Ann Schmidt

Cyanovirin-N Inhibits AIDS Virus Infections in Vaginal Transmission Models

The cyanobacterial protein cyanovirin-N (CV-N) potently inactivates diverse strains of HIV-1 and other lentiviruses due to irreversible binding of CV-N to the viral envelope glycoprotein gp120. In this study, we show that recombinant CV-N effectively blocks HIV-1(Ba-L) infection of human ectocervical explants. Furthermore, we demonstrate the in vivo efficacy of CV-N gel in a vaginal challenge model by exposing CV-N-treated female macaques (Macaca fascicularis) to a pathogenic chimeric SIV/HIV-1 virus, SHIV89.6P. All of the placebo-treated and untreated control macaques (8 of 8) became infected. In contrast, 15 of 18 CV-N-treated macaques showed no evidence of SHIV infection. Further, CV-N produced no cytotoxic or clinical adverse effects in either the in vitro or in vivo model systems. Together these studies suggest that CV-N is a good candidate for testing in humans as an anti-HIV topical microbicide.

Lipid-drug association enhanced HIV-1 protease inhibitor indinavir localization in lymphoid tissues and viral load reduction: A proof of concept study in HIV-2287-infected macaques

Analysis of indinavir levels in HIV-positive patients indicated that drug concentrations in lymph node mononuclear cells (LNMCs) were about 25–35% of mononuclear cells in blood. To enhance lymphatic delivery of anti-HIV drugs, a novel drug delivery strategy was designed consisting of lipid-associated indinavir (50–80 nm in diameter) complexes in suspension for subcutaneous (SC) injection. Due to the pH-dependent lipophilicity of indinavir, practically all the drug molecules are incorporated into lipid phase when formulated at pH 7.4 and 5:1 lipid-to-drug (m/m) ratio. At pH 5.5, about 20% of drugs were found in lipid–drug complexes. Effects of lipid association on the time course of plasma indinavir concentrations were determined in macaques (Macaca nemestrina) administered with either soluble or lipid-associated formulation of indinavir (10 mg/kg, SC). Results yielded about a 10-fold reduction in peak plasma concentration and a 6-fold enhancement in terminal half-life (t1/2&bgr; = 12 vs. 2 hours). In addition, indinavir concentrations in both peripheral and visceral lymph nodes were 250–2270% higher than plasma (compared with <35% with soluble lipid-free drug administration in humans). Administration of lipid-associated indinavir (20 mg/kg daily) to HIV-2287–infected macaques (at 30–33 weeks after infection) resulted in significantly reduced viral RNA load and increased CD4 T cell number concentrations. Collectively, these data indicate that lipid association greatly enhances delivery of the anti-HIV drug indinavir to lymph nodes at levels that cannot be achieved with soluble drug, provides significant virus load reduction, and could potentially reverse CD4 T cell depletion due to HIV infection.

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.

Infection of Macaca nemestrina neonates with HIV-1 via different routes of inoculation

Objectives:Receptive anal intercourse but not orogenital sex has been identified as a major risk factor for transmission of HIV-1. Recent studies using simian immunodeficiency virus (SIV) in rhesus macaques have demonstrated relatively efficient infection following oral administration, indicating that modes of transmission may vary between HIV-1 and SIV. Here, we investigate whether HIV-1 infection of macaques via the oral route is more efficient than via the rectal route. Design:Eleven Macaca nemestrina neonates were exposed to HIV-1 via different routes (four oral, two intravenous, and five rectal). One animal was orally inoculated with a sham inoculum and two control animals were not exposed. Methods:All animals were followed for virological signs of infection, and for pathogenesis associated with HIV-1 infection by general physical examinations, complete blood cell counts and lymphocyte subset analysis, and full necropsies. Results:Three out of five rectally exposed macaques and both of the intravenously inoculated animals became infected with HIV-1, whereas none of the orally exposed animals showed evidence of HIV-1 infection. Clinical observations following exposure included failure to thrive in the orally inoculated animals and low CD4/CD8 ratios in the rectally exposed macaques. Conclusions:The finding that, contrary to what has been reported for SIV, transmission of HIV-1 via the oral route is not more efficient than via the rectal route, indicates important biological differences between HIV-1 and SIV, with direct implications for the spread of HIV and associated AIDS, and for development of anti-HIV-1 vaccines.

Activation in vivo of retroperitoneal fibromatosis-associated herpesvirus, a simian homologue of human herpesvirus-8.

Retroperitoneal fibromatosis-associated herpesvirus of rhesus macaques (RFHVMm) is a gammaherpesvirus closely related to human herpesvirus-8 (HHV-8), which is thought to be a necessary cofactor for the development of Kaposis sarcoma (KS) in humans. Here, RFHVMm infection of rhesus macaques exposed to the D-type retrovirus simian retrovirus-2 (SRV-2) is described. Development of SRV-2 viraemia, infection with simian immunodeficiency virus or administration of cyclosporin A could result in persistent RFHVMm viraemia. From this, it is concluded that productive retrovirus infection or otherwise-induced immune suppression has the ability to activate this herpesvirus in vivo. Elevated levels of circulating interleukin-6, a cytokine that plays a central role in KS, were found in RFHVMm-viraemic animals. In viraemic animals, RFHVMm was found in tissues that are common sites for the development of AIDS-associated KS, especially the oral cavity. Together, these data suggest a common biology between RFHVMm infection of macaques and HHV-8 infection and pathogenesis in humans.

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 1 week 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.

Enhanced replication of HIV-1 in vivo in pigtailed macaques (Macaca nemestrina)

Non‐human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV‐1) and animals that are readily available for research. HIV‐1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow‐up period. These data suggest that a useful non‐human primate model for AIDS can be attained in pigtailed macaques (M. nemestrina).

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.

Suppression of maternal virus load with zidovudine, didanosine, and indinavir combination therapy prevents mother-to-fetus HIV transmission in macaques

&NA;Recently, we developed a maternal‐fetal macaque model using a highly pathogenic HIV‐2 strain, HIV‐2287, to study the time course of HIV transmission in utero. Most pregnant macaques (Macaca nemestrina) infected with HIV‐2287 (10‐103 infective doses) transmitted HIV to their fetuses, as verified by positive identification of virus‐infected mononuclear cells and free viral RNA in fetal blood. To determine whether an antiretroviral drug combination therapy composed of two dideoxynucleosides, azidothymidine (15 mg/kg) and dideoxyinosine (15 mg/kg), and a protease inhibitor, indinavir (25 mg/kg), could completely inhibit mother‐to‐fetus HIV transmission, we administered these drugs orally through gastric catheters to five pregnant macaques infected with 10 infective doses of HIV‐2287. Beginning 30 minutes after HIV inoculation, the dams were given the combination antiviral therapy three times daily until delivery by cesarean section. Drug treatment reduced the maternal virus load to a minimally detectable level but did not prevent primary HIV‐2287 infection. All fetal and infant blood samples were virus negative by internally controlled RNA polymerase chain reaction (QC‐RNA‐PCR) and virus coculture assays. Fetal and infant CD4+ T‐cell levels remained normal throughout the experiment. These findings strongly suggest that combination chemotherapy with azidothymidine, dideoxyinosine, and indinavir can suppress maternal viral load enough to prevent mother‐to‐fetus transmission of HIV.