Michael D. Lubeck

Nonranom association of parental genes in influenza A virus recombinants

Abstract Analysis of gene pairs in 40 recombinants derived from influenza A/PR/8/34 and A/HK/8/68 viruses demonstrates that reassortment of gens in recombinant viruses is not completely random with respect to all gene pairs. In particular, recombinants in which P1 and P2 and P2 and P3 genes were derived from the same strain were observed at frequencies much higher than expected. Conversely, recombinants in which P1 and P2 or P2 and P3 were derived from different strains were observed much less frequently than expected. The results suggest that these pairs of genes might be “linked”. Less significant “linkage” was observed within the P1 and NP, P1 and P3, and P3 and NS gene pairs. Examination of all other gene pairs revealed random derivation from the two parental viruses. These observations can be explained either by differential interaction of RNA segments or by a requirement for effective protein-protein interactions for gene products involved in RNA transcription and/or replication.

Efficacy of adenovirus-vectored respiratory syncytial virus vaccines in a new ferret model

In the absence of an adequate small animal model for testing the efficacy of adenovirus-vectored respiratory syncytial virus (RSV) vaccines, a ferret model was established for this purpose. Recombinant adenovirus types 4, 5 and 7 expressing the RSV fusion glycoprotein (F), the attachment glycoprotein (G) or both F and G were constructed previously. These recombinants contain a deletion of a large portion of the E3 region of the respective adenovirus vector. In addition, an Ad7(E3+)F recombinant virus which contains an intact E3 region was constructed to assess whether E3 region functions might enhance vaccine immunogenicity. Evaluation of these viruses in the ferret model demonstrated that Ad4 and Ad5 recombinants, administered intranasally to ferrets, induce stronger seroresponses to RSV than do Ad7 recombinant viruses. Ad7(E3+)F did not show enhanced immunogenicity relative to E3-deleted recombinant viruses. However, measurement of RSV infectivity in nasal washes, following intranasal RSV challenge, showed that five different vaccination regimens, Ad7F/Ad4F, Ad7G/Ad4G, Ad7FG/Ad4FG, Ad4F/Ad7(E3+)F and Ad5F/Ad4F, protected ferrets from RSV infection in a dose-dependent manner.

Evaluation of adenovirus type 4 and type 7 recombinant hepatitis B vaccines in dogs

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 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.

Antigenic variants of influenza viruses: Marked differences in the frequencies of variants selected with different monoclonal antibodies

Abstract Monoclonal antibodies against the hemagglutinin of two influenza A viruses and one influenza B virus were used to analyze the frequencies of antigenic variant subpopulations in cloned virus seeds. Antigenic variants selected with monoclonal antibodies specific for different antigenic determinants were obtained over a wide range of frequencies. With one monoclonal antibody directed to the hemagglutinin of A/PR/8/34 virus antigenic variants were isolated at a frequency of 10- 4.4 , whereas another monoclonal antibody, specific for the hemagglutinin of X-31 virus, completely neutralized all infectious virus in the X-31 virus seeds examined, indicating a frequency of antigenic variation, for one seed at least, of less than 10 −8.1 .

Enhanced immunogenicity of hepatitis B surface antigen by insertion of a helper T cell epitope from tetanus toxoid

The currently marketed hepatitis B vaccines in the U.S. are based on the recombinant major hepatitis B surface antigen (HBsAg) of hepatitis B virus. Although a large majority of individuals develop protective immunity to HBV-induced disease after three immunizations, routinely a small but a significant percentage of the human population does not respond well to these vaccines. In this report, we describe the generation of a novel HBsAg molecule containing a Th epitope derived from tetanus toxoid (TT). Using recombinant DNA technology. the TT Th epitope (TTe) was inserted into the HBsAg coding sequence. Using a recombinant adenovirus expression system, HBsAg TTe chimeric protein was produced in A549 cells and found to be secreted into culture medium as 22 nm particles. The chimeric HBsAg particles were readily purified by immunoaffinity chromatography and their immunogenicity was evaluated relative to native HBsAg produced in an adenovirus expression system. When evaluated in inbred and outbred strains of mice, HBsAg TTe was shown to enhance several-fold the anti-HBs response relative to native HBsAg. Further enhanced responses were observed in mice primed with TT. This highly immunogenic form of HBsAg has promise as an improved HBsAg subunit vaccine.

Adenovirus vectors for gene expression

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.

Isolation and characterization of a highly attenuated respiratory syncytial virus (RSV) vaccine candidate by mutagenesis of the incompletely attenuated RSV A2 ts-1 NG-1 mutant virus

Ts-1, a temperature sensitive (ts) mutant of RSV, was previously derived from RSV A2 virus by mutagenesis with 5-fluorouracil (5-FU). Ts-1 was attenuated for adult volunteers and seropositive children but retained a low level of virulence in seronegative infant vaccinees as indicated by the occurrence of upper respiratory tract disease. Ts-1 NG-1, a more defective derivative of ts-1, was produced by mutagenesis of ts-1 with nitrosoguanidine. However, ts-1 NG-1 still retained a low level of virulence for the upper respiratory tract and showed some genetic instability in chimpanzees. With renewed interest in the goal of developing a live, attenuated RSV vaccine, we have now attempted to further attenuate ts-1 NG-1 by mutagenesis with 5-FU and 5-azacytidine. Four mutants that are phenotypically different from the ts-1 NG-1 parental virus were identified. Each of the four mutants was more restricted in replication in BALB/c mice compared with the ts-1 NG-1 parental virus. One of the ts-1 NG-1 derivatives, termed A-20-4, which showed the lowest (35 degrees C) in vitro shutoff temperature and which was also completely restricted in replication in BALB/c mice, was selected for further evaluation in seronegative chimpanzees. A-20-4 did not cause rhinorrhea in chimpanzees but induced detectable titers of serum RSV neutralizing antibodies in 2 of 4 chimpanzees. Apparent complete protection to subsequent challenge with wild-type RSV was observed in each of the four chimpanzees previously immunized with A-20-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral Adenoviruses as the Carriers for Human Immunodeficiency Virus or Hepatitis B Virus Surface Antigen Genes

The development of virus vaccines based on live recombinant adenoviruses has received increasing attention in the recent past because live virus-vectored vaccines possess significant advantages for immunization against viral diseases relative to inactivated or subunit vaccines. A major strength of the adenovirus vector approach is immunization by oral administration of vaccine. An adenovirus recombinant vaccine is administered as an enteric-coated tablet, which ensures safe passage of virus to the intestines where asymptomatic replication occurs. Foreign viral proteins are thus expressed in the context of an infection of the gut. We believe that presentation of viral antigens in this manner is likely to evoke substantial humoral immunity, including secretory responses, as well as significant cell-mediated immunity. In addition, adenoviruses represent an attractive viral vector system for vaccine development because they exhibit several characteristics that allow for high expression of foreign viral genes. In this chapter several features of the adenovirus vector system as applied to its use for vaccination purposes will be reviewed, with an emphasis on the use of adenoviruses as vaccine vectors for diseases caused by hepatitis B virus (HBV) and human immunodeficiency virus (HIV).