Rational vaccine development and evaluation requires identifying and measuring the magnitude of epitope-specific CD8 T cell responses. However, conventional CD8 T cell epitope discovery methods are labor intensive and do not scale well. In this study, we accelerate this process by using an ultradense peptide array as a high-throughput tool for screening peptides to identify putative novel epitopes. In a single experiment, we directly assess the binding of four common Indian rhesus macaque MHC class I molecules (Mamu-A1*001, -A1*002, -B*008, and -B*017) to ∼61,000 8-mer, 9-mer, and 10-mer peptides derived from the full proteomes of 82 SIV and simian HIV isolates. Many epitope-specific CD8 T cell responses restricted by these four MHC molecules have already been identified in SIVmac239, providing an ideal dataset for validating the array; up to 64% of these known epitopes are found in the top 192 SIVmac239 peptides with the most intense MHC binding signals in our experiment. To assess whether the peptide array identified putative novel CD8 T cell epitopes, we validated the method by IFN-γ ELISPOT assay and found three novel peptides that induced CD8 T cell responses in at least two Mamu-A1*001–positive animals; two of these were validated by ex vivo tetramer staining. This high-throughput identification of peptides that bind class I MHC will enable more efficient CD8 T cell response profiling for vaccine development, particularly for pathogens with complex proteomes for which few epitope-specific responses have been defined.

For more than three decades it has been recognized that small amounts of vector saliva can significantly alter the infectivity of vector-borne pathogens and subsequent in vivo dynamics. Mouse and nonhuman primate models now serve as useful platforms to study Zika virus (ZIKV) pathogenesis, candidate therapies, and vaccines, but they rely on needle inoculation of virus: the effects of mosquito-borne infection on disease outcome have not been explored in these models. To model vector-borne transmission of ZIKV in nonhuman primates, we infected Aedes aegypti mosquitoes with ZIKV and allowed them to feed on four ZIKV-naive rhesus macaques. We compared ZIKV replication kinetics and tissue distribution between animals that were subcutaneously inoculated with 104 plaque-forming units of ZIKV and those that were exposed via mosquito bite. Here, we show that infection via mosquito bite delays ZIKV replication to peak viral loads in rhesus macaques. Importantly, in mosquito-infected animals ZIKV tissue distribution was limited to hemolymphatic tissues, female reproductive tract tissues, kidney, and liver, potentially emulating key features of human ZIKV infections, most of which are characterized by mild or asymptomatic disease. This newly developed system will be valuable for studying ZIKV disease because it more closely mimics human infection by mosquito bite than needle-based inoculations.

31Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We 32 infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The 33 pregnancy was complicated by preterm premature rupture of membranes (PPROM) and fetal 34 demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal 35 interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the 36 myometrial radial arteries. ZIKV RNA was disseminated throughout the fetus tissues and 37 maternal immune system at necropsy, as assessed by quantitative RT-PCR for viral RNA. 38Replicating ZIKV was identified in fetal tissues, maternal lymph node, and maternal spleen by 39 fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included 40 a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is 41 the first report of ocular pathology and prolonged viral replication in both maternal and fetal 42 tissues following congenital ZIKV infection in rhesus macaques. PPROM followed by fetal 43 demise and severe pathology of the visual system have not been described in macaque 44 congenital infection previously; further nonhuman primate studies are needed to determine if an 45 increased risk for PPROM is associated with congenital Zika virus infection. 46 47 Author summary 48 A ZIKV infection during pregnancy is associated with malformations in fetal development 49including, but not limited to, ocular and brain anomalies, such as microcephaly, and stillbirth. The 50 3 development of an accurate pregnancy model to study the effects of ZIKV will provide insight into 51 vertical transmission, ZIKV tissue distribution, and fetal injury and malformations. Non-human 52 primates closely resemble human in terms of the reproductive system, immunity, placentation 53 and pregnancy. Our study demonstrates that the rhesus macaque is a compelling model in which 54 to study ZIKV during pregnancy due to similar outcomes between the human and rhesus 55 macaque. These similarities include prolonged viremia, vertical transmission, adverse pregnancy 56 outcomes and fetal pathology, including defects in the visual system. 57 58