<i>Haemophilus influenzae </i>fusion protein and construction method and use thereof

Abstract

Provided are a fusion protein and a construction method thereof. The fusion method consists of: a Haemophilus influenzae protein D and a Hin47 (Htra) protein. The fusion protein can serve as a protein vehicle for a Haemophilus influenzae polysaccharide-protein conjugate vaccine, thereby increasing immunogenicity of a polysaccharide antigen.

Claims

1. A Haemophilus influenzae fusion protein obtainable by fusion of Haemophilus influenzae HiD protein and Haemophilus influenzae Hin47 protein, wherein the amino acid sequence of Hin47 is the sequence shown in SEQ ID No: 1, and the amino acid sequence of HiD is the sequence shown in SEQ ID No: 3.

2. The fusion protein of claim 1, wherein the fusion protein is obtainable by fusion of Hin47 and HiD in a 1:1 ratio through a linker, the linker enables the monomers of Hin47 protein and HiD protein to maintain their own unique conformations.

3. The fusion protein of claim 2, wherein the linker is G4S with the sequence shown in SEQ ID No: 2.

4. The fusion protein of claim 1, wherein the composition form of a gene for the fusion protein is (n)Hin47-linker-(n)HiD, (n)HiD-linker-(n)Hin47, -linker-HiD-(Hin47-linker-HiD)n-linker-Hin47-, -linker-Hin47-(HiD-linker-Hin47)n-linker-HiD-, wherein n is 1-3, representing the number of copies of the genes for the monomers.

5. The fusion protein of claim 1, wherein the composition form of the gene for the fusion protein is -linker-(Hin47-linker-HiD-linker)n-Hin47-, -linker-(HiD-linker-Hin47-linker)n-HiD-, wherein n is 1-3, representing the number of copies of the genes for the monomers.

6. The fusion protein of claim 1, wherein the fusion protein is used as a protein carrier in the preparation of a conjugate vaccine using a polysaccharide antigen as an active ingredient.

7. The fusion protein of claim 6, wherein the conjugate vaccine is against Haemophilus influenzae, pneumonia, meningitis, dysentery or typhoid.

8. The fusion protein of claim 1, wherein the fusion protein is used as a protein carrier for a Hia and Hib conjugate vaccine, wherein Hia stands for a capsular polysaccharide in Haemophilus influenzae serotype a, and Hib stands for a capsular polysaccharide in Haemophilus influenzae serotype b.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the testing of the expression of the fusion protein;

(2) FIG. 2 illustrates the purification process of the fusion protein Hin47-HiD;

(3) FIG. 3 illustrates the comparison between immunogenicity of Hin47 and HiD

(4) FIG. 4 illustrates the comparison of anti-Hin47 immunogenicity between Hin47 and Hin47-HiD, wherein, the immunization dose of the Hin47 monomer in the fusion protein Hin47-HiD is the same as that of the Hin47 antigen in the control group, wherein L, M and H indicate that the dose of the Hin47 monomer antigen (including Hin47 in control group) in fusion protein Hin47-HiD is 1 g, 5 g and 10 g, respectively;

(5) FIG. 5 illustrates the comparison of anti-HiD immunogenicity between HiD and Hin47-HiD, wherein the immunization dose of the HiD monomer in the fusion protein Hin47-HiD is the same as that of the HiD antigen in the control group, wherein L, M and H indicate that the dose of the HiD monomer antigen (including HiD in the control group) in the fusion protein Hin47-HiD is 1 g, 5 g and 10 g, respectively;

(6) FIG. 6 illustrates the purification process of Hia conjugate;

(7) FIG. 7 illustrates the purification process of Hib conjugate.

DETAILED DESCRIPTION OF THE INVENTION

(8) The technical solution of the present invention is further described below with reference to specific examples.

Example 1: Preparation of the Fusion Protein Hin47-HiD

(9) (1) Construction of the Hin47-HiD Expression Vector

(10) The Hin47-HiD fusion gene was synthesized by Life Technologies Corporation, wherein the selected linker was G4S (GGGGS), the fusion gene comprised one copy of the gene for Hin47 and one copy of the gene for HiD, with Hin47 in the upstream of HiD, and the designed restriction site in the 5 of the fusion gene being NdeI and in the 3 being BamHI, wherein the amino acid sequence of Hin47 was the sequence shown in SEQ ID No: 1, the amino acid sequence of the Linker was the sequence shown in SEQ ID No: 2, and the amino acid sequence of the HiD was the sequence shown in SEQ ID No: 3, see Table 1 below for details.

(11) TABLE-US-00002 TABLE1 Aminoacidsequence offusionproteinHin47-HiD Aminoacidsequence Hin47 MTLPSFVSEQNSLAPMLEKVQPAVVTLSVEGKAKVDSRSPFLD DIPEEFKFFFGDRFAEQFGGRGESKRNFRGLGSGVIINASKGY VLTNNAVIDEADKITVQLQDGREFKAKLVGKDELSDIALVQLE KPSNLTEIKFADSDKLRVGDFTVAIGNPFGLGQTVTSGIVSAL GRSTGSDSGTYENYIQTDAAVNRGNSGGALVNLNGELIGINTA IISPSGGNAGIAFAIPSNQASNLVQQILEFGQVRRGLLGIKGG ELNADLAKAFNVSAQQGAFVSEVLPKSAAEKAGLKAGDIITAM NGQKISSFAEIRAKIATTGAGKEISLTYLRDGKSHDVKMKLQA DDSSQLSSKTELPALDGATLKDYDAKGVKGIEITKIQPNSLAA QRGLKSGDIIIGINRQMIENIRELNKVLETEPSAVALNILRGD SNFYLLVQ Linker GGGGS HiD DPSSHSSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFA QQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKD GRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSH FRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDI AAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGM DLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVV KYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPY TVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLK GIK

(12) As can be seen from Table 1, the Hin47-HiD fusion protein contains 790 aa and has a molecular weight of 87 kD.

(13) The fusion gene was double-digested with NdeI and BamHI, ligated with a T vector double-digested with the same endonucleases using T4 DNA ligase, the resulting plasmid was transformed into DH5a competent cells, and extracted and verified by PCR and double-digestion.

(14) The T clone was double-digested with NdeI and BamHI to obtain the fusion gene, which was ligated with pET9a double-digested with the same endonucleases, the fusion gene on the expression plasmid was transformed into BL21 competent cells, and the expression vector was confirmed by double-digestion and PCR.

(15) The positive monoclonal colonies were picked, transferred to LB medium, cultured at 37 C. and shaken at 200 rpm overnight, transferred to 50 ml fresh LB medium, when OD.sub.600=0.6-0.7, IPTG at a final concentration of 1 mM was added, and after induction at 37 C. for 4 hours, the cell pellet was collected by centrifugation. The precipitate was redissolved with PBS and sonicated, and the supernatant was subjected to SDS-PAGE electrophoresis to analyze whether the protein was expressed, the result is shown in FIG. 1:

(16) FIG. 1 shows that the fusion protein was highly expressed in the supernatant.

(17) It should be noted that the techniques used in the construction of the expression vector for the fusion protein Hin47-HiD and the induced expression are routine experimental procedures known to those skilled in the art and are available in published literature and in books.

(18) (2) Purification of the fusion protein Hin47-HiD

(19) The bacterial cells expressing Hin47-HiD were grown overnight, and then transferred to fermentation medium. When OD.sub.600=0.6-0.7, IPTG was added to a final concentration of 1 mM, and after induction at 37 C. for 4 hours, the bacteria was subjected to centrifugation to collect precipitate.

(20) The precipitate was redissolved with PBS, sonicated, and centrifuged to collect the supernatant. The supernatant was passed through a Q column to collect the flowthrough liquid (the target protein flowed through), then the Q column flowthrough liquid was passed through a CHT column, and 150 mM PB+170 mM NaCl was used to wash the column, and 175 mM PB+1 M NaCl was used to elute the target protein. The purification process is advantageous since it has simple steps, can make use of conventional fillers, and can produce the target protein with the purity of 80% or higher with only two steps. The result is shown in FIG. 2.

(21) As can be seen from FIG. 2, the target protein with a purity of 80% or higher can be obtained by the two-step column purification.

Example 2: Study on Immunogenicity of the Fusion Protein Hin47-HiD

(22) BALB/C mouse was immunized with the fusion protein Hin47-HiD three times at intervals of two weeks to study its immunogenicity, with Hin47 and HiD as controls. Whole blood was collected 14 days after the last immunization, and serum antibody levels were tested.

(23) In the control groups, the immunization doses of the Hin47 or HiD protein antigen were 1 g, 5 g and 10 g, respectively, while in the immunization group of the fusion protein, the doses of the protein monomer were 1 g, 5 g and 10 g, respectively.

(24) The mice were immunized with Hin47 and HiD antigens in three doses of 1 g, 5 g and 10 g, respectively, and the antibody titers of the three immune sera were tested with the corresponding antigens, the results are shown in FIG. 3.

(25) Statistical Analysis:

(26) P(Hin47 1 g: HiD 1 g)=0.000<0.01

(27) P(Hin47 5 g: HiD 5 g)=0.000<0.01

(28) P(Hin47 10 g: HiD 10 g)=0.000<0.01

(29) The results showed that Hin47 generates a stronger immune response than HiD at the same dose.

(30) The immunogenicity of the fusion protein Hin47-HiD was studied in three doses, and the actual immunization dose of the protein monomer in the fusion protein was the same as that of the control antigen (Hin47 or HiD). In the comparison experiment for the immunogenicity between Hin47-HiD and Hin47, the three groups L, M and H represented the doses of Hin47 monomer antigen in the fusion protein of 1 g, 5 g and 10 g, respectively.

(31) The comparison of the immunogenicity between Hin47 and Hin47-HiD is shown in FIG. 4.

(32) Statistical Analysis:

(33) P(L group Hin47: Hin47-HiD)=0.2820> 0.05

(34) P(M group Hin47: Hin47-HiD)=0.2820> 0.05

(35) P(H group Hin47: Hin47-HiD)=0.350> 0.05

(36) The results show that the fusion protein Hin47-HiD does not affect the immunogenicity of Hin47.

(37) The immunogenicity of the fusion protein Hin47-HiD was studied in three doses, and the actual immunization dose of the protein monomer in the fusion protein was the same as that of the control antigen (Hin47 or HiD). In the comparison experiment for the immunogenicity between Hin47-HiD and HiD, the three groups L, M and H represented the doses of HiD monomer antigen in the fusion protein of 1 g, 5 g and 10 g, respectively.

(38) The comparison of the immunogenicity between HiD and Hin47-HiD is shown in FIG. 5.

(39) Statistical Analysis:

(40) P(L group HiD: Hin47-HiD)=0.000<0.01

(41) P(M group HiD: Hin47-HiD)=0.000<0.01

(42) P(H group HiD: Hin47-HiD)=0.000<0.01

(43) The results show that the Hin47-HiD fusion protein can effectively enhance the immunogenicity of HiD.

Example 3: Preparation of Hia and Hib Conjugate Bulks

(44) Polysaccharide-protein conjugates of polysaccharide Hia and Hib were prepared by using the same method. Specifically, after adding the capsular polysaccharide to CDAP for 30 seconds, 0.2 M TEA was added to adjust the pH to 9.5 for polysaccharide activation, and the activation was continued for 2.5 min, after the activation, the pH was adjusted to 9.0, Hin47-HiD fusion protein was added according to a ratio of polysaccharide to protein of 1:1, the reaction was carried out at room temperature for 1 hour, the resulting mixture was kept at 4 C. overnight, and then 2 M glycine was added to terminate the reaction; the resulting mixture was dialyzed to remove the reaction reagents, and the conjugate was separated and purified by CL-4B gel.

(45) The CL-4B gel separation result of Hia conjugate is shown in FIG. 6.

(46) The CL-4B gel separation result of Hib conjugate is shown in FIG. 7.

(47) TABLE-US-00003 TABLE 2 Testing results of Hia and Hib bivalent conjugate vaccine Polysac- Protein Polysac- Free Name of charide content charide/ polysac- Free conjugate content mg/ml mg/ml protein charide protein Hia 3.5 7.21 0.485 13% 4% conjugate bulk Hib 2.8 8.47 0.33 10% 2% conjugate bulk

Example 4: Production of the Finished Product of Hia and Hib Bivalent Conjugate Vaccine, ie, Conjugate Vaccine Against Haemophilus influenzae Serotype a and Serotype b

(48) Taking the production of 200 vials of the vaccine as an example, in theory, 100 ml of the final bulk was required to prepare 200 vials of the final lot. According to the concentration of the conjugate bulk, 0.57 ml of Hia conjugate bulk, 0.72 ml of Hib conjugate bulk, 1 ml of 1 M PB, and 50 ml of NS were required, supplied with 47.7 ml of WFI, to obtain the final bulk, each vial was then filled with 0.5 ml to obtain the finished product.

(49) The final lot contains 10 ug (polysaccharide content) of Hia conjugate and 10 g Hib conjugate, 0.45% NaCl, and 10 mM PB per dose.

Example 5: Study on the Immunogenicity of the Conjugate Vaccine Against Haemophilus influenzae Serotype a and Serotype b

(50) The immunogenicity of the conjugate vaccine against Haemophilus influenzae serotype a and serotype b (with Hin47-HiD as the carrier protein) was evaluated according to the protocol for the efficacy test of the conjugate vaccine against Haemophilus influenzae serotype b as described in the Chinese Pharmacopoeia. Specifically, 10 BALB/C mice with weight of 12-14 g were immunized with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b in human dose (containing Hia polysaccharide 2.5 g and Hib polysaccharide 2.5 g respectively), and 10 BALB/C mice were immunized with NS as the negative control, the immunization was performed 2 times at 2-week intervals, and eyeballs were removed to collect serum 14 days after the last immunization.

(51) The titers of antibodies in serum against both capsular polysaccharide serotype-a and serotype-b were measured by ELISA, and the measurement of the serum samples of NS-immunized mice was used as the CUT off value to calculate the rate of positive immune response in the vaccine group. The results are shown in Table 3.

(52) TABLE-US-00004 TABLE 3 Test results of immune responses with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b Coating Immune Name of conjugate vaccine antigen Dilution response rate conjugate vaccine against capsular 100 100% Haemophilus influenzae polysaccharide serotype a and serotype b serotype a capsular 100 100% polysaccharide serotype b

(53) The results show that: the fusion protein Hin47-HiD, as a protein carrier, can effectively stimulate the body to produce immune response to the polysaccharide antigens. In this example, the mice were immunized with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b in human dose, and the response rate after two immunizations was 100%, which reached the release standard of Chinese Pharmacopoeia for the immunization efficacy of the Hib conjugate vaccine product.

Example 6: Study on the Protection of the Conjugate Vaccine Against Haemophilus influenzae Serotype a and Serotype b

(54) The content of specific antibodies in serum can be effectively measured by ELISA, however, not all antibodies have bactericidal protection function, and serum bactericidal antibodies (SBA) can reflect the functional activity of antibodies, which directly reflects the protective effects of the vaccine.

(55) The test results of SBA in sera of mice immunized with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b are shown in Table 4.

(56) TABLE-US-00005 TABLE 4 Test results of SBA of the conjugate vaccine against Haemophilus influenzae serotype a and serotype b Bacterial Name of conjugate vaccine strain SBA conjugate vaccine against Haemophilus 1341 Haemophilus influenzae influenzae serotype a and serotype b serotype a Haemophilus 1560 influenzae serotype b

(57) In this example, the SBA in sera of mice immunized with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b was as high as 1300, which can effectively prevent infection from Haemophilus influenzae serotype a and Haemophilus influenzae serotype b.

(58) TABLE-US-00006 TABLE 5 Antibody titers against the carrier protein in the conjugate vaccine against Haemophilus influenzae serotype a and serotype b Titer of Titer of anti-Hin47 anti-HiD antigen antigen antibody antibody Name of conjugate vaccine (GMT/lgOD) (GMT/lgOD) conjugate vaccine against 6.34 5.65 Haemophilus influenzae serotype a and serotype b

(59) As can be seen from Table 5, the mice simultaneously produced antibodies to the carrier protein after being immunized with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b, wherein the titer of anti-HiD antigen antibody was 5.65 and that of anti-Hin47 antigen antibody was up to 6.34. These antibodies could effectively prevent infection of non-typeable Haemophilus influenzae.

(60) Protective results show that: after the mice are immunized with the conjugate vaccine against Haemophilus influenzae serotype a and serotype b, anti-Hia and anti-Hib polysaccharide antibodies can effectively prevent Haemophilus influenzae serotype a and Haemophilus influenzae serotype b, while antibodies against the carrier protein Hin47-HiD can effectively prevent non-typeable Haemophilus influenzae.

(61) The application principle of the present invention may be as follows: the polysaccharide-protein conjugate vaccine can promote the thymus-independent polysaccharide antigen to become thymus-dependent, thereby making the polysaccharide antigen suitable for immunoprophylaxis in populations such as infants and children. The first generation of the conjugate vaccine protein carrier is tetanus toxoid TT and diphtheria toxoid DT, the second generation of the conjugate vaccine protein carrier is a non-toxic protein such as diphtheria toxin mutants CRM; the present invention belongs to a new generation of conjugate vaccine protein carrier, which is characterized in that in addition to effectively improving the immunogenicity of the polysaccharide antigens, antibodies against the carrier protein itself can also resist certain diseases. Hin47 and HiD have been shown to prevent non-typeable Haemophilus influenzae, whereas the Hin47-HiD fusion protein developed by the present invention can significantly improve the immunogenicity of the weak antigen HiD without affecting the immunogenicity of Hin47, and more importantly, the Hin47-HiD fusion protein can be effective as a conjugate vaccine protein carrier. Therefore, in the present invention, a novel conjugate vaccine protein carrier is developed, the conjugate vaccine developed by using it as a carrier can prevent the bacterial infection corresponding to polysaccharide antigen, in addition, antibodies against the carrier protein itself can prevent non-typeable Haemophilus influenzae.

(62) The above detailed description of the Haemophilus influenzae fusion protein, the construction method and use thereof with reference to the above examples are intended to be illustrative and not limitative, and certain embodiments may be extended within the scope of the present invention. Therefore, any changes and modifications without departing from the general concept of the present invention should fall within the protection scope of the present invention.