FIG. 3.
Humoral immune responses in immunized hu-PBL-NOD/SCID mice. (A) Western blot analysis for HIV-1 gp120 Abs. The nitrocellulose membrane was probed with the following sera: (i) HIV-1-infected patient serum (1:1,000), (ii) pooled serum samples from mice that received DC transduced with HSV gp120MN/LAI (1:5 dilution), and (iii) pooled serum samples from mice that received DC transduced with a control amplicon vector (HSVlac). Sera were collected and pooled 7 days after DC immunization. Blots were incubated with the secondary Ab, a horseradish peroxidase-conjugated anti-human IgG, and developed with a chemiluminescence substrate. (B) IgG ELISA for HIV-1 envelope-reactive Abs in vaccinated mice. Mice were immunized with HSV-transduced DC; 7 days later, the animals were either challenged with infectious HIV-1 (strain HIV-1ADA or HIV-1LAI) or mock challenged (uninfected mice). Serum samples were then collected 14 days after challenge and analyzed by ELISA with an anti-human IgG Ab; the results are shown for pooled serum samples from six to eight mice. Sera from healthy, noninfected mice and HIV-infected patient sera were included in the assay as negative and positive controls, respectively (results not shown). (C) Neutralization assay results with sera from immunized mice. Sera from immunized mice were collected at day 7, following infusion of amplicon-transduced DC (HSV gp120MN/LAI or HSVlac in the immunized and control populations, respectively). Sera from a total of four mice per group were then pooled and tested for the ability to inhibit HIV infection of PHA-blasts at the indicated dilutions. Neutralization assays were performed with both LAI and ADA viral strains, with an initial MOI of 0.001 (102 TCID50 of cell-free virus was added to 105 PHA-blasts). On day 7 after viral infection, virus replication was measured by RT activity in cell-free culture supernatants.