The identification of regions of HIV-1 Env that are targeted by NAbs and an understanding of the immunogenicity of these regions in the setting of infection may guide the development of vaccine immunogens that elicit a protective humoral immune response. The SIV-infected rhesus monkey model of AIDS provides an important system for dissecting the targets of NAbs and assessing the evolution of the humoral immune response following vaccination and/or infection. ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early Benzenesulfonamide NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that thisenvvaccination regimen did not alter the kinetics or the breadth of NAbs during early contamination. == INTRODUCTION == Passive immunization studies in nonhuman primates and correlates of protection studies in both nonhuman primates and human vaccinees have exhibited that antibodies can contribute to preventing the acquisition of simian immunodeficiency computer virus (SIV) and human immunodeficiency computer virus type 1 (HIV-1) (1,10,1114,25,27,28,33,35,37,41). Therefore, considerable efforts are being directed toward identifying HIV-1 immunogens that elicit broadly neutralizing antibody (NAb) responses. The identification of regions of HIV-1 Env that are targeted by NAbs and an understanding of the immunogenicity of these regions in the setting of contamination may guideline the development of vaccine immunogens that elicit a protective humoral immune response. The SIV-infected rhesus monkey model of AIDS provides an important system for dissecting the targets of NAbs and assessing the evolution of the humoral immune response following vaccination and/or contamination. The emergence of variants of SIV that escape recognition by NAbs has been well documented in SIV-infected rhesus monkeys (4,5,31,32,38,40,45). Several NAb epitopes have been previously identified in the V1/V2 and V4 regions of Benzenesulfonamide SIV Env (2,6,9,1518,20,34,39,40). In addition, we have recently exhibited that mutations in variable regions 1 and 4 of SIVmac251 Env are responsible for the escape from recognition by NAbs that develop following mucosal contamination (3,45). However, the precise early neutralization antibody determinants during acute SIV infection have not been defined. The identification of epitopes that play a role in inducing protective immunity early in contamination is crucial for AIDS vaccine development. The primary objective for the present study was to identify the principal neutralization determinants of SIVmac251 during early contamination and assess whether prior vaccination with an Env immunogen altered the kinetics and specificity of the humoral immune response. Based on a previous study of longitudinalenvsequence analysis in rhesus monkeys that were mucosally infected with SIVmac251 (45), we hypothesized that the initial neutralizing antibody response against SIV is usually directed against the variable region 4 of Env. Furthermore, we hypothesized that prior immunization with an SIVmac Env immunogen alters the early neutralizing antibody kinetics and specificities that develop following infection. To test these hypotheses, we utilized a pseudovirion-based, TZM-bl reporter gene neutralization assay to characterize the early neutralizing antibody responses in a cohort of monkeys that were immunized with vaccine regimens that either did or did not include SIVmacenv. To elucidate the targets of the early NAb responses, we constructed a panel of SIVmac251 Env pseudoviruses that have specific site-directed mutations in Env, and we used these mutant viruses to probe sera from vaccinated monkeys infected with SIVmac251. == MATERIALS AND METHODS == == Monkeys. == Outbred, adult male Indian-origin rhesus monkeys were housed at an AAALAC-accredited institution (Bioqual, Rockville, MD). All procedures were performed with full protocol approvals from the Institutional Animal Care and Use Committee for Vaccine Research Center, NIAID, NIH, and in accordance with theGuide for the Care and Use of Laboratory Animals(30). All animal studies were approved by the Vaccine Research Center Animal Care and Use Committees. == Immunization and viral challenge. == Eight of 16 monkeys were part of a cohort that has been previously published (24). Eight monkeys were immunized with a plasmid DNA construct carrying SIVmac239gag/pol/nefon a schedule of 0, 4, and 8 weeks at a dose of 4 mg/plasmid/inoculation, followed Benzenesulfonamide by intramuscular PLCG2 immunization with a recombinant adenovirus 5 (rAd5) vector carryinggag/polat a dose of 1011particles at week 40. The 8 monkeys in theenvarm of the study received the same plasmid DNA construct carrying SIVmac239gag/pol/nefand an additional plasmid DNA construct carrying SIVmac239envgp140CFI (7) on the same 0-, 4-, and 8-week schedule with 4 mg/plasmid/inoculation. At week 40, the latter group was inoculated with one rAd5 vector carrying SIVmac239gag/poland another carrying SIVmac239envpg140 CFI, each at a dose of 1011particles. Following immunization, monkeys in both arms of the study were.