Emergence of simian immunodeficiency virus-specific cytotoxic CD4+ T cells and increased humoral responses correlate with control of rebounding viremia in CD8-depleted macaques infected with Rev-independent live-attenuated simian immunodeficiency virus.

Indian rhesus macaques infected with the Rev-independent live-attenuated SIVmac239 strains control viremia to undetectable levels, have persistent but low cellular and humoral anti-SIV responses, and show no signs of immune deficiency. To analyze the immune mechanisms responsible for viral control, five macaques infected at day 1 after birth were subjected to CD8(+) cell depletion at 6.7 y postinfection. This resulted in viremia increases to 3.7-5.5 log(10) RNA copies, supporting a role of CD8-mediated responses in the control of viral replication. The rebounding viremia was rapidly controlled to levels below the threshold of detection, and occurred in the absence of SIV-specific CD8(+) T cells and significant CD8(+) T cell recovery in four of the five animals, suggesting that other mechanisms are involved in the immunological control of viremia. Monitoring immune responses at the time of viral control demonstrated a burst of circulating SIV-specific CD4(+) T cells characterized as CD45RA(-)CD28(+)CD95(+)CCR7(-) and also granzyme B(+), suggesting cytotoxic ability. Control of viremia was also concomitant with increases in humoral responses to Gag and Env, including a transient increase in neutralizing Abs against the neutralization-resistant SIVmac239 in four of five animals. These data demonstrate that a combination of cellular responses mediated by CD4(+) T cells and humoral responses was associated with the rapid control of the rebounding viremia in macaques infected by the Rev-independent live-attenuated SIV, even in the absence of measurable SIV-specific CD8(+) T cells in the blood, emphasizing the importance of different components of the immune response for full control of SIV infection.

Chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects.

The reverse transcriptase (RT) inhibitor tenofovir (TFV) is highly effective in the simian immunodeficiency virus (SIV) macaque model of human immunodeficiency virus infection. The current report describes extended safety and efficacy data on 32 animals that received prolonged (>or=1- to 13-year) daily subcutaneous TFV regimens. The likelihood of renal toxicity (proximal renal tubular dysfunction [PRTD]) correlated with plasma drug concentrations, which depended on the dosage regimen and age-related changes in drug clearance. Below a threshold area under the concentration-time curve for TFV in plasma of approximately 10 microg x h/ml, an exposure severalfold higher than that observed in humans treated orally with 300 mg TFV disoproxil fumarate (TDF), prolonged TFV administration was not associated with PRTD based on urinalysis, serum chemistry analyses, bone mineral density, and clinical observations. At low-dose maintenance regimens, plasma TFV concentrations and intracellular TFV diphosphate concentrations were similar to or slightly higher than those observed in TDF-treated humans. No new toxicities were identified. The available evidence does not suggest teratogenic effects of prolonged low-dose TFV treatment; by the age of 10 years, one macaque, on TFV treatment since birth, had produced three offspring that were healthy by all criteria up to the age of 5 years. Despite the presence of viral variants with a lysine-to-arginine substitution at codon 65 (K65R) of RT in all 28 SIV-infected animals, 6 animals suppressed viremia to undetectable levels for as long as 12 years of TFV monotherapy. In conclusion, these findings illustrate the safety and sustained benefits of prolonged TFV-containing regimens throughout development from infancy to adulthood, including pregnancy.

Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection.

BACKGROUND: We reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251) mutants with a K65R mutation in reverse transcriptase (RT), and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what extent findings with SIV can be extrapolated to HIV-1 RT. Accordingly, to model HIV-1 RT responses, 12 macaques were inoculated with RT-SHIV, a chimeric SIV containing HIV-1 RT, and started on prolonged tenofovir therapy 5 months later. RESULTS: The early virologic response to tenofovir correlated with baseline viral RNA levels and expression of the MHC class I allele Mamu-A*01. For all animals, sensitive real-time PCR assays detected the transient emergence of K70E RT mutants within 4 weeks of therapy, which were then replaced by K65R mutants within 12 weeks of therapy. For most animals, the occurrence of these mutations preceded a partial rebound of plasma viremia to levels that remained on average 10-fold below baseline values. One animal eventually suppressed K65R viremia to undetectable levels for more than 4 years; sequential experiments using CD8+ cell depletion and tenofovir interruption demonstrated that both CD8+ cells and continued tenofovir therapy were required for sustained suppression of viremia. CONCLUSION: This is the first evidence that tenofovir therapy can select directly for K70E viral mutants in vivo. The observations on the clinical implications of the K65R RT-SHIV mutants were consistent with those of SIVmac251, and suggest that for persons infected with K65R HIV-1 both immune-mediated and drug-dependent antiviral activities play a role in controlling viremia. These findings suggest also that even in the presence of K65R virus, continuation of tenofovir treatment as part of HAART may be beneficial, particularly when assisted by antiviral immune responses.

Long lasting control and lack of pathogenicity of the attenuated Rev-independent SIV in rhesus macaques.

A cohort of 22 rhesus macaques of Indian origin infected as neonates, juveniles, or adults by Rev-independent strains of SIV was monitored over several years. After the initial acute phase, virus replication was controlled and plasma virus loads were persistently below the threshold of the assay. The animals were monitored for up to 7.6 years after infection for viral loads, cellular and humoral immune responses, hematological changes, and overall health and no signs of immune dysfunction or AIDS were observed. This study represents several years of additional observation compared to the previously published results, and indicates that the Rev-independent SIV clones tested do not cause AIDS-like progressive disease within 7.6 years from infection. All the animals showed persistent humoral and cellular SIV-specific immune responses, consistent with chronic infection. Different Rev-independent SIV strains showed similar properties and lack of pathogenicity. Multicolor flow cytometric analysis demonstrated preservation of the Central Memory subset of T cells in the attenuated SIV-infected animals. This study demonstrates a potent, long-lasting control of the Rev-independent attenuated SIV in macaques independent of the age at virus exposure.

Role of CD8+ cells in controlling replication of nonpathogenic Simian Immunodeficiency Virus SIVmac1A11.

Infection of macaques with the avirulent molecular clone SIVmac1A11 results in transient low viremia and no disease. To investigate if this low viremia is solely due to intrinsic poor replication fitness or is mediated by efficient immune-mediated control, 5 macaques were inoculated intravenously with SIVmac1A11. Three animals that were depleted of CD8+ cells at the start of infection had more prolonged viremia with peak virus levels 1 to 2 logs higher than those of 2 animals that received a non-depleting control antibody. Thus, CD8+ cell-mediated immune responses play an important role in controlling SIVmac1A11 replication during acute viremia.

Simian immunodeficiency virus (SIV) envelope quasispecies transmission and evolution in infant rhesus macaques after oral challenge with uncloned SIVmac251: increased diversity is associated with neutralizing antibodies and improved survival in previously immunized animals.

BACKGROUND: Oral infection of infant macaques with simian immunodeficiency virus (SIV) is a useful animal model to test interventions to reduce postnatal HIV transmission via breast-feeding. We previously demonstrated that immunization of infant rhesus macaques with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol and Env, or live-attenuated SIVmac1A11 resulted in lower viremia and longer survival compared to unimmunized controls after oral challenge with virulent SIVmac251 (Van Rompay et al., J. Virology 77:179-190, 2003). Here we evaluate the impact of these vaccines on oral transmission and evolution of SIV envelope variants. RESULTS: Limiting dilution analysis of SIV RNA followed by heteroduplex mobility assays of the V1-V2 envelope (env) region revealed two major env variants in the uncloned SIVmac251 inoculum. Plasma sampled from all infants 1 week after challenge contained heterogeneous SIV env populations including one or both of the most common env variants in the virus inoculum; no consistent differences in patterns of env variants were found between vaccinated and unvaccinated infants. However, SIV env variant populations diverged in most vaccinated monkeys 3 to 5 months after challenge, in association with the development of neutralizing antibodies. CONCLUSIONS: These patterns of viral envelope diversity, immune responses and disease course in SIV-infected infant macaques are similar to observations in HIV-infected children, and underscore the relevance of this pediatric animal model. The results also support the concept that neonatal immunization with HIV vaccines might modulate disease progression in infants infected with HIV by breast-feeding.

Tenofovir primes rhesus macaque cells in vitro for enhanced interleukin-12 secretion.

We investigated if the antiviral drug tenofovir has immunomodulatory effects in macaques, similar to those described in murine models. While in vivo experiments were complicated by high individual and temporal variability of immune parameters, tenofovir primed macaque peripheral blood mononuclear cells in vitro for enhanced IL-12 secretion following exposure to bacterial antigens.

Partial efficacy of a VSV-SIV/MVA-SIV vaccine regimen against oral SIV challenge in infant macaques.

Despite antiretroviral medications, the rate of pediatric HIV-1 infections through breast-milk transmission has been staggering in developing countries. Therefore, the development of a vaccine to protect vulnerable infant populations should be actively pursued. We previously demonstrated that oral immunization of newborn macaques with vesicular stomatitis virus expressing simian immunodeficiency virus genes (VSV-SIV) followed 2 weeks later by an intramuscular boost with modified vaccinia ankara virus expressing SIV (MVA-SIV) successfully induced SIV-specific T and B cell responses in multiple lymphoid tissues, including the tonsil and intestine [13]. In the current study, we tested the oral VSV-SIV prime/systemic MVA-SIV boost vaccine for efficacy against multiple oral SIVmac251 challenges starting two weeks after the booster vaccination. The vaccine did not prevent SIV infection. However, in vaccinated infants, the level of SIV-specific plasma IgA (but not IgG) at the time of challenge was inversely correlated with peak viremia. In addition, the levels of SIV-specific IgA in saliva and plasma were inversely correlated with viral load at euthanasia. Animals with tonsils that contained higher frequencies of SIV-specific TNF-α- or IFN-γ-producing CD8(+) T cells and central memory T cells at euthanasia also had lower viremia. Interestingly, a marked depletion of CD25(+)FoxP3(+)CD4(+) T cells was observed in the tonsils as well as the intestine of these animals, implying that T regulatory cells may be a major target of SIV infection in infant macaques. Overall, the data suggest that, in infant macaques orally infected with SIV, the co-induction of local antiviral cytotoxic T cells and T regulatory cells that promote the development of IgA responses may result in better control of viral replication. Thus, future vaccination efforts should be directed towards induction of IgA and mucosal T cell responses to prevent or reduce virus replication in infants.

Immunogenicity of viral vector, prime-boost SIV vaccine regimens in infant rhesus macaques: attenuated vesicular stomatitis virus (VSV) and modified vaccinia Ankara (MVA) recombinant SIV vaccines compared to live-attenuated SIV.

In a previously developed infant macaque model mimicking HIV infection by breast-feeding, we demonstrated that intramuscular immunization with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins provided partial protection against infection following oral inoculation with virulent SIV. In an attempt to further increase systemic but also local antiviral immune responses at the site of viral entry, we tested the immunogenicity of different orally administered, replicating vaccines. One group of newborn macaques received an oral prime immunization with a recombinant vesicular stomatitis virus expressing SIVmac239 gag, pol and env (VSV-SIVgpe), followed 2 weeks later by an intramuscular boost immunization with MVA-SIV. Another group received two immunizations with live-attenuated SIVmac1A11, administered each time both orally and intravenously. Control animals received mock immunizations or non-SIV VSV and MVA control vectors. Analysis of SIV-specific immune responses in blood and lymphoid tissues at 4 weeks of age demonstrated that both vaccine regimens induced systemic antibody responses and both systemic and local cell-mediated immune responses. The safety and immunogenicity of the VSV-SIVgpe+MVA-SIV immunization regimen described in this report provide the scientific incentive to explore the efficacy of this vaccine regimen against virulent SIV exposure in the infant macaque model.

Developing a neonatal HIV vaccine: insights from macaque models of pediatric HIV/AIDS.

PURPOSE OF REVIEW: This review analyzes recent findings from nonhuman primate models of HIV/AIDS that are most relevant to developing active neonatal vaccine strategies against HIV breast milk transmission. We focus on studies published from 2005 to early 2007 that have characterized simian immunodeficiency virus or simian/human immunodeficiency virus transmission and the efficacy of HIV vaccine strategies in neonatal macaques. RECENT FINDINGS: Nonhuman primate models of natural HIV breast milk transmission recapitulate many features of infection in human infants; however, the variation in timing and overall low rate of infection in these models precludes their use in conducting vaccine studies. Oral inoculation of infant macaques with defined viral inocula results in reliable transmission and is an efficient model for evaluating neonatal HIV vaccine strategies. All HIV vaccine strategies tested in neonatal macaques are immunogenic, but only a subset of these vaccines confer significant protection against virus acquisition or simian AIDS after oral challenge. SUMMARY: Candidate HIV vaccine strategies can elicit virus-specific humoral and cell-mediated immune responses in newborn primates; however, vaccine immunogenicity in infant macaques is not a reliable criterion for predicting a vaccine's efficacy against oral virus challenge exposure.

Evaluation of oral tenofovir disoproxil fumarate and topical tenofovir GS-7340 to protect infant macaques against repeated oral challenges with virulent simian immunodeficiency virus.

Simian immunodeficiency virus (SIV) infection of infant macaques is a useful animal model of pediatric HIV infection to evaluate the potential of chemoprophylactic regimens to reduce mother-to-infant transmission of HIV. Previous studies have demonstrated that short-term subcutaneous administration of the reverse transcriptase inhibitor tenofovir was highly effective in protecting newborn macaques against infection after a single high-dose oral inoculation with virulent SIVmac251. In the current study, we mimicked HIV transmission through breast-feeding by repeatedly feeding infant macaques low doses of SIVmac251. Topical administration of a low dose of the second-generation tenofovir prodrug GS-7340 did not have detectable prophylactic efficacy. Oral administration of tenofovir disoproxil fumarate (DF; 10 mg/kg SID) lowered the infection rate at birth, but had lower efficacy against virus infection at 4 weeks of age, most likely because drug levels became suboptimal relative to those obtained with the current tenofovir DF regimen in humans. These prophylactic results further underscore the relevance of the current tenofovir DF prevention trials in pediatric and adult populations.

Rapid virus dissemination in infant macaques after oral simian immunodeficiency virus exposure in the presence of local innate immune responses.

A vaccine to protect human immunodeficiency virus (HIV)-exposed infants is an important goal in the global fight against the HIV pandemic. Two major challenges in pediatric HIV vaccine design are the competence of the neonatal/infant immune system in comparison to the adult immune system and the frequent exposure to HIV via breast-feeding. Based on the hypothesis that an effective vaccine needs to elicit antiviral immune responses directly at the site of virus entry, the pattern of virus dissemination in relation to host immune responses was determined in mucosal and lymphoid tissues of infant macaques at 1 week after multiple oral exposures to simian immunodeficiency virus (SIV). The results show that SIV disseminates systemically by 1 week. Infant macaques can respond rapidly to virus challenge and mount strong innate immune responses. However, despite systemic infection, these responses are most pronounced in tissues close to the viral entry site, with the tonsil being the primary site of virus replication and induction of immune responses. Thus, distinct anatomic compartments are characterized by unique cytokine gene expression patterns. Importantly, the early response at mucosal entry sites is dominated by the induction of proinflammatory cytokines, while cytokines with direct antiviral activity, alpha/beta interferons, are only minimally induced. In contrast, both antiviral and proinflammatory cytokines are induced in lymphoid tissues. Thus, although infant macaques can respond quickly to oral viral challenge, the locally elicited immune responses at mucosal entry sites are likely to favor immune activation and thereby virus replication and are insufficient to limit virus replication and dissemination.

Structured treatment interruptions with tenofovir monotherapy for simian immunodeficiency virus-infected newborn macaques.

We demonstrated previously that prolonged tenofovir treatment of infant macaques, starting early during infection with virulent simian immunodeficiency virus (SIVmac251), can lead to persistently low or undetectable viremia even after the emergence of mutants with reduced in vitro susceptibility to tenofovir as a result of a K65R mutation in reverse transcriptase; this control of viremia was demonstrated to be mediated by the generation of effective antiviral immune responses. To determine whether structured treatment interruptions (STI) can induce similar immunologic control of viremia, eight newborn macaques were infected with highly virulent SIVmac251 and started on a tenofovir STI regimen 5 days later. Treatment was withdrawn permanently at 33 weeks of age. All animals receiving STI fared much better than 22 untreated SIVmac251-infected infant macaques. However, there was a high variability among animals in the viral RNA set point after complete drug withdrawal, and none of the animals was able to achieve long-term immunologic suppression of viremia to persistently low levels. Early immunologic and viral markers in blood (including the detection of the K65R mutation) were not predictive of the viral RNA set point after drug withdrawal. These results, which reflect the complex interactions between drug resistance mutations, viral virulence, and drug- and immune-mediated inhibition of virus replication, highlight the difficulties associated with trying to develop STI regimens with predictable efficacy for clinical practice.

Evaluation of passively transferred, nonneutralizing antibody-dependent cellular cytotoxicity-mediating IgG in protection of neonatal rhesus macaques against oral SIVmac251 challenge.

Previously, Ab-dependent cellular cytotoxicity (ADCC) was significantly correlated with reduced acute viremia upon intrarectal SIVmac251 challenge of immunized rhesus macaques. To directly assess ADCC protective efficacy, six neonatal macaques were infused s.c. with immune IgG (220 mg/kg) purified from the immunized animals and positive for ADCC and Ab-dependent cell-mediated viral inhibition (ADCVI) activities. Six neonates received control IgG. The neonates were challenged twice orally with 10(5) 50% inhibiting tissue culture-infective dose of SIVmac251 2 days post-IgG infusion. At challenge, plasma of neonates that received immune IgG did not neutralize SIVmac251 but had geometric mean ADCC titers of 48,130 and 232,850 against SIVmac251 -infected and gp120-coated targets, respectively. Peak ADCVI activity varied from 62 to 81%. ADCC activity declined with the 2-wk IgG half-life but was boosted at wk 4, together with de novo ADCC-mediating Abs in controls, by postchallenge viremia. ADCVI activity was similarly induced. No protection, assessed by viral burdens, CD4 counts, and time to euthanasia was observed. Possible factors contributing to the discrepancy between the previous correlation and lack of protection here include: the high oral challenge dose compared with the 400-fold lower intrarectal dose; the challenge route with regard to viral dissemination and distribution of infused IgG; insufficient NK effector activity and/or poor functionality in newborns; insufficient immune IgG; and the possibility that the previous correlation of ADCC with protection was augmented by cellular immune responses also present at challenge. Future studies should explore additional challenge routes in juvenile macaques using higher amounts of potent IgG preparations.

Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV.

An infant macaque model was developed to test pediatric vaccine candidates aimed at reducing HIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age, significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV or MVA-SIV. When infected, ALVAC-SIV- and MVA-SIV-vaccinated animals also had reduced viremia compared with unimmunized animals. The results of these investigations suggest that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection through breastfeeding.

Topical administration of low-dose tenofovir disoproxil fumarate to protect infant macaques against multiple oral exposures of low doses of simian immunodeficiency virus.

Simian immunodeficiency virus (SIV) infection of infant macaques is a useful animal model to determine whether topical (oral) administration of antiviral compounds to the nursing infant could reduce human immunodeficiency virus transmission through breast-feeding. The reverse-transcriptase inhibitor tenofovir was selected because of previous demonstrations that systemic drug levels are effective in preventing SIV infection. To mimic the multiple exposures to virus during breast-feeding, 14 infant macaques were fed 15 low doses of SIVmac251 without chemical restraint. Six animals were treated with placebo, and 2 groups of 4 animals received oral topical doses of tenofovir disoproxil fumarate (DF; equivalent to 0.037 mg of tenofovir/day). About half the animals of each group became infected. In a subsequent study, 2 oral inoculations of 4 juvenile macaques with a mixture of tenofovir DF and SIVmac251 induced persistent infection. Topical administration of low doses of tenofovir DF did not protect against oral SIV infection.

CD8+-cell-mediated suppression of virulent simian immunodeficiency virus during tenofovir treatment.

The ability of tenofovir to suppress viremia in simian immunodeficiency virus (SIV)-infected macaques for years despite the presence of virulent viral mutants with reduced in vitro susceptibility is unprecedented in this animal model. In vivo cell depletion experiments demonstrate that tenofovir's ability to suppress viremia during acute and chronic infection is significantly dependent on the presence of CD8+ lymphocytes. Continuous tenofovir treatment was required to maintain low viremia. Although it is unclear whether this immune-mediated suppression of viremia is linked to tenofovir's direct antiviral efficacy or is due to independent immunomodulatory effects, these studies prove the concept that antiviral immune responses can play a crucial role in suppressing viremia during anti-human immunodeficiency virus drug therapy.

Functional and morphological development of lymphoid tissues and immune regulatory and effector function in rhesus monkeys: cytokine-secreting cells, immunoglobulin-secreting cells, and CD5(+) B-1 cells appear early in fetal development.

Little is known regarding the timing of immune ontogeny and effector function in fetal humans and nonhuman primates. We studied the organization of lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus monkey fetuses during the second and third trimesters (65 to 145 days of gestation; term = 165 days). Immunoglobulin-secreting and cytokine-secreting cells were detected at day 80. The thymus, spleen, lymph nodes, and intestinal mucosa were examined for cells expressing CD3, CD5, CD20, CD68, p55, and HLA-DR. In the spleens of 65-day-old fetuses (early second trimester), the overwhelming majority of total lymphocytes were CD5(+) CD20(+) B-1 cells. The remaining lymphocytes were CD3(+) T cells. By day 80, splenic B and T cells were equal in number. Intraepithelial CD3(+) CD5(-) T cells and lamina propria CD20(+) CD5(+) B cells were present in the intestines of 65-day-old fetuses. By day 80, numerous CD20(+) CD5(+) B cells were present in the jejunums and colons and early lymphocyte aggregate formation was evident. The spleens of 80- to 145-day-old fetuses contained immunoglobulin M (IgM)-secreting cells, while IgA-, IgG-, interleukin-6-, and gamma interferon-secreting cells were numerous in the spleens and colons. Thus, by the second trimester, the lymphoid tissues of the rhesus monkey fetus have a complete repertoire of properly organized antigen-presenting cells, T cells, and B cells.

The clinical benefits of tenofovir for simian immunodeficiency virus-infected macaques are larger than predicted by its effects on standard viral and immunologic parameters.

Previous studies have demonstrated that tenofovir (9-[2-(phosphonomethoxy)propyl]adenine; PMPA) treatment is usually very effective in suppressing viremia in macaques infected with simian immunodeficiency virus (SIV). The present study focuses on a subset of infant macaques that were chronically infected with highly virulent SIVmac251, and for which prolonged tenofovir treatment failed to significantly suppress viral RNA levels in plasma despite the presence of tenofovirsusceptible virus at the onset of therapy. While untreated animals with similarly high viremia developed fatal immunodeficiency within 3-6 months, these tenofovir-treated animals had significantly improved survival (up to 3.5 years). This clinical benefit occurred even in animals for which tenofovir had little or no effect on CD4 and CD8 lymphocyte counts and antibody responses to SIV and test antigens. Thus, the clinical benefits of tenofovir were larger than predicted by plasma viral RNA levels and other routine laboratory parameters.

Rev-independent simian immunodeficiency virus strains are nonpathogenic in neonatal macaques.

The viral protein Rev is essential for the export of the subset of unspliced and partially spliced lentiviral mRNAs and the production of structural proteins. Rev and its RNA binding site RRE can be replaced in both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) by the constitutive RNA transport element CTE of the simian type D retroviruses. We used neonatal macaques as a sensitive animal model to evaluate the pathogenicity of a pair of SIV mutant strains generated from Rev-independent molecular clones of SIVmac239 which differ only in the presence of the nef open reading frame. After high primary viremia, all animals remained persistently infected at levels below the threshold of detection. All macaques infected as neonates developed normally, and none showed any signs of immune dysfunction or disease during follow-up ranging from 2.3 to 4 years. Therefore, the Rev-RRE regulatory mechanism plays a key role in the maintenance of high levels of virus propagation, which is independent of the presence of nef. These data demonstrate that Rev regulation plays an important role in the pathogenicity of SIV. Replacement of Rev-RRE by the CTE provides a novel approach to dramatically lower the virulence of a pathogenic lentivirus. These data further suggest that antiretroviral strategies leading to even a partial block of Rev function may modulate disease progression in HIV-infected individuals.