ADAM9 drives the immunosuppressive microenvironment by cholesterol biosynthesis-mediated activation of IL6-STAT3 signaling for lung tumor progression.

Chronic inflammation associated with lung cancers contributes to immunosuppressive tumor microenvironments, reducing CD8+ T-cell function and leading to poor patient outcomes. A disintegrin and metalloprotease domain 9 (ADAM9) promotes cancer progression. Here, we aim to elucidate the role of ADAM9 in the immunosuppressive tumor microenvironment. A bioinformatic analysis of TIMER2.0 was used to investigate the correlation of ADAM9 and to infiltrate immune cells in the human lung cancer database and mouse lung tumor samples. Flow cytometry, immunohistochemistry, and RNA sequencing (RNA-seq) were performed to investigate the ADAM9-mediated immunosuppressive microenvironment. The coculture system of lung cancer cells with immune cells, cytokine array assays, and proteomic approach was used to investigate the mechanism. By analyzing the human LUAD database and the mouse lung cancer models, we showed that ADAM9 was associated with the immunosuppressive microenvironment. Additionally, ADAM9 released IL6 protein from cancer cells to inhibit IL12p40 secretion from dendritic cells, therefore leading to dendritic cell dysfunction and further affecting T-cell functions. Proteomic analysis indicated that ADAM9 promoted cholesterol biosynthesis and increased IL6-STAT3 signaling. Mechanistically, ADAM9 reduced the protein stability of LDLR, resulting in reduced cholesterol uptake and induced cholesterol biosynthesis. Moreover, LDLR reduction enhanced IL6-STAT3 activation. We reveal that ADAM9 has a novel biological function that drives the immunosuppressive tumor microenvironment by linking lung cancer's metabolic and signaling axes. Thus, by targeting ADAM9 an innovative and promising therapeutic opportunity was indicated for regulating the immunosuppression of lung cancer.

SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters.

DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the coronavirus disease 2019 (COVID-19) pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared with TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant T helper 1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and the Omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.

Inhibition of ADAM9 promotes the selective degradation of KRAS and sensitizes pancreatic cancers to chemotherapy.

Kirsten rat sarcoma virus (KRAS) signaling drives pancreatic ductal adenocarcinoma (PDAC) malignancy, which is an unmet clinical need. Here, we identify a disintegrin and metalloproteinase domain (ADAM)9 as a modulator of PDAC progression via stabilization of wild-type and mutant KRAS proteins. Mechanistically, ADAM9 loss increases the interaction of KRAS with plasminogen activator inhibitor 1 (PAI-1), which functions as a selective autophagy receptor in conjunction with light chain 3 (LC3), triggering lysosomal degradation of KRAS. Suppression of ADAM9 by a small-molecule inhibitor restricts disease progression in spontaneous models, and combination with gemcitabine elicits dramatic regression of patient-derived tumors. Our findings provide a promising strategy to target the KRAS signaling cascade and demonstrate a potential modality to enhance sensitivity to chemotherapy in PDAC.

A broad-spectrum pneumococcal vaccine induces mucosal immunity and protects against lethal Streptococcus pneumoniae challenge.

Pneumococcal disease is a major threat to public health globally, impacting individuals across all age groups, particularly infants and elderly individuals. The use of current vaccines has led to unintended consequences, including serotype replacement, leading to a need for a new approach to combat pneumococcal disease. A promising solution is the development of a broad-spectrum pneumococcal vaccine. In this study, we present the development of a broad-spectrum protein-based pneumococcal vaccine that contains three pneumococcal virulence factors: rlipo-PsaA (lipidated form), rPspAΔC (truncated form), and rPspCΔC (truncated form). Intranasal immunization with rlipo-PsaA, rPspAΔC, and rPspCΔC (LAAC) resulted in significantly higher IgG titres than those induced by administration of nonlipidated rPsaA, rPspAΔC, and rPspCΔC (AAC). Furthermore, LAAC immunization induced the production of higher IgA titres in vaginal washes, feces, and sera in mice, indicating that LAAC can induce systemic mucosal immunity. In addition, administration of LAAC also induced Th1/Th17-biased immune responses and promoted opsonic phagocytosis of Streptococcus pneumoniae strains of various serotypes, implying that the immunogenicity of LAAC immunization provides a protective effect against pneumococcal infection. Importantly, challenge data showed that the LAAC-immunized mice had a reduced bacterial load not only for several serotypes of the 13-valent conjugate pneumococcal vaccine (PCV13) but also for selected non-PCV13 serotypes. Consistently, LAAC immunization increased the survival rate of mice after bacterial challenge with both PCV13 and non-PCV13 serotypes. In conclusion, our protein-based pneumococcal vaccine provides protective effects against a broad spectrum of Streptococcus pneumoniae serotypes.

A TLR9 agonist synergistically enhances protective immunity induced by an Alum-adjuvanted H7N9 inactivated whole-virion vaccine.

Antigen sparing is an important strategy for pandemic vaccine development because of the limitation of worldwide vaccine production during disease outbreaks. However, several clinical studies have demonstrated that the current aluminum (Alum)-adjuvanted influenza vaccines fail to sufficiently enhance immune responses to meet licensing criteria. Here, we used pandemic H7N9 as a model virus to demonstrate that a 10-fold lower amount of vaccine antigen combined with Alum and TLR9 agonist can provide stronger protective effects than using Alum as the sole adjuvant. We found that the Alum/CpG 1018 combination adjuvant could induce more robust virus-specific humoral immune responses, including higher total IgG production, hemagglutination-inhibiting antibody activity, and neutralizing antibody titres, than the Alum-adjuvanted formulation. Moreover, this combination adjuvant shifted the immune response toward a Th1-biased immune response. Importantly, the Alum/CpG 1018-formulated vaccine could confer better protective immunity against H7N9 challenge than that adjuvanted with Alum alone. Notably, the addition of CpG 1018 to the Alum-adjuvanted H7N9 whole-virion vaccine exhibited an antigen-sparing effect without compromising vaccine efficacy. These findings have significant implications for improving Alum-adjuvanted influenza vaccines using the approved adjuvant CpG 1018 for pandemic preparedness.

Co-delivery of a trimeric spike DNA and protein vaccine with aluminum hydroxide enhanced Th1-dominant humoral and cellular immunity against SARS-CoV-2.

Protein subunit vaccines have been used as prophylactic vaccines for a long time. The well-established properties of these vaccines make them the first choice for the coronavirus disease 2019 (COVID-19) outbreak. However, it is not easy to develop a protein vaccine that induces cytotoxic T lymphocyte responses and requires a longer time for manufacturing, which limits the usage of this vaccine type. Here, we report the combination of a recombinant spike (S)-trimer protein with a DNA vaccine-encoded S protein as a novel COVID-19 vaccine. The recombinant S protein was formulated with different adjuvants and mixed with the DNA plasmid before injection. We found that the recombinant S protein formulated with the adjuvant aluminum hydroxide and mixed with the DNA plasmid could enhance antigen-specific antibody titers, neutralizing antibody titers. We further evaluated the IgG2a/IgG1 isotype and cytokine profiles of the specific boosted T-cell response, which indicated that the combined vaccine induced a T-helper 1 cell-biased immune response. Immunized hamsters were challenged with severe acute respiratory syndrome coronavirus 2, and the body weight of the hamsters that received the recombinant S protein with aluminum hydroxide and/or the DNA plasmid was not reduced. Alternatively, those that received control or only the DNA plasmid immunization were reduced. Interestingly, after the third day of the viral load in the lungs, the viral challenge could not be detected in hamsters immunized with the recombinant S protein in aluminum hydroxide mixed with DNA (tissue culture infectious dose < 10). The viral load in the lungs was 109 , 106 , and 107 for the phosphate-buffered saline, protein in aluminum hydroxide, and DNA-only immunizations, respectively. These results indicated that antiviral mechanisms neutralizing antibodies play important roles. Furthermore, we found that the combination of protein and DNA vaccination could induce relatively strong CD8+ T-cell responses. In summary, the protein subunit vaccine combined with a DNA vaccine could induce strong CD8+ T-cell responses to increase antiviral immunity for disease control.

Recombinant lipidated FLIPr effectively enhances mucosal and systemic immune responses for various vaccine types.

Formyl peptide receptor-like 1 inhibitor protein (FLIPr) is an immune evasion protein produced by Staphylococcus aureus, and FLIPr is a potential vaccine candidate for reducing Staphylococcus aureus virulence and biofilm formation. We produced recombinant lipidated FLIPr (rLF) to increase the immunogenicity of FLIPr and showed that rLF alone elicited potent anti-FLIPr antibody responses to overcome the FLIPr-mediated inhibition of phagocytosis. In addition, rLF has potent immunostimulatory properties. We demonstrated that rLF is an effective adjuvant. When an antigen is formulated with rLF, it can induce long-lasting antigen-specific immune responses and enhance mucosal and systemic antibody responses as well as broad-spectrum T-cell responses in mice. These findings support further exploration of rLF in the clinic as an adjuvant for various vaccine types with extra benefits to abolish FLIPr-mediated immunosuppressive effects.

TLR9 and STING agonists cooperatively boost the immune response to SARS-CoV-2 RBD vaccine through an increased germinal center B cell response and reshaped T helper responses.

Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.

Humoral and cellular immunity in three different types of COVID-19 vaccines against SARS-CoV-2 variants in a real-world data analysis.

BACKGROUND: An effective vaccine response is currently a critical issue in the control of COVID-19. Little is known about humoral and cellular immunity comparing protein-based vaccine with other types of vaccines. The relevance of basal immunity to antibody production is also unknown. METHODS: Seventy-eight individuals were enrolled in the study. The primary outcome were the level of spike-specific antibodies and neutralizing antibodies measured by ELISA. Secondary measures included memory T cells and basal immunity estimated by flow cytometry and ELISA. Correlations for all parameters were calculated using the nonparametric Spearman correlation method. RESULTS: We observed that two doses of mRNA-based Moderna mRNA-1273 (Moderna) vaccine produced the highest total spike-binding antibody and neutralizing ability against the wild-type (WT), Delta, and Omicron variants. The protein-based MVC-COV1901 (MVC) vaccine developed in Taiwan produced higher spike-binding antibodies against Delta and Omicron variants and neutralizing ability against the WT strain than the adenovirus-based AstraZeneca-Oxford AZD1222 (AZ) vaccine. Moderna and AZ vaccination produced more central memory T cells in PBMC than the MVC vaccine. However, the MVC vaccine had the lowest adverse effects compared to the Moderna and AZ vaccines. Surprisingly, the basal immunity represented by TNF-α, IFN-γ, and IL-2 prior to vaccination was negatively correlated with the production of spike-binding antibodies and neutralizing ability. CONCLUSION: This study compared memory T cells, total spike-binding antibody levels, and neutralizing capacity against WT, Delta, and Omicron variants between the MVC vaccine and the widely used Moderna and AZ vaccines, which provides valuable information for future vaccine development strategies.

Correction: DNA vaccination induced protective immunity against SARS CoV-2 infection in hamsterss.

[This corrects the article DOI: 10.1371/journal.pntd.0009374.].

A flexible liposomal polymer complex as a platform of specific and regulable immune regulation for individual cancer immunotherapy.

BACKGROUND: The applicability and therapeutic efficacy of specific personalized immunotherapy for cancer patients is limited by the genetic diversity of the host or the tumor. Side-effects such as immune-related adverse events (IRAEs) derived from the administration of immunotherapy have also been observed. Therefore, regulatory immunotherapy is required for cancer patients and should be developed. METHODS: The cationic lipo-PEG-PEI complex (LPPC) can stably and irreplaceably adsorb various proteins on its surface without covalent linkage, and the bound proteins maintain their original functions. In this study, LPPC was developed as an immunoregulatory platform for personalized immunotherapy for tumors to address the barriers related to the heterogenetic characteristics of MHC molecules or tumor associated antigens (TAAs) in the patient population. Here, the immune-suppressive and highly metastatic melanoma, B16F10 cells were used to examine the effects of this platform. Adsorption of anti-CD3 antibodies, HLA-A2/peptide, or dendritic cells' membrane proteins (MP) could flexibly provide pan-T-cell responses, specific Th1 responses, or specific Th1 and Th2 responses, depending on the host needs. Furthermore, with regulatory antibodies, the immuno-LPPC complex properly mediated immune responses by adsorbing positive or negative antibodies, such as anti-CD28 or anti-CTLA4 antibodies. RESULTS: The results clearly showed that treatment with LPPC/MP/CD28 complexes activated specific Th1 and Th2 responses, including cytokine release, CTL and prevented T-cell apoptosis. Moreover, LPPC/MP/CD28 complexes could eliminate metastatic B16F10 melanoma cells in the lung more efficiently than LPPC/MP. Interestingly, the melanoma resistance of mice treated with LPPC/MP/CD28 complexes would be reversed to susceptible after administration with LPPC/MP/CTLA4 complexes. NGS data revealed that LPPC/MP/CD28 complexes could enhance the gene expression of cytokine and chemokine pathways to strengthen immune activation than LPPC/MP, and that LPPC/MP/CTLA4 could abolish the LPPC/MP complex-mediated gene expression back to un-treatment. CONCLUSIONS: Overall, we proved a convenient and flexible immunotherapy platform for developing personalized cancer therapy.

Omicron-specific mRNA vaccine induced cross-protective immunity against ancestral SARS-CoV-2 infection with low neutralizing antibodies.

The major challenge in COVID-19 vaccine effectiveness is immune escape by SARS-CoV-2 variants. To overcome this, an Omicron-specific messenger RNA (mRNA) vaccine was designed. The extracellular domain of the spike of the Omicron variant was fused with a modified GCN4 trimerization domain with low immunogenicity (TSomi). After immunization with TSomi mRNA in hamsters, animals were challenged with SARS-CoV-2 virus. The raised nonneutralizing antibodies or cytokine secretion responses can recognize both Wuhan S and Omicron S. However, the raised antibodies neutralized SARS-CoV-2 Omicron virus infection but failed to generate Wuhan virus neutralizing antibodies. Surprisingly, TSomi mRNA immunization protected animals from Wuhan virus challenge. These data indicated that non-neutralizing antibodies or cellular immunity may play a more important role in vaccine-induced protection than previously believed. Next-generation COVID-19 vaccines using the Omicron S antigen may provide sufficient protection against ancestral or current SARS-CoV-2 variants.

Formulation of SARS-CoV-2 Spike Protein with CpG Oligodeoxynucleotides and Squalene Nanoparticles Modulates Immunological Aspects Following Intranasal Delivery.

Nasal spray vaccination is viewed as a promising strategy for inducing both mucosal and systemic protection against respiratory SARS-CoV-2 coronavirus. Toward this goal, a safe and efficacious mucosal adjuvant is necessary for the transportation of the antigen across the mucosal membrane and antigen recognition by the mucosal immune system to generate broad-spectrum immune responses. This study describes the immunological aspects of SARS-CoV-2 spike (S)-protein after being formulated with CpG oligodeoxynucleotides (ODNs) and squalene nanoparticles (termed PELC). Following intranasal delivery in mice, higher expression levels of major histocompatibility complex (MHC) class II and costimulatory molecules CD40 and CD86 on CD11c+ cells were observed at the draining superficial cervical lymph nodes in the CpG-formulated S protein group compared with those vaccinated with S protein alone. Subsequently, the activated antigen-presenting cells downstream modulated the cytokine secretion profiles and expanded the cytotoxic T lymphocyte activity of S protein-restimulated splenocytes. Interestingly, the presence of PELC synergistically enhanced cell-mediated immunity and diminished individual differences in S protein-specific immunogenicity. Regarding humoral responses, the mice vaccinated with the PELC:CpG-formulated S protein promoted the production of S protein-specific IgG in serum samples and IgA in nasal and bronchoalveolar lavage fluids. These results indicate that PELC:CpG is a potential mucosal adjuvant that promotes mucosal/systemic immune responses and cell-mediated immunity, a feature that has implications for the development of a nasal spray vaccine against COVID-19.

Measurements of Anti-SARS-CoV-2 Antibody Levels after Vaccination Using a SH-SAW Biosensor.

To prevent the COVID-19 pandemic that threatens human health, vaccination has become a useful and necessary tool in the response to the pandemic. The vaccine not only induces antibodies in the body, but may also cause adverse effects such as fatigue, muscle pain, blood clots, and myocarditis, especially in patients with chronic disease. To reduce unnecessary vaccinations, it is becoming increasingly important to monitor the amount of anti-SARS-CoV-2 S protein antibodies prior to vaccination. A novel SH-SAW biosensor, coated with SARS-CoV-2 spike protein, can help quantify the amount of anti-SARS-CoV-2 S protein antibodies with 5 µL of finger blood within 40 s. The LoD of the spike-protein-coated SAW biosensor was determined to be 41.91 BAU/mL, and the cut-off point was determined to be 50 BAU/mL (Youden's J statistic = 0.94733). By using the SH-SAW biosensor, we found that the total anti-SARS-CoV-2 S protein antibody concentrations spiked 10−14 days after the first vaccination (p = 0.0002) and 7−9 days after the second vaccination (p = 0.0116). Furthermore, mRNA vaccines, such as Moderna or BNT, could achieve higher concentrations of total anti-SARS-CoV-2 S protein antibodies compared with adenovirus vaccine, AZ (p < 0.0001). SH-SAW sensors in vitro diagnostic systems are a simple and powerful technology to investigate the local prevalence of COVID-19.

Induction of high affinity monoclonal antibodies against SARS-CoV-2 variant infection using a DNA prime-protein boost strategy.

BACKGROUND: Calls for the coronavirus to be treated as an endemic illness, such as the flu, are increasing. After achieving high coverage of COVID-19 vaccination, therapeutic drugs have become important for future SARS-CoV-2 variant outbreaks. Although many monoclonal antibodies have been approved for emergency use as treatments for SARS-CoV-2 infection, some monoclonal antibodies are not authorized for variant treatment. Broad-spectrum monoclonal antibodies are unmet medical needs. METHODS: We used a DNA prime-protein boost approach to generate high-quality monoclonal antibodies. A standard ELISA was employed for the primary screen, and spike protein-human angiotensin-converting enzyme 2 blocking assays were used for the secondary screen. The top 5 blocking clones were selected for further characterization, including binding ability, neutralization potency, and epitope mapping. The therapeutic effects of the best monoclonal antibody against SARS-CoV-2 infection were evaluated in a hamster infection model. RESULTS: Several monoclonal antibodies were selected that neutralize different SARS-CoV-2 variants of concern (VOCs). These VOCs include Alpha, Beta, Gamma, Delta, Kappa and Lambda variants. The high neutralizing antibody titers against the Beta variant would be important to treat Beta-like variants. Among these monoclonal antibodies, mAb-S5 displays the best potency in terms of binding affinity and neutralizing capacity. Importantly, mAb-S5 protects animals from SARS-CoV-2 challenge, including the Wuhan strain, D614G, Alpha and Delta variants, although mAb-S5 exhibits decreased neutralization potency against the Delta variant. Furthermore, the identified neutralizing epitopes of monoclonal antibodies are all located in the receptor-binding domain (RBD) of the spike protein but in different regions. CONCLUSIONS: Our approach generates high-potency monoclonal antibodies against a broad spectrum of VOCs. Multiple monoclonal antibody combinations may be the best strategy to treat future SARS-CoV-2 variant outbreaks.

A DNA vaccine candidate delivered by an electroacupuncture machine provides protective immunity against SARS-CoV-2 infection.

A major challenge in the use of DNA vaccines is efficient DNA delivery in vivo. Establishing a safe and efficient electric transfer method is the key to developing rapid DNA vaccines against emerging infectious diseases. To overcome the complexity of designing new electric transfer machines for DNA delivery, a clinically approved electric transfer machine could be considered as an alternative. Here, we report an electroacupuncture machine-based method for DNA vaccine delivery after intramuscular injection of the COVID-19 DNA vaccine. The S gene of SARS-CoV-2 in the pVAX1 plasmid (pSARS2-S) was used as an antigen in this study. We optimized the clinically used electroacupuncture machine settings for efficient induction of the neutralizing antibody titer after intramuscular injection of pSARS2-S in mice. We found that pSARS2-S immunization at 40 Vpp for 3-5 s could induce high neutralizing antibody titers and Th1-biased immune responses. IFN-γ/TNF-α-secreting CD4+ and CD8+ T cells were also observed in the DNA vaccination group but not in the recombinant protein vaccination group. T-cell epitope mapping shows that the major reactive epitopes were located in the N-terminal domain (a.a. 261-285) and receptor-binding domain (a.a. 352-363). Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. In the preclinical toxicology study, blood biochemistry, hematology, and DNA persistence analysis reveal that the DNA delivery method is safe. Furthermore, the raised antisera could also cross-neutralize different variants of concern. These findings suggest that DNA vaccination using an electroacupuncture machine is feasible for use in humans in the future.

Low-Dose SARS-CoV-2 S-Trimer with an Emulsion Adjuvant Induced Th1-Biased Protective Immunity.

During the sustained COVID-19 pandemic, global mass vaccination to achieve herd immunity can prevent further viral spread and mutation. A protein subunit vaccine that is safe, effective, stable, has few storage restrictions, and involves a liable manufacturing process would be advantageous to distribute around the world. Here, we designed and produced a recombinant spike (S)-Trimer that is maintained in a prefusion state and exhibits a high ACE2 binding affinity. Rodents received different doses of S-Trimer (0.5, 5, or 20 µg) antigen formulated with aluminum hydroxide (Alum) or an emulsion-type adjuvant (SWE), or no adjuvant. After two vaccinations, the antibody response, T-cell responses, and number of follicular helper T-cells (Tfh) or germinal center (GC) B cells were assessed in mice; the protective efficacy was evaluated on a Syrian hamster infection model. The mouse studies demonstrated that adjuvating the S-Trimer with SWE induced a potent humoral immune response and Th1-biased cellular immune responses (in low dose) that were superior to those induced by Alum. In the Syrian hamster studies, when S-Trimer was adjuvanted with SWE, higher levels of neutralizing antibodies were induced against live SARS-CoV-2 from the original lineage and against the emergence of variants (Beta or Delta) with a slightly decreased potency. In addition, the SWE adjuvant demonstrated a dose-sparing effect; thus, a lower dose of S-Trimer as an antigen (0.5 µg) can induce comparable antisera and provide complete protection from viral infection. These data support the utility of SWE as an adjuvant to enhance the immunogenicity of the S-Trimer vaccine, which is feasible for further clinical testing.

Furin and TMPRSS2 Resistant Spike Induces Robust Humoral and Cellular Immunity Against SARS-CoV-2 Lethal Infection.

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

Monoclonal antibody targeting the conserved region of the SARS-CoV-2 spike protein to overcome viral variants.

Most therapeutic mAbs target the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Unfortunately, the RBD is a hot spot for mutations in SARS-CoV-2 variants, which will lead to loss of the neutralizing function of current therapeutic mAbs. Universal mAbs for different variants are necessary. We identified mAbs that recognized the S2 region of the spike protein, which is identical in different variants. The mAbs could neutralize SARS-CoV-2 infection and protect animals from SARS-CoV-2 challenge. After cloning the variable region of the light chain and heavy chain, the variable region sequences were humanized to select a high-affinity humanized mAb, hMab5.17. hMab5.17 protected animals from SARS-CoV-2 challenge and neutralized SARS-CoV-2 variant infection. We further identified the linear epitope of the mAb, which is not mutated in any variant of concern. These data suggest that a mAb recognizing the S2 region of the spike protein will be a potential universal therapeutic mAb for COVID-19.