Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies.

Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWT from which cenicriviroc-resistant strain KK652-67 was obtained was resistant to these NAbs. The V3 region of KK652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other.

Conformational epitope consisting of the V3 and V4 loops as a target for potent and broad neutralization of simian immunodeficiency viruses.

Inducing neutralizing antibodies (NAb) is the key to developing a protective vaccine against human immunodeficiency virus type 1 (HIV-1). To clarify the neutralization mechanism of simian immunodeficiency virus (SIV), we analyzed NAb B404, which showed potent and broad neutralizing activity against various SIV strains. In 4 SIVsmH635FC-infected macaques, B404-like antibodies using the specific VH3 gene with a long complementarity-determining region 3 loop and λ light chain were the major NAbs in terms of the number and neutralizing potency. This biased NAb induction was observed in all 4 SIVsmH635FC-infected macaques but not in 2 macaques infected with a SIV mix, suggesting that induction of B404-like NAbs depended on the inoculated virus. Analysis using Env mutants revealed that the V3 and V4 loops were critical for B404 binding. The reactivity to the B404 epitope on trimeric, but not monomeric, Env was enhanced by CD4 ligation. The B404-resistant variant, which was induced by passages with increasing concentrations of B404, accumulated amino acid substitutions in the C2 region of gp120. Molecular dynamics simulations of the gp120 outer domains indicated that the C2 mutations could effectively alter the structural dynamics of the V3/V4 loops and their neighboring regions. These results suggest that a conformational epitope consisting of the V3 and V4 loops is the target for potent and broad neutralization of SIV. Identifying the new neutralizing epitope, as well as specifying the VH3 gene used for epitope recognition, will help to develop HIV-1 vaccines.

Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail.

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 non-human 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 7,900-, 1,000-, 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.