26 research outputs found
Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells
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 population
High IFN-alpha responses associated with depletion of lymphocytes and natural IFN-producing cells during classical swine fever
During the acute phase of the viral hemorrhagic disease, classical swine fever (CSF), a severe hematologic depletion in primary lymphoid organs and depletion of peripheral blood T and B lymphocytes are observed. The onset of these pathologic events is before viremia and independent of leukocyte infection, indicating a host-mediated effect possibly through a cytokine storm. Here, we show that high serum levels of interferon- ¿ (IFN-¿) were found during this phase of CSF, detectable as early as 2 days postinfection and reaching maximum levels 3¿5 days postinfection (250¿1300 U/mL). This IFN-¿ response was related to the virulence of the viral strain used, with avirulent virus not inducing any detectable serum IFN-¿. A progressive depletion of natural IFN-producing cells/plasmacytoid dendritic cells (pDC), the likely in vivo source of IFN-¿, was also induced by the viral infection. An important finding was that the onset of severe lymphopenia was concomitant with the IFN-¿ responses, and all animals with serum IFN-¿ had depleted B and T lymphocytes. A statistically significant correlation between lymphocyte depletion and serum IFN-¿ indicates a relationship between the two events, which is supported by the known hematologic effects of high IFN-¿ doses in viv
Analysis of protective immunity against PRV infection in pigs using attenuated and inactivated PRV vaccines
Knowledge on the immune mechanisms of
protective immunity against Pseudorabies virus (PRV) infections in pigs is essential for the
development of new, biologically safe vaccines. To evaluate the role of
different immune mechanisms in relation to protection, we studied the humoral and
cell-mediated immune responses in pigs after vaccination with attenuated
or inactivated PRV vaccines and after challenge infection with wild type PRV. In addition, we
also studied the role of the adjuvant component in the induction of
protective immunity. Groups of 10-12 week old SPF pigs were intramuscularly vaccinated twice,
with an interval of 4 weeks. All vaccines were based on the Bartha strain.
Group 1 received the modified live vaccine in o/w adjuvant (MLV +A), group 2 the modified
live vaccine without adjuvant (MLV), group 3 the inactivated vaccine
with o/w adjuvant (IV +A) and group 4 the inactivated vaccine without adjuvant (IV). Group 5
was sham-vaccinated with PBS. Six weeks after the second vaccination,
all pigs were intranasally challenged
with plaque forming units of the virulent wild type
PRV strain NIA3 per animal to assess the protection. The animals were
screened for humoral and cellular immune responses, including Facscan analysis of the
proliferating cell compartment at regular intervals after vaccination
and challenge infection. Virus excretion, body weight, fever and clinical signs were assessed
after challenge infection. Pigs vaccinated with MLV +A, MLV or IV +A
induced high levels of neutralising antibodies in pigs whereas pigs vaccinated with IV
developed significantly lower levels of VN antibody titers. Pigs
vaccinated with MLV +A developed the strongest lymphoproliferation (LPT) responses followed
by pigs vaccinated with MLV. In contrast, pigs vaccinated with inactivated
vaccines developed weak LPT (IV +A) or very weak (IV) LPT responses. The analysis of the
proliferating cell compartment showed that MLV +A vaccinated pigs had
significantly more CD4/CD8/CD6
blast cells than the other groups of pigs, and
significantly more CD4/CD8/CD6 blast cells than the IV +A, IV
and sham vaccinated pigs. After challenge infection, MLV +A vaccinated pigs were protected
the best followed by MLV vaccinated pigs, as illustrated by the reduction
of virus excretion. Although pre-challenge VN antibody levels of IV +A vaccinated pigs were
comparable with antibody levels of MLV (+ or -A) vaccinated pigs, IV +A pigs
were less protected during the first week after challenge infection. Moreover, although
antibody levels between pigs vaccinated with IV +A and IV differed significantly,
both groups of pigs were equally poorly protected, confirming the lack of correlation between
VN antibody titres and early reduction in virus excretion. After challenge
infection, pigs vaccinated with MLV+A showed strongest LPT responses with high numbers of
both CD4/CD8/CD6 and
CD4/CD8/CD6 blast cells at day 3 post-challenge.
Pigs vaccinated with MLV showed intermediate LPT responses with high numbers of
CD4/CD8/CD6
blast cells whereas pigs vaccinated with IV+A showed weak LPT
responses with high numbers of CD4/CD8/CD6
blast cells. These data indicate that
protection and early reduction in virus excretion is not related to VN antibody
titers but to the strength of LPT responses,
further characterised by high numbers of both
CD4/CD8/CD6
and CD4/CD8/CD6
cell subsets. The use of the o/w adjuvant
led to stronger antibody and stronger LPT
responses after vaccination with MLV and IV with an
increase of the CD4/CD8/CD6 blast cell population
Phenotype of IL-2 or pseudorabies virus induced cytolytic cells in the pig
We investigated the phenotype
of IL-2 and pseudorabies virus (PRV) induced porcine cytolytic cells. Peripheral blood
mononuclear cells (PBMC) were isolated from PRV-immune and non immune minipigs.
The PRV-immune PBMC was restimulated in vitro with PRV and the non immune PBMC were
stimulated in vitro with IL-2. Thereafter, we separated these in vitro cultured
cells in various phenotypic subsets by the magnet-activated cell sorter after labelling
specific CD markers on these cells with magnetic beads. The cytolytic assays
with the separated cell subsets revealed that the cytolytic activity of the immune cells was
predominantly PRV specific and mediated by CD6CD8 T-lymphocytes (the presumed
classical cytolytic T-cell (CTL) phenotype in pigs). In addition to these PRV-specific CTL,
some natural killing (by CD6CD8) was also detected. In contrast, when PBMC were
stimulated with IL-2, cytolytic assays revealed that cytolytic activity was mainly mediated
by CD6CD8 lymphocytes. After PBMC were (re)stimulated in vitro, the frequencies
of various lymphocyte subsets induced by PRV or IL-2 were also analysed by FACScan. This
FACScan analysis demonstrated that after PRV stimulation of PBMC from PRV-immune
minipigs, more CD2/CD3,
CD6/CD8 and CD4/CD8 double positive and IgM positive lymphocytes, and
less CD8/CD3gd (double positive lymphocytes) proliferated, than after IL-2
stimulation of PBMC from non-immune minipigs. However, when we compared the frequencies of
lymphocyte subsets in immune PBMC after in vitro (re)stimulation with PRV or
with IL-2-induced frequencies, we only found small differences in frequency of various
lymphocyte subsets. In conclusion, we demonstrated that killing by IL-2 induced
cytolytic PBMC was mainly mediated by CD6CD8
lymphocytes (MHC-unrestricted killing) and
killing by PRV-induced cytolytic PBMC was mainly virus specific and mediated by
CD6CD8 lymphocytes
(MHC-restricted killing). These results make it clear that besides
MHC-unrestricted killing, PRV specific MHC-restricted killing can also be detected
in PRV-immune minipigs