Efficacy of intradermally administrated E2 subunit vaccines in reducing horizontal transmission of classical swine fever virus.

To investigate if intradermal (ID) vaccination and intramuscular (IM) vaccination result in a comparable reduction of horizontal transmission of classical swine fever virus (CSFV), two registered E2 subunit marker vaccines were examined. Vaccine A was a water-in-oil emulsion containing the E2 glycoprotein originating from the Alfort/Tübingen strain and vaccine B was a water-oil-water emulsion containing the E2 glycoprotein originating from the Brescia strain. Eight groups, of ten pigs each, were vaccinated with either vaccine A or B, intramuscularly (IM) or intradermally (ID). Two different vaccination-challenge intervals were used for each vaccine. Furthermore, one group was vaccinated with a tenfold ID dose of vaccine A and one non-vaccinated group served as a control group. Five pigs from each group were challenged with the moderately virulent CSFV strain Paderborn, while the remaining five pigs served as contacts. Using vaccine A, full transmission to all contact pigs in both ID vaccinated groups occurred. No virus transmission was observed when IM vaccinated pigs were challenged 14 days post-vaccination (14dpv) whereas only one out of five contact pig became infected when they were challenged 10dpv. Using vaccine B no virus transmission was observed when pigs were ID or IM vaccinated and challenged 10dpv. When challenged 3dpv full transmission occurred in the ID vaccinated group, whereas four out of five contact pigs became infected in the IM vaccinated group. This result indicates that ID vaccination does not result in better protection against horizontal CSFV transmission compared to IM vaccination, for the vaccines studied.

Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.

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- alpha (IFN-alpha) 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-alpha response was related to the virulence of the viral strain used, with avirulent virus not inducing any detectable serum IFN-alpha. A progressive depletion of natural IFN-producing cells/plasmacytoid dendritic cells (pDC), the likely in vivo source of IFN-alpha, was also induced by the viral infection. An important finding was that the onset of severe lymphopenia was concomitant with the IFN-alpha responses, and all animals with serum IFN-alpha had depleted B and T lymphocytes. A statistically significant correlation between lymphocyte depletion and serum IFN-alpha indicates a relationship between the two events, which is supported by the known hematologic effects of high IFN-alpha doses in vivo.

Comparison of immune responses after intra-typic heterologous and homologous vaccination against foot-and-mouth disease virus infection in pigs.

This study compares the immune responses and protection induced by intra-typic heterologous vaccination with that induced by homologous vaccination against challenge with foot-and-mouth disease virus (FMDV). Humoral and cell-mediated immune responses and protection against challenge with FMDV O Taiwan were examined in a non-vaccinated group, a group vaccinated with O Taiwan FMD vaccine and a group vaccinated with O Manisa FMD vaccine. Five pigs from each group were challenged with FMDV type O Taiwan 14 days after vaccination and five other pigs were contact-exposed to the inoculated pigs. Both homologous and heterologous vaccination protected against challenge with FMDV O Taiwan at 2 weeks after vaccination. In the heterologous vaccinated group, cross-neutralizing antibody titres against O Taiwan could be detected although the ratio 'r(1)' was 0.4, which was significantly smaller than the critical r-value. Cell-mediated immune responses were detected after both homologous and heterologous vaccination. Virus-induced in vitro lymphocyte (cross-) proliferation and production of both a Th1-type (IFN-gamma) and a Th2-type (IL-10) cytokine response were demonstrated in cultures of peripheral blood mononuclear cells (PBMC). The findings show that heterologous (emergency) vaccination can prevent clinical disease and shedding of virus. The induction of the cell-mediated immune responses after (heterologous) vaccination needs more research but data on these responses might provide additional tools for both vaccine choice and vaccine development.

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 populations.

Vaccine-induced T cell-mediated immunity plays a critical role in early protection against pseudorabies virus (suid herpes virus type 1) infection in pigs.

The aim of our study was to evaluate the relative importance of antibody and T cell-mediated immunity in protection against pseudorabies virus (suid herpes virus type 1) infection in pigs. We induced different levels of immune responses by using: (1) a modified live vaccine; (2) the same modified live vaccine with an oil-in-water (o/w) adjuvant; (3) an inactivated vaccine; and (4) the same inactivated vaccine with an o/w adjuvant. Subsequently, we challenged pigs with virulent pseudorabies virus (PRV). We demonstrated that best-protected pigs stood out by maintaining strong T cell-mediated immune (CMI) responses after challenge. Of the immune parameters tested, protection against virus shedding was correlated best with the magnitude of the IFN-gamma response of in vitro re-stimulated peripheral blood mononuclear cells (PBMC) with an additional role for PRV-specific IgG2 antibodies. The use of an o/w adjuvant resulted in higher antibody and CMI responses, in particular with an increased frequency of memory T helper blast cells of in vitro re-stimulated PBMC. However, this adjuvant-induced enhancement of the immune response had a limited additional effect on the efficacy of inactivated vaccines. This study suggests a major contribution of the CMI response in early protection against PRV infection and that PRV-induced IFN-gamma responses may serve as a suitable indicator for assessing the immune status of vaccinated pigs.

Vaccination of pigs two weeks before infection significantly reduces transmission of foot-and-mouth disease virus.

The objective of this study was to investigate whether and at what time interval could vaccination reduce transmission of foot-and-mouth disease virus (FMDV) among pigs. Reduction of virus transmission by vaccination was determined experimentally. Transmission of FMDV was studied in three groups of ten pigs: one non-vaccinated group and two groups that were vaccinated 7 days (-7 dpi) and 14 days before inoculation (-14 dpi), respectively. Five randomly selected pigs from each group were inoculated with FMDV type O Taiwan, while the other five pigs left in the groups were exposed to the inoculated pigs by direct contact. Clinical signs were recorded, virus isolation and RT-PCR were carried out on oropharyngeal fluid (OPF), and the neutralizing antibody titres and the antibody response against non-structural (NS) proteins of FMDV were determined. No virus transmission was observed in the -14 dpi group, whereas virus transmission was observed in all contact pigs affecting both the non-vaccinated and the -7 dpi group. The reproduction ratio R in the -14 dpi vaccinated group was significantly lower than that of the non-vaccinated group. This study confirms the potential of vaccination as an important tool to reduce transmission of FMDV.