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The isolation of influenza virus 80 years ago in 1933 very quickly led to the development of the first generation of live-attenuated vaccines. The first inactivated influenza vaccine was monovalent (influenza A). In 1942, a bivalent vaccine was produced after the discovery of influenza B. It was later discovered that influenza viruses mutated leading to antigenic changes. Since 1973, the WHO has issued annual recommendations for the composition of the influenza vaccine based on results from surveillance systems that identify currently circulating strains. In 1978, the first trivalent vaccine included two influenza A strains and one influenza B strain. Currently, there are two influenza B lineages circulating; in the latest WHO recommendations, it is suggested that a second B strain could be added to give a quadrivalent vaccine. The history of influenza vaccine and the associated technology shows how the vaccine has evolved to match the evolution of influenza viruses.

Influenza is an infection of humans beings and many animal species. It is caused by viruses which belong to the Orthomyxoviridae family. There are three types of influenza viruses A, B and C. The type A is the most pathogenic of all. The type is determined mainly by the nature of the nucleoprotein (NP), an antigen which does not greatly vary. On the contrary, the surface antigens, among which the haemagglutinin is the most important, are highly variable and their nature determines the sub-type of virus within the type A. The expressed mutations affecting the haemagglutinin are referred as antigenic drift and make virological surveillance necessary in order to annually assess the composition of the vaccine strains. The segmented nature of the genome of influenza viruses, makes possible the genetic reassortment of two different influenza viruses co-infecting one cell and produces a new hybrid virus. When such an event affects the haemagglutinin, the reassortment leads to an antigenic shift. In nature, it most certainly takes place in swine, between human and avian viruses. Whereas antigenic drift is a continuous and progressive phenomenon, antigenic shift occurs occasionally every 10 to 30 years. The emergence of a hybrid virus bearing a new haemagglutinin and thus belonging to a new human subtype, can be the starting point of the genesis of a pandemic, generally associated with a high mortality rate in humans. The participation of the pig is specially mentioned.

Neuraminidase is one of the two surface glycoproteins of influenza virions. In order to compare neuraminidases of the same subtype but isolated from different species (man, birds, pig), a new and simple method was adapted and optimized using peanut hemagglutinin. Results were very similar to those obtained with the classical method recommended by the WHO, using fetuin as a substrate. The technique was used to examine the relationship between animal and human neuraminidases belonging to serotypes N1 and N2. The results confirm the possible role of ducks as a reservoir for influenza viruses and the eventuality of interspecific exchanges.

A human T-cell line, B3, has been obtained by cloning spleen cells at limiting dilutions in the presence of influenza-A-virus-infected autologous cells. B3 cells were OKT 3+4+8-, E rosetting+, Sig- and were HLA-DR (+) after stimulation and HLA-DR (-) when resting. They proliferate specifically in the presence of influenza-virus-infected cells. Remarkable is that (a) the proliferation was obtained with viruses of both A and B types and (b) only autologous cells seem to be able to present the viral antigen to B3 cells.

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.

Influenza virus demonstrates several levels of antigenic variations, drifts and shifts, which help it to escape to herd immunity pressures. Recent examples show the importance of such variations in the succession and continuity of epidemiological manifestations. During the last few years, simultaneous circulation of several subtypes of influenza A was observed while in the past, one subtype was replacing the other. Thus in 1981, for the only subtype H3N2, A/Texas, A/Bangkok and A/intermediate Texas-Bangkok viruses were isolated in France at the same time in several foci. Furthermore, double infection with different subtypes in the same individual have been documented in man as well as in animals (birds). Related strains have also been isolated simultaneously in the same individual and cloning by plaque analysis makes it possible to evaluate easily this possibility in wild strains. The unexpected frequency of double infections or of variations of virus in feral animals or humans increases the possibility of genetic reassortments and participates for an important part to global variability of the virus.

Live or inactivated influenza virus is inoculated intratracheally to rabbit. After 4 days or more, lungs are washed with 50 ml of saline and fluid is examined for immunoglobulin and antibody content as well as for type and properties of free cells. IgA and IgG are detectable in the fluid but not IgM. Specific antiviral activity is associated only with IgA at the earliest stages. Free cells are so called 'alveolar macrophages' as demonstrated by shape and phagocytic or enzymatic properties. Their total numbers vary according to type of immunization: non-immunized 5.10-5. inactivated virus 2.10-6. live virus 10.10-6. A high percentage (50 to 90 percent) are positive in IgA IF test, only 10 percent with IgG test. Peroxydase electron microscopy shows intracellular presence of IgA.

In man influenza viruses induce a cytolytic T lymphocyte (CTL) activity directed against autologous or HLA-A or -B compatible target cells infected with the immunizing virus. While only type specific CTL are characterized in man, we report here experiments showing intertypic activities of human CTL from donors vaccinated with both A and B type influenza viruses. Their peripheral blood leucocytes (PBL) restimulated in vitro with live influenza virus of one type gave rise to both anti-A and -B activities, when non-infected or Sendaï infected target cells were not lysed. These intertypic activities were restricted by HLA-A or -B antigens and were inhibited by OKT3 antibody. When u.v.-inactivated viruses were used as restimulating antigen, no intertypic CTL were obtained. The results of competition experiments with cold targets show that no common antigens were recognized by anti-A and anti-B CTL. Moreover the restricting HLA-A or -B molecules seen in association with A or B types viruses appeared different in the same experiment, confirming that different antigens were probably involved for the agents of A and B subgroups. This influenza specific intertypic activity was therefore probably due to an intertypic stimulation of type specific CTL activities, possibly arising at the level of T helper cells.

Normally, under good survival conditions, the ciliary beating can be observed and recorded during hours after dissection of the respiratory epithelium. When laboratory animals have been immunized locally against influenza virus (under delayed type hypersensitivity conditions) the ciliary beating stops within 3 to 6 minutes when the same virus is put in contact with the sensitized mucosa. The specificity and the immunological conditions of this stopping have been analyed and the practical importance of this new test in cellular immunology is discussed.