Faculty Bibliography

We designed synthetic, epitope-focused immunogens that preferentially display individual neutralization epitopes targeted by cross-subtype anti-HIV V3 loop neutralizing monoclonal antibodies (mAbs). Vaccination of rabbits with these immunogens resulted in the elicitation of distinct polyclonal serum Abs that exhibit cross-subtype neutralization specificities mimicking the mAbs that guided the design. Our results prove the principle that a predictable range of epitope-specific polyclonal cross-subtype HIV-1 neutralizing Abs can be intentionally elicited in mammals by vaccination. The precise boundaries of the epitopes and conformational flexibility in the presentation of the epitopes in the immunogen appeared to be important for successful elicitation. This work may serve as a starting point for translating the activities of human broadly neutralizing anti-HIV-1 monoclonal antibodies (bNAbs) into matched immunogens that can contribute to an efficacious HIV-1 vaccine.

The RV144 HIV vaccine trial indicated that gp120 V2 antibodies were associated with lower risk of infection, thus the mapping of V2 epitopes can contribute to the design of an effective HIV vaccine. We solved the crystal structure of human monoclonal antibody (mAb) 2158 that targets a conformational V2 epitope overlapping the alpha4beta7 integrin binding site, and constructed a full-length model of V1V2. Comparison of computational energy stability to experimental ELISA results identified a hydrophobic core, stabilizing the V2 region for optimal 2158 binding, and residues that directly mediate side-chain interactions with mAb 2158. These data define the binding surface recognized by mAb 2158 and offer a structural explanation for why a mismatching mutation at position 181 (I181X) in the V2 loop is associated with a vaccine efficiency of 78% in the RV144 clinical vaccine trial.IMPORTANCE Correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the second variable region (V2) of HIV-1 gp120 was responsible for the modest protection observed in the trial. V2 is a highly variable and immunogenic region and structural information for its antigenic landscape will be important for a rational design of an effective HIV-1 vaccine. Using X-ray crystallography, computational design tools, and mutagenesis assays, we have carried out a detailed and systematic investigation of the epitope recognition of human V2 mAb 2158 and demonstrated that its epitope region overlaps the integrin binding site within V2. In addition, we proposed a structural based mechanism for mismatching of isoleucine at position 181 with increased vaccine efficacy seen in the RV144 vaccine trial.

HIV-1 envelope gp120 is the target for neutralizing antibodies (NAbs) against the virus. Various approaches have been explored to improve immunogenicity of broadly neutralizing epitopes on this antigen with limited success. We previously demonstrated that immunogenicity of gp120 and especially its V3 epitopes was enhanced when gp120 was co-administered as immune-complex vaccines with monoclonal antibodies (mAb) to the CD4-binding site (CD4bs). To define the mechanisms by which immune complexes influence V3 immunogenicity, we compared gp120 complexed with mAbs specific for the C2 region (1006-30), the V2 loop (2158), or the CD4bs (654), and found that the gp120/654 and gp120/2158 complexes elicited anti-V3 NAbs, but the gp120/654 complex was the most effective. gp120 complexed with 654 F(ab')2 was as potent, indicating that V3 immunogenicity is determined by the specificity of the mAb's Fab fragment used to form the complexes. Importantly, the gp120/654 complex not only induced anti-gp120 antibodies (Abs) to higher titers, but also of greater avidity. The Abs were cross-reactive with V3 peptides from most subtype B and some subtype C isolates. Neutralization was detected only against Tier-1 HIV-1 pseudoviruses, while Tier-2 viruses, including the homologous JRFL strain, were not neutralized. However, JRFL produced in the presence of a mannosidase inhibitor was sensitive to anti-V3 NAbs in the immune sera. These results demonstrate that the gp120/654 complex is a potent immunogen for eliciting cross-reactive functional NAbs against V3 epitopes, of which exposure is determined by the specific compositions of glycans shrouding the HIV-1 envelope glycoproteins.

The third variable region (V3) of HIV-1 gp120 plays a key role in viral entry into host cells; thus, it is a potential target for vaccine design. Human monoclonal antibody (mAb) 447-52D is one of the most broadly and potently neutralizing anti-V3 mAbs. We further characterized the 447-52D epitope by determining a high-resolution crystal structure of the Fab fragment in complex with a cyclic V3 and interrogated the antigen-antibody interaction by a combination of site-specific mutagenesis, isothermal titration calorimetry (ITC) and neutralization assays. We found that 447-52D's neutralization capability is correlated with its binding affinity and at 25 degrees C the Gibbs free binding energy is composed of a large enthalpic component and a small favorable entropic component. The large enthalpic contribution is due to (i) an extensive hydrogen bond network, (ii) a pi-cation sandwiching the V3 crown apex residue Arg315, and (iii) a salt bridge between the 447-52D heavy chain residue AspH95 and Arg315. Arg315 is often harbored by clade B viruses; thus, our data explained why 447-52D preferentially neutralizes clade B viruses. Interrogation of the thermodynamic signatures of residues at the antigen binding interface gives key insights into their contributions in the antigen-antibody interaction.