David M. Anderson

Thrombotic Microangiopathy in the HIV-2-Infected Macaque

Thrombotic microangiopathy (TMA) has been increasingly reported in human immunodeficiency virus (HIV)-infected humans over the past decade. The pathogenesis is unknown. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques (Macaca nemestrina), infected intravenously with a virulent HIV-2 strain, HIV-2(287), in addition to that of four uninfected control macaques. Necropsies were performed between 12 hours and 28 days after infection. HIV-2 antigen was detectable in peripheral blood mononuclear cell (PBMC) cocultures in all animals after 10 days of HIV-2 infection; a rapid decline in CD4(+) PBMC (<350/microliter) was seen in five of six animals 21 days and 28 days after infection. No macaque developed features of clinical AIDS. Typical lesions of human HIV-associated nephropathy were undetectable. Six of the 27 HIV-2-infected macaques demonstrated both histological TMA lesions (thrombi in glomerular capillary loops and small arteries, mesangiolysis) and ultrastructural lesions (mesangiolysis, subendothelial lucency, platelet thrombi in glomerular capillary lumina). Extrarenal thrombi were detected in the gastrointestinal and adrenal microvasculature of macaques that had developed renal TMA. None of the control animals demonstrated features of renal TMA at necropsy. In a retrospective analysis of kidneys obtained from 39 additional macaques infected with HIV-2(287), seven cases demonstrated TMA. In situ hybridization showed no detectable HIV-2 RNA in kidney sections of 65/66 HIV-2-infected macaques, including all 13 TMA cases. Expression of the chemokine receptor CXCR4, the putative coreceptor for HIV-2(287), was absent in intrinsic renal cells in all HIV-2-infected macaques. The HIV-2-infected macaque may be a useful model of human HIV-associated TMA. Our data do not support a role of direct HIV-2 infection of intrinsic renal cells as an underlying mechanism.

Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that causes rapid CD4+ cell depletion in Macaca nemestrina.

With few exceptions, humans are the only species known to develop acquired immunodeficiency syndrome (AIDS) after human immunodeficiency virus (HIV) infection. We report here that an isolate of HIV type 2, EHO, readily established persistent infection in 100% of Macaca nemestrina in three consecutive transmission studies. Of the eight infected animals, five showed persistently high virus load and six developed AIDS‐like diseases or CD4+ cell depletion within 4 years of infection. The pathology and clinical signs closely parallel those of HIV‐1 infection of humans, including lymphadenopathy, anemia, CD4+ cell depletion, and opportunistic infections. A cell‐free virus stock was established from the lymph nodes of an animal that developed AIDS‐like diseases. This virus, HIV‐2/287, was highly pathogenic in M. nemestrina, causing CD4+ cell depletion within 2–8 weeks post‐infection. While both HIV‐2 EHO and HIV‐2/287 use predominantly CXCR4, the latter shows greatly enhanced replicative capacity in macaque peripheral blood mononuclear cells (PBMCs). The establishment of a human immunodeficiency virus that causes rapid and reproducible CD4+ cell depletion in macaques could facilitate the study of HIV pathogenesis and the development of effective vaccines and therapy against AIDS.

Viral dynamics of early HIV infection in neonatal macaques after oral exposure to HIV-2287: an animal model with implications for maternal-neonatal HIV transmission.

A model of vertical HIV transmission was developed using oral HIV‐2287 exposure of newborn Macaca nemestrina. The minimal Animal Infectious Dose for this oral route was found to be 10‐fold higher than that for atraumatic viral transmission across other mucosal membranes (vaginal/rectal) of juvenile macaques. However, once infection was established, viral replication was rapid and plasma viremia could be detected by reverse‐transcriptase polymerase chain reaction and viral co‐culture within 1u2003week following exposure. No animal was resistant to infection and all macaques initially exposed to a subinfectious viral inoculum were subsequently infected by re‐exposure of mucosal membranes. Higher viral load during primary infection correlated with a more rapid CD4 depletion; however, all HIV‐2287‐infected animals developed CD4 depletion during the observation period. This animal model can now be used to study early viral replication in the presence and absence of anti‐retroviral agents to help identify conditions to reduce vertical HIV transmission in human newborns.

Characterization of a maternal-fetal HIV transmission model using pregnant macaques infected with HIV-2287

Abstract: To study mechanisms involved in mother‐to‐fetus transmission of human immunodeficiency virus (HIV) in utero, we have developed a chronically catheterized pregnant macaque model that permits simultaneous and sequential determination of virus in maternal and fetal blood and amniotic fluid during pregnancy. In this report, we have characterized this model using three groups of pregnant macaques designed to sample: (1) maternal blood, fetal blood, and amniotic fluid (n = 6); (2) maternal blood and amniotic fluid (n = 6); or (3) maternal blood only (n = 2). After inoculation with the highly pathogenic HIV‐2287, all pregnant macaques developed brief but intense viremias followed by precipitous CD4+ T‐cell declines within 2–3 weeks. While all the infants born to dams of the three groups were HIV positive, the degree of infection and outcome of HIV infection varied. All infants were shown to be HIV‐RNA‐positive by reverse transcriptase‐polymerase chain reaction (RT‐PCR). However, HIV‐infected cells were detected only in the blood of those born to dams enrolled in groups 1 and 2: most of these infants progressed to CD4+ T‐cell depletion. The infants in group 3 exhibited HIV‐RNA in plasma, although neither HIV‐infected cells nor CD4+ T‐cell depletion was detectable. However, all infants developed HIV‐2‐specific antibody at various levels by 2 months of age. Together, the data suggest that, while the degree of instrumentation may modulate intensity of virus transmission to fetus, the highly pathogenic HIV‐2287 exhibited a high frequency of virus transmission from the mother to fetus.

West Nile and St. Louis Encephalitis Virus Antibody Seroconversion, Prevalence, and Persistence in Naturally Infected Pig-Tailed Macaques (Macaca nemestrina)

ABSTRACT Pig-tailed macaques (Macaca nemestrina) naturally infected with West Nile virus were monitored from 1999 to 2005 to determine virus-specific antibody seroconversion, prevalence, and persistence. Antibodies persisted for up to 36 months, as detected by epitope-blocking enzyme-linked immunosorbent and hemagglutination inhibition assays. Exposure to cocirculating St. Louis encephalitis virus was evaluated by Western blotting and immunofluorescence assays.

Deep transcriptional sequencing of mucosal challenge compartment from rhesus macaques acutely infected with simian immunodeficiency virus implicates loss of cell adhesion preceding immune activation

ABSTRACT Pathology resulting from human immunodeficiency virus (HIV) infection is driven by protracted inflammation; the primary loss of CD4+ T cells is caused by activation-driven apoptosis. Recent studies of nonhuman primates (NHPs) have suggested that during the acute phase of infection, antiviral mucosal immunity restricts viral replication in the primary infection compartment. These studies imply that HIV achieves systemic infection as a consequence of a failure in host antiviral immunity. Here, we used high-dose intrarectal inoculation of rhesus macaques with simian immunodeficiency virus (SIV) SIVmac251 to examine how the mucosal immune system is overcome by SIV during acute infection. The host response in rectal mucosa was characterized by deep mRNA sequencing (mRNA-seq) at 3 and 12 days postinoculation (dpi) in 4 animals for each time point. While we observed a strong host transcriptional response at 3 dpi, functions relating to antiviral immunity were absent. Instead, we observed a significant number of differentially expressed genes relating to cell adhesion and reorganization of the cytoskeleton. We also observed downregulation of genes encoding members of the claudin family of cell adhesion molecules, which are coexpressed with genes associated with pathology in the colorectal mucosa, and a large number of noncoding transcripts. In contrast, at 12 dpi the differentially expressed genes were enriched in those involved with immune system functions, in particular, functions relating to T cells, B cells, and NK cells. Our findings indicate that host responses that negatively affect mucosal integrity occur before inflammation. Consequently, when inflammation is activated at peak viremia, mucosal integrity is already compromised, potentially enabling rapid tissue damage, driving further inflammation. IMPORTANCE The HIV pandemic is one of the major threats to human health, causing over a million deaths per year. Recent studies have suggested that mucosal antiviral immune responses play an important role in preventing systemic infection after exposure to the virus. Yet, despite their potential role in decreasing transmission rates between individuals, these antiviral mechanisms are poorly understood. Here, we carried out the first deep mRNA sequencing analysis of mucosal host responses in the primary infection compartment during acute SIV infection. We found that during acute infection, a significant host response was mounted in the mucosa before inflammation was triggered. Our analysis indicated that the response has a detrimental effect on tissue integrity, causing increased permeability, tissue damage, and recruitment of SIV target cells. These results emphasize the importance of mucosal host responses preceding immune activation in preventing systemic SIV infection.

The Nonhuman Primate as a Model for Biomedical Research

This chapter provides a brief summary of issues surrounding the utilization of nonhuman primates in biomedical research. Although a relatively small proportion of the total number of animals utilized in biomedical research, nonhuman primates occupy a unique position as the species most closely related to humans, and thus have the potential to provide highly relevant information regarding human health issues. Nonhuman primates are utilized across a wide diversity of research topics and examples are provided including infectious disease, neuroscience, and genomics. Pertinent information relating to ethical issues, species selection, housing, and specific pathogen-free status are provided as an overview of relevant issues associated with selection of nonhuman primate models. Selected references are provided as a reference for more comprehensive information relating to these topics.

Systemic and Intestinal Immune Responses to HIV-2287 Infection in Macaca nemestrina

Nonhuman primate models of human AIDS have been used successfully to evaluate candidate vaccines and infection intervention therapies. Successes of pathogenicity studies in primate models have been limited because of the varied infection outcomes and characteristic low number of study animals. The acutely pathogenic HIV-2(287)--Macaca nemestrina model has shown promise both in antiviral drug evaluation and in pathogenicity studies. Here we describe virus replication, spread, and host responses during the first 28 days of HIV-2(287) infection. Focusing on 18 macaques from a larger 27-macaque study, we report changing virus loads, CD4(+) cell depletions, and antibody responses both systemically and in the mucosa of the small intestine. After intravenous inoculation, blood and intestinal tissue were collected from pairs of macaques at 12 hr and 1, 2, 4, 6, 10, 14, 21, and 28 days postinfection. Specimens were examined for evidence of infection by quantitative cultures, in situ hybridization, lymphocyte subset monitoring, and antibody production. The data were presented serially as though all samples were collected from a single macaque. The highest blood virus loads were detected between days 10 and 14 and subsequently decreased through day 28. This coincided with a significant increase in ileum mucosa virus loads on day 10, which became undetectable by day 28. The lowest levels of CD4(+) cells were observed on days 21 and 28 in blood and ileum mucosa. CD4(+):CD8(+) cell ratios in blood and ileum dropped dramatically after day 10 to lowest levels by day 28. Intestinal virus loads were inversely correlated with CD4(+) cell and virus-specific antibody levels in the ileum after day 6. These results underscore the suitability of this model for pathogenicity studies as well as the importance of the intestinal lymphoid tissues as an initial site of virus replication and cell destruction during the acute, asymptomatic stage of AIDS development.

Rapid Shift from Virally Infected Cells to Germinal Center-Retained Virus after HIV-2 Infection of Macaques

Lymphoid tissues are the primary target during the initial virus dissemination that occurs in HIV-1-infected individuals. Recent advances in antiretroviral therapy and techniques to monitor virus load in humans have demonstrated that the early stages of viral infection and host response are major determinants of the outcome of individual infections. Relatively little is known about immunopathogenic events occurring during the acute phase of HIV infection. We analyzed viral dissemination within lymphoid tissues by in situ hybridization and by combined immunohistochemistry/in situ hybridization during the acute infection phase (12 hours to 28 days) in pig-tailed macaques (Macaca nemestrina), challenged intravenously with a virulent strain of HIV-2, HIV-2(287). Two stages in viral dissemination were clearly evident within the first 28 days after HIV-2(287) infection. First, a massive increase in individual HIV-2-infected cells, mostly CD3+ T lymphocytes and a smaller percentage of macrophages and interdigitating dendritic cells, was identified within lymph nodes which peaked on the 10th day after HIV-2 infection. A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection. Coincident with a marked reduction in individual HIV-2 RNA+ cells by day 14 postinfection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection. Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells. In this nonhuman primate model of HIV-associated immunopathogenesis, using a virus derived from a human pathogen, we identified a significant shift in the pattern of HIV-2 localization within a narrow time frame (day 10 to day 14). This shift in virus localization and behavior indicates that there may be a discrete but remarkably narrow window for therapeutic interventions that interrupt this stage in the natural course of HIV infection. Reproducibility and the accelerated time course of disease development make this model an excellent candidate for such intervention studies.