Development of a novel, guinea pig-specific IFN-γ ELISPOT assay and characterization of guinea pig cytomegalovirus GP83-specific cellular immune responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine

Abstract

The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model.

Keywords: CMV pp65; Congenital CMV infection; Cytomegalovirus vaccine; ELISPOT; Glycoprotein B; Guinea pig; Guinea pig cytokine; Guinea pig immunology; IFN-γ; Interferon gamma; Placenta; TORCH infection; pp65.

Fig. 1

Antibody response to MVA vaccination. (A) Timeline of vaccinations and blood draws. Animals were initially injected with 1 × 10⁸ pfu of either MVA-gB (4 animals) or MVA-GP83 (12 animals), and two additional boosts of the same dose were given at two-week intervals (▼). Blood was collected prior to the first vaccination, at the time of each subsequent boost, and at two weeks post-final vaccination (▽). Antibody titers expressed as log transformed data from end-point dilution ELISA assay as described in Section 2.7. (B) Mean ELISA titers of MVA-GP83 (n = 12) and MVA-gB (n = 4) groups are expressed as log-transformed data from the 3rd bleed post-vaccination (14 days following the third dose). Data depicted are results of end-point dilution ELISA assay as described in Section 2.7. Animals below the cut-off of the ELISA assay (1:80) were assigned a titer of 1:40 for statistical analyses. No detectable response was observed in pre-immune sera (data not shown). ELISA titers were significantly higher following vaccination with MVA-gB construct (p < 0.0005). ELISA responses from 4 GPCMV naïve animals challenged with GPCMV (1 × 10⁵ pfu inoculated sc) were also measured at day 28 (black bar). (C) Specificity of the immune response was determined by Western blot analysis. Antibody responses to GP83 and gB were measured using purified virions followed by immune blotting with rabbit polyclonal anti-GP83, mouse monoclonal anti-gB, pre-immune sera, and sera from the fourth bleed (representative animals shown for MVA-gB and MVA-GP83 vaccinated animals).

Fig. 2

Enumeration of IFN-γ excreting splenocytes in response to mitogen and peptide stimulation. Splenocytes were isolated from uninfected or infected animals at 28 dpi using a Ficoll gradient (A) Splenocytes were either treated with DMSO (no stimulus control), the mitogen ConA, or GP83 peptides and enumerated using ELISPOT assays specific for IFN-γ. Representative wells are shown. (B) Triplicate wells for each sample were counted and averaged for each animal. Results are shown in mean number of IFN-γ positive cells per 100,000 splenocytes for group (DMSO treated cells are shown in white and ConA treated cells are shown in gray; error bars = SEM). Splenocytes were isolated from animals at 28–32 days after the third vaccination using a Ficoll gradient. Results were quantitated as above for each vaccinated group. (C) DMSO treated cells are shown in white and ConA treated cells are shown in gray (error bars = SEM). (D) Harvested splenocytes were stimulated with pooled peptides that span the entire GP83 protein, as described in Section 2.10. Triplicate wells for each sample were counted and the mean for all animals (including non-responders) in both vaccine groups determined. Mean number of IFN-γ positive cells per 100,000 splenocytes for each vaccinated group is shown. Animals vaccinated with MVA-gB are shown in white and MVA-GP83 vaccinated animals are shown in gray (error bars = SEM; *p < 0.05).

Fig. 3

Mapping of T cell epitopes of GP83 using peptide pools. Splenocytes were stimulated with one of 24 different pools of peptides that contained 11–12 9-mers that span the GP83 protein. IFN-γ producing cells were counted in triplicate for each animal for each pool using ELISPOT. Peptides were considered to stimulate T cells if the response differed significantly from unstimulated cells (DMSO control). (A) Six animals vaccinated with MVA-GP83 responded to a single peptide contained in both pool 12 and pool 19. Representative data from one animal is shown. (B) The remaining 3 animals vaccinated with the MVA-GP83 construct displayed stimulation of T cells by multiple peptides (representative from one animal data shown). (C) No response was observed to any pool in any animals vaccinated with MVA-gB vaccine. Among the MVA-GP83 vaccinated animals, 3/12 were non-responders by this assay. A representative example from one animal is shown. *p < 0.05, **p < 0.005, ***p < 0.001.

Fig. 4

Characterization of the anti-GP83 T cell response generated by GPCMV infection. Splenocytes from uninfected and GPCMV infected animals were stimulated with one of 24 different pools of peptides that contained 11–12 9-mers that spanned the GP83 protein. IFN-γ producing cells were counted in triplicate for each animal for each pool using ELISPOT. Peptides were considered to stimulate T cells if the response differed significantly from unstimulated cells (DMSO control). (A) No response was observed to any pool in uninfected animals (representative data from one animal is shown). (B) Significant responses compared to unstimulated cells were engendered by multiple peptide pools followings stimulation of splenocytes isolated from GPCMV infected animals (representative data from one animal is shown; *p < 0.05).