Comparison of different prime-boost regimes with DNA and recombinant Orf virus based vaccines expressing glycoprotein D of pseudorabies virus in pigs.

Both DNA and Orf virus (ORFV; Parapox virus) based vaccines have shown promise as alternatives for conventional vaccines in pigs against pseudorabies virus (PRV) infection causing Aujeszky's disease. In the present study we evaluated the efficacy of different prime-boost regimes in pigs in terms of immunogenicity and protection against challenge infection with PRV. The different prime-boost regimes consisted of the homologous prime-boost regimes (DNA followed by DNA or ORFV followed by ORFV) and the heterologous prime-boost regimes (DNA followed by ORFV and ORFV followed by DNA), all based on glycoprotein D (gD) of PRV. Moreover, we compared the efficacy of the different prime-boost regimes with the efficacy of a conventional modified live vaccine (MLV). The different prime-boost regimes resulted in different levels of immunity and protection against challenge infection. Most effective was the regime of priming with DNA vaccine followed by boosting with the ORFV based vaccine. This regime resulted in strong antibody responses, comparable to the antibody responses obtained after prime-boost vaccination with a conventional MLV vaccine. Also with regard to protection, the prime DNA-boost ORFV regime performed better than the other prime-boost regimes. This study demonstrates the potential of a heterologous prime-boost vaccination strategy against PRV based on a single antigen, and that in the natural host, the pig.

A DNA vaccine coding for gB and gD of pseudorabies virus (suid herpes type 1) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines.

DNA vaccines are capable of priming the immune system of neonates in the presence of maternal antibodies. However, it is still not clear whether the extent of priming and protection against challenge infections induced by a DNA vaccine in maternally immune newborns is better than that induced by conventional vaccines. To study this, we used the pseudorabies virus (PRV) infection model in the natural host, the pig. We compared the efficacy of a DNA vaccine with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. We measured the priming of the immune response and the degree of protection against challenge infection for all vaccine types. We vaccinated piglets with or without maternal immunity twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naïve piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naïve piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not.

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.

The foot-and-mouth disease epidemic in The Netherlands in 2001.

An outbreak of foot-and-mouth disease (FMD) in Great Britain was reported on 21 February 2001, followed by an outbreak of FMD in The Netherlands a month later. This Dutch index outbreak occurred on a mixed, veal-calf/dairy-goat farm in Oene, in the central part of The Netherlands. The most-likely route of infection was the import of Irish veal-calves to this Dutch herd via an FMD-contaminated staging point in France. With hindsight, more herds seemed to be infected by the time the index outbreak was confirmed. The regular EU control measures were implemented, in combination with pre-emptive culling of herds within 1km of each outbreak. Nevertheless, more outbreaks of FMD occurred. Most of the virus infections on those farms were "neighborhood infections". Because the situation seemed out of control locally and the destruction capacity became insufficient, it was decided to implement an emergency vaccination strategy for all biungulates in a large area around Oene to stop further spread of the virus. All susceptible animals on approximately 1800 farms in this area were vaccinated. All farms subsequently were depopulated, starting from 2 weeks after vaccination. In total, 26 outbreaks were detected (the last outbreak on 22 April 2001). In total, approximately 260,000 animals were killed.

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus.

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.

Discrimination of different subsets of cytolytic cells in pseudorabies virus-immune and naive pigs.

We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6(+) CD8(bright+) CD4(-) (CTL phenotype), CD6(+) CD8(dull+) CD4(+) (the fraction containing memory T helper cells), CD6(+) CD8(-) CD4(+) (T helper cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(+) ( gammadelta-T cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(-) (NK phenotype) and CD6(-) CD8(-) CD4(-) gammadelta-T(-) or gammadelta-T(+). Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6(+) CD8(+) and CD4(+) CD8(+) and fewer CD6(-) CD8(+) and gammadelta-T(+) CD8(+) lymphocytes than IL-2 stimulation of naive PBMC (P<0.05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6(+) CD8(+) T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6(-) CD8(+) T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.

A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection.

Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.

Cytolytic function for pseudorabies virus-stimulated porcine CD4+ CD8dull+ lymphocytes.

We previously observed that pseudorabies (PRV) virus-specific killing in vitro was mediated by CD6+ CD8+ lymphocytes. Also a high percentage of CD4+ lymphocytes, among these CD6+ CD8+ lymphocytes, was observed. The purpose of this study was, therefore, to further characterize the killing ability of PRV-stimulated CD4+ CD8+ lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from blood of PRV-immune pigs and were stimulated in vitro with PRV. After 6 days, the frequency of CD4+ CD8+ lymphocytes in peripheral blood was determined by flow cytometry analyses. Lymphocytes were separated using a magnet-activated cell sorter or a FACSVantage SE, and the cytolytic activity of the isolated populations was determined. Flow cytometry analyses demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of 26% +/- 4% CD4+ CD8dull+ lymphocytes. We further demonstrated that killing by PRV-stimulated PBMC was mediated by CD4+ CD8dull+ T lymphocytes and CD4- CD8+ T lymphocytes (classic cytolytic T lymphocytes and natural killer cells). The CD4+ CD8dull+ T lymphocytes showed major histocompatibility complex (MHC) II-restricted PRV-specific killing. The CD4- CD8+ T lymphocytes showed both PRV-specific and natural killing. The CD4+ CD8dull+ lymphocytes, which are unique in the pig, seemed to have a more heterogeneous function than was earlier demonstrated. In conclusion, we demonstrated that PRV-specific CD4+ CD8dull+ lymphocytes are able to kill PRV-infected target cells in a MHC II-restricted manner.

Evaluation of newly developed immunoperoxidase monolayer assays for detection of antibodies against bovine herpesvirus 4.

This study describes the evaluation of immunoperoxidase monolayer assays (IPMAs) for detection of antibodies against bovine herpesvirus 4 (BHV4) DN-599 or BHV4 LVR 140 in sera of cattle. We compared the quality of these IPMAs with the quality of a BHV4 indirect enzyme-linked immunosorbent assay (ELISA). In addition, a preliminary serological survey of BHV4 antibodies was carried out to estimate the seroprevalence of BHV4 in Dutch cattle at different ages. The specificities of both BHV4 IPMAs were 1.00. The geometrical mean titers (detection limit) of the BHV4 IPMAs were twice as high as that of the BHV4 indirect ELISA. In experimentally infected cattle, BHV4 antibodies were detectable by IPMAs 16 to 18 days postinfection, which was almost 2 weeks earlier than in the indirect ELISA. The reproducibility of the BHV4 DN-599 IPMA (kappaD value, 0.92) and of the BHV4 LVR 140 IPMA (kappaD value, 0.87) were good. For field sera the overall agreement between the BHV4 indirect ELISA and the two BHV4 IPMAs, DN-599 and LVR 140, was 95 and 96%, respectively. The serological-survey study showed that the estimated seroprevalence of BHV4 in Dutch cattle was 16 to 18% and that the percentage of BHV4-positive animals varied by age category (between 6 and 43%). In summary, the two BHV4 IPMA formats have several advantages that make IPMA a useful alternative to the BHV4 indirect ELISA for detecting BHV4 antibodies in cattle.

Pitfalls in the application of enzyme-linked immunoassays for the detection of circulating trypanosomal antigens in serum samples.

The experimental infection of two goats with Trypanosoma vivax trypanosomes provided samples for analysis using parasitology techniques and antigen-detection enzyme-linked immunosorbent assays (ELISAs) for T. vivax, T. congolense and T. brucei. Clinical, parasitological and serological findings were monitored during the course of infection to identify problems in the application of these ELISAs. The data clearly showed that the ELISAs examined were entirely unsuitable for the reliable detection of trypanosomal antigen. Consequently, research strategies pertinent to the development of a new generation of both antigen and antibody ELISAs are outlined considering the problems encountered. These were (1) the reactivity of the reagents; (2) the specificity of the reagents; (3) the nature of the test sample, e.g. the compartmentalisation of trypanosomes between plasma, serum and red blood cells; (4) possible interference with the ELISA through immune complexing; and (5) the biology of the host/trypanosome relationship to gain an understanding of fluctuations in trypanosomes in the systemic circulation.

Vaccination of pigs against pseudorabies virus with plasmid DNA encoding glycoprotein D.

We analysed the ability of a plasmid carrying the gene encoding glycoprotein D (gD) of pseudorabies virus (PRV) to induce humoral and cell-mediated immune responses and assessed the protection provided by PRV-gD DNA vaccination against challenge infection with PRV. Immunization with plasmid PRV-gD induced neutralizing antibodies and lymphocyte proliferative responses both in mice and pigs. Moreover, when challenged with virulent PRV six weeks following the last immunization, PRV-gD DNA vaccinated pigs excreted virus for a significantly shorter period and showed less clinical symptoms than pigs vaccinated with a control plasmid. Thus, in the target animal, DNA vaccination with PRV-gD DNA induces protective immunity against challenge infection.

Effect of vaccination route and composition of DNA vaccine on the induction of protective immunity against pseudorabies infection in pigs.

Vaccination with naked DNA may be an alternative to conventional vaccines because it combines the efficacy of attenuated vaccines with the biological safety of inactivated vaccines. We recently showed that the vaccination with naked DNA coding for the immunorelevant glycoprotein D (gD) of pseudorabies virus (PRV) induced both antibody and cell-mediated immunity in pigs and provided protection against challenge infection. To determine whether the efficacy of the naked DNA vaccination against PRV could be improved, we compared three sets of variables. First, the efficacy of the naked DNA vaccine coding only for the immunorelevant gD was compared with a cocktail vaccine containing additional plasmids coding for two other immunorelevant glycoproteins, gB and gC. Second, the intramuscular route of vaccination was compared with the intradermal route. Third, the commonly used needle method of inoculation was compared with the needleless Pigjet injector method. Five groups of five pigs were vaccinated three times at 4-weeks intervals and challenged with the virulent NIA-3 strain of PRV 6 weeks after the last vaccination. Results showed that although the cocktail vaccine induced stronger cell-mediated immune responses than the vaccine containing only gD plasmid, both vaccines protected pigs equally well against challenge infection. Intradermal inoculation with a needle induced significantly stronger antibody and cell-mediated immune responses and better protection against challenge infection than intramuscular inoculation. Our data show that the route of administering DNA vaccines in pigs is important for an optimal induction of protective immunity.

Silent memory induction in maternal immune young animals.

Maternal immunity was shown to be an effector mechanism which does not include transfer of memory. 'Boosting' of maternal immunity by vaccination was not effective. Transferred maternal immunity negatively interfered with the induction of optimal protection by vaccination. Antibody formation was not observed after vaccination of maternally immune piglets. In contrast, induction of memory had occurred in animals under maternal immune suppression. Vaccination in young animals negatively interfered with or abrogated, effective maternal immune protection. There was no correlation between specific serum antibody titres in piglets and protection to PRV. Thus apart from protection provided by antibodies contributions of other soluble factors and the cellular immune compartment as represented in colostrum and/or milk were important for protection.

Establishment and characterization of porcine cytolytic cell lines and clones.

Although non-major-histocompatibility-complex-restricted cytolytic cells appear to significantly influence antiviral immunity in pigs, the phenotype and functional characteristics of these cells are not well defined. To allow a detailed analysis of these subsets, we established and characterized cell lines and clones of interleukin-2-activated (IL-2) cytolytic cells. Cell lines and clones were obtained from peripheral blood mononuclear cells of minipigs of the swine-leucocyte-antigen-complex (SLA) d/d haplotype. Cells were cultured in the presence of human recombinant IL-2 and cloned by double limiting dilution in the presence of gamma-irradiated L14 cells (a retrovirus immortalized B-lymphoblastoid cell line of the haplotype SLAd/d) or gamma-irradiated autologous peripheral blood mononuclear cells as feeder cells. Cytolytic cell lines and clones were characterized for their ability to kill different target cells and for their cell surface phenotype. All obtained clones expressed CD2 and CD8 and were negative for CD4. The following three subsets of cytolytic cells were identified: Subset 1) CD3- CD5- cells that killed K562 cells (a natural killer cell susceptible target cell line), as well as the pseudorabies virus (PRV)-infected or uninfected porcine kidney cells. These cells were considered to be typical natural killer cells. Subset 2) CD3 gamma/delta + CD5- T-cells that killed K562 cells and PRV virus-infected or uninfected porcine kidney cells, infected or uninfected L14 cells, and L14 cells constitutively expressing the PRV viral glycoprotein gB or gC. These cells were considered to be gamma/delta T-cells with natural killer activity. Subset 3) CD3 alpha/beta + CD5+ T-cells that killed L14 cells, PRV-infected L14 cells, and PRV gB- and gC-transfected L14 cells. These cells were possibly induced by the L14 feeder cells, used in the in vitro culture system. None of the cytolytic effector cells killed only MHC-matched viral infected cells. In conclusion, we describe a method to isolate, clone, and culture cytolytic cells from pigs. The clones could be cultured for 5 months, which allowed appropriate phenotypic and functional characterization of the various clones. Two of the subsets, CD3 gamma/delta T- and the natural killer cell subset may be involved in antiviral immunity in this species.

Virulence, immunogenicity and reactivation of seven bovine herpesvirus 1.1 strains: clinical and virological aspects.

Specific pathogen-free calves were inoculated intranasally with one of seven strains of bovine herpesvirus 1.1 (BHV 1.1) to identify a highly virulent strain for use in vaccination-challenge experiments. The calves were monitored clinically and virologically. Clear differences in virulence between the strains were observed. The Iowa strain was the most virulent; the four calves infected with the strain had the most severe clinical signs; two of them died and viraemia was detected in three of them. To evaluate the immunogenicity of the seven strains all the calves were challenged 16 weeks later with the Iowa strain. The calves of a control group showed the typical signs of a BHV1 infection, whereas all the other calves were protected against disease and shed little or no virus. Hence, the differences in virulence were not associated with differences in immunogenicity. After the calves had been treated with dexamethasone, differences were observed between the strains in the amount of virus that was excreted.

Standardisation and validation of enzyme-linked immunosorbent assay techniques for the detection of antibody in infectious disease diagnosis.

Enzyme-linked immunosorbent assay (ELISA) techniques for the detection of antibodies are now widely used throughout the world for the diagnosis of infectious diseases in veterinary medicine. Although many laboratories have independently developed ELISA techniques for their own purposes, little progress has been made with respect to the international standardisation and validation of these techniques. This lack of international conformity is of major concern to organisations such as the Office International des Epizooties (OIE), the United Nations Food and Agriculture Organisation (FAO), the World Health Organisation (WHO) and the International Atomic Energy Agency (IAEA) which are involved in the establishment of international guidelines and programmes for the control, surveillance and/or eradication of infectious diseases. In this regard, a Joint FAO/IAEA Meeting of Consultants was convened in Vienna in January 1992 to review aspects of ELISA data expression, primary reference standards, quality assurance and diagnostic validation. Based on the consensus derived from this meeting, the authors describe procedures which are recommended as a platform on which to build definitive guidelines for international standardisation of ELISA protocols and reagents, in cooperation with the OIE and the OIE Reference Laboratories.