Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in -vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and '5I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the. HA The utility of poxviruses as eukaryotic expression vectors can be categorized as follows: (i) definition of the regulatory events in the viral replication cycle by following the expression of a foreign gene from a variety of early or late promoters or by analysis of foreign genetic elements in a cytoplasmic location, (ii) as a eukaryotic expression vector for the synthesis of biological products, and (iii) in the construction of live recombinant vaccines directed against both human and veterinary infectious diseases.The biological activity of vaccinia recombinants expressing a cloned hemagglutinin (HA) gene from influenza virus as an example of the production of live vaccines by the' use of appropriately modified poxviruses is the subject of this communication.MATERIALS AND METHODS Construction of Chimeric Donor Plasmids for in Vivo Recombination. Plasmids were constructed, analyzed, and purified using standard techniques. Plasmids were constructed for insertion of plasmid pBR322, DNA into vaccinia as follows. Isolated vaccinia HindIII fragment F was circularized by ligation with T4 DNA ligase. This circularized fragment F was then cut with BamHI. The linear inverted fragment F was ligated to BamHI-cleaved pBR322 that had been treated with alkaline phosphatase (7) and used to transform (8) competent Escherichia coli RRL (9). Recombinant plasmids were screened by restriction analysis of minilysates (10). Two plasmids that contained the inverted HindIII fragment F in opposite orientation within pBR322 were designated pDP301A and pDP301B. A plasmid (pJZ102) containing the cDNA sequence of the HA gene of influenza strain A/PR/8/34 (HINI) inserted into the HindIII site of pBR322 was obtained from P. Palese (Mt. Sinai, New York). The HA sequence in pJZ102 was reversed at the HindIII site by cutting with HindIII and religating. Plasmids with the HA sequence in opposite orientation were then designated pJZL2XA and pJZ102B. For insertion of the HA sequence directly into the BamHI site within the HindIII fragment F of vaccinia, a plasmid containing a Pst I subclone of the HindIII fragment F was constructed using plasmid pBR325 (11). To have a plasmid that contained only a single BamHI site, the BamHI site of pBR325 was removed by cleaving pBR325 ...
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