Recombinant Human Papillomavirus Vaccine Composition and Use thereof

Abstract

Disclosed in the present disclosure are a recombinant human papillomavirus vaccine composition and a use thereof. Compared with other combinations of antigens and adjuvants, the new vaccine composition provided in the present invention has a more beneficial immune effect.

Claims

1. A recombinant human papillomavirus (HPV) vaccine formulation composition, comprising 9 types of VLPs, each formed by the assembly of one of the 9 types of major capsid proteins: HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, and 58L1, and a pharmaceutically acceptable combined adjuvant system.

2. The vaccine formulation composition according to claim 1, wherein the pharmaceutically acceptable combined adjuvant system is a combination of aluminum salt adjuvant and a CpG ODN adjuvant.

3. The vaccine formulation composition according to claim 1, wherein a content of L1 protein VLPs of each HPV type is between 2-60 μg.

4. The vaccine formulation composition according to claim 3, wherein contents of VLPs for HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, and 58L1 types are respectively 6-60 μg, 4-40 μg, 3-30 μg, 4-40 μg, 2-20 μg, 2-20 μg, 2-20 μg, 2-20 μg, and 2-20 μg.

5. The vaccine formulation composition according to claim 2, wherein the aluminum adjuvant is selected from aluminum hydroxide and aluminum phosphate.

6. The vaccine formulation composition according to claim 5, wherein the aluminum adjuvant is aluminum phosphate.

7. The vaccine formulation composition according to claim 5, wherein the aluminum phosphate has a PI value of 5 to 9.5.

8. The vaccine formulation composition according to claim 2, wherein a content of the aluminum adjuvant is approximately 225-1000 μg.

9. The vaccine formulation composition according to claim 8, wherein the content of the aluminum adjuvant is approximately 500 μg.

10. The vaccine formulation composition according to claim 2, wherein a content of the CpG ODN is approximately 200-1500 μg.

11. The vaccine formulation composition according to claim 10, wherein the content of the CpG ODN is approximately 250-1000 μg.

12. The vaccine formulation composition according to claim 2, wherein contents of VLPs for HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, and 58L1 types are respectively 6-60 μg, 4-40 μg, 3-30 μg, 4-40 μg, 2-20 μg, 2-20 μg, 2-20 μg, 2-20 μg, and 2-20 μg, a content of the aluminum adjuvant is approximately 225-1000 μg, and a content of the CpG ODN is approximately 250-1000 μg.

13. The vaccine formulation composition according to claim 12, wherein the contents of VLPs for HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, and 58L1 types are respectively 6-60 μg, 4-40 μg, 3-30 μg, 4-40 μg, 2-20 μg, 2-20 μg, 2-20 μg, 2-20 μg, and 2-20 μg, the content of the aluminum adjuvant is approximately 500 μg, and the content of the CpG ODN is approximately 500-1000 μg.

14. (canceled)

15. A test vaccine kit, comprising the vaccine formulation composition according to claim 1, wherein the kit comprises a vaccine administration device selected from a syringe device, a liquid ejection device, a powder device, and a nebulizer device.

16. The vaccine formulation composition according to claim 2, wherein the CpG ODN adjuvant is CpG7909.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1: Preparation of Vaccine Composition Containing Various Types of L1 VLP Antigen Proteins of HPV

[0043] In order to investigate the technical effect of the vaccine formulation composition provided by the present disclosure, the inventors of the present disclosure prepared the following various vaccine formulation compositions (0.5 ml/dose), each of formulation compositions contains various types of L1 VLP proteins of HPV (HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, and 58L1), an aluminum adjuvant, and a CpG ODN adjuvant. The specific preparation method was as follows: firstly, a stock solution of various types of to L1 VLP antigens of HPV was adsorbed on the aluminum adjuvant (aluminum phosphate adjuvant AP, purchased from Shanghai Zerun Biotechnology Co., Ltd.) to prepare adsorption samples having different ratios of various types of L1 VLP antigens of HPV/aluminum adjuvant (w/w) (here, the aluminum adjuvant content is substantially the content of aluminum element); and then different concentrations of CpG ODN samples (CpG 7909 or CpG 1018, both purchased from Guangzhou RiboBio Co., Ltd.) were added into the antigen/aluminum adsorption samples. After the above formulations were thoroughly mixed, if not administrated immediately, they were stored at 4° C. The specific ratios were as follows.

TABLE-US-00001 TABLE 1 aluminum HPV phosphate CpG ODN various types of L1 VLP of HPV (μg) antigen adjuvant adjuvant volume No. 16 18 6 11 31 33 45 52 58 dosage (AP)(μg) (μg) (μl) 1 60 40 30 40 20 20 20 20 20 1X 500 500 500 2 60 40 30 40 20 20 20 20 20 1X 500 1000 500 3 60 40 30 40 20 20 20 20 20 1X 500 0 500 4 6 4 3 4 2 2 2 2 2 0.1X.sup.  500 500 500 5 6 4 3 4 2 2 2 2 2 0.1X.sup.  500 1000 500 6 60 40 30 40 20 20 20 20 20 1X 500 1000 500 7 6 4 3 4 2 2 2 2 2 0.1X.sup.  500 1000 500 Note: The CpG ODN contained in vaccines Nos. 1, 2, 4, and 5 are CpG 7909, and the CpG ODN contained in vaccines Nos. 6 and 7 are CpG 1018.

Example 2

[0044] For the obtained recombinant human papillomavirus vaccine formulation compositions of Example 1 to be evaluated, the inventors carried out immunogenicity studies using BALB/c mice as an animal model. The immunogenicity of the vaccine formulation compositions provided by the present disclosure was investigated by using various types of L1 VLP proteins of HPV as antigens and using an aluminum adjuvant and CpG ODN as to adjuvants. 6-8-week-old BALB/c female mice were randomly divided into groups, 10 mice in each group. The vaccines prepared using various types of L1 VLP proteins of HPV in combination with adjuvants (Table 1) were administrated into mice through intramuscular injection, with an injection volume of 0.05 ml (i.e. 1/10 dose). A self-prepared vaccine group, a marketed vaccine Gardasil 9 group, and an adjuvant control group were set. Among them, 1-dose group was immunized on day 0, blood samples were collected on day 35; 2-dose group was immunized on days 0 and 21, and blood samples were collected on days 35 and 56; and 3-dose group was immunized on days 0, 21, and 42, and blood samples were collected on day 56. Pseudovirion-Based Neutralization Assay (PBNA) was used to detect the titers of neutralizing antibodies against various types of L1 VLP proteins of HPV in the serum, and the geometric mean titers (GMT) of the antibodies were calculated.

[0045] The immunogenicity evaluation method is a conventional technical means in to the art. As an example, the more specific operation method of the Pseudovirion-Based Neutralization Assay is as follows.

[0046] (1) HEK 293FT cells in a good proliferation condition were digested into individual cells with 0.25% Trypsin-EDTA, the cells were diluted to a suitable concentration with a DMEM complete medium, and the cells were counted by a cell counter.

[0047] (2) According to the cell count results, HEK 293FT cells were diluted to a density of 1.5×10.sup.5/ml with the DMEM complete medium and pre-plated in a 96-well cell culture plate, with each well added 100 μl of cell dilution, and incubated in an incubator at 37° C. and 5% CO.sub.2 until cell adhesion.

[0048] (3) An inactivated serum sample was diluted with the DMEM complete medium to an appropriate initial dilution, and then several gradients of consecutive two-fold dilutions were performed, with double wells for each dilution.

[0049] (4) The pseudovirus solution was diluted to log(TCID.sub.50/0.1 ml)=3.2±0.5 with the DMEM complete medium and mixed thoroughly.

[0050] (5) Into the serum dilution, equal volume of pseudovirus dilution was added, mixed thoroughly, and incubated at room temperature for 60 minutes. Positive control wells were also set, each well containing the DMEM complete medium and equal volume of pseudovirus dilution; and blank control wells were set, each well containing the DMEM complete medium.

[0051] (6) After the incubation, 100 μl of the serum-pseudovirus mixed liquid was accurately taken and added slowly and carefully to the pre-plated 96-well cell to plate.

[0052] (7) The resultant was incubated in an incubator at 37° C. and 5%002 for 72 hours, the result was observed using a fluorescence microscope, or a spot analyzer was used to read, analyze, and process the results.

[0053] The studying results are shown in Tables 2 to 5 below.

[0054] As shown in Table 2, addition of a CpG ODN adjuvant to a traditional 9-valent HPV vaccine containing only an aluminum adjuvant can significantly increase the neutralizing antibody titer in the serum of immunized mice, and GMT was increased by 2.64 to 13.93 times.

TABLE-US-00002 TABLE 2 Comparison of neutralizing antibody titers (GMT) in mouse serum on day 35 after immunization by one dose with each vaccine Group Group of Vaccine HPV Group of vaccine No. 1 No. 3 No. 1/No. 3 Type HPV1X+AP+CpG HPV1X+AP (fold) HPV 16 3719 4222 3.25 HPV 18 13719 3676 3.73 HPV 6 2599 746.4 3.48 HPV 11 2425 746.4 3.25 HPV 31 4222 649.8 6.50 HPV 33 4222 984.9 4.29 HPV 45 6859 857.4 8.00 HPV 52 9051 3430 2.64 HPV 58 19401 1393 13.93 Note: The formulas of vaccines Nos. 1 and 3 are shown in Table 1. Vaccine No. 3 does not contain CpG ODN, and the contents of the other components are the same as those of vaccine No

[0055] As shown in Table 3, the level of neutralizing antibodies on day 35 after immunization by one dose of vaccine No. 1 containing the combined adjuvant was compared with that on day 14 after immunization by two doses of the marketed vaccine Gardasil 9 (Merck). GMTs of HPV 16, 6, 11, and 52 were substantially equivalent to that for Gardasil 9, and GMTs of the other five types were better than that for Gardasil 9.

TABLE-US-00003 TABLE 3 Comparison of neutralizing antibody titers GMT in mouse serum on day 35 after immunization by one dose of the novel 9-valent HPV vaccine and day 14 after immunization by two doses of Gardasil 9 Group Vaccine No. 1 Gardasil 9 1 × HPV + AP + CpG 1 × HPV + AP No. 1/ HPV on day 35 after one on day 14 after Gardasil 9 Type dose two doses (fold) HPV 16 13719 20794 0.66 HPV 18 13719 7611 1.80 HPV 6 2599 3430 0.76 HPV 11 2425 1656 1.46 HPV 31 4222 2425 1.74 HPV 33 4222 828.2 5.10 HPV 45 6859 509.8 13.45 HPV 52 9051 11943 0.76 HPV 58 19401 11143 1.74 Note: The formula of vaccine No. 1 is shown in Table 1; the marketed vaccine Gardasil 9 does not contain CpG ODN, and the antigen dosage is the same as that of vaccine No. 1, that is, the antigen dosage of Gardasil 9 is the same as the 1 × antigen dosage of the present disclosure; and each group has 10 mice.

[0056] As shown in Table 4, the neutralizing antibody level on day 35 after immunization by two doses of vaccine No. 1 containing the combined adjuvant was compared with that on day 14 after immunization by three doses of the marketed vaccine Gardasil 9. The GMTs of HPV 6, 11, 31, and 52 were substantially equivalent to that for Gardasil 9, and the GMTs of the other five to types were better than that for Gardasil 9. In accordance with the comparison data of one dose to two doses in Table 3, the above results show that the novel 9-valent HPV vaccine containing the combined adjuvant provides better immunogenicity even if the number of doses for immunization is reduced.

TABLE-US-00004 TABLE 4 Comparison of neutralizing antibody titers GMT in mouse serum after immunizatior by two doses of the novel 9-valent HPV vaccine and after immunization by three doses of Gardasil 9 Group Vaccine No. 1 Gardasil 9 1 × HPV + AP + CpG 1 × HPV + AP HPV on day 35 after two on day 14 after No. 1/Gardasil 9 Type doses three doses (fold) HPV 16 81275 38802 2.09 HPV 18 32254 11143 2.89 HPV 6 14368 11143 1.29 HPV 11 5080 2986 1.70 HPV 31 6400 5572 1.15 HPV 33 18102 1715 10.56 HPV 45 32254 606.3 53.20 HPV 52 18102 14703 1.23 HPV 58 36204 16890 2.14 Note: The formula of vaccine No. 1 is shown in Table 1; the marketed vaccine Gardasil 9 does not contain CpG ODN, and the antigen dosage is the same as that of vaccine No. 1, that is, the antigen dosage of Gardasil 9 is the same as the 1 × antigen dosage of the present disclosure; and each group has 10 mice.

[0057] As shown in Table 5, the neutralizing antibody level on day 35 after immunization by one dose of vaccine No. 1 containing the combined adjuvant was compared with that on day 14 after immunization by two doses of the marketed vaccine Gardasil 9. The GMTs of HPV 16, 6, and 52 were substantially the same as those of Gardasil 9, and the GMTs of the other six types were better than those of Gardasil 9.

[0058] As shown in Table 5, in a case where the CpG ODN adjuvant was added to the traditional 9-valent HPV vaccine containing only an aluminum adjuvant, even if the HPV antigen content was reduced to 1/10 dosage of Gardasil 9, the to neutralizing antibody levels of the nine HPV types was still comparative to those for Gardasil 9.

TABLE-US-00005 TABLE 5 Comparison of neutralizing antibody titers GMT in mouse serum on day 35 after immunization by one dose of each vaccine Group HPV Vaccine No. 5 Group Gardasil 9 No. 5/Gardasil 9 type 0.1 × HPV + AP + CpG 1 × HPV + AP (fold) HPV 16 22286 19401 1.15 HPV 18 6400 5572 1.15 HPV 6 2111 800 2.64 HPV 11 1393 800 1.74 HPV 31 2786 1600 1.74 HPV 33 2786 1393 2.00 HPV 45 3676 800 4.60 HPV 52 1393 2111 0.66 HPV 58 6400 8445 0.76 Note: The formula of vaccine No. 5 is shown in Table 1. The marketed vaccine Gardasil 9 does not contain CpG ODN, and the aluminum adjuvant content is the same as that of vaccine No. 5. The dosage of 9 HPV types is 10 times that of vaccine No. 5, that is, the antigen dosage of Gardasil 9 is the same as the 1 × antigen dosage in the present disclosure.

Example 3

[0059] The inventors also investigated the immunogenicity of the novel 9-valent HPV vaccines with different CpG ODN contents (such as 500-1000 μg) and the immunogenicity of the novel 9-valent HPV vaccines containing different CpG to ODN types (such as CpG7909 and CpG1018). The results show that the novel 9-valent HPV vaccines of various formulations all have good immunogenicity. The experimental procedure in mice and neutralizing antibody detection method are the same as those in Example 2. The studying results are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Neutralizing antibody titers GMT in mouse serum on day 14 after immunization by two doses of each vaccine Group Vaccine No. 2 Vaccine No. 4 Vaccine No. 6 Vaccine No. 7 HPV 1 × HPV + AP + 0.1 × HPV + AP + 1 × HPV + AP + 0.1 × HPV + AP + type 1000CpG7909 500CpG7909 1000CpG1018 1000CpG1018 HPV 16 38802 44572 16890 11143 HPV 18 22286 11143 8445 9701 HPV 6 4850 3676 1600 1056 HPV 11 1838 1838 606.3 919 HPV 31 4850 2111 527.8 527.8 HPV 33 29407 11143 7352 2786 HPV 45 4850 6400 9701 4850 HPV 52 1393 1838 919 696.4 HPV 58 14703 11143 6400 8445 Note: the formulas of vaccines Nos. 2, 4, 6, and 7 vaccine are shown in Table 1. The mouse muscle immunization dosage is 1/10 dosage, and the immunization volume is 0.05 ml.

[0060] It can be seen from Tables 2 to 6 that the vaccine compositions of the present disclosure prepared according to the specific component and ratio have good immunological activity, especially when the contents of the various types of L1 VLP proteins of HPV (HPV 16L1, 18L1, 6L1, 11L1, 31L1, 33L1, 45L1, 52L1, and 58L1) are respectively 6-60 μg, 4-40 μg, 3-30 μg, 4-40 μg, 2-20 μg, 2-20 μg, 2-20 μg, 2-20 μg, and 2-20 μg, the content of the aluminum adjuvant is 500 to μg, and the content of the CpG ODN adjuvant is 500-1000 μg, the compositions have excellent immune effects, and can be used as a new generation of vaccine formulation composition and as a guide for dosage selection ranges of the antigen and combined adjuvant in the formulation.

[0061] All documents mentioned in the present disclosure are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present disclosure, those skilled in the art can make various changes or modifications to the present disclosure, and these equivalent forms also fall within the scope defined by the appended claims of the present application.