The second major class of viral vaccines consists of live viruses. The only FDA-licensed product against influenza is the cold-adapted attenuated vaccine. It is based on work originally done by Maassab's laboratory (5) and later by Murphy and colleagues (6). Influenza virus was passaged at 25°C in tissue culture (chicken kidney cells) and in embryonated eggs. This modified Jennerian approach resulted in a cold-adapted, temperature-sensitive, and highly attenuated master strain. The annually updated vaccine strains are generated in the laboratory by reassortment with viruses more closely related to the currently circulating ones. The resulting vaccine strains (both A and B types) are 6:2 reassortants with the 6 nonsurface protein genes derived from the cold-adapted master strains and the HA and NA from circulating A and B viruses, reflecting the changing antigenicity. These cold-adapted influenza virus vaccines are easily administered by nasal spray. They induce local mucosal neutralizing immunity and cell-mediated responses that may be longer lasting and more cross-protective than those elicited by chemically inactivated (killed) vaccine preparations. Vaccine efficacy in vaccine-naive children 6 months to 18 years of age is high (range 73%–96%). In children revaccinated for a second season, vaccine efficacy climbs to 82% to 100% (7).
Need for Improvement?
Despite the obvious efficacy of both killed and live influenza virus vaccines, there is room for new developments. Among the critical issues in developing new and better vaccines are the following: price per dose, speed of production, ease of production, choice of substrates to grow the virus in or to express viral antigens, cross-protection for variant strains, efficacy in general and in immunologically naive populations, safety, and acceptance by the regulatory agencies and the public.
New Adjuvants
Most of the current inactivated influenza virus vaccines do not contain an adjuvant. To stretch the available supply, antigen-sparing adjuvant approaches should be considered (8). Alum is an adjuvant that has been approved by the FDA for use in several vaccines. MF59, a proprietary adjuvant from Chiron (Emeryville, CA, USA), has also been successfully used in several countries (other than the United States). If, under adjuvant conditions, a fifth or a tenth of the antigenic mass currently present per vaccine dose (45 ?g of HA protein) would suffice to stimulate an adequate protective response, a big supply problem would be solved.
Many adjuvants are now under investigation. Liposome-like preparations containing cholesterol and viral particles (immune-stimulating complexes) have been successfully used in mice (9) by subcutaneous and intranasal administration. Another adjuvant strategy involves the use of heat-labile Escherichia coli toxin complexed with lecithin vesicles and killed trivalent influenza virus preparations for intranasal administration (10). Although this specific vaccine has been withdrawn because of Bell's palsy cases associated with its administration, similar approaches may become more acceptable in the future if these safety issues can be resolved. Much work is also currently being conducted on synthetic adjuvants, such as synthetic lipid A, muramyl peptide derivatives, and cationic molecules (11). Also, Ichinohe et al. showed that poly (I:C) is a promising new and effective intranasal adjuvant for influenza virus vaccines (12).
Genetically Engineered Live and Killed Influenza Virus Vaccines
As indicated, current FDA-licensed influenza vaccines are based on technologies developed in the 1960s and earlier. Through the breakthrough of reverse genetics techniques (13–15), infectious influenza viruses from plasmid DNAs transfected into tissue culture cells can now be rescued. This technology permits the construction of high-yield 6:2 seed viruses by mixing the 6 plasmid DNAs from a good-growing laboratory strain with the HA and NA DNAs obtained by cloning relevant genes from currently circulating viruses. Thus, within a 1- to 2-week period, the appropriate seed viruses could be generated for distribution to the manufacturers. The backbones of the 6:2 recombinant viruses could be prepared, tested, and distributed in advance. Similar approaches can be envisioned for the manufacturing of live, cold-adapted influenza virus vaccines. In this case, the backbone would consist of the 6 genes of the cold-adapted master strain. Again, the HA and NA of the currently circulating strains would be cloned and used for rescue in the plasmid-only reverse genetics system. Such an approach would have several advantages over the present manufacturing process. First, it would dramatically accelerate the timeframe for obtaining seed viruses for annual production and thus allow more time to select the appropriate antigenic seed strains. Second, it would standardize the seed viruses to be used. Regulatory agencies do not insist on a sequenced product to be given to humans but instead allow only partially characterized products for annual immunization. Third, DNA cloning may eliminate any adventitious agents present in the throat washings of the original isolate. Finally, in the case of the current highly pathogenic H5 strains, viruses with that HA (containing a multibasic HA1/HA2 cleavage site) kill embryonated eggs, making it difficult to use eggs as growth substrate. Also personnel involved in manufacturing those vaccines might be in danger of becoming infected. Thus, the HA of these virulent strains will need to be modified. Removal of the basic cleavage peptide by reverse genetics results in a virus that is attenuated for embryonated eggs, thus allowing high yields to be attained. Modification by reverse genetics results in a product that is easier to manufacture and safer to handle (this includes safety considerations for all persons working with the virus).
Live Influenza Virus Vaccines with Altered Nonstructural Protein 1 Genes
The ability to site specifically engineering changes in the influenza virus genome also allows us to consider novel vaccine approaches. We have demonstrated that the nonstructural protein 1 (NS1) of influenza viruses has interferon antagonist activity (16). Influenza viruses that lack NS1 cannot counter the interferon response of the host. Thus, infection of cells with a virus that lacks NS1 results in the induction of interferon and blockage of virus replication. When truncations are made in NS1, viruses are generated with an intermediate activity, which enables them to replicate in the host and also to induce an interferon response. By engineering a virus with intermediate virulence and ability to induce interferon, one can construct ideal influenza virus vaccines that are both attenuated and highly immunogenic (17–20). Interferon appears to be an excellent adjuvant that enhances production of immunoglobulins and contributes to the activation of dendritic cells required for antigen presentation (21–23). We thus believe that, per virus particle made or antigen molecule delivered, the immune response will be enhanced compared to that of conventional live or killed virus vaccines. This process should translate into lower doses of live virus vaccine required to induce a robust and protective immune response. If a hundredfold lower dose is required, many more people could have access to influenza virus vaccines. This issue is clearly of paramount importance in the e