Pretreatment with 3-methyladenine ameliorated Pseudomonas aeruginosa-induced acute pneumonia by inhibiting cell death of neutrophils in a mouse infection model.

Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide. Clinical isolates that are resistant to multiple antimicrobials make it intractable. The interactions between P. aeruginosa and host cell death have multiple effects on bacterial clearance and inflammation; however, the potential intervention effects remain to be defined. Herein, we demonstrated that intravenous administration of 3-methyladenine before, but not after, P. aeruginosa infection enhanced autophagy-independent survival, which was accompanied by a decrease in the bacterial load, alleviation of pathology and reduction in inflammatory cytokines, in an acute pneumonia mouse model. Interestingly, these beneficial effects were not dependent on neutrophil recruitment or phagocytosis, but on the enhanced killing capacity induced by inhibiting the cell death of 3-MA pretreated neutrophils. These findings demonstrate a novel protective role of 3-MA pretreatment in P. aeruginosa-induced acute pneumonia.

Evaluation of a bivalent recombinant vaccine candidate targeting norovirus and rotavirus: Antibodies to rotavirus NSP4 exert antidiarrheal effects without virus neutralization.

We previously found that when tandemly expressed with SR69A -VP8*, nonstructural protein 4 (NSP4) of the rotavirus Wa strain exerts a minor effect on elevating the antibody responses targeting the rotavirus antigen VP8* of the 60-valent nanoparticle SR69A -VP8* but could fully protect mice from diarrhea induced by the rotavirus strain Wa. In this study, we chose comparably less immunogenic norovirus 24-valent P particles with homogenous (i.e., VP8* from rotavirus) and heterogeneous (i.e., protruding domain of norovirus) antigens and in more challenging rotavirus SA11 strain-induced diarrhea mouse models to evaluate its main role in recombinant gastroenteritis virus-specific vaccines. The results showed that although as an adjuvant NSP4 exerted limited effects on the elevation of norovirus-specific or VP8*-specific neutralizing antibody production, as an antigen it could confer potent protection, particularly when synergized with VP8*, in rotavirus SA11 strain-induced diarrhea mouse models, possibly blocking the invasion of the intestinal wall by enterotoxin. NSP4 may be unnecessary for other recombinant vaccines as adjuvants, and its display mode should be evaluated specifically to avoid blocking coexpressed antigens in the norovirus P particles.

Comparison of immune responses induced by two or three doses of an alum-adjuvanted inactivated SARS-CoV-2 vaccine in mice.

Waning antibodies and rapidly emerging variants are challenges for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine development. Adjusting existing immunization schedules and further boosting strategies are under consideration. Here, the immune responses induced by an alum-adjuvanted inactivated SARS-CoV-2 vaccine in mice were compared among immunization schedules with two or three doses. For the two-dose schedule, a 0-28-day schedule induced 5-fold stronger spike-specific IgG responses than a 0-14-day schedule, with only a slight elevation of spike-specific cellular immunity 14 days after the last immunization. A third homologous boost 2 or 5 months after the second dose for the 0-28-day schedule slightly strengthened humoral responses (1.3-fold for the 0-1-3-month schedule, and 1.8-fold for the 0-1-6-month schedule) 14 days after the last immunization. Additionally, a third homologous boost (especially with the 0-1-3-month schedule) induced significantly stronger cell-mediated immunity than both two-dose immunization schedules for all indexes tested, with a response similar to that induced by a one-dose heterologous boost with BNT162b2 in clinical trials, according to cellular immunity analysis (1.5-fold). These T cell responses were Th2 oriented, with good CD4+ and CD8+ memory. These results may offer clues for applying a homologous boosting strategy for alum-adjuvanted inactivated SARS-CoV-2 vaccines.

SARS-CoV-2 S1 is superior to the RBD as a COVID-19 subunit vaccine antigen.

Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into a global pandemic within a matter of months. While subunit vaccines are one of the prominent options for combating coronavirus disease 2019 (COVID-19), the immunogenicity of spike protein-based antigens remains unknown. When immunized in mice, the S1 domain induced much higher IgG and IgA antibody levels than the receptor-binding domain (RBD) and more efficiently neutralized SARS-CoV-2 when adjuvanted with alum. It is inferred that a large proportion of these neutralization epitopes are located in the S1 domain but outside the RBD and that some of these are spatial epitopes. This finding indicates that expression systems with posttranslational modification abilities are important to maintain the natural configurations of recombinant spike protein antigens and are critical for effective COVID-19 vaccines. Further, adjuvants prone to a Th1 response should be considered for S1-based subunit COVID-19 vaccines to reduce the potential risk of antibody-dependent enhancement of infection.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

Two versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.

A rabies mRNA vaccine with H270P mutation in its glycoprotein induces strong cellular and humoral immunity.

Rabies is a lethal zoonotic disease that kills approximately 60,000 people each year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates its host-cell entry. RABV-G's pre-fusion conformation displays major known neutralizing antibody epitopes, which can be used as immunogen for prophylaxis. H270P targeted mutation can stabilize RABV-G in the pre-fusion conformation. Herein, we report the development of a highly promising rabies mRNA vaccine composed of H270P targeted mutation packaged in lipid nanoparticle (LNP), named LNP-mRNA-G-H270P. Humoral and cellular immunity of this vaccine were assessed in mice comparing to the unmodified LNP-mRNA-G and a commercially available inactivated vaccine using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. The results show the titer of RABV-G-specific IgG and virus-neutralization antibody titers (VNTs) in LNP-mRNA-G-H270P group were significant higher than those in LNP-mRNA-G and inactivated vaccine groups. Likewise, IFN-γ-secreting splenocytes, level of IL-2 in the supernatant of spleen cells, as well as IFN-γ-producing CD4+ T cells in LNP-mRNA-G-H270P group were significant higher than those in the other two vaccine groups. Hence, these results demonstrated that targeting the H270P mutation in RABV-G through an mRNA-LNP vaccine platform represents a promising strategy for developing a more efficacious rabies vaccine.

Comparison of Immune Responses between Inactivated and mRNA SARS-CoV-2 Vaccines Used for a Booster Dose in Mice.

A large amount of real-world data suggests that the emergence of variants of concern (VOCs) has brought new challenges to the fight against SARS-CoV-2 because the immune protection elicited by the existing coronavirus disease 2019 (COVID-19) vaccines was weakened. In response to the VOCs, it is necessary to advocate for the administration of booster vaccine doses to extend the effectiveness of vaccines and enhance neutralization titers. In this study, the immune effects of mRNA vaccines based on the WT (prototypic strain) and omicron (B1.1.529) strains for use as booster vaccines were investigated in mice. It was determined that with two-dose inactivated vaccine priming, boosting with mRNA vaccines could elevate IgG titers, enhance cell-mediated immunity, and provide immune protection against the corresponding variants, but cross-protection against distinct strains was inferior. This study comprehensively describes the differences in the mice boosted with mRNA vaccines based on the WT strain and the omicron strain, a harmful VOC that has resulted in a sharp rise in the number of infections, and reveals the most efficacious vaccination strategy against omicron and future SARS-CoV-2 variants.

Evaluation of the Immunological Efficacy of an LNP-mRNA Vaccine Prepared from Varicella Zoster Virus Glycoprotein gE with a Double-Mutated Carboxyl Terminus in Different Untranslated Regions in Mice.

Cell-mediated immunity (CMI) plays a key role in the effectiveness of varicella zoster virus (VZV) vaccines, and mRNA vaccines have an innate advantage in inducing CMI. Glycoprotein E (gE) has been used widely as an antigen for VZV vaccines, and carboxyl-terminal mutations of gE are associated with VZV titer and infectivity. In addition, the untranslated regions (UTRs) of mRNA affect the stability and half-life of mRNA in the cell and are crucial for protein expression and antigenic translational efficiency. In this study, three UTRs were designed and connected to the nucleic acid sequence of gE-M, which is double mutated in the extracellular region of gE. Then, mRNA with different nucleic acids was encapsulated in lipid nanoparticles (LNPs), forming three LNP-mRNA VZV vaccines, named gE-M-Z, gE-M-M, and gE-M-P. The immune response elicited by these vaccines in mice was evaluated at intervals of 4 weeks, and the mice were sacrificed 2 weeks after the final immunization. In the results, the gE-M-P group, which retains the nucleic acid sequence of gE-M and is connected to Pfizer/BioNTech's BNT162b2 UTRs, induced the strongest humoral immune response and CMI. Because CMI is crucial for protection against VZV and for the design of VZV vaccines, this study provides a feasible strategy for improving the effectiveness and economy of future VZV vaccines.

Comparison of the Immune Effects of an mRNA Vaccine and a Subunit Vaccine against Herpes Zoster Administered by Different Injection Methods.

Previous studies have shown that the herpes zoster subunit vaccine Shingrix™ performs well in clinical trials. However, the key ingredient in its adjuvant, QS21, is extracted from rare plants in South America, so vaccine production is limited. Compared with subunit vaccines, mRNA vaccines have the advantages of faster production and not requiring adjuvants, but currently, there is no authorized mRNA vaccine for herpes zoster. Therefore, this study focused on herpes zoster subunit and mRNA vaccines. We prepared a herpes zoster mRNA vaccine and compared the effects of vaccine type, immunization route, and adjuvant use on vaccine immunological efficacy. The mRNA vaccine was injected directly into mice via subcutaneous or intramuscular injection. The subunit vaccine was mixed with adjuvants before immunization. The adjuvants include B2Q or alum. B2Q is BW006S + 2395S + QS21. BW006S and 2395S are phosphodiester CpG oligodeoxynucleotides (CpG ODNs). Then, we compared the cell-mediated immunity (CIM) and humoral immunity levels of the different groups of mice. The results showed that the immune responses of mice inoculated with the mRNA vaccine prepared in this study were not significantly different from those of mice inoculated with the protein subunit vaccine supplemented with the B2Q. The mRNA vaccine-induced immune responses following subcutaneous or intramuscular injection, and the different immunization routes did not lead to significant differences in immune response intensity. Similar results were also observed for the protein subunit vaccine adjuvanted with B2Q but not alum. The above results suggest that our experiment can provide a reference for the preparation of mRNA vaccines against herpes zoster and has certain reference significance for the selection of the immunization route; that is, there is no significant difference in the immune response caused by subcutaneous versus an intramuscular injection, so the injection route can be determined according to the actual situation of individuals.

Ionizable Lipid Nanoparticles Enhanced the Synergistic Adjuvant Effect of CpG ODNs and QS21 in a Varicella Zoster Virus Glycoprotein E Subunit Vaccine.

Varicella zoster virus (VZV) causes two diseases: varicella upon primary infection and herpes zoster when latent viruses in the sensory ganglia reactivate. While varicella vaccines depend on humoral immunity to prevent VZV infection, cell-mediated immunity (CMI), which plays a therapeutic role in the control or elimination of reactivated VZV in infected cells, is decisive for zoster vaccine efficacy. As one of the most abundant glycoproteins of VZV, conserved glycoprotein E (gE) is essential for viral replication and transmission between ganglion cells, thus making it an ideal target subunit vaccine antigen; gE has been successfully used in the herpes zoster vaccine ShingrixTM on the market. In this report, we found that ionizable lipid nanoparticles (LNPs) approved by the Food and Drug Administration (FDA) as vectors for coronavirus disease 2019 (COVID-19) mRNA vaccines could enhance the synergistic adjuvant effect of CpG oligodeoxynucleotides (CpG ODNs) and QS21 on VZV-gE, affecting both humoral immunity and CMI. Vaccines made with these LNPs showed promise as varicella vaccines without a potential risk of herpes zoster, which identifies them as a novel type of herpes zoster vaccine similar to ShingrixTM. All of the components in this LNP-CpG-QS21 adjuvant system were proven to be safe after mass vaccination, and the high proportion of cholesterol contained in the LNPs was helpful for limiting the cytotoxicity induced by QS21, which may lead to the development of a novel herpes zoster subunit vaccine for clinical application.

LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with Shingrix™ in VZV-primed mice.

Latent varicella-zoster virus (VZV) may be reactivated to cause herpes zoster, which affects one in three people during their lifetime. The currently available subunit vaccine Shingrix™ is superior to the attenuated vaccine Zostavax® in terms of both safety and efficacy, but the supply of its key adjuvant component QS21 is limited. With ionizable lipid nanoparticles (LNPs) that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers, and oligodeoxynucleotides containing CpG motifs (CpG ODNs) approved by the FDA for a subunit hepatitis B vaccine as immunostimulators, we developed a LNP vaccine encapsulating VZV-glycoprotein E (gE) and CpG ODN, and compared its immunogenicity with Shingrix™ in C57BL/6J mice. The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix™ as determined by enzyme-linked immunosorbent assay (ELISA). Most importantly, the LNP vaccine induced comparable levels of cell-mediated immunity (CMI) that plays decisive roles in the efficacy of zoster vaccines to Shingrix™ in a VZV-primed mouse model that was adopted for preclinical studies of Shingrix™. Number of IL-2 and IFN-γ secreting splenocytes and proportion of T helper 1 (Th1) cytokine-expressing CD4+ T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix™ boosted mice. All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities, indicating the potential of LNP-CpG-adjuvanted VZV-gE as a more cost-effective zoster vaccine.

Th2-Oriented Immune Serum After SARS-CoV-2 Vaccination Does Not Enhance Infection In Vitro.

The relatively lower protection rate of the alum-adjuvanted inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines reminds us of the antibody-dependent enhancement (ADE) phenomenon observed in preclinical studies during the development of vaccines for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). In this study, using the S1 segment of the SARS-CoV-2 spike protein or inactivated whole SARS-CoV-2 virus as an antigen and aluminum as an adjuvant, the risk of ADE of infection with T helper 2 (Th2)-oriented immune serum from mice (N=6) and humans (N=5) was examined in immune cell lines, which show different expression patterns of Fc receptors. Neither the immune serum from alum-adjuvanted S1 subunit vaccines nor inactivated SARS-CoV-2 vaccination enhanced SARS-CoV-2 S pseudotyped virus infection in any of the tested cell lines in vitro. Because both of these Th2-oriented immune sera could block SARS-CoV-2 infection without ADE of infection, we speculate that the lower protection rate of the inactivated SARS-CoV-2 vaccine may be attributed to the lower neutralizing antibody titers induced or the pulmonary eosinophilic immunopathology accompanied by eosinophilic infiltration in the lungs upon virus exposure. Adjustment of the immunization schedule to elevate the neutralizing antibody levels and skew adjuvants toward Th1-oriented responses may be considered to increase the efficacies of both inactivated and spike protein-based subunit SARS-CoV-2 vaccines.

SQSTM1/p62 regulate breast cancer progression and metastasis by inducing cell cycle arrest and regulating immune cell infiltration.

The autophagy adaptor protein SQSTM1/p62 is overexpressed in breast cancer and has been identified as a metastasis-related protein. However, the mechanism by which SQSTM1/p62 contributes to breast cancer progression and tumor microenvironment remains unclear. This study revealed that silencing SQSTM1/p62 expression suppressed breast cancer progression via regulating cell proliferation and reshaping the tumor microenvironment (TME). Here, we found that SQSTM1/p62 was overexpressed in multiple human cancer tissue types and that was correlated with poor patient overall survival (OS) and disease-free survival (DFS). Moreover, we found that short-hairpin RNA (shRNA)-mediated knockdown of p62 expression significantly inhibited cell proliferation, migration, and invasion, and promoted cell death in vitro, as well as suppressed breast cancer growth and lung metastasis in vivo. In addition, flow cytometry analysis of splenocytes and tumor infiltrating lymphocytes (TILs) indicated that the numbers of CD8α+ interferon (IFN)-γ+ cells (CTLs) and CD4+IFN-γ+ (Th1) cells were increased while those of CD4+IL-4+ (Th2) cells, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) were decreased. RT-PCR analyses showed that the gene expression of Th1/Th2 cytokines changed in the tumor microenvironment. Silencing SQSTM1/p62 suppressed tumor cell lung metastasis. Together, our results provide strong evidence that silencing tumor cell SQSTM1/p62 inhibited tumor growth and metastasis through cell cycle arrest and TME regulation. This finding provides a novel molecular therapeutic strategy for breast cancer progression and metastasis treatment.

Antioxidant peptidomics reveals novel skin antioxidant system.

It is generally agreed that reactive oxygen species (ROS) contribute to skin aging, skin disorders, and skin diseases. Skin possesses an extremely efficient antioxidant system. This antioxidant activity is conferred by two systems: antioxidant enzymes and small molecules that can scavenge ROS by donating electrons. No gene-encoded secreted ROS scavengers have been reported. Amphibian skin is a multifunctional organ acting in defense, respiration, and water regulation, although it seems susceptible. Amphibian skins are easily harmed by biological or non-biological attacks such as microorganism infection or radiation injury. Among vertebrates, skins of amphibian are exposed to more dangers of radiation injury than others. Radiation toxicity occurs by directly attacking the genetic material and/or by generating ROS. In addition, amphibian skin respiration and inflammatory response also induce ROS generation. It is rational to hypothesize that amphibian skins should have potent free radical scavenging and radioprotective ability for their survival. Rana pleuraden is distributed in Southwest of China; it lives in the subtropical plateau (altitude around 2300 m) where there is strong ultraviolet radiation and long duration of sunshine. By peptidomics and genomics approaches, a large amount of antioxidant peptides belonging to 11 different groups with variable structures were isolated from the skin secretions of R. pleuraden. Their free radical scavenging and anti-inflammatory abilities were studied. All of these peptide share highly homologous preproregions, although mature antioxidant peptides have very divergent primary structures, suggesting the possibility of a common ancestor. Some peptides were also found to have multifunctional properties, such as combined antioxidant, anti-inflammatory, and antimicrobial activities. According to our knowledge, no gene-encoded specific antioxidant peptides have been reported except metallothionein. Our work possibly reveals a new skin antioxidant system. The current work also provides a large amount of peptide candidates with medical-pharmaceutical significance.

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog, Rana nigrovittata.

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents.

Immunogenicity of Varicella-Zoster Virus Glycoprotein E Formulated with Lipid Nanoparticles and Nucleic Immunostimulators in Mice.

Theoretically, the subunit herpes zoster vaccine ShingrixTM could be used as a varicella vaccine that avoids the risk of developing shingles from vaccination, but bedside mixing strategies and the limited supply of the adjuvant component QS21 have made its application economically impracticable. With lipid nanoparticles (LNPs) that were approved by the FDA as vectors for severe acute respiratory syndrome coronavirus 2 vaccines, we designed a series of vaccines efficiently encapsulated with varicella-zoster virus glycoprotein E (VZV-gE) and nucleic acids including polyinosinic-polycytidylic acid (Poly I:C) and the natural phosphodiester CpG oligodeoxynucleotide (CpG ODN), which was approved by the FDA as an immunostimulator in a hepatitis B vaccine. Preclinical trial in mice showed that these LNP vaccines could induce VZV-gE IgG titers more than 16 times those induced by an alum adjuvant, and immunized serum could block in vitro infection completely at a dilution of 1:80, which indicated potential as a varicella vaccine. The magnitude of the cell-mediated immunity induced was generally more than 10 times that induced by the alum adjuvant, indicating potential as a zoster vaccine. These results showed that immunostimulatory nucleic acids together with LNPs have promise as safe and economical varicella and zoster vaccine candidates.

Immune Responses to Varicella-Zoster Virus Glycoprotein E Formulated with Poly(Lactic-co-Glycolic Acid) Nanoparticles and Nucleic Acid Adjuvants in Mice.

The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy, yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunological adjuvant QS-21 still need to be improved. Based on poly(lactic-co-glycolic acid) (PLGA) delivery systems that are stable during the lyophilization and rehydration process and using a double-emulsion (w/o/w) solvent evaporation method, we designed a series of nanoparticles with varicella-zoster virus antigen glycoprotein E (VZV-gE) as an antigen and nucleic acids including polyinosinic-polycytidylic acid (Poly I:C) and phosphodiester CpG oligodeoxynucleotide (CpG ODN), encapsulated as immune stimulators. While cationic lipids (DOTAP) have more potential than neutral lipids (DOPC) for activating gE-specific cell-mediated immunity (CMI) in immunized mice, especially when gE is encapsulated in and presented on the surface of nanoparticles, PLGA particles without lipids have the greatest potential to induce not only the highest gE-specific IgG titers but also the strongest gE-specific CMI responses, including the highest proportions of interferon-γ (IFN-γ)- and interleukin-2 (IL-2)-producing CD4+/CD8+ T cells according to a flow cytometry assay and the greatest numbers of IFN-γ- and IL-2-producing splenocytes according to an enzyme-linked immunospot (ELISPOT) assay. These results showed that immune-stimulating nucleic acids together with the PLGA delivery system showed promise as a safe and economical varicella and zoster vaccine candidate.

Effects of Varicella-Zoster Virus Glycoprotein E Carboxyl-Terminal Mutation on mRNA Vaccine Efficacy.

Glycoprotein E (gE) is one of the most abundant glycoproteins in varicella-zoster virus and plays pivotal roles in virus replication and transmission between ganglia cells. Its extracellular domain has been successfully used as an antigen in subunit zoster vaccines. The intracellular C-terminal domain was reported to be decisive for gE trafficking between the endoplasmic reticulum, trans-Golgi network and endosomes and could influence virus spread and virus titers. Considering that the trafficking and distribution of mRNA vaccine-translated gE may be different from those of gE translated against the background of the viral genome (e.g., most gE in virus-infected cells exists as heterodimers with another glycoprotein, gI,), which may influence the immunogenicity of gE-based mRNA vaccines, we compared the humoral and cellular immunity induced by LNP-encapsulated mRNA sequences encoding the whole length of gE, the extracellular domain of gE and a C-terminal double mutant of gE (mutant Y569A with original motif AYRV, which targets gE to TGN, and mutants S593A, S595A, T596A and T598A with the original motif SSTT) that were reported to enhance virus spread and elevate virus titers. The results showed that while the humoral and cellular immunity induced by all of the mRNA vaccines was comparable to or better than that induced by the AS01B-adjuvanted subunit vaccines, the C-terminal double mutant of gE showed stable advantages in all of the indicators tested, including gE-specific IgG titers and T cell responses, and could be adopted as a candidate for both safer varicella vaccines and effective zoster vaccines.

An Established Th2-Oriented Response to an Alum-Adjuvanted SARS-CoV-2 Subunit Vaccine Is Not Reversible by Sequential Immunization with Nucleic Acid-Adjuvanted Th1-Oriented Subunit Vaccines.

A recently reported parallel preclinical study between a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine and an inactivated SARS-CoV-2 vaccine adjuvanted with alum showed pulmonary immunopathology typical of eosinophil accumulation in a mouse pneumonia model for the latter, which implied a potential role of cellular immunity in the difference in the protection rate between these two forms of vaccines. For those who have been vaccinated with alum-adjuvanted subunit or inactivated SARS-CoV-2 vaccines, whether the Th2 responses that have been established and the absence of induced cellular immunity could be changed is an open question. Using two heterologous boosts with Th1-oriented CpG ODN-adjuvanted S1-based SARS-CoV-2 subunit vaccines for mice that were primed with two doses of Th2-oriented alum-adjuvanted S1-based SARS-CoV-2 subunit vaccines, we demonstrated that established Th2 orientation could not be reversed to Th1 orientation and that no cellular immunity was induced, which should have been induced if the boosting vaccines were used as the prime vaccines. These results remind us that if widely administered alum-adjuvanted SARS-CoV-2 vaccines cannot overcome the challenge of coronavirus disease 2019 (COVID-19) and that if cellular immunity is important for the efficacy of SARS-CoV-2 vaccines in the future, the choice of more powerful heterologous boosting vaccine forms that can induce cellular immunity should be considered very carefully before application.

Effects of rotavirus NSP4 protein on the immune response and protection of the SR69A-VP8* nanoparticle rotavirus vaccine.

Rotavirus causes severe diarrhea and dehydration in young children. Even with the implementation of the current live vaccines, rotavirus infections still cause significant mortality and morbidity, indicating a need for new rotavirus vaccines with improved efficacy. To this end, we have developed an SR69A-VP8*/S60-VP8* nanoparticle rotavirus vaccine candidate that will be delivered parenterally with Alum adjuvant. In this study, as parts of our further development of this nanoparticle vaccine, we evaluated 1) roles of rotavirus nonstructural protein 4 (NSP4) that is the rotavirus enterotoxin, a possible vaccine target, and an immune stimulator, and 2) effects of CpG adjuvant that is a toll-like receptor 9 (TLR9) ligand and agonist on the immune response and protection of our SR69A-VP8*/S60-VP8* nanoparticle vaccine. The resulted vaccine candidates were examined for their IgG responses in mice. In addition, the resulted mouse sera were assessed for i) blocking titers against interactions of rotavirus VP8* proteins with their glycan ligands, ii) neutralization titers against rotavirus replication in cell culture, and iii) passive protection against rotavirus challenge with diarrhea in suckling mice. Our data showed that the Alum adjuvant appeared to work better with the SR69A-VP8*/S60-VP8* nanoparticles than the CpG adjuvant, while an addition of the NSP4 antigen to the SR69A-VP8*/S60-VP8* vaccine may not help to further increase the immune response and protection of the resulted vaccine.