Background paper. Progress towards a coronavirus recombinant DNA vaccine.
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Animals↗
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Protection induced by synthetic peptides corresponding to three serotypes in foot and mouth disease virus.
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Animals↗
Alphavirus-based expression systems.
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Alphavirus↗
Oral immunization of rabbits with VP60 particles confers protection against rabbit hemorrhagic disease.
Rabbit hemorrhagic disease virus (RHDV) causes more than 90% mortality in adult rabbits. In this study, the cDNA of the VP60 coding sequence of RHDV was cloned under the control of the polyhedrin and p10 promoters of baculovirus to be expressed in insect cells. The expression of RHDV VP60 under the control of the p10 promoter was 5-10 times higher than using the polyhedrin promoter. The p10-derived VP60 was able to assemble into virus-like particles (VLPs). RHDV VLPs were successfully used to protect rabbits against the disease even at doses as low as 0.5 micrograms when injected intramuscularly or subcutaneously. The ability to elicit an immune response was independent of the adjuvant or the route of immunization. Remarkably, oral administration of RHDV VLPs efficiently induced protecting antibodies to RHD at doses as low as 3 micrograms. The use of binary ethylenimine for the stabilization of the VLPs was decisive for eliciting a good oral immunity. This report demonstrates the potential use of these procapsids in obtaining RHD oral vaccines and opens the door to the use of these capsids for the prevention of the disease in wild animals. Therefore, a new, and potentially important application of recombinant VLPs in the induction of protective immunity by the oral route is foreseen.
Administration, Oral↗
Intercellular trafficking of the major tegument protein VP22 of bovine herpesvirus-1 and its application to improve a DNA vaccine.
Intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121-258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1-120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22-N.
Animals↗
Modified activity of a VP2-located neutralizing epitope on various vaccine, pathogenic and hypervirulent strains of infectious bursal disease virus.
Nine monoclonal antibodies (Mabs) to a vaccine strain of infectious bursal disease virus (IBDV) of intermediate virulence were characterized in Western-blot, radioimmunoprecipitation, ELISA additivity, and neutralization assays. At least two distinct serotype 1-specific conformation-dependent overlapping neutralizing antigenic domains were shown to be present on IBDV-VP2, and were respectively probed by Mabs 3 and 4, and by Mabs 6 and 7. Ten serotype 1 vaccine or pathogenic IBDV strains were tested for neutralization. Most mild or intermediate vaccine strains were efficiently neutralized by all Mabs, whereas US variant A, European pathogenic strain Faragher 52/70 and French hypervirulent isolate 89163 were not neutralized by Mabs 3 and 4. In addition, these two Mabs were shown to bind to the Faragher 52/70 strain, but not to the 89163 isolate, in an antigen-capture ELISA. These results suggest that a neutralizing epitope is possibly modified in European pathogenic IBDV strains, and that Mabs 3 and 4 may prove useful for antigenic differentiation between European classical and hypervirulent isolates.
Antibodies, Monoclonal↗
Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease.
The right hand end Nde I fragment 3 (90.8-100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.
Animals↗
Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins.
A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV.
African Swine Fever↗
Trypsin sensitivity of several human rhinovirus serotypes in their low pH-induced conformation.
Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.
Capsid↗
Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection.
Extracellular subviral particles produced by HeLa cells infected with a recombinant vaccinia virus encoding the prM and E genes of Japanese encephalitis virus (JEV) were purified and characterized. These particles contained the JEV prM/M and E proteins embedded in a lipid bilayer, and RNA was not detected in particles using the polymerase chain reaction and primers recognizing a part of the JEV E gene. The particles were uniformly spherical with a 20-nm diameter and had 5-nm projections on their surface. Mice that received a single inoculation of the purified extracellular particles emulsified with Freund's complete adjuvant were fully protected against 4.9 x 10(5) LD50 of JEV. Comparison of the neutralizing and hemagglutination-inhibiting antibody titers and radioimmunoprecipitation data showed that immunization with the particles induced an immune response similar to that following inoculation with the recombinant vaccinia virus.
Animals↗
Vaccination against feline immunodeficiency virus using fixed infected cells.
Crandell feline kidney cells and feline thymocytes, either feline immunodeficiency virus (FIV) infected or uninfected, were fixed with paraformaldehyde and used to vaccinate cats. The cells were mixed with a 30:70 water/mineral oil emulsion containing 250 micrograms ml-1 N-acetyl-D-glucosaminyl-beta-(1-4)-N-acetyl-muramyl-L-alanyl-D-isoglutam ine. Eighteen specific pathogen-free cats were vaccinated three times with 3-week intervals and challenged 21 days after the final boost with a low dose of the homologous FIV-UT113 strain. Eight out of ten cats that had received FIV-infected cell vaccines developed significant anti-FIV antibody titres to the envelope and core antigens. Neutralizing antibodies were detectable at the moment of challenge in the sera of these animals. Within 5 weeks after challenge 15 out of 18 cats became viraemic. Three animals, two that had been vaccinated with FIV-infected thymocytes and did not develop antibody, and one that had received an uninfected thymocyte preparation, remained uninfected for 6 months. Upon rechallenge of the three animals, two again resisted infection; these cats had been immunized with the infected and the uninfected thymocyte preparations, respectively.
Animals↗
Studies of fowlpox virus recombination in the generation of recombinant vaccines.
A p7.5/beta-galactosidase (7.5 lacZ) gene construct, cloned adjacent to the fowlpox virus (FPV) thymidine kinase (tk) gene was used as a marker to identify the products of recombination as 'blue' FPV plaques. The rFPVs were detected as early as 4 h after the introduction of plasmid DNAs and by 72 h post-infection (p.i.) for one transfer vector comprised 0.48% of the viral population. The proportion of rFPV increased linearly from 0.073% to 0.62% as the cumulative length of homologous sequences in the transfer vector increased from 0.73 to 4.5 kb. Two approaches using a second reporter gene, the Newcastle disease virus haemagglutinin-neuraminidase (NDV HN) gene were tested to differentiate between single and double cross-over events. In one, the HN gene was cloned into the FPV tk gene and the 7.5 lacZ cloned outside of the homologous region. Progeny of a single cross-over with FPV DNA generated an unstable plaque containing the HN gene and subsequent intramolecular recombination resulted in excision of the 7.5 lacZ and the generation of a stable 'white' plaque. For virus grown in CEF cells (tk+) in the presence of 5-bromo-deoxyuridine, only those viruses which contained a tk gene disrupted by the HN gene formed plaques. This approach allowed us to easily identify rFPV containing the HN gene but lacking 7.5 lacZ or other bacterial sequences. In a second approach, a double cross-over between rFPV DNA containing a stably expressed beta-galactosidase gene cloned into the tk gene (blue plaque) and plasmid DNA containing the HN gene flanked by tk sequences would allow transplacement of the 7.5 lacZ gene with the HN gene, and generating a white plaque. We were unable to generate recombinant viruses with the HN gene and which generated a white plaque, indicating that double cross-over events do not occur at a sufficiently high frequency in FPV.
Base Sequence↗
Novel approaches to foot and mouth disease vaccination.
In view of the problems associated with the use of conventional foot and mouth disease (FMD) vaccines, such as antigenic variability of the FMD virus, the cost of vaccination programmes and problems associated with vaccine storage, alternative approaches to the design of FMD virus are discussed. Identification of the antigenic site of the FMD virus and isolation or synthesis of the peptides which make up the antigenic site are considered for vaccine use.
Animals↗
Development of a vaccine for the prevention of AIDS, a critical appraisal.
The pathogenesis and clinical expression of HIV-1 infection in humans is considered in terms of classical pathogenetic studies of viral infections for which successful vaccines have been produced. The unique features of HIV pathogenesis are defined, and gaps in knowledge identified as a framework for considering designs for immune intervention. Envelope-derived candidate vaccines have been used in immunization and challenge experiments in SIV/macaque or HIV/chimpanzee models, presented either as vaccinia recombinant vectors or as subunits, singly or in sequence. These studies have been paralleled by clinical trials for safety and immunogenicity in seronegative individuals. Data generated will permit comparison of immune responses to specific antigens and delivery systems in animal models and in humans. In limited studies conducted under optimized conditions, non-human primates have been protected against virus challenge when immunized with some candidate vaccines or following passive transfer of high-titred antibody. Consideration of current information suggests that in order to prevent HIV infection it may be necessary to devise new strategies capable of inducing and maintaining high threshold titres of biologically relevant antibody as well as persistence of active cytotoxic T cells recognizing multiple epitopes.
AIDS Vaccines↗
Protection of rabbits against rabbit viral haemorrhagic disease with a vaccinia-RHDV recombinant virus.
In order to protect domestic and wild rabbits against RVHD, we constructed a recombinant vaccinia-RHDV virus, using the Copenhagen strain of the vaccinia virus. This recombinant virus expressed the RHDV capsid protein (VP60). Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in immunoprecipitation and indirect immunofluorescence with the antibodies raised against RHDV. The recombinant virus induced high level of RHDV specific antibodies in rabbits following immunization. Inoculations by both the intradermal and oral routes allow protection of animals against a challenge with virulent RHDV.
Administration, Oral↗
Immunogens of bovine viral diarrhea virus.
Bovine viral diarrhea virus (BVDV) is a ubiquitous pathogen of cattle that induces economically important diseases affecting multiple organ systems. In the United States, over 150 biological products are licensed for control of BVDV. These products contain live or killed BVDV, and many products contain other viruses or bacteria. Potency tests for these vaccines are based on animal inoculation and serology. For live virus vaccines, titration of viral infectivity in cell culture is an accepted alternative to animal inoculation. The immunogens in a killed virus vaccine may be measured by enzyme linked immunoabsorbent assay. Immunogens of BVDV that stimulate a protective immune response have not been conclusively identified. Epitopes on a putative viral envelope glycoprotein, gp53, are involved in viral neutralization. Other viral glycoproteins, gp48 and gp25, are immunogenic but epitopes on these proteins do not stimulate production of antibodies that efficiently neutralize virus. Progress in developing meaningful in vitro assays for quantitation of BVDV immunogens awaits identification of viral proteins that stimulate a protective immunity.
Animals↗
Immunogens of rotaviruses.
Rotaviruses cause gastroenteritis in neonates of many animal species including cattle, swine, horses, dogs, cats, chickens and turkeys. Rotavirions are nonenveloped, are about 75 nm in diameter, have a double capsid, and contain 11 double-stranded RNA segments as their genome. Several antigenically distinct groups of rotaviruses have been identified and have been alphabetically designated as A through G. Group A rotaviruses were the first group of rotaviruses isolated and are the most commonly detected rotaviruses in diarrheic animals. Group A rotaviruses have two surface proteins, VP4 and VP7, both of which are important in serotype determination and in inducing neutralizing antibodies and protective immunity. Multiple serotypes of group A rotavirus based on glycoprotein VP7 (designated as G types) and based on VP4 (P types) have been identified. The immune response to rotaviruses is essentially serotype specific, however, cross-reactive or heterotypic epitopes have also been identified. Currently acceptable methods for immunogen quantitation include the induction of neutralizing antibody in host or laboratory animals. The in vivo efficacy of vaccines against rotavirus-associated gastroenteritis remains the standard method against which in vitro methods must be compared. Several animal models have been developed which could potentially be used in evaluating the efficacy of candidate vaccines. Monoclonal antibodies to rotavirus immunogens are also currently available and serve as valuable reagents for in vitro quantitation of rotaviral immunogens.
Animals↗
Influence of dose and route of inoculation on responses of chickens to recombinant fowlpox virus vaccines.
The influence of dose and route of inoculation on responses of chickens to vaccination with recombinant fowlpox viruses (rFPVs) expressing an influenza haemagglutinin (HA) (FPV-HA) and the infectious bursal disease virus (IBDV) VP2 antigen (FPV-VP2) has been evaluated. Antibody responses to influenza and fowlpox virus were generated following vaccination via the wing web by subcutaneous inoculation or skin scarification. Intranasal and conjunctival inoculation failed to induce antibodies to FPV or influenza. Following direct intratracheal inoculation antibodies developed to influenza but not FPV. Dose response studies with the FPV-HA and FPV-VP2 recombinants showed that good responses to FPV and the vaccine antigen could be generated over a wide (10000 fold) dose range following wing web inoculation. The responses generated by the FPV-VP2 recombinant over this vaccine dose range protected against IBDV infection of the bursae following challenge with the Australian IBDV 002/73 isolate. These data suggest that effective application of rFPVs for poultry vaccination may be restricted to wing web and parenteral routes of inoculation.
Animals↗