Human adenovirus vectors containing intact or largely deleted E3 region were used to construct adenovirus-hepatitis B recombinant viruses (Ad-HepB) and shown to produce substantial amount of recombinant protein, hepatitis B surface antigen (HBsAg), in tissue culture. Previously we showed that these viruses were able to elicit good anti-HBs antibodies in a dog model. In the present study, the Ad-HepB viruses were evaluated for replication and immunogenicity in chimpanzees which sustain permissive infection by human adenoviruses. Recombinants containing entire E3 region showed better replication pattern than their E3 deleted counterparts as evidenced by longer duration and high titers of virus shedding. The effect of E3 region was also seen in the antibody titers against HBsAg in that the E3 containing viruses showed better response than the E3 deleted viruses. The importance of E3 region for the development of adenovirus vectored vaccines is further discussed.

Recombinant human adenovirus (Ad) type 4-, 5-, and 7-vectored vaccines expressing either the HIV env or gag-protease genes were tested for immunogenicity in three chimpanzees. The first phase of the vaccination protocol consisted of a primary and two booster immunizations with Ad-HIVs by the oral route of administration, followed by a single booster immunization with Gag and/or Env subunit vaccines. The second phase of the vaccination protocol consisted of intranasal administration of Ad-HIVs previously administered by the oral route. Following the first phase adenovirus was shed into stools for only 1-7 days and modest type-specific anti-adenovirus neutralizing antibody titers were induced. Strong anti-Env binding antibody responses were detected in all three animals following the second oral booster immunization. One chimpanzee responded with a low-titered type-specific neutralizing antibody response to HIV. Cell-mediated immune responses to Env were not detected after the primary vaccination, but were detected following all booster immunizations. Administration of the Gag subunit vaccine boosted both humoral and cell-mediated immune responses to Gag antigens. In contrast, the Env subunit vaccine boosted cellular but not humoral immune responses. In the second phase of the vaccination protocol, both virus shedding and anti-adenovirus responses were enhanced. All three chimpanzees responded to the intranasal administration of Ad7-HIVs with boosted anti-HIV serum responses, including low-titered type-specific neutralizing antibodies, elicited anti-HIV antibodies at secretory sites, and stimulated cell-mediated immune responses to both Gag and Env antigens.

The effect of rev (art/trs) gene on the level of HIV-1 envelope (env) expression using recombinant adenovirus was investigated. Recombinant adenoviruses expressing either the envelope or the rev gene of the human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the gene into an expression cassette. The expression cassette contained the adenovirus type 7 major late promoter, followed by leader 1 of the adenovirus tripartite leader and a portion of intron between leaders 1 and 2, leaders 2 and 3, and a hexon polyadenylation signal. The cassette was then inserted at the terminal region between the E4 and ITR regions of the adenovirus 7 genome with a concomitant E3 region deletion (80-87 m.u.). A549 cells infected with the recombinant virus containing the env gene produced the envelope glycoproteins gp160, gp120, and gp41. HIV-1 envelope gene expression was greatly enhanced (20- to 50-fold) in the cells that were simultaneously infected with the recombinant adenovirus containing the rev gene as measured by ELISA and Western blotting. Interestingly, this effect was observed despite the lack of the 5' down splice site for rev and seems to be post-transcriptional. Another recombinant adenovirus which contains both the rev and the env genes was constructed by inserting the rev gene in the deleted E3 region and the env gene in the terminal cassette. This double recombinant virus expressed high levels of env antigen in A549 cells similar to those attained upon co-infection with two separate recombinant viruses containing the rev or env gene. Furthermore, the rev gene nucleotide sequence could be altered without altering the amino acid sequence and its sequences truncated by 17 amino acids from the C-terminus had no effect of rev function.

Click to increase image sizeClick to decrease image sizeKey Words: recombinant viral vaccinesrecombinant adenovirus vectorsadenovirus vectors in AIDS vaccines

We report here the cloning and sequencing of the major late promoter (MLP) and the tripartite leader (TPL) from simian adenovirus type 30 (sAd30) and the comparison of the sAd30 nucleotide (nt) sequence with that of human adenoviruses (hAd). The nt sequence homology between sAd30 and hAd2 is 75% from -66 to +190 relative to the cap site. This sAd30 MLP segment contains the upstream regulatory sequence element, TATA box, and downstream regulatory sequence elements that are homologous to hAd MLP. The sAd30 upstream regulatory sequence has a small palindromic DNA sequence GTCACGTGAC, and the TATA box contains the sequence of ATAAA instead of TATAAA. The sAd30 TPL was located on the sAd30 genome as identified by sequence homology with the hAd counterpart. The splice sites of TPL introns were confirmed by sequence analysis of cDNAs synthesized from sAd30-infected cells. There is a 74.2% nt sequence homology between the TPL of sAd30 and hAd2. The conservation of these sequence elements during evolution of Ad suggests that they are essential for the transcription and translation of Ad ML transcripts.

A DNA sequence coding for the second kringle of tissue-type plasminogen activator (t-PA) was synthesised chemically and inserted just before the double-kringle region of the t-PA gene. This tris-kringle PA or hybrid F gene was expressed in a BPV-based expression vector and the expression product was purified to homogeneity. The specific activity of the protein on fibrin-agar plate was about 250 000 iu/mg compared to ∼ 500 000 iu/mg for rt-PA. Analysis of the protein by SDS-PAGE showed that it was present mainly in the single chain form and displayed doublet bands of Mr 68 000 and 73 000 Da, probably due to differential glycosylation. Although hybrid F showed tighter binding to lysine-Sepharose, its binding to a fibrin clot was comparable to that of t-PA. Hybrid F and t-PA were found to have comparable amidolytic activities toward a synthetic chromogenic substrate, S-2288, suggesting that the extra kringle in hybrid F did not affect its catalytic function. However, hybrid F exhibited considerably lower activity in fibrin-dependent plasminogen activation characterised by higher Km and lower kcat values than the native t-PA. This shows that addition of an extra kringle in t-PA somehow interferes in the ternary complex formation with fibrin and plasminogen and thereby adversely affects its fibrin-stimulated activity.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.

A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene.

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.