Study On A Novel Pneumococcal Protein-polysaccharide Conjugate Vaccine Based On Biotin-streptavidin

Streptococcus pneumoniae is a gram-positive pathogen widely distributed in nature,which often causes pneumococcal diseases such as sinusitis,otitis media,bacteremia and meningitis.Infants,the elderly,and patients with underlying diseases are the main infected population.It is estimated that hundreds of thousands of children die of pneumococcal disease every year.In 2018,there were about 294,000 children under five years old who died of pneumococcal infection globally.Most of these cases came from developing countries with backward economic and sanitary conditions.Antibiotics are the first choice for the treatment of pneumococcal diseases,but Streptococcus pneumoniae has gradually resistant to commonly used antibiotics,which increases the difficulty of treatment and makes the development of pneumonia vaccines extremely important.The pneumococcal polysaccharide vaccine PPV23 can induce T cell-independent immune response,but it has no immunological memory and is difficult to induce protective immunity in infants under 2 years old.Pneumococcal polysaccharide conjugate vaccine PCVs combines capsular polysaccharides with protein carriers to transform capsular polysaccharide antigens from T cell-independent antigens to T celldependent antigens,which make infants have good antibody responses and immune memory after immunization,its technology is complicated and expensive,so it is difficult to be widely used in the world.Above all,with the use of these two vaccines based on capsular polysaccharide of Streptococcus pneumoniae,the disease caused by the non-vaccine-covered serotype of Streptococcus pneumoniae is increasing year by year.Therefore,it is urgent to develop a pneumonia vaccine that does not rely on Streptococcus pneumoniae capsular polysaccharide and can induce protective immunity in susceptible people of all ages.In this study,a new type of pneumonia protein conjugate vaccine was prepared.Through the non-covalent effect of streptavidin-biotin,the candidate protein of pneumonia vaccine evaluated in the laboratory was linked to the pneumococcal capsular polysaccharide,the defects of low serotype coverage,serotype substitution and inability to produce protective immunity for infants were overcome,the processes is simple and have more opportunity to be promoted and used on a global scale to form herd immunity.This research is divided into the following two parts:1.Preparation of a novel pneumococcal protein-polysaccharide conjugate vaccineThe core streptavidin sequence with higher stability after expression and biotin binding ability under extreme conditions were selected and express with pneumonia vaccine candidate proteins PspA4 and PsaA-PspA23 evaluated in the laboratory.the fusion proteins PspA4-SA and PsaA-PspA23-SA of core streptavidin were purified by affinity chromatography,gel filtration chromatography and ion exchange chromatography.The polysaccharide with hydroxyl groups was activated by CDAP to form cyanate esters,reacted with the amino groups carried by PEG3-biotin to synthesize the biotinylated polysaccharide Bio-CPS4,which is characterized by infrared broad spectrum.By means of the non-covalent interaction between streptavidin and biotin,the fusion proteins PspA4-SA and PsaA-PspA23-SA are incubated with biotinylated polysaccharide Bio-CPS4 to obtain the proteoglycan conjugate PspA4-SA-Bio-CPS4 and PsaA-PspA23-SA-Bio-CPS4,which were characterized by HPLC.Molecular sieve was used to remove the free protein in the conjugate,free polysaccharide and total sugar of PspA4-SA-Bio-CPS4 and PsaA-PspA23-SA-BioCPS4 were detected by anthrone sulfate method,and immunized according to a certain proportion.2.Effectiveness evaluation of a novel pneumococcal protein-polysaccharide conjugate vaccineThe mice were divided into proteoglycan conjugate experimental group,protein alone group,polysaccharide alone group,positive control PCV13 group and negative control PBS group,the mice were immunized with corresponding immunogens.The titer and typing of antigen-specific antibody were detected in the immunized serum.The spleen cells of the immunized mice were stimulated with PspA4 and PsaA-PspA23 protein antigens to detect Th1,Th2 and Th17 cytokines,The opsonized phagocytosis experiment was used to evaluate the effective antibody level induced by the polysaccharide conjugate vaccine.ATCC BAA334(serotype 4,PspA family 2 subclass3)whose serotype and PspA subclass are both within the protection range of the conjugate vaccine,and ATCC 6308(serotype 8,PspA family 1 subclass 1)whose serotype and PspA subclass are not protected by the conjugate vaccine are selected to challenge mice to test the protective efficacy of the vaccine in mice,as well as crossreactivity and broad-spectrum.In summary,a novel pneumonia protein polysaccharide conjugate vaccine was successfully prepared in this study.In the in vivo and in vitro evaluation of the vaccine,the mice immunized with the conjugate produced high-titer antibodies,and the cytokine test proved that the humoral and cellular immunity induced by the conjugate vaccine were stronger than those of protein or polysaccharide alone.Finally,under the lethal dose of Streptococcus pneumoniae infection,the highest survival rate of mice immunized with the conjugate can reach 90%,which also proves that the vaccine has cross-protection and broad-spectrum.