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.

Progesterone implants enhance SIV vaginal transmission and early virus load.

Simian immunodeficiency virus (SIV) can cross the intact vaginal epithelium to establish a systemic infection in macaques (mac). Using this SIVmac model, we found that subcutaneous progesterone implants, which could mimic hormonally based contraceptives, thinned the vaginal epithelium and enhanced SIV vaginal transmission 7.7-fold over that observed in macaques treated with placebo implants and exposed to SIV in the follicular phase of the menstrual cycle. Progesterone treatment also increased the number of SIV DNA-positive cells in the vaginal lamina propria as detected by in situ polymerase chain reaction analysis. Moreover, plasma viral RNA was elevated for the first three months in macaques with progesterone implants, and three of the progesterone-treated macaques developed relatively rapid disease courses. This study shows that SIV genital infection and disease course are enhanced by subcutaneous implants containing progesterone when compared with the rate of vaginal transmission in the follicular phase.

Antigenic modulation of Friend virus erythroleukemic cells in vitro by serum from mice with dormant erythroleukemia.

Friend leukemia virus (FLV) erythroleukemic cells cultured in medium containing FLV-immune serum from dormant FLV-infected mice undergo modulation of FLV cell surface antigens. Modulation was determined by an increased resistance to FLV antibody-mediated complement-dependent lysis and was associated temporally with the capping of FLV-immune complexes at the cell surface. Modulated cells regained their susceptibility to FLV antibody-mediated complement-dependent lysis when transferred to medium containing normal mouse serum. After 48 h of culture in FLV-immune serum, 26% of the FLV erythroleukemic cells were devoid of FLV cell surface antigens as demonstrated by immunofluoresence. Antigenic modulation occurred to a greater extent in cells maintained in logarithmic growth than in cells in GO or resting phase. FLV-antigenic modulation is discussed as a possible mechanism by which antibody induces and maintains FLV-transformed cells in a dormant state.

Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques.

We used the simian immunodeficiency virus (SIV)/rhesus macaque model to study events that underlie sexual transmission of human immunodeficiency virus type 1 (HIV-1). Four female rhesus macaques were inoculated intravaginally with SIVmac251, and then killed 2, 5, 7, and 9 d later. A technique that detected polymerase chain reaction-amplified SIV in situ showed that the first cellular targets for SIV were in the lamina propria of the cervicovaginal mucosa, immediately subjacent to the epithelium. Phenotypic and localization studies demonstrated that many of the infected cells were likely to be dendritic cells. Within 2 d of inoculation, infected cells were identified in the paracortex and subcapsular sinus of the draining internal iliac lymph nodes. Subsequently, systemic dissemination of SIV was rapid, since culturable virus was detectable in the blood by day 5. From these results, we present a model for mucosal transmission of SIV and HIV-1.

Natural infection of a homozygous delta24 CCR5 red-capped mangabey with an R2b-tropic simian immunodeficiency virus.

A homozygous 24-bp deletion (Delta24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Delta24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Delta24CCR5/ Delta24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Delta24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by beta-chemokines. At 86.6%, the Delta24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Delta24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Delta24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Delta24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host.

Effects of in vivo CD8(+) T cell depletion on virus replication in rhesus macaques immunized with a live, attenuated simian immunodeficiency virus vaccine.

The role of CD8(+) T lymphocytes in controlling replication of live, attenuated simian immunodeficiency virus (SIV) was investigated as part of a vaccine study to examine the correlates of protection in the SIV/rhesus macaque model. Rhesus macaques immunized for >2 yr with nef-deleted SIV (SIVmac239Deltanef) and protected from challenge with pathogenic SIVmac251 were treated with anti-CD8 antibody (OKT8F) to deplete CD8(+) T cells in vivo. The effects of CD8 depletion on viral load were measured using a novel quantitative assay based on real-time polymerase chain reaction using molecular beacons. This assay allows simultaneous detection of both the vaccine strain and the challenge virus in the same sample, enabling direct quantification of changes in each viral population. Our results show that CD8(+) T cells were depleted within 1 h after administration of OKT8F, and were reduced by as much as 99% in the peripheral blood. CD8(+) T cell depletion was associated with a 1-2 log increase in SIVmac239Deltanef plasma viremia. Control of SIVmac239Deltanef replication was temporally associated with the recovery of CD8(+) T cells between days 8 and 10. The challenge virus, SIVmac251, was not detectable in either the plasma or lymph nodes after depletion of CD8(+) T cells. Overall, our results indicate that CD8(+) T cells play an important role in controlling replication of live, attenuated SIV in vivo.

Nucleotide sequence of SRV-1, a type D simian acquired immune deficiency syndrome retrovirus.

Simian acquired immune deficiency syndrome (SAIDS) in the macaque genus of monkeys at the California Primate Research Center is apparently caused by infection by a type D retrovirus. The complete nucleotide sequence (8173 base pairs) of a molecular clone of the prototype SAIDS virus isolate, SRV-1, reveals a typical retrovirus structure with long terminal repeats (346 base pairs) and open reading frames for the gag (663 codons), pol (867 codons), and env (605 codons) genes. SRV-1 also has a separate open reading frame of 314 codons between the gag and pol genes that defines the viral protease gene (prt) and a short open reading frame of unknown significance downstream from the env gene. The SRV-1 protease region shows a high degree of homology to its counterpart in the hamster intracisternal A-type particle genome; both these protease genes are about twice as long as the analogous region of other retroviruses. SRV-1 has no notable similarity in either genetic organization or sequence to the human AIDS retroviruses.

Simian AIDS: isolation of a type D retrovirus and transmission of the disease.

A type D retrovirus related to but distinct from Mason-Pfizer monkey virus was isolated in vitro from the blood of two rhesus monkeys (Macaca mulatta) with simian acquired immunodeficiency syndrome (SAIDS). Three juvenile rhesus monkeys that were injected intravenously with tissue culture fluids containing this virus developed SAIDS after 2 to 4 weeks.

Transmission of simian acquired immunodeficiency syndrome (SAIDS) with blood or filtered plasma.

Simian acquired immunodeficiency syndrome (SAIDS), a disease clinically and pathologically similar to acquired immunodeficiency syndrome in humans, was transmitted from diseased rhesus monkeys (Macaca mulatta) to normal monkeys by inoculation with heparinized whole blood or plasma that had been passed through filters of 0.45 micrometer pore size. This suggests that the causative agent is small and most probably a virus. No viruses, however, were isolated by standard cell culture techniques from the blood or filtered plasma which caused SAIDS. Both cellular and humoral immunity were markedly depressed in animals with advanced SAIDS.

Protection against vaginal SIV transmission with microencapsulated vaccine.

Although protection in animal models against intravenous challenges with simian immunodeficiency virus (SIV) has been reported, no previous vaccines have protected against a heterosexual route of infection. In this study, five of six macaques were protected against vaginal challenge when immunized with formalin-treated SIV in biodegradable microspheres by the intramuscular plus oral or plus intratracheal route. Oral immunization alone did not protect. After a second vaginal challenge, three of four intramuscularly primed and mucosally boosted macaques remained protected. The data suggest that protection against human immunodeficiency virus vaginal transmission could be provided by microsphere-based booster vaccines when used to immunize women who are systemically primed.

Parallel evolution of CCR5-null phenotypes in humans and in a natural host of simian immunodeficiency virus.

The C-C chemokine receptor CCR5 in humans and rhesus macaques (Macaca mulatta) serves as the primary coreceptor for cellular entry by macrophagetropic strains of human immunodeficiency virus type 1 (HIV-1) and all reported strains of simian immunodeficiency virus (SIV) [1-6]. Humans homozygous for a 32 bp deletion allele of CCR5, resulting in a null phenotype, are highly resistant to infection by HIV-1 [7-9], prompting development of therapies and vaccines targeting CCR5. We now report a novel deletion allele of CCR5, with an allele frequency of 0.04, in sooty mangabey monkeys (Cercocebus torquatus atys), a natural host of SIV (SIVsmm) [10]. The mutant protein was not expressed at the cell surface and accordingly did not function as a viral coreceptor. Primary activated lymphocytes from mangabeys heterozygous for the deletion allele expressed significantly less CCR5 on the cell surface. Moreover, SIV seroprevalence and viremia were comparable among CCR5 heterozygotes and wild-type animals. Parallel evolution of CCR5-null alleles in humans and sooty mangabeys suggests that similar negative selection pressures have acted against CCR5, as would occur during epidemics of infectious agents that require CCR5 for pathogenesis. Sooty mangabeys bred to homozygosity for the deletion allele will be useful for experimental studies on the context-dependent role of CCR5 in host defense and microbial pathogenesis.

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.

Natural history of endemic type D retrovirus infection and acquired immune deficiency syndrome in group-housed rhesus monkeys.

A 2.5-year epidemiologic study of a breeding group of rhesus monkeys (Macaca mulatta), which is a focus of endemic simian acquired immunodeficiency syndrome (SAIDS), demonstrated a strong association between the occurrence of SAIDS and infection with a type D retrovirus, SAIDS retrovirus serotype 1 (SRV-1). Of 23 healthy "tracer" juvenile rhesus monkeys, 19 (83%) died with SAIDS within 9 months of introduction into the resident SAIDS-endemic population. In contrast, 21 healthy "sentinel" juvenile rhesus monkeys placed in the same outdoor enclosure but denied physical contact with the SAIDS-affected group by a 10-foot-wide "buffer zone" remained free of SRV-1, SRV-1 antibody, and disease for 2.5 years. The SAIDS-specific mortality rate was significantly higher in juveniles than in adults. In repeated serologic testing, the overall prevalence of SRV-1 antibody ranged from 68 to 85%. Antibody prevalence increased with age. Seroconversion was found to be a poor indicator of infection rate, as approximately 50% of virus-positive juvenile monkeys had no antibody detectable by enzyme-linked immunosorbent assay. Repeated viral isolations from all animals revealed 1) SRV-1 viremia with clinical SAIDS; 2) persistent viremia and viral shedding in apparently healthy animals; 3) transient viremia and clinical recovery; 4) intermittent viremia, suggesting activation of latent infections; and 5) viremia in a 1-day-old infant, suggesting transplacental transmission. The prevalence of SRV-1 antibody in SAIDS-free breeding groups of rhesus monkeys was 4%. The seroprevalence of antibodies against human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus (HIV), and simian immunodeficiency virus (SIV; formerly STLV-III) was uniformly low or absent in both SAIDS-free and SAIDS-affected groups of rhesus monkeys, demonstrating that these retroviruses are not etiologically linked to SAIDS at the California Primate Research Center.

Inapparent carriers of simian acquired immune deficiency syndrome type D retrovirus and disease transmission with saliva.

Simian acquired immune deficiency syndrome (SAIDS) type D retrovirus (SRV) was isolated from saliva, urine, and peripheral blood mononuclear cells of a 6-year-old healthy rhesus monkey (Macaca mulatta) seronegative for antibodies to human T-lymphotropic virus (HTLV) type I, HTLV type III, and simian T-lymphotropic virus type III (STLV-III), identified as an inapparent SAIDS carrier in retrospective epidemiologic studies. This animal was linked to 34 cases of SAIDS over a 3-year period. Two juvenile rhesus monkeys inoculated iv with the SRV-containing saliva from this carrier became persistently infected with the retrovirus and developed SAIDS after 4-6 weeks. Both animals seroconverted to SRV, but neither had detectable preinoculation or postinoculation antibodies against HTLV type I, HTLV type III, or STLV-III. One of these animals died of SAIDS with disseminated cytomegalovirus infection after 24 weeks, and the other remains alive with persistent SRV viremia, generalized lymphadenopathy, and splenomegaly after a transient immunosuppression. Major clinical and pathological features associated with the newly described STLV-III were not observed. SRV was subsequently identified in saliva of 2 additional healthy carriers as well as monkeys with SAIDS. The findings of a carrier state in SAIDS and evidence for saliva transmission of the probable causative virus further support the usefulness of this animal model of nononcogenic immunosuppressive retroviral disease.

Immunodeficiency in rhesus monkeys associated with the original Mason-Pfizer monkey virus.

The Mason-Pfizer monkey virus (MPMV) was reisolated from a cryopreserved sample of the original MPMV-containing rhesus breast carcinoma, and complete integrated MPMV provirus was detected in chromosomal DNA of this tumor. Reanalysis of the in vivo pathogenicity and molecular character of MPMV reisolated from the rhesus breast tumor and analysis of the original MPMV after long-term in vitro propagation in human and rhesus cells show that the original MPMV produces an acquired immunodeficiency similar to that caused by the recently described simian acquired immune deficiency syndrome type D retroviruses, and the MPMV genome and its immunosuppressive effect in vivo have remained stable despite prolonged in vitro passage in human and rhesus cells.

New advances in vaccine delivery systems.

Successful application of the next generation of vaccines will require that protection be induced with a minimal number of administrations, and that a practical approach to inducing immunity at mucosal surfaces be developed. For these reasons, vaccine-containing microspheres were formulated from the biodegradable and biocompatible copolymer poly(DL-lactide-co-glycolide) [DL-PLG]. Subcutaneous immunization of mice with 1- to 10-microns microspheres containing a toxoid vaccine of staphylococcal enterotoxin B (SEB) induced a 500-fold potentiation of the circulating antitoxin response. Strong adjuvant activity was dependent on the microspheres being no more than 10 microns in diameter and required that the antigen was within the particles. The rate of DL-PLG biodegradation is a function of the ratio of lactide to glycolide, and the co-injection of SEB toxoid microspheres formulated with two different DL-PLG ratios stimulated both a primary and an anamnestic secondary antitoxin response. When it was administered by the oral or intratracheal (IT) route, microencapsulated SEB toxoid was found to be effective in the induction of concurrent circulating and disseminated mucosal antibody responses. Female rhesus macaques immunized with a microencapsulated simian immunodeficiency virus (SIV) vaccine produced high levels of circulating anti-SIV antibodies, and following oral or IT boosting, specific antibodies were found in vaginal wash fluids. Vaginal challenge with viable homologous SIV resulted in the infection of three out of four nonimmunized but only one out of seven microsphere-immunized macaques. Thus, DL-PLG microspheres are a promising approach to the delivery of vaccines, combining adjuvant activity with controlled release and effective presentation to mucosally associated lymphoid tissues (MALT).

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.

Vaginal transmission of SIV: assessing infectivity and hormonal influences in macaques inoculated with cell-free and cell-associated viral stocks.

Cell associated and cell-free simian immunodeficiency virus (SIV) were used to investigate transmission of SIV across the vaginal mucosa of rhesus macaques. The intact vaginal epithelium was found to be a strong but penetrable barrier to cell-free SIV infection. We found that 10,000-fold more cell-free SIV was needed to infect 100% of the macaques by the vaginal route when compared to the dose needed to infect 100% by the intravenous (i.v.) route. Like cell-free SIV, cell-associated SIV was an efficient means of transmission if given by the i.v. route; as few as 2 SIV-infected peripheral blood mononuclear cells (PBMC) were infectious inoculum. However, macaques were resistant to cell-associated SIV when exposed by the vaginal route; 10,000 SIV-infected PBMC failed to infect vaginally inoculated macaques. It was also found that vaginal transmission of cell-free SIV to macaques increased during the luteal phase of the menstrual cycle compared to the follicular phase. Results with this animal model predict that cell-free human immunodeficiency virus (HIV) is likely to be the more efficient mode of HIV vaginal transmission and that susceptibility may vary during the menstrual cycle.