Background: HIV-1-specific broadly neutralizing antibodies (BnAbs), such as the membrane proximal external region (MPER) antibodies 2F5 and 4E10, contain long H chain complementarity determining region 3 (CDR-H3) which is encoded by DH gene segment, that lacks tyrosine and includes patches of hydrophobic and charged amino acids. However, B cells bearing Igs with charged or hydrophobic CDR-H3s are normally culled from mature B cell subsets. Thus, elucidation of mechanisms that underlie the difficulty in generating HIV-1 can be seen as a part of fundamental need to better understand how to distinguish self and non-self receptors that can neutralize pathogenic antigens without self-inflicting dame to the host.
Methods: Mice-BALB/c mice cohort limited to the use of single DH gene segments were generated. ΔD-DFL is a human-like repertoire control. ΔD-DµFS promotes the use of hydrophobic amino acid CDR-H3. The ΔD-iD is charged amino acid enriched CDR-H3. Immunization-each strain of 10 mice was immunized with HIV-1 JR-FL gp140 protein. Epitope Identification-Two group of mice (No. 4 & 5) of each strain were selected (prior to immunization, and after the 2nd and 4th immunizations) for PEPperPRINT Chip to detect their epitopes on HIV-1 JR-FL gp140. Serum Assay-Binding ability was examined by HIV-1 envelope protein ELISA. Blocking activity was examined by competitive ELISA with soluble CD4 (sCD4) and 2F5, 2G12 antibodies.
Results: 1. We obtained evidence of strong and clear polyclonal responses to immunization with JR-FL gp140. As a general rule, the heterogeneity of the anti-JR-FL gp140 response varied by DH genotype with ΔD-DFL>Wild Type (WT)>>ΔD-DμFS, ΔD-iD. Conversely, the intensity of the response was greatest in the ΔD-iD.
2. Linear response showed by PEPperPRINT chip was not identified with the response to natural HIV-1 epitopes, however, the ΔD-iD favored charged epitopes that is inconsistent with our hypothesis.
3. Anti-JR-FL gp140 antibodies present in the sera of the DH altered did not block HIV-1 envelope CD4, 2F5, 2G12 binding site. This suggests reduced affinity, stability or on rates, or increased off rates for Igs obtained from DH altered mice.
Conclusions: The pattern of epitope recognition and antigen binding in the response to HIV-1 JR-FL gp140, in part, is on DH gene segment sequence. Restrictions imposed by natural selection of DH sequence may underlie some of the difficulty that patients experience in HIV-1 neutralizing antibody.