Hsp110-mediated enhancement of CD4+ T cell responses to the envelope glycoprotein of members of the family Flaviviridae in vitro does not occur in vivo

Abstract

The use of heat shock proteins (HSP) to enhance activation of the immune response to chaperoned antigen is being explored for immunotherapy. Hsp110 chaperones large protein substrates more effectively than Hsp70, offering the potential to use complex antigens containing multiple epitopes in HSP-based vaccines. In this study, we investigated the ability of recombinant bovine Hsp110 to chaperone E2 glycoprotein, the major envelope protein of bovine viral diarrhea virus (BVDV) and the dominant target of neutralizing antibodies. Hsp110 formed complexes with E2, as demonstrated by immunoprecipitation. When monocytes from BVDV-immunized cattle were stimulated with these complexes and incubated with autologous CD4(+) T cells, enhanced levels of proliferation were observed. To determine the ability of these complexes to improve immunogenicity in vivo, cattle were vaccinated with either Hsp110-E2 complex or E2 only, combined with Quil-A adjuvant. In contrast to the in vitro data, cellular and humoral responses to E2 were greater in the E2-only vaccination group, indicating that complex formation had actually reduced the immunogenicity of E2. This study highlights the need for further understanding of the means by which HSP complexes are endocytosed and processed in vivo to enable the design of successful vaccine strategies.

FIG. 1.

Bovine Hsp110 chaperones large proteins. (A) Aggregation assay. Luciferase protein (Luc) was incubated either alone or in the presence of Hsp110 or BSA at 43°C for 30 min. Aggregation was monitored by measuring the increase in optical density at 320 nm. The data show the means of two independent experiments. (B) Western blot showing immunoprecipitation (IP) of E2 alone and Hsp110-E2 complexes formed at 56°C or at room temperature (RT), using a rabbit anti-Hsp110 or preimmune Ab conjugated to protein A-Sepharose beads. The proteins were resolved on a 10% SDS-PAGE gel, followed by immunoblotting with anti-V5-HRP antibody to detect V5-tagged E2 protein. The experiment was performed twice with similar results.

FIG. 2.

Proliferation of E2-specific CD4⁺ T cells is enhanced following treatment of monocytes with Hsp110-E2 complexes. (A to F) Monocytes were incubated with E2 or Hsp110-E2 complexes and autologous CD4⁺ T cells from a BVDV-immune animal for 5 days before being pulsed with [³H]thymidine, incubated for a further 16 h, and harvested. Incorporated radioactivity was determined by liquid scintillation counting and expressed as cpm. The culture conditions were as follows: increasing concentrations of E2 only or Hsp110-E2 complexes formed at 56°C with a constant concentration of 500 ng/ml Hsp110 (results are expressed as the means of triplicate determinations plus standard deviations [SD]) (A); increasing concentrations of Hsp110 only or Hsp110-E2 complex formed at 56°C with a constant concentration of 5 ng/ml E2 (results are expressed as the means of triplicate determinations plus SD) (B); 5 ng/ml E2 and 500 ng/ml Hsp110 or Hsp110-E2 complexes formed at 56°C using the same final protein concentrations (results are expressed as the means of 10 independent experiments plus standard errors of the mean [SEM]) (C); experiment performed as in panel C, but complexes were formed at room temperature (results are expressed as the means of six independent experiments plus SEM) (D); experiment performed as in panel C, but as a control, monocytes were also incubated with a combination of noncomplexed E2 and Hsp110 (results are expressed as the means of three independent experiments plus SEM) (E); experiment was performed as in panel C, but after 72 h, supernatants were harvested and assayed for gamma interferon production (results are expressed as the means of three independent experiments plus SEM) (F).*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA followed by a Bonferroni posthoc test.

FIG. 3.

Vaccination with E2 alone results in higher cellular responses than vaccination with Hsp110-E2 complexes. Cattle (three per group) were vaccinated with E2 or Hsp110-E2 complexes and boost vaccinated 21 days later. One animal remained unvaccinated (control). PBMC (2 × 10⁵ per well) were cultured weekly in the presence of 100 ng/ml E2 (A) or 2 μg/ml PWM as a positive control (B). After 5 days, the cells were pulsed with [³H]thymidine and incubated for a further 16 h before being harvested. Incorporated radioactivity was determined by liquid scintillation counting and expressed as cpm.

FIG. 4.

Vaccination with E2 alone results in higher humoral responses than Hsp110-E2 complexes. (A) At day 28, serum samples were analyzed by indirect ELISA for E2-specific IgG antibody titers. Results are expressed as the mean of duplicate determinations for each animal. (B) At day 25, the number of E2-specific IgG plasma cells in the blood was determined using a B cell ELISPOT assay. Results are expressed as the mean of duplicate determinations for each animal. *, P < 0.05; **, P < 0.01; one-way ANOVA followed by Bonferroni posthoc test.

FIG. 5.

Cytokine profiles in culture supernatants from PBMC of vaccinated animals. Supernatants from PBMC collected at day 28 and stimulated with Hsp110 (500 ng/ml) or E2 (100 ng/ml) for 72 h were assayed by capture ELISA for gamma interferon (IFN-γ) (A) or IL-10 (B) production. Results are expressed as the means of duplicate determinations for each vaccination group plus SEM.