Billie B. Davison

A Preliminary Evaluation of Recombinant Adeno-Associated Virus Biodistribution in Rhesus Monkeys After Intrahepatic Inoculation In Utero

The ability to deliver genes to fetuses in utero may prove crucial for those genetic diseases that are associated with severe fetal morbidity and for which there is no effective postnatal therapy. In utero therapy may be especially useful in diseases that affect the central nervous system because the immature blood-brain barrier may facilitate gene delivery to neural target cells. We investigated whether in utero inoculation of recombinant adeno-associated virus (rAAV) into rhesus monkey fetuses would be a useful method of gene delivery, especially to the central nervous system. When the monkeys were sacrificed after birth, we found vector genomes distributed in many tissues, including the brain and peripheral blood. Pericapillary astrocytes expressing transgene products were detected by immunohistochemistry. In addition, we occasionally found vector genomes in the maternal blood. No adverse clinical or pathologic effects were observed in the inoculated monkeys. We concluded that (1) in utero intrahepatic inoculation of rAAV is a potentially safe and useful method of delivering genes to many fetal tissues; (2) astrocytes may be the cell type most easily targeted in the central nervous system (CNS) after systemic administration; and (3) the potential of inadvertent gene transfer to the mother must be considered.

GENOTYPIC ANALYSIS OF INFANT MACAQUES INFECTED TRANSPLACENTALLY AND ORALLY

Abstract: The SIV‐infected macaque provides an excellent model to study factors involved in maternal‐fetal transmission of HIV. In our prenatal transmission studies, female macaques were inoculated intravenously during midgestation with either SIV/DeltaB670 or a combination of SIV/DeltaB670 and the macrophage‐tropic molecular clone SIV/17E‐Fr. The females harbored a genetically diverse virus population at parturition, whereas a single genotype from the maternal quasispecies was identified in the infants. One of two variants was transplacentally transmitted to the infants, SIV/17E‐Fr or B670‐Cl 12, a genotype contained within the SIV/DeltaB670 inoculum. Both of these variants have been identified in the central nervous system of macaques that have developed encephalitis and they replicate in vitro on primary rhesus macrophages. These results suggest a critical role for macrophages in fetal infection in utero. In our perinatal transmission studies we have evaluated the viral genotypes found in two newborn macaques infected orally with SIV/DeltaB670 and in one infant infected via amniotic inoculation in late gestation. More than one viral genotype was identified in each infant, moreover, each infant harbored different genotypes. These results suggest different mechanisms are responsible for viral infection via these routes.

Placental Histopathologic Changes Associated with Subclinical Malaria Infection and Its Impact on the Fetal Environment

Microscopic examination of placental tissue can provide an accurate assessment of malaria infection during pregnancy. In this cross-sectional study of 193 women in Iquitos, Peru, 1.0% and 6.6% had parasites in the peripheral blood as detected by microscopy and polymerase chain reaction, respectively. However, 22% had placental malaria pigment indicating past, subclinical infections. Placental tissues with pigment from 24 cases were matched by gravidity and month of delivery to 24 controls and histopathologically examined. Cases had significantly higher number of monocytes in the intervillous space (44.7 versus 25.5; P = 0.012). Pigmented monocytes in fetal vessels were present in 33.3% of cases. This study demonstrated that subclinical malarial infection occurred frequently in pregnant women and is associated with increased presence of monocytes in the placenta. Pigmented monocytes in fetal vessels suggest parasites can breach the placental barrier and enter the fetal circulation.

The Role of Soluble Tumor Necrosis Factor Receptor Types I and II and Tumor Necrosis Factor–α in Malaria during Pregnancy

In a prospective study of rhesus monkeys inoculated with Plasmodium coatneyi or saline on an infection/gestational timeline, we determined the serum levels of tumor necrosis factor-alpha (TNF-alpha), soluble tumor necrosis factor receptor type I (sTNFR-I), and soluble tumor necrosis factor receptor type II (sTNFR-II) in peripheral blood throughout primigravid pregnancy, malaria infection, and a combination of the two. Our goal was to determine the association between levels of TNF-alpha and of its 2 soluble receptors and the course of pregnancy and/or malaria and infant outcome. We found that any detectable level of TNF-alpha was always associated with fetal death and that the sTNFRs may be important for fetal protection, possibly through neutralizing the toxic effects of TNF-alpha. Our findings also showed that increased levels of sTNFR-II were associated specifically with malaria and not with normal pregnancy or even pregnancy with low birth weight due to other causes. In contrast, increases in sTNFR-I levels during the later half of normal pregnancies indicate that sTNFR-I may be important in regulating TNF-alpha levels in preparation for normal labor and delivery.

Alterations in the Profile of Blood Cell Types during Malaria in Previously Unexposed Primigravid Monkeys

Malaria in nonimmune, primigravid women threatens both mother and fetus. We used the Plasmodium coatneyi/rhesus monkey model to examine factors associated with this. Clinical and immunologic responses during the blood stage of chronic malaria (4 months) were evaluated in 8 malaria-naive primigravid (PMI) and 8 age-matched nulligravid (NMI) infected monkeys, compared with those in 8 primigravid, noninfected control monkeys. Although parasitemia levels were similar, recrudescence was more frequent and prolonged, and anemia was more severe in PMI than in NMI monkeys. During infection, CD2+, CD4+, and CD8+ lymphocyte levels were higher in NMI than in PMI monkeys. Monocyte and neutrophil levels were lower in PMI than in NMI monkeys. During chronic, untreated malaria, NMI monkeys had a B lymphocyte count 23 times greater than that of PMI monkeys. Pregnancy-induced immunomodulation, defined as a lack of appropriate cellular responses to malaria, was indiscernible until the immune system was challenged by a pathogen.

Altered Immune Responses in Rhesus Macaques Co-Infected with SIV and Plasmodium cynomolgi: An Animal Model for Coincident AIDS and Relapsing Malaria

Background Dual epidemics of the malaria parasite Plasmodium and HIV-1 in sub-Saharan Africa and Asia present a significant risk for co-infection in these overlapping endemic regions. Recent studies of HIV/Plasmodium falciparum co-infection have reported significant interactions of these pathogens, including more rapid CD4+ T cell loss, increased viral load, increased immunosuppression, and increased episodes of clinical malaria. Here, we describe a novel rhesus macaque model for co-infection that supports and expands upon findings in human co-infection studies and can be used to identify interactions between these two pathogens. Methodology/Principal Findings Five rhesus macaques were infected with P. cynomolgi and, following three parasite relapses, with SIV. Compared to macaques infected with SIV alone, co-infected animals had, as a group, decreased survival time and more rapid declines in markers for SIV progression, including peripheral CD4+ T cells and CD4+/CD8+ T cell ratios. The naïve CD4+ T cell pool of the co-infected animals was depleted more rapidly than animals infected with SIV alone. The co-infected animals also failed to generate proliferative responses to parasitemia by CD4+ and CD8+ T cells as well as B cells while also having a less robust anti-parasite and altered anti-SIV antibody response. Conclusions/Significance These data suggest that infection with both SIV and Plasmodium enhances SIV-induced disease progression and impairs the anti-Plasmodium immune response. These data support findings in HIV/Plasmodium co-infection studies. This animal model can be used to further define impacts of lentivirus and Plasmodium co-infection and guide public health and therapeutic interventions.

Hematologic and lymphocyte immunophenotypic reference values for normal rhesus monkey (Macaca mulatta) umbilical cord blood; gravidity may play a role in study design

Abstract:  Hematology and flow cytometry reference values for rhesus umbilical cord blood (UCB) were established in 17 healthy infant rhesus monkeys delivered by elective cesarean section 10 days preterm. The infants were born to age matched, singly caged primigravid or secundigravid dams. The hematology and flow cytometry values were determined by automated cell counter and by FACS. No significant differences were observed with respect to infant gender. With respect to gravida, the primigravid infants had a significantly higher percentage (P= 0.05) of CD20+ B lymphocytes in UCB.