CN109550046B - Combined vaccine for adsorbing acellular pertussis-poliomyelitis-b haemophilus influenzae and preparation method thereof - Google Patents
Combined vaccine for adsorbing acellular pertussis-poliomyelitis-b haemophilus influenzae and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a combined vaccine for adsorbing acellular pertussis-poliomyelitis-b haemophilus influenzae and a preparation method thereof. The combined vaccine provided by the invention comprises acellular pertussis antigens, diphtheria antigens, tetanus antigens, inactivated poliovirus and Haemophilus influenzae type b antigens; the poliovirus is a Sabin strain. The combined vaccine prepared by the low virulent strain Sabin of the poliovirus has higher biological safety, small adverse reaction of the vaccine and low production cost. By optimizing the antigen composition, the adjuvant and the stabilizer of the combined vaccine, the immunogenicity of various antigens in the combined vaccine provided by the invention is fully exerted, and the combined vaccine has higher stability and is suitable for popularization and application.
Description
Technical Field
The invention relates to the field of medical biotechnology and biological products, in particular to an adsorption acellular pertussis-poliomyelitis-b type haemophilus influenzae combined vaccine and a preparation method thereof.
Background
Pertussis, diphtheria, tetanus, poliomyelitis and haemophilus influenzae type b (Hib) infections are infectious diseases that are seriously detrimental to human health, especially the life health of infants and young children, and vaccination is the most cost-effective measure to prevent these infectious diseases.
Since 1997, the use of pertussis-diphtheria-tetanus-poliomyelitis-Haemophilus influenzae type b (DTaP-IPV/Hib) combination vaccines has begun in Canada and some countries in Europe and more than twenty countries have now listed them as a planned immunization. The DTaP-IPV/Hib combined vaccine can simultaneously prevent five serious threats including pertussis, diphtheria, tetanus, poliomyelitis and b-type haemophilus influenzae infection, and infant infection and infectious diseases, and greatly simplifies the immune procedure.
Currently, the only DTaP-IPV/Hib combined vaccine approved to be on the market in China is Pentaxim TM produced by SanofiPasteur (SP), in the combined vaccine, the strain selected by the IPV vaccine is Salk strain, but the Salk strain belongs to a strong wild strain and has strong toxicity, so the World Health Organization (WHO) encourages more manufacturers to use the Sabin strain to produce inactivated poliomyelitis vaccine (sIPV). So far, in DTaP-IPV/Hib combined vaccines which are on the market all over the world, the strains selected by IPV are Salk strains mostly. Therefore, the development of DTaP-IPV/Hib combined vaccine with higher safety and stability is of great significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a combined vaccine for adsorbing acellular pertussis-poliomyelitis-haemophilus influenzae type b and a preparation method thereof.
The combined vaccine of the diphtheria-pertussis-tetanus, poliomyelitis and haemophilus influenzae type b is prepared by adopting the low-virulent strain Sabin strain of poliovirus (IPV), and compared with the currently widely used high-virulent strain Salk strain, the low-virulent strain Sabin strain has higher safety.
The invention provides an adsorption acellular pertussis-poliomyelitis-b haemophilus influenzae combined vaccine, which comprises acellular pertussis antigens, diphtheria antigens, tetanus antigens, inactivated poliovirus and b haemophilus influenzae antigens; the poliovirus is a Sabin strain.
The Sabin strains are divided into different virus subtypes and the poliovirus described herein may include any subtype of the Sabin strain.
Preferably, in the present invention, the poliovirus includes type I, type II and type III of Sabin strain.
Although the Sabin strain has an advantage of high safety, the Sabin strain is far less immunogenic as a low virulent strain than the virulent strain Salk which is widely used in the prior art. In order to improve the immunogenicity of the Sabin strain and at the same time avoid its interference with other antigens in the combined vaccine as much as possible, in the present invention, each 0.5mL of the combined vaccine comprises the following components:
sabin strain I poliovirus 7.5-15 DU;
type II Sabin strain poliovirus 22.5-45 DU;
type III Sabin strain poliovirus 22.5-45 DU;
preferably, the antigen ratio of the type I Sabin strain poliovirus to the type II Sabin strain poliovirus is 1: 2-1: 3.
in the present invention, the acellular pertussis antigen includes one or more of pertussis toxin PT, pertussis filamentous hemagglutinin FHA, and pertactin PRN.
The diphtheria antigen includes diphtheria toxoid DT.
The tetanus antigen comprises tetanus toxoid TT.
The Haemophilus influenzae type b antigen includes capsular polysaccharide of Haemophilus influenzae type b.
Preferably, in the present invention, the acellular pertussis antigens are pertussis toxin PT, pertussis filamentous hemagglutinin FHA, and pertactin PRN;
the diphtheria antigen is diphtheria toxoid DT.
The tetanus antigen is tetanus toxoid TT.
In particular, the combination vaccines of the present invention include pertussis toxin PT, pertussis filamentous hemagglutinin FHA and pertactin PRN, diphtheria toxoid DT, tetanus toxoid TT, capsular polysaccharides of haemophilus influenzae type b, and inactivated poliovirus Sabin type I, II and III viruses.
In order to better promote the adsorption and immunogenicity of the antigen in the combined vaccine, the combined vaccine also comprises an adjuvant, and the adjuvant comprises aluminum hydroxide.
Preferably, in the preparation process of the combined vaccine, various antigen components are respectively adsorbed by aluminum hydroxide and then mixed;
in the combined vaccine, the content of the aluminum hydroxide is 1.0-2.0 mg/mL.
Further, the inventors have found that the stability of the combination vaccine can be further improved by adding phosphate buffer, but excessive phosphate causes the decrease of the adsorption rate of the antigen protein, and the combination vaccine also comprises phosphate buffer in the present invention. Preferably, in the combined vaccine, the content of the phosphate buffer solution is 2-6mmol/mL of the combined vaccine.
In the present invention, the phosphate buffer may be a phosphate buffer commonly used in the art, including but not limited to phosphate buffers consisting of potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and the like.
In a preferred embodiment of the present invention, the phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate.
Furthermore, the inventors found that the addition of M199 culture medium commonly used in cell culture in the prior art has a significant enhancing effect on the virus stability in combination vaccines, in particular the antigen stability of IPV Sabin strains, and therefore, in the present invention, the combination vaccine further comprises M199 culture medium.
Preferably, the combined vaccine contains 5-20% of M199 culture medium concentrated solution; the M199 culture medium concentrated solution is 10 XM 199 culture medium.
In order to minimize interference between antigen components and to improve the immunopotency and stability of the vaccine, as a preferred embodiment of the present invention, each 0.5mL of the combination vaccine comprises the following components:
in order to make the combination vaccine better suitable for Chinese, in the present invention, the Haemophilus influenzae type b is preferably MH200201 strain (preliminary study of lyophilized Haemophilus influenzae type b conjugate vaccine, J.International biologies, 6 months 2014, vol 37, 3 rd).
On the other hand, the invention also provides a preparation method of the combined vaccine, which comprises the following steps:
(1) respectively preparing inactivated virus liquid, acellular pertussis stock solution, diphtheria stock solution, tetanus stock solution and b type haemophilus influenzae stock solution of type I, type II and type III Sabin strain poliovirus;
(2) respectively and independently adsorbing inactivated virus liquid, acellular pertussis stock solution, diphtheria stock solution and tetanus stock solution of type I, type II and type III Sabin strain poliovirus by using an aluminum hydroxide adjuvant;
(3) mixing the obtained aluminum hydroxide adjuvant adsorption solution;
(4) and (4) adding an M199 culture medium into the mixed solution obtained in the step (3), fully and uniformly mixing, adding a phosphate buffer solution, and adjusting the pH value to 6.0-7.0.
Preferably, the haemophilus influenzae type b stock solution in the step (1) can be independently packaged without being adsorbed by an aluminum hydroxide adjuvant, or without adding an M199 culture medium and a phosphate buffer solution, and forms a DTaP-IPV/Hib quintuplet vaccine with the DTaP-IPV.
According to the needs, the combined vaccine provided by the invention can be prepared into common formulations in the field such as injection, freeze-drying agent and the like by adding auxiliary materials allowed in the field.
The invention has the beneficial effects that: the invention adopts the low virulent strain Sabin strain of poliovirus (IPV) to prepare the acellular pertussis-tetanus-Inactivated Poliomyelitis (IPV) -b type haemophilus influenzae combined vaccine (DTaP-IPV/Hib), compared with the IPV virulent strain Salk strain used in the prior art, the combined vaccine provided by the invention has the advantages of high biological safety, small adverse reaction of the vaccine, low production cost and important significance for preventing poliomyelitis. In addition, the b-type haemophilus influenzae antigen adopted by the invention is derived from MH200201 strain isolated from Chinese, so that the combined vaccine provided by the invention can generate better immune protection for Chinese.
Aiming at the combined vaccine with the specific antigen combination provided by the invention, the composition and the content of each antigen in the combined vaccine are optimized, so that the mutual interference among the antigens is reduced, the full play of the immunogenicity of each antigen is promoted, and the immune efficacy of the vaccine to five pathogens is obviously improved and the stability of a vaccine product is effectively improved by screening and optimizing proper combined vaccine adjuvants and vaccine stabilizers.
Drawings
FIG. 1 shows the effect of adding phosphate buffer solutions with different concentrations on the pH stability of the DTaP-IPV/Hib combination vaccine in example 3 of the present invention, wherein the addition amounts of the phosphate buffer solutions are 2mmol, 4mmol, 6mmol, 8mmol and 10mmol, respectively.
FIG. 2 shows the effect of different concentrations of 10 XM 199 medium on the thermostability of IPV type I D antigen in example 4 of the present invention.
FIG. 3 shows the effect of different concentrations of 10 XM 199 medium on the thermostability of IPV type II D antigen in example 4 of the present invention.
FIG. 4 is a graph showing the effect of different concentrations of 10 XM 199 medium on the thermostability of IPV type III D antigen in example 4 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the preparation of the combination vaccine is as follows, unless otherwise specified:
(1) according to the requirements of the 2010 version of the pharmacopoeia of the people's republic of China, acellular pertussis antigens (PT, FHA and PRN), Tetanus Toxoid (TT) and Diphtheria Toxoid (DT), inactivated liquid of poliovirus of Sabin strains I, II and III and a haemophilus influenzae type b are respectively prepared, wherein the pertussis antigens are prepared by using bordetella pertussis; the tetanus toxin is prepared by adopting clostridium tetani; diphtheria antigen is prepared by diphtheria bacillus; preparing inactivated poliovirus solutions of type I, type II and type III Sabin strains, wherein the type I, type II and type III strains of the Sabin strains of the poliovirus are adopted (CN 201110123949.7); preparing a haemophilus influenzae type b antigen a haemophilus influenzae type b MH200201 strain is adopted (preliminary study on freeze-dried haemophilus influenzae type b conjugate vaccine, journal of international biological systems, 6 months 2014, volume 37, stage 3);
(2) respectively and independently adsorbing inactivated virus liquid of type I, type II and type III Sabin strain poliovirus, pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertactin PRN, diphtheria toxoid DT and tetanus toxoid TT prepared in the step (1) with a proper amount of aluminum hydroxide adjuvant; after adsorption, the obtained antigen adsorption solution is uniformly mixed, 10 XM 199 culture medium is added for mixing and stirring, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution is added for mixing and stirring, and the pH is adjusted to be 6.5.
(3) And (3) packaging the type b haemophilus influenzae capsular polysaccharide stock solution independently without the treatment of the step (2).
Example 1 analysis of IPV neutralizing antibody levels of combination vaccines for antigen content of different Sabin strains
In order to analyze the influence of the antigen content of different Sabin strains on the IPV immune effect of the combined vaccine, three DTaP-IPV/Hib combined vaccine test articles (test articles 1, 2 and 3) containing D antigens with different concentrations of I, II and III are prepared, the antigen content of the three combined vaccine test articles is shown in Table 1, and a commercially available IPV vaccine is used as a reference article. Wistar rats are immunized with 10 test products and control products respectively, each test product and control product is injected with 0.5mL, blood is collected 21 days after immunization, serum is separated, the titer of poliomyelitis neutralizing antibodies in the rats is detected by a cell method, and an ED50 value is calculated.
TABLE 1 antigen content of three combination vaccine test articles
The detection results of the titer of the type I, type II and type III poliomyelitis neutralizing antibodies are shown in tables 2, 3 and 4, and the results show that the ED50 values of the type I, type II and type III poliomyelitis neutralizing antibodies in rats immunized by the three DTaP-IPV/Hib combined vaccine test products are not lower than those of the control product, so that the detection results of the titer of the poliomyelitis neutralizing antibodies of the three DTaP-IPV/Hib combined vaccine test products meet the quality standard, wherein the titer of the antibodies of the test product 1 and the test product 2 is higher than that of the test product 3.
TABLE 2 rat IPV type I neutralizing antibody assay
TABLE 3 rat IPV type II neutralizing antibody assay
TABLE 4 rat IPV type III neutralizing antibody assay
Example 2 analysis of the protective efficacy of DTaP-IPV/Hib combination vaccines containing different concentrations of aluminium hydroxide
DTaP-IPV/Hib combination vaccines containing aluminum hydroxide adjuvants of different concentrations were prepared by optimizing the adjuvants of the DTaP-IPV/Hib combination vaccine based on the test article 1 of example 1, wherein the aluminum hydroxide content of each 0.5mL preparation was 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL and 2.5mg/mL, respectively, and the in vivo immunopotency test of pertussis, diphtheria, tetanus, antibody positive conversion rate of Hib, and IPV was performed to analyze the immune effect of the combination vaccine. Diphtheria, tetanus, pertussis and Hib efficacy experiments were performed according to the Baikuaike and Hib titer detection method in the third part of the 'Chinese pharmacopoeia' 2015 edition. The method comprises the steps of immunizing wistar rats respectively by IPV (infectious bursal disease) with original-fold, 1/3-fold, 1/9-fold and 1/27-fold 4 dilutions, injecting 10 rats in each group with each dilution of 0.5mL, collecting blood after 21 days of immunization, separating serum, detecting the titer of neutralizing antibodies by a cell method, and calculating an ED50 value. The results of diphtheria antibody titer are shown in table 5, tetanus titer is shown in table 6, pertussis titer is shown in table 7, IPV neutralizing antibody results are shown in table 8, and Hib antibody detection results are shown in table 9. The result shows that when the content of aluminum hydroxide in the DTaP-IPV/Hib combined vaccine is 0.5mg/mL, the antibody titer of diphtheria immunity and the antibody titer of IPV immunity do not meet the quality standard, when the content of aluminum hydroxide is 1.0-2.0mg/mL, the immunity titers of various antigens meet the quality standard, and when the content of aluminum hydroxide is continuously increased, the immunity titer of the vaccine tends to be reduced.
TABLE 5 Diptheria immunization Titers of DTaP-IPV/Hib combination vaccine
TABLE 6 tetanus Immunity Titers of DTaP-IPV/Hib combination vaccine
TABLE 7 pertussis immune titers for DTaP-IPV/Hib combination vaccine
TABLE 8 IPV immunopotency of DTaP-IPV/Hib combination vaccine
TABLE 9 Hib immunopotency of DTaP-IPV/Hib combination vaccine
EXAMPLE 3 Effect of phosphate buffer on the antigen protein adsorption Rate and pH stability of DTaP-IPV/Hib combination vaccine
In addition to the DTaP-IPV/Hib combination vaccine containing 1.5mg/mL of the aluminum hydroxide adjuvant as sample 1 in example 1, the effect of the aluminum hydroxide adjuvant on the adsorption rate of the antigen protein and the pH stability when 2mmol, 4mmol, 6mmol, 8mmol, and 10mmol of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 6.0 to 7.0) was added to each mL of the DTaP-IPV/Hib combination vaccine was examined. The protein adsorption rate analysis is shown in table 10. After the phosphate buffer solution is added, the protein adsorption rate is gradually reduced along with the increase of the concentration of the phosphate buffer solution, when the phosphate buffer solution is added at a concentration of 8mmol/mL, the protein adsorption rate is reduced to be below 90%, and when the phosphate buffer solution is added at a concentration of 2-6mmol/mL, the adsorption rate is above 90%, so the concentration of the phosphate buffer solution added in the combined vaccine should not be higher than 6 mmol/mL. The DTaP-IPV/Hib combined vaccine added with phosphate with different concentrations is placed at 37 ℃ and pH monitoring is carried out, wherein the monitoring time points are 0 day, 7 days, 14 days and 21 days. The result is shown in figure 1, the pH value of the DTaP-IPV combined vaccine continuously rises along with the prolonging of the standing time, when the concentration of phosphate radical is 2-6mmol/mL, the pH value of the sample rises rapidly in 0-7 days, the average rising is about 0.2, the rising amplitude in 7-14 days is small, and the pH value tends to be stable; and when the phosphate content is 8mmol/mL and 10mmol/mL, the pH value of the sample is continuously increased within 0-14 days of standing, and the stability of the vaccine is adversely affected.
TABLE 10 influence of the amount of phosphate buffer added on the adsorption rate of antigen proteins
Example 4 Effect of M199 Medium on the stability of combination vaccines
M199 culture media with different concentrations are added into the prepared mixed IPV adsorption solution of the type I, type II and type III Sabin strains, and the influence of the adsorption solution on the stability of virus antigens is examined. Different volumes of M199 culture medium concentrated solution (10 XM 199 culture medium, namely concentrated M199 culture medium with all components having final concentration 10 times of that of the conventional M199 culture medium) are added, a control group without the addition of M199 and a test group with 5% and 10% (volume percentage content) of M199 concentrated solution added to each 0.5ml of IPV adsorption solution are respectively arranged, the IPV adsorption solution with different concentrations of M199 is placed at 37 ℃, the time points are 0 day, 7 days and 14 days, and the content of the D antigen in the IPV adsorption solution is respectively determined. The detection results of the type I, type II and type III D antigen contents of IPV are shown in fig. 2, fig. 3 and fig. 4, respectively, and when no M199 is added, the content of D antigen is continuously decreased with the increase of the standing time, and when IPV adsorption solution is left at 37 ℃ for 14 days, the D antigen in the original solution of poliovirus type I, type II and type III is decreased by 20% to 30%. When the addition amount of concentrated M199 in the IPV adsorption solution is 5% and 10%, the contents of IPV D antigens of I type, II type and III type are basically stable within 0-1 day, and after the IPV adsorption solution is placed for 14 days at 37 ℃, the contents of the D antigens of I type, II type and III type are not obviously reduced, and the result shows that the addition of the M199 culture medium has an important effect on the stability of the poliovirus antigen.
Example 5 in vivo immunopotency assay for DTaP-IPV/Hib combination vaccine
Through optimization of the embodiments 1-4, the optimal component formula which can enable various antigen components to fully and stably exert immune activity when the Sabin low virulent strain is adopted to prepare the DTaP-IPV/Hib combined vaccine is obtained. In this example, the following optimal formulations of the DTaP-IPV/Hib combination vaccine obtained by the above screening were taken as an example (test group), and the formulation of the DTaP-IPV/Hib combination vaccine (0.5mL) in the test group was as follows, by analyzing the immune potency of the DTaP-IPV/Hib combination vaccine and comparing the immune potency with that of the currently commercially available DTaP-IPV/Hib combination vaccine prepared using the strain of the virulent strain salak:
at present, no DTaP-IPV/Hib quintuplet vaccine developed by China exists in the domestic market, so that the imported DTaP-IPV/Hib quintuplet vaccine sold in the market at present is selected as a contrast to be compared with an IPV neutralizing antibody of the combined vaccine, and the IPV in the imported quintuplet vaccine is prepared from a Salk virulent strain. The detection method of the IPV neutralizing antibody comprises the steps of immunizing wistar rats with 4 dilutions of original times, 1/3 times, 1/9 times and 1/27 times respectively, injecting 0.5mL of 10 antibodies in each group, collecting blood and separating serum 21 days after immunization, detecting the neutralizing antibody titer by a cell method, and calculating an ED50 value. The experimental results are shown in table 11, and the results show that compared with the control group combined vaccine, the IPV titer in the quintuplet vaccine (experimental group) of the invention is obviously higher than the I type in vivo efficacy result, and compared with the II type and the III type, the IPV titer has no obvious difference.
TABLE 11 comparison of the immunization potency of DTaP-IPV/Hib quintuplet vaccine with that of Salk virulent strain quintuplet vaccine
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. An adsorption acellular pertussis-poliomyelitis-haemophilus influenzae type b combination vaccine, characterized in that each 0.5mL of the combination vaccine consists of the following components: sabin strain I poliovirus 7.5-15 DU; type II Sabin strain poliovirus 22.5-45 DU; type III Sabin strain poliovirus 22.5-45 DU; pertussis toxin PT20-40 mug; 15-35 mug of pertussis filamentous hemagglutinin FHA; b, 5-20 mug Bordetella pertussis adhesin PRN; diphtheria toxoid DT10-15 Lf; tetanus toxoid TT 2-5 Lf; 10-30 microgram of haemophilus influenzae type b capsular polysaccharide; 0.5-1.0mg of aluminum hydroxide; 1-3mmol of phosphate buffer solution; 5% -10% of 10 XM 199 culture medium;
in the combined vaccine, the antigen ratio of the type I Sabin strain poliovirus to the type II Sabin strain poliovirus is 1: 2-1: 3;
in the preparation process of the combined vaccine, type I, type II and type III Sabin strain poliovirus, pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertactin PRN, diphtheria toxoid DT and tetanus toxoid TT are respectively adsorbed by aluminum hydroxide and then mixed;
the preparation method of the combined vaccine comprises the following steps:
(1) preparing inactivated virus liquid of Sabin strains poliovirus of type I, type II and type III, pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertactin PRN, diphtheria toxoid DT, tetanus toxoid TT and haemophilus influenzae type b capsular polysaccharide;
(2) separately adsorbing inactivated virus liquid of Sabin strain poliovirus I, II and III, pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertactin PRN, diphtheria toxoid DT and tetanus toxoid TT by using an aluminum hydroxide adjuvant;
(3) mixing the obtained aluminum hydroxide adjuvant adsorption solution;
(4) adding an M199 culture medium into the mixed solution obtained in the step (3), fully and uniformly mixing, adding a phosphate buffer solution, and adjusting the pH value to 6.0-7.0;
the Haemophilus influenzae type b capsular polysaccharide in the step (1) does not need to be adsorbed by an aluminum hydroxide adjuvant, and does not need to be added with an M199 culture medium and a phosphate buffer solution for treatment.
2. A method for preparing the combination vaccine of claim 1, comprising the steps of:
(1) preparing inactivated virus liquid of Sabin strains poliovirus of type I, type II and type III, pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertactin PRN, diphtheria toxoid DT, tetanus toxoid TT and haemophilus influenzae type b capsular polysaccharide;
(2) separately adsorbing inactivated virus liquid of Sabin strain poliovirus I, II and III, pertussis toxin PT, pertussis filamentous hemagglutinin FHA, pertactin PRN, diphtheria toxoid DT and tetanus toxoid TT by using an aluminum hydroxide adjuvant;
(3) mixing the obtained aluminum hydroxide adjuvant adsorption solution;
(4) adding an M199 culture medium into the mixed solution obtained in the step (3), fully and uniformly mixing, adding a phosphate buffer solution, and adjusting the pH value to 6.0-7.0;
the Haemophilus influenzae type b capsular polysaccharide in the step (1) does not need to be adsorbed by an aluminum hydroxide adjuvant, and does not need to be added with an M199 culture medium and a phosphate buffer solution for treatment.
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