AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: failure to protect and possible enhancement of challenge infection by four cell-based vaccines prepared with autologous lymphoblasts

Abstract

Immunogenicity and protective activity of four cell-based feline immunodeficiency virus (FIV) vaccines prepared with autologous lymphoblasts were investigated. One vaccine was composed of FIV-infected cells that were paraformaldehyde fixed at the peak of viral expression. The other vaccines were attempts to maximize the expression of protective epitopes that might become exposed as a result of virion binding to cells and essentially consisted of cells mildly fixed after saturation of their surface with adsorbed, internally inactivated FIV particles. The levels of FIV-specific lymphoproliferation exhibited by the vaccinees were comparable to the ones previously observed in vaccine-protected cats, but antibodies were largely directed to cell-derived constituents rather than to truly viral epitopes and had very poor FIV-neutralizing activity. Moreover, under one condition of testing, some vaccine sera enhanced FIV replication in vitro. As a further limit, the vaccines proved inefficient at priming animals for anamnestic immune responses. Two months after completion of primary immunization, the animals were challenged with a low dose of homologous ex vivo FIV. Collectively, 8 of 20 vaccinees developed infection versus one of nine animals mock immunized with fixed uninfected autologous lymphoblasts. After a boosting and rechallenge with a higher virus dose, all remaining animals became infected, thus confirming their lack of protection.

FIG. 1.

Experimental plan. Groups of five cats (four in the case of the mock-1 vaccine) were given five subcutaneous doses of the immunogens mixed 1:1 with incomplete Freund’s adjuvant in a total volume of 2 ml (small arrows). The immunogens used and their FIV contents are shown in Table 1. Two months after completion of this primary immunization, all cats were challenged intravenously (large arrows) with 5 CID₅₀ of homologous virus (plasma of infected cats). The animals that after 7 months were still virus negative were given a booster dose of their respective immunogens and, after 2 additional months, challenged again with 10 CID₅₀ of the same virus.

FIG. 2.

Effects of AT2 treatment on FIV properties. (A) Effect on infectivity. The FIV stock used for vaccine production was incubated at 4°C for 2 h with the indicated concentrations of AT2 and then titrated for residual infectivity in MBM cell cultures. Values shown are the viral titers obtained at day 8 of culture expressed as TCID₅₀. (B) Effect on electrophoretic mobility. Viral samples treated at 4°C for 2 h with no AT2 (lane 1) or with 300 μM AT2 (lane 2) were electrophoresed and then probed with FIV-immune cat serum, followed by peroxidase-conjugated anti-cat IgG polyclonal antibody. (C) Effect on ability to bind PLB. A total of 5 × 10⁵ PLB was mixed with the indicated amounts of FIV, which was either native (▪), inactivated with 300 μM AT2 (), or AT2-inactivated and then disrupted by sonication (□). After 2 h at 4°C, the PLB were pelleted and examined for bound FIV RNA copies by quantitative TM-PCR. The results are expressed as the percent viral RNA copies found associated with the cells relative to the number of input copies. (D) Dose curve of AT2-FIV binding to PLB. Indicated amounts of AT2-FIV were incubated with 5 × 10⁵ PLB which, after 2 h at 4°C, were pelleted and examined as described above for bound viral RNA. The results are expressed as the numbers of viral RNA copies found associated with the cells.

FIG. 3.

FIV-binding IgG antibodies in vaccinated and mock-immunized cats, as determined by ELISA with purified and disrupted whole FIV (A and C) or lectin-purified gp derived thereof (B and D) as the test antigen. (A and B) Responses to primary immunization. (C and D) Responses of animals that had escaped infection after the first virus challenge to a booster given 9 months after the completion of primary immunization. Symbols represent geometric means, and bars indicate 95% confidence limits. For symbols without bars, the limits lie within the symbols. Arrowheads indicate immunogen doses. Immunogen: ▪, FC^PLB; ♦, ID-1; ▴, ID-2; •, ID-3; □, mock-1; ○, mock-2.

FIG. 4.

FIV neutralization curves with day-of-challenge vaccine sera as determined by two different procedures. In the standard procedure (solid lines), sera were reacted with FIV at 4°C for 1 h before inoculation into MBM cell cultures, which were then immediately incubated at 37°C. In the other procedure (dashed lines), aimed at facilitating antibody interaction with ID epitopes, MBM cell cultures were reacted with FIV at 4°C for 1 h, inoculated with cold test sera, further incubated at 4°C for 1 h, and finally transferred to 37°C. All sera were preadsorbed with MBM cells prior to testing.

FIG. 5.

Plasma viremia and proviral and infectious cell burdens in the PBMC of vaccinated and mock-immunized cats that became infected after the first (A) and second (B) FIV challenges.