Previously we demonstrated that fetal alcohol exposure attenuates hypoxic cerebral vasodilation in fetal and neonatal sheep. One mechanism may be altered expression of brain vasoactive substances. We hypothesized that early fetal alcohol exposure alters the number of fetal neurons expressing vasoactive intestinal peptide (VIP), a potent cerebral vasodilator. Thirteen pregnant ewes received daily i.v. infusions of alcohol (1.5 g/kg) or saline on days 30-54 of gestation (term = 145 days). Fourteen fetal brains (6 alcohol-exposed, 8 saline control) were obtained on gestational day 126. Using unbiased stereology, we counted immunohistochemically-labeled VIP neurons in one half of each forebrain with an optical fractionator. The total NeuN-labeled neurons were similarly counted. Alcohol-exposed fetal sheep brains had fewer VIP-immunopositive neurons per hemisphere, 14.6 x 10(6), compared to saline controls, 19.8 x 10(6). The total neuron number was not different, 1.19 x 10(9) versus 1.23 x 10(9) respectively, indicating a selective decrease in VIP neurons as a result of alcohol exposure. In sheep, alcohol exposure early in gestation is associated with fewer VIP-producing neurons later in gestation compared to saline controls; therefore, alcohol-related changes in the number of VIP-expressing neurons may be responsible in part for the attenuated hypoxic cerebral vasodilation described in fetal and neonatal sheep exposed to alcohol earlier in gestation.

We report the assessment and validation of an NS1 epitope-blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to West Nile virus (WNV) in macaques. Sera from naturally infected Macaca nemestrina were tested by ELISA and plaque reduction neutralization test (PRNT). Results were correlated with hemagglutination inhibition (HAI) data. Our results demonstrate that the blocking ELISA rapidly and specifically detects WNV infection in M. nemestrina. In addition, the diagnostic value of 7 commercially available immunoassays (PanBio immunoglobulin [Ig] M ELISA, PanBio IgG ELISA, PanBio immunofluorescence assay (IFA), InBios IgG ELISA, InBios IgM ELISA, Focus Diagnostics IgG ELISA, and Focus Diagnostics IgM ELISA) in M. nemestrina was evaluated and compared with that of the epitope-blocking ELISA. The PanBio IgG ELISA was found to effectively diagnose WNV exposure in M. nemestrina. Further, PanBio IFA slides are fast and reliable screening tools for diagnosing flaviviral exposure in M. nemestrina.

Our goal was to determine if a multiplex technique using a fluorescent bead-based flow cytometric assay could yield results comparable to traditional enzyme-linked immunosorbent assay (ELISA) in terms of sensitivity, specificity, cross-reactivity, and throughput. We applied both techniques to serologic screening of specific pathogen-free macaques, for type D simian retrovirus, simian T-lymphotropic virus, Cercopithicine herpesvirus 1, and simian immunodeficiency virus, and found a high correlation between the bead-based multiplex assay and ELISA. The multiplex assay demonstrated greater sensitivity with no loss in specificity when compared to the ELISA. A lower false-positive rate with the multiplex assay decreased the number of confirmatory Western blots required. Using the multiplex assay, we were able to screen samples for 4 viruses simultaneously in the time it took to perform a single-virus ELISA, resulting in a faster turnaround time and higher throughput. The multiplexed assay provided greater sensitivity, increased stability, and better performance than ELISA.

We evaluated four priming-boosting vaccine regimens for the highly pathogenic simian human immunodeficiency virus SHIV89.6P in Macaca nemestrina. Each regimen included gene gun delivery of a DNA vaccine expressing all SHIV89.6 genes plus Env gp160 of SHIV89.6P. Additional components were two recombinant vaccinia viruses, expressing SHIV89.6 Gag-Pol or Env gp160, and inactivated SHIV89.6 virus. We compared (i) DNA priming/DNA boosting, (ii) DNA priming/inactivated virus boosting, (iii) DNA priming/vaccinia virus boosting, and (iv) vaccinia virus priming/DNA boosting versus sham vaccines in groups of 6 macaques. Prechallenge antibody responses to Env and Gag were strongest in the groups that received vaccinia virus priming or boosting. Cellular immunity to SHIV89.6 peptides was measured by enzyme-linked immunospot assay; strong responses to Gag and Env were found in 9 of 12 vaccinia virus vaccinees and 1 of 6 DNA-primed/inactivated-virus-boosted animals. Vaccinated macaques were challenged intrarectally with 50 50% animal infectious doses of SHIV89.6P 3 weeks after the last immunization. All animals became infected. Five of six DNA-vaccinated and 5 of 6 DNA-primed/particle-boosted animals, as well as all 6 controls, experienced severe CD4(+)-T-cell loss in the first 3 weeks after infection. In contrast, DNA priming/vaccinia virus boosting and vaccinia virus priming/DNA boosting vaccines both protected animals from disease: 11 of 12 macaques had no loss of CD4(+) T cells or moderate declines. Virus loads in plasma at the set point were significantly lower in vaccinia virus-primed/DNA-boosted animals versus controls (P = 0.03). We conclude that multigene vaccines delivered by a combination of vaccinia virus and gene gun-delivered DNA were effective against SHIV89.6P viral challenge in M. nemestrina.

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.

We assessed four prime‐boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using β‐galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six‐plasmid vaccine capable of producing non‐infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.

We determined if the genetic adjuvants, granulocyte-macrophage colony stimulating factor (GM-CSF) and B7-2, could improve the immunogenicity and efficacy of an HIV-2 DNA vaccine. The vaccine consisted of the HIV-2 tat, nef, gag, and env genes synthesized using optimized codons and formulated with cationic liposomes. Baboons (Papio cynocephalus hamadryas) were immunized by the intramuscular, intradermal, and intranasal routes with these expression constructs and challenged with HIV-2UC2 by the intravaginal route. In the first month after HIV-2 vaginal challenge, the baboons receiving the HIV-2 DNA vaccine with or without the genetic adjuvants had significant reductions in the viral loads in the peripheral blood mononuclear cells (PBMC) (P=0.028) while the reductions in their plasma viremia were suggestive of a protective effect (P=0.1). These data demonstrate that partial protection against HIV-2 vaginal challenge, as measured by reduced viral load, can be achieved using only a DNA vaccine formulation.

A model of vertical HIV transmission was developed using oral HIV-2(287) 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-2(287)-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.

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.

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-2(287), 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-2(287) exhibited a high frequency of virus transmission from the mother to fetus.

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.