Figure 5. Induction of MPER-specific HIV NAbs by immunization with HA-C14S/gp41 DNA and VLP vaccines.

Guinea pigs were immunized as described in Figure 3A and sera collected at 2 weeks after the fourth immunization were analyzed for the levels of MPER-specific antibody responses by ELISA as well as their neutralizing activity against a conserved epitope in MPER that was recognized by the monoclonal antibody 4E10 by a pseudovirion neutralization assay. A. Antibody responses against the MPER were determined by ELISA using the 2F5/4E10 peptide (ELLELDKWASLWNWFNITNW, a synthetic peptide corresponding to a segment in the MPER) as coating antigens, and expressed as the O.D. values for each serum sample at 1∶200 dilutions. SIV-4E10 (B) or SIVmac239 (C) pseudovirions were incubated with individual serum samples from vaccinated guinea pigs at 1∶50 dilutions in 10% FCS-DMEM for 1 hr at 37°C and then added to TZM-bl cells seeded in a 96-well plate. Two days after infection, the medium was removed and the cells were fixed and stained for β-galactosidase expressing cells. Neutralizing activity was expressed as the percentage of reduction in the number of β-galactosidase expressing cells in sample wells compared to those in control wells (% neutralization). D. Peptide competition of SIV-4E10 pseudovirus neutralization by sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines. Guinea pig sera (Group 1 and Group 2) were diluted in 10% FCS-DMEM at 1∶40 and pre-incubated with 0.1 ug of the 2F5/4E10 peptide (ELLELDKWASLWNWFNITNW) or a control peptide (AMQMLKETI, corresponding to a segment in the HIV Gag protein) at 37°C for 1 hr as indicated, and then used in neutralization assay (at a final dilution of 1∶50) against the SIV-4E10 pseudovirus. Group 1, HA-C14S/gp41 DNA; Group 2, HA-C14S/gp41 VLP.