GALLO CALDERON marina beatriz
congresos y reuniones científicas
Plant biotechnology approaches for a peptide vaccine design against Canine Parvovirus and Canine Distemper Virus
Congreso; PEGS -The Essential Protein Engineering Summit; 2015
During the last few years, an increasing number of cases of sick dogs showing clinical symptoms compatible with Canine parvovirus (CPV) and Canine distemper virus (CDV) were reported in Argentina. Since many of these animals had received at least one vaccination, concerns were raised among breeders, owners and veterinary practitioners about the ability of the current commercially available vaccines to protect their pups. This situation was also noted in other countries around the world. CPV is a non-enveloped virus which belongs to the genus Parvovirus and carries a single stranded DNA genome of approximately 5,200 nucleotides (nt). The viral genome is enclosed into an icosahedral capsid made up of a combination of two proteins, Viral Polypeptide 1 and 2 (VP1 and VP2) and two non-structural proteins. About 90% of the capsid proteins are VP2, and most of the B cell epitopes are located on this protein. A few years after the discovery of the first characterized CPV2 strain (1978), new antigenic variants appeared. Currently, the original CPV2 strain is not circulating in dog population, although it is still present in vaccine formulations, whereas the variants CPV2a, CPV2b and CPV2c are distributed worldwide. On the other hand, CDV, which belongs to the genus Morbillivirus within the family Paramyxoviridae, is an enveloped virus containing a non-segmented, single-stranded, negative RNA genome of approximately 15,000 nt. This genome encodes the following proteins: matrix, fusion, hemagglutinin, nucleocapsid (NP), polymerase and phosphoprotein. NP epitopes from influenza virus (Orthomyxoviridae) and measles virus which belongs to the same genus of CDV, are recognized by cytotoxic T cells producing a Th1 response profile that plays an important role in recovery from the infection. We have developed molecular methods to detect these two viruses in rectal swab and clotted blood samples. Sequence analysis of the virus strains present in local samples showed that these strains are genetically distant from vaccine strains. In particular, we reported the high prevalence of CPV2c strain in Argentina as it was observed in other regions of the world. Therefore, the development of new vaccine formulations against CPV and CDV in a short period of time is required. VP2 from CPV and NP from CDV are good candidates as vaccine antigens due to its immunogenic properties. We propose a plant-based transient expression platform of VP2 and NP to produce these potential immunogens. This platform ensures the rapidly production of enough amounts of protein that allows the performance of several studies to analyse the antigenicity and immunogenicity of the recombinant proteins. Genes of full length VP2 and NP of local and vaccine strains were amplified by RT PCR. The primers used in the amplification reaction include the Kozak sequence and S2S sequence at ยด5 end. The amplified product were digested, purified and cloned into pENTR4 vector. Finally, these constructs and the pK7WG2 binary vector were recombined using the GATEWAY technology (Invitrogen). We obtained several constructs carrying the VP2 or NP coding regions that were introduced into Agrobacterium tumefaciens EHA101 competent cells by electroporation. The transgenes were transformed into the tobacco plants (Nicotiana tabacum) to express the heterologous proteins