Enhancement of intranasalmucosal immunization of mucosal vaccines by ultrasonic treatment

The pathogens of most infectious diseases invade the host through mucosal sites,and immunization with mucosal vaccines is the best means of combating these infectious diseases.Oral delivery and nasal delivery are the most common methods of mucosal vaccination.However,the delivery process is inefficient,and mucosal vaccination is ineffective because the vaccine formulation is easily and rapidly removed and has difficulty in crossing the mucosal surface.In this paper,we investigated whether the mucosal immune response could be enhanced by ultrasound facilitation of nasal mucosal delivery of vaccine preparations.For this purpose,we used manganese dioxide(MnO2)as the vaccine carrier/adjuvant,coated with chitosan oligosaccharide(COS)to enhance mucosal adsorption,and further physically adsorbed model antigen ovalbumin(OVA)to construct a nanoparticulate vaccine formulation MnO2@COS@OVA.Ultrasound treatment was found to promote antigen delivery and recruitment of dendritic cells(DCs)and macrophages as well as T-cell infiltration in nasal mucosal tissues through nasal mucosal immunization studies.With ultrasound assistance,MnO2@COS@OVA particles promoted the maturation of DCs in vitro and in vivo and promoted the production of effector memory T cells in vivo and cytokine secretion by splenocytes in vitro.In particular,ultrasound treatment significantly increased the levels of secretory IgA antibodies in the nasal mucosa and genital tract mucosa of experimental mice.In addition,the experimental data showed that the MnO2@COS@OVA particles had good biocompatibility and caused no significant damage to the nasal mucosal and vital organ tissue.These data suggest that ultrasound treatment can promote the induction of efficient immune responses to mucosal vaccines and provide new ideas for the opening and clinical translation of mucosal vaccines.

A triple-RBD-based mucosal vaccine provides broad protectionagainst SARS-CoV-2 variants of concern

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broadspectrum protection against the initial infection and thereby curb the transmission potential.Here,we designed a chimeric tripleRBD immunogen,3Ro-NC,harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold.3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern.Notably,intranasal immunization with 3RoNC plus the mucosal adjuvant KFD(3Ro-NC+KFDi.n)elicits coordinated mucosal IgA and higher neutralizing antibody specificity(closer antigenic distance)against the Omicron variant.In Omicron-challenged human ACE2 transgenic mice,3Ro-NC+KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung(85.7-fold)and the nasal turbinate(13.6-fold).Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies.Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection,pathology and transmission potential.

Mucosal vaccine delivery:A focus on the breakthrough of specific barriers

Mucosal vaccines can effectively induce an immune response at the mucosal site and form the first line of defense against microbial invasion.The induced mucosal immunity includes the proliferation of effector T cells and the production of IgG and IgA antibodies,thereby effectively blocking microbial infection and transmis sion.However,after a long period of development,the transformation of mucosal vaccines into clinical use is still relatively slow.To date,fewer than ten mucosal vaccines have been approved.Only seven mucosal vaccines against coronavirus disease 2019(COVID-19) are under investigation in clinical trials.A representative vaccine is the adenovirus type-5 vectored COVID-19 vaccine(Ad5-nCoV) developed by Chen and coworkers,which is currently in phase Ⅲ clinical trials.The reason for the limited progress of mucosal vaccines may be the complicated mucosal barriers.Therefore,this review summarizes the characteristics of mucosal barriers and highlights strategies to overcome these barriers for effective mucosal vaccine delivery.

Recent progress in application of nanovaccines for enhancing mucosal immune responses

Mucosal vaccines that stimulate both mucosal and systemic immune responses are desirable,as they could prevent the invading pathogens at their initial infection sites in a convenient and userfriendly way. Nanovaccines are receiving increasing attention for mucosal vaccination due to their merits in overcoming mucosal immune barriers and in enhancing immunogenicity of the encapsulated antigens.Herein, we summarized several nanovaccine strategies that have been reported for enhancing mucosal immune responses, including designing nanovaccines that have superior mucoadhesion and mucus penetration capacity, designing nanovaccines with better targeting efficiency to M cells or antigen-presenting cells, and co-delivering adjuvants by using nanovaccines. The reported applications of mucosal nanovaccines were also briefly discussed, including prevention of infectious diseases, and treatment of tumors and autoimmune diseases. Future research progresses in mucosal nanovaccines may promote the clinical translation and application of mucosal vaccines.

Mucosal delivery of nanovaccine strategy against COVID-19 and its variants

Despite the global administration of approved COVID-19 vaccines(e.g.,ChAdOx1 nCoV-19?,mRNA-1273?,BNT162b2?),the number of infections and fatalities continue to rise at an alarming rate because of the new variants such as Omicron and its subvariants.Including COVID-19 vaccines that are licensed for human use,most of the vaccines that are currently in clinical trials are administered via parenteral route.However,it has been proven that the parenteral vaccines do not induce localized immunity in the upper respiratory mucosal surface,and administration of the currently approved vaccines does not necessarily lead to sterilizing immunity.This further supports the necessity of a mucosal vaccine that blocks the main entrance route of COVID-19:nasal and oral mucosal surfaces.Understanding the mechanism of immune regulation of M cells and dendritic cells and targeting them can be another promising approach for the successful stimulation of the mucosal immune system.This paper reviews the basic mechanisms of the mucosal immunity elicited by mucosal vaccines and summarizes the practical aspects and challenges of nanotechnology-based vaccine platform development,as well as ligand hybrid nanoparticles as potentially effective target delivery agents for mucosal vaccines.

Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector(VSV_(MT))in golden syrian hamster

COVID-19 is caused by coronavirus SARS-CoV-2.Current systemic vaccines generally pro-vide limited protection against viral replication and shedding within the airway.Recombinant VSV(rVSV)is an effective vector which inducing potent and comprehensive immunities.Currently,there are two clinical trials investigating COVID-19vaccines based on VSV vectors.These vaccines were developed with spike protein of WA1 which administrated intramuscularly.Although intranasal route is ideal for activating mucosal immunity with VSV vector,safety is of concern.Thus,a highly attenuated rVSV with three amino acids mutations in matrix protein(VSV_(MT))was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern.It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently.VSV_(MT) indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein.With a single-dosed intranasal inoculation of rVSV_(ΔGMT)-S_(Δ21),potent SARS-CoV-2specific neutraliza-tion antibodies could be stimulated in animals,particularly in term of mucosal and cellular immunity.Strikingly,the chimeric VSV encoding S_(Δ21) of Delta-variant can induce more potent immune responses compared with those encoding S_(Δ21) of Omicron-or WA1-strain.VSV_(MT) is a promising platform to develop a mucosal vaccine for countering COVID-19.

A micro-sized vaccine based on recombinant Lactiplantibacillus plantarum fights against SARS-CoV-2 infection via intranasal immunization

COVID-19 has globally spread to burden the medical system.Even with a massive vaccination,a mucosal vaccine offering more comprehensive and convenient protection is imminent.Here,a micro-sized vaccine based on recombinant Lactiplantibacillus plantarum(rLP)displaying spike or receptor-binding domain(RBD)was characterized as microparticles,and its safety and protective effects against SARS-CoV-2 were evaluated.We found a 66.7%mortality reduction and 100%protection with rLP against SARS-CoV-2 in a mouse model.The histological analysis showed decreased hemorrhage symptoms and increased leukocyte infiltration in the lung.Especially,rLP:RBD significantly decreased pulmonary viral loads.For the first time,our study provides a L.plantarum-vectored vaccine to prevent COVID-19 progress and transmission via intranasal vaccination.

Mucosal immune responses in the lung during respiratory infection:The organization and regulation of iBALT structure

The local immune defenses of respiratory system play a crucial role in safeguarding against pathogens and eliminating infected cells.In this article,we review the current knowledge regarding the establishment and regulation of local im-mune responses within the lungs,with a particular focus on the formation of inducible bronchus-associated lymphoid tissue(iBALT),which has demonstrated a capacity to mount protective immune responses against several pathogens,including influenza virus,severe acute respiratory syndrome coronavirus(SARS-CoV)/SARS-CoV-2,and Mycobacte-rium tuberculosis(Mtb).Furthermore,we explore the development and regulation of long-term immune memory within the lungs,which may be facilitated by iBALT.This review aims to provide a summary of potential targets within iBALT for pathogen defense and vaccine design.

Innate endogenous adjuvants prime to desirable immune responses via mucosal routes

Vaccination is an effective strategy to prevent infectious or immune related diseases, which has made remarkable contribution in human history. Recently increasing atten- tions have been paid to mucosal vaccination due to its multiple advantages over conventional ways. Subunit or peptide antigens are more reasonable immunogens for mucosal vaccination than live or attenuated pathogens, however adjuvants are required to augment the immune responses. Many mucosal adjuvants have been developed to prime desirable immune responses to different etiologies. Compared with pathogen derived adjuvants, innate endogenous molecules incorporated into mucosal vaccines demonstrate prominent adjuvanticity and safety. Nowadays, cytokines are broadly used as mucosal adjuvants for participation of signal transduction of immune responses, activation of innate immunity and polarization of adaptive immunity. Desired immune responses are promptly and efficaciously primed on basis of specific interactions between cytokines and corresponding receptors. In addition, some other innate molecules are also identified as potent mucosal adjuvants. This review focuses on innate endogenous mucosal adjuvants, hoping to shed light on the development of mucosal vaccines.

The construction of in vitro nasal cavity-mimic M-cell model,design of M cell-targeting nanoparticles and evaluation of mucosal vaccination by nasal administration

In order to better evaluate the transport effect of nanoparticles through the nasal mucosa,an in vitro nasal cavity-mimic model was designed based on M cells.The differentiation of M cells was induced by co-culture of Calu-3 and Raji cells in invert model.The ZO-1 protein staining and the transport of fluorescein sodium and dexamethasone showed that the inverted co-culture model formed a dense monolayer and possessed the transport ability.The differentiation of M cells was observed by upregulated expression of Sialyl Lewis A antigen(SLAA)and integrinβ1,and down-regulated activity of alkaline phosphatase.After targeting M cells with iRGD peptide(cRGDKGPDC),the transport of nanoparticles increased.In vivo,the co-administration of iRGD could result in the increase of nanoparticles transported to the brain through the nasal cavity after intranasal administration.In the evaluation of immune effect in vivo,the nasal administration of OVA-PLGA/iRGD led to more release of IgG,IFN-γ,IL-2 and secretory IgA(sIgA)compared with OVA@PLGA group.Collectively,the study constructed in vitro M cell model,and proved the enhanced effect of targeting towards M cell with iRGD on improving nasal immunity.

Intranasal and oral vaccination with protein-based antigens： advantages, challenges and formulation strategies

Most pathogens initiate their infections at the human mucosal surface. Therefore, mucosal vaccination, especially through oral or intranasal administration routes, is highly desired for infectious diseases. Meanwhile, protein-based antigens provide a safer alternative to the whole pathogen or DNA based ones in vaccine development. However, the unique biopharmaceutical hurdles that intranasally or orally delivered protein vaccines need to overcome before they reach the sites of targeting, the relatively low im- munogenicity, as well as the low stability of the protein antigens, require thoughtful and fine-tuned mucosal vaccine formulations, including the selection of immunostimulants, the identification of the suitable vaccine delivery system, and the determination of the exact composition and manufacturing conditions. This review aims to provide an up-to-date survey of the protein antigen-based vaccine formulation development, including the usage of immunostimulants and the optimization of vaccine delivery systems for intranasal and oral administrations.