Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells

Abstract

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations.

Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs

“…Subsequently, mature and activated DC subsets would then be involved in induction of adaptive immune responses, illustrated by punctual IFN-γ and IL-18 secretions in serum. This was in agreement with CTL stimulation by CSFV-infected mature mo-DC [14] and with the initiation of cellular immune responses described in pigs surviving infection [35]. Additionally, the fact that high proportions of both DC subsets expressed IL-10 in the spleen could reflect the starting of humoral immune response, in accordance with the detection of secreting IgM E2 + B cells the first days post-infection [39].…”

“…It was shown that CSFV replicates in mo-DC without cytopathological effect but prevents their activation and thereby the secretion of IFN-α, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 [4,13]. CSFV also blocks mo-DC maturation by inhibiting MHC-II up-regulation, although these cells remain able to present E2 viral envelop glycoprotein to cytotoxic T lymphocytes [14]. Similarly, CSFV replicates in NIPC [4].…”

Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

“…CD107a is expressed on the cell surface following activation-induced degranulation, and it has been described as a marker for cytotoxic CD8 T cell activity whose expression has been associated with loss of perforin following antigen stimulation (34). The data presented here suggest that CSFV-specific IFN-␥-expressing cells may also have cytotoxic capacity and consolidate our previous results where we showed that IFN-␥ expression was restricted to CD8 T cells expressing intracellular perforin (7) and other earlier studies that demonstrated CSFV-specific cytotoxic activity by T cell cultures isolated from vaccinated pigs (8)(9)(10). While IFN-␥-secreting T EM cells have been implicated in protection against a variety of intracellular pathogens, simultaneous production of IFN-␥, TNF-␣, and IL-2 detected at the single-cell level by multiparameter flow cytometry has been correlated with enhanced vaccine-induced protection (45)(46)(47)(48)(49)(50)(51).…”

“…Moreover, it has recently been shown that there is a close temporal correlation between the induction of CSFV-specific T cell IFN-␥ responses and rapid protection induced by a C-strain vaccine (6). In addition to the secretion of IFN-␥, which has been shown to exert direct antiviral effects on CSFV in vitro (7), vaccine-induced CSFV-specific T cells have also been shown to have the capacity to lyse infected cells with specificities mapped to the structural protein E2 and the nonstructural protein NS3 (8)(9)(10). Flow cytometric studies have identified both CD4 and CD8 T cells as the cellular source of CSFV-specific IFN-␥, with the latter coexpressing the cytolytic molecule perforin (7,11).…”

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens

“…CTL assays. A standard chromium ( 51 Cr) release assay was used to measure antigen-specific T cell cytotoxicity as previously described (37).…”