Utilization of Feline ELISpot to Evaluate the Immunogenicity of a T Cell-Based FIV MAP Vaccine

Abstract

The prototype and the commercial dual-subtype feline immunodeficiency virus (FIV) vaccines conferred protection against homologous FIV strains as well as heterologous FIV strains from the vaccine subtypes with closely related envelope (Env) sequences. Such protection was mediated by the FIV neutralizing antibodies (NAbs) induced by the vaccines. Remarkably, both prototype and commercial FIV vaccines also conferred protection against heterologous FIV subtypes with highly divergent Env sequences from the vaccine strains. Such protection was not mediated by the vaccine-induced NAbs but was mediated by a potent FIV-specific T-cell immunity generated by the vaccines (Aranyos et al., Vaccine 34: 1480-1488, 2016). The protective epitopes on the FIV vaccine antigen were identified using feline interleukin-2 (IL-2) and interferon-γ (IFNγ) ELISpot assays with overlapping FIV peptide stimulation of the peripheral blood mononuclear cells (PBMC) from cats immunized with prototype FIV vaccine. Two of the protective FIV peptide epitopes were identified on FIV p24 protein and another two protective peptide epitopes were found on FIV reverse transcriptase. In the current study, the multiple antigenic peptides (MAPs) of the four protective FIV peptides were combined with an adjuvant as the FIV MAP vaccine. The laboratory cats were immunized with the MAP vaccine to evaluate whether significant levels of vaccine-specific cytokine responses can be generated to the FIV MAPs and their peptides at post-second and post-third vaccinations. The PBMC from vaccinated cats and non-vaccinated control cats were tested for IL-2, IFNγ, and IL-10 ELISpot responses to the FIV MAPs and peptides. These results were compared to the results from CD4 and CD8 T-cell proliferation to the FIV MAPs and peptides. Current study demonstrates that IL-2 and IFNγ ELISpot responses can be used to detect memory responses of the T cells from vaccinated cats after the second and third vaccinations.

Immunogenicity and Efficacy of a Novel Multi-Antigenic Peptide Vaccine Based on Cross-Reactivity between Feline and Human Immunodeficiency Viruses

“…The peptides that showed a significant increase in CD8 + T-cell responses both in feline and human infected PBMC were selected for generating immunogens for an animal study, as described previously [17,18]. The four peptides of the same kind were tagged with palmitic acid or Tat peptide using three–five lysine residues [16] (Figure 1) to enhance intracellular permeability of these peptides for efficient antigen presentation and loading on MHCI molecule. The 8–12-week-old SPF cats were used to test the immunogenicity and protection as depicted in Figure 1.…”

“…The protective FIV peptide epitopes were selected by their potential to induce high levels of T-cell proliferation and cytokine production as the polyfunctional T-cell activities for both FIV-vaccinated cats and HIV + human subjects. The CD4 + and CD8 + T-cell proliferation were determined by the flow cytometry (fluorescence-activated cell sorter [FACS])-based carboxyfluorescein diacetate succinimide ester (CFSE) proliferation analyses with a positive threshold of ≥0.5% CFSE low as previously described [16]. IFNγ and IL-2 production were measured by IFNγ and IL-2 ELISpot analyses using feline or human IFNγ and IL-2 ELISpot module kits from R&D Systems (Minneapolis, MN) using manufacturer’s protocol [16].…”

“…The CD4 + and CD8 + T-cell proliferation were determined by the flow cytometry (fluorescence-activated cell sorter [FACS])-based carboxyfluorescein diacetate succinimide ester (CFSE) proliferation analyses with a positive threshold of ≥0.5% CFSE low as previously described [16]. IFNγ and IL-2 production were measured by IFNγ and IL-2 ELISpot analyses using feline or human IFNγ and IL-2 ELISpot module kits from R&D Systems (Minneapolis, MN) using manufacturer’s protocol [16]. The positive threshold for ELISpot analyses was ≥50 spot forming units (SFU)/10 6 peripheral blood mononuclear cells (PBMC).…”

“…Immune analyses were performed at 3-6 weeks post-second vaccination depending on the vaccine groups, and three and six weeks post-third vaccination for all vaccine groups. Immune analyses consisted of CD4 + and CD8 + T-cell proliferation using FACS-based CFSE-proliferation with a threshold of 0.5% CFSE low as previously described [16,17,18], and IL-2 and IFNγ ELISpot analyses for production of these cytokines with a positive threshold of 50 SFU/10 6 PBMC as previously described [16]. In addition, at six weeks post-third vaccination before the challenge, additional blood samples were collected for PBMC to perform FIV peptide- or MAP-specific perforin, GrzA, GrzB, IL-2, and IFNγ mRNA production analyses.…”