Canine parvovirus type 2 vaccine protects against virulent challenge with type 2c virus.

The ability of dogs vaccinated with a live attenuated CPV type 2 (Nobivac Intervet) vaccine to resist challenge with a current CPV2c isolate was investigated. Six SPF beagle dogs were given the minimum recommended course of vaccination, comprising a single inoculation of vaccine (Nobivac Lepto+Nobivac Pi) at 8-10 weeks of age followed 3 weeks later with a parvovirus vaccine in combination with distemper, adenovirus and parainfluenza virus (Nobivac DHPPi) and a repeat leptospirosis vaccine. Six control dogs were kept unvaccinated. All animals were challenged orally with a type 2c isolate of CPV and monitored for clinical signs, virus shedding, white blood cell fluctuations and serological responses. All vaccinated dogs were fully protected; showing no clinical signs nor shedding challenge virus in the faeces, in contrast to control animals, which displayed all the typical signs of infection with pathogenic CPV and shed challenge virus in the faeces.

Onset of immunity following in ovo delivery of avian metapneumovirus vaccines.

Avian metapneumovirus (aMPV) is an important cause of disease in chickens and turkeys. As infection can occur early in life and spread of the virus throughout a flock is rapid, an early onset of immunity post-vaccination would be advantageous. We have studied the serological immune response and the onset of protective immunity of an aMPV vaccine delivered to chickens via the in ovo route compared to oculonasal delivery at day old. A 1000-fold lower dose delivered in ovo to chicken specific pathogen free (SPF) embryos, than vaccination at day old, provided a significantly higher antibody response. In the presence of maternally derived antibody (MDA), there was no significant difference in antibody response between the vaccination routes. However, the onset of immunity (OOI) for the vaccine delivered to MDA positive chicken embryos was 5 days post-hatch in comparison to 8 days post-hatch for the same dose of vaccine given at day old indicating that chicks would be protected against disease earlier in the field if vaccinated by the in ovo route. In further experiments the OOI for a turkey vaccine delivered to MDA positive turkey embryos was shown to be 8 days post-hatch.

The efficacy of an avian metapneumovirus vaccine applied simultaneously with infectious bronchitis and Newcastle disease virus vaccines to specific-pathogen-free chickens.

Avian metapneumovirus (aMPV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) are important respiratory pathogens of chickens. To achieve early posthatch protection against all three diseases it would be helpful to deliver live aMPV, IBV, and NDV vaccines simultaneously at 1 day of age. However, previous work has indicated that the efficacy of aMPV vaccines may be affected when codelivered with IBV or NDV vaccines. The efficacy of an aMPV vaccine when codelivered to chickens in a trivalent combination with an NDV and an IBV vaccine was examined. The serological antibody response to the aMPV vaccine given with the IBV and NDV vaccine was significantly lower than when the aMPV vaccine was given alone. However, the aMPV vaccine did not affect the serological response to the IBV and NDV vaccines. Irrespective, the efficacy of the aMPV vaccine was not affected based on clinical signs postchallenge. This is the first report showing aMPV, IBV, and NDV vaccines can be codelivered without affecting the efficacy of the aMPV vaccine.

Molecular mimicry and ankylosing spondylitis: possible role of a novel sequence in pullulanase of Klebsiella pneumoniae.

Molecular mimicry has been shown between two sequences of Klebsiella pneumoniae pulD secretion protein (DRDE) with HLA-B27 (DRED) and pulA (pullulanase) enzyme (Gly-X-Pro) with types I, III and IV collagen respectively. IgG antibody levels in AS patients were elevated against 16mer synthetic peptides of HLA-B27 and pulD by enzyme immunosorbent assay (ELISA) compared to controls (P < 0.001). ELISA assays against K. pneumoniae grown in the absence and presence of pullulan demonstrated significant levels of IgA antibody in AS patients compared to controls (P < 0.001). Increased IgA and IgG antibody levels to pulA and types I and IV collagen were observed in AS patients compared to controls (P < 0.001). These observations could be relevant in the sequence of molecular events in AS.

Cloning, expression and immunogenicity of the avian pneumovirus (Colorado isolate) F protein.

The F protein of the Colorado isolate of avian pneumovirus (APV), expressed from a DNA plasmid, was recognized by antiserum to both A and B subgroup APVs. After two intramuscular injections of turkeys with this plasmid, a homologous antibody response was detected by enzyme-linked immunosorbent assay. This antibody also recognized subgroup A APV. However, there was no neutralization of the Colorado isolate or of subgroup A or B viruses. Although no significant clinical protection was detected following homologous challenge of poults, an anamnestic serological response was seen, suggesting that a systemic antibody response but no local mucosal immunity was induced.

Expression of chicken interleukin-2 by turkey herpesvirus increases the immune response against Marek's disease virus but fails to increase protection against virulent challenge.

As Marek's disease virus continues to evolve towards greater virulence, more efficacious vaccines will be required in the future. We expressed chicken interleukin-2 (IL-2) from a turkey herpesvirus (HVT) in an attempt to increase the efficacy of HVT as a vaccine against Marek's disease. The recombinant IL-2/HVT was safe for in ovo vaccination, although it replicated less in the birds compared with the parent HVT strain. Expression of IL-2 increased the neutralizing antibody response against HVT but did not increase the protection against virulent Marek's disease virus challenge.

A recombinant turkey herpesvirus expressing chicken interleukin-2 increases the protection provided by in ovo vaccination with infectious bursal disease and infectious bronchitis virus.

In ovo vaccination remains an attractive option for the mass application of vaccines to poultry, ensuring a uniform application of vaccine in a cost-effective manner. However, the number of vaccines that can be delivered safely by this method is limited. Several infectious bursal disease virus (IBDV) vaccines can be given in ovo though most are delivered post-hatch and there are no currently licensed embryo-safe infectious bronchitis virus (IBV) vaccines. Reduction in the dose of vaccines given in ovo is one possibility to ensure embryo safety though efficacy can be reduced when low doses are used. We have investigated the use of embryo-safe IBDV and IBV vaccines and the effects of co-delivery of a turkey herpesvirus recombinant expressing bioactive chicken IL-2 (IL-2/HVT). Co-delivery of the IL-2/HVT with low doses of the IBDV or IBV vaccines significantly increased the antibody response against these viruses. In addition the protection against challenge with virulent IBDV or IBV was increased significantly. This suggests that the co-delivery of IL-2/HVT with low doses of other vaccines in ovo may be one method to increase the number of vaccines that can be given safely and efficaciously via in ovo vaccination.

Safety and efficacy of an infectious bronchitis virus used for chicken embryo vaccination.

Commercial vaccines for in ovo vaccination have not yet been developed for infectious bronchitis virus (IBV), the major coronavirus in the poultry industry. Recombinant IBVs based on the Beaudette strain expressing the Beaudette spike protein (Beau-R) or that from the virulent M41 strain (BeauR-M41(S)) were assessed for their potential as prototype vaccines for application to 18-day-old embryos. Pathogenicity was assessed by observing the effect on hatchability, and/or the production of nasal discharge and/or the effects on ciliary activity in the trachea at various time points post hatch. In contrast to commercial IBV vaccines given in ovo, the Beau-R and BeauR-M41(S) strains did not reduce hatchability or cause nasal discharge, and caused minimal damage to the ciliated epithelium of the trachea. The presence of the spike protein from a virulent virus did not increase the pathogenicity of the virus according to the criteria used. Assessment of the BeauR-M41(S) strain for efficacy showed that it protected up to 90% of chicks against challenge with virulent IB virus (M41) in a dose dependent manner. Further egg passage of the BeauR-M41(S) strain (BeauR-M41(S) EP10) did not increase its pathogenicity though it did improve its efficacy, based on serology and protection against a virulent challenge. BeauR-M41(S) EP10 was more efficacious than BeauR-M41(S) protecting more birds against virulent challenge and providing a better serological antibody response. BeauR-M41(S) EP10 induced a serological response similar to that of a commercial vaccine given at day-old though the commercial vaccine provided slightly higher efficacy. These results are promising for the development of embryo safe efficacious IBV vaccines for in ovo application.

Priming in vivo and quantification in vitro of class I MHC-restricted cytotoxic T cells to human papilloma virus type 11 early proteins (E6 and E7) using immunostimulating complexes (ISCOMs).

Immunostimulating complexes (ISCOMs) efficiently deliver soluble antigen into both the cytosolic (endogenous) and endosomal (exogenous) pathways of antigen processing. Cytosolic delivery to antigen-presenting cells (APCs) may therefore be useful for the stimulation and assay of class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) in vitro. In this study, mice were immunized with ISCOMs containing fusion proteins of the E6 or E7 early proteins of human papilloma virus type 11 (HPV 11) to elicit CTL. These CTL were then restimulated in vitro using APCs pulsed with the same ISCOMs, prior to cytotoxicity assay using syngeneic target cells infected with recombinant vaccinia viruses. In this way, antigen-specific, MHC-restricted lysis by CD8+ cells was detected. However, this was dependent on the use of low density splenocytes as APCs for restimulation in vitro. Limiting dilution analyses showed a direct correlation between the CTL responder frequency and the number of times the animals were immunized in vivo. We conclude that in lieu of infectious virus, the use of ISCOMs to mediate antigen delivery to APCs in vitro can be used to quantitate CTL activity. This may have applications in monitoring vaccine efficacy, particularly to viruses such as HPV, which cannot be presently obtained as infectious virus in sufficient quantity for CTL propagation and assay.

Human cytotoxic T lymphocytes stimulated by endogenously processed human papillomavirus type 11 E7 recognize a peptide containing a HLA-A2 (A*0201) motif.

Cytotoxic T lymphocytes (CTL) may play an important role in the control of human papillomavirus (HPV)-induced anogenital neoplasias, but have been difficult to study owing to the difficulty in obtaining sufficient quantities of infectious virus. To address this we have stimulated human HPV-specific CTL in vitro using low-density cells (LDC) from peripheral blood mononuclear cells (PBMC). Low-density cells were used to present synthetic peptides, or endogenously processed peptides expressed from recombinant vaccinia viruses, to high-density PBMC (predominantly lymphocytes) for 6 days. Cytotoxic T lymphocytes stimulated with endogenously processed HPV 11 E7 recognized the synthetic HLA-A2 (A*0201) motif-containing nonamer, 4-12. In reciprocal experiments, CTL stimulated with this peptide in vitro recognized targets expressing endogenously processed E7. The responses in each case were A2 restricted and peptide specific. Two additional A2 motif-containing nonamers from HPV 6b E7 (21-30 and 47-55) also elicited peptide-specific, A2-restricted CTL. The data illustrate the potential that in vitro stimulation with LDC has in understanding CTL responses to experimentally problematic viral systems such as HPV, and may offer a route to specific immunotherapy of HPV-associated lesions.