Use of an In Vivo FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

Abstract

Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

Single-Dose Vaccination with a Hepatotropic Adeno-associated Virus Efficiently Localizes T Cell Immunity in the Liver with the Potential To Confer Rapid Protection against Hepatitis C Virus

“…However, the immunodominant CD8 ϩ T cell epitopes of NS5B in BALB/c mice, necessary to synthesize a tetramer in order to detect NS5B-specific CD8 ϩ T cells, are unknown. Consequently, we modified a fluorescent target array (FTA) assay (32)(33)(34) to map the most immunodominant T H and CD8 ϩ T cell epitopes of NS5B in vivo in mice vaccinated with cytolytic DNA encoding NS5B (pVAX-NS5B-PRF). The assay introduces fluorescently labeled bar-coded autologous naive splenocytes pulsed with viral peptides (i.e., the FTA) into previously immunized mice to evaluate the magnitude and/or avidity of T H cell and cytotoxic CD8 ϩ T cell responses in vivo (32)(33)(34).…”

“…Consequently, we modified a fluorescent target array (FTA) assay (32)(33)(34) to map the most immunodominant T H and CD8 ϩ T cell epitopes of NS5B in vivo in mice vaccinated with cytolytic DNA encoding NS5B (pVAX-NS5B-PRF). The assay introduces fluorescently labeled bar-coded autologous naive splenocytes pulsed with viral peptides (i.e., the FTA) into previously immunized mice to evaluate the magnitude and/or avidity of T H cell and cytotoxic CD8 ϩ T cell responses in vivo (32)(33)(34). Traditional peptide-based mapping and T cell stimulation assays such as enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) analyses do not distinguish CD4 ϩ and CD8 ϩ T cell responses and/or involve prolonged culture/manipulation of T cells in vitro.…”

“…Clinical data suggest that the presence of T H cells that could mobilize CD8 ϩ T cell responses is a robust correlate of clearance from primary HCV infection (9). T H cells recognize cognate peptide:major histocompatibility complex class II (MHC-II) molecules presented on B (B220 ϩ ) cells and deliver activation signals (i.e., costimulation) to B cells resulting in the upregulation of CD69 on antigen-presenting B220 ϩ cells (29,33,34). Therefore, the geometric mean fluorescent intensity (GMFI) of CD69 expression on peptide-pulsed B220 ϩ targets in an FTA is a direct measure of the T H cell responses in vivo (29,33,34), and this analysis was used to map the immunodominant T H cell epitope within P3 of NS5B (Fig.…”

“…Our in vivo FTA-based epitope mapping analysis also led to the primary development of a functional K d NS5B 451-459 tetramer and the identification of NS5B 225-240 and NS5B 495-512 immunodominant peptides for the analysis of NS5B-specific T H cell responses in BALB/c mice. We have previously reported that the FTA is a high-throughput and versatile technique to measure the magnitude and avidity of CD4 ϩ T H cell and CD8 ϩ killer T cell responses in vivo (32)(33)(34). FTA overcomes many drawbacks associated with manipulating T cells and their functional parameters in vitro, which are common in conventional T cell stimulation assays such as ELISpot and ICS.…”

Protection of mice against the highly pathogenic VVIHD-J by DNA and fowlpox recombinant vaccines, administered by electroporation and intranasal routes, correlates with serum neutralizing activity

“…The development of live-attenuated or inactivated mucosal vaccines M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 should therefore meet the needs for better protection against pathogens that penetrate through mucosal membranes (Neutra & Kozlowski, 2006). Several studies have demonstrated that combined systemic and mucosal prime/ boost immunization can enhance both the humoral and cellular arms of immune responses (Ranasinghe et al, 2006;Srivastava et al, 2008), and different immune outcomes have resulted from combinations of poxvirus vectors using prime/ boost vaccination regimens (Ranasinghe et al, 2006;Wijesundara et al, 2014). Moreover, vaccinations in which DNA priming is followed by a recombinant viral vaccine boost can elicit greater immunity when compared to the use of single immunogens (Lu, 2009;Radaelli et al, 2003;Radaelli et al, 2007;Wang et al, 2008).…”