| Summary: | An effective HIV-1 vaccine will likely elicit broadly neutralizing antibodies (bnAbs). However, development of vaccine immunogens that induce bnAbs remains a challenging goal. Understanding the somatic maturation pathways of known broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) may help vaccine immunogen design. All known HIV-1 bnmAbs are highly somatically matured, and the putative germline antibodies of the known HIV-1 bnmAbs lack measurable binding activity to HIV-1 envelope glycoprotein (Env).
Based on these observations, we hypothesize that somatic maturation of known HIV-1 bnmAbs may be initiated by primary immunogens which may not be related to HIV-1 Env; such primary immunogens trigger the somatic maturation of the germline antibodies and induce intermediate antibodies that bind to HIV-1 Env and further mature to bnAbs upon HIV-1 infection or Env vaccination. The main objective of my study is to identify intermediate antibodies of bnmAb b12 in uninfected and infected human individuals, as well as in uninfected rhesus macaques, the model animals for vaccine development.
I constructed two nonimmune cDNA antibody VH1 scFv libraries using the mRNAs isolated from pooled PBMCs of 200 uninfected healthy human individuals and one uninfected rhesus macaque, respectively, and identified 5 and 10 possible b12 intermediate immunoglobulin heavy chain V-segments (IGHVs) from the human and macaque nonimmune libraries, respectively.
454 deep sequencing of the VHs and VLs in the nonimmune and two immune human cDNA Fab libraries confirmed the existence of b12 intermediate IGHVs, but we did not find further maturation of the b12 intermediate IGHVs in HIV-1-infected human individuals. Further sequence analysis revealed the extremely low frequency of the VHs with exactly the same V(D)J recombination as b12, which may explain the lack of further maturation of the intermediate IGHVs of b12 in HIV-1-infected humans.
Characterization of HIV-1 Env trimer may aid in Env-based vaccine immunogen design. Therefore, I investigated the importance of Env variable loops in Env-mediated viral function. A panel of gp160JRFL loop deletion/replacement mutants were constructed and tested. The results indicate that, besides the CD4 binding loop and V3, V1V2 and loop D are also critical for virus entry into permissive cells. Deletion of variable V4 or V5 loop or replacement of V4 or V5 loop with a flexible linker of the same length abolish Env cell surface display, which may result from the conformational changes of the V4 or V5 loop deletion or replacement Env proteins. V4 or V5 deletion or replacement knocks out the CD4 binding site and CD4-induced site on Env, but enhances the exposure of the membrane-proximal external region (MPER) and N-trimer structure.
In summary, my study demonstrated the existence of intermediate b12 IGHVs in uninfected and HIV-1-infected humans and rhesus macaques. These intermediate antibody fragments may be used to identify primary immunogens that initiate b12 somatic maturation. My study also showed the importance of Env variable loops for Env structural integrity and Env-mediated viral function. The enhanced exposure of the MPER in gp160JRFL ΔV4 or ΔV5 may be further explored for vaccine development to induce MPER-specific bnAbs. === published_or_final_version === Microbiology === Doctoral === Doctor of Philosophy
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