In the present study, by building on the previous development of a DC-SIGN-targeting virus-like structure (VLS) vaccine platform and a comprehensive characterization of SARS-CoV-2 structural biology, particularly insights into the role of the nucleocapsid (N) protein in eliciting cytotoxic T lymphocyte (CTL) responses during infection, we designed a SARS-CoV-2 virion-mimetic structural vaccine that encapsulates an mRNA encoding the spike S1 antigen complexed with N protein complexes, with S1 proteins loaded on its surface. This characterized virion-mimetic structural vaccine not only induces the production of high-efficiency antibodies against both the spike and N proteins but also elicits robust S1-specific and N-specific CTL responses in animal models. Furthermore, the generated antibodies exhibit cross-reactive neutralizing activity against multiple SARS-CoV-2 variants and provide protective immunity against challenge with mutant viruses in immunized hosts. This SARS-CoV-2 virion-mimetic structure effectively recapitulates natural infection pathways, comprehensively activating the innate immune system and thereby creating an optimal microenvironment for eliciting potent and broad-spectrum adaptive immune responses.

Poliomyelitis and hand, foot, and mouth disease (HFMD) are important significant pediatric infectious diseases that can be prevented by the inactivated poliovirus vaccine (IPV) and enterovirus 71 (EV71) vaccines, respectively. This study investigated immune responses to sequential or co-administration of Sabin strain-based IPV (sIPV) with EV71 vaccines in mice. Sequential immunization induced an anti-EV71 neutralizing antibody GMT of 128, which was nominally significantly higher than that observed after combined immunization (GMT 91). Combined immunization elicited anti-poliovirus type I and type II GMTs of 119 and 152, respectively, which were nominally significantly higher than those induced by sequential immunization (type I: GMT 78; type II: GMT 111). Co-immunization showed potential additive effects, enhancing neutralizing antibody titers against type I and II polioviruses and promoting concurrent Th1 and Th2 activation. These findings support the potential of combined vaccination strategies to improve protection against poliomyelitis and EV71-associated disease.

Rabies virus (RABV) is a zoonotic pathogen with an almost 100% case-fatality rate and limited post-exposure prophylaxis options. mRNA-based antigen delivery represents a promising approach but requires further optimization. We prepared a cationic lipid nanoparticle (LNP) delivery system using the thin-film hydration method and characterized its physicochemical properties and delivery efficiency. An mRNA encoding a fusion antigen comprising rabies virus glycoprotein (G) and herpes simplex virus type 2 infected cell polypeptide 35 (ICP35), lacking canonical 5' and 3' untranslated region sequences, was constructed. In vitro protein expression of the G-ICP35 mRNA was confirmed. Immunogenicity was evaluated in BALB/c mice following three intramuscular immunizations with G-ICP35 mRNA, RABV G/N proteins, or their combination. Tail vein blood samples were collected on days 7, 21, and 39 after the final immunization, and rabies virus-specific antibody titers and T-cell responses were assessed. The cationic LNP formulation efficiently mediated intracellular delivery and expression of the G-ICP35 mRNA. In vivo, the mRNA-LNP formulation induced detectable humoral and cellular immune responses, including rabies virus-specific IgG production and antigen-specific T-cell activation. Notably, co-immunization with mRNA and subunit vaccines further enhanced immune responses compared with either vaccine alone, achieving peak rabies virus-specific IgG titers of 694.5 IU/mL. These findings demonstrate the feasibility of a DCChol-based mRNA-LNP formulation for fusion antigen delivery and support further investigation of combined mRNA and subunit vaccine strategies.

Human cytomegalovirus (HCMV) is an important human pathogen and paradigm for subversion of host intrinsic, innate, and adaptive immunity. Necroptosis is a critical intrinsic antiviral defence that directly eliminates infected cells, preventing viral spread. However, a systematic analysis to comprehensively identify HCMV necroptosis antagonists is lacking. Here, we screened HCMV block deletion viruses lacking clusters of ‘accessory’ genes non-essential for viral replication, cell lines stably expressing individual genes in the candidate block, and single-gene deletion viral mutants. This identified pUS22 as a novel necroptosis inhibitor and the first viral protein to repress transcription of the key necrosome component RIPK3. pUS22 interacts with and inhibits transcription factors Sp1 and Sp3, which would otherwise activate RIPK3 transcription by binding a core proximal promoter element. As a consequence of reduced RIPK3 protein abundance, phosphorylation of terminal necroptosis mediator MLKL is inhibited, limiting cell death and enhancing viral replication and spread.

The adaptive immune protection elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has been proven to control the severity of novel coronavirus disease 2019 (COVID-19). However, the contributions of innate lymphoid cells formed from immunization are poorly defined in vaccine evaluation. Here, we highlight how the natural killer (NK) and macrophage (Mϕ) cells’ response, primed by the inactivated COVID-19 vaccine, is crucial to preventing lung injury. We propose that a specific subset of NK cells, marked CD56dimCD16+NKG2C+, along with M2-like Mϕ, CD8+ T cells, are important in defending against SARS-CoV-2. Our studies using a rhesus macaque model showed that this orchestration of protection was depicted as a trajectory of adaptive NK and Mϕ cell responses from circulating peripheral blood mononuclear cells to the lungs. Through single-cell RNA sequencing and mass cytometry (cytometry by time-of-flight) analysis, we also identified the significance of adaptive CD56dimCD16+CD57+NKG2C+ NK cells and classical monocytes with chemotaxis traits in orchestrating T cell immunity in humans. Interestingly, our findings show a deficiency of these adaptive cells in older participants post-booster vaccination, leading to potentially inadequate protection. This study discusses the evaluation of vaccines at the innate immune level, which can contribute to the development of successful vaccines.

Herpes simplex virus type 1 (HSV-1) is a very concerning pathogen due to its ability to persist in the host’s nervous system and continuously interfere with the immune system, which complicates treatment. Therefore, the development of an effective HSV-1 vaccine is crucial. In this study, we focused on an HSV-1 mutant strain, M6, which includes several deleted genes associated with viral infection virulence and latent infection function, and explored its infection of macrophages and immunological characteristics. The study found that both the attenuated strain M6 and the wild-type strain infect macrophages through the binding of the gD protein to the HVEM receptor on the macrophage surface. Compared to the wild-type strain, the attenuated M6 strain induced a milder immune response, characterized by the lower expression of immune signaling molecules and inflammatory cytokine levels. Upon reintroducing macrophages infected with the two strains into mice, the M6 strain induced lower levels of inflammatory cytokines and higher levels of chemokines in spleen cells and also slightly lower humoral and cellular immune responses than the wild-type strain. Further histopathological analysis revealed that mice in the attenuated M6 group showed more stable body weight changes and milder pathological damage in immune organs such as the liver, spleen, and lymph nodes. In conclusion, the attenuated M6 strain exhibits good immunogenicity and mild pathological side effects, suggesting its potential as an effective immunogen.

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD50 of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL−1 nAb by day 7; two doses peaked at approximately 1000 IU mL−1. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL−1 within 1 week), and sustains potent CD8+ CTL and CD4+ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate.

Based on a dual-cationic lipid nanoparticle (LNP) platform, we developed a novel rabies virus (RABV) nucleocapsid-like nanostructure (NLS) vaccine. The antigen comprises two nucleocapsid components: a mRNA encoding the RABV glycoprotein (G-mRNA) and a recombinant RABV nucleoprotein (RABV-N) expressed in Escherichia coli (E. coli). These components are co-encapsulated within dual-cationic LNPs to form the RABV NLS vaccine. Compared with the G-mRNA vaccine and inactivated vaccine (INV), the NLS vaccine elicits significantly stronger humoral and cellular immune responses. Seven days after a single dose, the NLS vaccine elicited neutralizing antibody titers of ~10 IU mL-1. Following two doses on days 0 and 21, the peak neutralizing antibody titer reached 1,000 IU mL-1, and 100% of mice survived a challenge with 25 LD₅₀ of the fixed RABV strain on day 14 post-boost.

The constant mutation of SARS-CoV-2 has led to the continuous appearance of viral variants and their pandemics and has improved the development of vaccines with a broad spectrum of antigens to curb the spread of the virus. The work described here suggested a novel vaccine with a virus-like structure (VLS) composed of combined mRNA and protein that is capable of stimulating the immune system in a manner similar to that of viral infection. This VLS vaccine is characterized by its ability to specifically target dendritic cells and/or macrophages through S1 protein recognition of the DC-SIGN receptor in cells, which leads to direct mRNA delivery to these innate immune cells for activation of robust immunity with a broad spectrum of neutralizing antibodies and immune protective capacity against variants. Research on its composition characteristics and structural features has suggested its druggability. Compared with the current mRNA vaccine, the VLS vaccine was identified as having no cytotoxicity at its effective application dosage, while the results of safety observations in animals revealed fewer adverse reactions during immunization.

Recent studies have indicated that sequentially administering SARS-CoV-2 vaccines can result in increased antibody and cellular immune responses. In this study, we compared homologous and heterologous immunization strategies following two doses of inactivated vaccines in a mouse model. Our research demonstrates that heterologous sequential immunization resulted in more immune responses displayed in the lymph node germinal center, which induced a greater number of antibody-secreting cells (ASCs), resulting in enhanced humoral and cellular immune responses and increased cross-protection against five variant strains. In further single B-cell analysis, the above findings were supported by the presence of unique B-cell receptor (BCR) repertoires and diversity in CDR3 sequence profiles elicited by a heterologous booster immunization strategy.

Trivalent oral poliovirus vaccine (tOPV) has been withdrawn and instead an inactivated poliovirus vaccine (IPV) and bivalent type 1 and type 3 OPV (bOPV) sequential immunization schedule has been implemented since 2016, but no immune persistence data are available for this polio vaccination strategy. This study aimed to assess immune persistence following different polio sequential immunization schedules. Venous blood was collected at 24, 36, and 48 months of age from participants who had completed sequential schedules of combined IPV and OPV in phase III clinical trials. The serum neutralizing antibody titers against poliovirus were determined, and the poliovirus-specific antibody-positive rates were evaluated. A total of 1104 participants were enrolled in this study. The positive rates of poliovirus type 1- and type 3-specific antibodies among the sequential immunization groups showed no significant difference at 24, 36, or 48 months of age. The positive rates of poliovirus type 2-specific antibody in the IPV-IPV-tOPV group at all time points were nearly 100%, which was significantly higher than the corresponding rates in other immunization groups (IPV-bOPV-bOPV and IPV-IPV-bOPV). Immunization schedules involving one or two doses of IPV followed by bOPV failed to maintain a high positive rate for poliovirus type 2-specific antibody.

The circulating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variant presents an ongoing challenge for surveillance and detection. It is important to establish an assay for SARS-CoV-2 antibodies in vaccinated individuals. Numerous studies have demonstrated that binding antibodies (such as S-IgG and N-IgG) and neutralizing antibodies (Nabs) can be detected in vaccinated individuals. However, it is still unclear how to evaluate the consistency and correlation between binding antibodies and Nabs induced by inactivated SARS-CoV-2 vaccines. In this study, serum samples from humans, rhesus macaques, and hamsters immunized with inactivated SARS-CoV-2 vaccines were analyzed for S-IgG, N-IgG, and Nabs. The results showed that the titer and seroconversion rate of S-IgG were significantly higher than those of N-IgG. The correlation between S-IgG and Nabs was higher compared to that of N-IgG. Based on this analysis, we further investigated the titer thresholds of S-IgG and N-IgG in predicting the seroconversion of Nabs. According to the threshold, we can quickly determine the positive and negative effects of the SARS-CoV-2 variant neutralizing antibody in individuals. These findings suggest that the S-IgG antibody is a better supplement to and confirmation of SARS-CoV-2 vaccine immunization.

Hand, foot, and mouth disease (HFMD) is caused by the enterovirus family, which includes EV-A71 and more than 10 other members. The disease involves a complex pathological mechanism that includes intricate and finely-tuned interactions between these pathogens and the host. The research on vaccines that can effectively be used to prevent HFMD caused by major pathogens suggested that the viruses presenting with the same structure but different antigenic traits in response to the immune system interactions enable to interact dynamically to cell surface receptors and to lead to similar pathological outcome through diverse mechanisms. This suggests that further understanding of the whole process of signal stimulation by viral antigen molecules and innate immune receptor molecules could improve our recognition about the events of pathological injury to the body and the characterization of antiviral immune responses with phenotypic differences during the pathogenesis. The accumulated data about process of interaction between virus structure and host in molecular level might provide the theoretical and technical support for next generation of vaccine against HFMD for public health initiatives.

Improved vaccination requires better delivery of antigens and activation of the natural immune response. Here we report a lipid nanoparticle system with the capacity to carry antigens, including mRNA and proteins, which is formed into a virus-like structure by surface decoration with spike proteins, demonstrating application against SARS-CoV-2 variants. The strategy uses S1 protein from Omicron BA.1 on the surface to deliver mRNA of S1 protein from XBB.1. The virus-like particle enables specific augmentation of mRNAs expressed in human respiratory epithelial cells and macrophages via the interaction the surface S1 protein with ACE2 or DC-SIGN receptors. Activation of macrophages and dendritic cells is demonstrated by the same receptor binding. The combination of protein and mRNA increases the antibody response in BALB/c mice compared with mRNA and protein vaccines alone. Our exploration of the mechanism of this robust immunity suggests it might involve cross-presentation to diverse subsets of dendritic cells ranging from activated innate immune signals to adaptive immune signals. This paper presents a virus-like lipid nanoparticle decorated with spike proteins capable of carrying antigens, including mRNA and proteins, for vaccination against SARS-CoV-2 variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike (S) protein is a critical viral antigenic protein that enables the production of neutralizing antibodies, while other structural proteins, including the membrane (M), nucleocapsid (N) and envelope (E) proteins, have unclear roles in antiviral immunity. In this study, S1, S2, M, N and E proteins were expressed in 16HBE cells to explore the characteristics of the resultant innate immune response. Furthermore, peripheral blood mononuclear cells (PBMCs) from mice immunized with two doses of inactivated SARS-CoV-2 vaccine or two doses of mRNA vaccine were isolated and stimulated by these five proteins to evaluate the corresponding specific T-cell immune response. In addition, the levels of humoral immunity induced by two-dose inactivated vaccine priming followed by mRNA vaccine boosting, two homologous inactivated vaccine doses and two homologous mRNA vaccine doses in immunized mice were compared. Our results suggested that viral structural proteins can activate the innate immune response and elicit a specific T-cell response in mice immunized with the inactivated vaccine. However, the existence of the specific T-cell response against M, N and E is seemingly insufficient to improve the level of humoral immunity.

Background: Herpes simplex virus 1 (HSV-1), an important human pathogen, is capable of latent infection in neurons and productive (lytic) infection in other tissue cells. Once infected with HSV-1, the immune system of the organism cannot eliminate the virus and carries it lifelong. HSV-1 possesses approximately 150 kb of double-stranded linear genomic DNA and can encode at least 70 proteins and 37 mature microRNAs (miRNAs) derived from 18 precursor miRNAs (pre-miRNAs). Summary: These HSV-1-encoded miRNAs are widely involved in multiple processes in the life cycle of the virus and the host cell, including viral latent and lytic infection, as well as host cell immune signaling, proliferation, and apoptosis. Key Message: In this review, we focused primarily on recent advances in HSV-1-encoded miRNA expression, function, and mechanism, which may provide new research ideas and feasible research methods systemically and comprehensively.

(1) Background: As the COVID-19 pandemic enters its fourth year, it continues to cause significant morbidity and mortality worldwide. Although various vaccines have been approved and the use of homologous or heterologous boost doses is widely promoted, the impact of vaccine antigen basis, forms, dosages, and administration routes on the duration and spectrum of vaccine-induced immunity against variants remains incompletely understood. (2) Methods: In this study, we investigated the effects of combining a full-length spike mRNA vaccine with a recombinant S1 protein vaccine, using intradermal/intramuscular, homologous/heterologous, and high/low dosage immunization strategies. (3) Results: Over a period of seven months, vaccination with a mutant recombinant S1 protein vaccine based on the full-length spike mRNA vaccine maintained a broadly stable humoral immunity against the wild-type strain, a partially attenuated but broader-spectrum immunity against variant strains, and a comparable level of cellular immunity across all tested strains. Furthermore, intradermal vaccination enhanced the heterologous boosting of the protein vaccine based on the mRNA vaccine. (4) Conclusions: This study provides valuable insights into optimizing vaccination strategies to address the ongoing challenges posed by emerging SARS-CoV-2 variants.

To provide a basis for further optimization of the polio sequential immunization schedule, this study evaluated the effectiveness of booster immunization with one dose of bivalent oral poliovirus vaccine (bOPV) at 48 months of age after different primary polio immunization schedules. At 48 months of age, one dose of bOPV was administered, and their poliovirus types 1–3 (PV1, PV2, and PV3, respectively)-specific neutralizing antibody levels were determined. Participants found to be negative for any type of PV-specific neutralizing antibody at 24, 36, or 48 months of age were re-vaccinated with inactivated polio vaccine (IPV). The 439 subjects who received a bOPV booster immunization at the age of 48 months had lower PV2-specific antibody levels compared with those who received IPV. One dose of IPV during basic polio immunization induced the lowest PV2-specific antibody levels. On the basis of our findings, to ensure that no less than 70% of the vaccinated have protection efficiency, we recommend the following: if basic immunization was conducted with 1IPV + 2bOPV (especially Sabin strain-based IPV), a booster immunization with IPV is recommended at 36 months of age, whereas if basic immunization was conducted with 2IPV + 1bOPV, a booster immunization with IPV is recommended at 48 months of age. A sequential immunization schedule of 2IPV + 1bOPV + 1IPV can not only maintain high levels of antibody against PV1 and PV3 but also increases immunity to PV2 and induces early intestinal mucosal immunity, with relatively good safety. Thus, this may be the best sequential immunization schedule for polio in countries or regions at high risk for polio.

A new lipid system broadening the capacity of antigen carriage, including mRNA and protein, is established, and an assembled virus-like-structure (VLS) encapsulated mRNA of S1 protein from XBB.1 and loaded S1 protein from omicron BA.1 on the surface is identified. This characterized VLS enables to specifically augment mRNA expression in human respiratory epithelial cells and macrophages via its loaded S1 protein on the surface interacting with ACE2 or DC-SIGN molecules of cells. It also archives effective mRNA expression in mouse dendritic cells (DCs) and macrophages via surface S1 protein binding to DC-SIGN molecules followed by the activation of DCs and macrophages in mice. The intensive antibody response against viral variants in Balb/c mice immunized by VLS was observed to be greater than those immunized by mRNA or protein vaccine alone, while ACE +/+ mice and hamsters immunized with VLS were observed to be more effective for restraining viral replication of the Omicron or Wuhan strain challenge. The mechanism of this robust immunity elicited by VLS was found being involved in the integrated effect of diverse signals from activated innate immunity to the adaptive immune system.