Cross-protective efficacy and safety of an adenovirus-based universal influenza vaccine expressing nucleoprotein, hemagglutinin, and the ectodomain of matrix protein 2.

It is necessary to develop universal vaccines that act broadly and continuously to combat regular seasonal epidemics of influenza and rare pandemics. The aim of this study was to find the optimal dose regimen for the efficacy and safety of a mixture of previously developed recombinant adenovirus-based vaccines that expressed influenza nucleoprotein, hemagglutinin, and ectodomain of matrix protein 2 (rAd/NP and rAd/HA-M2e). The vaccine efficacy and safety were measured in the immunized mice with the mixture of rAd/NP and rAd/HA-M2e intranasally or intramuscularly. The minimum dose that would be efficacious in a single intranasal administration of the vaccine mixture and cross-protective efficacy against various influenza strains were examined. In addition, the immune responses that may affect the cross-protective efficacy were measured. We found that intranasal administration is an optimal route for 107 pfu of vaccine mixture, which is effective against pre-existing immunity against adenovirus. In a study to find the minimum dose with vaccine efficacy, the 106 pfu of vaccine mixture showed higher antibody titers to the nucleoprotein than did the same dose of rAd/NP alone in the serum of immunized mice. The 106 pfu of vaccine mixture overcame the morbidity and mortality of mice against the lethal dose of pH1N1, H3N2, and H5N1 influenza infections. No noticeable side effects were observed in single and repeated toxicity studies. We found that the mucosal administration of adenovirus-based universal influenza vaccine has both efficacy and safety, and can provide cross-protection against various influenza infections even at doses lower than those previously known to be effective.

A universal recombinant adenovirus type 5 vector-based COVID-19 vaccine.

A universal recombinant adenovirus type-5 (Ad5) vaccine against COVID19 (Ad-US) was constructed, and immunogenicity and broad-spectrum of Ad5-US were evaluated with both intranasal and intramuscular immunization routes. The humoral immune response of Ad5-US in serum and bronchoalveolar lavage fluid were evaluated by the enzyme-linked immunosorbent assay (ELISA), recombinant vesicular stomatitis virus based pseudovirus neutralization assay, and angiotensin-converting enzyme-2 (ACE2) -binding inhibition assay. The cellular immune response and Th1/Th2 biased immune response of Ad5-US were evaluated by the IFN-γ ELISpot assay, intracellular cytokine staining, and Meso Scale Discovery (MSD) profiling of Th1/Th2 cytokines. Intramuscular priming followed by an intranasal booster with Ad5-US elicited the broad-spectrum and high levels of IgG, IgA, pseudovirus neutralizing antibody (PNAb), and Th1-skewing of the T-cell response. Overall, the adenovirus type-5 vectored universal SARS-CoV-2 vaccine Ad5-US was successfully constructed, and Ad5-US was highly immunogenic and broad spectrum. Intramuscular priming followed by an intranasal booster with Ad5-US induced the high and broad spectrum systemic immune responses and local mucosal immune responses.

Comparative safety analysis of mRNA and adenoviral vector COVID-19 vaccines: a nationwide cohort study using an emulated target trial approach.

OBJECTIVE: This nationwide cohort study compared the incidence of adverse events of special interest (AESIs) between adenoviral vector-based (ChAdOx1) and mRNA-based (BNT162b2 or mRNA-1273) coronavirus disease 2019 (COVID-19) vaccines. METHODS: A targeted trial emulation study was conducted using data from the National Health Insurance Service database. Vaccinees aged 18-85 years who had received at least one dose of ChAdOx1 or an mRNA-based vaccine were identified. The 42-day risks of AESIs were calculated. RESULTS: A total of 1 767 539 ChAdOx1 vaccinees were matched exactly with mRNA vaccinees according to their risk factors. The 42-day risks of adverse events were low (∼0 to 176 events per 100 000 persons in both vaccine groups), and the incidence rates of AESIs were comparable between the two platforms, except for a higher occurrence of acute cardiac injury (incidence rate ratio [IRR], 1.22; 95% CI, 1.10-1.35), myocarditis or pericarditis (IRR, 2.14; 95% CI, 1.14-4.04), and arrhythmia (IRR, 1.46; 95% CI, 1.24-1.71) in mRNA vaccinees. The incidence of Guillain-Barré syndrome (IRR, 0.20; 95% CI, 0.06-0.69), vasovagal syncope (IRR, 0.77; 95% CI, 0.62-0.97), radiculopathy (IRR = 0.59, 95% CI, 0.41-0.84), and aseptic arthritis (IRR, 0.81; 95% CI, 0.70-0.93) was significantly lower in mRNA-based vaccinees compared with ChAdOx1 vaccinees. DISCUSSION: A remarkable platform-dependent difference was observed in the safety profiles of COVID-19 vaccines, particularly for myocarditis or pericarditis and Guillain-Barré syndrome. However, the overall risk of AESIs was low for both vaccine platforms.

Two Point Mutations in the Glycoprotein of SFTSV Enhance the Propagation Recombinant Vesicular Stomatitis Virus Vectors at Assembly Step.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen for which approved therapeutic drugs or vaccines are not available. We previously developed a recombinant vesicular stomatitis virus-based vaccine candidate (rVSV-SFTSV) by replacing the original glycoprotein with Gn/Gc from SFTSV, which conferred complete protection in a mouse model. Here, we found that two spontaneous mutations, M749T/C617R, emerged in the Gc glycoprotein during passaging that could significantly increase the titer of rVSV-SFTSV. M749T/C617R enhanced the genetic stability of rVSV-SFTSV, and no further mutations appeared after 10 passages. Using immunofluorescence analysis, we found that M749T/C617R could increase glycoprotein traffic to the plasma membrane, thus facilitating virus assembly. Remarkably, the broad-spectrum immunogenicity of rVSV-SFTSV was not affected by the M749T/C617R mutations. Overall, M749T/C617R could enhance the further development of rVSV-SFTSV into an effective vaccine in the future.

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses.

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8+ T cell responses. Moreover, E2-E1-LNP induced CD8+ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.

A Simplified SARS-CoV-2 Mouse Model Demonstrates Protection by an Oral Replicon-Based mRNA Vaccine.

A mouse model of SARS-CoV-2 that can be developed in any molecular biology lab with standard facilities will be valuable in evaluating drugs and vaccines. Here we present a simplified SARS-CoV-2 mouse model exploiting the rapid adenoviral purification method. Mice that are sensitive to SARS-CoV-2 infection were generated by transducing human angiotensin-converting enzyme 2 (hACE2) by an adenovirus. The expression kinetics of the hACE2 in transduced mice were assessed by immunohistochemistry, RT-PCR, and qPCR. Further, the ability of the hACE2 to support viral replication was determined in vitro and in vivo. The hACE2 expression in the lungs of mice was observed for at least nine days after transduction. The murine macrophages expressing hACE2 supported viral replication with detection of high viral titers. Next, in vivo studies were carried out to determine viral replication and lung disease following SARS-CoV-2 challenge. The model supported viral replication, and the challenged mouse developed lung disease characteristic of moderate interstitial pneumonia. Further, we illustrated the utility of the system by demonstrating protection using an oral mRNA vaccine. The multicistronic vaccine design enabled by the viral self-cleaving peptides targets receptor binding domain (RBD), heptad repeat domain (HR), membrane glycoprotein (M) and epitopes of nsp13 of parental SARS-CoV-2. Further, Salmonella and Semliki Forest virus replicon were exploited, respectively, for gene delivery and mRNA expression. We recorded potent cross-protective neutralizing antibodies in immunized mice against the SARS-CoV-2 delta variant. The vaccine protected the mice against viral replication and SARS-CoV-2-induced weight loss and lung pathology. The findings support the suitability of the model for preclinical evaluation of anti-SARS-CoV-2 therapies and vaccines. In addition, the findings provide novel insights into mRNA vaccine design against infectious diseases not limiting to SARS-CoV-2.

The Recombinant Eg.P29-Mediated miR-126a-5p Promotes the Differentiation of Mouse Naive CD4+ T Cells via DLK1-Mediated Notch1 Signal Pathway.

Cystic echinococcosis (CE) is a zoonotic parasitic disease spread worldwide caused by Echinococcus granulosus (Eg), which sometimes causes serious damage; however, in many cases, people are not aware that they are infected. A number of recombinant vaccines based on Eg are used to evaluate their effectiveness against the infection. Our previous report showed that recombinant Eg.P29 (rEg.P29) has a marvelous immunoprotection and can induce Th1 immune response. Furthermore, data of miRNA microarray in mice spleen CD4+ T cells showed that miR-126a-5p was significantly elevated 1 week after immunization by using rEg.P29. Therefore, in this perspective, we discussed the role of miR-126a-5p in the differentiation of naive CD4+ T cells into Th1/Th2 under rEg.P29 immunization and determined the mechanisms associated with delta-like 1 homolog (DLK1) and Notch1 signaling pathway. One week after P29 immunization of mice, we found that miR-126a-5p was significantly increased and DLK1 expression was decreased, while Notch1 pathway activation was enhanced and Th1 response was significantly stronger. The identical conclusion was obtained by overexpression of mmu-miR-126a-5p in primary naive CD4+ T cells in mice. Intriguingly, mmu-miR-126a-5p was significantly raised in serum from mice infected with protoscolex in the early stages of infection and markedly declined in the late stages of infection, while has-miR-126-5p expression was dramatically reduced in serum from CE patients. Taken together, we show that miR-126a-5p functions as a positive regulator of Notch1-mediated differentiation of CD4+ T cells into Th1 through downregulating DLK1 in vivo and in vitro. Hsa-miR-126-5p is potentially a very promising diagnostic biomarker for CE.

Identification of Tumor Antigens and Immune Subtypes of Glioblastoma for mRNA Vaccine Development.

The use of vaccines for cancer therapy is a promising immunotherapeutic strategy that has been shown to be effective against various cancers. Vaccines directly target tumors but their efficacy against glioblastoma multiforme (GBM) remains unclear. Immunotyping that classifies tumor samples is considered to be a biomarker for immunotherapy. This study aimed to identify potential GBM antigens suitable for vaccine development and develop a tool to predict the response of GBM patients to vaccination based on the immunotype. Gene Expression Profiling Interactive Analysis (GEPIA) was applied to evaluate the expression profile of GBM antigens and their influence on clinical prognosis, while the cBioPortal program was utilized to integrate and analyze genetic alterations. The correlation between antigens and antigen processing cells was assessed using TIMER. RNA-seq data of GBM samples and their corresponding clinical data were downloaded from the Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) for further clustering analysis. Six overexpressed and mutated tumor antigens (ARHGAP9, ARHGAP30, CLEC7A, MAN2B1, ARPC1B and PLB1) were highly correlated with the survival rate of GBM patients and the infiltration of antigen presenting cells in GBMs. With distinct cellular and molecular characteristics, three immune subtypes (IS1-IS3) of GBMs were identified and GBMs from IS3 subtype were more likely to benefit from vaccination. Through graph learning-based dimensional reduction, immune landscape was depicted and revealed the existence of heterogeneity among individual GBM patients. Finally, WGCNA can identify potential vaccination biomarkers by clustering immune related genes. In summary, the six tumor antigens are potential targets for developing anti-GBMs mRNA vaccine, and the immunotypes can be used for evaluating vaccination response.

Inactivated Rabies Virus Vectored MERS-Coronavirus Vaccine Induces Protective Immunity in Mice, Camels, and Alpacas.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emergent coronavirus that has caused frequent zoonotic events through camel-to-human spillover. An effective camelid vaccination strategy is probably the best way to reduce human exposure risk. Here, we constructed and evaluated an inactivated rabies virus-vectored MERS-CoV vaccine in mice, camels, and alpacas. Potent antigen-specific antibody and CD8+ T-cell responses were generated in mice; moreover, the vaccination reduced viral replication and accelerated virus clearance in MERS-CoV-infected mice. Besides, protective antibody responses against both MERS-CoV and rabies virus were induced in camels and alpacas. Satisfyingly, the immune sera showed broad cross-neutralizing activity against the three main MERS-CoV clades. For further characterization of the antibody response induced in camelids, MERS-CoV-specific variable domains of heavy-chain-only antibody (VHHs) were isolated from immunized alpacas and showed potent prophylactic and therapeutic efficacies in the Ad5-hDPP4-transduced mouse model. These results highlight the inactivated rabies virus-vectored MERS-CoV vaccine as a promising camelid candidate vaccine.

Immunogenicity of COVID-19 mRNA vaccines in immunocompromised patients: a systematic review and meta-analysis.

BACKGROUND: Immunocompromised (IC) patients are at higher risk of severe SARS-CoV-2 infection, morbidity, and mortality compared to the general population. They should be prioritized for primary prevention through vaccination. This study aimed to evaluate the efficacy of COVID-19 mRNA vaccines in IC patients through a systematic review and meta-analysis approach. METHOD: PubMed-MEDLINE, Scopus, and Web of Science were searched for original articles reporting the immunogenicity of two doses of mRNA COVID-19 vaccines in adult patients with IC condition between June 1, 2020 and September 1, 2021. Meta-analysis was performed using either random or fixed effect according to the heterogeneity of the studies. Subgroup analysis was performed to identify potential sources of heterogeneity. RESULTS: A total of 26 studies on 3207 IC patients and 1726 healthy individuals were included. The risk of seroconversion in IC patients was 48% lower than those in controls (RR = 0.52 [0.42, 0.65]). IC patients with autoimmune conditions were 54%, and patients with malignancy were 42% more likely to have positive seroconversion than transplant recipients (P < 0.01). Subgroup meta-analysis based on the type of malignancy, revealed significantly higher proportion of positive seroconversion in solid organ compared to hematologic malignancies (RR = 0.88 [0.85, 0.92] vs. 0.61 [0.44, 0.86], P = 0.03). Subgroup meta-analysis based on type of transplantation (kidney vs. others) showed no statistically significant between-group difference of seroconversion (P = 0.55). CONCLUSIONS: IC patients, especially transplant recipients, developed lower immunogenicity with two-dose of COVID-19 mRNA vaccines. Among patients with IC, those with autoimmune conditions and solid organ malignancies are mostly benefited from COVID-19 vaccination. Findings from this meta-analysis could aid healthcare policymakers in making decisions regarding the importance of the booster dose or more strict personal protections in the IC patients.

SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Identification of Leptospiral Protein Antigens Recognized by WC1+ γδ T Cell Subsets as Target for Development of Recombinant Vaccines.

Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. γδ T cells play a role in the protective immune response in livestock species against Leptospira, while human γδ T cells also respond to Leptospira. Thus, activation of γδ T cells has emerged as a potential component in the optimization of vaccine strategies. Bovine γδ T cells proliferate and produce gamma interferon (IFN-γ) in response to vaccination with inactivated leptospires, and this response is mediated by a specific subpopulation of the WC1-bearing γδ T cells. WC1 molecules are members of the group B scavenger receptor cysteine-rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and γδ TCR coreceptors, the WC1 system has been proposed as a novel target to engage γδ T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ γδ T cells and identify two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific γδ T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+ γδ T cells respond. Identification of protein antigens will enhance our understanding of the role γδ T cells play in the leptospiral immune response and in recombinant vaccine development.

Chemically modified mRNA beyond COVID-19: Potential preventive and therapeutic applications for targeting chronic diseases.

Chemically modified mRNA represents a unique, efficient, and straightforward approach to produce a class of biopharmaceutical agents. It has been already approved as a vaccination-based method for targeting SARS-CoV-2 virus. The COVID-19 pandemic has highlighted the prospect of synthetic modified mRNA to efficiently and safely combat various diseases. Recently, various optimization advances have been adopted to overcome the limitations associated with conventional gene therapeutics leading to wide-ranging applications in different disease conditions. This review sheds light on emerging directions of chemically modified mRNAs to prevent and treat widespread chronic diseases, including metabolic disorders, cancer vaccination and immunotherapy, musculoskeletal disorders, respiratory conditions, cardiovascular diseases, and liver diseases.

CD19+IgD+CD27- Naïve B Cells as Predictors of Humoral Response to COVID 19 mRNA Vaccination in Immunocompromised Patients.

Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19. We aimed to analyze B-cell subsets in these patients to identify potential predictors of humoral vaccination response. Patients (n=120) suffering from hematologic malignancies or other causes of immunodeficiency and healthy controls (n=79) received a full vaccination series with an mRNA vaccine. B-cell subsets were analyzed prior to vaccination. Two independent anti-SARS-CoV-2 immunoassays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed three to four weeks after the second vaccination. Seroconversion occurred in 100% of healthy controls, in contrast to 67% (RBD) and 82% (TSP) of immunocompromised patients, while only 32% (RBD) and 22% (TSP) achieved antibody levels comparable to those of healthy controls. The number of circulating CD19+IgD+CD27- naïve B cells was strongly associated with antibody levels (ρ=0.761, P<0.001) and the only independent predictor for achieving antibody levels comparable to healthy controls (OR 1.07 per 10-µL increase, 95%CI 1.02-1.12, P=0.009). Receiver operating characteristic analysis identified a cut-off at ≥61 naïve B cells per µl to discriminate between patients with and without an optimal antibody response. Consequently, measuring of naïve B cells in immunocompromised hematologic patients could be useful in predicting their humoral vaccination response.

Tumor-antigens and immune landscapes identification for prostate adenocarcinoma mRNA vaccine.

Prostate adenocarcinoma (PRAD) is a leading cause of death among men. Messenger ribonucleic acid (mRNA) vaccine presents an attractive approach to achieve satisfactory outcomes; however, tumor antigen screening and vaccination candidates show a bottleneck in this field. We aimed to investigate the tumor antigens for mRNA vaccine development and immune subtypes for choosing appropriate patients for vaccination. We identified eight overexpressed and mutated tumor antigens with poor prognostic value of PRAD, including KLHL17, CPT1B, IQGAP3, LIME1, YJEFN3, KIAA1529, MSH5 and CELSR3. The correlation of those genes with antigen-presenting immune cells were assessed. We further identified three immune subtypes of PRAD (PRAD immune subtype [PIS] 1-3) with distinct clinical, molecular, and cellular characteristics. PIS1 showed better survival and immune cell infiltration, nevertheless, PIS2 and PIS3 showed cold tumor features with poorer prognosis and higher tumor genomic instability. Moreover, these immune subtypes presented distinguished association with immune checkpoints, immunogenic cell death modulators, and prognostic factors of PRAD. Furthermore, immune landscape characterization unraveled the immune heterogeneity among patients with PRAD. To summarize, our study suggests KLHL17, CPT1B, IQGAP3, LIME1, YJEFN3, KIAA1529, MSH5 and CELSR3 are potential antigens for PRAD mRNA vaccine development, and patients in the PIS2 and PIS3 groups are more suitable for vaccination.

Pediatric COVID-19 patients in South Brazil show abundant viral mRNA and strong specific anti-viral responses.

COVID-19 manifests as a milder disease in children than adults, but the underlying mechanisms are not fully characterized. Here we assess the difference in cellular or humoral immune responses of pediatric and adult COVID-19 patients to see if these factors contribute to the severity dichotomy. Children's non-specific immune profile is dominated by naive lymphocytes and HLA-DRhighCX3CR1low dendritic cells; meanwhile, children show strong specific antibody and T cell responses for viral structural proteins, with their T cell responses differing from adults by having weaker CD8+TNF+ T cells responses to S peptide pool but stronger responses to N and M peptide pools. Finally, viral mRNA is more abundant in pediatric patients. Our data thus support a scenario in which SARS-CoV-2 infected children contribute to transmission yet are less susceptible to COVID-19 symptoms due to strong and differential responses to the virus.

Antibody-Mediated Targeting of Antigens to Intestinal Aminopeptidase N Elicits Gut IgA Responses in Pigs.

Many pathogens enter the host via the gut, causing disease in animals and humans. A robust intestinal immune response is necessary to protect the host from these gut pathogens. Despite being best suited for eliciting intestinal immunity, oral vaccination remains a challenge due to the gastrointestinal environment, a poor uptake of vaccine antigens by the intestinal epithelium and the tolerogenic environment pervading the gut. To improve uptake, efforts have focused on targeting antigens towards the gut mucosa. An interesting target is aminopeptidase N (APN), a conserved membrane protein present on small intestinal epithelial cells shown to mediate epithelial transcytosis. Here, we aimed to further optimize this oral vaccination strategy in a large animal model. Porcine APN-specific monoclonal antibodies were generated and the most promising candidate in terms of epithelial transcytosis was selected to generate antibody fusion constructs, comprising a murine IgG1 or porcine IgA backbone and a low immunogenic antigen: the F18-fimbriated E. coli tip adhesin FedF. Upon oral delivery of these recombinant antibodies in piglets, both mucosal and systemic immune responses were elicited. The presence of the FedF antigen however appeared to reduce these immune responses. Further analysis showed that F18 fimbriae were able to disrupt the antigen presenting capacity of intestinal antigen presenting cells, implying potential tolerogenic effects of FedF. Altogether, these findings show that targeted delivery of molecules to epithelial aminopeptidase N results in their transcytosis and delivery to the gut immune systems. The results provide a solid foundation for the development of oral subunit vaccines to protect against gut pathogens.