Comparative Immunogenicity Analysis Of Microneedle Intradermal Versus Intramuscular Administration Of SARS-CoV-2 Recombinant S1 Protein Immunogen In Mice

Objective:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused significant damage to healthcare systems globally,and vaccines are the most effective way to prevent and control its spread.The SARS-CoV-2 S1 protein is a promising candidate for vaccine development,given its vital role in forming the viral capsid and facilitating virus binding and entry into host cells.While traditional intramuscular(IM)vaccines have proven to be an effective means of inducing antibody responses,intradermal(ID)injection may provide better suppression of SARS-CoV-2 infection.This study aimed to compare the immunogenicity of intradermal and intramuscular immunization with a SARS-CoV-2 S1 recombinant protein vaccine in mice to evaluate their potential for inducing humoral and cellular immune responses.Methods:112 female BALB/c mice were randomized into 14 groups,each consisting of 8 mice.The groups were as follows:a control group that received an adjuvant injection only,S1/-5ug group,S1-Al(OH)3-5ug group,S1-/-10ug group,Sl-Al(OH)3-10ug group,S1-/20ug group,and S1-Al(OH)3-20ug group.The protein to adjuvant mass ratio was maintained at 1:10,and the mice were immunized via intramuscular and microneedle intradermal injections.The immunization program consisted of three immunizations at 0,2,and 4 weeks.Cellular immune responses were compared using an unpaired t-test and the Kruskal-Wallis test followed by Dunn’s multiple comparison,and any P value less than 0.05 was considered a significant difference.Enzyme-linked immunosorbent assay was utilized to compare binding antibody levels of IgG,IgG1,and IgG2a induced by different immunization doses and routes.Additionally,the levels of neutralizing antibodies were assessed using SARS-CoV-2 wild type(WT)and Omicron(BA.5)pseudovirus,followed by the evaluation of cellular immune responses through enzyme-linked immunospot assay.Results:1.Both intramuscular injection and microneedle intradermal injection of SARS-CoV-2 S1 protein induced robust levels of IgG,IgG1,and IgG2a antibodies in mice.With the same immunization dosage,the adjuvant significantly enhanced the immunogenicity of recombinant S1 protein.In the intramuscular immunization group receiving 10μg and the 10μg+Al(OH)3 group,IgG(geometric mean titer,GMT:32000<724077,P<0.0001)showed a significant increase.Similarly,in the microneedle intradermal immunization group receiving 10μg and the 1Oμg+Al(OH)3 group,IgG(GMT:1217750<2896310,P<0.01)exhibited a notable elevation.Comparing the same immunization dosage,the microneedle intradermal injection induced higher titers of IgG,IgG1,and IgG2a antibodies compared to the intramuscular immunization.The intramuscular immunization group with 10μg and the microneedle intradermal immunization group with 10μg showed significant differences in IgG(GMT:32000<1217750,P<0.0001),IgG1(GMT:80474<631333,P<0.001),and IgG2a(GMT:50<4525.5,P<0.01).Furthermore,the microneedle intradermal immunization demonstrated a pronounced dose-sparing effect,as the microneedle immunization group with 10μg showed significant differences compared to the intramuscular immunization group with 20μg for IgG(GMT:1217750>76109,P<0.001).2.Microneedle intradermal immunization in mice significantly induced high levels of interferon-gamma(IFN-y)and interleukin-4(IL-4)in spleen cells.The group receiving 20μg of microneedle intradermal immunization exhibited the highest level of IFN-y(370.7±42.36 immune spots),while the group receiving 10μg+Al(OH)3 microneedle intradermal immunization showed the highest level of IL-4(416.7±51.83 immune spots).Compared to intramuscular immunization,microneedle intradermal immunization induced a higher and more balanced cellular immune response.The ratio of IgG1/IgG2a induced by microneedle intradermal immunization was lower than that of intramuscular immunization.Furthermore,the dose-sparing effect was reflected in the levels of IFN-γ and IL-4.The microneedle immunization group with 10μg showed significant differences compared to the intramuscular immunization group with 20μg for IFN-γ(199.7>88.7,P<0.05)and IL-4(310>210,P<0.01).3.Both immunization methods resulted in higher levels of neutralizing antibodies against the wild-type pseudovirus compared to Omicron BA.5.The microneedle intradermal immunization with 10μg+Al(OH)3 demonstrated a geometric mean titer(GMT)of 922 for neutralizing antibodies against the wild-type virus and 99 against Omicron BA.5.In the serum of mice immunized without the adjuvant,a weak neutralizing antibody response was detected.For the same immunization dosage group,the microneedle intradermal immunization induced stronger neutralizing antibody levels than intramuscular immunization.Significant differences were observed in the neutralizing antibody levels between the intramuscular immunization group receiving 10μg and the microneedle intradermal immunization group(GMT:67<384,P<0.01).Conclusion:Intradermal immunization with SARS-CoV-2 S1 recombinant protein induces a stronger protective immune response than intramuscular immunization.ID immunization induces higher IgG,IgG1,and IgG2a antibody titers and a more balanced cellular immune response than IM immunization.ID immunization also has a dose-sparing effect.