Louis N. Martin

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow–up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Normal T-Cell Turnover in Sooty Mangabeys Harboring Active Simian Immunodeficiency Virus Infection

ABSTRACT Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) remain healthy though they harbor viral loads comparable to those in rhesus macaques that progress to AIDS. To assess the immunologic basis of disease resistance in mangabeys, we compared the effect of SIV infection on T-cell regeneration in both monkey species. Measurement of the proliferation marker Ki-67 by flow cytometry showed that mangabeys harbored proliferating T cells at a level of 3 to 4% in peripheral blood irrespective of their infection status. In contrast, rhesus macaques demonstrated a naturally high fraction of proliferating T cells (7%) that increased two- to threefold following SIV infection. Ki-67+ T cells were predominantly CD45RA−, indicating increased proliferation of memory cells in macaques. Quantitation of an episomal DNA product of T-cell receptor α rearrangement (termed α1 circle) showed that the concentration of recent thymic emigrants in blood decreased with age over a 2-log unit range in both monkey species, consistent with age-related thymic involution. SIV infection caused a limited decrease of α1 circle numbers in mangabeys as well as in macaques. Dilution of α1 circles by T-cell proliferation likely contributed to this decrease, since α1 circle numbers and Ki-67+ fractions correlated negatively. These findings are compatible with immune exhaustion mediated by abnormal T-cell proliferation, rather than with early thymic failure, in SIV-infected macaques. Normal T-cell turnover in SIV-infected mangabeys provides an explanation for the long-term maintenance of a functional immune system in these hosts.

Necropsy Findings in Rhesus Monkeys Experimentally Infected with Cultured Simian Immunodeficiency Virus (SIV)/Delta

Lesions induced in rhesus monkeys by different isolates of simian immunodeficiency virus (SIV)/Delta were studied at necropsy. Four groups of monkeys were inoculated with SIV/Delta isolated from other experimentally infected rhesus monkeys, while one group was inoculated with SIV/Delta from an asymptomatic mangabey monkey. Three rhesus isolates and the mangabey isolate were virulent, killing 75–100% of infected monkeys. One rhesus isolate, which had been extensively passaged in vitro, was attenuated but was restored to virulence by single animal passage. Clinically, infected monkeys had lymphadenopathy, splenomegaly, diarrhea, and a rash. Most monkeys died of enteric disease. The following lesions were seen: weight loss, thymic atrophy, lymphoid atrophy, bone marrow hyperplasia, encephalitis, colitis, amyloidosis, hepatitis, glomerulosclerosis, and the presence of syncytial cells. One Rh Epstein-Barr virus (EBV)-related lymphoma occurred. Opportunistic agents were identified: cytomegalovirus, adenovirus, Cryptosporidia, and Pneumocystis. Shigella and Campylobacter often caused colitis.

Infection and AIDS in Adult Macaques After Nontraumatic Oral Exposure to Cell-Free SIV

Unprotected receptive anal intercourse is a well-recognized risk factor for infection with human immunodeficiency virus-type 1 (HIV-1). Isolated human case reports have implicated HIV-1 transmission by oral-genital exposure. Adult macaques exposed nontraumatically to cell-free simian immunodeficiency virus (SIV) through the oral route became infected and developed acquired immunodeficiency syndrome (AIDS). The minimal virus dose needed to achieve systemic infection after oral exposure was 6000 times lower than the minimal dose required to achieve systemic infection after rectal exposure. Thus, unprotected receptive oral intercourse, even in the absence of mucosal lesions, should be added to the list of risk behaviors for HIV-1 transmission.

The replicative capacity of rhesus macaque peripheral blood mononuclear cells for simian immunodeficiency virus in vitro is predictive of the rate of progression to AIDS in vivo.

Survival of rhesus macaques (Macaca mulatta) experimentally infected with simian immunodeficiency virus (SIV) varies significantly from animal to animal. Some animals die within 2 months while others survive for more than 5 years, even when identical inocula are used. This diversity in survival creates a significant problem in the design of therapeutic and vaccine trials using the SIV-macaque model because the use of small numbers of animals may provide results that are misleading. Identifying an in vitro assay that could determine the survival of monkeys prior to infection would prove extremely useful for stratifying experimental groups. Analysis of the survival of a cohort of 59 control animals obtained from over a decade of vaccine and therapeutic trials has demonstrated that the ability of peripheral blood mononuclear cells (PBMC) from a naïve animal to produce virus in vitro was highly predictive of disease progression in vivo following experimental inoculation. Animals classified in vitro as high producers of virus progressed to disease significantly more rapidly than animals classified as either low (P=0.002) or intermediate (P=0.013) producers of virus. The hierarchy of high and low virus production was maintained in purified CD4(+) T cell cultures, indicating that this phenotype is an intrinsic property of the CD4(+) T cell itself. These findings should significantly aid in the design of vaccine and therapeutic trials using the SIV-macaque model. Furthermore, since these studies suggest that the rate of virus replication is controlled by innate characteristics of the individual, they provide new insight into the pathogenesis of AIDS.

Oral Transmission of Primate Lentiviruses

Oral transmission of human immunodeficiency virus type 1 (HIV-1) is well documented in children who become infected postnatally through breast milk. In contrast, epidemiologic surveys have yielded conflicting data regarding oral HIV-1 transmission among adults, even though case reports have described seroconversion and the development of AIDS in adults whose only risk was oral-genital contact. To study oral virus transmission in primate models, we exposed rhesus macaques of various ages to cell-free simian immunodeficiency virus (SIV), including uncloned and molecularly cloned viruses. In neonates, viremia and AIDS developed after nontraumatic oral exposure to several SIV strains. Furthermore, chimeric simian human immunodeficiency viruses containing the HIV-1 envelope can also cross intact upper gastrointestinal mucosal surfaces in neonates. In adult macaques, infection and AIDS have resulted from well-controlled, nontraumatic, experimental oral exposure to different strains of SIV. These findings have implications for the risks of HIV-1 transmission during oral-genital contact.

Chromatographic fractionation of rhesus monkey (Macaca mulatta) IgG subclasses using DEAE cellulose and protein A-Sepharose

Rhesus monkey serum was applied to a protein A-Sepharose column at pH 7.3. IgG was not detectable in the unbound effluent. Bound protein was eluted with a pH gradient of citrate-phosphate buffer. Two major elution peaks reproducibly resulted at pH 4.85-4.9 and pH 4.35-4.43, respectively, with only slight variability among individuals. Since the immunoelectrophoretic mobility of IgG in the peak at pH 4.85-4.9 was slower than that of the peak at pH 4.35-4.43, serum was first fractionated by ion exchange chromatography into 2 fractions: the first did not bind to DEAE cellulose in 0.01 M phosphate buffer pH 7.8, the second did bind and was eluted by an NaCl gradient. Each DEAE fraction was then further fractionated on protein A-Sepharose. IgG in the first DEAE fraction bound to protein A at pH 7.3 and eluted in a single peak at pH 4.72-5.0. IgG in the second DEAE fraction also bound to protein A, but eluted in 2 peaks at pH 4.65-5.1, and 4.25-4.6, respectively. All 3 IgG fractions eluted in the same position from an A5m column, had immunodiffusion reactions of identity with anti-human gamma chain and were composed of similar heavy and light chains judged by SDS polyacrylamide gel electrophoresis. However, IgG eluting from protein A at the low pH (4.25-4.6) had a reaction of partial identity with other IgG fractions when tested by immunodiffusion against rabbit anti-rhesus gamma chain, suggestive of an antigenically different subclass. Analysis of rabbit antisera prepared against the 3 IgG fractions confirmed the occurrence of at least 3 antigenically distinct rhesus monkey IgG subclasses. These 3 subclasses have been provisionally designated IgG I, IgG II and IgG III.

Cyclosporin A Modulation of Early Virologic and Immunologic Events during Primary Simian Immunodeficiency Virus Infection in Rhesus Monkeys

Virologic and immunologic effects of immunomodulation during primary simian immunodeficiency virus (SIV) infection were examined in monkeys treated with cyclosporin or vehicle for 32 days beginning 5 days before SIV inoculation. Duration of antigenemia decreased in 5 of 7 treated monkeys, 2 having delayed onset and peak of antigenemia. Although proviral DNA levels in blood and lymph nodes and infected cell numbers in lymph nodes were transiently decreased, levels were similar to those in controls by day 14. The CD4:CD8 ratio and percentage of CD4+ CD29+ cells decreased in controls 14 days after inoculation, but this decrease was delayed in treated monkeys. Two treated monkeys demonstrated rapid disease, with progressive antigenemia preceding early deaths 90-96 days after inoculation. Nevertheless, immunomodulation influenced the kinetics of primary SIV infection in some monkeys, supporting the rationale of careful exploration of the strategy of interference with the heightened state of cellular activation together with direct antiretroviral therapy in human immunodeficiency virus infection.

Lentivirus-induced Pulmonary Lesions in Rhesus Monkeys (Macaca mulatta) Infected with Simian Immunodeficiency Virus

Necropsy reports from 28 rhesus monkeys that had been experimentally infected with simian immunodeficiency virus (SIV) and that were free of cytomegalovirus were reviewed. Lung sections from 24 of these monkeys that had no etiologic agent other than SIV detected in the lung were studied in detail by histopathologic, immunohistochemical, and electron microscopic examination and by in situ hybridization. Fourteen of the monkeys were part of a serial euthanasia study, while others were euthanatized after they became moribund. The following lesions were detected: perivascular inflammation, vasculitis, interstitial pneumonia, syncytial cells, hemorrhage, fibrin exudation, and pleural fibrosis. Perivascular inflammation was the most frequent lesion and occurred as early as 2 weeks after inoculation. Severe pneumonia and numerous syncytial cells were seen only in animals euthanatized because they had become moribund. The lesions appeared to be directly due to SIV infection. SIV antigens, RNA, and virions were detected in syncytial cells and macrophages by immunohistochemical examination, in situ hybridization, and transmission electron microscopic examination, respectively. The amount of virus present was correlated with the severity of the lesions. The SIV-induced lesions were different from those of the lymphocytic interstitial pneumonia, which occurs in human immunodeficiency virus-infected children and in ovine lentivirus-infected sheep and goats.

Direct Inoculation of Simian Immunodeficiency Virus from Sooty Mangabeys in Black Mangabeys (Lophocebus aterrimus): First Evidence of AIDS in a Heterologous African Species and Different Pathologic Outcomes of Experimental Infection

ABSTRACT A unique opportunity for the study of the role of serial passage and cross-species transmission was offered by a series of experiments carried out at the Tulane National Primate Research Center in 1990. To develop an animal model for leprosy, three black mangabeys (BkMs) (Lophocebus aterrimus) were inoculated with lepromatous tissue that had been serially passaged in four sooty mangabeys (SMs) (Cercocebus atys). All three BkMs became infected with simian immunodeficiency virus from SMs (SIVsm) by day 30 postinoculation (p.i.) with lepromatous tissue. One (BkMG140) died 2 years p.i. from causes unrelated to SIV, one (BkMG139) survived for 10 years, whereas the third (BkMG138) was euthanized with AIDS after 5 years. Histopathology revealed a high number of giant cells in tissues from BkMG138, but no SIV-related lesions were found in the remaining two BkMs. Four-color immunofluorescence revealed high levels of SIVsm associated with both giant cells and T lymphocytes in BkMG138 and no detectable SIV in the remaining two. Serum viral load (VL) showed a significant increase (>1 log) during the late stage of the disease in BkMG138, as opposed to a continuous decline in VL in the remaining two BkMs. With the progression to AIDS, neopterin levels increased in BkMG138. This study took on new significance when phylogenetic analysis unexpectedly showed that all four serially inoculated SMs were infected with different SIVsm lineages prior to the beginning of the experiment. Furthermore, the strain infecting the BkMs originated from the last SM in the series. Therefore, the virus infecting BkMs has not been serially passaged. In conclusion, we present the first compelling evidence that direct cross-species transmission of SIV may induce AIDS in heterologous African nonhuman primate (NHP) species. The results showed that cross-species-transmitted SIVsm was well controlled in two of three BkMs for 2 and 10 years, respectively. Finally, this case of AIDS in an African monkey suggests that the dogma of SIV nonpathogenicity in African NHP hosts should be reconsidered.