Title:  Influenza virus vaccine composition

United States Patent:  6,372,223

Assignee:  Baxter Aktiengesellschaft (Vienna, AT)

Filed:  June 12, 2001

PCT NO:  PCT/AT99/00223

102(e) Date:  June 12, 2001

PCT PUB. Date:  March 23, 2000

Foreign Application Priority Data:  Sep 15, 1998[AT] (1555/98)

This invention describes an influenza virus vaccine containing an influenza virus antigen obtained from a cell culture, with an influenza virus antigen content between 1 .mu.g and 5 .mu.g per dose and aluminum as an adjuvant as well as a method for its preparation.

Description of the Invention

The present invention concerns an influenza virus vaccine composition with a reduced influenza virus antigen content and with aluminum as an adjuvant. In addition, the present invention concerns the use of the vaccine composition in the production of a drug and for the induction of an effective immune response in higher vertebrates, in particular in humans.

Influenza virus infections pose an increasing risk to the health, particularly to the health of the elderly and of persons suffering from chronic diseases, since the infection in these groups of persons frequently leads to an increase in the mortality rate. Since the introduction of an inactive influenza vaccine containing inactive virus material from infected embryonated chicken eggs in the 1940s, the risk and the course of the infection have improved and the mortality rate in the elderly has decreased.

For a vaccine which leads to a positive ratio between the vaccine dose and the IgG antibody response to be effective, health authorities recommend a vaccine dose between 10 .mu.g and 15 .mu.g of HA (hemagglutination) antigen per dose.

The short-term production of large quantities of antigen for the manufacture of vaccine during a pandemic, in particular by means of the method using embryonated chicken eggs which has been used so far, is not only labor-intensive and requires a supply of large quantities of eggs but, due to the short intervals of time between identifying the virus type and making the vaccine available, also requires a considerable logistic effort. In addition, due to the increasing awareness that especially the group of people at risk should be vaccinated early on, the future demand for an effective vaccine will increase more and more.

Based on present estimates, the effectiveness of a human influenza virus vaccine is in a range from 30% to 80%. To increase the effectiveness, it has been proposed that the vaccine dose be increased. Studies carried out by Palache et al. (1993, Vaccine 11, pp. 3-9) and Palache et al. (1993, Vaccine 11, pp. 892-908), however, show that an increase in the vaccine dose is not always sufficient to increase the antibody response and to protect those vaccinated since the degree of the antibody response is highly dependent on the antigen. Although it was found that there is a tendency toward an increased immune response if a higher antigen dose is used, this tendency is less pronounced above a range from 10 .mu.g to 15 .mu.g and often does not justify the side effects caused by the high vaccine dose.

Other approaches to increasing the immune response, in particular in the elderly, targeted using additional adjuvants. Thus, antigen preparations containing mineral oil emulsions did indeed elicit an immune response that was superior to that of vaccines without this adjuvant; however, they were also responsible for more severe side effects (Fukumi et al., 1967, In: Symposium Series. Immunobiology Standard, Vol. 5, p. 237 ff., Karger, Basel, New York).

In clinical studies involving humans, aluminum, the only adjuvant allowed for use in humans, did not provoke a greater immunogenicity of influenza virus antigen although the immune response in mice had been increased by the adjuvant (Davenport et al., 1968, J. Immunol. 100, pp. 1139-1140, Nicholson et al., 1979, J. Biol. Stand. 7, pp. 123-136, Bachmayr et al., 1976, Split and subunit vaccines. In: Influenza: Virus, Vaccines, Strategy (Ed. P. Selby), Academic Press, New York, pp. 149-152, Jennings et al., 1981, J. Hyg. 81, pp.1-16). Studies carried out by Skea et al. (1993, Vaccine 11, pp. 1018-1026) to examine the increase in the immune response to an influenza virus vaccine also showed that aluminum compounds by themselves are not very effective adjuvants for influenza virus antigen. But Skea et al. (cit. loc.), by increasing the adhesion of HA antigen to aluminium by specific anti-influenza virus HA antibodies, were able to elicit a 1500 times higher immogenicity in mice and a 5 times higher immunogenicity in rabbits compared to aluminum by itself. Based on this, they reasoned that the adjuvant activity of aluminum can be considerably improved by increasing the physical adhesion between the antigen and the adjuvant. So far, data from experiments in higher mammals or humans, however, have not yet been made available.

A number of experiments in which adjuvants with an acceptable reactivity were tested has been carried out. Thus, immunostimulating complexes (ISCOMS.TM.), oil-in-water adjuvants (Coulter et al., 1998, Vaccine 16, pp. 1243-1253), Vaxcel.TM., TiterMax.TM., Syntex, AlPO₄, Freund's complete and incomplete adjuvant (Robuccio et al., 1995, Lab. Animal Sci. 45, pp. 420-426), poly(amidoamine) dendrimer (WO 97/28809), and MF59 (Keitel et al., 1993, Vaccine 11, pp. 909-913, Martin, 1997, Biologicals 25, pp. 209-213) were tested for their adjuvant activity on influenza virus HA antigen. It was found that the adjuvants used increase the immune response to different degrees. On the other hand, depending on the adjuvant concentration used, they also cause more or less severe side effects.

An additional problem that arises especially when groups of high-risk individuals, such as persons suffering from allergies and patients with asthma, are vaccinated with the conventional influenza virus vaccine from chicken eggs relates to the fact that these individuals are particularly prone to developing side effects, such as allergic reactions to chicken proteins. In many cases, even a small quantity of chicken protein was enough to provoke life-threatening, hypersensitive, allergic reactions. As a result, it became the general practice to dilute the influenza virus vaccine at a ratio of 1:10 to reduce the quantity of chicken protein that was administered along with the vaccine. Studies carried out by Guarnaccia et al. (1990, Ann. Allergy 65, pp. 218-221), however, showed that the reduction of the normally administered quantity of influenza virus antigen also leads to a considerable reduction of the immune response and therefore recommended that quantity of antigen not be reduced since such a reduction fails to ensure an adequate protection of the patients against a viral infection.

Thus, the problem to be solved by the present invention is to make available a vaccine composition which does not have the disadvantages described above, such as high antigen dose, adjuvants which provoke side effects, or allergic reactions to chicken proteins.

This problem is solved according to the present invention by making available an influenza virus vaccine containing influenza virus antigen obtained from a cell culture, with a maximum of 1 .mu.g to 5 .mu.g of influenza virus antigen per dose and with aluminum as an adjuvant.

In corroboration of the results obtained by Guarnaccia et al. (1990, Ann. Allergy 65, pp. 218-221) it was found that a reduction of the influenza antigen quantity in a vaccine dose also leads to a decreased immune response in mice. Also, in agreement with the results obtained by Davenport et al. (1968, J. Immunol. 100, pp. 1139-1140, Hjorth et a., 1997, Vaccine 15, pp. 541-546), it was possible to show that with a vaccine composition which contains influenza virus antigen that has been isolated by conventional means from infected embryonated chicken eggs and, at the same time, aluminum as an adjuvant, the HA titer is increased. But with a vaccine dose with a content of antigen that has been reduced to as much as 1/10 of the normally adequate antigen dose (1.5 .mu.g), the addition of aluminum led to an HA titer that is approximately as high as the vaccine containing 10 times the quantity of antigen (Table 1).

Surprisingly, however, it was found that the same quantity of an antigen of the same influenza virus strain that was isolated from a cell culture infected with influenza virus induced 2 times as high an antibody titer as the antigen that is isolated from chicken eggs. In addition, it was possible to show that the addition of aluminum to a preparation containing antigen that has been isolated from a cell culture increases the immune response of the antigen only insignificantly, if at all, when a normally high antigen dose is administered, whereas during an immunization with a preparation that contains a considerably lower dose of this antigen and aluminum as an adjuvant, the HA titer is even higher than that of a higher antigen dose (Table 1) and thus leads to an increased immune response.

Therefore, the adjuvant activity of aluminum for influenza virus antigen that has been isolated from a cell culture is considerably higher than for an antigen that has been isolated from embryonated eggs. This was all the more surprising in that it was generally known to those skilled in the art that at an antigen content of .ltoreq.10 .mu.g of antigen per dose, the immune response is considerably reduced (Guarnaccia et al., 1990, Ann. Allergy 65, pp. 218-221) and that aluminum has only a week adjuvant effect on influenza antigen.

In addition, it could not have been foreseen that especially an influenza virus antigen preparation, which had been produced from purified antigen from a cell culture, and the combination of a reduced influenza virus antigen content and aluminum as an adjuvant would lead to a considerably higher immune response than that that can be obtained with a preparation with a content of .gtoreq.10 .mu.g of antigen per dose without adjuvant. As a result of the reduction of the antigen content in the vaccine and the presence of aluminum as an adjuvant, it was possible to obtain a 10-fold increase in the immunogenicity of the influenza virus antigen that had been isolated from the cell culture.

According to a special aspect of the present invention, the influenza virus vaccine according to the present invention contains 1 .mu.g to 2.5 .mu.g of antigen per dose. Especially recommended is a vaccine formulation according to the present invention with an influenza virus antigen content of 1.5 pg per dose.

The influenza virus vaccine according to the present invention contains aluminum, preferably in the form of aluminum hydroxide (Al(OH)₃) or aluminum phosphate (AlPO₄). In the vaccine formulation, the concentration of aluminum can preferably reach a final strength of 0.05% to 0.5%.

In addition to the increased immunogenicity of the preparation, the special advantage of the vaccine formulation according to the present invention is to be seen in the fact that, as a result of (i) the reduced antigen content and (ii) the use of an adjuvant which has been tested over many years and which has already been approved for use in humans, it is nearly completely free from side effects.

In addition, from an antigen quantity normally required for one dose of vaccine, it is possible to produce up to 10 times as many doses of vaccine (15 .mu.g per dose vs. 1.5 .mu.g per dose). As a result, not only the industrial expenditure required in the production of the antigen is reduced but at the same time, it is possible to solve the problems which may arise if a pandemic were to break out, namely of rapidly making available several million doses of vaccine.

In the context of the present invention, it was not only possible to show that the vaccine according to the present invention induces an improved immune response in mice but experiments with chimpanzees also showed that the vaccine preparation according to the present invention is effective in higher mammals as well. This was especially surprising since it was not to be expected that (i) the immunoadsorbent, i.e., aluminum, would have the effect of improving the immune response in higher mammals and that (ii) the reduction of the antigen content would provoke a considerably higher immune response in chimpanzees. At the same time, these experiments also showed that the preparation according to the present invention is nearly completely free from any side effects.

In particular, it was found that the purified influenza virus antigen preparation which was obtained from the cell culture does not only fulfill all of the criteria listed in the CPMP guidelines which must be met by all effective virus vaccines but also that the antigen in the vaccine, compared to a conventional vaccine from eggs, has a considerably higher immunogenicity and, in addition, does not contain chicken proteins which are responsible for potential allergic reactions.

The experiments also show that the increase in the HA titer in chimpanzees which had been immunized with influenza virus antigen isolated from a cell culture was not only considerably higher than in animals which had been immunized with the conventional vaccine with the same dose of antigen but also that the immune response after immunization with a vaccine with a lower quantity of antigen (1-5 .mu.g of antigen per dose) and an adjuvant . . . [ungrammatical sentence or text missing] . . . after 90 days, the HA titer was in part one third higher than or twice as high as when a higher quantity of antigen without an adjuvant was administered (Table 4).

According to a special aspect of the present invention, the vaccine according to the present invention preferably contains influenza virus antigen which is produced and isolated from a cell culture that has been infected with influenza virus. The cell culture can be a Vero cell culture that has been cultivated in a serum- or protein-free medium, with the influenza virus antigen preferably being obtained according to the method described in the International Patent No. WO 96/15231. After purification by means of continuous density-gradient centrifugation and DNase treatment, a solution containing influenza virus that has been obtained using this particular method is obtained in the form of a purified, concentrated virus antigen preparation. This concentrated preparation can be used in the subsequent production of the vaccine according to the present invention.

Another aspect of this invention relates to a method for the production of an influenza virus vaccine for higher mammals, in particular for humans, which comprises the following steps

infecting a cell culture with influenza virus

cultivating the infected cells

harvesting the viruses produced

purifying the virus preparation

producing a concentrated virus preparation

diluting the virus preparation to an antigen content between 1 .mu.g to 5 .mu.g of antigen per dose and adding aluminum as an adjuvant in a concentration between 0.05% to 0.5%.

The production of the influenza virus antigen for the vaccine is preferably carried out in a serum- or protein-free cultivated Vero cell culture as described in International Patent No. WO 96/15231. The cells infected with influenza virus are cultivated until the desired virus titer is obtained, and the viruses are isolated from the supernatant portion of the culture. It was found that the purification of the virus preparations obtained is especially important. In this context, one purification method has proved to be especially suitable; it comprises the steps of a saccharose gradient DNase treatment, and diafiltration and sterile filtration. Even after the preparation that has been purified according to this method is diluted to 1/10 of the antigen content administered in one conventional dose of vaccine and aluminum as an adjuvant is added, it still provokes an immune response that is higher than the vaccine preparation containing no adjuvant but an antigen content of 15 .mu.g of antigen per dose which had normally been considered necessary to be effective.

Another aspect of the present invention relates to the use of an influenza virus preparation with a maximum antigen content between 1 .mu.g to 5 .mu.g of antigen per dose and with aluminum as an adjuvant for the prophylaxis against influenza virus infections.

In this context, the use according to the present invention to prevent an influenza virus infection in humans is to be especially preferred. Since, due to the absence of egg-specific proteins, the probability of an allergic reaction to these proteins is excluded, the vaccine according to the present invention is especially suitable for administration to groups of persons who are high-risk individuals, such as asthmatics and allergy sufferers, as well as to individuals with an immunodeficiency and to the elderly.

Based on the tests on chimpanzees carried out in the context of the present invention, it was found that especially the influenza virus antigen which had been obtained from a cell culture and to which aluminum as an adjuvant had been added induces a better immune response and thus is a better immunogen than an antigen that has been prepared by means of conventional methods. Furthermore, the vaccine according to the present invention has the advantage that it is available in a form free from chicken protein and therefore does not have the side effects generally connected with this protein.

Claim 1 of 8 Claims

What is claimed is:

1. An influenza virus vaccine comprising an influenza virus antigen isolated from a cell culture and having an influenza virus antigen content between 1 .mu.g and 5 .mu.g/dose and with aluminum as an adjuvant.

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