Unique V3 loop sequence derived from the R2 strain of HIV-type 1 elicits broad neutralizing antibodies.

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies. In this study, DNA vaccines were constructed to express the gp120 subunit of Env from the isolate HIV-1(R2) using both wild-type and codon-optimized gene sequences. Three copies of the murine C3d were added to the carboxyl terminus to enhance the immunogenicity of the expressed fusion protein. Mice (BALB/c) vaccinated with DNA plasmid expressing the gp120(R2) using codon-optimized Env sequences elicited high-titer anti-Env antibodies regardless of conjugation to C3d. In contrast, only mice vaccinated with DNA using wild-type gp120(R2) sequences fused to mC3d(3), had detectable anti-Env antibodies. Interestingly, mice vaccinated with DNA expressing gp120(R2) from codon-optimized sequences elicited antibodies that neutralized both homologous and heterologous HIV-1 isolates. To determine if the unique sequence found in the crown of the V3 loop of the Env(R2) was responsible for the elicitation of the cross-clade neutralizing antibodies, the codons encoding for the Pro-Met (amino acids 313-314) were introduced into the sequences encoding the gp120(ADA) (R5) or gp120(89.6) (R5X4). Mice vaccinated with gp120(ADA)-mC3d(3)-DNA with the Pro-Met mutation had antibodies that neutralized HIV-1 infection, but not the gp120(89.6)-mC3d(3)-DNA. Therefore, the use of the unique sequences in the Env(R2) introduced into an R5 tropic envelope, in conjunction with C3d fusion, was effective at broadening the number of viruses that could be neutralized. However, the introduction of this same sequence into an R5X4-tropic envelope was ineffective in eliciting improved cross-clade neutralizing antibodies.

Multiple residues in the extracellular domains of CCR3 are critical for coreceptor activity.

Human immunodeficiency virus type 1 (HIV-1) binds to the human CD4 (hCD4) and a coreceptor to enter permissive human cells. The chemokine receptors, hCCR5 and hCXCR4, are the primary coreceptors used by HIV-1 isolates in vivo, however, hCCR3 has been implicated as a coreceptor for HIV infection of the central nervous system. To determine the domains and amino acids important in hCCR3 coreceptor activity, chimeras between the permissive hCCR3 and the non-permissive rhesus macaque CCR3 (RhCCR3) were constructed and assessed for coreceptor activity for two R5 strains of HIV-1 (YU-2 and ADA) and one R5X4 strain (89.6). Even though three extracellular domains of CCR3 participated in coreceptor activity for the two R5 isolates (ECD-1, ECD-3, and ECD-4), for the R5X4 isolate, ECD-4, and to a lesser extent ECD-3, were critical for coreceptor activity. In addition, residues 13 and 20 in ECD-1, residue 179 in ECD-3, and residue in 271 in ECD-4 of CCR3 were identified for HIV-1 envelope-mediated entry for R5 isolates. In contrast, all the residues on ECD-4 appeared necessary for coreceptor activity for HIV-1(89.6). Therefore, multiple residues on multiple extracellular domains of hCCR3 are important for coreceptor activity for HIV-1.