Comparative Study On Novel CpG Molecules In Enhancing Vaccine Immune Efficacy

Background Adjuvants are also known as immunomodulators or immune boosters.As an additive to a vaccine,when injected into the body before or mixed with an antigen,it enhances the body’s immune response to the antigen or changes the type of immune response,and is a non-specific immune booster that is not antigenic in itself.The ideal adjuvant enhances the immune response and thus provides the body with better protection.From the discovery of aluminium adjuvants to the present,there are various substances known to have adjuvant activity,which can be broadly classified according to their chemical properties as follows: inorganic salt-based adjuvants(aluminium adjuvants),emulsion-based adjuvants(MF59),water-soluble adjuvants(saponins),adjuvants for particulate antigen delivery systems(AS01),adjuvants targeting pattern recognition receptors,etc.There are currently only seven approved adjuvants for human use.However,a growing number of studies have found that the majority of them still have more than a few drawbacks.For example,aluminium adjuvants are often effective in increasing serum antibody levels,but their ability to cause injection site reactions and to induce cellular immunity is weak,limiting their use to some extent.MF59 adjuvant induces a stronger humoral immune response than aluminium adjuvant,but it suffers from greater toxicity and is not suitable for use as a human vaccine adjuvant.Saponins have been used as vaccine adjuvants for a long time and promote antigen-specific antibody CD4+ and CD8+ helper T cell responses,but their mechanism of action is unclear and may be a safety concern as a human vaccine adjuvant.GSK’s recombinant herpes zoster vaccine Shingrix(CHO cells),developed using AS01 B as an adjuvant and approved in China in May 2019,contains QS-21 in its composition,which is haemolytic and needs to be used with cholesterol and liposomal forms to eliminate side effects.Currently,CpG molecules have great potential.CpG is an agonist of the pattern recognition receptor,TLR9.Its mechanism of action is to activate TLR9 and trigger the expression of downstream inflammation-related genes,thus achieving the effect of activating and enhancing the immune response.CpG can be classified into fore types,A,B,C and D,depending on its structure.CpG1018,which has been approved for use,belongs to CpG type B.Compared to traditional vaccines,the new hepatitis B vaccine,which was launched in the US in 2017,not only simplifies the vaccination process due to its combination with CpG1018,but also offers more significant protection to people in different age groups.However,the high clinical use of CpG1018 poses certain safety risks and is exclusively monopolised by Dynavax,which only authorises individual pharmaceutical companies to use it,which has led to a restricted field of vaccine development and research.Therefore,the search for other more efficient and safe CpG adjuvants is of great importance for vaccine development.Methods Six different CpG-ODN sequences,named CpG1-6,were designed in different combinations by sequence search and comparison to identify human-mouse homologous CpG motifs.After the corresponding six CpG molecules were obtained by artificial synthesis,they were combined with SARS-Co V-2 recombinant subunit vaccine respectively,and the marketed adjuvant CpG1018 was used as a positive control.BALB/c was immunized twice at two-week intervals and orbital blood was taken weekly for Ig G1 and Ig G2 a indicators after immunization.Mice were executed three weeks after the two immunisations and eye blood was taken for binding antibody inhibition;spleen lymphocytes were taken for ELISPOT testing.Mice were immunized with different doses of CpG adjuvant,and the total Ig G titres of the conjugated antibodies and the inhibition rate of the conjugated antibodies were measured and compared to screen the optimal dose of CpG adjuvant for its immune boosting effect.In order to explore the immune enhancing effect of novel adjuvant molecules on therapeutic vaccines,this study simultaneously observed the effect of adjuvants on the level of cellular immunity and biological effects of a therapeutic human papillomavirus type 16 recombinant adenovirus vector vaccine(Ad5-HPV16-E6E7).In the experiments on the effect of adjuvants on cellular immunity to the therapeutic vaccine,CpG molecules were combined with Ad5-HPV16-E6E7 vaccine in a single immunisation of C57 mice,and the level of antigen-specific lymphocyte activation was measured 12 days later with the aid of the ELISPOT method.In a study of the effect on biological effects,the effect of CpG molecules on the tumour suppressive effect of the vaccine was examined by using the TC-1tumour cell transplantation model,observing C57 mice transplanted with TC-1 cells 24 hours after vaccination with Ad5-HPV16-E6E7 vaccine containing CpG molecules,and by the tumour formation and subsequent death of the mice.Results Two weeks after initial immunization,Ig G2 a levels were also significantly higher in the CpG6 group compared to the CpG1018 group as a control,and serum Ig G2 a levels were significantly higher in the CpG6 mice compared to the CpG1018 group.Similarly,the level of Ig G1 in the CpG6 group was also significantly higher than that in the CpG1018 group.And after the enhanced immunization,the serum levels of Ig G1 and Ig G2 a were significantly increased in all groups,and the serum Ig G2 a levels in the CpG6 group were always higher than those in the CpG1018 group.Statistically significant differences between groups in the rate of inhibition of conjugated antibodies against the prototype strain after 14 days of initial immunisation and 21 days of booster immunisation(F=13.660,12.580,19.376 and 19.376,P all<0.001);two weeks after the initial immunization in a 10-fold dilution,the serum inhibition rate of binding antibodies against the prototype strain was significantly higher in the CpG6 group(64.67±20.4)% of mice than in the CpG1018 group(27.84±20.23)% of mice(t=2.70,P=0.031);three weeks after the enhanced immunization,the inhibition rate of the CpG6 mice was significantly higher than that of the CpG1018 mice(70.84±17.20;52.61±14.22)% in sera diluted 1000-fold,both against the prototype(89.71±4.83)% strain and the Delta(79.04±6.32)% strain(t=2.36,P=0.046;t=3.80,P=0.005).ELISPOT test results showed that there was no significant difference in the number of cells specific for secreted IL-2(1030.44±291.33;874.75±402.28;915.74±270.03;743.68 ± 332.07)(t=0.76,P=0.469;t=0.78,P=0.459)and IFN-γ(t=0.70,P=0.503;t=0.90,P=0.395)in the splenocytes of the CpG1018 and CpG6 groups of mice against the prototype(947.62±300.92;795.06±332.09;825.67±326.11;678.53±269.60)and Delta strains.In a dose-effect assay exploring adjuvant after initial immunisation,there was no significant difference in total Ig G titres of bound antibodies and inhibition of bound antibodies between mice serum at 4-6 weeks after initial immunisation 5μg and 10μg,and serum Ig G concentrations and inhibition of bound antibodies were higher in mice injected with CpG6 compared to CpG1018.The effect on cellular immunity to therapeutic vaccine was tested in an ELISPOT assay to detect effector T cells producing specific IFN-γ in immunised mouse spleen lymphocytes stimulated by a mixed peptide pool of HPV16 E7.The highest number of specific spots was found in the VAC+CpG6 group(vaccine+CpG6),followed by the vaccine+CpG1018,vaccine alone and blank groups.Although no significant difference was shown in the study,the overall level still indicated that CpG6 was superior to CpG1018 in terms of immune enhancement.In the biological effect observation experiment,none of the mice in the Control group(high-dose vaccine group)grew tumours;all of the mice in the blank group developed tumours;and the VAC group(vaccine alone)had a tumour rate of 80%.In the adjuvant group,the VAC+CpG6(vaccine+CpG6)group had the lowest tumour rate;the VAC+CpG1018(vaccine+CpG1018)group had a relatively weak effect and there was mouse mortality.Conclusion Among a series of novel CpG molecules,we designed CpG6 in combination with the SARS-Co V-2 recombinant subunit vaccine to produce a more rapid humoral immune response in mice after initial vaccine immunization,and neither humoral nor cellular immunity was weaker than the marketed CpG1018 adjuvant after booster immunization.A lower dose(5 μg)of CpG6 was sufficient to induce an effective humoral immune response in the initial immunisation,and at low doses CpG6 showed a stronger immune boosting capacity than CpG1018 adjuvant.In terms of its effect on the efficacy of therapeutic vaccines,the novel CpG molecule designed in this experiment,CpG6,significantly enhanced the cellular immune response to the vaccine and significantly reduced the tumour formation rate in mice.The novel CpG6 molecule is expected to be a potential alternative adjuvant for vaccine use.