Inactivated influenza virus vaccine for nasal or oral administration

The invention relates to a vaccine composition containing at least one inactive influenza virus antigen and aluminum as an adjuvant for nasal or oral use for prophylaxis of influenza virus infections.

Influenza virus infections represent an ever greater health risk, especially in older people and in people with chronic diseases, since the infection often leads to an increased mortality rate in these groups of people. Since the introduction of inactivated influenza vaccines, containing inactivated virus material from infected incubating eggs, in the 1940s, the risk and the course of the infection, as well as the mortality rate in older people, have decreased.

Previous inactivated influenza virus vaccines from eggs are for parenteral administration

Humans license and induce anti-HA-IgG antibodies in serum. The cross-protection g heterologous influenza viruses is primarily due to the Kri reactivity of IgA antibodies in a natural infection (Liew et al., 1984, Eur. J. Immunol. 14: 350-356) was therefore in the development of new The method of immunization against influenza virus infections attempts to stimulate the production of the mucosal IgA immune response.

To this end, a number of developments have been developed for intranasal or oral administration of fluenzavirus vaccines. For example, the administration of an inactivated virus vaccine (Waldman et al., 1968, Nature 218: 594-595), an inactivated vaccine combined with carbo: vinyl polymer (Oka et al., 1990, Vaccine 8: 573-576) or with pertussis toxin B oligomer (Oka al., 1994, Vaccine 12: 1255-1258), a split virus vaccine with cholera toxin, E. coli heat-lab enterotoxin or liposomes (Tamura et al., 1992, J. Immunol. 149: 981-988, Komasse et al. Vaccine 16: 248-254, de Haan, 1995, Vaccine 13: 155-162), an emulsion -inactivated Vakt (Avtushenko et al., 1996, J. Biotechnol. 44: 21-28) or a cold-adapted live attenuated influenza virus vaccine (Belshe et al., 1998, N. Engl. J. Med. 338: 1405 -1412) not only to induce HAI-IgG antibodies in the serum but also secretory IgA antibodies of the cosal membrane.

Inactivated viruses as vaccines applied orally or nasally, however, have to be given in high concentrations in order to lead to a significant increase in antibodies. The negation of inactivated influenza virus or antigen in side-effect-free and commercially reasonable doses therefore does not lead to a satisfactory immune response when administered nasally or orally without the addition of a vans (Chen et al., 1989, Current Topics in Microblogy and Immunology: 146: 101-106, Couch et al., 1997, J. Infect. Dis. 176: 38-44). For example, the optimal induction of the immune response when an emulsion-inactive vaccine is administered orally requires an antigen content between 66 μg antigen / dose and 384 μg antigen / dose 35 (Avtushenko et al., 1996, J. Biotechnol. 44: 21-28) . This dose is thus far above that of an activated vaccine for parenteral administration, which is around 15 μg antigen / dose

Although cholera toxin, E. coli heat-labile toxin, and pertussis toxin are effective adjuj; If influenza antigen is administered orally or nasally, they are not used for human use because of the toxic side effects. The only adjuvant that has so far been approved for use in humans is aluminum.

A cold-adapted, live-attenuated influenza virus for nasal administration that is currently in clinical studies is based on virus antigens, of which reassortants have to be produced annually by means of different methods, the genes for the hemagglutih and neuramidase antigens of the corresponding influenza A. - or B strain can be transferred to an attenuated I 45 th, cold-adapted master virus strain. This method is very time-consuming and labor-intensive. In addition, there is the risk that reversion will reverse the attenuated virus from a virulent virus and thus trigger viraemia. When live viruses are immunized, they also spread further in the body of the vaccinated person. Also, when using cold-adapted viruses, it is always necessary to keep the virus vaccine at half-freezing point, if possible at -20 ° C, which makes it essential to maintain the cold chain so that the vaccine can be stored sufficiently.

The production of the virus reassortants and the replication of the vaccine viruses take place in the egg, which means that there is a risk of contaminating, infectious!

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AT 407 958 B because they represent infectious material and therefore a high safety standard must be observed.

It is therefore an object of the present invention to provide an influenza virus vaccine composition which does not have the disadvantages described above and which effectively induces an IgA and IgG immune response in mammals.

The object is achieved according to the invention by the use of a vaccine composition containing at least one inactivated influenza virus or influenza virus antigen and aluminum as an adjuvant for nasal or oral administration. The composition described is particularly suitable as a vaccine for prophylaxis against influenza virus infections.

It has been shown in the context of the present invention that an inactivated influenza virus vaccine containing aluminum as an adjuvant, which is administered nasally or orally, triggers an effective IgG and IgA immune response in mammals. This was particularly surprising because previous attempts to develop effective influenza virus vaccines have found that the adjuvant effect of aluminum to increase the immunogenicity of the antigen is very low even with a parenterally administered vaccine (Davenport et al., 1968, J. Immunol 100: 1139-1140).

It has also been found that the nasal or oral use of the vaccine composition according to the invention achieves a significantly higher IgG and IgA titre and a higher HAITiter in mammals than in previously known vaccine formulations which either only contain inactivated influenza viruses or inactivated viruses with cholera toxin or live viruses (Table 1).

The use according to the invention is therefore particularly suitable for inducing a protective mucosal IgA and a systemic IgG immune response.

Since aluminum is the only adjuvant approved for use in humans, the use according to the invention of the vaccine combination of inactivated influenza virus and aluminum has the great advantage that it is unproblematic for direct use in humans. In addition to the increased immunoreactivity of the vaccine composition when administered nasally or orally, the particular advantage of the use according to the invention is that the vaccine is completely free of side effects by using an adjuvant that has been tried and tested over many years and is approved for use in humans.

For the use according to the invention, the composition aluminum can preferably be in the form of aluminum hydroxide (Al (OH)₃) or aluminum phosphate (AlPO₄) contain. The concentration of aluminum in the vaccine is preferably in a final concentration of 0.05% to 0.5%.

The amount of influenza virus antigen in the vaccine is the usual amount of antigen for a vaccine dose. The amount of antigen contained in a vaccination dose is preferably between 1.5 μg antigen / dose to 50 μg antigen / dose in humans.

The influenza virus antigen can be prepared and purified from infected eggs using conventional methods.

However, the virus antigen is preferably obtained from an infected cell culture, as described, for example, in WO 96/15231. Particularly preferred for the use according to the invention for producing an influenza virus vaccine is an influenza virus antigen which is obtained from a VERO cell culture infected with influenza virus and grown in a serum- and protein-free medium. The virus antigen obtained from the infected cell culture is first inactivated with formalin and can then subsequently be obtained as a purified, concentrated virus antigen preparation by means of continuous density centrifugation in the gradient, DNAse treatment and dia- and sterile filtration. This concentrated preparation can then be used together with aluminum as an adjuvant for the use according to the invention for producing a vaccine which can be administered nasally and orally.

A particular advantage in the production of the vaccine is that the virus material is inactivated before cleaning, which results in a much higher purity of the antigen preparation compared to the cleaning of attenuated live viruses.

A particular advantage when using influenza virus antigen, obtained from a serum and protein-free cell culture infected with influenza virus, is the absence of egg-specific proteins which may trigger an allergic reaction to these proteins

AT 407 958 B can. The use according to the invention is therefore suitable for the prophylaxis of influenza virus infections, in particular in groups of people who belong to a high-risk group, such as asthmatics, allergy sufferers, but also people with immunosuppression and in older men.

The vaccine can be applied in various ways.

According to one embodiment of the invention, administration takes place intranasally via the mukoi route. The intranasal administration of the vaccine composition can be formulated, for example, in liquid form as nasal drops, spray or for inhalation, as a powder, cream or as emulsions.

The composition can contain various additives such as stabilizers, Pi or preservatives.

For simple application, the vaccine composition is preferably in one package appropriate container for the distribution of the antigen in the form of nasal drops or an aerc; contain.

According to a further embodiment of the invention, the administration takes place orally, the vaccine, for example, in the form of a tablet or encased in a gelatin capsule of a microcapsule, which simplifies oral administration. The manufacture of these application forms is within the general knowledge of a person skilled in the art.

The invention is explained in more detail with the aid of the following examples, although it is not restricted to examples.

Example 1:

Manufacture of an influenza virus vaccine preparation

Influenza virus was obtained from protein-free Vero cell culture, infected with Influenza A or BV soot strain, according to WO 96/15231 or by conventional methods from allantoic fluid of infected incubated chicken eggs.

To produce an inactivated influenza virus preparation from cell culture, formalin (final concentration 0.025%) was added to the supernatant of an infected Vero cell culture and the 30 viruses were inactivated at 32 ° C. for 24 hours. This material was purified by zonal centrifugation in or around a continuous 0-50% sucrose gradient, DNAse treatment, diafiltration and Si filtration. The cleaned material was stored at -70 ° C. The end product was tested for residual contamination and the following criteria were defined for each dose:

Hemagglutinin content:

Protein content:

Sucrose content:

Formalin content:

Benzonase content:

Residual DNA (VERO):

Endotoxin content:

Pyrogen:

> 15 µg HA per strain £ 250 µg <200 mg ⁵ 5 μ9 ng £ 100 pg <100 EU free run

Example 2:

Intranasal immunization of mice with various influenza virus preparations

The antigen preparations from Example 1 were diluted in PBS to an HA antigen content of 15 pg / ml and optionally with AI (OH)₃ up to a final concentration of 0.2% or adjuvanted with cholera toxin. To produce a preparation for intranasal immunization of mice, the solution was adjusted to the corresponding amount of antigen with PBS, optionally containing AI (OH)₃ or cholera toxin, diluted.

4 Balb / c mice each were injected intranasally with various influenza virus preparations, with 50 μl of a solution containing influenza virus antigen and optionally adjuvant being dripped into the mice's nasal cavities. The 1st immunization took place on day 0, the 2nd on day 7 and the 3rd on day 14. On day 28, the IgG, IgA and HAI were determined from serum, saliva and lung lavage.

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AT 407 958 B

Table 1 shows the scheme of intranasal immunization of the individual groups of mice with different influenza virus preparations.

Table 1

Immunization schedule for intranasal immunization of mice

<td>Group Balb / c mice</td><td>antigen</td><td>dose</td><td>route</td>

<td></td><td></td><td></td><td></td>

<td>1st group</td><td>inactivated Viruses out VERO cells</td><td>1 pg HA</td><td>50 pl / in</td>

<td></td><td></td><td></td><td></td>

<td>2nd group</td><td>inactivated Viruses out infected eggs</td><td>1 pg HA</td><td>50 pl / in</td>

<td></td><td></td><td></td><td></td>

<td>3rd group</td><td>Live viruses from VERO cells</td><td>5x10^e OATH₅₀</td><td>50 pl / in</td>

<td>4th group</td><td>Live viruses from infected eggs</td><td>5x10 ° EID₅₀</td><td>50 pl / in</td>

<td></td><td></td><td></td><td></td>

<td>5th group</td><td>VERO mock preparation</td><td>5% of 1 pg</td><td>50 pl / in</td>

<td></td><td></td><td></td><td></td>

<td>6th group</td><td>Eggs mock preparation</td><td>5% of 1 pg</td><td>50 pl / in</td>

Example 3

Determination of the IgA titer in saliva, lung lysate and serum, the IgG titer and the HAI titer in the serum

On day 28 after immunization, samples were taken from the animals' saliva, lungs and serum and the antibody titer was determined in the individual samples.

Saliva samples were collected by injection of 0.5 ml PBS into the mouse oral cavity and tested for the presence of IgA antibodies.

To prepare lung lysate samples, the mice were sacrificed, the chest opened, the lungs removed and flushed with PBS. The lungs were then minced and the homogenate centrifuged to remove cell tissue. The supernatant was collected and stored at -20 ° C. until it was tested for IgA antibodies.

The IgG and IgA antibody titers were determined using a commercial influenza virus type A and B ELISA test (Genzyme Virotech). After incubation for 2 hours at 37 ° C, the specific IgG and IgA antibodies were conjugated with goat anti-mouse IgG or IgA (Pharmigen) and

AT 407 958 Β chromogenic substrate containing H₂O₂ and O-phenylenediamine, detected.

To determine the HAI titer, blood was taken from the mice and the sepi obtained in the influenza A or B hemagglutination test (HAI titer) according to the method of Palmer e (1975, Advanced laboratory technicals for immunological diagnostic, US Dept. Health. Ed. Fare, PHS Atlanta, Immunology Ser. No. 6, Procedural guide, part 2, haemagglutination - in tion test, pp. 25-62).

Table 2 shows a summary of the determination of the IgA antibody titer in the Spei lung lysate and serum and the IgG antibody titer and HAI titer in the serum. The data clearly shows that an inactivated whole virus vaccine without adjuvant stimulates neither the IgA nor the immune response. In contrast, immunization with live virus or in fourth whole virus adjuvanted with cholera toxin leads to an increase in IgA, IgG and HAI in the mice, as well as after immunization with inactivated whole virus adjuvanted with Aliji nium, with the latter vaccine the IgG- immune response in serum was even the highest of the preparations tested. The highest HAI titre was also measured in the inactivated vaccine with aluminum.

The results show that intranasal immunization with inactivated influenza virus cine adjuvanted with aluminum induces a slightly increased igA immune response and a significantly higher IgG immune response in mammals compared to previously known influenza virus vaccine preparations, without the disadvantages of a live virus or vaccine a vaccine containing a non-approved adjuvant such as Chol Toxin.

Example 4:

Storage stability of the vaccine composition at different temperatures around al. Wellibichel, show

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For stability studies, the monovalent bulks (MB) of the influenza virus from Johannesburg 82, Nanchang and B / Harbin were gerted for 12 months at + 4 ° C, -20 ° C and -80 ° C. After 6 and 12 months, the specific haemagglutination test (HA) content of Johannesburg 82 (MB / J / 0197), Nanchang (MB / N / 0197) and B / Harbin (MB / H / 0397) without Plurj) and Johannesburg 82 (MB / J / 0297 / P), Nanchang (MB / N / 0297P) and B / Harbin (MB / H / 0497P) Pluronic using SRD (single radial immunodiffusion) assay according to Wood et al., 1977, J. Biol S dard 5: 237-247 was determined and the deviation from the initial value was calculated in percent.

The trivalent bulks (TVB) 410197 (without Pluronic) and 4102997P (with Pluronic) were stored for 12 months at + 4 ° C and then tested in the SRD. In addition, the retention patterns of these MBs and TVBs, which were stored at rap temperature for sterility tests, were analyzed in the SRD after 12 months.

The results of the MB's are in table 3 and the results of the TVB's in table 4 because} &lt;represents:

Storage of the MBs at + 4 ° C and -80 ° C for 1 year shows almost no decrease in the specific HA content compared to the initial value in the case of Johannesburg 82 and chang. The B / Harbin preparations seem to be less stable, they drop by approx. 25% Pluronic) and approx. 40% (with Pluronic). Storage at -20 ° C seems to have a significant effect on the stability, with Johannesburg 82 the values ​​drop by 27% (without Pluronic) 11% (with Pluronic), with Nanchang by 9% (without Pluronic) and 19% respectively. (with Pluronic) and for B / Ha} 1 by 34% (without Pluronic) and 47% (with Pluronic). The results of the storage at room temperature show an astonishing stability of the preparation. In Johannesburg 82 approximately the original HA content can still be detected, in Nanchang approx. 65% and at B / Harbin approx. 4 Storage of the TVBs at + 4 ° C for 1 year again shows no significant differences in the HA content. The stability of the TVB's at room temperature for 1 year differs for the strains: in Johannesburg 82 there are no significant differences in the HA content, in b chang it decreases slightly (approx. 10%) and in B / Harbin it decreases by about one third.

The preparations are overall very stable, even when stored at room temperature, and there are no significant differences between the preparations with and without Pluronic ge

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<td></td><td></td><td>Φ O.</td><td> &lt;</td><td>O</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>O</td><td>O</td><td>O</td><td>O</td><td>O</td>

<td></td><td></td><td rowspan="2">co</td><td></td><td></td><td>O</td><td>O</td><td>O</td><td>O</td><td>O</td><td></td><td>O</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>uZ</td><td>V</td><td></td><td>CM</td><td></td><td></td><td>CM</td><td></td><td>M.</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td>

<td colspan="2">ω r-</td><td></td><td></td><td></td><td>to</td><td></td><td></td><td>CO</td><td></td><td></td><td></td><td></td><td> &lt;0</td><td></td><td></td><td>CO</td>

<td></td><td rowspan="2">CO &gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>s.</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td>X</td><td></td><td></td><td>X</td><td></td><td></td><td></td><td></td><td>X</td><td></td><td></td><td>X</td>

<td colspan="2"> 3</td><td></td><td></td><td></td><td>O</td><td>CD</td><td></td><td>n</td><td></td><td></td><td></td><td></td><td>O</td><td>CD</td><td></td><td>O</td>

<td colspan="2"></td><td></td><td></td><td></td><td></td><td>H</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H"</td><td></td><td rowspan="2"></td>

<td colspan="2"> &lt;</td><td></td><td></td><td> |</td><td> &lt;</td><td>O</td><td> 1</td><td> &lt;</td><td></td><td></td><td> 1</td><td> 1</td><td> &lt;</td><td>O</td><td> 1</td>

<td colspan="2">E.</td><td></td><td></td><td></td><td>X</td><td></td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">E.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">CO</td><td></td><td></td><td></td><td>z</td><td></td><td>z</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">ω</td><td></td><td></td><td></td><td> -5</td><td></td><td> —3</td><td></td><td>z</td><td></td><td>z</td><td></td><td></td><td></td><td> 1</td><td></td>

<td colspan="2"></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>iu</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"></td><td></td><td></td><td>Cl&gt;</td><td></td><td></td><td rowspan="2">CD</td><td></td><td>c /&gt;</td><td></td><td>ω</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"></td><td></td><td></td><td>c</td><td></td><td></td><td></td><td> 3</td><td></td><td> 3</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">ω σ&gt; ο c</td><td></td><td></td><td>-Vakzi</td><td>ΈΓ © ;&gt;</td><td></td><td>Vaccir</td><td>activated)</td><td>Ό C.</td><td></td><td>md-Vir</td><td></td><td>JmT O O 2</td><td></td><td>O O</td><td></td>

<td colspan="2">Ε</td><td></td><td></td><td> 2</td><td>CO</td><td></td><td>1 D&gt;</td><td>CO</td><td>Φ £}</td><td> 2</td><td>Φ JO</td><td></td><td> 2</td><td></td><td>s</td><td></td>

<td></td><td rowspan="2">Ε</td><td></td><td></td><td>ω</td><td>c</td><td></td><td>o&gt;</td><td>c</td><td>Φ</td><td>Φ</td><td>Φ</td><td></td><td>Φ</td><td></td><td rowspan="2">LU</td><td></td>

<td></td><td></td><td></td><td> &gt;</td><td></td><td></td><td>LU</td><td></td><td>—I</td><td> &gt;</td><td></td><td></td><td> &gt;</td><td></td><td></td>

J: Johannesburg 82 (A / H1N1), N: Nanchang (A / H3N2), H: B / Harbin, nb = not determined

AT 407 958 B

Table 3

Storage stability of MB's influenza vaccine of the 1997/98 season I.

<td></td><td>Lot</td><td>storage</td><td>0 months</td><td>6 months</td><td>12 months</td><td></td>

<td rowspan="2">Johannesburg 82</td><td>MB / J / 0197</td><td>+ 4 ° C -20 ° C -80 ° C RT</td><td>184pg</td><td>204pg [111%] 182pg [99%] 210pg [114%]</td><td>189pg [103%] 134pg [73%] 187pg [102%] 152pg [83%]</td><td></td>

<td>MB / J / 0297P</td><td>+ 4 ° C -20 ° C -80 ° C RT</td><td>198pg</td><td>230pg [116%] 202pg [102%] 226pg [114%]</td><td>207pg [105%] 177pg [89%] 212pg [107%] 161 pg [81%]</td><td></td>

<td rowspan="2">Nanchang</td><td>MB / N / 0197</td><td>+ 4 ° C -20 ° C -80 ° C RT</td><td>126pg</td><td>130μg [103%] 124pg [98%] 143pg [114%]</td><td>131pg [104%] 115pg [91%] 132pg [105%] 83pg [66%]</td><td></td>

<td>MB / N / 0297P</td><td>+ 4 ° C -20 ° C -80 ° C RT</td><td>140pg</td><td>128pg [91%] 139pg [99%] 143pg [102%]</td><td>134pg [96%] 113pg [81%] 150pg [107%] 90 pg [64%]</td><td></td>

<td rowspan="4">B / Harbin</td><td rowspan="2">MB / H / 0397</td><td>+ 4 ° C -20 ° C -80 ° C</td><td>116μς</td><td>89pg [77%] 101pg [87%] 97pg [84%]</td><td>83pg [72%] 76pg [66%] 88pg [76%]</td><td></td>

<td>RT</td><td>324μς</td><td> -</td><td>148pg [46%]</td><td></td>

<td rowspan="2">MB / H / 04 / 97P</td><td>+ 4 ° C -20 ° C -80 ° C</td><td>146pg</td><td>95pg [65%] 108pg [74%] 105pg [72%]</td><td>87pg [60%] 77pg [53%] 89pg [61%]</td><td></td>

<td>RT</td><td>374μς</td><td> -</td><td>159pg [43%]</td><td></td>

RT: room temperature P: with Pluronic

Details of the specific haemagglutination (HA) content per ml and in brackets, details of the HA content in relation to the initial value in percent of the

Table 4

Storage stability Influenza vaccine of the 1997/98 season il

Storage of the TVB's (trivalent bulk) at + 4 ° C and at room temperature

<td rowspan="3">tribe</td><td rowspan="3">Lot</td><td rowspan="3">Pluronic</td><td colspan="3">storage</td><td></td>

<td rowspan="2">0 months</td><td colspan="2">12 months</td><td></td>

<td>+ 4 ° C</td><td>Room temperature</td><td>¹</td>

<td>Johannesburg 82</td><td> 410198</td><td> -</td><td>16.8 pg</td><td>17.5pg [104%]</td><td>15.8pg [94%]</td><td></td>

<td>Nanchang</td><td> 410198</td><td> -</td><td>15.9pg</td><td>16.3pg [103%]</td><td>14.1pg [89%]</td><td></td>

<td>B / Harbin</td><td> 410198</td><td> -</td><td>16.3pg</td><td>14.1pg [87%]</td><td>10.6pg [65%]</td><td></td>

AT 407 958 B

<td rowspan="3">tribe</td><td rowspan="3">Lot</td><td rowspan="3">Pluronic</td><td colspan="3">storage</td>

<td rowspan="2">0 months</td><td colspan="2">12 months</td>

<td>+ 4 ° C</td><td>Room temperature</td>

<td>Johannesburg 82</td><td>410298P</td><td> +</td><td>16 ^ g</td><td>17.4pg [103%]</td><td>17.3µ₉ [102%]</td>

<td>Nanchang</td><td>410298P</td><td> +</td><td>15 ^ g</td><td>13 ^ g [90%]</td><td>13 ^ g [90%]</td>

<td>B / Harbin</td><td>410298P</td><td> +</td><td>14 ^ g</td><td>14.1pg [97%]</td><td> 9,7_M.g [67%]</td>

Details of the specific haemagglutination (HA) content per dose (= 0.5 ml) and, in brackets, details of the change in the HA content from the initial value in percent