Orf Virus-Based Vaccine Vector D1701-V Induces Strong CD8+ T Cell Response against the Transgene but Not against ORFV-Derived Epitopes

Abstract

The potency of viral vector-based vaccines depends on their ability to induce strong transgene-specific immune response without triggering anti-vector immunity. Previously, Orf virus (ORFV, Parapoxvirus) strain D1701-V was reported as a novel vector mediating protection against viral infections. The short-lived ORFV-specific immune response and the absence of virus neutralizing antibodies enables repeated immunizations and enhancement of humoral immune responses against the inserted antigens. However, only limited information exists about the D1701-V induced cellular immunity. In this study we employed major histocompatibility complex (MHC) ligandomics and immunogenicity analysis to identify ORFV-specific epitopes. Using liquid chromatography-tandem mass spectrometry we detected 36 ORFV-derived MHC I peptides, originating from various proteins. Stimulated splenocytes from ORFV-immunized mice did not exhibit specific CD8+ T cell responses against the tested peptides. In contrast, immunization with ovalbumin-expressing ORFV recombinant elicited strong SIINFEKL-specific CD8+ T lymphocyte response. In conclusion, our data indicate that cellular immunity to the ORFV vector is negligible, while strong CD8+ T cell response is induced against the inserted transgene. These results further emphasize the ORFV strain D1701-V as an attractive vector for vaccine development. Moreover, the presented experiments describe prerequisites for the selection of T cell epitopes exploitable for generation of ORFV-based vaccines by reverse genetics.

Keywords: CD8+ T cells; MHC class I; ORFV; Orf virus; Parapoxvirus; epitopes; immune response; immunopeptidome; mass spectrometry; vaccine; viral vector.

Figure 1

Transgene-specific CD8+ T cell response induced by the ORFV vector. H-2K^b C57BL/6 mice (n = 6) were immunized i.m. two times with V12-Ova-D12-GFP or negative control V-D12-mCherry. Ova-specific CD8+ T cell response in individual mice was determined one week after the second administration. (A) Frequency of specific cytotoxic T lymphocytes of the total CD8+ T cells in the spleen was assessed by Ova_257-264 dextramer staining. (B) Percentage of Ova_257-264 SIINFEKL peptide-specific CD8+ T cells producing the indicated cytokines was determined by intracellular cytokine staining. (C) Pie chart shows the extent of simultaneous CD107a, TNF-α, IFN-γ and IL-2 production by the Ova-specific CD8+ T cells. Frequencies are shown as means ± SEM. Ova, ovalbumin; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon-gamma; IL-2, interleukin-2.

Figure 2

HeLa-K^b represent target cells for the identification of ORFV-derived major histocompatibility complex (MHC) class I peptides. (A) Cell viability and infection rate following 20 h exposure to ORFV. HeLa-K^b cells were infected with indicated multiplicity of infection (MOI). Percentages of viable cells after staining with Zombie Aqua dye and infected cells by mCherry expression were determined by flow cytometry. The data represent the means ± SEM for three independent experiments. (B) H-2K^b surface expression after ORFV infection with indicated MOI. Twenty hours post infection absolute numbers of H-2K^b molecules on the cell surface were determined by flow cytometry using the quantitative indirect immunofluorescence kit. Data shown are means ± SEM from three replicates. (C,D) Epitope presentation by HeLa-K^b cells. Cells were infected with V12-Ova-D12-GFP or negative control V-D12-mCherry with MOI 5 or loaded with synthetic Ova_257-264 SIINFEKL peptide. Twenty hours after infection cell surface staining was performed using anti-mouse SIINFEKL bound H-2K^b antibody followed by flow cytometry analysis. ORFV infection was determined by GFP or mCherry expression. Data are representative of two independent experiments. * p < 0.05; ni, non-infected cells; gMFI, geometric mean fluorescence intensity; Ova, ovalbumin.

Figure 3

Peptide-specific T cell responses by IFN-ƴ ELISPOT in ORFV immunized mice. H-2K^b C57BL/6 mice (n = 6) were immunized twice with V12-Ova-D12-GFP or control ORFV by i.m. route. Ova_257-264 SIINFEKL- and ORFV-derived H-2K^b peptide-specific responses were determined seven days after the second administration. (A) Numbers of IFN-ƴ spot forming units (SFUs) after stimulation of splenocytes from individual mice with synthetic peptide pools (Pools 1–4) or Ova_257-264 SIINFEKL-peptide. Spot counts of >1000 were set to 1000 because of inaccurate spot count due to technical limitations. Dotted and solid lines indicate the mean SFUs in negative control wells within control ORFV or V12-Ova-D12-GFP group respectively. Data are shown as means ± SEM of all analysed samples in two independent technical replicates. (B) Representative IFN-ƴ ELISPOT assay showing splenocyte responses of control ORFV or V12-Ova-D12-GFP immunized mice. PHA, phytohemagglutinin; Ova, ovalbumin; IFN-γ, interferon-gamma.

Figure 4

CD8+ T cell responses to the individual ORFV-derived peptides by intracellular cytokine staining (ICS) in ORFV immunized mice. H-2K^b C57BL/6 mice (n = 6) were immunized twice with V12-Ova-D12-GFP or control ORFV by i.m. route. One week after the second administration specific cytotoxic T lymphocyte responses to the individual ORFV-derived peptides within pooled splenocytes were evaluated using ICS. Percentages of CD107a, TNF-α, IFN-γ and IL-2 expressing CD8+ T cells after stimulation with synthetic peptides (A) 1–8, (B) 9–16, (C) 17–24 and (D) 25–32, control ORFV or Ova_257-264 SIINFEKL peptide. Data are shown as means ± SEM of three independent technical replicates. PHA, Phytohemagglutinin; ns, not significant; Ova, ovalbumin; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon-gamma; IL-2, interleukin-2.