Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection

The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.Avian H5N1 influenza viruses, currently circulating mainly in southeast Asia, are likely to cause the next influenza pandemic (18, 26, 37). The supply of embryonated eggs for traditional influenza vaccine production may be endangered in this case. Efforts to produce inactivated H5N1 vaccines in permanent cells have resulted in large-scale manufacturing, for instance, in Vero cells (21). This approach, based either on fermentation of H5N1 wild-type (wt) viruses (21) or on viruses attenuated by reverse genetics (9, 31), is the most straightforward strategy for egg-independent, rapid vaccine production.A further approach that may result in more widely available, egg-independent H5 vaccines is the use of recombinant viral vectors expressing protective antigens. Promising protection results were obtained so far with adenovirus-based vectors in mouse models (13, 14). Adenovirus vectors are usually produced in permanent complementing cell lines (11) and have been widely used in clinical trials. Cancellation of a recent trial involving human immunodeficiency virus adenovirus vectors due to suspected enhancement of disease, however, may complicate the future use of these vectors (41). Poxvirus vectors, including recombinant modified vaccinia virus Ankara (MVA) (1, 43), constitute a further class of vectors that have been used to express H5N1 influenza virus antigens (5, 22, 44, 46). Usually, however, the large-scale production of MVA is carried out in primary chicken cells, since these are the most efficient production substrates and are also accepted by regulators. In a pandemic, this production platform may not be available because permanent nontumorigenic avian cell lines are currently not available for production.In this study, we used a permanent cell line, modified Vero cells, to produce nonreplicating vaccinia virus vectors expressing the H5 hemagglutinin (HA), the major influenza virus protective antigen. The defective vaccinia virus (dVV) vectors are safe due to their lack of replication capacity in normal hosts, while they share the superior immunizing properties of poxviral live vaccines (15, 33). Previously, a permanent cell line based on rabbit kidney cells was engineered to express the essential vaccinia virus D4R gene encoding the enzyme uracil-DNA-glycosylase. This cell line allowed the construction of replication-deficient vaccinia virus vectors (15). In this work, a complementing system based on Vero cells was established and used to produce the defective vaccinia virus vector dVV-HA5. The vector was used to immunize mice and was compared to an inactivated whole-virus (whv) vaccine and to replicating control viruses. The dVV-HA5 candidate vaccine induced neutralizing antibodies and full protection, similar to results with an inactivated whv vaccine. Further, it is important to ensure that the immune responses generated by a pandemic influenza vaccine give long-lived, broad, cross-clade protection. While antibody responses to influenza virus provide protective immunity, T-cell responses are also thought to play an important role in clearance of and recovery from infections. Thus, a vaccine which can produce both effective humoral and T-cell responses would be advantageous. A vaccinia virus vector-based pandemic influenza vaccine has the potential to provide this advantage.