Molecular epidemiology of simian immunodeficiency virus SIVsm in U.S. primate centers unravels the origin of SIVmac and SIVstm.

Retrospective molecular epidemiology was performed on samples from four sooty mangabey (SM) colonies in the United States to characterize simian immunodeficiency virus SIVsm diversity in SMs and to trace virus circulation among different primate centers (PCs) over the past 30 years. The following SIVsm sequences were collected from different monkeys: 55 SIVsm isolates from the Tulane PC sampled between 1984 and 2004, 10 SIVsm isolates from the Yerkes PC sampled in 2002, 7 SIVsm isolates from the New Iberia PC sampled between 1979 and 1986, and 8 SIVsm isolates from the California PC sampled between 1975 and 1977. PCR and sequencing were done to characterize the gag, pol, and env gp36 genes. Phylogenetic analyses were correlated with the epidemiological data. Our analysis identified nine different divergent phylogenetic lineages that cocirculated in these four SM colonies in the Unites States in the past 30 years. Lineages 1 to 5 have been identified previously. Two of the newly identified SIVsm lineages found in SMs are ancestral to SIVmac251/SIVmac239/SIVmne and SIVstm. We further identified the origin of these two macaque viruses in SMs from the California National Primate Research Center. The diversity of SIVsm isolates in PCs in the United States mirrors that of human immunodeficiency virus type 1 (HIV-1) group M subtypes and offers a model for the molecular epidemiology of HIV and a new approach to vaccine testing. The cocirculation of divergent SIVsm strains in PCs resulted in founder effects, superinfections, and recombinations. This large array of SIVsm strains showing the same magnitude of diversity as HIV-1 group M subtypes should be extremely useful for modeling the efficacy of vaccination strategies under the real-world conditions of HIV-1 diversity. The genetic variability of SIVsm strains among PCs may influence the diagnosis and monitoring of SIVsm infection and, consequently, may bias the results of pathogenesis studies.

Molecular epidemiology of simian T-cell lymphotropic virus type 1 in wild and captive sooty mangabeys.

A study was conducted to evaluate the prevalence and diversity of simian T-cell lymphotropic virus (STLV) isolates within the long-established Tulane National Primate Research Center (TNPRC) colony of sooty mangabeys (SMs; Cercocebus atys). Serological analysis determined that 22 of 39 animals (56%) were positive for STLV type 1 (STLV-1). A second group of thirteen SM bush meat samples from Sierra Leone in Africa was also included and tested only by PCR. Twenty-two of 39 captive animals (56%) and 3 of 13 bush meat samples (23%) were positive for STLV-1, as shown by testing with PCR. Nucleotide sequencing and phylogenetic analysis of viral strains obtained demonstrated that STLV-1 strains from SMs (STLV-1sm strains) from the TNPRC colony and Sierra Leone formed a single cluster together with the previously reported STLV-1sm strain from the Yerkes National Primate Research Center. These data confirm that Africa is the origin for TNPRC STLV-1sm and suggest that Sierra Leone is the origin for the SM colonies in the United States. The TNPRC STLV-1sm strains further divided into two subclusters, suggesting STLV-1sm infection of two original founder SMs at the time of their importation into the United States. STLV-1sm diversity in the TNPRC colony matches the high diversity of SIVsm in the already reported colony. The lack of correlation between the lineage of the simian immunodeficiency virus from SMs (SIVsm) and the STLV-1sm subcluster distribution of the TNPRC strains suggests that intracolony transmissions of both viruses were independent events.

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.

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.

Classic AIDS in a sooty mangabey after an 18-year natural infection.

Prevailing theory holds that simian immunodeficiency virus (SIV) infections are nonpathogenic in their natural simian hosts and that lifelong infections persist without disease. Numerous studies have reported that SIV-infected sooty mangabeys (SMs; Cercocebus atys) remain disease free for up to 24 years despite relatively high levels of viral replication. Here, we report that classic AIDS developed after an 18-year incubation in an SM (E041) with a natural SIVsm infection. Unlike that described in previous reports of SIV-related disease in SMs, the SIVsm infecting E041 was not first passaged through macaques; moreover, SM E041 was simian T-cell leukemia virus antibody negative. SM E041 was euthanized in 2002 after being diagnosed with severe disseminated B-cell lymphoma. The plasma virus load had been approximately the same for 16 years when a 100-fold increase in virus load occurred in years 17 and 18. Additional findings associated with AIDS were CD4(+)-cell decline, loss of p27 core antibody, and loss of control of SIVsm replication with disseminated giant cell disease. These findings suggest that the time to development of AIDS exceeds the average lifetime of SMs in the wild and that the principal adaptation of SIV to its natural African hosts does not include complete resistance to disease. Instead, AIDS may develop slowly, even in the presence of high virus loads. However, a long-term relatively high virus load, such as that in SM E041, is consistent with AIDS development in less than 18 years in humans and macaques. Therefore, the results also suggest that SMs have a special mechanism for resisting AIDS development.

Evidence for a rhesus monkey model of asymptomatic tuberculosis.

Rhesus monkeys (RM) were inoculated intrabronchially with graded doses of Mycobacterium tuberculosis (MTB) strains Erdman and H37Rv in an effort to produce a model of asymptomatic tuberculosis infection. Erdman strain produced active disease within 7-11 weeks regardless of dose. Low doses of H37Rv resulted in asymptomatic infections; high doses produced active disease within 11 weeks. Over a 4-month period of post-inoculation study, MTB culture-filtrate protein (CFP)-stimulated bronchoalveolar lavage cells (BALC) and blood mononuclear cells (PBMC) from monkeys with active disease (30 cfu Erdman-inoculated) or asymptomatic infection (200 cfu H37Rv-inoculated) produced similar significant quantities of mRNA encoding for IFN-gamma or TNF-alpha, but insignificant quantities of IL-4 mRNA. Differences were observed in antigen-induced in vitro blastogenic responses and serum anti-lipoarabinomannan (LAM) antibody responses in animals with active compared with asymptomatic MTB infections. The results indicate that RM are a good model for the study of asymptomatic tuberculosis infections using low doses of H37Rv.

A brief history of the discovery of natural simian immunodeficiency virus (SIV) infections in captive sooty mangabey monkeys.

Experimental leprosy studies using Mycobacterium leprae inoculum isolated from a sooty mangabey monkey (SMM) resulted in the accidental discovery that SMM's asymptomatically carry simian immunodeficiency virus (SIV) that is pathogenic in macaques. We showed that the SMM virus, SIVDelta, was antigenically related to SIVmac, which had been identified in macaques, and to the human immunodeficiency virus (HIV). Similar asymptomatic natural SIV infections had been reported in African green monkeys (AGM). Our results together with observations of others led us to propose that both SIVmac and SIVDelta originated in SMM and that SIV emerged in humans as a result of early African nonhuman primate SIV trans-species infections in humans.

Spectrum of manifestations of Mycobacterium tuberculosis infection in primates infected with SIV.

To characterize the manifestations of coinfection with M. tuberculosis and SIV infection, we studied 12 SIV-infected rhesus monkeys, six of which were infected intrabronchially with a low dose of Mycobacterium tuberculosis H37Rv. In the six coinfected animals, M. tuberculosis antigen-stimulated lung and blood cells produced high concentrations of IFN-gamma but not IL-4 8-16 weeks after infection. Of the three coinfected animals with high levels of plasma viremia, two developed disseminated tuberculosis and the other died of bacterial peritonitis. Of three coinfected animals with moderate levels of plasma viremia, two had no clinical or radiographic evidence of tuberculosis or progressive SIV infection for 6 months after infection. At neuropsy, pulmonary granulomata were observed and acid-fast organisms or M. tuberculosis were present. These clinical, immunologic and pathologic findings are consistent with those in humans with latent tuberculosis infection (LTBI), and suggest that a model of LTBI in SIV-infected primates can be developed. Such a model will permit delineation of the immunologic and microbial factors that characterize LTBI in HIV-infected persons.

Noninvasive detection of new simian immunodeficiency virus lineages in captive sooty mangabeys: ability to amplify virion RNA from fecal samples correlates with viral load in plasma.

The sooty mangabey (SM) (Cercocebus atys) is the natural host of a simian immunodeficiency virus, termed SIVsm, which gave rise to human immunodeficiency virus type 2. Data on the geographic distribution, prevalence, and genetic diversity of SIVsm in the wild remains limited. To address this issue, noninvasive strategies based on screening SM fecal and urine specimens for SIVsm-specific antibodies and virion RNA (vRNA) were developed, and the results were correlated with viral loads in plasma. Twenty-three SIVsm-infected and 27 uninfected SMs were evaluated. Time-matched urine, fecal and plasma samples were collected over a 2-month period from 16 captive naturally infected SMs. The remaining 7 infected and 27 uninfected SMs were sampled once. Each specimen was subjected to enhanced chemiluminescence-Western blot analysis and nested reverse transcriptase (RT) PCR. The results showed that urine was highly sensitive (96%) and specific (100%) for detection of SIVsm antibodies, while fecal detection was much less sensitive (16%). Conversely, vRNA detection was more sensitive in feces (50%) than in urine (2%) samples. Fecal-vRNA detection correlated with viral loads in plasma (P < 0.002). SMs with detectable fecal vRNA had a mean viral load in plasma of 458,006 copies/ml, while those with undetectable fecal vRNA had a mean viral load in plasma of 29,428 copies/ml. Moreover, for every log increase in the viral load in plasma, the odds of detecting virus in fecal samples increased 87-fold. Genetic diversity of SIVsm in the SM colony was characterized by sequencing partial gag (846 bp) and gp43 (439 bp) fragments. Surprisingly, four new SIVsm lineages were identified, two of which were initially detected by fecal RT-PCR. This study documents the suitability of noninvasive methods for the detection and molecular characterization of new SIV variants. These assays will be useful for studying the phylogeny and epidemiology of SIVsm infections in the wild, and they hold promise as tools for investigating natural SIV infections in endangered nonhuman primates.

Under-explored experimental topics related to integral mycobacterial vaccines for leprosy.

Many leprosy vaccine studies have utilized live or killed whole mycobacteria, such as Bacille Calmette-Guérin, Indian Cancer Research Center (ICRC) bacilli and Mycobacterium w either alone or in combination with killed Mycobacterium leprae. For Bacille Calmette-Guérin, the vaccine dose is generally that which gives the largest delayed-type hypersensitivity response with minimal side effects. The doses of other integral mycobacterial vaccines appear to be arbitrarily chosen. Hypotheses governing immunologic responses to complex antigens predict that the doses used may be too high, resulting in protection of some individuals and increasing the susceptibility of other individuals to leprosy. The natural history of an individual's prior exposure to environmental mycobacteria will affect the outcome of protective vaccination using a given dose of mycobacterial vaccine in the individual.

Antileprosy protective vaccination of sooty mangabey monkeys with BCG or BCG plus heat-killed mycobacterium leprae: immunologic observations

Groups of sooty mangabey monkeys (SMM) were vaccinated and boosted with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or BCG + low-dose (LD) or high-dose (HD) heat-killed M. leprae (HKML), or were unvaccinated. Prior to and following vaccination-boosting and subsequent M. leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were immunologically observed longitudinally for approximately 3 years. SMM [multibacillary (MB) leprosy-prone as a species] were not protected clinically by BCG or BCG + HKML, although the disease progress was slowed by vaccination with BCG alone. The longitudinal immune response profiles to BCG or BCG + HKML in SMM showed that: 1) vaccination with BCG or BCG + HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to ML antigens, which returned to baseline post-boosting and post-live ML challenge; 2) BCG + LD HKML-vaccinated groups gave the largest blastongenic response (SI = 23) followed by the BCG + HD HKML group (SI = 14.5) and by the BCG-only vaccinated group (SI = 3.6); 3) significantly diminished numbers of blood CD4+ (helper) and CD4+CD29+ (helper-inducer) T-cell subsets were observed longitudinally in all ML-challenged groups compared to controls regardless of whether they had been vaccinated or not; 4) CD8+ (suppressor) T-cell numbers remained longitudinally constant, on average, in all ML-challenged groups (vaccinated or not) compared to controls; 5) there was a significant decrease in the CD4+:CD8+ ratio over time in all ML-challenged groups (vaccinated or not); 6) vaccination with BCG or BCG + LD or HD HKML resulted in significantly increased numbers of CD4+CD45RA+ (suppressor-inducer) T cells longitudinally compared to the unvaccinated, ML-challenged control group; and 7) over time, vaccination with BCG + HKML followed by live ML-challenge produced higher IGM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody response ratios than BCG-only vaccinated, ML-challenged monkeys or unvaccinated, ML-challenged SMM, consistent with prior observations that IgG anti-PGL-I responses correlate with resistance to and protection from clinical leprosy and IgM anti-PGL-I responses correlate with increased susceptibility.

Antileprosy protective vaccination of rhesus monkeys with BCG or BCG plus heat-killed mycobacterium leprae: immunologic observations

Groups of rhesus monkeys were vaccinated and boosted with Mycobacterium bovis bacillus Calmette Guerin (BCG) or BCG plus low-dose (LD) or high-dose (HD) heat-killed M. leprae (HKML), or were unvaccinated. Prior to and following vaccination-boosting and subsequent M. leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were observed longitudinally for approximately 3 years. Vaccination with BCG plus HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to lepromin, which returned to baseline post-boosting and post-live-ML-challenge, minimally reappearing significantly 2 years post-ML-challenge. Vaccination with BCG failed to stimulated positive blastogenic responses to lepromin before ML-challenge but small, marginally positive, intermittent responses were seen post-ML-challenge. Compared to the unvaccinated ML-challenged group, significant increases in the numbers of blood CD4+ and CD8+ T-cell subsets and an increased CD4+:CD8+ ratio were observed in both BCG plus HKML-vaccinated, ML-challenged groups, but not in the BCG-only-vaccinated, ML-challenged group. CD4+CD29+ and CD4+CD45RA+ subset numbers increased significantly over time in only the BCG plus LD HKML-vaccinated, ML-challenged group. Compared to unvaccinated, ML-challenged groups, vaccination with BCG or BCG plus HKML followed by ML-challenge produced lower IgM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody ratios and protected rhesus monkeys from clinical leprosy, consistent with prior observations that low IgM:IgG anti-PGL-I responses correlated with resistance to and protection from leprosy.