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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 adenovirus (Ad)-human immunodeficiency virus (HIV) vaccines expressing HIVIIIB Env and Gag proteins were evaluated for immunogenicity in chimpanzees following intranasal administration. When Ad7-, Ad4-, and Ad5-vectored vaccines were administered sequentially at 0, 24, and 52 weeks, respectively, to three chimpanzees, the inoculations resulted in limited virus replication in the nasopharynx, but extensive Ad-HIV replication occurred in the intestine. High-titered IgG serum antibody responses to Env and Gag that were nonneutralizing were induced following booster administration of Ad4-HIV recombinant viruses. Following the Ad5-HIV booster, low levels of neutralizing antibodies as well as V3 loop antibodies were induced in all three chimpanzees that persisted for several months. Administration of a gp160 subunit vaccine (baculovirus derived) in SAF-m 24 weeks later boosted broadly neutralizing serum antibodies that peaked within 1 month of the injection. Two additional subunit boosters 19 and 37 weeks later were progressively less effective at stimulating serum neutralizing antibody responses. Substantial local immune responses were induced in nasal, vaginal, and salivary secretions following the third Ad-HIV intranasal immunization. These responses were further boosted with the gp160 subunit vaccine, which also stimulated production of rectal antibodies. The predominant responses in all secretions tested were of the IgG isotype, although some IgA responses were also detected. Strong blastogenic responses to HIV recombinant Env and Gag proteins were induced after each immunization.

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.

Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates.

Recombinant hepatitis B virus vaccines based on adenovirus (Ad) vectors Ad4 and Ad7 have been prepared. However, immunogenicity testing of such vaccines in experimental animals is difficult because these human adenoviruses exhibit a highly restricted host range. In this study, the dog was evaluated as a model for screening Ad4- and Ad7-vectored vaccines. Intratracheal inoculation of dogs with Ad4 and Ad7 induced substantial type-specific humoral immune responses that were significantly higher than responses obtained following pharyngeal or oral inoculations. Inoculation of dogs with recombinant Ad7 and Ad4 vaccines expressing hepatitis B surface antigen (HBsAg) elicited large antibody responses to HBsAg (anti-HBs). Substantial secondary anti-HBs responses were produced upon sequential immunizations with heterotypic Ad7 and Ad4 recombinant vaccines. These data thus indicate that the dog is a useful model for evaluating immune responses to vaccines based on Ad4 and Ad7 vectors.

Adenovirus type 7 vaccine strain was engineered to express foreign antigens from both the E 3 early promoter in the E 3 region and the major late promoter inserted between the E 4 region and the right inverted terminal repeat. This multiple expression vector was used to express hepatitis B core antigen (HBcAg), hepatits B e antigen (HBeAg), and hepatitis B surface antigen (HBsAg). The gene inserted in the E 3 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 E 3 region, the surface antigen gene of hepatitis B virus was inserted behind the major late promoter in the E 4 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 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.

High yielding adenovirus (Ad)-hepatitis B recombinant (Ad-Hep B) viruses were prepared by insertion of the hepatitis B surface antigen (HBsAg) gene into the early region 3 (E3 region) of Ad4 or Ad7 vectors containing intact or largely deleted E3 regions. Both E3-deleted and non-deleted recombinants produced about six- to eight-fold higher amounts of HBsAg than previously reported recombinants. These recombinant viruses were evaluated for immunogenicity in dogs which sustain abortive lung infections by Ad4 and Ad7. Recombinants containing E3 deletions elicited 10- to 12-fold stronger anti-HBs primary responses than previously evaluated low yield recombinants. Further immunizations with heterotypic Ad-Hep B recombinants induced substantial anti-HBs booster responses as well as anti-‘a’ epitope responses. In contrast, recombinant viruses containing intact E3 regions induced only weak or negligible anti-HBs responses, although such viruses induced strong antibody responses to the Ad vectors. The immunogenicity of high-yielding Ad recombinants correlated with temporal expression of HBsAg and thus the dog represents a valuable model for evaluation of immune responses to heterologous proteins that are expressed early and that do not require efficient DNA replication. Recombinants expressing HBsAg late in the infectious cycle require further testing in the fully permissive chimpanzee model. This study establishes that the E3-deleted high yield Ad4 and Ad7 recombinants represent promising live oral hepatitis B vaccine candidates.