Development of a Novel Canine Parvovirus Vaccine Capable of Stimulating Protective Immunity in Four-Week-Old Puppies in the Face of High Levels of Maternal Antibodies.

Many highly effective vaccines have been developed to protect dogs against disease caused by canine parvovirus, but despite this vaccine interference by maternally derived antibodies continues to cause immunisation failure. To help overcome this limitation we have developed a novel, recombinant canine parvovirus type 2c vaccine strain, based on the structural and non-structural elements of an established type 2 vaccine. This novel CPV-2c vaccine strain has unique efficacy in the field, it is able to induce sterilising immunity in naïve animals 3 days after vaccination and is able to overcome very high levels of maternally derived antibodies from 4 weeks of age-thus closing the immunity gap to canine parvovirus infection in young puppies. The vaccine strain, named 630a, has been combined with an established canine distemper virus Onderstepoort vaccine strain to produce a new bivalent vaccine (Nobivac DP PLUS), intended to immunise very young puppies in the face of high levels of maternally derived antibody. Here, we describe the onset of immunity and maternal antibody interference studies that support the unique efficacy of the strain, and present overdose studies in both dogs and cats that demonstrate the vaccine to be safe.

Generation and immunogenicity of virus-like particles based on mink enteritis virus capsid protein VP2 expressed in Sf9 cells.

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.

Isolation and sequence analysis of the complete H gene of canine distemper virus from domestic dogs in Henan Province, China.

Eighteen canine distemper virus (CDV) isolates were obtained from clinical samples in Henan province, China, between 2012 and 2016. These viruses could not be recognized by 1A4, a monoclonal antibody specific for the H protein of CDV vaccine strains. The complete haemagglutinin (H) genes of all 18 isolates were sequenced, and phylogenetic analysis showed that they segregated into two clusters within the Asia-1 genotype. Moreover, the H genes of four viruses were found to lack a potential N-glycosylation site at position 309, which is the most conserved site within the Asia-1 genotype of CDV, and a novel potential N-glycosylation site (amino acids 517-519) was found in strain HL013, which has not been reported previously. These results will help in achieving a better understanding of the evolution of CDV in China.

Genetic characterization of parvoviruses in domestic cats in Henan province, China.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) infections are highly contagious and cause serious enteric diseases, with high fatality rates of cats and dogs. Given the importance of cats as a potential source of genetic diversity for parvoviruses, parvovirus strains detected in cats with gastroenteritis signs were analysed, and molecular characterisation, sequence analysis and phylogeny were evaluated on the VP2 gene. The results showed that FPV, new CPV-2a, and CPV-2 are co-circulating in the cat population in Henan province of China. Moreover, CPV-2 strains (F2016020, and F2016021) with Ser297Ala substitution in VP2 protein was for the first time detected in cats with clinical gastroenteritis. This study provided new important findings about the evolutionary of parvovirus infection in domestic cats.

Potential for broad-spectrum protection against feline calicivirus using an attenuated myxoma virus expressing a chimeric FCV capsid protein.

It has previously been demonstrated that recombinant myxoma viruses expressing FCV capsid protein are capable of eliciting protective responses against virulent FCV challenge, following vaccination, in cats. An attempt was made to produce a bivalent myxoma recombinant expressing the capsid protein genes of both FCV strains F9 and LS015. The FCV capsid protein genes were inserted into the myxoma growth factor gene (MGF) locus, and the serine protease inhibitor (SERP 2) gene locus. Subsequent recombination between myxoma-FCV viruses resulted in a recombinant expressing a chimeric form of the capsid protein. Nonetheless, cats immunised with this myxoma-FCV recombinant demonstrate high levels of serum neutralising antibodies against both F9 and LS015 strains. Such a chimeric vaccine may provide effective protection against a wide range of FCV strains.

Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein.

Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma-FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma-FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat.

Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection.

Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria.

E3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants. This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus. Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs. A second construct, containing a different promoter, showed delayed growth and genome instability which, based on the size difference between these inserts, suggests a maximum packaging size of 106 to 109% wild-type genome size for CAV-1.