Vaccine composition with aluminium hydroxide nanoparticles

Abstract

A vaccine composition comprising at least one antigen and one adjuvant, characterized in that the adjuvant comprises sterile-filterable nanoparticles comprising pseudo-boehmite and polyacrylate.

Claims

1. A vaccine composition comprising at least one antigen and an adjuvant, wherein the adjuvant comprises nanoparticles comprising pseudo-boehmite and polyacrylate, and wherein the size of the nanoparticles is such that they are able to pass through a sterilizing filter of 220 nm size pore.

2. The vaccine composition as claimed in claim 1, wherein the core of the nanoparticle consists essentially of pseudo-boehmite and the polyacrylate is located essentially at the surface of the nanoparticles.

3. The vaccine composition as claimed in claim 1, wherein the pseudo-boehmite is at least 90% of the mass of the nanoparticles.

4. The vaccine composition as claimed claim 1, wherein the size of the nanoparticles is less than 300 nm.

5. The vaccine composition as claimed in claim 1, wherein the composition comprises at least one influenza antigen.

6. The vaccine composition as claimed in claim 1, wherein the composition comprises at least one tetanus protein.

7. A method of making a vaccine composition comprising combining nanoparticles comprising pseudo-boehmite and polyacrylate with at least one antigen, wherein the size of the nanoparticles is such that they are able to pass through a sterilizing filter of 220 nm size pore.

8. The method of claim 7, wherein the vaccine composition is formulated for intradermal administration.

9. The method of claim 8, wherein the vaccine composition comprises at least one influenza antigen.

10. A process for preparing a vaccine composition as claimed in claim 1, the process comprising, preparing nanoparticles comprising pseudo-boehmite and polyacrylate, wherein the size of the nanoparticles is such that they are able to pass through a sterilizing filter of 220 nm size pore, filtering said nanoparticles by means of a sterilizing filter, adding at least one vaccine antigen to said nanoparticles, and, optionally, performing an additional filtration.

11. The vaccine composition as claimed in claim 2, wherein the size of the nanoparticles is less than 220 nm.

12. The vaccine composition as claimed in claim 11, wherein the pseudo-boehmite is at least 90% of the mass of the nanoparticles.

13. The vaccine composition as claimed in claim 12, wherein the composition comprises at least one influenza antigen.

14. The vaccine composition as claimed in claim 13, wherein the composition comprises at least one tetanus protein.

15. The method of claim 7, wherein the size of the nanoparticles is less than 220 nm.

16. The method as claimed in claim 15, wherein the pseudo-boehmite is at least 90% of the mass of the nanoparticles.

17. The method as claimed in claim 16, wherein the composition comprises at least one influenza antigen.

18. The method as claimed in claim 16, wherein the composition comprises at least one tetanus antigen.

Description

EXAMPLE 1: SYNTHESIS OF A COLLOIDAL SUSPENSION OF PSEUDO-BOEHMITE NANOPARTICLES

(1) Sodium polyacrylate (NaPa) of molecular mass 2100 supplied by Fluka and aluminum chloride hexahydrate (AlCl.sub.3.6H.sub.2O) supplied by Avocado were used.

(2) 9 g of NaPa were dissolved in 75 ml of aqueous 0.1 M AlCl.sub.3 solution. The resulting mixture was stirred vigorously at room temperature for 24 hours. The pH of the mixture was then between 5.5 and 5.9. 5 M sodium hydroxide NaOH was then added dropwise until a pH of 10.5 was obtained, and the mixture was then stirred for a further 10 minutes. When the pH increase reached 9.0-9.5, the solution became slightly turbid, which corresponds to the precipitation of the pseudo-boehmite nanoparticles. To obtain the final nanoparticles, the suspension obtained at pH 10.5 was transferred into an autoclave and heated at 160° C. with gentle stirring (15 rpm) for 3 hours. Next, the colloidal suspension was washed by dialysis against 5 L of distilled water using a Sartorius Slice 200 benchtop machine equipped with a 30 kDa polyether polysulfone membrane. The resulting colloidal suspension was stored in polypropylene bottles. The size measurements performed showed that a single population of nanoparticles was present, the size of which ranged from 15 to 40 nm (numerical distribution), with a polydispersity index of 0.19 (intensity analysis). A sample of solid material was obtained by centrifugation at 25 000 rpm for 1 hour and dried at 80° C. overnight, for physicochemical analytical purposes.

(3) The sample was analyzed by X-ray diffraction by means of a Stoe-Stadi-P diffractometer using the Kα1 line of copper (λ=1.5406 Å). The radiation is perfectly monochromatic by virtue of the presence of a front monochromator consisting of a germanium crystal. For this analysis, the samples, ground beforehand, are placed in Lindemann tubes (0.3 mm in diameter). The capillary tube, placed on a goniometric head, is rotated and the diffractogram is recorded in Debye-Scherrer mode using a short linear detector of PSD type (position-sensitive detector) able to cover an angular range of 11° (2θ).

(4) The X-ray diffraction analysis of the dried sample gave a result characteristic of pseudo-boehmite.

(5) The nanoparticles obtained were able to be sterile filtered through a 0.2 μm Millipore PVDF membrane, with a minimal loss of material (3.1%) determined by atomic absorption.

EXAMPLE 2: SYNTHESIS OF NANOPARTICLES OF PSEUDO-BOEHMITE AND OF POLYACRYLATE USING A SOLUTION OF SODIUM POLYACRYLATE OF MOLAR MASS 60 000

(6) Sodium polyacrylate (NaPa) of molecular mass 60 000 supplied by Polysciences in the form of an aqueous 35% (w/w) solution and aluminum chloride hexahydrate (AlCl.sub.3.6H.sub.2O) supplied by Fluka were used.

(7) In order to obtain 36 g of polymer, 102.86 g of polymer solution which contained 66.86 g of water were taken.

(8) 7.2429 g of AlCl.sub.3 were placed in a beaker and 225.89 g of water were added thereto. This aluminum solution was added to the polymer solution, so as to obtain 300 ml of a 0.1 M AlCl.sub.3 solution.

(9) The mixture obtained was white.

(10) This mixture was stirred vigorously for 24 hours.

(11) After 24 hours, the pH of the mixture was 6.1. 5 M sodium hydroxide NaOH was then added dropwise up to a pH of 10.2.

(12) The mixture was homogenized with stirring for 1 hour and then placed with stirring (1400 rpm) in an autoclave and heated at 160° C. for 3 hours.

(13) The solution obtained was slightly opalescent, and was analyzed to show that it consisted of nanoparticles having by X-ray diffraction peaks characteristic of pseudo-boehmite, and a mean size of about 100 nm, compatible with sterilizing filtration.

(14) The solution was able to be stored for more than 1 year while maintaining the integrity of the nanoparticles, which did not reaggregate.

EXAMPLE 3: PREPARATION OF A VACCINE COMPOSITION COMPRISING NANOPARTICLES OF ALUMINUM HYDROXIDE AND PURIFIED TETANUS PROTEIN, AND FILTRATION TEST

(15) A vaccine composition was prepared by adding 10 μl of a preparation of purified tetanus protein assayed at a concentration of 1200 flocculation units/ml of saline solution to 4.5 ml of a pseudo-boehmite nanoparticle suspension prepared according to example 1.

(16) The mixture was stirred moderately and then passed through a PVDF membrane (supplied by Millipore) mounted on a 5 ml plastic syringe.

(17) Measurement of the size of the nanoparticles was performed both before and after the addition of tetanus protein to the nanoparticles; the same size profile was obtained, whether in the presence or absence of protein, which demonstrates that the tetanus protein does not lead to aggregation of the nanoparticles.

(18) Similarly, it was determined that the filtration led to a 3% loss of aluminum, which is entirely acceptable from an industrial viewpoint.

(19) It may thus be concluded that the vaccine compositions according to the invention may be filtered without excessive loss of material on a 0.22 μm sterilizing filter.

EXAMPLE 4: REACTOGENICITY TEST ON MICE OF A COMPOSITION ACCORDING TO THE INVENTION COMPRISING NANOPARTICLES AND INFLUENZA ANTIGENS, ADMINISTERED INTRADERMALLY

(20) Female BALB/c mice were used as animal model for this test, in order to evaluate the local reactogenicity of compositions according to the invention, compared with standard aluminum suspensions.

(21) In this test, the antigen consisted of a trivalent influenza vaccine known as Flu ID stock comprising the inactivated fragmented strains A/Solomon/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004 at a rate of 150 μg/ml of HA per strain.

(22) Four groups of 10 mice received at an interval of 3 weeks, intradermally, into the inner face of the ear, a sub-optimal dose of 30 μl (i.e. 0.3 μg of HA/strain) of vaccine in the presence or absence of an adjuvant.

(23) The administered compositions were prepared in the following manner: Group A: Flu ID alone; 54 μl of Flu ID stock were diluted in 756 μl of PBS buffer. Group B: Flu ID+AlOOH; the following were successively added: 54 μl of Flu ID stock 604 μl of PBS buffer 152 μl of a commercial suspension of AlOOH containing 8.01 mg/ml of aluminum (Alhydrogel®) The mixture was stirred moderately for 2 hours. Group C: Flu ID+AlPO.sub.4; the following were successively added: 54 μl of Flu ID stock 463 μl of PBS buffer 293 μl of a commercial suspension of AlPO.sub.4 containing 4.15 mg/ml of aluminum (AdjuPhos®) The mixture was stirred moderately for 2 hours. Group D: Flu ID+nanoAlOOH; the following were successively added: 54 μl of Flu ID stock 5 μl of H.sub.2O 76 μl of 10-fold concentrated PBS buffer 675 μl of a pseudo-boehmite nanoparticle suspension, prepared in the manner described in example 1 and comprising 1.8 mg of Al/ml. The mixture was stirred moderately for 2 hours at room temperature.

(24) The mice were monitored each day, and the edemas, the erythemas and the lesions appearing on the ear were graded on an unofficial scale, for 2 weeks after each injection.

(25) Irrespective of the formulation tested, no significant erythema was observed. Similarly, no edema was reported.

(26) However, white/reddish nodules were noted at the point of injection, appearing in the case of the mice which received the compositions comprising standard aluminum, whether with aluminum hydroxide or with aluminum phosphate. However, surprisingly and very interestingly, no nodules were visible on the mice which received a composition according to the invention.

(27) The results relating to the number of mice presenting nodules after administration of the adjuvants comprising the aluminum of the prior art are illustrated in table 1 below:

(28) TABLE-US-00001 TABLE 1 After the first injection (D0) Adjuvant D1 D2 D3 D4 D7 D8 D9 D10 D11 D15 AlOOH 0/10 0/10 0/10 0/10 6/10 10/10 10/10 10/10 10/10 8/10 AlPO.sub.4 0/10 1/10 2/10 3/10 6/10  9/10  9/10  9/10 10/10 9/10 After the repeat injection (D21) Adjuvant D22 D23 D24 D25 D28 D29 D30 D31 D32 D35 D39 AlOOH 5/10 9/10 10/10 9/10 10/10 10/10 10/10 10/10 10/10  8/10  9/10 AlPO.sub.4 9/10 9/10  8/10 7/10  9/10 10/10 10/10 10/10 10/10 10/10 10/10

(29) It is particularly interesting to note that, despite the fact that the amounts of aluminum are the same (45 μg) in all the groups, formulating the aluminum in nanoparticles according to the invention makes it much better tolerated.

EXAMPLE 5: IMMUNOGENICITY TEST ON MICE OF A COMPOSITION ACCORDING TO THE INVENTION COMPRISING NANOPARTICLES AND INFLUENZA ANTIGENS, ADMINISTERED INTRADERMALLY

(30) This test evaluated the immunogenicity of the compositions according to the invention compared with compositions comprising standard aluminum, either AlOOH or AlPO.sub.4, but also with regard to a composition not comprising any aluminum but only polymer. In this test, as in the preceding example, the antigen consisted of a trivalent influenza vaccine known as Flu ID stock comprising the inactivated fragmented strains A/Solomon/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004 at a rate of 150 μg/ml of HA per strain. The batch of Flu ID stock vaccine used for this test was the same as that used in the preceding example.

(31) The test was performed on female BALB/c mice divided into five groups of 9.

(32) Each mouse received, at an interval of 3 weeks, intradermally, into the inner face of the ear, a sub-optimal dose of 30 μl (i.e. 0.3 μg of HA/strain) of a composition as described below, as a function of the group to which it belonged.

(33) The administered compositions were prepared in the following manner: Group A: Flu ID alone; 54 μl of Flu ID stock were diluted in 756 μl of PBS buffer. Group B: Flu ID+AlPO.sub.4; the following were successively added: 54 μl of Flu ID stock 463 μl of PBS buffer 293 μl of a commercial suspension of AlPO.sub.4 containing 4.15 mg/ml of aluminum (AdjuPhos®) The mixture was vortexed for 10 seconds. Group C: Flu ID+AlOOH; the following were successively added: 54 μl of Flu ID stock 595 μl of PBS buffer 161 μl of a commercial suspension of AlOOH containing 7.53 mg/ml of aluminum (Alhydrogel®) The mixture was vortexed for 10 seconds. Group D: Flu ID+pseudo-boehmite nanoparticles; the following were successively added: 54 μl of Flu ID stock 5 μl of H.sub.2O 76 μl of 10-fold concentrated PBS buffer 675 μl of a pseudo-boehmite nanoparticle suspension, prepared in the manner described in example 1 and comprising 1.8 mg of Al/ml. The mixture was vortexed for 10 seconds. Group E: Flu ID+polymer; the following were successively added: 54 μl of Flu ID stock 5 μl of H.sub.2O 76 μl of 10-fold concentrated PBS buffer 675 μl of a solution containing 17 mg/ml of NaPa (sodium polyacrylate) of molar mass 2100 supplied by Fluka. The mixture was vortexed for 10 seconds.

(34) The mice were monitored throughout the test.

(35) Blood samples were taken from each mouse at D42, i.e. 3 weeks after the second injection, by sectioning of the carotid vein. The samples were treated in order to isolate the serum to perform the humoral response tests.

(36) The spleens were also taken from six mice per group in order to perform the cell response tests.

(37) The tests that were performed are ELISA assays, hemagglutination inhibition tests, and also ELISPOT assays.

(38) The ELISA assays were performed in a conventional manner, in order to determine the amounts of serum IgG1 and IgG2a specific for the strain A/H1N1. The antibody detection threshold is 20 (1.3 log 10) ELISA units. All the titers are expressed in log 10 of ELISA units. The geometrical mean and the corresponding 95% confidence interval were calculated for each group of animals.

(39) The hemagglutination inhibition test makes it possible to assess the functional antibodies present in the serum of the immunized animals. It measures the capacity of the induced antibodies to inhibit the hemagglutination of hen red blood cells by the influenza virus studied. The hemagglutination inhibition (HI) titer is the inverse of the final dilution for which no hemagglutination is observed. The geometrical mean and the corresponding 95% confidence interval were calculated for each group of animals. This was done with regard to each of the three strains present in the administered composition.

(40) The ELISPOT assays are performed using freshly isolated spleen cells, incubated overnight and restimulated with a mixture of the three strains of the vaccine composition or with a nonamer peptide corresponding to a class I epitope (epitope CD8) of the NP protein. The cell responses are expressed as the number of cells secreting influenza-specific IL-5 or IFN-γ, for 10.sup.6 splenocytes.

(41) During the restimulation with the influenza-specific peptide, no CD8+ T cells secreting IL-5 or IFN-γ were detected by ELISPOT, irrespective of the group of mice under consideration.

(42) As regards the in vitro restimulation with the influenza antigens, the results obtained are represented in table 2 below, in which the 95% confidence intervals are indicated in parentheses:

(43) TABLE-US-00002 TABLE 2 Group under Number of cells secreting Number of cells secreting consideration IL-5/10.sup.6 splenocytes IFN-γ/10.sup.6 splenocytes A: Flu ID 24 [12-48] 2 [0-17] alone B: Flu ID + 17 [6-45] 2 [0-32] AlPO.sub.4 C: Flu ID + 46 [19-115] 13 [5-38] AlOOH D: Flu ID + 197 [79-491] 79 [30-205] nanoparticles E: Flu ID + 80 [42-151] 8 [1-81] polyacrylate

(44) These results show that, surprisingly, by means of the nanoparticles according to the invention, it is possible to obtain a cell response, especially stimulation of the cells secreting IL-5 and also of the cells secreting IFN-γ.

(45) From a statistical viewpoint, according to a mixed model with Dunnett adjustment, the nanoparticles are considered to have significantly increased the number of cells secreting IL-5 and IFN-γ by 8.2 times (p<0.001) and by 39.5 times (p=0.002), respectively, which is not the case for the aluminum-based adjuvants of the prior art.

(46) It is also noted that polyacrylate alone increased the secretion of IL-5 by only 3.3 times (p=0.027), which is at the significance limit.

(47) The results relating to the humoral response tests are collated in table 3 below, in which, in each case, the 95% confidence intervals are given in parentheses.

(48) TABLE-US-00003 TABLE 3 Group under HI against HI against IgG1 against IgG2a against consideration H1N1 H3N2 HI against B H1N1 (log10) H1N1 (log10) A: Flu ID 101 [52-194] 127 [66-244] 17 [6-46] 4.8 [4.4-5.2] 4.3 [3.9-4.6] alone B: Flu ID + 1881 [1096-3229] 1097 [654-1842] 593 [301-1165] 6.4 [6.2-6.7] 4.4 [3.9-4.9] AlPO.sub.4 C: Flu ID + 1185 [637-2207] 1185 [603-2331] 640 [353-1161] 6.4 [6.1-6.6] 4.1 [3.4-4.8] AlOOH D: Flu ID + 1185 [676-2078] 1613 [1017-2558] 508 [228-1130] 6.1 [5.9-6.4] 5.1 [4.7-5.4] nanoparticles C: Flu ID + 93 [49-177] 148 [90-243] 13 [4-38] 5.0 [4.8-5.3] 4.1 [3.7-4.5] polyacrylate

(49) From a statistical viewpoint, the increase of the HI titers and of the IgG1 titers obtained with the nanoparticles was considered significant with regard to the titers obtained with the vaccine alone according to a mixed model with Dunnett adjustment (11.7 times for the anti-H1N1 HI titer with p<0.001; 12.7 times for the anti-H3N2 HI titer with p<0.001; 29.9 times for the anti-B titer with p<0.001; 20.0 times for the anti-H1N1 IgG1 titer with p<0.001 and 6.3 times for the anti-H1N1 IgG2a titer with p=0.008).

(50) These results show the capacity of the nanoparticles according to the invention to induce a humoral response after two intradermal immunizations. It may be noted that the polymer alone does not have this adjuvant power.

(51) Moreover, only the nanoparticles according to the invention lead to an increase in the IgG2a response, which means that the immune response profile is more oriented toward a response of TH1 type than for the other aluminum-based adjuvants.