Although broadly neutralizing HIV antibodies are detected in infected (and sometimes uninfected) humans, immunization with HIV-1 antigens generally do not elicit broadly neutralizing antibodies. Clinical studies found that GBV-C E2 antibody is associated with improved survival in HIV and GBV-C co-infected individuals, and E2 antibody neutralizes HIV-1 infectivity by reducing virus entry in vitro. To determine if GBV-C E2 elicits broadly neutralizing HIV monoclonal antibodies (MAbs), we immunized mice with either E2 (lacking C-terminal transmembrane domain) or a synthetic peptide we previously demonstrated is involved in HIV entry and generated anti-E2 MAbs against both antigens. Here, we examined the interactions of both MAbs for their ability to inhibit HIV-1 infectivity, precipitate HIV-1 particles, and bind HIV-specific structural protein antigens.
GBV-C E2 protein was expressed in CHO cells, and a synthetic peptide generated of the 17 amino acid E2 region involved in HIV entry. Mice were immunized with 25 ug E2 protein or peptide in IFA four times prior to sacrifice, and hybridomas generated. One anti-E2 and one anti-peptide hybridoma cell line (8H2 and 1C4 respectively) were identified from more than 2,000 independent cultures using a capture E2 ELISA. HIV-1 envelope proteins (gp120, gp140 and gp41), gp41 peptides (MPER and T-20), and X4- and R5-tropic HIV-1 isolates representing clades A, B, and D were studied.
Both GBV-C anti-E2 MAbs precipitated HIV-1 particles and neutralized X4 and R5-tropic HIV isolates from diverse geographic regions representing three clades (IC50 ranging from 2.5 to 7.5 ug/mL). These antibodies did not neutralize mumps or yellow fever viruses, demonstrating specificity. 8H2 reacted with HIV-1 gp140 and gp41 proteins, but not gp120 using two types of ELISA methods. Both 8H2 and 1C4 recognized the HIV-1 gp41 fusion (MPER) peptide in immunoblot assays.
Although HIV-1 antigens do not elicit broadly neutralizing HIV-1 antibodies, GBV-C E2 and an E2 peptide elicited HIV-1 neutralizing MAbs in mice. Since GBV-C E2 antibodies are associated with prolonged survival in three clinical studies, these data illustrate the potential for a novel antigen to incorporate into HIV-1 vaccine strategies.