Knut Elbers

The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex

A field study was conducted to investigate the effect of vaccination against porcine circovirus type 2 (PCV2) in pigs suffering from porcine respiratory disease complex (PRDC). A total of 1542 pigs were allocated randomly into two treatment groups at approximately 20 days of age. Groups received either a Baculovirus-expressed recombinant PCV2 Open Reading Frame (ORF) 2 vaccine or placebo by single intramuscular injection. Median onset of PCV2 viraemia and respiratory signs occurred when animals were 18 weeks old. Vaccination reduced the mean PCV2 viral load by 55-83% (p < 0.0001) and the mean duration of viraemia by 50% (p < 0.0001). During the period of study (from 3 to 25 weeks of age) vaccinated animals exhibited a reduced mortality rate (6.63% vs. 8.67%, difference -2.04%; p = 0.1507), an improved average daily weight gain (649 g/day vs. 667 g/day; difference +18 g/day; p < 0.0001) and a reduced time to market (164.8 days vs. 170.4 days; difference -5.6 days; p < 0.0001). The effects on performance were greatest in the 8-week period between the onset of PCV2 viraemia and the end of finishing. These data demonstrate that vaccination against PCV2 alone can significantly improve the overall growth performance of pigs in a multi-factorial, late occurring disease complex such as PRDC.

Reduction of PMWS-associated clinical signs and co-infections by vaccination against PCV2

The effects of a single-dose recombinant Porcine circovirus type 2 (PCV2) open reading frame 2 (ORF2) subunit vaccine were studied in a post-weaning multisystemic wasting syndrome (PMWS)-affected pig herd. A total of 1519 3-week-old piglets were allocated randomly into two treatment groups and either vaccinated against PCV2 or treated with a placebo. Study animals were followed from the time of vaccination until the end of finishing. Onset of PCV2 viraemia and clinical signs of PMWS (wasting, cough, dyspnoea, pallor and lethargy) were observed when animals were approximately 9-10 weeks old. Compared to placebo-treated animals, vaccinated animals had a significantly reduced PCV2 viral load and duration of viraemia (p < 0.0001). This reduction in viraemia was not affected by the level of maternal anti-PCV2 antibodies present at the time of vaccination. During the period of viraemia (10-26 weeks of age) vaccinated animals exhibited a 53% reduction in mortality rate (p = 0.0010), a 4.84 kg higher body weight gain (p < 0.0001) and a significant reduction in clinical signs (p < or = 0.0004). Furthermore, lung samples of vaccinated animals had a considerably reduced number of co-infections with PRRSV and Mycoplasma hyorhinis than lung samples of placebo-treated animals. These data indicate that vaccination against PCV2 alone protects pigs from clinical signs and co-infections associated with PMWS.

CLASSICAL SWINE FEVER VIRUS-SPECIFIC CYTOTOXIC T LYMPHOCYTES AND IDENTIFICATION OF A T CELL EPITOPE

Classical swine fever virus (CSFV)-specific cytotoxic T lymphocytes (CTL) were derived from peripheral blood mononuclear leukocytes of immunized NIH-minipigs (MHC d/d haplotype) after in vitro restimulation with infectious CSFV. Their cytotoxic activity was determined against CSFV-infected target cells obtained from simian virus 40 (SV40) large T antigen-transfected immortalized kidney cells of a syngeneic miniature swine. Experiments with separated effector cell populations revealed that the CSFV-specific cytotoxic activity was mediated by CD4(-)CD6+CD8+ MHC class I-restricted T lymphocytes. Infection of target cells with various vaccinia virus/CSFV recombinants led to the identification of a major antigenic site for CSFV-specific CTL near the cleavage site between the non-structural proteins p80 (NS3) and p10 (NS4a). Using synthetic overlapping nonapeptides which covered this protein region the sequence ENALLVALF is the first sequence to be identified as an MHC class I-restricted T cell epitope recognized by CSFV-specific CTL.

Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting Erns RNase and Npro protease.

ABSTRACT Different genetically engineered mutants of bovine viral diarrhea virus (BVDV) were analyzed for the ability to establish infection in the fetuses of pregnant heifers. The virus mutants exhibited either a deletion of the overwhelming part of the genomic region coding for the N-terminal protease Npro, a deletion of codon 349, which abrogates the RNase activity of the structural glycoprotein Erns, or a combination of both mutations. Two months after infection of pregnant cattle with wild-type virus or either of the single mutants, the majority of the fetuses contained virus or were aborted or found dead in the uterus. In contrast, the double mutant was not recovered from fetal tissues after a similar challenge, and no dead fetuses were found. This result was verified with a nonrelated BVDV containing similar mutations. After intrauterine challenge with wild-type virus, mutated viruses, and cytopathogenic BVDV, all viruses could be detected in fetal tissue after 5, 7, and 14 days. Type 1 interferon (IFN) could be detected in fetal serum after challenge, except with wild-type noncytopathogenic BVDV. On days 7 and 14 after challenge, the largest quantities of IFN in fetal serum were induced by the Npro and RNase-negative double mutant virus. The longer duration of fetal infection with the double mutant resulted in abortion. Therefore, for the first time, we have demonstrated the essential role of both Npro and Erns RNase in blocking interferon induction and establishing persistent infection by a pestivirus in the natural host.

The live attenuated bovine viral diarrhea virus components of a multi-valent vaccine confer protection against fetal infection.

Fetal infection with bovine virus diarrhea virus (BVDV) causes severe economic loss and virus spread in cattle. This study investigated the ability of modified live BVDV I and II components of a commercially available modified live virus (MLV) vaccine (Breed-Back FP 10, Boehringer Ingelheim Vetmedica Inc.) to prevent fetal infection and abortion, and therefore the birth of persistently infected animals. Heifers immunized with vaccine 4-8 weeks before insemination showed no adverse effects. All vaccinated animals had seroconverted to BVDV 4 weeks after immunization. Pregnant heifers were divided into two vaccination and two control groups and challenged with type I or II BVDV on days 60-90 of gestation. Seroconversion, clinical signs, immunosuppression, viremia, mortality, abortion rate, and fetal infection were studied. Post-challenge, 6/11 (type I challenged) and 8/11 (type II challenged) vaccinated heifers were free from clinical signs of BVD. Post-challenge clinical signs noted in the vaccinated groups were mild to moderate, while all unvaccinated controls had clinical signs ranging from moderate to severe. Viremia was not detected post-challenge in any of the vaccinated heifers. However, 100% of the controls were BVDV viremic on at least 1 day post-challenge. One of 22 vaccinated heifers had transient leukopenia, whereas 2/8 and 6/7 unvaccinated heifers in control groups I and II, respectively, had transient leukopenia. Type II BVDV infection led to abortion or death in 86% of unvaccinated heifers. The corresponding vaccinated group showed no deaths or abortions. All control group fetuses were infected with BVDV. The test vaccine gave 91% (type I BVDV challenged) and 100% (type II BVDV challenged) protection from fetal infection. This vaccine is safe and effective against fetal infection, abortion (type II BVDV) and the birth of persistently infected animals.

Deletion of gene 52 encoding glycoprotein M of equine herpesvirus type 1 strain RacH results in increased immunogenicity

The immunogenicity of equine herpesvirus type 1 (EHV-1) strain RacH was compared to a RacH virus in which gene 52 encoding glycoprotein M (gM) was interrupted by insertion of LacZ (HDeltagM-Ins) and a RacH with 75% of gene 52 was deleted and replaced by LacZ (HDeltagM-HS). HDeltagM-Ins failed to produce full-length gM, but the carboxy-terminal portion was still expressed. No gM expression was detected in HDeltagM-HS-infected cells. Mice were immunised once with 1x10(3) to 1x10(5) plaque-forming units (PFU) of RacH or mutant viruses and challenged with virulent RacL11 virus 29 days later. A dose-dependence of protection was observed in RacH-immunised mice, and following immunisation with 1x10(4) or 1x10(3) PFU body weight losses and increased virus titres in lungs were observed after challenge infection. HDeltagM-HS-immunised mice were completely protected even after immunisation with 1x10(3) PFU. Mice immunised with 1x10(3) PFU of HDeltagM-Ins but not the higher doses showed signs of disease after challenge infection.

Introduction to RNA Vaccines

RNA vaccines are attractive, because they exhibit characteristics of subunit vaccines and live-attenuated vectors, including flexible production and induction of both humoral and cellular immunity. While human proof-of-concept for RNA vaccines is still pending, the nascent field of RNA therapeutics has already attracted substantial industry and government funding as well as record investments of private venture capital. Most recently, the WHO acknowledged messenger RNA (mRNA) as a new therapeutic class. In this chapter, we briefly review key developments in RNA vaccines and outline the contents of this volume of Methods in Molecular Biology.