Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies.

The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, others, and we have shown that passively infused antibody can protect against an intravenous challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. After controlling the macaque estrous cycle with progesterone, anti-HIV-1 neutralizing monoclonal antibodies 2F5 and 2G12, and HIV immune globulin were tested. Whereas all five control monkeys displayed high plasma viremia and rapid CD4 cell decline, 14 antibody-treated macaques were either completely protected against infection or against pathogenic manifestations of SHIV-infection. Infusion of all three antibodies together provided the greatest amount of protection, but a single monoclonal antibody, with modest virus neutralizing activity, was also protective. Compared with our previous intravenous challenge study with the same virus and antibodies, the data indicated that greater protection was achieved after vaginal challenge. This study demonstrates that antibodies can affect transmission and subsequent disease course after vaginal SHIV-challenge; the data begin to define the type of antibody response that could play a role in protection against mucosal transmission of HIV-1.

Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

Fetal antigens on the surface of human lymphoid cells.

Human B-lymphoblastoid cells established in long-term culture from healthy adults carry surface components that are normally found in human fetal tissues at about 10 wk of age. These antigens are strongly expressed on neoplastic B lymphocytes but not on thymocytes or a cultured T-cell line. They are carried by a small subpopulation of normal adult peripheral blood lymphocytes as well.

Control of SIV rebound through structured treatment interruptions during early infection.

In a randomized controlled trial with acute simian immunodeficiency virus (SIV)-infected macaques, both highly active antiretroviral therapy (HAART) and HAART with fixed-schedule structured treatment interruption (STI-HAART; alternating 3 weeks on and 3 weeks off therapy) suppressed viral load. In the STI-HAART group, T cell virus-specific immune response (VIR) and control of viral rebound increased concurrently during subsequent interruptions. In contrast, VIR did not increase and SIV rebounded after permanent treatment withdrawal in all animals on continuous HAART. Fixed-schedule STI-HAART appears to be an effective alternative to continuous HAART for the early treatment of retroviral infection.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

The effect of accounting for biarticularity in hip flexor and hip extensor joint torque representations.

Subject-specific torque-driven models have ignored biarticular effects at the hip. The aim of this study was to establish the contribution of monoarticular hip flexors and hip extensors to total hip flexor and total hip extensor joint torques for an individual and to investigate whether torque-driven simulation models should consider incorporating biarticular effects at the hip joint. Maximum voluntary isometric and isovelocity hip flexion and hip extension joint torques were measured for a single participant together with surface electromyography. Single-joint and two-joint representations were fitted to the collected torque data and used to determine the maximum voluntary joint torque capacity. When comparing two-joint and single-joint representations, the single-joint representation had the capacity to produce larger maximum voluntary hip flexion torque (larger by around 9% of maximum torque) and smaller maximum voluntary hip extension torque (smaller by around 33% of maximum torque) with the knee extended. Considering the range of kinematics found for jumping movements, the single-joint hip flexors had the capacity to produce around 10% additional torque, while the single joint hip extensors had about 70% of the capacity of the two-joint representation. Two-joint representations may overcome an over-simplification of single-joint representations by accounting for biarticular effects, while building on the strength of determining subject-specific parameters from measurements on the participant.

Infection of feline embryo adherent cells with feline leukemia virus: feline oncornavirus-associated cell membrane antigen expression and morphologic transformation.

Feline embryo adherent cells were infected with the Richard or Kawakami-Theilen strains of feline leukemia virus (FeLV) and examined for feline oncornavirus-associated cell membrane antigen (FOCMA), viral group-specific antigen (gsa) production, and in vitro evidence of transformation. As early as 10 days after infection, when more than half of the infected cells were gsa positive, FOCMA was detected on 5-10 percent of the cells. Transitory morphologic alterations (epithelioid appearance and rounding) were first noted in most cultures around 20-30 days post infection. At this time, approximately 50% of the cells in infected cultures expressed FOCMA. Morphologic characteristics of transformed fibroblastic cells (rounded shape, disordered alignment, and low adhesion to substratum), as well as enhanced agglutinability by plant lectins and ability to grow in agar, were demonstrated in one of four FeLV-infected, FOCMA-positive cultures. Findings showed that FOCMA may be expressed in FeLV-infected monolayer cells independent of transformation as assessed by in vitro criteria.

Increased chemotaxis of leukocytes from mice bearing tumors.

Increased chemotaxis toward activated serum was demonstrated by leukocytes from bone marrow, spleen, and peripheral blood of tumor-bearing mice as compared with those of normal mice. Increased chemotaxis was correlated with duration of tumor growth, which, in turn, was correlated with size of tumor. Upon surgical tumor removal, chemotaxis fell to normal levels unless metastasis had occurred, in which case, the extent of chemotaxis was correlated with the volume of the metastasis. Increased chemotaxis was seen in relation to the growth of a mammary adenocarcinoma as well as that of a chemically induced fibrosarcoma. The major chemotactant in serum was shown to be complement derived, and sera from tumor-bearing and normal animals were equally effective. The presence of a tumor resulted in an increase in the percentage of polymorphonuclear cells in the bone marrow. The numbers of cells which migrated were independent of the cellular composition of the bone marrow in normal mice. In contrast, a negative correlation was found between the percentage of lymphocytes present and the number of bone marrow cells which migrated toward activated serum when the cells originated in a tumor-bearing mouse. The data suggested the increased chemotaxis was a property of the cells rather than the soluble substances in the serum.

Fetal antigens in nonneoplastic conditions.

During studies that showed the presence of fetal antigens on the surface of human malignant melanoma tumor cells, polyvalent antisera specific for human fetal tissues of varying ages were developed. These reagents demonstrated varying patterns of expression of fetal antigens at different ages in various tissues of the human fetus. The possibility that nonneoplastic adult cells showing either maturation arrest or excessive proliferation also might express fetal antigens led to studies of human bone marrow. Although normal bone marrow cells expressed low levels of fetal antigens, large amounts were seen on bone marrow cells of patients with anemias due to iron, B12, or folic acid deficiencies, as well as on those with leukemia. Moreover, normal adult tissues adapted to long-term culture also expressed fetal antigens. After 3 weeks in organ culture adult human skin showed morphological changes similar to those seen in fetal periderm and strongly expressed fetal antigens. In addition, lymphoblasts in long-term cultured human lymphoid cell lines established from normal donors also carried surface fetal antigens. These latter antigens were shared with neoplastic B-cells (chronic lymphocytic leukemia) but not with T-cells. Their expression varied with the cell cycle. The reexpression of fetal antigens on malignant cells is thought to signal a basic derangement in the control of differentiation which is considered to be peculiar to neoplasia. However, these studies indicate that normal adult cells also may reexpress fetal antigens under circumstances unrelated to neoplasia but associated with either maturation arrest or rapid and excessive proliferation.

Inhibition of erythroid colony-forming cells by a Mr 15,000 protein of feline leukemia virus.

The effects of concentrated ultraviolet-inactivated feline leukemia virus (FeLV), the purified Mr 15,000 envelope protein (p15E) of FeLV, or the purified Mr 27,000 structural protein (p27) of FeLV on feline bone marrow mononuclear cells were studied in vitro in methylcellulose cultures. Whole virus and purified viral proteins were from the Kawakami-Theilen isolate of FeLV, which induces erythroid aplasia in cats. Bone marrow mononuclear cells from FeLV-negative young adult cats were preincubated with a medium control, ultraviolet-inactivated whole virus, or the p15E or p27 of FeLV, incubated in methylcellulose cultures for 2 days, and then observed for the formation of colony-forming units-erythroid (CFU-E) and colony-forming units-granulocyte/macrophage. The ultraviolet-inactivated Kawakami-Theilen isolate of FeLV at concentrations of 10 or 20 micrograms of viral protein/5 X 10(4) cells suppressed CFU-E to 66 to 56% of control values but had no significant effect on proliferation of colony-forming units-granulocyte/macrophage. p15E at concentrations of 0.1 to 0.2 micrograms/5 X 10(4) cells decreased CFU-E numbers to 0 to 1% of control values, whereas the same concentration of p27 did not alter CFU-E growth when compared with controls. Neither p15E nor p27 had a significant effect on growth of colony-forming units-granulocyte/macrophage. The erythrosuppressive effects of whole virus and an envelope-derived protein but not a structural core protein suggest that FeLV envelope proteins are important in the selective inhibition of erythrogenesis observed in vivo in FeLV-infected cats.

Characteristics of the CD8+ lymphocytosis during primary simian immunodeficiency virus infections.

OBJECTIVE: To investigate the source of the expanded blood CD8+ subsets during an acute primary simian immunodeficiency virus (SIV) infection of macaques and the potential role of these cells in disease progression. DESIGN AND METHODS: The primary CD8+ lymphocytosis, which occurs at 1-2 weeks following infection with SIVsmm/PBj-14, was examined in rhesus and cynomolgus macaques. Extensive subset analysis of the expanded blood CD8+ cell pool in a rhesus macaque was compared phenotypically with those in thymus, lymph nodes, spleen, ileum and lung washouts obtained at necropsy during blood lymphocytosis. The influence of the primary CD8+ cells expansion on disease progression was assessed at days 175-679 post-infection in long-term PBj-14 survivors staged according to immunological, virological and histopathological changes in their lymphoid organs. RESULT: The very rapid and transient blood lymphocytosis following infection consisted of two distinct CD45RA(low), CD8+ and CD28-, lymphocyte function-associated antigen (LFA)-1(high), CD45RA(high), CD8+ populations. These populations were present in low levels in thymus, lymph and spleen but were highly represented in mucosal tissues, such as long washout, in which CD28- LFA-1(high) CD45RA(high) CD8+ cells comprised 86% of CD8+ cells, and gut, which was predominantly CD45RA(low) CD28- CD8+ cells. A comparison of progressor and non-progressor PBj-14-infected rhesus and cynomolgus macaques also indicated that the existence or magnitude of a blood CD8+ lymphocytosis during the acute phase of infection did not by itself appear to influence or be predictive of disease progression. CONCLUSION: The marked blood CD8+ lymphocytosis observed during acute SIV infection did not result from expansion of virus-specific precursors in peripheral lymph node and did not appear to influence the rate of disease progression. The findings provide a novel explanation for the primary CD8+ cell lymphocytosis and invoke a mechanism whereby virus-induced cytokine/chemokine production in mucosal sites initiate the transient migration of a pre-existing CD8+ population into the blood from compartments such as lung and gut. Such results suggest that the magnitude of lymphocytosis may depend on the level of viral replication in mucosal tissues and the presence of other infections, for example, cytomegalovirus.

Rhodopsin determinations in C57BL/6J-pallid strain mice.

The influence of light environment on rhodopsin concentration per eye was determined in littermate pigmented and nonpigmented C57BL/6J-pallid gene mice reared under cyclic light or continuous dark environments. Attempts to exacerbate a congenital manganese deficiency in pallid strain mice included dietary deprivation and supplementation with manganese and exposure to intense light followed by the determination of rhodopsin recovery rates in darkness. Homozygous pallid mice (pa/pa) reared in cyclic light had rhodopsin levels which were significantly lower than heterozygous (+/pa) or homozygous (+/+) black control mice. Dark-rearing resulted in a significant increase in rhodopsin per eye in pallid strain mice and equivalent levels in adult mice, but young pallid strain mice did not achieve the same rhodopsin concentration as young +/+ mice. Although dietary manganese deprivation or supplementation did not significantly alter rhodopsin levels among pallid mice, the deficient diet resulted in lower rhodopsin per eye in the young +/+ control animals. The recovery of rhodopsin in darkness following intense light exposure was equal and complete within 24 hr for most genotypes. However, recovery by pallid mice after 24 hr was significantly lower than by pigmented or albino genotypes.

Vaccine-elicited immune responses prevent clinical AIDS in SHIV(89.6P)-infected rhesus monkeys.

Accumulating evidence has demonstrated the importance of cytotoxic T lymphocytes (CTLs) and helper T lymphocytes in controlling HIV-1 replication. We have elicited immune responses in rhesus monkeys utilizing DNA vaccines augmented by the administration of IL-2/Ig, a fusion protein consisting of interleukin-2 and the Fc portion of IgG2. These vaccine-elicited immune responses did not prevent infection following a high-dose intravenous challenge with SHIV(89.6P) but did control viremia to nearly undetectable levels and prevented immunodeficiency and clinical disease. In contrast, control monkeys developed high levels of viremia and exhibited a rapid loss of CD4(+) T cells, significant clinical disease progression, and death in half of the animals by day 140 following challenge. Vaccine approaches that elicit immune responses capable of reducing plasma viral loads, but not capable of inducing sterilizing immunity, may still provide substantial clinical benefits.

Infection of rhesus and cynomolgus macaques with a rapidly fatal SIV (SIVSMM/PBj) isolate from sooty mangabeys.

A variant of simian immunodeficiency virus (SIVSMM/PBj), isolated from a chronically infected pig-tailed macaque has been shown in previous studies to produce acutely fatal disease uniformly in pig-tailed macaques and in some rhesus macaques. The present study extends investigation of SIVSMM/PBj pathogenesis in rhesus and cynomolgus monkeys. Cynomolgus and rhesus macaques were found to be uniformly susceptible to infection, but as previously reported, the rhesus were found to not be uniform in their response during the acute disease. Homogenized tissues from a rhesus that died acutely from SIVSMM/PBj were passaged to 6 rhesus monkeys in an attempt to increase lethality. Five of 6 rhesus monkeys receiving intravenous inoculation of either spleen (10(3) TCID50) or lymph node (10(5) TCID50) homogenate developed acute disease; 4 died (days 8-10), 1 recovered, and one rhesus remained asymptomatic. Three of 3 cynomolgus macaques and 4 of 4 pig-tailed macaques receiving the same inoculum died acutely within 9 days. Clinical disease in macaques that died was characterized by diffuse lymphadenopathy within 5 days of inoculation and severe diarrhea beginning 1 to 3 days before death. Anorexia, lymphopenia (< 1000 cells/mm3), and mild hypoalbuminemia preceded onset of diarrhea by 24 h. Viral p27 was detected in circulation by day 6 postinfection, with all animals dying acutely having detectable serum p27 and no detectable humoral response. Acute lethality was attributed to severe metabolic acidosis (pH < 7.20) which was observed 24-48 h prior to death in the pig-tailed and cynomolgus macaques. Immunohistochemistry revealed numerous SIV antigen-positive lymphocytes and macrophages in the lymph nodes, spleen, gut-associated lymphoid tissues and gastrointestinal lamina propria. Histopathologic lesions included marked to severe hyperplasia of the T-cell-dependent areas in lymphoid tissues and diffuse nonulcerative lymphohistiocytic gastroenteritis. Surviving rhesus developed strong humoral immune responses to the major SIV proteins.

Decline in the CD4+ lymphocyte population in the blood of SIV-infected macaques is not reflected in lymph nodes.

Although loss of CD4+ lymphocytes in peripheral blood is a standard criterion for evaluating the course of HIV disease, little is known about changes within lymphoid organs, which contain the bulk (> 50%) of the body's lymphocytes. Because such studies are feasible only by using non-human primates, we have examined lymph nodes (LNs), spleen, and blood from monkeys infected with two isolates of simian immunodeficiency virus (SIV). During both the acute and chronic phases of these infections, characteristic reductions in the blood CD4+ cell levels are not reflected in LN, where the CD4+ pool remains within normal levels. However, when circulating CD4/CD8 ratios have consistently fallen to approximately 0.5, striking decreases in the percentage of CD4 cells (CD4%) and CD4/CD8 ratios in LN occur concomitantly with dramatic increases in viral antigen expression on follicular dendritic cells within LN germinal centers (GCs). The data suggest that loss from the total T cell pool in minimal until the final stages of SIV and HIV disease and that the immunological deterioration of LN is the event that precipitates the increased susceptibility to infections and progression to AIDS.

Viral DNA burden and decline in percentage of CD4-positive cells in the lymphoid compartment of SIV-infected macaques.

The decline in CD4+ cells and increased viral DNA and RNA burden in the blood of human immunodeficiency virus (HIV)-infected individuals have been used as closely related correlates of disease progression. However, little is known about levels of total or unintegrated viral DNA in lymphoid tissue of HIV-infected patients and how they relate to CD4+ cell decline or disease progression. Exploiting the similarities between HIV- and simian immunodeficiency virus (SIV)-induced disease, we examined lymphoid organs and peripheral blood from SIV-infected macaques for total (pol) and unintegrated 2-LTR circular viral DNA by polymerase chain reaction (PCR). Two SIV isolates (SIVmac/251 and SIVmne/E11S) that differ markedly in their biological and clinical properties were studied. The results indicate that total viral DNA burdens vary considerably between isolates. There was no strong association between total viral DNA levels and CD4% in lymphoid tissues when isolates were compared and death was not associated with any particular level of viral pol DNA. In contrast, accumulation of unintegrated viral DNA was closely associated with decline in CD4/CD8 ratios in lymphoid organs and AIDS. The appearance of both pol and unintegrated viral DNA in thymus of infected macaques also emerged as one of the single best correlates or possible predictors of advanced disease yet studied. Their roles in pathogenesis are discussed.

Association of interleukin-6 in the pathogenesis of acutely fatal SIVsmm/PBj-14 in pigtailed macaques.

Infection with a variant of simian immunodeficiency virus (SIVsmm/PBj-14) causes death in juvenile pigtailed macaques within 8 days of infection. The primary pathology is localized to the lymphoid tissues of the gut and spleen. Although the virus is present, the lesions are most consistent with acute reactive inflammation. We studied the serum and tissues for evidence of acute cytokine production often associated with acute inflammation. One factor, IL-6, was found to be significantly increased (> 1000-fold) over all other measured cytokines in all the pigtailed macaques who died acutely. Increased levels of IL-6 were found both in the serum and in the inflamed tissues. mRNA for IL-6 was found in the tissues with the highest protein levels of IL-6. The marked increase in IL-6 and IL-6 mRNA correlated with the virus levels in the tissues and serum as determined by viral isolation, immunohistochemistry, and Northern blot analysis. These findings suggest that the underlying pathogenesis of primary tissue damage, necrosis, and death by PBj-14 is the induction of cytokine production. Although the presence of the virus may be critical for the initiation of these events, the intense inflammatory reaction is associated with the cause of death.

Effect of PMPA and PMEA on the kinetics of viral load in simian immunodeficiency virus-infected macaques.

In this study we compared the effect of postexposure treatment of the acyclic nucleoside analogs 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) and 9-(2-phosphonylmethoxypropyl)-adenine (PMPA) on the kinetics of viral load in the blood and lymph nodes of rhesus macaques chronically infected with SIVmac251 for 18 weeks. Two of the four macaques treated with PMPA (20 mg/kg per day) for 28 consecutive days had demonstrable reductions in viral loads of 1.5 and 3 logs. Three of four macaques given the same dosing regimen of PMEA had viral load reductions ranging from 1.25 to 2.8 logs. Furthermore, treatment with either drug caused a reduction in virus burden in the lymph nodes by 2 weeks posttreatment. However, in both PMEA- and PMPA-treated animals, viral loads rebounded to day of treatment levels by 2 weeks after termination of treatment. The extent to which viral load was suppressed was similar for both drugs. In contrast, viral loads in three of four mock-treated animals remained persistently high throughout the study. This study has demonstrated that postexposure treatment with these acyclic nucleoside analogs could modulate the kinetics of viral load reduction in some animals.

HIV-1 infection in pigtailed macaques.

Four pigtailed macaques were inoculated with autologous cells expressing low levels of human immunodeficiency virus type 1 (HIV-1). During the first 10 weeks, infectious virus was recovered from peripheral blood mononuclear cells (PBMCs) and lymph nodes from three of the animals. Subsequently, HIV-1 DNA was frequently detected in uncultured PBMCs from all three animals, and virus was isolated from one of them at weeks 38 and 61. The fourth animal, which was rechallenged at week 10 with cell-free virus isolated from one of the others, never became virus isolation positive, but harbored HIV-1 proviral genomes. These virus infections were accompanied by the development of varied HIV-1-specific humoral immune responses. Antibodies to gp160 were first apparent at week 8 in the three initially infected animals and persisted. The animal from whom virus was isolated at late times also developed persisting antibodies to HIV-1 p24 and gp120. Antibodies to gp120 and gp160 became apparent in the rechallenged animal at 1 week following reinoculation, but they waned with time. In vivo passage of the virus was attempted at week 6. One recipient pigtailed macaque and one recipient cynomolgus monkey failed to become detectably infected following transfusion of virus-positive blood and lymph node cells. The long-term presence of HIV-1-specific antibodies and proviral genomes in these animals, and the recovery of infectious virus more than 1 year following inoculation, are indicative of persistent infection, and confirm previous reports that pigtailed macaques are susceptible to HIV-1.

Deletion of the nef gene abrogates the ability of SIV smmPBj to induce acutely lethal disease in pigtail macaques.

Simian immunodeficiency virus (SIV) infection in macaque species is typically associated with the development of a progressive immunodeficiency disease, similar to human AIDS, resulting in death of animals in months to years after infection. In contrast, a variant virus, termed SIVsmmPBj, induces an acute disease in macaques, resulting in death in 5 to 14 days after infection. Previously, we have shown that several viral determinants contribute to the pathogenesis of this disease. The present study was undertaken to evaluate the role of Nef in the pathogenesis of SIVsmmPBj-induced acute disease. A molecular clone of SIVsmmPBj was generated that contains a deletion in the nef coding region (PBj6.6 delta nef). Virus derived from this molecular clone was tested with the parental virus, PBj6.6, in replication studies in pigtail macaque and rhesus macaque peripheral blood mononuclear cells (PBMCs). In general, PBj6.6 delta nef displayed markedly reduced replication abilities when compared with PBj6.6; the only exception being in stimulated pigtail macaque PBMCs, where replication kinetics were nearly identical. In addition, PBj6.6 delta nef was unable to induce the proliferation of peripheral blood mononuclear cells (PBMCs) in vitro, a unique characteristic of acutely pathogenic SIVsmmPBj. Inoculation of this virus into pigtail macaques resulted in infection, but did not result in any detectable acute disease. These studies suggest that Nef is an important viral determinant in the pathogenesis of SIVsmmPBj-induced disease, and further suggest that Nef plays a significant role in viral replication in vivo.