AN ELITE NEUTRALIZER MONKEY DEVELOPS RESISTANT VIRAL VARIANTS. Neutralization resistant SHIVAD8 variants that emerged in an infected macaque elite neutralizer targeting the HIV 1 gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV 1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan. ANTIBODY MEDIATED IMMUNOTHERAPY OF MACAQUES CHRONICALLY INFECTED WITH SHIV SUPPRESSES VIREMIA. Neutralizing antibodies (NAbs) can confer immunity to primate lentiviruses by blocking infection in macaque models of AID. However, earlier studies of anti-HIV 1 NAbs administered to infected individuals or humanized mice, reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV 1 monoclonal antibodies (mAbs), possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These NAbs target different regions of the HIV 1 envelope glycoprotein including the CD4 binding site (bs), glycans located in the V1/V2, V3, and V4 regions, and the membrane proximal external region of gp41. We have examined two of the new antibodies, directed to the CD4 bs and the V3 region (3BNC117 and 10-1074 respectively) for their ability to block infection and suppress viremia in macaques infected with the R5 tropic SHIVAD8 virus, which emulates many of the pathogenic and immunogenic properties of HIV 1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected monkeys, the 10-1074 antibody caused a rapid decline in virus loads to undetectable levels for 4 to 7 days, followed by virus rebound during which neutralization resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viremia for 3 to 5 weeks in some long-term chronically SHIV infected animals with low CD4+ T cell levels. A second cycle of anti-HIV 1 mAb therapy, administered to two previously treated animals, successfully controlled virus rebound. These results suggest that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1 infected individuals experiencing immune dysfunction. THE PRIMARY ROLE OF VPX IN SIV INFECTED MACAQUES IS TO SUPPRESS SAMHD1 IN CD4+ MEMORY T CELLS. The Vpx accessory protein is encoded by HIV 2, related SIVsm strains, SIVmnd, and SIVrcm. Vpx has been reported to antagonize restriction imposed by Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in cells of myeloid lineage (dendritic cells, monocytes, and macrophages) and in quiescent CD4+ T cells 1-4. Previous studies have shown that SIVmac239 Vpx deletion mutants exhibit an attenuated replication phenotype in inoculated macaques 5,6. Thus, it remains to be determined whether endogenous SAMHD1 must also be suppressed in memory CD4+ T cells to maintain the high levels of set-point viremia attending pathogenic infection in vivo. Here we show that SIVmac Vpx defective viruses were deficient in degrading SAMHD1 in cultured CD4+ T lymphocytes and exhibited an attenuated replication phenotype during infections of ConA activated rhesus blood lymphocytes. In monkeys inoculated with wt SIVmac239, levels of endogenous SAMHD1 were markedly reduced in memory CD4+ T cells collected on day 9 PI compared to the levels measured in uninfected animals. When macaques were inoculated by the mucosal route with the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying the Gln76Ala Vpx point mutation, which affects DCAF1/SAMHD1 interaction, virus acquisition, progeny virion production during the acute infection, and the maintenance of set point viremia were greatly attenuated. Revertant viruses emerging in animals carried either an Ala76Ser substitution, at the original site of the Vpx mutation, or an Ile32Thr second site change, a likely contact point of Vpx with the c-terminal domain of DCAF1. The viral revertants exhibited an augmented replication phenotype both in vitro and in vivo and were able to degrade SAMHD1. Taken together, these results indicate that SAMHD1 restricts SIV replication in T lymphocytes and Vpx counteracts this effect by degrading endogenous SAMHD1 in memory CD4 T cells to optimize viral infections in vivo. ENV-SPECIFIC GERMINAL CENTER REACTIONS AND FOUNDER VIRUS CO-EVOLVE DURING THE GENERATION OF BROAD NEUTRALIZING ANTIBODIES IN SHIV-AD8 INFECTION. Broadly neutralizing antibodies (bNAbs) protect against human immunodeficiency virus type 1 (HIV-1) infection, yet how they are generated during chronic infection remains unclear. Here, we investigated the relationship between the virus, envelope-specific TFH cells, envelope-specific B cells, and the generation of bNAbs during SHIVAD8 infection. We found that the frequency of Env-specific TFH cells and Env-specific IgG+ Germinal Center (GC) B cells correlated with broad plasma neutralization. The Env-specific TFH cells in monkeys with good neutralization had a gene expression profile skewed towards TFH, and away from TH1 or T follicular regulatory cells. Broader antibody neutralization was also associated with greater affinity maturation in bone marrow Env-specific IgG+ memory B cells, high viral loads and evolution of transmitted founder Env sequences. These results demonstrate the importance of immunological and virological co-evolution for the generation of bNAbs, in particular the contribution of antigen-specific TFH and GC B cell reactions.
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