Expression and immunogenicity of the spike glycoprotein of porcine respiratory coronavirus encoded in the E3 region of adenovirus.
The full length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen to an amount of approximately 33 micrograms per 10(6) cells, as determined by ELISA. The antigen was cell-associated except in the late phase of the infection, when a low amount (4 micrograms per 10(6) cells) was released in the culture supernatant. The cell-associated antigen consisted of 2 polypeptides of 160 K and 175 K, respectively. The 160 K polypeptide comigrated with the authentic S' precursor from PRCV-infected cells. The 175 K polypeptide had the same mobility as the authentic mature S protein from PRCV-infected cells and from PRCV released in the supernatant. The extracellular recombinant antigen corresponded with the 175 K mature protein. Immunofluorescent staining gave evidence that some recombinant S protein was exposed on the cell surface; it also showed that the protein was recognized by conformation-specific anti-S monoclonal antibodies. Piglets, immunized oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV-challenge.
Callebaut
P
P
Laboratory of Virology, Faculty of Veterinary Medicine, University of Gent, Belgium.
Pensaert
M
M
D000260:Adenoviruses, Human / Q000235:genetics / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017934:Coronavirus / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D018352:Coronavirus Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D017931:DNA Primers; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000276:immunology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012141:Respiratory Tract Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D064370:Spike Glycoprotein, Coronavirus; D013552:Swine; D013553:Swine Diseases; D013737:Testis / Q000821:virology; D013997:Time Factors; D014158:Transcription, Genetic; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000276:immunology*; D014765:Viral Vaccines; D014779:Virus Replication
D017931:DNA Primers; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus