Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene.

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.

Co-expression of hepatitis B virus antigens by a non-defective adenovirus vaccine vector.

Adenovirus type 7 vaccine strain was engineered to express foreign antigens from both the E3 early promoter in the E3 region and the major late promoter inserted between the E4 region and the right inverted terminal repeat. This multiple expression vector was used to express hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), and hepatitis B surface antigen (HBsAg). The gene inserted in the E3 region was derived from the core gene of the hepatitis B virus genome. When the precore region was present, an immunoreactive group of proteins with molecular weights ranging from 15,000 to 19,000 was secreted into the media. Velocity sedimentation centrifugation of media and lysates from cells infected with recombinants containing the core gene with the precore region resulted in peaks of HBeAg at the top of the gradient where authentic HBeAg should be found. In addition to the core gene in the E3 region, the surface antigen gene of hepatitis B virus was inserted behind the major late promoter in the E4 region resulting in an adeno-hepatitis recombinant virus capable of expressing both the core gene and the HBsAg cells. Cells infected with the adeno-hepatitis recombinants could also be stained with peroxidase-conjugates after reacting to antibody against HBcAg. Inoculation of dogs with the recombinant viruses which contained the core gene, with and without the precore sequence, resulted in a significant antibody response to HBcAg/HBeAg. The dogs also produced a significant antibody response to HBsAg as well as neutralizing antibody to adenovirus.

Adenovirus vaccine vectors expressing hepatitis B surface antigen: importance of regulatory elements in the adenovirus major late intron.

Adenovirus types 4 and 7 are currently used as live oral vaccines for prevention of acute respiratory disease caused by these adenovirus serotypes. To investigate the concept of producing live recombinant vaccines using these serotypes, adenovirus types 4 (Ad4) and 7 (Ad7) were constructed that produce HBsAg upon infection of cell cultures. Ad4 recombinants were constructed that express HBsAg from a cassette inserted 135 bp from the right-hand terminus of the viral genome. The cassette contained the Ad4 major late promoter followed by leader 1 of the tripartite leader, the first intervening sequence between leaders 1 and 2, leaders 2 and 3, the HBsAg gene, and tandem polyadenylation signals from the Ad4 E3B and hexon genes. Using this same cassette, a series of Ad4 recombinants expressing HBsAg were constructed with deletions in the intervening sequence between leaders 1 and 2 to evaluate the contribution of the downstream control elements more precisely. Inclusion of regions located between +82 and +148 as well as +148 and +232 resulted in increases in expression levels of HBsAg in A549-infected cells by 22-fold and 44-fold, respectively, over the levels attained by an adenovirus recombinant retaining only sequences from +1 to +82, showing the importance of these elements in the activation of the major late promoter during the course of a natural Ad4 viral infection. Parallel increases were also observed in steady-state levels of cytoplasmic HBsAg-specific mRNA. When similar Ad7 recombinant viruses were constructed, these viruses also expressed 20-fold more HBsAg due to the presence of the intron. All Ad4 and Ad7 recombinants produced HBsAg particles containing gp27 and p24 which were secreted in the medium. When dogs were immunized intratracheally with one of these Ad7 recombinants, they seroconverted to both Ad7 and HBsAg to a high level.

Expression of HBV surface antigen or HIV envelope protein using recombinant adenovirus vectors.

Recombinant adenoviruses were constructed that contained either the HBsAg coding sequence or the HIV envelope protein coding sequence. The recombinant adenoviruses can replicate normally in cultured human cells. Cells infected with the adenovirus-HBV recombinant secreted HBsAg into the tissue culture medium. This HBsAg had immunological and physical properties similar to those of the 22-nm particles found in human serum. Expression of HIV envelope protein in cells infected with the adenovirus-HIV recombinant was demonstrated using cytoimmunofluorescence and immunoprecipitation. A hamster model was developed to evaluate the immunogenic properties of adenovirus-HBV recombinants. Hamsters inoculated intranasally with live adenovirus-HBV recombinant produced antibody against both adenovirus and hepatitis B virus surface antigen.

Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes.

Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322. Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined. The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids. Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide. The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively. Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al. (1984, J. Virol. 51, 860-862). A strong prokaryotic promoter sequence was also found between residues 434 and 462. A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable. The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology. Four of these regions showed variation between serotypes.