Title: Inactivated influenza virus vaccine for nasal or oral application
United States Patent: 6,635,246
Issued: October 21, 2003
Inventors: Barrett; Noel (Klosterneuburg/Weidling, AT); Kistner; Otfried (Vienna, AT); Gerencer; Marijan (Vienna, AT); Dorner; Friedrich (Vienna, AT)
Assignee: Baxter Healthcare S.A. (Zurich, CH)
Appl. No.: 913400
Filed: December 5, 2001
PCT Filed: February 1, 2000
PCT NO: PCT/AT00/00023
PCT PUB.NO.: WO00/47222
PCT PUB. Date: August 17, 2000
Abstract
The invention relates to nasal or oral administration of a compound containing inactivated influenza virus antigen and aluminum as adjuvant for the prophylaxis of influenza virus infections. Said vaccine is especially suitable for inducing a mucosal IgA immune response and systemic IgG immune response.
BRIEF SUMMARY OF THE INVENTION
The problem is solved 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 suitable in particular as a vaccine for the prophylaxis of influenza virus infections.
In the context of the present invention, it was shown that an inactivated influenza virus vaccine containing aluminum as adjuvant for nasal or oral administration triggers an effective IgG as well as IgA immune response in mammals. This was especially surprising because with the approaches to date towards the development of effective influenza virus vaccines it was found that the adjuvant effect of aluminum in elevating the immunogenicity of the antigen is very slight, even in a vaccine for parenteral administration (Davenport et al., 1968, J. Immunol. 100:1 139-1140).
Furthermore, it was found that with the nasal or oral application of the vaccine composition according to the invention a considerably higher IgG and IgA titer as well as a higher HAI titer is achieved in mammals than with the vaccine formulations known to date that contain either only inactivated influenza viruses, inactivated viruses with cholera toxin, or live viruses (Table 1).
Therefore, the application according to the invention is suitable in particular for the induction of a protective mucosal IgA and a systemic IgG immune response.
Since aluminum is the only adjuvant approved for application in humans, the application, according to the invention, of the vaccine combination of inactivated influenza virus and aluminum has the great advantage that it can be administered directly to humans without any problem. The special advantage of the use according to the invention, therefore, aside from the elevated immunoreactivity of the vaccine composition for nasal or oral administration is that through use of an adjuvant that has been tested over a number of years and whose application to humans is approved, the vaccine is completely free of side effects.
DETAILED DESCRIPTION OF THE INVENTION
For use according to the invention, the composition can contain aluminum preferably in the form of aluminum hydroxide (Al(OH)3) or aluminum phosphate (AlPO4). In this case, the concentration of the aluminum is preferably in a final concentration of 0.05% to 0.5%.
The influenza virus antigen quantity in the vaccine in this case is the customary antigen quantity for a vaccine dose. Preferably, the antigen quantity that is contained in a vaccine dose is between 1.5 .mu.g antigen/dose to 50 .mu.g antigen/dose in humans.
The influenza virus antigen can be produced from infected eggs via conventional methods, and purified.
Preferably, however, the virus antigen is obtained from an infected cell culture, such as described, for example, in WO 96/15231. Particularly preferred for the use according to the invention to produce an influenza virus vaccine is an influenza virus antigen that is obtained from a VERO cell culture infected with influenza virus that is cultured in a serum and protein-free medium. The virus antigen obtained from the infected cell culture is first inactivated with formalin and can then be obtained as a purified, concentrated virus antigen preparation by means of continuous density gradient centrifugation, DNAse treatment, diafiltration, and sterile filtration. This concentrated preparation can then be used together with aluminum as an adjuvant for the use according to the invention to produce a vaccine for nasal or oral administration.
A special advantage in the production of the vaccine is that the virus material is inactivated before purification, and so in comparison to the purification of attenuated live viruses, a considerably higher degree of purity of the antigen preparation is achieved.
A particular advantage in the use of influenza virus antigens obtained from a serum and protein-free cell culture infected with influenza virus is the absence of egg-specific proteins that could trigger an allergic reaction against these proteins. Therefore, the use according to the invention is especially suitable for the prophylaxis of influenza virus infections, particularly in populations that constitute higher-risk groups, such as asthmatics, those with allergies, and also people with suppressed immune systems and the elderly. The vaccine can be applied in different ways.
According to one embodiment of the invention, the intranasal administration is via the mucosal route. The intranasal administration of the vaccine composition can be formulated, for example, in liquid form as nose drops, spray, or suitable for inhalation, as powder, as cream, or as emulsion.
The composition can contain a variety of additives, such as stabilizers, buffers, or preservatives.
For simple application, the vaccine composition is preferably supplied in a container appropriate for distribution of the antigen in the form of nose drops or an aerosol.
According to another embodiment of the invention, the administration is oral and the vaccine may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule, which simplifies oral application. The production of these forms of administration is within the general knowledge of a technical expert.
The invention will be explained in more detail on the basis of the following examples, whereby it is not limited to the examples.
EXAMPLES
Example 1 Production of an influenza virus vaccine preparation.
Influenza virus was obtained from a protein-free VERO cell culture infected with influenza A or B virus strain, according to WO 96/15231 or according to conventional methods from allantoic fluid from infected, incubated chicken eggs.
For the production of an inactivated influenza virus preparation from cell culture, the supernatant of an infected VERO cell culture to which formalin (final concentration 0.025%) was added, and the viruses were inactivated at 32oC. for 24 h. This material was purified by zonal centrifugation in a continuous 0-50% sucrose gradient, DNAse treatment, diafiltration, and sterile filtration. The purified material was stored at -70oC. The final product was tested for residual contamination and the following criteria were established per dose:
Hemagglutinin content: .gtoreq.15 .mu.g HA per strain
Protein content: .ltoreq.250 .mu.g
Sucrose content: .ltoreq.200 mg
Formalin content: .ltoreq.5 .mu.g
Benzonase content: .ltoreq.5 ng
Residual DNA (VERO): .ltoreq.100 pg
Endotoxin content: .ltoreq.100 EU
Pyrogen: free
Example 2 Intranasal immunization of mice with different influenza virus preparations.
The antigen preparations from Example 1 were diluted in PBS to an HA antigen content of 15 .mu.g/mL and optionally Al(OH)3 added to a final concentration of 0.2%, or cholera toxin. For the production of a preparation for intranasal immunization of mice, the solution was diluted to the appropriate quantity of antigen with PBS, optionally containing Al(OH)3 or cholera toxin.
Four Balb/c mice each received intranasal immunization with different influenza virus preparations, and in each case, 50 .mu.L of a solution containing influenza virus antigen and optionally an adjuvant were administered drop-wise into the nostrils of the mice. The first immunization was given on Day 0, the second on Day 7, and the third on Day 14. On the 28th day the IgG, IgA, and HAI titers in serum, saliva, and pulmonary lavage were determined.
Table 1 shows the plan of the intranasal immunization of the individual
mice groups with different influenza virus preparations.
TABLE 1
Vaccination plan for the intranasal immunization of mice
Group Balc/c mice Antigen Dose Route
1. Group Inactivated whole 1 .mu.g HA 50 .mu.l/i.n.
viruses from VERO
cells
2. Group Inactivated whole 1 .mu.g HA 50 .mu.l/i.n.
viruses from
infected eggs
3. Group Live viruses from 5 x 106 EID50 50
.mu.l/i.n.
VERO cells
4. Group Live viruses from 5 x 106 EID50 50
.mu.l/i.n.
infected eggs
5. Group VERO mock 5% of 1 .mu.g 50 .mu.l/i.n.
preparation
6. Group Egg mock 5% of 1 .mu.g 50 .mu.l/i.n.
preparation
Example 3 Determination of the IgA titer in the saliva, pulmonary lysate, and serum, as well as of the IgG titer and HAI titer in the serum.
On Day 28 after immunization, saliva, pulmonary, and serum specimens were taken from the animals, and the antibody titer in the individual specimens was determined.
Saliva specimens were collected by injection of 0.5 mL PBS into the oral cavity of the mouse, and the presence of IgA antibodies was tested.
To produce pulmonary lysate specimens, the mice were killed, the thorax opened, and the lungs removed and washed with PBS. Subsequently, the lungs were ground and the homogenate centrifuged in order to remove cell tissue. The supernatant was collected and stored at -20oC. until testing for IgA antibodies.
The IgG and IgA antibody titer was determined with a commercial influenza virus Type A and B ELISA test (Genzyme Virotech). After a 2 h incubation at 37oC., the specific IgG and IgA antibodies were detected with conjugated goat anti-mouse IgG or IgA (Pharmigen) and chromogenic substrate containing H2 O2 and o-phenyldiamine.
To determine the HAI titer, blood was taken from the mice, and the serum obtained was tested in the influenza A or B hemagglutination test (HAI titer) according to the method of Palmer et al. (1975, Advanced laboratory technicals [sic; techniques] for immunological diagnostic, U.S. Dept. Health. Ed. Welfare. P.H.S. Atlanta, Immunology Ser. No. 6, Procedural guide, part 2, hemagglutination inhibition test, pp. 25-62).
Table 2 shows a summary of the determination of the IgA antibody titer in the saliva, pulmonary lysate, and serum, and the IgG antibody titer as well as HAI titer in the serum. The data clearly indicate that neither the IgA nor the IgG immune response is stimulated by an inactivated whole virus vaccine without adjuvant. On the other hand, if the immunization is done with live virus or inactivated whole virus to which cholera toxin has been added, an increase of the IgA, IgG, and HAI titers takes place in the mice, just as after immunization with inactivated whole virus to which aluminum has been added, whereby with the latter vaccine, the IgG immune response in the serum was actually the highest of all preparations tested. Likewise, the highest HAI titer was measured for the inactivated vaccine with aluminum.
The results show that the intranasal immunization with inactivated influenza virus vaccine to which aluminum has been added induces a slightly increased IgA immune reaction in comparison to the known influenza virus vaccine preparations, and a considerably higher IgG immune response in mammals, without having the disadvantages of a live virus vaccine or a vaccine to which an adjuvant has been added that has not been approved for application to humans, such as cholera toxin.
Example 4 Storage stability of the vaccine composition at different temperatures
For stability investigations, the monovalent bulks (MB) of the influenza virus vaccination strains Johannesburg 82, Nanchang and B/Harbin were stored for 12 months at +4oC., -20oC., and -80oC. After 6 and 12 months, the specific hemagglutination test (HA) content of the MBs Johannesburg 82 (MB/J/0197), Nanchang (MB/N/0197), and B/Harbin (MB/H/0397) without Pluronic, as well as Johannesburg 82 (MB/J/0297P), Nanchang (MB/N/0297P), and B/Harbin (MB/H/0497P) with Pluronic was determined by means of a SRD (single radial immunodiffusion) assay according to Wood et al., 1977, J. Biol. Standard 5:237-247, and the deviation from the initial value was calculated in percent.
The trivalent bulks (TVB) 410197 (without Pluronic) and 4102997P (with Pluronic) were also stored for 12 months at +4oC., and then tested in the SRD assay. Furthermore, the reserve samples of these MBs and TVBs that had been stored at room temperature for sterility testing were analyzed in the SRD assay after 12 months.
The results of the MBs are given in Table 3, and the results of the TVBs in Table 4:
The storage of the MBs at +4oC. and -80oC. for 1 year show practically no reduction of the specific HA content compared with the initial value in the case of Johannesburg 82 and Nanchang. The B/Harbin preparations appear to be less stable; they drop by about 25% (without Pluronic) and about 40% (with Pluronic). Storage at -20oC. appears to have a significant influence on the stability; for Johannesburg 82, the values drop by 27% (without Pluronic) or 11% (with Pluronic), for Nanchang by 9% (without Pluronic) or 19% (with Pluronic), and for B/Harbin by 34% (without Pluronic) or 47% (with Pluronic). The results of storage at room temperature indicate an astounding stability of the preparation. For Johannesburg 82, approximately 80% of the original HA content can still be detected, for Nanchang ca. 65%, and for B/Harbin about 45%. Storage of the TVBs at +4oC. for 1 year again shows no significant difference in the HA content. The stability of the TVBs at room temperature for 1 year differs in the 3 strains: for Johannesburg 82 there is no significant difference in the HA content, for Nanchang a slight reduction (about 10%), and for B/Harbin a drop of approximately one third.
Overall, the preparations are very stable, even with storage at room temperature, and there is no significant difference between the preparations with and without Pluronic.
TABLE 2
Intranasal immunization of mice with different influenza preparations,
and determination of the IgA titer in
the saliva, pulmonary lysate, and serum and the IgG titer as
well as HAI titer in the serum
Titer
IgA
IgG HAI
Saliva Pulmonary lysate Serum
Serum Johann 82 Nanchang B/Harbin
Immunogen Strain Adjuvant Flu A Flu B Flu A Flu B Flu A Flu B
Flu A Flu B A/H1N1 A/H3N2 B
Vero Vaccine J, N, H -- <10 <10 <10 <10 <10
<10 800 100 80 80 20
(inactivated) Al(OH)3 40 10 320 40 160
<10 102.400 3.200 1.280 640 160
CTB 20 10 n.b. n.b. 80 <10
51.200 12.800 640 640 160
Egg vaccine J, N, H -- 10 10 10 <10 <10 <10
1,600 100 160 160 40
(inactivated Al(OH)3 40 20 320 40 160
10 51.200 6.400 640 320 160
Live virus, N -- 20 <10 n.b. n.b. 160 <10
51.200 <10 80 640 <10
Vero
Live virus, N -- 40 <10 n.b. n.b. 160 <10
51.200 <10 80 320 20
egg
Vero Mock -- <10 <10 <10 <10 <10
<10 <10 <10 80 160 20
-- Al(OH)3 <10 <10 n.b. n.b. <10
<10 <10 <10 80 160 <10
CTB <10 <10 n.b. n.b. <10
<10 <10 <10 80 160 <10
Egg Mock -- -- <10 <10 <10 <10 <10
<10 <10 <10 80 160 20
-- Al(OH)3 <10 <10 <10 <10 <10
<10 <10 <10 80 160 20
J: Johannesburg 82 (A/H1N1),
N: Nanchang (A/H3N2),
H: B/Harbin,
n.b. = not determined
TABLE 3
Storage stability of the MBs of influenza vaccine for the season 1997/98 I
Lot Storage 0 Months 6 Months 12 Months
Johannesburg 82 MB/J0197 +4oC. 184 .mu.g 204 .mu.g [111%] 189
.mu.g [103%]
-20oC 182 .mu.g [99%] 134
.mu.g [73%]
-80oC. 210 .mu.g [114%] 187
.mu.g [102%]
RT -- 152 .mu.g
[83%]
MB/J/0297P +4oC. 198 .mu.g 230 .mu.g [116%] 207
.mu.g [105%]
-20oC. 202 .mu.g [102%] 177
.mu.g [89%]
-80oC. 226 .mu.g [114%] 212
.mu.g [107%]
RT -- 161 .mu.g
[81%]
Nanchang MB/N0197 +4oC. 126 .mu.g 130 .mu.g [103%] 131
.mu.g [104%]
-20oC. 124 .mu.g [98%] 115
.mu.g [91%]
-80oC. 143 .mu.g [114%] 132
.mu.g [105%]
RT -- 83 .mu.g
[66%]
MB/N/0297P +4oC. 140 .mu.g 128 .mu.g [91%] 134
.mu.g [96%]
-20oC. 139 .mu.g [99%] 113
.mu.g [81%]
-80oC. 143 .mu.g [102%] 150
.mu.g [107%]
RT -- 90 .mu.g
[64%]
B/Hardin MB/H/0397 +4oC. 116 .mu.g 89 .mu.g [77%] 83
.mu.g [72%]
-20oC. 324 .mu.g 101 .mu.g [87%] 76
.mu.g [66%]
-80oC. 97 .mu.g [84%] 88
.mu.g [76%]
RT -- 148 .mu.g
[46%]
MB/H/04/97P +4oC. 146 .mu.g 95 .mu.g [65%] 87
.mu.g [60%]
-20oC. 374 .mu.g 108 .mu.g [74%] 77
.mu.g [53%]
-80oC. 105 .mu.g [72%] 89
.mu.g [61%]
RT -- 159 .mu.g
[43%]
RT: Room temperature P: with Pluronic
Data on the specific hemagglutination (HA) content given per mL and in
brackets, data on the HA content compared to the initial value given in
percent
TABLE 4
Storage stability of the influenza vaccine for the season 1997/98 II
Storage of the TVBs (trivalent bulk) at +4oC and at room
temperature
Storage
12 months
Room
Strain Lot Pluronic 0 Months +4oC.
temperature
Johannesburg 82 410198 - 16.8 .mu.g 17.5 .mu.g [104%] 15.8
.mu.g [94%]
Nanchang 410198 - 15.9 .mu.g 16.3 .mu.g [103%] 14.1
.mu.g [89%]
B/Harbin 410198 - 16.3 .mu.g 14.1 .mu.g [87%] 10.6 .mu.g
[65%]
Johannesburg 82 410298P + 16.9 .mu.g 17.4 .mu.g [103%] 17.3
.mu.g [102%]
Nanchang 410298P + 15.4 .mu.g 13.9 .mu.g [90%] 13.9 .mu.g
[90%]
B/Harbin 410298P + 14.5 .mu.g 14.1 .mu.g [97%] 9.7 .mu.g
[67%]
Data on the specific hemagglutination (HA) content per dose (= 0.5 mL), and
in brackets data on the change of the HA content compared to the initial
value given in percent
Claim 1 of 20 Claims
What is claimed is:
1. A method of provoking an immune response in a subject against influenza, wherein the method comprises nasally or orally administering to the subject a composition comprising an effective amount of (i) influenza virus antigen obtained from a mammalian cell culture comprising protein-free media and (ii) an aluminum salt, wherein the composition is free of media and egg proteins.
____________________________________________
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full
patent.
| [
Outsourcing Guide
] [
Cont. Education
] [
Software/Reports
] [
Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ] [ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ] [ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ] [ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ] [ Site Map ] |