Identification and characterization of vaccinia virus genes encoding proteins that are highly antigenic in animals and are immunodominant in vaccinated humans

Abstract

Vaccinia virus (W) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gtll expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of W, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins. * Corresponding author. MATERIALS AND METHODS Materials. Restriction endonucleases, modifying enzymes, isopropyl- ,-D-thiogalactopyranoside (IPTG), and protein A were obtained from Bethesda Research Laboratories, Inc., New England Biolabs, or Boehringer Mannheim Biochemicals. The radioisotopes [3H]thymidine, [35S]methionine, 125I, and [32P]dATP were from Amersham or ICN. Goat anti-rabbit peroxidase and goat anti-human peroxidase were from Organon Teknika, Cappel. Cloning, mapping, and DNA sequence analysis. DNA manipulations were carried out as previously described (31). DNA fragments obtained by digestion of viral and phage 386

Immunogenicity and protection efficacy of monovalent and polyvalent poxvirus vaccines that include the D8 antigen

“…Antibody raised against recombinant D8 protein neutralized IMV, soluble D8 protein inhibited viral absorption to the cells in a competitive manner and modified poxviruses with inactivated D8 demonstrated dramatically reduced infectivity (Hsiao et al, 1999). Immunization with recombinant D8 protein induced vaccinia-specific antibodies and proliferation responses (Demkowicz et al, 1992). Immunization with D8 DNA vaccine induced partial protection against intranasal challenge with IHD strain (Pulford et al, 2004).…”

“…D8L gene in vaccinia virus encodes an IMV envelope protein that is involved in cell-surface binding and adsorption of IMV to the cells (Maa et al, 1990). Antibodies to D8 were detected in mice after immunization with vaccinia virus (Demkowicz et al, 1992). Replication of vaccinia in brain tissues was significantly reduced when D8L gene was inactivated in recombinant viruses (Chernos et al, 1993).…”

Expression of N-methyl-D-aspartate receptors using vaccinia virus causes excitotoxic death in human kidney cells

“…Polyacrylamide gel electrophoresis and immunodetection of proteins transferred to Immobilon-P membranes (Millipore, Bedford, MA) were performed by standard procedures using the antibodies mentioned above or a rabbit polyclonal anti-vaccinia virus serum [Demkowicz et al, 1992] kindly supplied by Dr. M. Esteban. This serum recognizes both early and late viral proteins.…”

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

“…Antisera. The rabbit polyclonal anti-P4a (referred to in previous work as anti-62K), 14K, 39K, 15K, and 21K protein sera have been previously described (12,44,46), as has the polyclonal serum raised against live VV (45). Rabbit polyclonal antisera against P4b and VP8 were kindly provided by D. Hruby (University of Oregon).…”