Study On Periodic Mesoporous Organosilicon As A Nanoadjuvant For Subunit Vaccine And Its Immune Mechanism

Viral infections continue to be the leading cause of death among children and infants globally and are also the second leading cause of death among adults.Vaccines represent one of the most effective medical interventions currently available for prevention and control of viral infections,significantly reducing the morbidity and mortality associated with infections.Adjuvants,as an indispensable component of vaccines,play a crucial role in enhancing the immunogenicity and efficacy of vaccines.However,existing adjuvants have limitations in inducing effective immune responses against the complex and variable pathogens.In recent years,the emerging of nanoadjuvants has offered a new approach.These adjuvants not only efficiently target antigen proteins to antigen-presenting cells(APCs)through their carrier effects but also exert adjuvant effects to further activate immune cells.Nanoadjuvants explored to date include liposomes,calcium phosphate,mesoporous silica,among others.Although these nano-adjuvants have shown certain potential,their ability to induce efficient,broad-spectrum,and long-lasting immune responses still requires further enhancement.Periodic Mesoporous Organosilica(PMO)is an organic/inorganic hybrid nanocomposite material that has garnered attention for its excellent biocompatibility and carrier properties,particularly in the field of drug delivery where it has shown promising results.In previous studies conducted by our research group on tumor immunotherapy,we found that PMO could effectively promote the activation of innate immune cells such as macrophages.Building on this foundation,this thesis focuses on investigating the carrier and adjuvant properties of PMO,as well as its ability and mechanism to assist in inducing effective immune responses against highly variable pathogens such as Human Immunodeficiency Virus(HIV-1)and Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2),with the aim of exploring the potential application of PMO as a nanoadjuvant for subunit vaccines.The thesis first examines the carrier properties of PMO and PMO(N+)(amino-functionalized PMO,which is designed to adsorb more antigens).It was found that both PMO and PMO(N+)could effectively adsorb the HIV-1 gp120 trimer antigen ZCM to form vaccine formulations PMO-ZCM and PMO(N+)-ZCM,with PMO(N+)showing higher efficiency and stability in antigen adsorption.Electron microscopy,zeta potential,and particle size analyses indicated that PMO-ZCM and PMO(N+)-ZCM possess physical properties similar to natural viruses,presenting as nano-sized vaccines that approximate the size of viruses.Immunization of mice via the subcutaneous route revealed that both PMO-ZCM and PMO(N+)-ZCM showed significant improvements in persistence at the injection site and accumulation in lymph nodes compared to conventional subunit vaccines.Subsequently,the adjuvant properties of PMO and PMO(N+)were studied both in vitro and in vivo.Flow cytometric analysis and cytokine expression assays confirmed that PMO(N+)could effectively activate dendritic cells(DCs)both in vitro and in vivo,whereas PMO was more effective in activating macrophages.This suggests that while both PMO and PMO(N+)can effectively activate the innate immune response,they exhibit a certain selectivity in the activation properties towards different immune cells.Research indicates that the germinal center(GC)response is crucial for vaccine-induced broad-spectrum and highly effective humoral responses.To further explore the capability of PMO and PMO(N+)to induce GC responses,we conducted immunofluorescence and flow cytometry analyses on the lymph nodes of mice immunized with the vaccines,revealing rapid and effective formation of larger GCs and an increased number of GC B cells in the lymph nodes of mice immunized with PMO-ZCM within a short period(within 2 weeks).Isolating primary B cells from mice and incubating them with PMO and PMO(N+)showed that PMO could directly activate resting B cells and assist in further activation of the B cell receptor(BCR),whereas PMO(N+)neither assisted in BCR activation nor directly activated B cells.Transcriptomic sequencing analysis of the PMO activation mechanism revealed that Toll-like receptor(TLR)and C-type lectin receptor(CLR)pathways were effectively enriched during activation.Further analysis using signal pathway inhibitors and protein expression changes confirmed that PMO mainly activates resting B cells through the CLR pathway rather than the TLR pathway.In subsequent immunization experiments,we used the most effective adjuvant currently used in HIV subunit vaccine research,aluminum adjuvant(in its optimal formulation),as a control to evaluate the immunogenicity of PMO-ZCM and PMO(N+)-ZCM in mice.Results showed that the PMO adjuvanted vaccines induced higher titers and more durable Ig G antibodies than the aluminum adjuvanted vaccines,though there was no significant difference in the levels of neutralizing antibodies produced by both.Moreover,the PMO adjuvanted vaccines also induced a significant cellular immune response.Meanwhile,we found that the PMO(N+)adjuvanted vaccines,despite having a stronger antigen-binding capacity,were inferior to the PMO adjuvanted vaccines in inducing both humoral and cellular immune responses.Our comprehensive immunological analysis of lymph nodes and spleen tissues in mice post-vaccination revealed that PMO adjuvanted vaccines could effectively activate follicular helper T cells(Tfh),GC B,and memory B cells(MBC)in lymph nodes,as well as influence the dynamics of regulatory cells such as regulatory T cells(Treg).Additionally,these vaccines significantly activated CD4~+and CD8~+T cells in the spleen and promoted the release of various cytokines that regulate immune responses.In contrast,the level of immune cell activation in the lymph nodes and spleen by PMO(N+)adjuvanted vaccines was significantly lower than that by PMO adjuvanted vaccines.Subsequently,we also immunized mice with SARS-CoV-2 RBD protein using PMO as an adjuvant,further evaluating the immunogenicity and GC response characteristics of PMO adjuvanted COVID-19 vaccines.The results further corroborated the main findings and conclusions from the HIV-1 vaccine research.These studies indicate that PMO,as a vaccine adjuvant,can effectively induce GC responses and higher levels of antigen-specific humoral and cellular immune responses by directly activating the innate immunity of B cells.In the analysis of immune cell activation in lymph nodes post-vaccination,we observed that PMO adjuvanted vaccines significantly increased the activation levels of Treg,which could potentially limit the level of vaccine-induced immune responses.Based on this hypothesis,we further explored whether inhibiting Treg cells could enhance the immune activation capability of PMO adjuvanted COVID-19 vaccines in vivo.In the experiments,we inhibited Treg cells using anti-CD25 monoclonal antibodies(mAb)at different time points before and after vaccine immunization.The results showed that inhibiting Treg,regardless of the approach,further increased GC B cells,induced stronger GC responses,and enhanced the levels of broadly neutralizing antibodies(bn Abs).Notably,we found that injecting anti-CD25 mAb one day before each immunization not only induced stronger humoral immune responses but also significantly enhanced cellular immune responses.These studies suggest that relieving the suppression by Treg cells could help further unleash the potential of PMO to activate B cells and certain helper T cells,thereby enhancing the overall immune response.In summary,this thesis confirms the efficacy of PMO as a nanoadjuvant for subunit vaccines and reveals its adjuvant properties,functions,and mechanisms of action through a series of in vitro and in vivo analyses as follows:PMO can effectively adsorb subunit antigens and deliver them to APCs,forming a vaccine-like"depot"at the injection site.This depot prevents the rapid clearance of subunit antigens by innate immune cells and efficiently promotes their accumulation in lymph nodes;PMO possesses the capability to activate traditional APCs and can also directly activate resting B cells through the CLR signaling pathway,thereby rapidly inducing potent GC responses;Animal immunization experiments demonstrate that vaccines adjuvanted with PMO can induce durable humoral immune responses and robust cellular immune responses.Further studies indicate that inhibiting Treg at specific time points can effectively enhance the GC response induced by PMO-adjuvanted vaccines and significantly strengthen both humoral and cellular immune responses.The research presented in this thesis not only offers a novel nanoadjuvant with broad application potential but also contributes to a deeper understanding of the relationship between vaccine-induced GC responses and broad-spectrum,highly effective humoral immune responses.This,in turn,provides a foundation and rationale for the design of vaccines against infectious diseases.