Design And Construction Of DNA Nanovaccine For Indcuing Mucosal Immune Responses Against SARS-CoV-2

BackgroundThe coronavirus disease 2019（COVID-19）pandemic caused by severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）has caused more than 6 million deaths and 100 million infections worldwide,which has posed a huge and continuous threat to the world.SARS-Co V-2,which invades the host through the mucosa of the respiratory tract,may seed in the initial reservoir and continue to spread between individuals.Most importantly,all the currently authorized COVID-19 vaccines are inoculated via parenteral route,which predominantly elicit systemic immunity dominated by serum Ig G antibodies without conferring mucosal immunity.The virus may still replicate at the initial infection site（such as respiratory mucosa）and cause local infection.Continuous detoxification may cause the risk of infection of others.This might be partially reflected by the fact that a resurgence of individuals infected by SARS-Co V-2 has increased recently even in region initiated a large-scale vaccination campaign.Secretory immunoglobulin A（s Ig A）and pulmonary resident memory T cells(T_RM)are suggested to be key components in this first-line of defense.Therefore,the ability to mount efficient mucosal immune responses is essential to eliminate the pathogen at an earlier stage of infection.In contrast,DNA vaccines,which proved to be helpful in controlling the pandemic,would offer substantial advantages over virus-based vaccines,including safe profiles,ease of manufacture with low costs,and stability at room temperature.With the development of interdisciplinary of medicinal chemistry,nanotechnology and vaccinology,the study on the modification of nucleic acid delivery vectors based on the chemical modification to promote the protection or the improvement of therapeutic effects of DNA vaccine has attracted extensive attention.The Poloxamine704（T704）is proved to be able to mediate the high expression of gene drugs in mouse and large animal models in many studies.It is found that the hydrophilic polyethylene oxide（PEO）chain outside the structure of the T704is similar to the structure of polyethylene glycol（PEG）,which is the key chemical group for its function.It can form a dense hydrophilic shell layer on the surface of nanoparticles,which is conducive to increasing the stability of nanoparticles.The peptide can condense DNA in various degrees by changing the amount of arginine contained.In a previous study,we developed a self-assembled peptide-poloxamine nanoparticle（pDNA/PP-s Np）delivery system that is specifically designed for efficient delivery of nucleic acids across the mucus layer of the respiratory tract.Here,we evaluated pSpike formulated in PP-s Np（pSpike/PP-s Np）for mucosal applications,which can stimulate a comprehensive and balanced mucosal immune response against SARS-Co V-2,and provides complete protection against lethal SARS-Co V-2 challenge.Objectives1.To optimize the pDNA/PP-s Np.2.To identify the ability of pSpike/PP-s Np in inducing mucosal immune response.3.To clarify the protective effect and mechanism of pSpike/PP-s Np in complete protection against lethal SARS-Co V-2 challenge.Methods1.Preparation and transfection efficiency of pDNA/PP-s Np for in vitro study（1）Design and optimization of pDNA/PP-s NpFor in vitro study,T704 solution at a certain concentration（defined as the w/w ratio between T704 and pDNA）in nuclease-free water was mixed with an equal volume of peptide solution（concentration of peptide defined as the N/P ratio;namely,the ratio between nitrogen residues in peptide and nucleic acid phosphate groups）were appied using similiar method.pDNA solution（0.04 mg/m L）in same volume was mixed with the above T704/peptide solution using the same mixer.The complex was incubated for 15 min at room temperature before further use.pDNA/PP-s Np complex encoding firefly luciferase（i.e.p FLuc/PP-s Np）were prepared.And the protein expression of firefly luciferase in cells was observed by bioluminescence imaging by Firefly Luciferase Reporter Gene Assay Kit to determine the optimized formulation.（2）Characterization of pDNA/PP-s NpSize measurements were performed using dynamic light scattering（DLS）on a Malvern Zetasizer Nano-ZS.The morphology of pDNA/PP-s Np formulations were investigated by transmission electron microscopeand scanning electron microscope.The cell viability was measured by CCK-8 assay.（3）Cellular uptake of pDNA/PP-s NpFor cellular uptake investigation,the Cy5 conjugated pDNA was prepared according to manual instructions.DC2.4 and MH-S Cells were incubated with Cy5-pDNA/PP-s Np for 4h.The cells were futher collected for the analysis of mean fluorescence intensity（MFI）by flow cytometry to investigate the cellular uptake of pDNA/PP-s Np.2.Investigation of transfection efficiency and biocompatibility of pDNA/PP-s Np in vivo（1）Preparation and characterization of pDNA/PP-s Np for in vivo studyFor in vivo applications,T704 stock solution（10 mg/m L）prepared in nuclease-free water was mixed with an equal volume of peptide solution（0.667 mg/m L）using a self-desigened microfluidic mixer.pDNA solution（0.6 mg/m L）in double volumes were mixed with the above T704/peptide solution using the same mixer.The complex was incubated for 15 min at room temperature and filtration by 0.22μm before further use.Size measurements were performed using dynamic light scattering（DLS）on a Malvern Zetasizer Nano-ZS.The morphology of pDNA/PP-s Np formulations were investigated by transmission electron microscopeand scanning electron microscope.The biocompatibility was measured by hematoxylin-eosin staining（H&E）staining of lung pathology.（2）Capability of efficient mucus penetrating of pDNA/PP-s NpCy5-DNA-containing PP-s Np were prepared as described above.Leica SP8 microscope was used to record the motion of pDNA/PP-s Np in mucus network.Confocal images was used to investigate the mucus-penetration properties of Cy5-pSpike/PP-s Np through airway mucus of mice 48 h after the administration.（3）Evaluation of the in vivo transfection of pDNA/PP-s NpImages of in vivo bioluminescence induced by p FLuc/PP-s Np was detected at 24h,48h,72h and 7day,respectively to value the in vivo transfection of p FLuc/PP-s Np.The expression of the SARS-Co V-2 spike gene specific-m RNA in lung of mice intratracheally administered pSpike/PP-s Np,measured by RT-q PCR 48h after transfection.Subcellular localization of Spike protein was observed by immunohistochemical staining of lung of mice treated by pSpike/PP-s Np via pulmonary route.3.Immune response and protection evaluation of pSpike/PP-s Np（1）The evaluation of mucosal and humoral immunityMice were immunized on day 0 and boosted with the same dose on day 14 and 28,respectively.Each anesthetized mouse intratracheally received 50μL of pSpike/PP-s Np formulation containing 15μg pSpike.Serum and BALF were collected 35 days after priming,and SARS-Co V-2 Spike protein specific Ig G antibodies in mouse serum and s Ig A antibodies in BALF were measured by ELISA.Pseudovirus neutralizing antibody titers in serum and BALF were also detected.（2）The evaluation of T cell responses and memory-biased immunityFlow cytometry analysis and ELISpot assay were used to investigate the cellular immune responses and secretion of IFN-γand IL-4 activated via mucosal immunization of pSpike/PP-s Np.The memory-biased immunity within pulmonary mucosal sites was also detected by flow cytometry.（3）The immune protection against lethal SARS-Co V-2 infectionA SARS-Co V-2 C57MA14 strain（which causes severe respiratory symptoms and mortality in mice）was applied in the challenge study,that causes severe respiratory symptoms,and mortality to BALB/c mice.Immunized BALB/c mice were challenged intranasaly with 50LD50 SARS-Co V-2 C57MA14 on day 40 post initial immunization.Body weight and survival were recorded,and the lungs and turbinates were collected for subsequent viral loads detection.Results1.Characterization,transfection efficiency and toxicity evaluation of pDNA/PP s NpFor in vitro study,T704 stock solution（1.5 mg/m L）prepared in nuclease-free water was mixed with an equal volume of peptide solution（0.667 mg/m L）using a self-desigened microfluidic mixer.pDNA solution（0.04 mg/m L）in same volume were mixed with the above T704/peptide solution using the same mixer.The complex was incubated for 20 min at room temperature before further use.The average particle size was 72.3±2.1 nm,Zetapotential was21.4±0.9 m V,PDI was 0.179±0.015.TEM showed that the size of pDNA/PP-s Np particles was about 50-80 nm,with smooth spherical surface and no obvious aggregation.pDNA/PP-s Np displayed the most efficient transfection in several cell lines（DC2.4,Calu-3,16HBE and BEAS-2B）and no significant cytotoxicity on the viability of different cell lines.2.In vivo evaluation of the transfection efficiency and biocompatibility of pDNA/PP-s NpFor in vivo applications,T704 stock solution（10 mg/m L）prepared in nuclease-free water was mixed with an equal volume of peptide solution（0.667 mg/m L）using a self-desigened microfluidic mixer.pDNA solution（0.6 mg/m L）in double volumes were mixed with the above T704/peptide solution using the same mixer.The complex was incubated for 20 min at room temperature and filtration by 0.22μm before further use.Electron microscopy showed that pDNA/PP-s Np had a spherical morphology with uniform size distribution with 116.6±1.7nm,PDI with 0.183±0.011 and Zeta with-34.7±0.5 m V.pDNA/PP-s Np can be stored at room or high temperatures and remains stable.pDNA/PP-s Np had a great ability to promote the maturation of BMDC and penetrate the airway mucus.Flow cytometry analysis and immunofluorescence sections suggest that considerable pSpike uptake and expression mediated by PP-s Np could be clearly observed in pulmonary DCs without any tissue inflammation or damage was observed by hematoxylin-eosin staining（H&E）staining of lung pathology.3.pSpike/PP-s Np elicits potent T cell responses and memory-biased immunityThe highest Ig G titer within serum from pSpike/PP-s Np group reached 1/51200 on day 35.The titer ratio of Ig G2a/Ig G1 reveals that pSpike/PP-s Np tends to induce a Type 1 T helper cell（Th1）-biased immune response.Most importantly,only pSpike/PP-s Np induced high levels of spike-specific s Ig A antibody in BALF with most samples could reach an endpoint titer of 1/128.The neutralization titer(ND₅₀)of serum and BALF samples from pSpike/PP-s Np group approached～1/1875 and～1/73,respectively.A significantly higher level of IFN-γand IL-4secretion was detected in pulmonary lymphocytes,T_EM cells or T_CM cells,were also significantly activated via the immunization of pSpike/PP-s Np.4.pSpike/PP-s Np completely prevents lethal SARS-Co V-2 infection in the upper and lower respiratory tractsThe pSpike/PP-s Np vaccine demonstrated remarkable protective efficacy as evidenced by a 100%survival rate at the predetermined end（14 days post-challenge）.The body weights of pSpike/PP-s Np vaccinated mice decreased slightly（<10%）in the first 3 days post-challenge but gradually recovered to a level displayed by the control group（without challenge）.Moreover,significant lower levels of viral RNA were detected in the turbinates and lungs of pSpike/PP-s Np treated mice,and the viral RNA loads in turbinates and lungs decreased with time and were almost undetectable 14 days post-challenge.significant numbers of CD4⁺TRM cells were identified exclusively in pulmonary sections of pSpike/PP-s Np treated mice after challenge,and the NAb titers against SARS-Co V-2 C57MA14 strain in serum of pSpike/PP-s Np immunized mice were elevated more than 5 times 14 days after challenge.Conclusions1.The peptide can condense DNA through positive and negative charges.The hydrophilic polyethylene oxide（PEO）chain outside the structure of the T704 is similar to the structure of polyethylene glycol（PEG）,which can form a dense hydrophilic shell layer on the surface of nanoparticles and be conducive to increasing the stability of nanoparticles.Here,we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine（pSpike/PP-s Np）.pSpike/PP-s Np has good quality characteristics,superior gene-transfection and favorable biocompatibility in vitro and in vivo.2.pSpike/PP-s Np holds the capability of efficient mucus penetrating and mediates the specific uptake and transfection of pSpike by pulmonary DCs.pSpike/PP-s Np can effectively and transiently activate innate immunity without any tissue inflammation,which indicates that pSpike/PP-s Np is suitable for DNA vaccine carrier and is an effective delivery system design strategy.3.pSpike/PP-s Np promotes systemic and mucosal immune responses,characterized by mucosal Ig A secretion,high levels of neutralizing antibodies,and resident memory phenotype T-cell responses in the lungs of mice.4.pSpike/PP-s Np completely eliminates SARS-Co V-2 infection in both upper and lower respiratory tracts and enables 100%survival rate of mice following lethal SARS-Co V-2challenge.And the NAb and T_RM mediated protective effects were actively involved in the protection of mice from lung lesions and the complete elimination of infected SARS-Co V-2 in the whole respiratory tract.Our findings indicate pSpike/PP-s Np might be a promising platform to prevent SARS-Co V-2 transmission and help to curtail the spread of pandemic.