CN105963691B

CN105963691B - Streptococcus pneumoniae vaccine

Info

Publication number: CN105963691B
Application number: CN201610524959.4A
Authority: CN
Inventors: 查文娟
Original assignee: 查文娟
Current assignee: Nuosa Union (Beijing) Biomedical Technology Co., Ltd
Priority date: 2016-07-03
Filing date: 2016-07-03
Publication date: 2020-05-01
Anticipated expiration: 2036-07-03
Also published as: CN105963691A

Abstract

The invention discloses a pneumococcal vaccine and a preparation method thereof, wherein the pneumococcal vaccine is prepared by mixing an S3CS recombinant antigen or a mutant protein antigen thereof with an adjuvant. The vaccine can be used for immunization, improves the protection of the body against streptococcus pneumoniae, and can produce specific antibodies. The preparation method is simple and is suitable for the requirement of large-scale industrial production.

Description

Streptococcus pneumoniae vaccine

Technical Field

The invention relates to a vaccine, in particular to a streptococcus pneumoniae vaccine.

Background

Infections caused by pneumococci (the lung chain) are a major cause of morbidity and mortality worldwide. Pneumonia, febrile bacteremia and meningitis are the most common manifestations of invasive pneumococcal disease, and bacterial spread in the respiratory tract can lead to middle ear infections, sinusitis or recurrent bronchitis. Non-invasive manifestations are usually less severe, but more common, than invasive diseases. The likelihood of pneumococcal disease onset during influenza is further increased due to the spread of antibiotic-resistant infections, and pneumococcal pneumonia often follows influenza infection. The disease caused by streptococcus pneumoniae has become an important public health problem worldwide. Pneumococci have become the first killer of children worldwide.

The fatality rate of pneumonia in China is 16.4%, wherein the fatality rate of middle-aged and old people over 50 years old and infants under 1 year old is respectively as high as 28.6% and 22.0%. The carriage rate of pneumococcus in healthy children in China is high, and data statistics shows that the carriage rate of pneumococcus in healthy children in northern regions is 24.2%, and the carriage rate of pneumococcus in southern regions is 31.3%. Which is a significant cause of death in children under the age of 5. The main reasons are that the development of the immune system of infants is not complete and the immunity is weak. And the younger the infant, the weaker the immunity.

The pneumococcus has about 90 serotypes (strains), and the statistics of China show that the serotypes of the pneumococcus infected strains are 5,6, 19, 23, 14, 2 and 4 in turn. A study in which 860 pneumococcal isolates were collected showed that 109 (12.7%) showed serogroup 6 with 100% erythromycin resistance in pneumococcal serotype 6 in china, with types 6A, 6B and 6C of 62 (56.9%), 38 (34.9%) and 9 (8.2%) respectively.

23-valent pneumococcal vaccines produced by China biotechnology group Chengdu biological product research institute are prepared by selecting 23 most common pathogenic bacteria (1, 2,3,4,5,6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), fermenting, culturing, separating and purifying various polysaccharides on pneumococcal capsules respectively, and mixing the polysaccharides in equal proportion to prepare the vaccine.

Bacterial polysaccharides are a thymus-independent antigen, which differs from thymus-dependent antigens primarily in that the former do not require the help of T lymphocytes to produce antibodies. Polysaccharide vaccines therefore suffer from the following problems: (1) only weak immune response can be generated in the bodies of young animals or infants, even no immune response can be generated, and the immune response is enhanced along with the increase of the age; (2) generating low affinity antibodies; (3) only produces transient immune response, and does not have immune memory and immune enhancement effect during repeated inoculation; (4) immune tolerance is easy to generate; (5) the common adjuvant has no easy effect of immune enhancement on the antigen. The protective rate of the 23-valent polysaccharide vaccine for invasive lung chain infection is 50-70%. And can only be used for inoculation of people over 2 years old, and the peak onset age of pneumonia is 6-12 months old.

A number of specific polypeptides having antigenicity are described in PCT patent No. W098/18930, published 5/7 of 1998 entitled "antigens and vaccines for Streptococcus Pneumoniae" (Streptococcus Pneumoniae antigens and vaccines), but there is no report on the biological activity of these polypeptides.

Thus, the urgent need to use streptococcal antigens as components of vaccines for the prevention and/or treatment of streptococcal infections is becoming increasingly important.

Disclosure of Invention

The invention provides a streptococcus pneumoniae vaccine, wherein the protein amino acid sequence in the vaccine is shown as SEQ ID NO:1 is shown.

Other aspects of the invention provide vectors comprising a polynucleotide of the invention operably linked to an expression control region, host cells transfected with said vectors and methods of producing a polypeptide comprising culturing said host cells under conditions suitable for expression of the gene.

In another aspect, novel polypeptides encoded by the polynucleotides of the invention are provided.

According to one aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide having at least 95% identity to a polypeptide selected from the group consisting of SEQ ID NO. 1.

According to one aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide having at least 90% identity to a polypeptide selected from the group consisting of SEQ ID NO. 1.

According to one aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide having at least 85% identity to a polypeptide selected from the group consisting of SEQ ID NO. 1.

According to one aspect of the invention, there is provided an isolated polynucleotide encoding a polypeptide having at least 80% identity to a polypeptide selected from the group consisting of SEQ ID NO. 1.

In a particular embodiment, the invention also relates to a chimeric polypeptide comprising one or several polypeptides as defined in the conjugated protein or fragments, analogues or derivatives thereof.

The coupling protein is coupled and combined protein which is diphtheria toxoid, tetanus toxoid, carrier protein CRM197, hemophilus influenzae surface protein HiD, pertussis Prn surface protein, pertussis Fha antigen and/or streptococcus pneumoniae surface protein PspA.

In a specific embodiment, the polypeptide or chimeric polypeptide according to the invention is antigenic.

In a specific embodiment, the polypeptide or chimeric polypeptide according to the invention is capable of eliciting an immune response in an individual.

In a particular embodiment, the invention also relates to polypeptides capable of producing antibodies with binding specificity for a polypeptide or chimeric polypeptide as defined in the present invention.

An antibody that is "binding-specific" means that the antibody is capable of recognizing and binding to a particular polypeptide, but does not substantially recognize and bind to other molecules in a sample, such as a biological sample that naturally contains the selected peptide. The specific binding capacity of the selected polypeptide as antigen can be determined by enzyme-linked immunosorbent assay (ELISA).

According to the present invention, the polypeptide includes both polypeptides and chimeric polypeptides. Also included are polypeptides that have been altered in biological and pharmacological properties by fusion with other compounds, for example, polyethylene glycol to increase half-life; fused to leader or secretory sequences to

The purification is convenient; also prepro (prepro) sequences and fusions to pro (pro) sequences and (poly) saccharides.

Furthermore, where some amino acid regions are polymorphic, it may be more desirable to alter one or more particular amino acids to more effectively mimic different epitopes on different strains of Streptococcus.

The mutation is obtained by screening nearly 800 mutations possibly by the applicant.

The mutation site is shown in SEQ ID NO:1, or a pharmaceutically acceptable salt thereof. F23V (indicating the substitution of the F amino acid at position 23 with V), K42C, P60T, K92S, P122D, E133F, D180S, F189H, K243S, I304G, L320Y, V329P, F332K, I339V, I352M, F361W, K369P, S372K, F380M, L401E.

In addition, polypeptides of the invention can be modified by phthalylation of the terminal amino group (e.g., acetylphthalylation or thioacetic acid phthalylation) and terminal carboxyphthalylation (e.g., with ammonia or methylamine), to provide stability and enhance hydrophobicity for attachment or binding to a support or other molecule.

According to another aspect, the present invention provides a vaccine composition comprising at least one or several streptococcal polypeptides of the present invention in admixture with a pharmaceutically acceptable carrier diluent or adjuvant. Suitable adjuvants include oils, such as Freund's complete or incomplete adjuvant; salts, such as AlK (S04)2, AINa (S04)2, A1NH4(S04) a, silica, kaolin; carbon polynucleotides such as poly IC and poly AU. Preferred adjuvants include Querca (QuilA) and aluminum hydrogel (Alhydrogel). The vaccine of the present invention can be administered parenterally by injection, bolus infusion, nasopharyngeal absorption, cutaneous absorption, or buccal administration. Pharmaceutically acceptable carriers also include tetanus toxoid.

The vaccine compositions of the invention are useful for treating or preventing streptococcal infections and/or diseases and conditions caused by streptococcal infection as described in p.r.murry (eds.), e.j.baron, m.a.pfaller, f.c.tenover and r.h.yolken et al, Manual of clinical microbiology, ASM Press, Washington, d.c. 6 th edition, 1995, page 1482 (which is incorporated herein by reference).

In one embodiment, the vaccine composition of the present invention is used to treat and prevent meningitis, otitis media, bacteremia and pneumonia. In a particular embodiment, the vaccine composition of the invention is used for the treatment and prevention of infection with and/or disease and condition following infection with streptococci, in particular with streptococcus pneumoniae, group a streptococci (streptococcus pyogenes) [ group aspergillus (pyogenes) ], group B streptococci (GBS or streptococcus agalactiae) [ group bstreptrepus (GBS or agalactiae) ], dysgalactiae (dysgalactiae), papilla bacteria (uberis), agrostis (nocardia) and staphylococcus aureus (streptococcus aureus). In a specific embodiment, the infection by streptococcus is streptococcus pneumoniae.

In a particular embodiment, the vaccine is administered to individuals at risk of streptococcal infection, such as infants, the elderly and immunocompromised individuals.

As used herein, "individual" includes mammals. In one embodiment the mammal is a human.

The vaccine composition is preferably administered in a unit dose of about 0.001 to 100p, g/kg (antigen/body weight), more preferably 0.01 to 1Opg/kg, most preferably 0.1 to 1p, g/kg, with one to three administrations occurring at one to six week intervals between immunizations.

The effect of sense: the invention provides a protein with better immunogenicity, a vaccine prepared from the protein can provide better immune protection capability of streptococcus pneumoniae, the protein is simple to prepare, has low cost, has no side effect on human bodies, and is suitable for large-area popularization.

Detailed Description

EXAMPLE 1 expression of proteins

And (2) taking DNA of streptococcus pneumoniae as a template, and amplifying by designing a primer to obtain the DNA of SEQ ID NO:1, and then recovering the PCR product from the agarose gel by using QIAquick gel extraction kit of QIAGEN (Chatworth, CA), digesting the PCR product with Superlinker vector pSL301(Invitrogen, San Diego, CA), and recovering the PCR product from the agarose gel by using QIAquick gel extraction kit of QIAGEN (Chatworth, CA), ligating the restriction-digested genomic DNA fragment with the restriction-digested pSL301 vector, and ligating the ligated product with a plasmid of 5a E.coli, and purifying the recombinant plasmid of pSL301 (SL 301) containing the target gene by using QIAGEN kit, and sequencing and identifying the recombinant plasmid to be correct.

The target gene is constructed into a pET-32c (+) expression vector (Novagen, Madison, WT) through enzyme digestion connection, then is transformed into BL21 cells for prokaryotic expression, a protein form expressed by a supernatant is obtained through conventional expression, and the target protein with the concentration of 100mg/mL is obtained through column purification.

Example 2 obtaining of muteins

Introduction of mutation points

(1) Designing a corresponding mutagenesis primer according to a corresponding mutation site, and mutating an amino acid site corresponding to a wild type on the corresponding S3CS gene by adopting a PCR (polymerase chain reaction) site-specific mutation method; the mutation site is shown in SEQ ID NO:1, or a pharmaceutically acceptable salt thereof. F23V (indicating the substitution of the F amino acid at position 23 with V), K42C, P60T, K92S, P122D, E133F, D180S, F189H, K243S, I304G, L320Y, V329P, F332K, I339V, I352M, F361W, K369P, S372K, F380M, L401E.

(2) After the PCR product is recovered and purified by glue, carrying out enzyme digestion by using restriction enzyme, connecting the product with a plasmid pSL301 vector fragment subjected to the same enzyme digestion, and transforming escherichia coli DH5 α competent cells;

(3) and identifying the recombinant plasmid: and (3) identifying the recombinant plasmid by enzyme digestion and PCR (polymerase chain reaction) methods, and carrying out sequencing inspection, thereby obtaining the mutated nucleotide sequence.

The mutated S3CS gene was expressed according to the expression method of example 1, and the protein concentration was adjusted to 100mg/mL after purification.

Example 3 evaluation of antibody-producing Effect of pneumococcal protein vaccine

12-14g female 3-week-old BALB/c. pups, randomly divided into 23 groups of 20 mice each, were intraperitoneally injected with a conjugate vaccine formulated according to example 1 and 2 protein with Al (OH)3 as an immunoadjuvant, a commercial 13-valent pulmonary chain conjugate vaccine, a blank control (PBS), respectively.

Injections were given 3 times per week 0, 2, 4 of the program, each mouse containing 0.5ml of protein (0.5ug protein). Blood was collected from each experimental group 7 days after the injection of the third needle, and serum was separated and stored at-20 ℃ for future use.

For the detection of the protein antibody titer, the protein was dissolved in 0.05mol/L carbonate buffer solution of pH9.6 by indirect ELISA, coated with an ELISA plate at a concentration of 20ug/ml, and blocked with 2% BSA solution. During the experiment, the serum to be detected is diluted by 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, 102400, 204800 and 409600 times, then is added into an ELISA plate, the ELISA plate is placed at 37 ℃ for reaction for 40min, and after the ELISA plate is washed conventionally, a goat-anti-mouse secondary antibody marked by horseradish peroxidase is added. Buffer without serum was also added as a negative control. The color is developed by using tetraaminobenzidine, and the A value is read at the wavelength of 450nm by using an enzyme-labeling instrument. The ratio of the A value in each well to the A value in the negative well was calculated, but the largest dilution factor when the ratio was more than 2.1 was the antibody titer in serum, the titers of 20 mice were geometrically averaged, and the log value was calculated at the bottom of 20, and the results are shown in Table 1.

It is seen from the data that the protein vaccine of the present invention and the corresponding mutant protein vaccine have significantly enhanced immune response to lung chain polysaccharide in mice, and also have enhanced ability to produce antibodies compared to the commercially available vaccine.

Example 4 evaluation of Effect of immunized mice

1) The first immunization, 2mg of S3CS or its mutated recombinant protein antigen is diluted with PBS, and Al (OH)3 with the concentration of 1mg/mL is added; injecting with No. 5 semi-type needle, subcutaneous injection of inguinal, plantar and dorsal parts at two sides, with injection amount of l00uL for each BALB/C mouse, and setting positive control group, negative control group and blank control group;

2) the second immunization, the second immunization is carried out on the 14 th day, the immunization components are the same, the amount of the protein antigen injected is 1/2 of the first immunization, and the immunization route is the same as the above;

3) the third immunization, the third immunization is carried out on day 21, the immunization components are the same as above, the amount of the protein antigen injected is the same as that of the second immunization, and the immunization way is the same as above;

on day 14, a lethal dose is adopted, the streptococcus pneumoniae strain WU2 viable bacteria is injected into tail vein to carry out a virus challenge experiment, the amount of the bacteria injected into each BALB/C mouse is 1.5 × 109CFU, and the survival rate of each group of mice is counted after 14 days of observation. The results are shown in Table 2.

Table 2 shows; the average immune protection rate of the negative control group and the blank control group is 0 percent respectively, and the antigen protein or the mutant protein thereof has the protection characteristic of about 100 percent and has improved effect compared with the vaccine in the prior art.

Evaluation of safety

After the challenge experiment, the mice immunized by the protein are continuously cultured for 1 month, and are dissected, so that the organs of the mice are normal and have no variation. This is sufficient to show that the vaccine of the present invention is safe and reliable.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; it is intended that the following claims be interpreted as including all such alterations, modifications, and equivalents as fall within the true spirit and scope of the invention.

Sequence listing

The beautiful silk of (110)

Streptococcus pneumoniae vaccine < 120 >

〈160〉1

〈210〉1

〈211〉887

〈212〉PRT

Artificial sequence of < 213 >

〈400〉S3CS

1 MYTFILMLLD FLKNHDFHFF MLFFVFILIR WAVIYFHAVR YKSYSCSVSD EKLFSSVIIP

61 VVDEPLNLFE SVLNRISRHK PSEIIVVING PKNERLLKLC HDFNEKLENN MTPIQCYYTP

121 VPGKRNAIRV GLEHVDSQSD ITVLVDSDTV WTPRTLSELL KPFVCDKKIG GVTTRQRIFD

181 PERNLVTMFA NLLEEIRAEG SMKAMSVTGK VGCLPGRTIA FRTEILRECI HEFMNETFMG

241 FHKEVSDDRS LTNLTLKKGY KTVMQDTSVV YTDAPTSWKK FIRQQLRWAE GSQYNNLKMT

301 PWMIRNAPLM FFIYFTDMIL PMLLISFGVN IFLLKILNIT TVVYTASWLE IILYVLFGMI

361 FSFGGRNFKA MSRMKWYYVF LIPVFIIVLS IIMCPIRLLG LMRCSDDLGW GTRNLIEGGG

421 SGGGSGGGSG GGSGGGSMSK YKELAKNTGI FALANFSSKI LIFLLVPIYT RVLTTTEYGF

481 YDLVYTTIQL FVPILTLNIS EAVMRFLMKD GVSKKSVFSI AVLDIFIGSI AFALLLLVNN

541 LFSLSDLISQ YSIYIFVIFV FYTLNNFLIQ FSKGIDKIGV TAISGVISTA VMLAMNVILL

601 VVFDWGLLGF FIANVCGYVI PCIYIVSRLR LWELFEIKID KKLQWEMVYY ALPLVLNILS

661 WWVNNTSDRY IVTAIVGIQA SAIISVAYKI PQILSTISAI FIQSWQISAI KIQEDKSDTT

721 FVSNMLLYYN ALLLIIASGI ILFVKPISNI LFGISFYSAW ELVPFLIISS LFNAISGCIG

781 AIMGAKMDTH NIAKSALVGM IANIILNIVL TFLMGPQGIT ISTLIASFLI FYMRKDSVKE

841 INSETYRAIY LSWILLVVEA CLLIYMDFII GALIAMVINL FLLKDVI

Claims

1. A streptococcus pneumoniae vaccine comprising an antigenic protein and an adjuvant, wherein the antigenic protein is SEQ ID NO:1, or a sequence as set forth in SEQ ID NO:1, and specific mutation sites of the sequence subjected to point mutation are F23V, K42C, P60T, K92S, P122D, E133F, D180S, F189H, K243S, I304G, L320Y, V329P, F332K, I339V, I352M, F361W, K369P, S372K, F380M or L401E.