Site-directed mutagenesis carrier protein and purpose thereof to vaccine preparation

Abstract

The invention relates to a site-directed mutagenesis and site-directed modification protein antigen, and also relates to a site-directed mutagenesis and site-directed modification method of the protein antigen. The method comprises the following steps of introducing unnatural amino acids into specific sites of the protein antigen in a site-directed way by using a gene codon extension technology; and performing site-directed modification with the protein antigen by using the unnatural amino acids and a modifier, wherein the modifier is a receptor agonist such as tripalmitoyl-S-glyceryl cysteineand monophosphoryl lipid A. The invention further relates to application of the site-directed mutagenesis and site-directed modification protein antigen, such as a purpose as a vaccine and the like.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular, the invention relates to a protein with site-directed mutation and site-directed modification and its use in preparing vaccines, especially meningococcal multivalent vaccines. Background technique [0002] Meningococcal meningitis (meningococcal meningitis) caused by Neisseria meningococcus (Nm) infection is a worldwide acute respiratory infectious disease, which still seriously endangers human health. It's children. Neisseria meningitidis can be divided into 13 serogroups according to the differences in their capsular polysaccharide structures, and all serogroups can be pathogenic. Among them, the diseases caused by Neisseria meningitidis group A, B, C, Y, and W135 accounted for more than 95% of Neisseria meningitidis-related diseases. [0003] Among them, various monovalent (A or C), bivalent (A\C) and quadrivalent (ACYW135) vaccines based on meningococcal capsular polysacchar...

AI Technical Summary

Problems solved by technology

However, there is still no international universal meningococcal group B OMV vaccine

[0017] 2. Limitations of the protective coverage of OMV vaccines

MenB OMV vaccine has been successfully used to control the epidemic and outbreak of the disease, but its effectiveness is limited to the control of vaccine-type and subtype MenB disease outbreaks, and its effectiveness is limited for diseases caused by different types and subtypes of MenB

[0020] 3. The production process of MenB OMV affects its antigenicity

[0023] Early research put too much emphasis on protein purity, and used a more complex physical and chemical combination of repeated processes, which destroyed the natural conformation of OMV and weakened the rich antigenic sites, thus affecting the immunogenicity and antibody specificity of the vaccine , leading to poor protection and persistence of immunity

[0024] 4. "Broad-spectrum" group B meningococcal vaccines (such as Novartis 4C MenB, Bexsero) are difficult to achieve broad protection

Different countries have different types of Nm PorA in Group B, and the coverage of PorA strains of 4CMenB vaccine is affected by the PorA types of different Group B strains, which has limitations

[0028] The fHbp in the 4C MenB vaccine is Variant 1, and the effectiveness of immune serum against fHbp Variant 1 strains can reach 95%, but the protection against Variant 2 or 3 strains is only 56%

[0031] ① Group B has more Nm types and subtypes, and the cross protection between them is weak;

[0033] ③ The complexity and excessive purification of foreign vaccine preparation processes have changed the three-dimensional natural conformation of bacterial antigens (protein second, third and fourth spatial conformations), and the induced antibodies lack specificity against the invasion of natural epidemic strains;

[0034] ④ The multi-step purification process also leads to the loss, damage, and singleness of abundant antigenic sites, which affects its antigenicity

However, the above prior art is only limited to site-directed mutation of unnatural amino acids to specific sites of the target protein, without further modification

[0037] The pre-modified recombinant protein fHBP of the present invention has been proven to prevent the infection of group B meningococcus. Wyeth's Bivalent fHBp (r-fHBp) is a bivalent protein vaccine containing fHbp1 and 3, which has been used in adolescents It has been proved that it has a bactericidal effect, but in the infant group, the proportion of people with side effects such as fever and local redness increased significantly. It is speculated that the degree of lipidation is not uniform, leading to increased side effects

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