The Evolving History of Influenza Viruses and Influenza Vaccines

Claude Hannoun


Expert Rev Vaccines. 2013;12(9):1085-1094. 

In This Article

The Early Inactivated Vaccines

The first research programs attempting to develop an inactivated influenza vaccine, outside of the USSR, were conducted in England and in the USA at about the same time. However, it was at the start of the Second World War that the programs significantly developed in the setting of military projects, with the more or less official objective of protecting their troops while on mission.[6]

The relatively simple methods developed by Burnet in Australia for culturing the virus on chick embryos, involving inoculation into the allantoic cavity, made it easy to obtain sufficient amounts of the virus strains for the vaccine.[7] The vaccine development was undertaken independently on both sides of the Atlantic at the same time. However, the American scientists had more resources as the work was performed by the army. Thomas Francis, head of the US Army Commission, and his colleagues decided to use a vaccine prepared from allantoic fluid containing high concentrations of virus that had been purified and inactivated with formalin.[8] With an inactivated virus vaccine, the amount of antigen required to induce immunity is much greater than that for a live-attenuated virus vaccine, because unlike the live-attenuated virus, the inactivated virus does not replicate in the recipient. Thus, being able to obtain large quantities of virus in the allantoic fluid overcame this problem and an inactivated vaccine with a sufficiently high concentration of antigen could be prepared, after a simple, rapid purification process. The A/PR8 (H1N1) strain used, which was isolated in Puerto Rico in 1934, had a high replication potential in eggs, which enabled the required huge quantities of virus to be obtained.

It had undergone a very long adaptation process involving 77 passages in mice; 717 passages in cell culture; 30 passages in chick embryos; five passages in ferrets; and an additional 50 passages in chick embryos.

In 1940, for the first time, a different influenza virus was discovered and isolated. It was antigenically different from the influenza A(PR8) virus, but had the same properties in terms of culture in eggs. This virus was named Influenza B and the inactivated influenza vaccine had to be bivalent to provide protection against both types of influenza viruses (Figure 1). Thus, in 1942, 10,000 doses of the first bivalent vaccine containing the A/PR8 and B/Lee virus strains were administered in humans for testing. This bivalent vaccine contained 0.5 ml of virus concentrated from 5 ml of allantoic fluid containing influenza A and the same amount of influenza B. One half of the influenza A allantoic fluid contained the A/PR8 strain and the other half contained the Weiss strain, a strain that had been isolated more recently and that was slightly different from A/PR8. This precaution suggests some anticipation of the phenomenon of strain variation. These first clinical trials demonstrated a good serological response to both influenza A and B. After two doses, there was an eight- to ninefold increase in antibody titer, measured by the hemagglutination inhibition assay. However, at this time, there was no definitive confirmation of clinical protection, because the influenza epidemic during the 1942–1943 winter was mild.[9]

Figure 1.

Evolution of influenza viruses and corresponding evolution of influenza vaccines.

During the 1943–1944 season when the epidemic started in early November, the trial was repeated and 6,263 subjects were vaccinated.[10] This time the results showed that only 2.2% of the vaccinated subjects had clinically assessed influenza disease compared with 7.1% of those not vaccinated, an efficacy of 69%. No deaths occurred in either group. These trials carried out by the U.S. Army led to the conclusion that vaccination reduced the incidence of both mild and severe clinical episodes of influenza and influenza-related mortality. As was recorded in the final report: "Toxic reactions to vaccine were considered unimportant. Two cases were presumed to be due to a sensitization reaction to the egg component in one patient and an urticarial rash in another. Systemic reactions occurred in 2–3% of those vaccinated and were similar to the mild reactions observed after typhoid vaccination."

In 1944, Stanley described in detail the preparation and properties of influenza virus vaccine produced in embryonated hen eggs, concentrated and purified by differential centrifugation and inactivated by different procedures.[11] This well-defined protocol became the technique that was used by commercial manufacturers. A vaccine was manufactured according to this procedure containing A(PR8)/A(Weiss) and B(Lee) strains (a total of 2 mg of viral material), inactivated with formalin without adjuvant. Evaluation in mice showed the immunogenic properties of this type of inactivated vaccine. This bivalent vaccine started to be used widely in 1944, particularly for the American troops in Europe, and was then extended to civilians in 1945. Improvements in the production, safety and strain selection processes were then made, which led to the improved performance of influenza vaccine.