Influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness.

In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process-from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.

[Development of influenza vaccines against newly emerging A/H5N1 virus].

Emergence of highly virulent influenza A/H5N1 viruses in Hong Kong in 1997 posed a threat of pandemic and brought an urgent need to develop a suitable seed virus for vaccine production. The virulence of the H5N1 viruses to chicken embryos should hamper the efficient production of the vaccine. In addition, potential virulence to humans raised safety issue in manufacturing vaccine. Toward vaccine development, one approach is to use an avirulent avian influenza virus antigenically similar to the virulent ones as a surrogate vaccine strain. The other approach is based on the attenuation of pathogenicity of virulent H5N1 virus by genetic engineering of the hemagglutinin gene and selection of a gene constellation. The reverse genetics technique can make the latter approach possible. Candidate strains suitable for vaccine production could be prepared by using either approach.

[Development of influenza vaccines against pandemics].

Influenza vaccine plays a crucial role as an effective measure for control of worldwide influenza pandemics. Currently licensed inactivated influenza vaccines can be used for the pandemic situation. Surveillance to isolate animal influenza viruses as vaccine candidate strains greatly contributes to the rapid production of vaccine. The reverse genetics approach can afford to attenuate a highly virulent virus by genetic engineering of any viral genes for the use of vaccine strain. The other approach to develop influenza vaccine should be encouraged to overcome the disadvantage of currently used inactivated vaccine.

Avirulent Avian influenza virus as a vaccine strain against a potential human pandemic.

In the influenza H5N1 virus incident in Hong Kong in 1997, viruses that are closely related to H5N1 viruses initially isolated in a severe outbreak of avian influenza in chickens were isolated from humans, signaling the possibility of an incipient pandemic. However, it was not possible to prepare a vaccine against the virus in the conventional embryonated egg system because of the lethality of the virus for chicken embryos and the high level of biosafety therefore required for vaccine production. Alternative approaches, including an avirulent H5N4 virus isolated from a migratory duck as a surrogate virus, H5N1 virus as a reassortant with avian virus H3N1 and an avirulent recombinant H5N1 virus generated by reverse genetics, have been explored. All vaccines were formalin inactivated. Intraperitoneal immunization of mice with each of vaccines elicited the production of hemagglutination-inhibiting and virus-neutralizing antibodies, while intranasal vaccination without adjuvant induced both mucosal and systemic antibody responses that protected the mice from lethal H5N1 virus challenge. Surveillance of birds and animals, particularly aquatic birds, for viruses to provide vaccine strains, especially surrogate viruses, for a future pandemic is stressed.

[Structure and function of influenza virus neuraminidase].

Crystallographic studies of neuraminidase and neuraminidase-sialic acid complexes enabled to design new potent antiviral drugs against influenza. In addition, recent advance in reverse genetics of influenza virus provides us a better understanding of interrelationship between structure and function of the neuraminidase. Progress in elucidating the relationship between structure and function will further contribute to the control of influenza.

In vivo induction of cytotoxic T lymphocytes specific for a single epitope introduced into an unrelated molecule.

Cytotoxic T lymphocytes (CTLs) recognise antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules on virus-infected cells. The formation and transportation of antigenic peptides to class I MHC in the cells are multi-step reactions known as antigen processing. In order to design a good DNA vaccine, it is important to dissect the specificity of antigen processing. Here we describe the construction of an epitope-based plasmid vector as a device to investigate antigen processing in transfected cells. The epitope-based plasmid vector was constructed by insertion of an epitope-encoding minigene into the lacZ gene. We used a CTL epitope on influenza A virus nucleoprotein (NP366-374 epitope) as a model. Upon transfection, the epitope-based plasmid vector induced the expression of NP epitope antigenically as well as immunogenically. Immunization of mice with plasmid-transfected cells was able to induce NP epitope-specific CTLs in vivo. Moreover, the plasmid vector functioned as a gene vaccine; NP epitope-specific CTLs were primed in vivo upon transfection of the vector into dermis by electroporation. The results suggest that this epitope-based DNA delivery system may provide a new strategy for in vivo induction of epitope-specific CTLs to investigate antigen processing and presentation.

Glycosylation of neuraminidase determines the neurovirulence of influenza A/WSN/33 virus.

The neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus has previously been shown to be associated with neurovirulence in mice and growth in Madin-Darby bovine kidney (MDBK) cells. Nucleotide sequence analysis has indicated that the NA of WSN virus lacks a conserved glycosylation site at position 130 (corresponding to position 146 in the N2 subtype). To investigate the role of this carbohydrate in viral pathogenicity, we used reverse genetics methods to generate a Glyc+ mutant virus, in which the glycosylation site Asn-130 was introduced into the WSN virus NA. Unlike the wild-type WSN virus, the Glyc+ mutant virus did not undergo multicycle replication in MDBK cells in the absence of trypsin, presumably because of lack of cleavage activation of infectivity. In contrast, revertant viruses derived from the Glyc+ mutant were able to replicate in MDBK cells without exogenous protease. Nucleotide sequence analysis revealed that the NAs of the revertant viruses had lost the introduced glycosylation site. In contrast to wild-type and revertant viruses, the Glyc+ mutant virus was not able to multiply in mouse brain. These results suggest that the absence of a glycosylation site at position 130 of the NA plays a key role in the neurovirulence of WSN virus in mice.

Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses.

We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter.

Evolutionary pathways of N2 neuraminidases of swine and human influenza A viruses: origin of the neuraminidase genes of two reassortants (H1N2) isolated from pigs.

The complete nucleotide sequences of the neuraminidase (NA) genes of two reassortant (H1N2) and two H3N2 influenza A viruses isolated from pigs were determined and phylogenetic relationships between these and previously reported N2 NA genes were investigated. On the basis of pairwise nucleotide sequence identity, the NA genes of two reassortants, A/sw/Kanagawa/2/78 and A/sw/Ehime/1/80, were most closely related to those of human influenza A virus strains isolated in 1972 and the earliest available swine H3N2 influenza A viruses, respectively. Phylogenetic trees showed that the NA genes can be segregated into three groups, including lineages for (i) swine strains, (ii) the earliest human strain and (iii) recent human strains. The evolutionary tree for the 11 nucleotide and amino acid sequences suggested that the NAs of A/sw/HK/4/76 and A/sw/Kanagawa/2/78 belong to the lineage for recent human viruses. In contrast, the NA genes of the A/sw/HK/3/76 and H1N2 reassortant A/sw/Ehime/1/80 viruses were found to be of a swine lineage. The swine virus NA genes were further characterized by the cocirculation of two distinct lineages. Although the rates of synonymous (silent) substitutions for the swine and human viruses were nearly identical (0.00946 to 0.00884 per site per year), the rate of non-synonymous (amino acid changing) substitutions for swine virus NA genes was about 60% of that for the human virus.

Homotypic and heterotypic protection against influenza virus infection in mice by recombinant vaccinia virus expressing the haemagglutinin or nucleoprotein of influenza virus.

Recombinant vaccinia virus expressing the influenza virus haemagglutinin (HA) or nucleoprotein (NP) genes from A/SW/Hong Kong/1/74 (H1N1) under the control of a hybrid promoter containing the P7.5 early promoter element and promoter of the gene encoding the major protein of cowpox virus A type inclusion body was constructed to investigate protective immunity against homologous and heterologous viruses in mice. These recombinant vaccinia viruses produced authentic influenza virus HA and NP in infected cells. The recombinant vaccinia virus-influenza virus HA conferred efficient subtype-specific protection although mice challenged with heterologous influenza viruses underwent initial infection. By contrast, immunization with the recombinant vaccinia-influenza virus NP limited virus multiplication in the lungs against challenge infection with all H1N1 and H3N2 influenza viruses examined, although less efficiently. These results will prompt the re-examination of the possibility of using the recombinant vaccinia virus-influenza virus NP as a cross-protective vaccine.

Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses.

The hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.

Characterization of antibody and cytotoxic T lymphocyte responses to human influenza virus H3 haemagglutinin expressed from the haemagglutinin locus of vaccinia virus.

Antibody and cytotoxic T lymphocyte (CTL) responses to the haemagglutinin (HA) of human H3N2 influenza virus were analysed, using recombinant vaccinia viruses containing the influenza HA gene inserted into the HA gene locus of vaccinia virus. The recombinant vaccinia viruses elicited a high haemagglutination inhibiting (HI) antibody response to the homologous influenza virus in mice. In addition, HI antibody generated by the recombinant vaccinia virus reacted with antigenic variants of human H3N2 influenza virus in a manner similar to that elicited by the HA vaccine. Mice with a high response to influenza virus HA vaccine were highly responsive to the HA expressed from the recombinant vaccinia virus, as measured by HI antibody production. The immunogenicity of the influenza virus HA expressed by the recombinant seems to be attributable to the intrinsic immunogenicity of the HA molecule. The recombinants primed mice for an influenza virus H3-specific CTL response and primed CTLs recognized the target cells in a subtype-specific manner. The results indicate that a recombinant vaccinia virus derived by the insertion of a foreign gene into its HA gene locus is a potent live vaccine not only for eliciting a high antibody response but also for priming a specific CTL response.

Biological and immunological characterization of influenza virus haemagglutinin expressed from the haemagglutinin locus of vaccinia virus.

Plasmid vectors were constructed to facilitate the insertion and expression of a foreign gene in the haemagglutinin (HA) gene locus of vaccinia virus. Five unique cloning sites adjacent to the P7.5 promoter of vaccinia virus permit the rapid insertion of a foreign sequence coding for a protein into these plasmids. This vector system provides a simple procedure to select recombinant viruses because they can be readily identified on the basis of their HA-defective phenotype. Recombinant vaccinia viruses expressing influenza virus HA were constructed to characterize the possible use of this system. The recombinant viruses did express the influenza HA through the authentic pathway of biosynthesis. In addition to having immunological characteristics similar to the authentic influenza HA, the expressed HA was found to possess haemagglutinating, haemadsorption and acid-inducible fusion activities. These findings demonstrate the usefulness of this eukaryotic vector system.

Synthesis of proteins in Escherichia coli immunoreactive with sera from individuals infected with human T-cell leukemia virus type I.

The env-pX IV fused gene of human T-cell leukemia virus type I (HTLV-I) was inserted into lac promoter-directed expression vectors for production of viral proteins in bacteria. Resulting recombinant plasmids, pK13 and pK15, directed synthesis of fused proteins of 59 kDa (Env-p40x) and 100 kDa (Gag-Env-p40x), respectively. Western blot analysis showed that these proteins were reactive with sera of patients with adult T-cell leukemia (ATL) and retained multiple antigenic determinants of viral proteins. In combination with recombinant Gag protein [S. Itamura, K. Shigesada, M. Imai, N. Kobayashi, T. Hamakado, T. Harada and M. Hatanaka, Gene 38, 57-64 (1985)], these bacterially synthesized proteins may provide a useful tool for differential diagnosis of ATL by detecting serum antibodies against individual viral proteins and for analysis of viral gene functions.

Construction of a new plasmid vector that can express cloned cDNA in all translational reading frames.

Construction of a bacterial expression vector, pSI4001, is described. The vector contains the lac promoter-operator and three sets of ribosome-binding sites (RBSs) tandemly arranged in all possible reading frames. cDNA can be directly cloned downstream from these translational start points in the fixed and proper orientation by using the method of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289]. The open reading frame of any cDNA inserted may be automatically aligned in phase with either of the three ATG start codons, thus enabling its expression with a maximum theoretical probability of unity. Fusion with the lacZ gene (coding for beta-galactosidase) has shown that at least two of the three translation initiation sites exhibit high expression capacities and the remaining one can also function at a lower but significant rate. We used the vector to construct a bovine pituitary cDNA library, from which clones coding for prolactin were detected by immunological screening with an efficiency as high as two in three clones. The construction with triple RBSs should also provide a unique experimental model to study the regulation of overlapping translations.

Genomic expressions of human T-lymphotropic virus (HTLV-I).

Human T-lymphocyte cell line termed MT-2 is producing persistently HTLV-I virion and has a strong potential to transform human T-lymphocytes when cocultivated. The virion of HTLV-I (MT-2) was isolated and its RNA was extracted to analyze the gene and gene products of HTLV-I. HTLV (MT-2) virion RNA was translated in a rabbit reticulocyte lysate system in vitro in which a gag precursor polyprotein (p53) and a putative gag-prt fusion protein (p76) were synthesized from a full length 35S RNA. The full length provirus, HTLV-I (MT-2), was molecularly cloned and its genomic expression was examined transiently and permanently by transfecting in human lymphoid and non-lymphoid cells. The cloned provirus expressed the same virological activities as observed in naturally occurring infection of the virus. A new protease gene of HTLV-I was found and its function of the gene product was studied.

Expression of the gag gene of human T-cell leukemia virus type I in Escherichia coli and its diagnostic use.

An expression plasmid, pHY202, was constructed which directs the synthesis of a fusion protein encoded by the gag sequence of human T-cell leukemia virus type I (HTLV-I) inserted into the lacZ' gene. Escherichia coli cells harboring pHY202 produced the 43-kDal LacZ'-Gag fusion protein with a yield of approx. 0.3% of total soluble proteins. The fusion protein is specifically recognized by monoclonal antibodies against the Gag proteins p19 and p24, and could be applicable for the diagnosis of HTLV-I infection, because almost all sera from HTLV-I carriers gave a positive response in the enzyme-linked immunosorbent assay (ELISA) employing the LacZ'-Gag hybrid protein purified by immunoaffinity column chromatography.