKT frequencies showed a significantly greater CD4+ T lymphocyte decline post SIV infection. The disparate effect of SIV infection on NKT function in mangabeys and macaques could be a manifestation of their differential susceptibility to AIDS. Alternately, these data also raise the possibility that loss of anti-inflammatory NKT function promotes chronic immune activation in pathogenic SIV infection, while intact NKT function helps to protect natural hosts from developing immunodeficiency and aberrant immune activation.
Author Summary
Several African nonhuman primate species such as sooty mangabeys are naturally infected with SIV and maintain high levels of viral replication without developing AIDS. SIV-infected natural hosts do not show evidence of increased chronic immune activation, a feature that distinguishes them from AIDS-susceptible SIV-infected Asian macaques. In this study we compared natural killer T (NKT) lymphocytes, a unique subset of innate T lymphocytes with anti-inflammatory properties, in AIDS-resistant and AIDS-susceptible hosts. Sooty mangabey NKT cells retained normal functionality following SIV infection and were more potent than macaque NKT cells in their ability to produce interferon-γ and secrete anti-inflammatory cytokines. In contrast, NKT cells of SIV-infected macaques were markedly hypo-functional with regards to secretion of anti-inflammatory and effector cytokines and showed an association between loss of CD4+ NKT cells and increased immune activation. These findings suggest that dysfunctional NKT cells may promote increased immune activation in AIDS-susceptible hosts while intact effector and anti-inflammatory NKT cells could help to prevent immunodeficiency and increased immune activation in natural hosts.
doi:10.1371/journal.ppat.1002928
PMCID: PMC3447755  PMID: 23028326
5.  Similar Impact of CD8+ T Cell Responses on Early Virus Dynamics during SIV Infections of Rhesus Macaques and Sooty Mangabeys 
PLoS Computational Biology  2010;6(8):e1000901.
Despite comparable levels of virus replication, simian immunodeficiency viruses (SIV) infection is non-pathogenic in natural hosts, such as sooty mangabeys (SM), whereas it is pathogenic in non-natural hosts, such as rhesus macaques (RM). Comparative studies of pathogenic and non-pathogenic SIV infection can thus shed light on the role of specific factors in SIV pathogenesis. Here, we determine the impact of target-cell limitation, CD8+ T cells, and Natural Killer (NK) cells on virus replication in the early SIV infection. To this end, we fit previously published data of experimental SIV infections in SMs and RMs with mathematical models incorporating these factors and assess to what extent the inclusion of individual factors determines the quality of the fits. We find that for both rhesus macaques and sooty mangabeys, target-cell limitation alone cannot explain the control of early virus replication, whereas including CD8+ T cells into the models significantly improves the fits. By contrast, including NK cells does only significantly improve the fits in SMs. These findings have important implications for our understanding of SIV pathogenesis as they suggest that the level of early CD8+ T cell responses is not the key difference between pathogenic and non-pathogenic SIV infection.
Author Summary
Simian immunodeficiency viruses (SIV) are typically non-pathogenic in their natural hosts. However, if the same virus infects a non-natural host it often leads to AIDS-like symptoms. Therefore, comparing SIV infections in these two types of host might help explain the pathogenesis of SIV in non-natural hosts and thereby also that of HIV. We combined mathematical modeling with data on the levels of virus and immune cells early in infection, and compared both non-pathogenic SIV infections of sooty mangabeys and pathogenic SIV infection of rhesus macaques with respect to how the virus grows in them and to what extent it is controlled by the immune system. We found that the impact of the immune system on early virus replication is remarkably similar in both species. In particular, for both species virus replication can only be explained by the effect of CD8+ T cells. These findings have important implications for our understanding of SIV pathogenesis as they suggest that the impact of the early immune responses is not the key difference between pathogenic and non-pathogenic SIV infection.
doi:10.1371/journal.pcbi.1000901
PMCID: PMC2928736  PMID: 20865048
6.  Target Cell Availability, Rather than Breast Milk Factors, Dictates Mother-to-Infant Transmission of SIV in Sooty Mangabeys and Rhesus Macaques 
PLoS Pathogens  2014;10(3):e1003958.
Mother-to-infant transmission (MTIT) of HIV is a serious global health concern, with over 300,000 children newly infected in 2011. SIV infection of rhesus macaques (RMs) results in similar rates of MTIT to that of HIV in humans. In contrast, SIV infection of sooty mangabeys (SMs) rarely results in MTIT. The mechanisms underlying protection from MTIT in SMs are unknown. In this study we tested the hypotheses that breast milk factors and/or target cell availability dictate the rate of MTIT in RMs (transmitters) and SMs (non-transmitters). We measured viral loads (cell-free and cell-associated), levels of immune mediators, and the ability to inhibit SIV infection in vitro in milk obtained from lactating RMs and SMs. In addition, we assessed the levels of target cells (CD4+CCR5+ T cells) in gastrointestinal and lymphoid tissues, including those relevant to breastfeeding transmission, as well as peripheral blood from uninfected RM and SM infants. We found that frequently-transmitting RMs did not have higher levels of cell-free or cell-associated viral loads in milk compared to rarely-transmitting SMs. Milk from both RMs and SMs moderately inhibited in vitro SIV infection, and presence of the examined immune mediators in these two species did not readily explain the differential rates of transmission. Importantly, we found that the percentage of CD4+CCR5+ T cells was significantly lower in all tissues in infant SMs as compared to infant RMs despite robust levels of CD4+ T cell proliferation in both species. The difference between the frequently-transmitting RMs and rarely-transmitting SMs was most pronounced in CD4+ memory T cells in the spleen, jejunum, and colon as well as in central and effector memory CD4+ T cells in the peripheral blood. We propose that limited availability of SIV target cells in infant SMs represents a key evolutionary adaptation to reduce the risk of MTIT in SIV-infected SMs.
Author Summary
Currently 2.5 million children are infected with HIV, largely as a result of mother-to-child transmission, and there is no effective vaccine or cure. Studies of Simian Immunodeficiency Virus (SIV) infection of nonhuman primate species termed “natural hosts” have shown that mother-to-infant transmission of SIV in these animals is rare. Natural hosts are African monkey species that are naturally infected with SIV in the wild but do not develop AIDS. We sought to understand the mechanism by which natural hosts are protected from mother-to-infant transmission of SIV, aiming to translate our findings into novel strategies to prevent perinatal HIV infection. We found that natural host sooty mangabey infants have extremely low levels of target cells for SIV infection in lymphoid and gastrointestinal tissues. Direct comparison of infant sooty mangabeys and infant rhesus macaques (non-natural host species with high SIV transmission rates) confirmed that natural hosts have significantly lower levels of SIV target cells compared with non-natural hosts. Analysis of the breast milk of sooty mangabeys and rhesus macaques revealed similar levels of virus and ability to inhibit SIV infection. Our study provides evidence for target cell restriction as the main mechanism of protection from mother-to-infant SIV transmission in natural hosts.
doi:10.1371/journal.ppat.1003958
PMCID: PMC3946396  PMID: 24604066
7.  TRIM5 alpha Drives SIVsmm Evolution in Rhesus Macaques 
PLoS Pathogens  2013;9(8):e1003577.
The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5TFP/TFP or TRIM5Cyp/TFP. Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5Cyp. Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIMTFP alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.
Author Summary
Human immunodeficiency virus (HIV) resulted from the transmission of simian immunodeficiency viruses (SIV) from nonhuman primates followed by adaptation and expansion as a pandemic in humans. This required the virus to overcome a variety of intrinsic host restriction factors in humans in order to replicate efficiently. Similarly, SIV encounters restriction factors upon cross-species transmission between nonhuman primates, specifically from a natural host species such as sooty mangabey monkeys to rhesus macaques. Previously we observed significant differences in the levels of virus replication of SIV among rhesus macaques due to subtle differences in one of these restriction factors, TRIM5 among individual macaques. Although a restrictive version of TRIM5 resulted in lower viremia, we also observed that the virus spontaneously mutated in the viral capsid gene and that these mutations were associated with escape from TRIM5 restriction. In the present study, we found that introduction of these escape mutations into the parental virus confers resistance to TRIM5 both in tissue culture and in macaques. These studies provide direct evidence that TRIM5 is a critical factor influencing the cross-species transmission of SIV in primates.
doi:10.1371/journal.ppat.1003577
PMCID: PMC3749954  PMID: 23990789
8.  Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2 
PLoS Pathogens  2009;5(5):e1000429.
Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIVmac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIVsmm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.
Author Summary
Tetherin was recently identified as a host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes the antiviral effects of tetherin by expressing Vpu, which mediates the degradation of tetherin. While tetherin has broad activity against diverse types of viruses, only a few of the primate AIDS viruses express Vpu. Simian immunodeficiency virus (SIV) does not have a vpu gene. Since SIV infection of the rhesus macaque is an important animal model for AIDS vaccine development, we set out to determine how SIV overcomes restriction by tetherin in this species. We found that the SIV Nef protein could counteract rhesus macaque tetherin, but not human tetherin. Conversely, the HIV-1 Vpu protein counteracted human tetherin, but not rhesus tetherin. The specificity of Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. These observations identify a role for the SIV Nef protein in counteracting tetherin, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.
doi:10.1371/journal.ppat.1000429
PMCID: PMC2673686  PMID: 19436700
9.  ADCC Develops Over Time during Persistent Infection with Live-Attenuated SIV and Is Associated with Complete Protection against SIVmac251 Challenge 
PLoS Pathogens  2012;8(8):e1002890.
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 SIVmac251 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.
Author Summary
Live-attenuated vaccines can prevent simian immunodeficiency virus (SIV) infection upon experimental challenge of rhesus macaques. Although safety considerations preclude vaccinating humans with live-attenuated HIV-1, it may be possible to replicate the types of immunity induced by live-attenuated SIV through an alternative approach. Thus, identifying the immune responses underlying protection by live-attenuated SIV and understanding their induction would provide guidance for HIV-1 vaccine design. An important role for the maturation of virus-specific antibody responses could explain the time-dependent development of protection by live-attenuated SIV. However, antibodies that block the entry of the challenge virus into cells are usually undetectable. Antibodies can also direct the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). Here we show that live-attenuated SIV induces progressive increases in ADCC over time, and that the development of these antibodies is dependent upon the persistent replication of the vaccine strain. In two different experiments, the animals immunized with live-attenuated SIV that remained uninfected after pathogenic SIV challenge had higher measures of ADCC than those that became infected. Our results suggest that antibodies contribute to protection by live-attenuated SIV, and that persistent stimulation of antibody responses may be essential for HIV-1 vaccines to induce high ADCC activity.
doi:10.1371/journal.ppat.1002890
PMCID: PMC3426556  PMID: 22927823
10.  Distinct host cell proteins incorporated by SIV replicating in CD4+ T Cells from natural disease resistant versus non-natural disease susceptible hosts 
Retrovirology  2010;7:107.
Background
Enveloped viruses including the simian immunodeficiency virus (SIV) replicating within host cells acquire host proteins upon egress from the host cells. A number of studies have catalogued such host proteins, and a few have documented the potential positive and negative biological functions of such host proteins. The studies conducted herein utilized proteomic analysis to identify differences in the spectrum of host proteins acquired by a single source of SIV replicating within CD4+ T cells from disease resistant sooty mangabeys and disease susceptible rhesus macaques.
Results
While a total of 202 host derived proteins were present in viral preparations from CD4+ T cells from both species, there were 4 host-derived proteins that consistently and uniquely associated with SIV replicating within CD4+ T cells from rhesus macaques but not sooty mangabeys; and, similarly, 28 host-derived proteins that uniquely associated with SIV replicating within CD4+ T cells from sooty mangabeys, but not rhesus macaques. Of interest was the finding that of the 4 proteins uniquely present in SIV preparations from rhesus macaques was a 26 S protease subunit 7 (MSS1) that was shown to enhance HIV-1 'tat" mediated transactivation. Among the 28 proteins found in SIV preparations from sooty mangabeys included several molecules associated with immune function such as CD2, CD3ε, TLR4, TLR9 and TNFR and a bioactive form of IL-13.
Conclusions
The finding of 4 host proteins that are uniquely associated with SIV replicating within CD4+ T cells from disease susceptible rhesus macaques and 28 host proteins that are uniquely associated with SIV replicating within CD4+ T cells from disease resistant sooty mangabeys provide the foundation for determining the potential role of each of these unique host-derived proteins in contributing to the polarized clinical outcome in these 2 species of nonhuman primates.
doi:10.1186/1742-4690-7-107
PMCID: PMC3012658  PMID: 21162735
11.  The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable. 
Journal of Virology  1997;71(5):3641-3651.
Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.
PMCID: PMC191513  PMID: 9094638
12.  In Vivo Replication Capacity Rather Than In Vitro Macrophage Tropism Predicts Efficiency of Vaginal Transmission of Simian Immunodeficiency Virus or Simian/Human Immunodeficiency Virus in Rhesus Macaques 
Journal of Virology  1998;72(4):3248-3258.
We used the rhesus macaque model of heterosexual human immunodeficiency virus (HIV) transmission to test the hypothesis that in vitro measures of macrophage tropism predict the ability of a primate lentivirus to initiate a systemic infection after intravaginal inoculation. A single atraumatic intravaginal inoculation with a T-cell-tropic molecular clone of simian immunodeficiency virus (SIV), SIVmac239, or a dualtropic recombinant molecular clone of SIV, SIVmac239/1A11/239, or uncloned dualtropic SIVmac251 or uncloned dualtropic simian/human immunodeficiency virus (SHIV) 89.6-PD produced systemic infection in all rhesus macaques tested. However, vaginal inoculation with a dualtropic molecular clone of SIV, SIVmac1A11, resulted in transient viremia in one of two rhesus macaques. It has previously been shown that 12 intravaginal inoculations with SIVmac1A11 resulted in infection of one of five rhesus macaques (M. L. Marthas, C. J. Miller, S. Sutjipto, J. Higgins, J. Torten, B. L. Lohman, R. E. Unger, H. Kiyono, J. R. McGhee, P. A. Marx, and N. C. Pedersen, J. Med. Primatol. 21:99–107, 1992). In addition, SHIV HXBc2, which replicates in monkey macrophages, does not infect rhesus macaques following multiple vaginal inoculations, while T-cell-tropic SHIV 89.6 does (Y. Lu, P. B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045–3050, 1996). These results demonstrate that in vitro measures of macrophage tropism do not predict if a SIV or SHIV will produce systemic infection after intravaginal inoculation of rhesus macaques. However, we did find that the level to which these viruses replicate in vivo after intravenous inoculation predicts the outcome of intravaginal inoculation with each virus.
PMCID: PMC109796  PMID: 9525652
13.  ALVAC-SIV-gag-pol-env-Based Vaccination and Macaque Major Histocompatibility Complex Class I (A*01) Delay Simian Immunodeficiency Virus SIVmac-Induced Immunodeficiency 
Journal of Virology  2002;76(1):292-302.
T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIVmac251 (561). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIVmac251 (561) or SIVSME660. However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV89.6P or SHIVKU2, respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.
doi:10.1128/JVI.76.1.292-302.2002
PMCID: PMC135699  PMID: 11739694
14.  Tenofovir treatment augments anti-viral immunity against drug-resistant SIV challenge in chronically infected rhesus macaques 
Retrovirology  2006;3:97.
Background
Emergence of drug-resistant strains of human immunodeficiency virus type 1 (HIV-1) is a major obstacle to successful antiretroviral therapy (ART) in HIV-infected patients. Whether antiviral immunity can augment ART by suppressing replication of drug-resistant HIV-1 in humans is not well understood, but can be explored in non-human primates infected with simian immunodeficiency virus (SIV). Rhesus macaques infected with live, attenuated SIV develop robust SIV-specific immune responses but remain viremic, often at low levels, for periods of months to years, thus providing a model in which to evaluate the contribution of antiviral immunity to drug efficacy. To investigate the extent to which SIV-specific immune responses augment suppression of drug-resistant SIV, rhesus macaques infected with live, attenuated SIVmac239Δnef were treated with the reverse transcriptase (RT) inhibitor tenofovir, and then challenged with pathogenic SIVmac055, which has a five-fold reduced sensitivity to tenofovir.
Results
Replication of SIVmac055 was detected in untreated macaques infected with SIVmac239Δnef, and in tenofovir-treated, naïve control macaques. The majority of macaques infected with SIVmac055 experienced high levels of plasma viremia, rapid CD4+ T cell loss and clinical disease progression. By comparison, macaques infected with SIVmac239Δnef and treated with tenofovir showed no evidence of replicating SIVmac055 in plasma using allele-specific real-time PCR assays with a limit of sensitivity of 50 SIV RNA copies/ml plasma. These animals remained clinically healthy with stable CD4+ T cell counts during three years of follow-up. Both the tenofovir-treated and untreated macaques infected with SIVmac239Δnef had antibody responses to SIV gp130 and p27 antigens and SIV-specific CD8+ T cell responses prior to SIVmac055 challenge, but only those animals receiving concurrent treatment with tenofovir resisted infection with SIVmac055.
Conclusion
These results support the concept that anti-viral immunity acts synergistically with ART to augment drug efficacy by suppressing replication of viral variants with reduced drug sensitivity. Treatment strategies that seek to combine immunotherapeutic intervention as an adjunct to antiretroviral drugs may therefore confer added benefit by controlling replication of HIV-1, and reducing the likelihood of treatment failure due to the emergence of drug-resistant virus, thereby preserving treatment options.
doi:10.1186/1742-4690-3-97
PMCID: PMC1769512  PMID: 17184540
15.  Isolation and Characterization of a Neuropathogenic Simian Immunodeficiency Virus Derived from a Sooty Mangabey 
Journal of Virology  1998;72(11):8841-8851.
Transfusion of blood from a simian immunodeficiency virus (SIV)- and simian T-cell lymphotropic virus-infected sooty mangabey (designated FGb) to rhesus and pig-tailed macaques resulted in the development of neurologic disease in addition to AIDS. To investigate the role of SIV in neurologic disease, virus was isolated from a lymph node of a pig-tailed macaque (designated PGm) and the cerebrospinal fluid of a rhesus macaque (designated ROn2) and passaged to additional macaques. SIV-related neuropathogenic effects were observed in 100% of the pig-tailed macaques inoculated with either virus. Lesions in these animals included extensive formation of SIV RNA-positive giant cells in the brain parenchyma and meninges. Based upon morphology, the majority of infected cells in both lymphoid and brain tissue appeared to be of macrophage lineage. The virus isolates replicated very well in pig-tailed and rhesus macaque peripheral blood mononuclear cells (PBMC) with rapid kinetics. Differential replicative abilities were observed in both PBMC and macrophage populations, with viruses growing to higher titers in pig-tailed macaque cells than in rhesus macaque cells. An infectious molecular clone of virus derived from the isolate from macaque PGm (PGm5.3) was generated and was shown to have in vitro replication characteristics similar to those of the uncloned virus stock. While molecular analyses of this virus revealed its similarity to SIV isolates from sooty mangabeys, significant amino acid differences in Env and Nef were observed. This virus should provide an excellent system for investigating the mechanism of lentivirus-induced neurologic disease.
PMCID: PMC110301  PMID: 9765429
16.  Variability in a dominant block to SIV early reverse transcription in rhesus monkey cells predicts in vivo viral replication and time to death 
Virology Journal  2010;7:79.
While it has long been appreciated that there is considerable variability in host containment of HIV/SIV replication, the determinants of that variability are not fully understood. Previous studies demonstrated that the degree of permissivity of a macaque's peripheral blood mononuclear cells (PBMC) for infection with simian immunodeficiency virus (SIV) in vitro predicted that animal's peak plasma virus RNA levels following SIV infection in vivo. The present study was conducted to define the mechanisms underlying the variable intrinsic susceptibility of rhesus monkey PBMC to SIVsmE660 infection. In a cohort of 15 unrelated Indian-origin rhesus monkeys, infectability of PBMC of individual animals with SIVsmE660, as defined by tissue culture infectious dose (TCID50), varied by more than 3 logs and was a stable phenotype over time. Susceptibility of a monkey's PBMC to wild type SIVsmE660 infection correlated with the susceptibility of that monkey's PBMC to infection with VSV-G pseudotyped SIVsm543-GFP. Moreover, the permissivity of an individual monkey's PBMC for infection with this construct correlated with the permissivity of a B-lymphoblastoid cell line (B-LCL) generated from PBMC of the same animal. We found that the degree of intrinsic resistance of monkey B-LCL correlated with the copy number of early reverse transcription (ERT) SIV DNA. The resistance of monkey B-LCL to SIVsmE660 replication could be abrogated by preincubation of cells with the SIV virus-like particles (VLPs) and SIV resistance phenotype could be transferred to a SIV susceptible B-LCL through cell fusion. Finally, we observed a positive correlation between susceptibility of monkey B-LCL to SIV infection with a VSV-G pseudotyped SIV-GFP construct in vitro and both the peak plasma virus RNA levels in vivo and time to death following wild type SIV infection. These findings suggest that a dominant early RT restricting factor that can be saturated by SIV capsid may contribute to the variable resistance to SIV infection in rhesus monkey B-LCL and that this differential intrinsic susceptibility contributes to the clinical outcome of an SIV infection.
doi:10.1186/1743-422X-7-79
PMCID: PMC2874537  PMID: 20416115
17.  Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6 
Journal of Internal Medicine  2009;265(1):67-77.
Genescà M, McChesney MB, Miller CJ (Center for Comparative Medicine and California National Primate Research Center, University of California, Davis, CA, USA). Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6 (Review).
The recently failed clinical efficacy trial of an acquired immunodeficiency syndrome (AIDS) vaccine that elicits antiviral CD8+ T-cell responses has emphasized the challenge of producing an effective vaccine against human immunodeficiency virus (HIV). In the simian immunodeficiency virus (SIV)/rhesus monkey model of AIDS, live-attenuated lentivirus ‘vaccines’ provide the best protection from uncontrolled viral replication and clinical disease after pathogenic SIV challenge. This review summarizes a recent series of studies in which we show that after vaginal SIV challenge of rhesus macaques immunized with an attenuated lentivirus protection from uncontrolled viral replication is primarily mediated by CD8+ T cells in the vaginal mucosa. Immunization with a chimeric simian/human immunodeficiency virus (SHIV) results in a systemic infection that induces a moderate population of SIV-specific CD8+ and CD4+ T cells with cytolytic potential in the vaginal mucosa. Depletion of CD8+ T cells at the time of SIV challenge completely abrogates the protection mediated by prior infection with attenuated SHIV. Further after vaginal SIV challenge, the only significant expansion of SIV-specific T cells occurs in the vagina in these animals. No significant expansion of T-cell responses was observed in systemic lymphoid tissues. Thus, the presence of SIV-specific CD8+ T cells in the vagina on the day of vaginal SIV challenge and a modest expansion of local effector T cells is sufficient to stop uncontrolled SIV replication. It seems that T-cell based vaccine strategies that can elicit mucosal effector CD8+ T-cell populations and avoid inducing systemic T-cell proliferation upon exposure to HIV have the greatest potential for mimicking the success of live-attenuated lentiviral vaccines.
doi:10.1111/j.1365-2796.2008.02051.x
PMCID: PMC3401014  PMID: 19093961
caspase-3; CD107; cytolytic T cells; Ki67; T-cell activation; vagina
18.  Monocyte/macrophage trafficking in acquired immunodeficiency syndrome encephalitis: Lessons from human and nonhuman primate studies 
Journal of neurovirology  2008;14(4):318-326.
Here the authors discuss evidence in human and animal models supporting two opposing views regarding the pathogenesis of human immunodeficiency virus (HIV) in the central nervous system (CNS): (1) HIV infection in the CNS is a compartmentalized infection, with the virus-infected macrophages entering the CNS early, infecting resident microglia and astrocytes, and achieving a state of latency with evolution toward a fulminant CNS infection late in the course of disease; or alternatively, (2) events in the periphery lead to altered monocyte/macrophage (MΦ) homeostasis, with increased CNS invasion of infected and/or uninfected MΦs. Here the authors have reevaluated evidence presented in the favor of the latter model, with a discussion of phenotypic characteristics distinguishing normal resident microglia with those accumulating in HIV encephalitis (HIVE). CD163 is normally expressed by perivascular MΦs but not resident microglia in normal CNS of humans and rhesus macaques. In agreement with other studies, the authors demonstrate expression of CD163 by brain MΦs in HIVE and simian immunodeficiency virus encephalitis (SIVE). CNS tissues from HIV-sero positive individuals with HIVE or HIV-associated progressive multifocal leukoencephalopathy (PML) were also examined. In HIVE, the authors further demonstrate colocalization of CD163 and CD16 (FcγIII recptor) gene expression, the latter marker associated with HIV infection of monocyte in vivo and permissivity of infection. Indeed, CD163+ MΦs and microglia are often productively infected in HIVE CNS. In SIV infected rhesus macaques, CD163+ cells accumulate perivascularly, within nodular lesions and the parenchyma in animals with encephalitis. Likewise, parenchymal microglia and perivascular MΦs are CD163+ in HIVE. In contrast to HIVE, CD163+perivascular and parenchymal MΦs in HIV-associated PML were only associated with areas of demyelinating lesions. Interestingly, SIV-infected rhesus macaques whose viral burden was predominantly at 1 × 106 copies/ml or greater developed encephalitis. To further investigate the relationship between CD163+/CD16+ MΦs/microglia in the CNS and altered homeostasis in the periphery, the authors performed flow-cytometric analyses of peripheral blood mononuclear cells (PBMCs) from SIV-infected rhesus macaques. The results demonstrate an increase in the percent frequency of CD163+/CD16+ monocytes in animals with detectable virus that correlated significantly with increased viral burden and CD4+ T-cell decline. These results suggest the importance of this monocyte subset in HIV/SIV CNS disease, and also in the immune pathogenesis of lentiviral infection. The authors further discuss the potential role of CD163+/CD16+ monocyte/MΦ subset expansion, altered myeloid homeostasis, and potential consequences for immune polarization and suppression. The results and discussion here suggest new avenues for the development of acquired immunodeficiency syndrome (AIDS) therapeutics and vaccine design.
doi:10.1080/13550280802132857
PMCID: PMC2728912  PMID: 18780233
CD163; CD16; HIVE; macrophage monocyte; SIVE
19.  APOBEC3G Polymorphism as a Selective Barrier to Cross-Species Transmission and Emergence of Pathogenic SIV and AIDS in a Primate Host 
PLoS Pathogens  2013;9(10):e1003641.
Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary “arms-race” between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary “arms-race” hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.
Author Summary
APOBEC3G is a host factor that can inhibit replication of primate lentiviruses, including HIV-1, HIV-2, and the related simian immunodeficiency viruses (SIVs) of African primates. As a consequence, primate lentiviruses encode a protein, called Vif, which can induce degradation of APOBEC3G. Given its antiviral role, APOBEC3G may be an important genetic barrier to interspecies jumping of primate lentiviruses. To study this possibility, we asked whether APOBEC3G affected transmission of SIV from sooty mangabeys (SIVsm) to rhesus macaques and subsequent emergence of pathogenic SIVmac in the 1970s. We found that APOBEC3G of sooty mangabeys and rhesus macaques have divergent protein sequences, and that the Vif proteins of SIVsm (Vif-SIVsm) cannot counteract rhesus macaque APOBEC3G. We mapped Vif-SIVsm resistance to a specific substitution in the N-terminal domain of rhesus APOBEC3G, in which a highly conserved tyrosine is replaced by leucine-arginine (Y→LR). We also identified a viral counter-adaptation, found in the Vif proteins of all SIVmac strains, which specifically confers the ability to antagonize APOBEC3G of rhesus macaques. This change was most likely selected during adaptation of SIV to its new host. Together, these results demonstrate that APOBEC3G can serve as a critical genetic determinant of interspecies transmission of primate immunodeficiency viruses.
doi:10.1371/journal.ppat.1003641
PMCID: PMC3789815  PMID: 24098115
20.  Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239 
PLoS Pathogens  2009;5(1):e1000272.
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 SIVmac239 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 SIVmac239. 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 SIVmac239 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.
Author Summary
AIDS vaccine candidates based on recombinant DNA and/or viral vectors stimulate potent cellular immune responses. However, the extent of protection achieved by these vaccines has so far been disappointing. While live, attenuated strains of SIV afford more reliable protection in animal models, there are justifiable safety concerns with the use of live, attenuated HIV-1 in humans. As an experimental vaccine approach designed to uncouple immune activation from ongoing virus replication, we developed a genetic system for producing strains of SIV that are limited to a single cycle of infection. We compared repeated versus prime-boost vaccine regimens with single-cycle SIV for the ability to elicit protective immunity in rhesus macaques against a strain of SIV that is notoriously difficult to control by vaccination. Both vaccine regimens afforded significant containment of virus replication after challenge. Nevertheless, the extent of protection achieved by immunization with single-cycle SIV was not as good as the protection typically provided by persistent infection of animals with live, attenuated SIV. These observations have important implications for the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may ultimately be necessary for achieving the robust protection afforded by live, attenuated SIV.
doi:10.1371/journal.ppat.1000272
PMCID: PMC2621341  PMID: 19165322
21.  Rhesus Macaque Resistance to Mucosal Simian Immunodeficiency Virus Infection Is Associated with a Postentry Block in Viral Replication 
Journal of Virology  2002;76(12):6016-6026.
Elucidation of the host factors which influence susceptibility to human immunodeficiency virus or simian immunodeficiency virus (SIV) infection and disease progression has important theoretical and practical implications. Rhesus macaque 359, a vaccine control animal, resisted two successive intravaginal challenges with SIVmac251 and failed to seroconvert. Here, after an additional intrarectal SIVmac32H challenge, macaque 359 remained highly resistant to infection. Viral RNA (106 copies/ml) was observed in plasma only at week 2 postchallenge. Virus isolation and proviral DNA were positive only once at week eight postchallenge. The animal remained seronegative and cleared SIV in vivo. Its blood and lymph node cells obtained at 49 weeks after intrarectal challenge did not transmit SIV to a naive macaque. We found that the resistance of macaque 359 to SIV infection was not due to a high level of CD8+ suppressor activity but to an inherent resistance of its CD4+ T cells. To elucidate the basis for the unusually strong resistance of macaque 359 to SIV infection in vivo and in vitro, we investigated early events of viral infection and replication in CD4+ cells of macaque 359, including expression and mutation screening of SIV coreceptors and analysis of viral entry and reverse transcription. Mutation screening revealed no genetic alteration in SIV coreceptors. PCR analysis revealed a significant delay in production of early in vitro reverse transcription intermediates in macaque 359 cells compared to susceptible controls, but cell fusion assays showed that SIV entered the CD4+ CCR5+ cells of macaque 359 as readily as cells of macaques susceptible to SIV infection. Our results suggest that the resistance of macaque 359 to SIV infection is due to a postentry block in viral replication and implicate a cellular inhibitory mechanism in its CD4+ T cells. Identification of this host mechanism will help further elucidate the biochemistry of reverse transcription and may suggest therapeutic strategies. Determining the prevalence of this host resistance mechanism among macaques may lead to better design of SIV pathogenesis and vaccine studies.
doi:10.1128/JVI.76.12.6016-6026.2002
PMCID: PMC136199  PMID: 12021334
22.  SIVsm Quasispecies Adaptation to a New Simian Host 
PLoS Pathogens  2005;1(1):e3.
Despite the potential for infectious agents harbored by other species to become emerging human pathogens, little is known about why some agents establish successful cross-species transmission, while others do not. The simian immunodeficiency viruses (SIVs), certain variants of which gave rise to the human HIV-1 and HIV-2 epidemics, have demonstrated tremendous success in infecting new host species, both simian and human. SIVsm from sooty mangabeys appears to have infected humans on several occasions, and was readily transmitted to nonnatural Asian macaque species, providing animal models of AIDS. Here we describe the first in-depth analysis of the tremendous SIVsm quasispecies sequence variation harbored by individual sooty mangabeys, and how this diverse quasispecies adapts to two different host species—new nonnatural rhesus macaque hosts and natural sooty mangabey hosts. Viral adaptation to rhesus macaques was associated with the immediate amplification of a phylogenetically related subset of envelope (env) variants. These variants contained a shorter variable region 1 loop and lacked two specific glycosylation sites, which may be selected for during acute infection. In contrast, transfer of SIVsm to its natural host did not subject the quasispecies to any significant selective pressures or bottleneck. After 100 d postinfection, variants more closely representative of the source inoculum reemerged in the macaques. This study describes an approach for elucidating how pathogens adapt to new host species, and highlights the particular importance of SIVsm env diversity in enabling cross-species transmission. The replicative advantage of a subset of SIVsm variants in macaques may be related to features of target cells or receptors that are specific to the new host environment, and may involve CD4-independent engagement of a viral coreceptor conserved among primates.
Synopsis
Why do some infectious agents establish successful cross-species transmission while others do not? Despite the clear potential for diseases harbored by animals to become emerging human pathogens, this question remains unanswered. Certain simian immunodeficiency viruses (SIVs) responsible for the human HIV-1 and HIV-2 epidemics have succeeded in infecting new host species, including humans. This study provides clues to how an SIV adapts to a new host in an experimental cross-species transmission. Indeed, many emerging diseases are caused by highly mutation-prone RNA viruses like SIV, which exist not as a single species, but rather as a population of genetic variants within a single infection. The presence of numerous viral variants in an infected animal increases the chance that variants with the ability to enter into or multiply in a new host species are present. This study describes how an SIV population from a natural reservoir host, the sooty mangabey, adapts to a new monkey species, the rhesus macaque. A limited subset of SIV variants containing unique viral surface proteins appears well suited to multiply in the new host. This study documents how viral variation facilitates cross-species transmission, and highlights the particular importance of immunodeficiency virus envelope variants in infecting new hosts.
doi:10.1371/journal.ppat.0010003
PMCID: PMC1238738  PMID: 16201015
23.  Breaking Barriers to an AIDS Model with Macaque-Tropic HIV-1 Derivatives 
Biology : open access journal  2012;1(2):134-164.
The development of an animal model of human immunodeficiency virus type 1 (HIV-1)/AIDS that is suitable for preclinical testing of antiretroviral therapy, vaccines, curative strategies, and studies of pathogenesis has been hampered by the human-specific tropism of HIV-1. Although simian immunodeficiency virus (SIV) or HIV-1/SIV chimeric viruses (SHIVs)-rhesus macaque models are excellent surrogates for AIDS research, the genetic differences between SIV or SHIV and HIV-1 limit their utility as model systems. The identification of innate retro viral restriction factors has increased our understanding about blockades to HIV-1 replication in macaques and provided a guide for the construction of macaque-tropic HIV-1 clones. However, while these viruses replicate in macaque cells in vitro, they are easily controlled and have not caused AIDS in host animals, indicating that we may not fully understand the restrictive barriers of innate immunity. In this review, we discuss recent findings regarding HIV-1 restriction factors, particularly as they apply to cross-species transmission of primate lentiviruses and the development of a macaque model of HIV-1/AIDS.
doi:10.3390/biology1020134
PMCID: PMC3546514  PMID: 23336082
HIV-1; SIV; Macaca nemestrina; AIDS; cross-species; tropism; innate restriction
24.  Breaking Barriers to an AIDS Model with Macaque-Tropic HIV-1 Derivatives 
Biology  2012;1(2):134-164.
The development of an animal model of human immunodeficiency virus type 1 (HIV-1)/AIDS that is suitable for preclinical testing of antiretroviral therapy, vaccines, curative strategies, and studies of pathogenesis has been hampered by the human-specific tropism of HIV-1. Although simian immunodeficiency virus (SIV) or HIV-1/SIV chimeric viruses (SHIVs)-rhesus macaque models are excellent surrogates for AIDS research, the genetic differences between SIV or SHIV and HIV-1 limit their utility as model systems. The identification of innate retroviral restriction factors has increased our understanding about blockades to HIV-1 replication in macaques and provided a guide for the construction of macaque-tropic HIV-1 clones. However, while these viruses replicate in macaque cells in vitro, they are easily controlled and have not caused AIDS in host animals, indicating that we may not fully understand the restrictive barriers of innate immunity. In this review, we discuss recent findings regarding HIV-1 restriction factors, particularly as they apply to cross-species transmission of primate lentiviruses and the development of a macaque model of HIV-1/AIDS.
doi:10.3390/biology1020134
PMCID: PMC3546514  PMID: 23336082
HIV-1; SIV; Macaca nemestrina; AIDS; cross-species; tropism; innate restriction
25.  TRIM5 Suppresses Cross-Species Transmission of a Primate Immunodeficiency Virus and Selects for Emergence of Resistant Variants in the New Species 
PLoS Biology  2010;8(8):e1000462.
Cross-species transmission of simian immunodeficiency virus from sooty mangabeys (SIVsm) into rhesus macaques, and subsequent emergence of pathogenic SIVmac, required adaptation to overcome restriction encoded by the macaque TRIM5 gene.
Simian immunodeficiency viruses of sooty mangabeys (SIVsm) are the source of multiple, successful cross-species transmissions, having given rise to HIV-2 in humans, SIVmac in rhesus macaques, and SIVstm in stump-tailed macaques. Cellular assays and phylogenetic comparisons indirectly support a role for TRIM5α, the product of the TRIM5 gene, in suppressing interspecies transmission and emergence of retroviruses in nature. Here, we investigate the in vivo role of TRIM5 directly, focusing on transmission of primate immunodeficiency viruses between outbred primate hosts. Specifically, we retrospectively analyzed experimental cross-species transmission of SIVsm in two cohorts of rhesus macaques and found a significant effect of TRIM5 genotype on viral replication levels. The effect was especially pronounced in a cohort of animals infected with SIVsmE543-3, where TRIM5 genotype correlated with approximately 100-fold to 1,000-fold differences in viral replication levels. Surprisingly, transmission occurred even in individuals bearing restrictive TRIM5 genotypes, resulting in attenuation of replication rather than an outright block to infection. In cell-culture assays, the same TRIM5 alleles associated with viral suppression in vivo blocked infectivity of two SIVsm strains, but not the macaque-adapted strain SIVmac239. Adaptations appeared in the viral capsid in animals with restrictive TRIM5 genotypes, and similar adaptations coincide with emergence of SIVmac in captive macaques in the 1970s. Thus, host TRIM5 can suppress viral replication in vivo, exerting selective pressure during the initial stages of cross-species transmission.
Author Summary
The human immunodeficiency viruses HIV-1 and HIV-2 originated from cross-species transmission of simian immunodeficiency viruses (SIVs) from chimpanzees (SIVcpz) and sooty mangabeys (SIVsm), respectively. A related virus, SIVmac, causes AIDS-like pathogenesis in rhesus macaques; like HIV-2, SIVmac is the product of a cross-species jump of SIVsm from sooty mangabeys. The primate TRIM5 gene encodes a factor with potent antiviral activity when tested in the laboratory, and TRIM5 proteins are thought to play a role in restricting the movement of viruses between species in nature. In this study, we show that genetic variation in the TRIM5 gene of rhesus macaques heavily influences the outcome of cross-species transmission of SIVsm and that emergence of SIVmac in rhesus macaques in the 1970s required adaptations to circumvent the genetic barrier imposed by the rhesus macaque TRIM5 gene. Our results confirm the hypothesis that TRIM5 can influence the process of cross-species transmission and emergence of viruses related to HIV-1 and HIV-2 and serve as a striking illustration of how host genes can influence virus evolution.
doi:10.1371/journal.pbio.1000462
PMCID: PMC2927514  PMID: 20808775

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