Identification of CD4+ T cell epitopes in C. burnetii antigens targeted by antibody responses

Abstract

Coxiella burnetii is an obligate intracellular gram-negative bacterium that causes acute Q fever and chronic infections in humans. A killed, whole cell vaccine is efficacious, but vaccination can result in severe local or systemic adverse reactions. Although T cell responses are considered pivotal for vaccine derived protective immunity, the epitope targets of CD4(+) T cell responses in C. burnetii vaccination have not been elucidated. Since mapping CD4(+) epitopes in a genome with over 2,000 ORFs is resource intensive, we focused on 7 antigens that were known to be targeted by antibody responses. 117 candidate peptides were selected from these antigens based on bioinformatics predictions of binding to the murine MHC class II molecule H-2 IA(b). We screened these peptides for recognition by IFN-γ producing CD4(+) T cell in phase I C. burnetii whole cell vaccine (PI-WCV) vaccinated C57BL/6 mice and identified 8 distinct epitopes from four different proteins. The identified epitope targets account for 8% of the total vaccination induced IFN-γ producing CD4(+) T cells. Given that less than 0.4% of the antigens contained in C. burnetii were screened, this suggests that prioritizing antigens targeted by antibody responses is an efficient strategy to identify at least a subset of CD4(+) targets in large pathogens. Finally, we examined the nature of linkage between CD4(+) T cell and antibody responses in PI-WCV vaccinated mice. We found a surprisingly non-uniform pattern in the help provided by epitope specific CD4(+) T cells for antibody production, which can be specific for the epitope source antigen as well as non-specific. This suggests that a complete map of CD4(+) response targets in PI-WCV vaccinated mice will likely include antigens against which no antibody responses are made.

Figure 1. IgG antibody production for selected C. burnetii proteins is MHC-II dependent.

Seven recombinant proteins were generated and tested by ELISA for IgG reactivity of PI-WCV vaccinated C57BL/6 mice, C57BL/6 MHCII deficient (H2-Ab ^−/−) mice and PBS vaccinated C57BL/6 mice sera, 14-day post second vaccination. C. burnetii lysate was included as a positive control. PI-WCV vaccinated B6 mice generated detectable IgG responses against all antigens tested, while MHCII deficient mice showed no detectable IgG responses against individual recombinant proteins and only weak responses against C. burnetii lysate. *P<0.05, **P<0.01, ***P<0.001 comparing to PBS vaccinated B6 mice.

Figure 2. CD4⁺ T cells recognition of PI-WCV derived H-2 I-A^b epitopes in IFN-γ ICCS assays.

CD4⁺ T cells recognition of positive peptides identified by IFN-γ ELISPOT were tested in ICCS assay. Ten ug of each peptide was used to stimulate 2×10⁶ lymphocytes from three mice immunized with PI-WCV in the context of IFA and CpG 10 days earlier. Pool 8 used a mixture of eight peptides, which represents 8 different epitopes. A representative experiment of four total experiments is shown in (A). Percentages of IFNγ producing CD4⁺ T cells following stimulation with lysed Nine Mile phase I C. burnetii and peptides are shown. A peptide was considered positive if the average of the individual experiments resulted in at least >1 SD above background (Medium +DMSO). Panel B shows the average frequency and standard deviation of epitope specific IFNγ producing CD4⁺ T cells from four-independent experiments.

Figure 3. Selective protein-specific CD4⁺ T cell help to B cells after peptide and PI-WCV vaccination.

Four group of C57BL/6 mice (n = 5) were primed with three C. burnetii CD4⁺ epitopes (CBU 0383_69–83,CBU 1157_177–191 and CBU 1910_43–57) in the context of IFA and CpG or adjuvant alone, followed by PI-WCV vaccination 10 days later. (A) IgG antibody responses to three individual C. burnetii proteins after twice peptide vaccination. (B) At 28 days post PI-WCV vaccination, splenocytes from peptide and PI-WCV immunized mice were stimulated with peptides and lysed Nine Mile Phase I C. burnetii respectively, for 20 hours. Samples were subsequently stained for intracellular IFNγ and CD40L. Gated CD4⁺ lymphocytes are shown, and quantified percentages represent CD4⁺IFNγ⁺CD40L⁺ T cells. (C) Frequency of peptide specific IFN-γ producing CD4⁺ T cells at 28 days post PI-WCV vaccination. (D) IgG antibody responses to each C. burnetii protein at 0 days, 7days, 14 days, 21 days and 28 days post PI-WCV vaccination. *P<0.05, **P<0.01, ***P<0.001