Study Of Dengue Peptide Vaccine Based On Spherical Nucleic

【Background】The single-stranded positive-stranded RNA virus known as the dengue virus(DENV)is carried by arthropods and is responsible for dengue fever,dengue hemorrhagic fever,and dengue shock syndrome.Because to the potential of infection,about half of the world’s population is now at risk from DENV.The Antibody Dependent Enhancement(ADE)impact raises the risk of severe illness and fatality following a second infection with the heterotypic virus,despite the fact that the disease brought on by DENV is relatively self-limiting.There is an urgent need to create a safe and effective vaccine to stop infection and the spread of the disease because there is no known therapeutic medication for the dengue virus.Only one live attenuated vaccine(Cy D-TDV)is now approved for clinical use in several nations,although clinical trials have demonstrated that Cy D-TDV is only safe and effective in seropositive people who have already contracted dengue virus.In contrast,seronegative people who receive the vaccine for the first time are at an increased risk of developing severe dengue fever.As a result,research and ongoing vaccine development are still effective ways to stop dengue virus infection.Peptide vaccines can stimulate site-specific antibodies and immune responses,thus eliminating the risk of adverse reactions associated with vaccines.Although peptides still have disadvantages such as poor immunogenicity and poor metabolic stability,attaching them to nanoparticles can effectively overcome these disadvantages.Spherical nucleic acids,an emerging nanomaterial,can be used as carriers for peptide or protein delivery.In antiviral studies,spherical nucleic acids not only can attach the corresponding antigenic epitopes,but also greatly improve the immune response of the body due to the formation of adsorbed nucleic acids on their surface,usually from immunostimulatory nucleic acids.Dengue peptide vaccines based on spherical nucleic acid carriers are expected to be vaccine candidates against dengue virus infection.【Objective】The safety and efficacy of current dengue vaccines are not clinically satisfactory,which requires a new approach to design an effective dengue vaccine that does not cause adverse reactions.In this study,we screened a set of shared antigenic epitopes against four serotypes of dengue by genetic testing of four serotypes of DEVN,and designed a dengue peptide vaccine based on a spherical nucleic acid vector accordingly.The aim is to obtain a dengue peptide vaccine with high antigen delivery capacity,high safety and high immune effect level.【Methods】(1)Construction of peptide vaccineThe coupling of Au NP at 30 nm with the sulfhydrylated oligonucleotide sequence(CSV-1A)was successfully detected by UV spectrophotometry to form a spherical nucleic acid structure;the coupling of the cysteine-containing peptide with the sulfhydrylated Cp G was detected by agarose gel electrophoresis test;the morphology,size and electrical properties of the peptide vaccine were detected by transmission electron microscopy,particle size and zeta potential;the optimal coupling concentration of the peptide was detected by residual fluorimetry.(2)Screening of optimal culture conditions for bone marrow-like dendritic cells(BMDCs)The optimal culture concentration was determined by measuring the expression of CD11 c,a surface marker of BMDCs,using an immunofluorescence(IF)assay after BMDCs were grown in media with various amounts of growth factors.(3)Uptake of peptide vaccines by BMDCs and induction of their maturationLaser confocal microscopy was used to measure the amount of antigen taken up by BMDCs and to detect the expression of co-stimulatory molecules on their surface.RT-PCR was used to detect the expression of co-stimulatory molecules at the molecular level on the surface of BMDCs,and an enzyme-linked immunosorbent assay was used to detect the secretion of the cytokine IL-12p70 in the culture supernatant of BMDCs(ELISA).(4)Evaluation of immune effects and in vivo safetyBy using an ELISA to measure the targeted antibody Ig G level,the safety of the peptide vaccination was assessed in vivo by looking at pathological sections of each organ.【Results】(1)In this study,a dengue peptide vaccine(SNA-TBB)based on a spherical nucleic acid vector was constructed.Au NP was used as the core of the spherical nucleic acid,and CSV-1A was the nucleic acid attached to the surface of the core to form a spherical nucleic acid structure.The cysteine-containing peptide TBB was coupled to sulfhydrylated Cp G by disulfide bonding.CSV-1A and Cp G were coupled to the spherical nucleic acid by base complementary pairing.The characterization results showed that SNA-TBB has a particle size of about 50 nm,a smooth spherical surface,and good dispersion.(2)The growth and CD11 c expression of BMDCs were compared in the medium supplemented with different concentrations of growth factors,and the optimal growth and CD11 c expression were screened when GM-CSF was 10 ng/ml and IL-4 was 20 ng/ml;the peptide vaccine based on spherical nucleic acid vector did not produce cytotoxicity to BMDCs and could effectively deliver antigens.SNA-TBB was effective in stimulating the expression of co-stimulatory molecules CD80 and CD86 on the surface of BMDCs at both molecular and protein levels,and also promoted the secretion of IL-12p70 from BMDCs.(3)SNA-TBB induced high levels of antigen-specific Ig G antibodies in immunized mice,and no abnormal cell death,tissue reaction or inflammatory infiltration was observed in the heart,spleen,lung and kidney of immunized mice by case sections.In conclusion,we developed a highly safe delivery strategy for peptide vaccines that supports BMDC activation and maturation and can trigger tailored humoral immune responses.【Research significance】In this study,a spherical nucleic acid carrier-based delivery system was successfully constructed,which can effectively deliver antigen to BMDCs and stimulate the maturation and activation of BMDCs.This system can effectively deliver antigen to BMDCs,stimulate the maturation and activation of BMDCs,and solve the problem of poor immunogenicity of peptides while demonstrating a good safety profile.As an emerging third-generation vaccine technology,peptide vaccine provides a new research idea and direction for the research of safe and effective dengue vaccine.