Kazuhisa Yoshimura

Persistence of HIV-1 latent reservoir cells during antiretroviral therapy is a major obstacle for curing HIV-1. Latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells; however, there are few useful models for evaluating LRA activity in vitro. Here, we established a long-term cell culture system harboring thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo. A combination of an LRA and antiretroviral therapy (ART) significantly reduced viral rebound upon treatment interruption. Experimental investigation and mathematical modeling demonstrated that addition of LRA to ART induced latency-reversing effect and contributed to the eradication of replication competent HIV-1. The widely distributed intact provirus elimination (WIPE) assay will be useful for optimizing therapeutics against HIV-1 latency and investigating mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.

Introduction Human immunodeficiency virus type-1 (HIV-1) is responsible for the AIDS pandemic. HIV-1 exhibits extremely high levels of genetic diversity (Rambaut et al., 2004) indicating that rapidly changing genetic variation can confer on the virus the capacity to escape the immune system and anti-retroviral therapy (ART). The HIV-1 components presenting the highest degree of sequence diversity are the surface-expressed viral envelope glycoproteins (Env), which are prime targets for both entry inhibitors and neutralizing antibodies (NAbs) (Goulder and Watkins,2008). The function of Env is to facilitate the entry of HIV-1 into the target cell, a process mediated by recognition of the CD4 receptor and coreceptor (usually CCR5 or CXCR4) on the cellular membrane (Dalgleish et al.,1984;Klatzmann et al.,1984;Choe et al.,1996;Deng et al.,1996;Doranz et al.,1996;Dragic et al.,1996;Feng et al.,1996). Env is composed of the surface glycoprotein, gp120, and the transmembrane glycoprotein, gp41, which associate as a non-covalent complex to form a single subunit of a trimeric viral envelope spike (Wyatt and Sodroski, 1998). Gp120 is responsible for binding to CD4 and the coreceptor, whereas gp41 anchors the Env machinery at the viral membrane and induces membrane fusion during viral entry (Freed and Martin,1995;Bazan et al.,1998). Inhibitors that block either the interaction of Env with the CD4 receptor, the coreceptor, or the fusion reaction have been identified. Currently, two entry inhibitors have been licensed for clinical use, the fusion inhibitor, enfuvirtide (T-20) (Robertson,2003), and the CCR5 inhibitor, maraviroc (MVC) (Dorr et al.,2005;Gulick et al.,2008). As with any anti-retroviral drug, HIV can develop resistance to enfuvirtide and MVC. The main mechanism of resistance to enfuvirtide is the selection of changes in a 10 amino acid motif between residues 36 and 45 in the HR1 region of gp41 that forms part of the binding site of enfuvirtide (Greenberg and Cammack,2004). On the other hand, clinical resistance to MVC involves different genetic alterations in env giving rise to highly divergent Env phenotypes (Roche et al.,2013). Potential molecular mechanisms of resistance to MVC include tropism switching to CXCR4-using (X4) viruses (Westby et al.,2006;Raymond et al.,2015), increased kinetics of the entry step (Reeves et al., 2002; Putcharoen et

CD4 mimics are small molecules that inhibit the protein–protein interaction between gp120 and CD4, which is a key interaction for the entry of human immunodeficiency virus (HIV) into host immune cells. In the present study, mono‐cyclohexyl‐type CD4 mimics were designed to form hydrophobic and electrostatic interactions with Val430 and Asp368 located in the entrance of the Phe43 cavity of gp120, the interaction site of CD4. YIR‐329, a novel 1‐azaspiro[5.5]undecane derivative with a cyclohexyl ring attached to the piperidine ring, exhibited only slightly weaker anti‐HIV activity than a previously described lead HAR‐171, and modeling results indicated the formation of advantageous interactions by the para‐chlorophenyl moiety of YIR‐329. To introduce an electrostatic interaction with Asp368, derivatives with a guanidino group on the piperidine nitrogen atom were synthesized. Mono‐cyclohexyl‐type CD4 mimics with a guanidino group, such as YIR‐819 (N¹‐(4‐chlorophenyl)‐N²‐(1‐(2‐(N‐(amidino)glycinamide)ethyl)‐2‐cyclohexylpiperidin‐4‐yl)oxalamide) and YIR‐821 (1‐(2‐(5‐guanidinovaleramide)ethyl derivative of YIR‐819), were identified that exhibit approximately fivefold more potent anti‐HIV activity than YIR‐329. In combination with a neutralizing antibody, their anti‐HIV activities were augmenting. Modeling results suggest that these compounds interact effectively with Val430 and either Asp368 or Asp474 in the gp120 Phe43 cavity. YIR‐819 and YIR‐821 represent useful lead compounds for the further development of HIV‐1 entry inhibitors and could potentially be useful for co‐administration with neutralizing antibodies for the treatment of HIV infection and AIDS.

The initiation of drug therapy results in a reduction in the human immunodeficiency virus type 1 (HIV-1) population, which represents a potential genetic bottleneck. The effect of this drug-induced genetic bottleneck on the population dynamics of the envelope (Env) regions has been addressed in several in vivo studies. However, it is difficult to investigate the effect on the env gene of the genetic bottleneck induced not only by entry inhibitors but also by non-entry inhibitors, particularly in vivo. Therefore, this study used an in vitro selection system using unique bulk primary isolates established in the laboratory to observe the effects of the antiretroviral drug-induced bottleneck on the integrase and env genes. Env diversity was decreased significantly in one primary isolate [KP-1, harbouring both CXCR4 (X4)- and CCR5 (R5)-tropic variants] when passaged in the presence or absence of raltegravir (RAL) during in vitro selection. Furthermore, the RAL-selected KP-1 variant had a completely different Env sequence from that in the passage control (particularly evident in the gp120, V1/V2 and V4-loop regions), and a different number of potential N-glycosylation sites. A similar pattern was also observed in other primary isolates when using different classes of drugs. This is the first study to explore the influence of anti-HIV drugs on bottlenecks in bulk primary HIV isolates with highly diverse Env sequences using in vitro selection.

The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1) infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of nonhuman primates with simian immunodeficiency virus (SIV) is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb) B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7900-fold, 1000-fold, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.