Design And Immunological Study Of HIV-1 Nano-vaccines Based On Silica/calcium Phosphate Carrier And Adeno-associated Viral Vector

Human immunodeficiency virus type 1（HIV-1）remains one of the major threats to global health.Due to issues such as drug-resistant strains,insufficient compliance,and high cost of combination antiretroviral therapy,eradication of HIV-1 epidemic still requires broad and effective prophylactic vaccines.However,a licensed vaccine against HIV-1 has yet to be created.Because of the exceptional evolutionary capacity of HIV-1 Env,the global diversity of HIV-1 has become one of the most important difficulties in the development of HIV-1 vaccines.An ideal HIV-1 vaccine would elicit antibodies which can broadly neutralize field isolates of HIV-1.Therefore,induction of broadly neutralizing antibody（b NAb）through active or passive immunization in vivo is a potentially efficacious approach for the prevention of HIV-1 infection.This article consists of the following two parts:Part one:Surface-functionalized silica-coated calcium phosphate nanoparticles efficiently deliver DNA-based HIV-1 trimeric envelope vaccines for genetic immunization against HIV-1The prevention of HIV-1 infection is a crucial task that needs to be addressed due to the absence of a licensed vaccine against HIV-1.Compared to traditional protein vaccines,DNA vaccines can induce a long-term cellular immune response in addition to a humoral immune response in hosts to provide subsequent protective immunity against HIV-1.DNA vaccines present a promising alternative approach to combat HIV-1 infection due to their excellent safety profile,lack of severe side effects,and relatively rapid fabrication.Traditional vaccines composed of gp120 monomers or peptide fragments have been indicated to lack protective efficacy mediated by inducing HIV-1-specific neutralizing antibodies in clinical trials.Therefore,DNA vaccines expressing native-like trimeric HIV-1 antigens may be promising vaccine candidates for inducing NAb responses.Vaccines comprising the soluble BG505 SOSIP.664 trimer fused to the GCN4-based isoleucine zipper or bacteriophage T4 fibritin foldon motif with excellent simulation of the native HIV-1 envelope were chosen as trimer-based vaccine platforms in this study.Despite the various advantages of DNA vaccines,the immunogenicity and protection against HIV-1 induced by DNA vaccines are limited due to the poor uptake of these vaccines by antigen-presenting cells and their ready degradation by DNases and lysosomes.To address some of these issues of naked DNA vaccines,the development of an effective DNA-based delivery system is a promising strategy.Commonly used gene delivery vectors include non-viral vectors and viral vectors.Calcium phosphate nanoparticles are one of the most favorable antigenic carriers due to their high biocompatibility and good biodegradability.To address these issues of naked DNA vaccines,we described the feasibility of Cp G-functionalized silica-coated calcium phosphate nanoparticles（SCPs）for efficiently delivering DNA-based HIV-1trimeric envelope vaccines against HIV-1.Our results showed that SCP-based DNA immunization could significantly induce both broad humoral immune responses and potent cellular immune responses compared to naked DNA vaccination in vivo.Additionly,subcutaneous vaccination of the nano-vaccines induced significantly stronger immune responses than intramuscular vaccination.The nano-vaccines could induce neutralizing antibody responses against a wide panel of HIV-1 Env pseudoviruses,including tier 1 pseudoviruses and partial tier2 pseudoviruses.In conclusion,this study is the first to assess the feasibility of Cp G-functionalized SCPs for efficient delivery of DNA vaccines expressing native-like trimeric HIV-1antigens.The candidate nanoparticles showed enhanced immunogenicity,inducing both broad humoral immune responses and robust cellular immune responses.The use of Cp G-functionalized SCPs for delivering DNA-based HIV-1 trimeric envelope vaccines may be a promising strategy for the prevention of HIV-1 infection.Neutralizing activity of Cp G-functionalized SCPs against HIV-1 tier 2 pseudoviruses were not ideal.Since active immunization strategy was difficult to deal with the global diversity of HIV-1,passive immunization based on b NAbs was considered as a potentially viable strategy.Part two:Broad and potent bispecific neutralizing antibody gene delivery using adeno-associated viral vectors for passive immunization against HIV-1Since neutralizing antibody responses induced by active immunization in vivo were not ideal,passive immunization with human monoclonal antibodies emerge as a potentially viable strategy for the prevention of HIV-1 infection.However,b NAbs generally used for monotherapy(IC₈₀>5μg/m L)have limited breadth and potency and neutralize only 70～90%of all HIV-1 strains.To address the need for broader coverage of the HIV-1 epidemic and enhance the potency of b NAbs,we fused the single-chain variable antibody fragment（sc Fv）of b NAbs（PG9,PGT123,or NIH45-46）with full-length ibalizumab（i Mab）in an sc Fv-monoclonal antibody tandem format to construct bispecific broadly neutralizing antibody（Bib NAbs）in this study.However,short-term passive immunization requires continuous injections over long-term periods,which requires repeated hospital visits and cold chain transportation.Commonly used gene delivery vectors include non-viral vectors and viral vectors.Adeno-associated viral（AAV）vectors have been shown to consistently express transgenes for years in various animal models.To address this challenge of repeated passive administration,we described the feasibility of Bib NAb gene delivery mediated by recombinant adeno-associated virus 8（r AAV8）for generating long-term expression in this study.Our results showed that the expressed Bib NAbs targeting two distinct epitopes exhibited neutralizing activity against a panel of 20 HIV-1 pseudoviruses in vitro and in vivo,including tier 1 pseudoviruses and tier 2 pseudoviruses.After a single administration of the r AAV8-Bib NAb,the expression and neutralizing activity of the Bib NAbs in serum were sustained for at least 24 weeks.The concentration of the Bib NAbs reached a maximum 8 weeks post immunization,the maximum antibody concentration was up to 27.13±4.76μg/m L.In conclusion,this study demonstrated the feasibility of i Mab-based Bib NAb gene delivery mediated by r AAV8 as a means of generating continuous HIV-1 neutralizing activity.After a single administration of the r AAV8 vector,the expression and neutralizing activity of Bib NAbs in serum were sustained for a long period of time.To the best of our knowledge,very few studies have been published on Bib NAb gene delivery using r AAV8 vectors against HIV-1.Bib NAb gene delivery using r AAV8vectors may be promising for passive immunization against HIV-1 infection.In summary,this article is the first to evaluate the feasibility of Cp G-functionalized SCPs for delivering DNA-based HIV-1 trimeric envelope vaccines and r AAV8-mediated gene delivery of i Mab-based HIV-1 Bib NAbs.The former can induce both broad humoral immune responses and potent cellular immune responses,while the latter can maintain potent neutralizing antibody levels in vivo.Both nano-vaccines are promising candidates for preventing HIV-1 infection.