METHOD FOR PREPARING A VACCINE ANTIGEN, RESULTING VACCINE ANTIGEN AND USES

Abstract

The present invention relates to a method for preparing a vaccine antigen, which includes a step of fragmenting a biological membrane associated with said vaccine antigen by treating said biological membrane with at least one calixarene of formula (II): wherein: X is a —(CH2)-CO2Y group and Y is an alkaline metal or one of the pharmaceutically acceptable salts thereof, wherein said resulting vaccine antigen also includes a fragment of the biological membrane associated with said antigen. The present invention also relates to a vaccine that can be produced by implementing the method, including a calixarene of formula (II) in carrier format, with a quantity of 0.1 to 1,000 μg in the total weight of the vaccine. The present invention further relates to the use of a calixarene as defined above for the preparation of a vaccine or a vaccine antigen, and to the vaccine for use as a drug in the treatment or prevention of an infectious disease.

##STR00001##

Claims

1. A process for preparing a vaccine antigen, comprising a step of fragmenting a biological membrane associated with said vaccine antigen by bringing said biological membrane into contact with at least one calixarene of formula (II): ##STR00005## in which: X is a —(CH.sub.2)—CO.sub.2Y group, and Y is an alkali metal, or a pharmaceutically acceptable salt thereof, wherein said vaccine antigen obtained also comprises a fragment of the biological membrane, said fragment being associated with said antigen.

2. The process as claimed in claim 1, wherein Y is sodium.

3. The process as claimed in claim 1, wherein said biological membrane is derived from a prokaryotic or eukaryotic organism or from a virus.

4. The process as claimed in claim 1, wherein said biological membrane is derived from an isolated plant or animal cell or from an embryonated egg.

5. The process as claimed in claim 1, wherein said vaccine antigen comprises a membrane protein of viral or microbiological origin.

6. The process as claimed in claim 5, wherein said viral origin is chosen from the group comprising: the orthomyxoviridae virus family, the paramyxoviridae virus family, the coronaviridae virus family, the flaviviridae virus family, the Rhabdoviridae virus family, the Togaviridae virus family, the herpesviridae virus family, the poxviridae virus family and the retroviridae virus family.

7. The process as claimed in claim 6, wherein said flu viruses are chosen from influenza viruses A and influenza viruses B.

8. The process as claimed in claim 1, also comprising a step of dialyzing said vaccine antigen.

9. A vaccine that can be produced by carrying out the process as defined in claim 1, comprising a calixarene of formula (II) in excipient format in an amount of from 0.1 to 1000 μg in the total weight of the vaccine.

10. The vaccine as claimed in claim 9, wherein said vaccine antigen comprises a membrane protein of viral or microbiological origin.

11. The use of a calixarene as defined in claim 1, for preparing a vaccine or a vaccine antigen.

12. The vaccine as claimed in claim 9, for application thereof as a medicament in the treatment or prevention of an infectious disease.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0160] FIG. 1 represents the CALX133ACE immunization protocol with challenge in mice, at D−42 (42 days before the infection), D−21 (21 days before the infection), DO (day of the infection and of the blood sample), D4 (4 days after the infection) and D14 (14 days after the infection).

[0161] FIG. 2 represents the measurement of the HAI (Hemagglutination Inhibition) titer at D−42 (“Preliminary”), D−21 (“Sample 2”) and DO (“Sample 3”), of mice infected with H1N1 pdm (10.sup.e3) and optionally immunized with (from left to right): Fluviral® 1/5 vaccine, Triton X100 negative control, CALX133ACE negative control, 3 μg antigen Triton X100, 3 μg antigen-1 dose Triton X100, 3 μg antigen CALX133ACE and 0.5 μg antigen CALX133ACE.

[0162] FIG. 3 represents the measurement of the MN (microneutralization) titer at D−42 (“Preliminary”), D−21 (“Sample 2”) and DO (“Sample 3”), of mice infected with H1N1 pdm (10.sup.e3) and optionally immunized with (from left to right): Fluviral® 1/5 vaccine, Triton X100 negative control, CALX133ACE negative control, 3 μg antigen Triton X100, 3 μg antigen-1 dose Triton X100, 3 μg CALX133ACE antigen and 0.5 μg CALX133ACE antigen.

[0163] FIG. 4 represents the measurement of weight (in g) of the mice immunized and infected with H1N1 pdm (10.sup.e3 IU), from DO to D14, for the mice to which were administered: the Fluviral® vaccine, dose 1/5 (diamonds), the negative control Triton X100 Neg. C. (squares), the negative control CALX133ACE Neg. C (triangles), 3 μg antigen extracted with Triton X100 (cross), 3 μg antigen extracted with CALX133ACE (star), and 0.5 μg antigen extracted with CALX133ACE (circle).

EXAMPLES

Example 1: Protocol for Extracting the Influenza Vaccine Antigens Using the CALX133ACE and CALX1103ACE Molecules

[0164] The CALX133ACE and CALX1103ACE molecules (CALX1103ACE being a calixarene in which R.sup.4 is a C.sub.10 alkyl group) are evaluated for their ability to extract the influenza vaccine antigens (produced from a production of the vaccine seed X-179a, H1N1 pandemic 2009) and they are compared with a detergent used by vaccine producers (Triton X100) and two commercial detergents (FC12 and C12E8). Several concentrations, pH and extraction temperature conditions were evaluated.

[0165] The quantitative and qualitative evaluation of the vaccine antigen extraction was carried out using the industrial reference tests for measurement of hemagglutinating activity (hemagglutination test: Hirst, G. K. 1942. The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J. Exp. Med. 75:47-64 ([22]) and Salk, J. E. 1944. Simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immunol. 49:87-98 ([23])) and quantification (SRID; A quantitative, single-radial-diffusion test for immunological studies with influenza virus. Schild G C, Henry-Aymard M, Pereira H G. J Gen Virol. 1972 August; 16(2):231-6 ([24])) of the antigens extracted.

[0166] The conditions tested were those mentioned in table I:

TABLE-US-00001 TABLE I pH Condition Concen- PO.sub.4 Contact No. Molecule tested tration Temperature buffer time 1 Triton X100 4% Ambient 7.95 1 h 2 Triton X100 4% 37° C. 7.95 1 h 3 CALX133ACE 1% Ambient 7.95 1 h 4 CALX133ACE 1% 37° C. 7.95 1 h 5 CALX1103ACE 1% Ambient 7.95 1 h 6 CALX1103ACE 1% 37° C. 7.95 1 h 7 CALX133ACE 1%  4° C. 7.95 2 h 8 CALX1103ACE 1%  4° C. 7.95 2 h 9 Triton X100 4% 37° C. 7.95 1 h 10 Triton X100 4% 37° C. 6.74 1 h

[0167] After extraction, dialysis of the fragmented antigens (supernatant SN and extraction pellet) against 1×PBS was carried out in the conventional industrial production process (for example [9], [18]) in order to reduce the salt and detergent concentration of the extraction products. Each batch was thus dialyzed. It was determined that the Triton X100 was not completely removed during the dialysis. However, it appeared that the CALX133ACE molecule could be dialyzed, suggesting that it could be present only in trace amounts in a marketed possible vaccine product.

[0168] After dialysis, the extraction products were aliquoted and titrated for their hemagglutinating activity (HA). The data are given in table II below:

TABLE-US-00002 TABLE II Condition Titer in HAU/50 μl No. Molecule tested Concentration Temperature pH PO.sub.4 buffer Contact time Supernatant Pellet 10  Triton X100 4% 37° C.  6.74 1 h 128 16 1 Triton X100 4% Ambient 7.95 1 h 128 64 2 Triton X100 4% 37° C.  7.95 1 h 128 16 B CALX133ACE 0.1%   4° C. 7.95 2 h 128 4096 7 CALX133ACE 1% 4° C. 7.95 2 h 512 2 3 CALX133ACE 1% Ambient 7.95 1 h 512 4 4 CALX133ACE 1% 37° C.  7.95 1 h 128 0 D CALX1103ACE 0.1%   4° C. 7.95 2 h 64 128 8 CALX1103ACE 1% 4° C. 7.95 2 h 1024 32 5 CALX1103ACE 1% Ambient 7.95 1 h 512 32 6 CALX1103ACE 1% 37° C.  7.95 1 h 512 16

[0169] The results indicate significant hemagglutinating (HA) titers (512 and 1024 respectively) for the fragmented antigens (SN) obtained after extraction with the CALX133ACE and CALX1103ACE molecules at the concentration of 1% under conditions at 4° C. and at pH=7.95. These titers are significantly higher than those obtained for the antigens extracted (SN) according to the conventional protocol using Triton X100.

[0170] These results are all the more significant since (i) the hemagglutinating titer obtained is dependent on the CALX133ACE molecule concentration used (0.1% vs 1%) and (ii) the HA titer of the residues of non-extracted antigens (pellet) is inversely proportional to and lower than that obtained with Triton X100. The extraction temperature appears to be an important parameter; the temperature of 4° C. appears to be the optimal temperature for extraction of the vaccine antigens using the CALX133ACE and CALX1103ACE molecules.

[0171] These first results clearly indicated that the CALX133ACE molecules were suitable for the extraction of influenza vaccine antigens from batches of virus produced in the allantoic production system used by vaccine manufacturers. These CALX133ACE and CALX1103ACE molecules allow an extraction which is more efficient than that of the conventional one using Triton X100, from a qualitative point of view (more antigens extracted, higher hemagglutinating activity, native profile of antigen oligomerization).

Example 2: Optimization of the Fragmentation of the Vaccine Antigens and Quantification Thereof for Carrying Out an Efficacy Test in a Murine Model

[0172] The viral production was carried out by inoculation of 11-day-old embryonated hen's eggs with viral inocula (vaccine seed) followed by concentration and purification. The fragmentation was carried out according to the conventional industrial method using Triton X100 (4%), in comparison with the use of CALX133ACE molecules.

[0173] The qualitative evaluation of the vaccine antigen extracted is carried out by means of the industrial reference test, which is the measurement of the hemagglutinating activity of said vaccine antigens (activity of the main antigen, hemagglutinin, titer given in UHA/50 ml). The quantitative evaluation is carried out by means of the industrial reference technique, also used by the drug authorities for the validation and marketing authorization of vaccine batches: Single Radial ImmunoDiffusion (SRID, amount of HA in mg/ml). Finally, the quantification of HA by iTRAQ mass spectrometry makes it possible to obtain a relative quantification between the various samples to be compared in an animal test (amount of HA in mg/ml).

[0174] The molecules and conditions that were chosen for the fragmentation were: [0175] Triton X100 at 4% for 1 h at 37° C. [0176] The CALX133ACE molecule at 1% for 2 h at 4° C.

[0177] The CALX133ACE molecules do not cause any red blood cell hemolysis in the absence of flu antigens during the hemagglutination test. Furthermore, the molecules are dialyzable, thereby implying that it will be possible to limit the CALX133ACE to trace amounts in the sample.

Example 3: Animal Experiments

[0178] In a first experiment, several batches of antigens were produced under the optimal production conditions described above, using Triton X100 or the CALX133ACE molecule as splitting agent. Table III below is a summary of the fragmented materials used for the animal test.

TABLE-US-00003 TABLE III Volume to be injected per mouse Approximate HA concentration [HA] corrected (μl) Experimental % residual sample volume Titer in (μg/ml) by SRID by ITRAQ For 0.5 μg of HA For 2.5 μg of condition detergent (μl) UHA/50 μl quantif ITRAQ ratio (μg/ml) Ag HA Ag Split Triton X100 0.36 900 1024 144.83 Split Triton 144.83 3.45 17.26 4% X100/split Split 0.03 800 4096 54.08 CALX133ACE 101.28 4.94 24.68 CALX133ACE C = 1.43 1% Supernatant 0.19 1000 1024 119.21 Supernatant 119.21 20.97 Triton X100 4% Triton X100/S CALX133ACE C = 0.93 Supernatant 0.04 850 1024 48.41 128.64 19.43 CALX133ACE 1%

[0179] In this first experiment, it was planned to test two amounts of HA antigens for the splits (0.5 and 2.5 μg) and just one for the supernatants obtained by ultracentrifugation of the splits. For each condition, 12 Balb.c mice were immunized twice (on DO and then on D21) intramuscularly (100 μl total). Several blood samples were taken from the animals on DO, D21, D42 and D63 (cardiac punctures and euthanasia). After each blood sample series, the sera from one and the same group were harvested and mixed so as to have sufficient material for carrying out a hemagglutination inhibition (HAI) test in duplicate and also a microneutralization test in duplicate.

[0180] The results obtained are given in table IV below:

TABLE-US-00004 TABLE IV Preliminary Sample 2 Sample 3 Cardiac puncture Microneutralization Microneutralization Microneutralization HAI Microneutralization HAI (MN) HAI (MN) HAI (MN) (mean) (MN) Group 1 split <10 <10 <10 <10 <10 20 <10 <10 Triton X100 0.5 μg of Ag Group 2 split <10 <10 <10 <10 80 80 29.17 40 g Triton X100 2.5 μg of Ag Group 3 split <10 <10 <10 <10 40 80 25.83 40 CALX133ACE 0.5 μg of Ag Group 4 split <10 <10 <10 <10 80 160 36.67 80 CALX133ACE 2.5 μg of Ag Group 5 <10 <10 <10 <10 40 40 19.17 40 supernatant Triton X100 2.5 μg Group 6 <10 <10 <10 <10 <10 20 0.83 20 supernatant CALX133ACE 2.5 μg

[0181] The experiment with antigens generated by CALX133ACE fragmentation was validated (good tolerance of the mice, quality and amount of the sera taken, robustness of the HAI results with validated (+) and (−) controls).

[0182] The HAI and microneutralization results are coherent and indicate that the antigen split generated by CALX133ACE fragmentation induces a significant antibody response (sample 3 and cardiac puncture), unlike the antigens purified by ultracentrifugation of the splits (denoted “supernatant” in tables III and IV), since these purified antigens are no longer in a membrane context, thereby validating the process for producing and fragmenting the vaccine antigens using the CALX133ACE molecules in qualitative and quantitative terms.

[0183] Furthermore, a significant difference emerges in favor of the CALX133ACE process with respect to the Triton X100 process as regards the injected dose at 0.5 μg (sample 3 and cardiac puncture). The same tendency is found with the dose of 2.5 μg, but the difference in HAI is not significant (sample 3 and cardiac puncture).

[0184] Furthermore, it appears that the antibody response induced by a dose of 0.5 μg of antigens generated by the CALX133ACE process is similar to that induced with 2.5 μg of antigens generated by the Triton X100 process (sample 3 and cardiac puncture). The latter result is important for the invention since it emphasizes the possibility of obtaining an antibody response with 5 times fewer antigens when they are generated with the CALX133ACE molecule.

Example 4: Immunization of Mice with CALX133ACE: Evaluation in Mice of the Protective Capacity of the Vaccine Antigens Generated

[0185] In a second experiment, several batches of antigens were produced under the optimal production conditions described above, using Triton X100 or the CALX133ACE molecule as splitting agent. Table V below is a summary of the fragmented materials used for this second animal experiment. It was planned to immunize twice Balb/c mice with the CALX133ACE or Triton X100 antigenic preparations and then to infect them with the A/H1N1 California/7/2009 strain (H1N1 pdm09) in order to verify the protection in the animal.

[0186] Group 1: 12 mice (positive control) immunized i.m. (intramuscular) with 1/5 of a dose of Fluviral® (corresponding to 3 μg of HA per antigen).

[0187] Group 2: 12 negative control mice (“Ctrl”) immunized i.m. with the Triton X100 procedure without antigen (6 mice) or the CALX133ACE procedure without antigen (6 mice).

[0188] Group 3: 12 mice immunized i.m. with 3 μg of the HA preparation with Triton X100.

[0189] Group 4: 12 mice immunized i.m. with 3 μg of the HA preparation with CALX133ACE.

[0190] Group 5: 12 mice immunized i.m. with 0.5 μg of the HA preparation with CALX133ACE.

[0191] The immunization protocol is represented in FIG. 1.

[0192] Table V below is a summary of the fragmented materials used for this test.

TABLE-US-00005 TABLE V Volume to be % of injected per mouse detergent % of % of [HA] [HA] (μl) in the detergent detergent Approximate by corrected For Experimental crude after injected into sample HA titer SRID ITRAQ by ITRAQ For 0.5 μg 3 μg of condition sample adjustment the mouse volume (μl) (UHA/50 μl) (μg/ml) ratio (μg/ml) of Ag Ag Split Triton 0.15 0.22 0.120 1650 4096 108.88 Split 108.88 27.55 X100 4% CALX133 Split 0.03 0.03 0.004 or 3500 16384 68.37 ACE/Split 82.30 6.08 36.45 CALX133ACE 0.022 Triton 1% X100 T = 0.61 Ctrl-Triton 2 0.22 0.120 850 — — — — 27.55 X100 4% Ctrl- 0.02 0.03 0.022 670 — — — — 36.45 CALX133ACE 1%

[0193] The mice were sacrificed on D+4 (virus replication peak), with measurement of their pulmonary viral titer.

[0194] The pulmonary viral titer of the mice that were immunized and infected with H1N1 pdm09 (10.sup.e3 IU), on D+4, was measured after euthanasia and indicated in table VI below.

TABLE-US-00006 TABLE VI Pulmonary titer Mice log/ml pfu/ml pfu/g Fluviral ® 1-9 <10e2 Vaccine 1/5 1-10 <10e2 1-11 <10e2 1-12 <10e2 Ctrl Triton 2-4 9   9.3 × 10e8 4.88 × 10e9 X100 2-6 8.5 2.9 × 10e8 1.71 × 10e9 Ctrl 2-7 7.7 5.4 × 10e7  3.7 × 10e8 CALX133ACE 2-11 8.6 4.1 × 10e8 2.09 × 10e9 Split Triton 3-1 <10e2 X100 3 μg of 3-11* 4.4 2.6 × 10e4 1.71 × 10e5 Ag 3-12 <10e2 Split 4-7 <10e2 CALX133ACE 4-8 <10e2 3 μg of Ag 4-9 <10e2 4-10 <10e2 Split 5-6 <10e2 CALX133ACE 5-7 <10e2 0.5 μg of Ag 5-8 <10e2 5-9 <10e2 *Just 1 immunization

[0195] The weight of the Balb/c mice was monitored after intranasal infection with 10.sup.e3 IU/mouse of influenza virus A H1N1 pdm09 (pandemic) and is described in FIG. 4.

[0196] Measurement of the Protection by HAI

[0197] The following groups of mice were tested: [0198] mice immunized with the Fluviral® vaccine 1/5, [0199] mice immunized with Triton X100 only, [0200] mice immunized with CALX133ACE, [0201] mice immunized with the viral antigen prepared with Triton X100, 3 μg, [0202] mice immunized with the vaccine antigen extracted with CALX133ACE, 3 μg, [0203] mice immunized with the vaccine antigen extracted with CALX133ACE, 0.5 μg.

[0204] These mice are infected with H1N1 pdm (10.sup.e3).

[0205] The results of the HAI test on the sera of the mice that were immunized and infected with H1N1 pdm (10.sup.e3) are given in table VI.

TABLE-US-00007 TABLE VII A/California/7/2009 Number of Sample Sample mice Preliminary 2 3 Group 1 Fluviral ® 12 <10 20 160 Vaccine 1/5 Group 2-1 Ctrl Triton 6 <10 <10 10 X100 Group 2-2 Ctrl 6 <10 <10 <10 CALX133ACE Group 3-1 Split Triton 10 <10 10 80 X100 3 μg of Ag Group 4 Split 12 <10 20 320 CALX133ACE 3 μg of Ag Group 5 Split 12 <10 20 160 CALX133ACE 0.5 μg of Ag Control + (A/California) 1280 Control − (A/Perth) <10 D-42 D-21 D0

[0206] The measurement of the protection by HAI is shown in FIG. 2.

[0207] Measurement of the Protection by MN (Microneutralization)

[0208] The following groups of mice are tested: [0209] mice immunized with the Fluviral® vaccine 1/5, [0210] mice immunized with Triton X100 only, [0211] mice immunized with CALX133ACE, [0212] mice immunized with the viral antigen prepared with Triton X100, 3 μg, [0213] mice immunized with the vaccine antigen extracted with CALX133AC, 3 μg [0214] mice immunized with the vaccine antigen extracted with CALX133ACE, 0.5 μg.

[0215] These mice are infected with H1N1 pdm (10.sup.3).

[0216] The MN tests on the sera of the mice that were immunized and infected with H1N1 pdm (10.sup.3) are summarized in table VIII below.

TABLE-US-00008 TABLE VIII 17/06/2014 A/California/7/2009 Number of Sample Sample mice Preliminary 2 3 Group 1 Fluviral 12 <10 20 160 Vaccine 1/5 Group 2-1 Ctrl Triton 6 <10 <10 <10 X100 Group 2-2 Ctrl 6 <10 <10 <10 CALX133ACE Group 3-1 Split Triton 10 <10 20 40 X100 3 μg of Ag Group 4 Split 12 <10 20 320 CALX133ACE 3 μg of Ag Group 5 Split 12 <10 20 160 CALX133ACE 0.5 μg of Ag D-42 D-21 D0

[0217] The measurement of the protection by MN is shown in FIG. 3.

[0218] Protection of the Mice

[0219] FIG. 5 represents the monitoring of the weight of the mice that were immunized and infected with H1N1 pdm (10.sup.3 IU).

[0220] In all the immunized groups (Fluviral, Triton X100 and CALX133ACE): no weight loss or mortality.

[0221] Only the mice in the 2 negative control groups died.

CONCLUSION

[0222] HAI and MN Tests:

[0223] The immunization is as effective with 0.5 μg of antigen split/CALX133ACE as with 3 μg of Fluviral®.

[0224] The immunization is more effective with 3 μg of antigen split/CALX133ACE as with 3 μg of antigen split/Triton X100.

[0225] Protection:

[0226] The protection is as effective with 0.5 μg of antigen split/CALX133ACE as with 3 μg of Fluviral or 3 μg of antigen split/Triton X100.