Most influenza virus vaccines used in the United States and Europe consist of embryonated egg-grown and formaldehyde-inactivated preparations, which, after purification, are chemically disrupted with a nonionic detergent (for example, Triton X-100). The split virus preparations show lower pyrogenicity than whole virus vaccines. In general, 1 dose for adults contains the equivalent of 45 μg HA (15 μg HA for each of the 3 antigenic components). This dose is approximately the amount of purified virus obtained from the allantoic fluid of 1 infected embryonated egg. If 100 million doses of killed influenza virus vaccine are prepared, the manufacturer has to procure 100 million embryonated eggs. Clearly, this manufacturing process is dependent on the timely availability of embryonated eggs and the vaccine seed strains to be used in a particular season. Most of these prototype seed strains are provided to the manufacturers by government agencies, which create high-yielding strains through classical reassortment with a high-yielding laboratory strain, A/PR/8/34, following the procedures designed by Kilbourne (4). Unfortunately, only (high-yielding) influenza A viruses can be made in this way, and even with the A types, the 6:2 reassortants (HA and NA from recently circulating strains and the remaining 6 genes from A/PR/8/34 virus) are sometimes not easily obtained. This time-consuming process of reassortment is then followed by repeated passaging of the strain in embryonated eggs to allow for egg adaptation and growth enhancement. Influenza B virus prototype strains with good growth characteristics are usually obtained by direct and repeated passaging in embryonated eggs without attempting to generate reassortants. Although the manufacturing process is time-consuming, these killed influenza A and B virus vaccines are the workhorses for vaccination against influenza and have been shown time and again to be highly effective.

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).

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.

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 event of a new pandemic virus. Moreover, a live virus vaccine may give protective immunity in immunologically naive populations after a single administration, while killed virus vaccines may require high antigenic doses and a prime-boost regimen to protect against a pandemic strain. It may turn out that only live influenza virus vaccines can provide the necessary protection in case of a new pandemic. Because live influenza virus vaccines appear to be more effective in immunologically naive populations and they can be intranasally administered, they would represent a more economical way of vaccinating large numbers of people.

Influenza viruses continue to undergo antigenic drift, which is mostly reflected in accumulating changes in the HA. This fact requires us to change the vaccine formulation or at least to reexamine the seed strains on an annual basis. Unfortunately, predicting the evolutionary change of the viral HA has not been reliable (29). Thus, short of developing 20/20 foresight, predicting strain variation or the emergence of a particular pandemic strain (avian or otherwise) is unlikely (30). A more realistic approach is the design of more cross-protective vaccines for use in interpandemic years and during pandemics. Neirynck et al. have designed vaccines based on the conserved extracellular portion of the M2 protein fused to the hepatitis B core protein (31). Such an immunogen may induce a cross-reactive response in the vaccinated host. Similarly, immunization with the NA antigen is likely to induce responses that are more cross-reactive than those by the more variable HA (32). In both cases, however, protection will require immune responses that are more vigorous than what is seen after natural infection. Antibodies against NA and M2 proteins in infected humans are generally not protective. Thus vaccines consisting of NA or M antigens would need to be made to induce a dramatically enhanced immune response. Alternatively, genetically engineered viruses could be generated, which would express several variant antigens or epitopes, thereby achieving a more cross-protective immunization. Chimeric HA recombinant viruses that express an additional 140 amino acids have recently been described (33). Such genetically engineered viruses may present several conserved immunogenic epitopes on the viral surface, which would be a first step toward a more universal influenza vaccine.

Technologies are now in place to design and construct new influenza virus vaccines that have the potential to be cheaper and more cross-protective than current vaccine preparations, while at the same time being equally safe. The greatest problems for new and better vaccines appear to be associated with regulatory hurdles and the lack of an adequate market. Regarding the bureaucratic restrictions levied on vaccines by licensing agencies, the message has to come through "that small risks have to be tolerated for larger ones to be avoided" (34). Also, the message needs to be disseminated to the general public that vaccines have the best cost-benefit ratio of any medical treatment and that limitations of the tort law should be considered where vaccines are concerned. The public often views vaccines and prophylactic treatments in general as being of low priority. Many people also believe they should be free. Thus, the absence of a robust commercial market is a major difficulty, resulting in slow progress for research and development of new influenza vaccines and in dangerously thin supply lines. In fact, we are far from being prepared to deal with regular influenza outbreaks, and adequate measures to cope with a pandemic outbreak are only now being considered, but are not yet in place (35,36; and http://www.washingtonpost.com/wp-dyn/content/article/2005/11/01/AR2005110101100.html).