1B), and a gradual loss of total circulating CD4+T cells occurred in most animals (Fig

1B), and a gradual loss of total circulating CD4+T cells occurred in most animals (Fig. 4151 targeted more neutralization-resistant HIV-1 isolates. These results Isatoribine indicate that this SHIVAD8macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains. A major challenge in HIV vaccine research has been the development of immunogens capable of eliciting potent, broadly acting, neutralizing antibodies (NAbs). It is now appreciated that 1030% of HIV-1infected individuals produce cross-reactive NAbs of significant breadth (16). Less than 1% of such persons, so-called elite neutralizers, produce potent cross-cladeneutralizing activity, but only 23 y after virus acquisition (6). Although the emergence of broadly reacting antiHIV-1 NAbs in elite neutralizers has been associated with multiple rounds of somatic hypermutation (7), little is known about vaccine strategies able to elicit such antibodies. Longitudinal studies of HIV-1 infected persons have suggested that set-point plasma virus loads, CD4+T-cell levels, duration Rabbit Polyclonal to SNX4 of the infection, or antibody-binding avidity may contribute to the development of cross-reacting NAbs (5,8,9). It is also possible that this induction of such antibodies depends on unique gp120 epitopes associated with specific HIV-1 strains and individual B-cell repertoires or is simply a random process (10,11). A nonhuman primate model capable of generating cross-reactive antiHIV-1neutralizing activity could provide answers to some of these questions and contribute to the development of an effective prophylactic vaccine. In this regard, we recently reported that one rhesus monkey, inoculated with an uncloned preparation of the R5-tropic SHIVAD8, developed broad, potent, and glycan-specific NAbs with cross-reacting activity against virus isolates from different HIV-1 clades comparable to that described for elite HIV-1 neutralizers (12). In this study, we describe the construction of a pathogenic SHIVAD8molecular clone (SHIVAD8-EO). During its characterization, we discovered that eight of eight SHIVAD8-EOinfected animals generated cross-reactive NAbs directed against a SHIV carrying an envelope glycoprotein derived from a different HIV-1 isolate (namely HIV-1DH12). Because this result suggested that a majority of monkeys infected with the SHIVAD8-EOfamily of viruses might be able to generate NAbs against heterologous HIV-1 isolates, plasma samples collected from a cohort of 11 rhesus macaques, infected with either uncloned SHIVAD8swarm stocks or SHIVAD8molecular clones, were tested for their capacity to neutralize a panel of clade A, B, and C HIV-1 isolates. All of the plasmas from this group of 11 animals neutralized several tier 1A and 1B clade B HIV-1 strains. Three of the 11 macaques also generated >1:100 IC50neutralization titers against some tier 2 clade B HIV-1 isolates, and one of the three produced significant NAb titers against some clade A and C isolates. Taken together, these findings indicate that SHIVAD8is usually uniquely immunogenic during infections of rhesus monkeys and may be a particularly useful reagent for identifying viral determinants that drive B-cell maturation resulting in cross-reactive antiHIV-1 NAbs. == Results == == Contamination of Rhesus Monkeys with the SHIVAD8-EOMolecular Clone. == Although Isatoribine we had previously reported the construction and characterization of the R5-tropic SHIVAD8and had prepared uncloned SHIVAD8swarm stocks as challenge viruses for vaccine experiments (13,14), we had not obtained a pathogenic SHIVAD8molecular clone, capable of durably maintaining chronic virus contamination and inducing clinical immunodeficiency in inoculated animals. This was accomplished, as described inMaterials and Methods, by amplifying SHIVAD8vpuandenvgenes from the plasmas of several SHIVAD8infected monkeys and inserting individual amplicons into the genetic background of SIVmac239 (Figs. S1andS2). One of the resulting clones (SHIVAD8-EO) exhibiting robust replication kinetics in cultured rhesus peripheral blood mononuclear cells (PBMC) was inoculated into Isatoribine 11 animals: 4 by the i.v. (5,000 or 500 TCID50) and 7 by the intrarectal (5,000 or 1,000 TCID50) routes. Similar to uncloned SHIVAD8derivatives (13,14), the levels of set point viremia in macaques infected with the SHIVAD8-EOmolecular clone varied widely (102to >105RNA copies/mL) (Fig. 1A). Memory CD4+T cells at an effector site [recovered by bronchoalveolar lavage (BAL)] declined markedly in all of the monkeys during the acute phase of the SHIVAD8-EOinfection (Fig. 1B), and a gradual loss of total circulating CD4+T cells occurred in most animals (Fig. 1C). Monkey DC0L was euthanized.