Optimization of PCR for quantification of simian immunodeficiency virus genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA.

INTRODUCTION: Quantification of plasma viral load (PVL) is used to monitor disease progression in SIV-infected macaques. This study was aimed at optimizing of performance characteristics of the quantitative PCR (qPCR) PVL assay. METHODS: The PVL quantification procedure was optimized by inclusion of an exogenous control hepatitis C virus armored RNA (aRNA), a plasma concentration step, extended digestion with proteinase K, and a second RNA elution step. Efficiency of viral RNA (vRNA) extraction was compared using several commercial vRNA extraction kits. Various parameters of qPCR targeting the gag region of SIVmac239, SIVsmE660, and the LTR region of SIVagmSAB were also optimized. RESULTS: Modifications of the SIV PVL qPCR procedure increased vRNA recovery, reduced inhibition and improved analytical sensitivity. The PVL values determined by this SIV PVL qPCR correlated with quantification results of SIV RNA in the same samples using the 'industry standard' method of branched-DNA (bDNA) signal amplification. CONCLUSIONS: Quantification of SIV genomic RNA in plasma of rhesus macaques using this optimized SIV PVL qPCR is equivalent to the bDNA signal amplification method, less costly and more versatile. Use of heterologous aRNA as an internal control is useful for optimizing performance characteristics of PVL qPCRs.

Cyclic changes in the vaginal epithelium of normal rhesus macaques.

Studies in nonhuman primates indicate that changes in the thickness and integrity of the vaginal epithelium affect the transmission rates of HIV-1, but few studies have examined the normal variations that may occur in the vagina of normal macaques as a result of aging or changes in the menstrual cycle. This study was conducted to determine if differences occur in the thickness of the vaginal mucosa with age or menses. Vaginal mucosal thickness was compared in 46 rhesus macaques grouped as juvenile (1-3 years old), mature cycling (3-21 years old), and geriatric (> 21 years old). Epithelia of mature cycling macaques were also compared at different stages of the menstrual cycle. Older females (> 21 years) had the thinnest and least keratinized epithelium of all groups, followed by the youngest females (< 3 years). The vaginal epithelium was also thinner in cycling macaques during menses compared to the follicular stage. In addition, young, geriatric, or cycling macaques during menses had minimal keratinization. We hypothesize that normal physiologic changes in the vaginal epithelium of women occur with age and menses, which may affect a woman's susceptibility to HIV-1 transmission and other sexually transmitted diseases. Also, age and menstrual cycle should be considered when designing vaginal transmission experiments in rhesus macaques.

Molecular evidence for deep phylogenetic divergence in Mandrillus sphinx.

Mandrills (Mandrillus sphinx) are forest primates indigenous to western central Africa. Phylogenetic analysis of 267 base pairs (bp) of the cytochrome b gene from 53 mandrills of known and 17 of unknown provenance revealed two phylogeographical groups, with haplotypes differentiated by 2.6% comprising seven synonymous transitions. The distribution of the haplotypes suggests that the Ogooué River, Gabon, which bisects their range, separates mandrill populations in Cameroon and northern Gabon from those in southern Gabon. The haplotype distribution is also concordant with that of two known mandrill simian immunodeficiency viruses, suggesting that these two mandrill phylogroups have followed different evolutionary trajectories since separation.

Longitudinal follow up of SIVmac pathogenesis in rhesus macaques of Chinese origin: emergence of B cell lymphoma.

Two subspecies of rhesus (Rh) macaques, the Chinese (Ch) and Indian (Ind) subspecies were infected intravenously with 100TCID50 SIVmac239. CD4+, CD8+ T cells, plasma viral loads, depletion of intestinal lymphocytes with memory phenotype, humoral immune responses and clinical courses were monitored for 600 days. The pathogenesis of SIVmac was also compared with primary human immunodeficiency virus (HIV) infection of humans. Plasma viral loads in Ch Rh were lower in the acute and chronic phases compared with Ind Rh. SIVmac pathogenesis in Ch Rh was closer to virus loads in untreated HIV infected humans. Ch Rh had higher CD4/CD8 ratios, stronger antibody responses and interestingly, less depletion of intestinal memory CCR5+ CD4+ T lymphocytes compared with Ind Rh. One Ch Rh developed B cell origin lymphoma at 570 days post-infection, the first such report in this subspecies. Three of four Ind Rh developed AIDS within 6 months. The findings indicate that Ch Rh are more resistant to SIVmac pathogenesis compared with Ind Rh and that Ch Rh paralleled HIV-1 infections in untreated adult humans. The SIVmac infected Ch Rh subspecies are an acceptable model for HIV/AIDS.

Endometrial hyperplasia, polyps, and adenomyosis associated with unopposed estrogen in rhesus monkeys (Macaca mulatta).

The purpose of this study was to evaluate the effects of estrogen and progesterone on the vaginal mucosa and their role in vaginal transmission of simian immunodeficiency virus. Incidentally, endometrial hyperplasia was observed in estrogen-treated monkeys at necropsy. Six adult female rhesus monkeys (Macaca mulatta) were ovariectomized and 120 days later received two subcutaneous implants, each containing 200 mg estradiol. The animals were sacrificed 17-27 months later and the uterus examined at necropsy. All the monkeys had simple endometrial hyperplasia, some with polyps or adenomyosis, at the time of necropsy. The severity of the changes correlated with the time between implantation and necropsy. The lesions were similar to endometrial hyperplasia caused by unopposed estrogen in women, but occurred over a time period that is suitable for experimental manipulation. Rhesus monkeys could be used as a model to test the safety of various combinations of sex steroids for the prevention of postmenopausal symptoms in women.

The mucosal immune system: primary target for HIV infection and AIDS.

Despite intensive research, several questions remain regarding the pathogenesis of infection with HIV-1. Recently, it has been shown that simian immunodeficiency virus (SIV) selectively targets and destroys specific subsets of CD4+ T cells that are abundant in mucosal tissues but rare in peripheral lymphoid tissues. This finding could be highly relevant in explaining a major paradox in the infection and elimination of CD4+ T cells during HIV infection: the progressive decline in the number of CD4+ T cells in the blood, despite the paucity of HIV-infected cells in this tissue. This article discusses the hypothesis that infection with HIV and SIV, and the resulting disease, is governed by the state of cellular activation and the expression of chemokine receptors by specific subsets of CD4+ T cells residing in mucosal lymphoid tissues, rather than those found in the peripheral blood or lymph nodes.

An effective AIDS vaccine based on live attenuated vesicular stomatitis virus recombinants.

We developed an AIDS vaccine based on attenuated VSV vectors expressing env and gag genes and tested it in rhesus monkeys. Boosting was accomplished using vectors with glycoproteins from different VSV serotypes. Animals were challenged with a pathogenic AIDS virus (SHIV89.6P). Control monkeys showed a severe loss of CD4+ T cells and high viral loads, and 7/8 progressed to AIDS with an average time of 148 days. All seven vaccinees were initially infected with SHIV89.6P but have remained healthy for up to 14 months after challenge with low or undetectable viral loads. Protection from AIDS was highly significant (p = 0.001). VSV vectors are promising candidates for human AIDS vaccine trials because they propagate to high titers and can be delivered without injection.

Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro.

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV(162P4). Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.

A divergent simian immunodeficiency virus from sooty mangabey with an atypical Tat-TAR structure.

SIVsm, the simian immunodeficiency virus that naturally infects sooty mangabeys in West Africa, is the closest lentiviral relative of human immunodeficiency virus type 2 (HIV-2). To determine the genetic characteristics of SIVsm in its natural host, we sequenced the full-length genome of SIVsmSL92b, a primary isolate obtained from a pet sooty mangabey in Sierra Leone. SIVsmSL92b proved to be the most divergent member of the HIV-2/SIVsm lineage found thus far, having as much as 35% nucleotide divergence from other HIV-2 genomes. A phylogenetic association between SIVsmSL92b and HIV-2 PA subtype E, which had been previously revealed by the analysis of partial gag sequences, was extended to the pol gene. SIVsmSL92b showed several divergent features, including a short Tat protein of 104 residues and an atypical TAR structure. Specifically, only one of the duplicate TAR elements contained the conserved hexanucleotide loop sequence CUGGGX important for Tat-cyclin T1 binding. These features suggested that the mechanism of SIVsmSL92b Tat and TAR interaction differed from that described for HIV-2. Taken together, these findings indicated that the structural diversity within the HIV-2/SIVsm lineage was greater than previously appreciated.

Wild Mandrillus sphinx are carriers of two types of lentivirus.

Mandrillus sphinx, a large primate living in Cameroon and Gabon and belonging to the Papionini tribe, was reported to be infected by a simian immunodeficiency virus (SIV) (SIVmndGB1) as early as 1988. Here, we have identified a second, highly divergent SIVmnd (designated SIVmnd-2). Genomic organization differs between the two viral types; SIVmnd-2 has the additional vpx gene, like other SIVs naturally infecting the Papionini tribe (SIVsm and SIVrcm) and in contrast to the other SIVmnd type (here designated SIVmnd-1), which is more closely related to SIVs infecting l'hoest (Cercopithecus lhoesti lhoesti) and sun-tailed (Cercopithecus lhoesti solatus) monkeys. Importantly, our epidemiological studies indicate a high prevalence of both types of SIVmnd; all 10 sexually mature wild-living monkeys and 3 out of 17 wild-born juveniles tested were infected. The geographic distribution of SIVmnd seems to be distinct for the two types: SIVmnd-1 viruses were exclusively identified in mandrills from central and southern Gabon, whereas SIVmnd-2 viruses were identified in monkeys from northern and western Gabon, as well as in Cameroon. SIVmnd-2 full-length sequence analysis, together with analysis of partial sequences from SIVmnd-1 and SIVmnd-2 from wild-born or wild-living mandrills, shows that the gag and pol regions of SIVmnd-2 are closest to those of SIVrcm, isolated from red-capped mangabeys (Cercocebus torquatus), while the env gene is closest to that of SIVmnd-1. pol and env sequence analyses of SIV from a related Papionini species, the drill (Mandrillus leucophaeus), shows a closer relationship of SIVdrl to SIVmnd-2 than to SIVmnd-1. Epidemiological surveys of human immunodeficiency virus revealed a case in Cameroon of a human infected by a virus serologically related to SIVmnd, raising the possibility that mandrills represent a viral reservoir for humans similar to sooty mangabeys in Western Africa and chimpanzees in Central Africa.

Serial human passage of simian immunodeficiency virus by unsterile injections and the emergence of epidemic human immunodeficiency virus in Africa.

There is compelling evidence that both human immunodeficiency virus (HIV) types emerged from two dissimilar simian immunodeficiency viruses (SIVs) in separate geographical regions of Africa. Each of the two HIVs has its own simian progenitor and specific genetic precursor, and all of the primates that carry these SIVs have been in close contact with humans for thousands of years without the emergence of epidemic HIV. To date no plausible mechanism has been identified to account for the sudden emergence in the mid-20th century of these epidemic HIVs. In this study we examine the conditions needed for SIV to complete the genetic transition from individual human SIV infections to epidemic HIV in humans. The genetic distance from SIV to HIV and the mutational activity needed to achieve this degree of adaptation to human hosts is placed within a mathematical model to estimate the probabilities of SIV completing this transition within a single SIV-infected human host. We found that the emergence of even one epidemic HIV strain, following a single human exposure to SIV, was very unlikely. And the probability of four or more such transitions (i.e. HIV-1 groups M, O and HIV-2 subtypes A and B) occurring in a brief period is vanishingly small. We conclude that SIV cannot become a zoonosis, but requires adaptive mutations to become HIV. Some modern event must have aided in the transition of SIV to HIV. Our research indicates that serial passage of partially adapted SIV between humans could produce the series of cumulative mutations sufficient for the emergence of epidemic HIV strains. We examined the rapid growth of unsterile injections in Africa beginning in the 1950s as a biologically plausible event capable of greatly increasing serial human passage of SIV and generating HIV by a series of multiple genetic transitions. We conclude that increased unsterile injecting in Africa during the period 1950-1970 provided the agent for SIV human infections to emerge as epidemic HIV in the modern era.

Constitutively dead, conditionally live HIV-1 genomes. Ex vivo implications for a live virus vaccine.

An effective vaccine against AIDS is unlikely to be available for many years. As we approach two decades since the first identification of human immunodeficiency virus, type 1 (HIV-1), currently, only one subunit vaccine candidate has reached phase 3 of clinical trials. The subunit approach has been criticized for its inability to elicit effectively cytotoxic T-lymphocyte (CTL) response, which is felt by many to be needed for protection against HIV-1 infection. In subhuman primates, a live attenuated simian immunodeficiency virus (SIV) vaccine candidate, capable of inducing CTL, has been found to confer prophylactic immunity sufficient to prevent simian AIDS. Because replication competent (live) attenuated viruses could over time revert to virulence, such a live attenuated approach has largely been dismissed for HIV-1. Here, we describe the creation of constitutively dead conditionally live (CDCL) HIV-1 genomes. These genomes are constitutively defective for the Tat/TAR axis and are conditionally dependent on tetracycline for attenuated replication with robust expression of viral antigens. Our results suggest that CDCL genomes merit consideration as safer "live" attenuated HIV-1 vaccine candidates.

Evolution of endogenous retrovirus-like elements of the woolly mammoth (Mammuthus primigenius) and its relatives.

Endogenous retrovirus-like elements characterizable by a leucine tRNA primer (ERV-Ls) are reiterated genomic sequences known to be widespread in mammals, including humans. They may have arisen from an ancestral foamy virus-like element by successful germ line infection followed by copy number expansion. However, among mammals, only primates and rodents have thus far exhibited high copy number amplification and sequence diversification. Conventionally, empirical studies of proviral amplification and diversification have been limited to extant species, but taxa having good Quaternary fossil records could potentially be investigated using the techniques of "ancient" DNA research. To examine evolutionary parameters of ERV-Ls across both time and taxa, we characterized this proviral class in the extinct woolly mammoth (Mammuthus primigenius) and living elephants, as well as extant members of the larger clade to which they belong (Uranotheria, a group containing proboscideans, sirenians, hyraxes, and their extinct relatives). Ungulates and carnivores previously analyzed demonstrated low copy numbers of ERV-L sequences, and thus it was expected that uranotheres should as well. Here, we show that all uranothere taxa exhibit unexpectedly numerous and diverse ERV-L sequence complements, indicating active expansion within this group of lineages. Selection is the most parsimonious explanation for observed differences in ERV-L distribution and frequency, with relative success being reflected in the persistence of certain elements over a variety of sampled time depths (as can be observed by comparing sequences from fossil and extant elephantid samples).

Age-dependent changes in T cell homeostasis and SIV load in sooty mangabeys.

Sooty mangabeys (Cercocebus atys) showed age-dependent changes in T cell regeneration. Younger animals had a high percentage of CD4+ CD45RA + T cells and a high concentration of T cell receptor excisional circles (TRECs) in peripheral blood, which indicated active thymopoiesis. In contrast, older animals had an increased T cell turnover, which suggested that most T cell production occurred in the periphery. In addition, the number of peripheral CD4+ T cells naturally decreased with age. Non-pathogenic SIVsm infection did not significantly change the T cell proliferation rate or the TREC concentration, though it did cause a moderate loss of peripheral CD4 + T cells. The viral load correlated negatively with age, which could be accounted for by the reduced availability of CD4 + target cells in older mangabeys. Thus, the number of susceptible target cells may be a limiting factor in natural SIV infection.

Interactions between Mycobacterium leprae and simian immunodeficiency virus (SIV) in rhesus monkeys.

Groups of rhesus monkeys were inoculated with: 1) simian immunodeficiency virus (SIV)B670 alone; 2) Mycobacterium leprae alone; 3) SIV plus M. leprae on the same day; and 4) M. leprae 2 weeks after SIV. Animals were monitored at intervals for virus loads, antibody responses to M. leprae glycolipid antigens and to SIV Gp120, T-cell CD4+ and CD4+ CD29+ subset percentages, leprosy and acquired immunodeficiency syndrome (AIDS) clinical symptoms. Five out of six animals developed leprosy in each co-inoculated group, compared to one out of six in the M. leprae-only-inoculated group, indicating that M. leprae/SIV co-infection increases the susceptibility to leprosy, regardless of the timing of the two infections. Animals in the co-infected group that received M. leprae 2 weeks after SIV had a significantly slower rate of AIDS progression and long-term survival was significantly greater (three out of six) compared to the group inoculated with SIV alone (zero out of seven). All M. leprae-only-inoculated animals (six out of six) survived. Post-SIV-inoculation, a rapid decrease in the percentages of CD4 + and CD4 + CD29 + T-cells was observed in the SIV-only-inoculated group that was significantly blocked by co-inoculation with M. leprae 2 weeks after SIV, but not by SIV on the same day. The virus load set point was increased by approximately two logs in the group inoculated with M. leprae and SIV on the same day compared to SIV 2 weeks prior to M. leprae or the SIV-only-inoculated group. The results indicate that M. leprae, inoculated 2 weeks after SIV, decreased the pathogenicity of SIV compared to inoculation of M. leprae and SIV on the same day or SIV alone. The decreased pathogenicity correlated with a diminished loss of CD4 + and CD4 + CD29 + T-cell subsets in the group inoculated with M. leprae 2 weeks after SIV compared to the group inoculated with SIV alone. IgG antibody responses to M. leprae-specific cell wall phenolic glycolipid-I antigen were inhibited by 2-week-prior or same-day SIV co-inoculation compared to M. leprae-only inoculated animals. The IgG anti-lipoarabinomannan antibody response was enhanced in the group inoculated with M. leprae and SIV on the same day compared to the groups inoculated with M. leprae alone or SIV 2 weeks prior to M. leprae. Antibody responses to SIV Gp120 antigen were unimpaired in both co-inoculated groups compared to SIV-only-inoculated groups. The antibody results show that the immune responses to SIV and M. leprae are interrelated in SIV/M. leprae co-infected animals.

Estrogen protects against vaginal transmission of simian immunodeficiency virus.

Postmenopausal women and women who use injectable, progestin-based contraceptives are at increased risk of human immunodeficiency virus (HIV) infection, suggesting that progesterone and estrogen affect HIV-1 vaginal transmission. To evaluate the individual roles of these sex hormones in vaginal transmission, ovariectomized female macaques were treated with either progesterone or estrogen followed by intravaginal inoculation with SIVmac. All 6 untreated control macaques and 5 (83%) of 6 progesterone-treated animals became infected following intravaginal SIV inoculation. Conversely, none of 6 estrogen-treated macaques was infected. Vaginal subepithelial inoculation of estrogen-treated animals resulted in infection, which shows that the block occurred at the vaginal epithelium and/or lumen. These data suggest that estrogen-deficient women are at increased risk of HIV infection, because their vaginal microenvironments are rendered more susceptible. Moreover, topical vaginal estrogen therapy may be an effective means of reducing HIV vaginal transmission in these high-risk groups.

Use of inhibitors to evaluate coreceptor usage by simian and simian/human immunodeficiency viruses and human immunodeficiency virus type 2 in primary cells.

We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative coreceptor that is expressed in the cells of some individuals. SIV(mac)239 and SIV(mac)251 (from macaques) were also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors. The replication in human PBMC of SIV(rcm) (from a red-capped mangabey), a virus which uses CCR2 but not CCR5 for entry, was blocked by TAK-779, suggesting that CCR2 is indeed the paramount coreceptor for this virus in primary cells.