Localization of infection: neonatal rhesus macaques after oral viral challenge

Introduction: HIV+ mothers who are not on antiretroviral therapy have up to a 40% chance of passing HIV onto their children. This results in 150,000-200,000 cases of mother-to-child HIV transmission every year. Pediatric HIV transmission occurs most commonly through breast milk. The site of viral entry in breastfed infants is unknown. The overall goal of our study is to identify which tissues in the oral mucosa and gastrointestinal (GI) tract are susceptible to viral infection and to identify the initial viral target cells. Additionally, our study examines whether the passive transfer of antibodies to children through breast milk affects viral transmission. Methods: Two rhesus macaques less than one week of age were orally challenged with a mixture of a non-replicating reporter virus (LiCh) and replication-competent virus SHIV1157ipd3N4, which encodes HIV Clade C envelope four times per day for two days. Animals were sacrificed and tissues were harvested at 96 hours after initial challenge. Tissues were examined for foci of LiCh transduced cells by luciferase activity by an In Vivo Imaging System (IVIS). Tissues were then cryosectioned and analyzed by nested PCR and microscopy for further analysis of infection. Serum viral loads were measured by ELISA. Two additional animals were given 600ug/ml non-neutralizing IgG during the oral challenge with LiCh and SHIV1157ipd3N4 to examine possible effects of passively transferred IgG through breast milk. Results: At 96 hours, we were able to identify foci of LiCh transduced cells in the upper GI tract by IVIS. Additionally, mCherry DNA was found in the tongue of both macaques and in the stomach of one macaque. Nested PCR for gag DNA demonstrated that secondary SHIV1157ipd3N4 infection had spread throughout the oral cavity, esophagus, stomach, spleen, liver, and gut of the macaques at 96 hours after challenge. SHIV1157ipd3N4 infected cells were detected in the spleen by microscopy. Ongoing microscopy experiments are phenotyping initial viral target cells by mCherry signal and SHIV1157ipd3N4 infected cells throughout the oral cavity and GI tract. Infected macaques had a viral load about 200,000 copies/ml. Non-neutralizing IgG treatment reduced the viral load at least 10-fold; one animal had a viral load of 10,000 copies/ml, and the second animal had undetectable an viral load. Conclusions: These results at 96 hours and previous work suggest that immune cells within the oral cavity, esophagus, and transformation zone to the stomach are susceptible to primary infection. Additionally, our data suggest that the entire GI tract is highly susceptible to secondary viral infection. Preliminary studies suggest non-neutralizing IgG may slow secondary viral infection. Enrollment of additional animals is currently underway.