CD40.SARS.CoV2 VACCINE, BUT NOT mRNA, INDUCES SPECIFIC CD8+ T MEMORY STEM CELLS

Background: Vaccination plays a major role in controlling SARS-CoV2 infection but faces the issue of short-term protection. Beyond the generation of Abs, induction of memory CD8+T cells with stem cell-like (Tscm) properties is essential for long-term immunity to viruses. We have designed a sub-unit CD40.CoV-2 vaccine which targets Spike (S) and nucleocapsid (N) regions from SARS-CoV-2 to antigen presenting cells with comparable immunogenicity and protective effect than mRNA BNT162b2 (Pfizer-BioNTech) in preclinical models (Coleon S. EBioMed 2022). We hypothesized that CD40.CoV2 vaccine will elicit CD8+ Tscm cells. Method(s): CD40.CoV2 vaccine is a fully humanized mAb fused to RBD (aa 318-541) and N (aa 276-411). Humanized (hu) NSG mice (HIS-mice) (n=6/ group) received: i) CD40.CoV2 (10 mug equal to 1.3 mug of RBD, i.p.) +/- poly-ICLC (TLR3 agonist;50mug) or ii) BNT162b2 (1mug, i.m.);iii) IgG4.CoV2 (10mug, i.p.) as non-CD40-targeting control. Phenotype and function of splenic S and N-specific T cells were assessed at W5. Result(s): The CD40.CoV2 vaccine +/- poly-CLC induced significant S and N-specific Th1 huCD4+, cytokines-secreting huCD8+ T cells and RBD-specific IgG-switched huB cells as compared to mock injections and non-targeted IgG4.CoV2. CD40.CoV-2 vaccine +/- adjuvant induced higher frequencies of huCD8+ Tscm (CD95+ CD45RA+ CD62L+ ;median, (IQR) 22.4% (12.3-27.4) and 23% (20.7-29.1) +/- adjuvant, respectively) and central memory (TCM;CD45RA- CD62L+) CD8+ T cells (2.7% (2.3-6.2) and 5.1% (3.8-7.8) +/- adjuvant, respectively). In contrast, BNT162b2 induced predominantly effector memory (TEM, CD45RA- CD62L- ;median, (IQR) 63.1% (47.3-72.3)) but not Tscm (1.6% (0.9-6.6)) (figure). CD40.CoV-2 induced huCD8+ Tscm cells exhibit ;i) a higher proliferation index than TCM and TEM;ii) a functional profile secreting TNF and IFNgamma after restimulation with RBD or N peptides;cardinal features of Tscm cells. Conclusion(s): The CD40.SARS.CoV2, but not BNT162b2 vaccine, stimulates selective enrichment in S-and N-specific CD8+ Tscm cells that support longlasting anti-viral immunity. CD40.CoV2 sub-unit is under clinical development as a booster vaccine aimed to maintain durable anti-viral T and humoral responses. (Figure Presented).

Delivery of spike-RBD by bacterial type three secretion system for SARS-CoV-2 vaccine development.

COVID-19 pandemic continues to spread throughout the world with an urgent demand for a safe and protective vaccine to effectuate herd protection and control the spread of SARS-CoV-2. Here, we report the development of a bacterial vector COVID-19 vaccine (aPA-RBD) that carries the gene for the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Live-attenuated strains of Pseudomonas aeruginosa (aPA) were constructed which express the recombinant RBD and effectively deliver RBD protein into various antigen presenting cells through bacterial type 3 secretion system (T3SS) in vitro. In mice, two-dose of intranasal aPA-RBD vaccinations elicited the development of RBD-specific serum IgG and IgM. Importantly, the sera from the immunized mice were able to neutralize host cell infections by SARS-CoV-2 pseudovirus as well as the authentic virus variants potently. T-cell responses of immunized mice were assessed by enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) assays. aPA-RBD vaccinations can elicit RBD-specific CD4+and CD8+T cell responses. T3SS-based RBD intracellular delivery heightens the efficiency of antigen presentation and enables the aPA-RBD vaccine to elicit CD8+T cell response. Thus, aPA vector has the potential as an inexpensive, readily manufactured, and respiratory tract vaccination route vaccine platform for other pathogens.

Vaccination for SARS-CoV-2: a review

The present overview describes the various vaccines in use to prevent SARS-CoV-2 infection and COVID-19 disease. Their action is based on the natural defense mechanisms of the human body against viral infections. The effectiveness of the vaccines is described and details of the cellular and humoral immune response as measured following infection and following vaccination are reported. The immune response is waning in time. Side effects of vaccination exist, but are minor when compared to COVID-19 disease itself. Due to waning of the antiviral immunity and the continuing emergence of virus variants, regular repeat vaccinations and the development of stronger vaccines, ideally with coverage of an extended antigen spectrum, seem necessary. Copyright © 2022 Tijdschrift voor Geneeskunde en Gezondheidszorg. All rights reserved.

COVID-19 INFECTION MAY BE TRIGGER for DEVELOPMENT of IMMUNE-MEDIATED DISEASES (IMDS)

Background: As considerable evidence indicates viruses play an important role in the pathogenesis of infammatory rheumatic diseases as environmental factors. The most prominent pathogenic viruses which have been proposed in the triggering and initiation of autoimmune diseases include Parvovirus B19, Epstein-Barr-virus (EBV), Cytomegalovirus (CMV), Herpes virus-6, HTLV-1, Hepatitis A and C virus, and Rubella virus1. It is possible that COVID-19 infection is also a trigger. Because SARS-CoV-2 infection can break immune tolerance and trigger autoimmune responses, it is also likely to induce clinical autoimmunity2. Objectives: Find out a possible association between Covid-19 infection and development of IMDs. Methods: We analyzed data of 21 patients (Male 4/19 %/, female 17/81%/, mean age 45.5 ± 13,9 years), who were admitted to Rheumatology department of 'Mikayelyan' University Hospital after Covid-19 infection with newly diagnosed IMDs from June till December 2021. All of included had never had such kind of disorder before. EULAR/ACR criteria were used for diagnosis and assessment of disease activity. Results: After SARS-CoV-2 infection some patients presented with preserved fever, high levels of CRP and ESR, had rash and arthritis. Particularly, 3 (14.3%) developed systemic lupus erythematosus, 3(14.3%)-antiphospholipid syndrome, 4 (19%)-rheumatoid arthritis, 2(9.5%)-spondyloarthritis, 3 (14.3%)-sarcoidosis, 4 (19%)-erythema nodosum, 1 (4.8%)-small-vessel vasculitis, 1 (4.8%)-undifferentiated arthritis, and 1(4.8%)-Tietze syndrome. 11 (52.4%) experienced severe course of Covid-19 with pneumonia and respiratory failure, in 10 (47.6%) patients the course of disease was mild. We've found a signifcant association between severe course of Covid-19 and development of erythema nodosum. (p< 0.05). Also an association between female gender and severe course of Covid-19 was determined (p<0,05). Conclusion: In acute progression of the COVID-19 along with development of antiviral immunity, a dysregulated response of immune system may occur, represented by the marked cytokine release syndrome, macrophage activation, and systemic hyperinfammation.3 We analyzed the data of patients who didn't have any typical symptom of rheumatic diseases before coronavirus infection, therefor, on our opinion, virus played an important role to induce clinical autoimmun-ity and autoinfammation and subsequently-IMDs. Possibly, Covid-19 infection may be included in the group of trigger viruses for.

Immunotherapy: OFF-THE-SHELF PARTIAL HLA MATCHING SARS-COV-2 ANTIGEN SPECIFIC T CELL THERAPY: A NEW POSSIBILITY FOR COVID- 19 TREATMENT

Background & Aim: Background. Immunological characteristics of COVID-19 show pathological hyperinflammation associated with lymphopenia and dysfunctional T cell responses. These features provide a rationale for restoring functional T cell immunity in COVID-19 patients by adoptive transfer of SARS-CoV-2 specific T cells. Methods, Results & Conclusion: Methods. To generate SARS-CoV-2 specific T cells, we isolated peripheral blood mononuclear cells from 7 COVID-19 recovered and 13 unexposed donors. Consequently, we stimulated cells with SARS-CoV-2 peptide mixtures covering spike, membrane and nucleocapsid proteins. Then, we culture expanded cells with IL-2 for 21 days. We assessed immunophenotypes, cytokine profiles, antigen specificity of the final cell products. Results. Our results show that SARS-CoV-2 specific T cells could be expanded in both COVID-19 recovered and unexposed groups. Immunophenotypes were similar in both groups showing CD4+ T cell dominance, but CD8+ and CD3+CD56+ T cells were also present. Antigen specificity was determined by ELISPOT, intracellular cytokine assay, and cytotoxicity assays. One out of 14 individuals who were previously unexposed to SARS-CoV-2 failed to show antigen specificity. Moreover, ex-vivo expanded SARS-CoV-2 specific T cells mainly consisted of central and effector memory subsets with reduced alloreactivity against HLA-unmatched cells suggesting the possibility for the development of third-party partial HLA-matching products. Conclusion. In conclusion, our findings show that SARS-CoV-2 specific T cell can be readily expanded from both COVID-19 and unexposed individuals and can therefore be manufactured as a biopharmaceutical product to treat severe COVID-19 patients.

RITUXIMAB-ASSOCIATED B-CELL DEPLETION RESULTING IN RECURRENT COVID-19 PNEUMONIA

CASE: A 64 year old female with a history of rheumatoid arthritis (RA) and hypertension presented to the hospital with shortness of breath. Her RA had been well controlled with prednisone 5mg daily and rituximab infusions every 6 months, last given three months prior. SARS-CoV-2 PCR was positive and she was treated with dexamethasone. Two months later, she presented to the emergency department with recurrent shortness of breath. At this time, her cycle threshold was 29.82 and CT chest demonstrated bilateral air bronchograms. No improvement was noted with antibiotics, BAL showed 500 WBCs, 70% lymphocytes and biopsy of the left lung showed chronic interstitial inflammation, consistent with patchy organizing pneumonia. She improved with a course of prednisone 30mg for five weeks. Four months later, she was admitted for worsening shortness of breath. At this time, she was noted to be SARS-CoV-2 PCR negative and bronchoscopy showed no growth on cultures. One month later (7 months from initial hospitalization) she was readmitted with hypoxia. Cycle time was 19 indicating high viral load, SARS-CoV-2 PCR positive, nucleocapsid antibody positive and spike IgG antibody negative thus suggesting reinfection with lack of immune response to vaccination. She was treated with remdesivir with minimal improvement. Due to concern for reinfection, she was treated with baricitinib and casirivimab-imdevimab monoclonal antibody treatment with improvement. CD19 count was <10 with <0.5%, consistent with B cell depletion. IMPACT/DISCUSSION: Rituximab, an anti-CD20 monoclonal antibody that results in B-cell depletion, is commonly used to treat rheumatoid arthritis. While it is essential for disease control, it also risks compromising antiviral immunity, re-infection to SARS-CoV-2, and impaired vaccine efficacy (1). Our case describes a patient with rheumatoid arthritis treated with rituximab infusion, and her subsequent prolonged COVID-19 re-infection with multiple hospitalizations. B-Cell depletion from rituximab infusion leading to a prolonged COVID-19 course is a rare and relatively new phenomenon that is being reported over the course of the pandemic. Given her intermittent negative testing, high viral load, and lack of response to prior vaccination, it is likely that her presentation represented true re-infection in the setting of insufficient antibody production due to rituximab, further supported by improvement with monoclonal antibody treatment. As demonstrated by this patient, monoclonal antibodies were particularly effective in clinical improvement given her own lack of antibody response. CONCLUSION: While rituximab may be safe for many patients, it may result in significant, prolonged B-cell depletion. This depletion may increase the risk of reinfection, affect vaccine response and antibody formation. Administration of monoclonal antibody treatment should be considered in immunosuppressed and B-cell depleted patients with COVID-19 infection.

Immune response to COVID-19 vaccination in patients with B-cell malignancies after CD19 CART cell therapy

Introduction: Patients with hematologic malignancies are at an increased risk of morbid/mortality from COVID-19. Our prospective clinical study evaluated immune responses to COVID-19 mRNA vaccines in patients with B-cell lymphoma who had received CD19-directed chimeric antigen receptor (CAR) T cell therapy. Methods: We measured SARS-CoV-2 neutralizing activity of plasma from 18 patients and 4 healthy controls (HC) and antibody titers against viral spike proteins (S1, S2, RBD) including their delta variants using an enzyme-linked immunoassay (ELISA). We measured B cell subpopulations in the patients' blood using flow cytometry. Results: We found that the peripheral blood plasma from 3/4 HCs showed substantial SARS-CoV-2 neutralizing activity already at 4 weeks after the first dose of COVID-19 mRNA vaccine while none of the CD19 CART patients evidenced any antibody-mediated neutralizing activity at the same point in time. At 4 weeks after receiving the second dose of the vaccine, all 4 HCs showed complete neutralizing activity. In marked contrast, only 1 of 14 CART patients evidenced any relevant antibody-mediated SARS-CoV-2 neutralizing activity. Assessing whether a globally insufficient antibody-mediated immunity was the underlying cause of the lack of a response to the COVID-19 vaccine in our CART patients, we found that IgG antibody levels against common microbial and viral antigens like influenza, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), and tetanus toxoid, were comparable to those observed in HCs. However, while at 4 weeks post second dose of the vaccine the HCs showed high levels of vaccine-induced IgG antibody titers against all the viral spike proteins (S1, S2, RBD), including the delta variants of the S1 and RBD proteins, the vast majority of our CART patients did not evidence any SARS-CoV-2-specific antibodies. Importantly, a third booster vaccination did not lead to an improvement in the antiviral immunity in the 4 CART patients analyzed. When we assessed B cell subpopulations in the blood of patients and HCs, we found that prior treatments had completely eradicated all CD19+/CD20+ B cells in the patients while numbers of long-lived memory plasma cells were comparable to those of HCs. Conclusions: In this study, 17 of 18 patients with lymphoma who received CD19 CART therapy had very poor immunoreactivity to 1-3 doses of mRNA-based COVID-19 vaccines. Importantly, antibody responses to common recall antigens were preserved, suggesting impaired immune response primarily against novel antigens like SARS-COV-2. This lack of immunoreactivity against novel antigens was probably based on the eradication of earlier-stage B cell subpopulations after treatment with anti-CD19 and anti-CD20 immunotherapies.

Redirection of anti-virus immunity towards anti-tumor immunity using nanoparticle-induced immune tolerance for oncolytic adenovirus therapy

Recent clinical observations that some coronavirus infections induced complete remissions in lymphoma patients emphasized again the potential of cancer virotherapy. Infection of cancer cells with oncolytic viruses reshapes the tumor microenvironment by activating anti-viral and anti-tumor immunity. A phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD (DNX-2401) to treat recurrent malignant gliomas demonstrated activation of CD8+ T-cells and significant clinical benefits for a subset of patients. However, both anti-virus and anti-tumor immune responses are contingent on the activation of respective clones of CD8+ T-cells, which compete for clonal expansion. Thus, overexpansion of T-cells against viral antigens reduces the frequency of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for viral antigens will decrease anti-viral immunity and in turn derepress anti-tumor immunity, resulting in enhanced efficacy of cancer virotherapy. In this work, we used nanoparticles encapsulating adenoviral antigens E1A, E1B and hexon that distributed to liver resident macrophages (P<0.0001) and induced peripheral immune tolerance. Functional experiments to restimulate immune cells with viral or tumor antigens showed that injection of nanoparticles induced virus-specific immune tolerance and redirected the focus of the immune response towards tumor peptides as measured by interferon-gamma secretion (P<0.0001). Co-culture experiments also showed increased activation of immune cells against fixed tumor cells after nanoparticle treatment (P<0.0001). Reduction of virus-specific T-cells and concurrent expansion of tumor-specific T-cell clones were further confirmed with E1A or OVA tetramers (P<0.05). Flow cytometry analysis suggested increased anti-tumor responses were due to differences in T-cell clones and not due to other immune populations including natural killer cells or myeloid-derived suppressor cells (P=0.3). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens in tumor-bearing mice increased the overall survival and doubled the percentage of long-term survivors compared to virus treatment alone. Our data should propel the development of a future clinical trial aiming to maximize the potential of anti-tumor immunity during cancer virotherapies.

The role of platelets in antiviral immunity

The main role of platelets is traditionally assigned to participation in hemostasis reactions. In recent years, the data have appeared on the non-hemostatic platelet-related role and their active participation in inflammatory reactions. These platelet functions are predetermined by their ability to activate and secrete various immunomodulatory cytokines and chemokines. In addition, activated platelets can directly interact with viral receptors. Recently, there has been growing the knowledge regarding platelet-related regulation of diverse cell types. The result of this interaction is, among others, the formation of platelet-leukocyte aggregates, the focusing of neutrophils at the sites of injury, and generation of a scaffold for developing extracellular traps. Thus, platelets are not only participants in coagulation processes, but also important players in the inflammatory process. This lecture details the issues of platelets controlling and modulating host response to viral infection, as well as potential targets for therapeutic intervention.

Puerarin: A Potential Therapeutic for SARS-CoV-2 and Hantavirus Co-Infection.

Patients with Hantavirus-caused epidemic hemorrhagic fever (EHF) are at risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is currently no validated EHF/SARS-CoV-2 strategy. Several studies have recently shown Puerarin, a natural product, has potent antiviral properties. The goal of present study was to determine the mechanism of puerarin in patients with EHF/COVID-19. We use network pharmacology and bioinformatics to investigate the possible pharmacological targets, bioactivities, and molecular mechanisms of puerarin in the treatment of patients with EHF/SARS-CoV-2. The study investigated the pathogenesis of COVID-19 and EHF and the signaling pathway impacted by puerarin. 68 common genes linked to puerarin and EHF/SARS-CoV-2 were discovered during the investigation. By using protein-protein interaction (PPI) network, we identified RELA, JUN, NF-B1, NF-B2, and FOS as potential therapeutic targets. The bioactivity and signaling pathways of puerarin have also been demonstrated in the treatment of EHF and COVID-19. According to present study, puerarin could reduce excessive immune responses and inflammation through the NF-B, TNF, and HIF-1 signaling pathways. This study explored the potential therapeutic targets and mechanisms of Puerarin in the treatment of EHF/COVID-19.

IL-25 Inhibits Airway Anti-Viral Immunity and Promotes Virus Exacerbation of Allergic Airways Disease

Rationale We have previously reported blocking the IL-25 receptor (IL-17RB) prevented viral increased allergic airways inflammation and this was associated with reduced lung viral load. To investigate IL-25 regulation of airway anti-viral immunity we hypothesised that IL-25 directly inhibits airway epithelial cell (AEC) type I/III interferon expression and antibody blockade of IL-25 in vivo boosts lung interferon expression and reduces lung viral load in parallel with reduced type 2 airway inflammation. Methods In vitro Immunofluorescence was used to visualise epithelial IL-25 and IL- 17RB proteins in endobronchial biopsies from patients with asthma and healthy subjects and in AEC differentiated at ALI. AEC from n = 14 donors with asthma were differentiated at the air-liquid interface (ALI) and infected with RV-A1, MOI=0.1. A subset of AECs was treated with anti-IL-25 mAb (LNR125) before infecting with RV-A1 or human coronavirus 229E. Differentiated AEC from healthy donors were treated with recombinant IL-25 protein and infected with RV-A1. Nanostring immune transcriptomic data expressed as digital mRNA counts for exact copy number or was expressed as log2 fold change ratio against -log10 Bejamini-Yekutieli-corrected p-values. In vivo 6- 8-week-old, BALB/c mice sensitised and intranasally challenged daily for 3 days with ovalbumin to induced allergic airways disease. A single subcutaneous injection of 250 μg LNR125 was administered during ovalbumin challenge. Mice were then infected i.n. with RV-A1, 6 hours after final allergen challenge. On day 1 and day 7 post-infection, BAL were collected, lung lobe tissue was collected for viral RNA and cytokine expression. Results IL-25 and IL-17RB were constitutively expressed at the apical surface of airway epithelium in biopsies and AEC cultures. RV infection increased IL-25 expression by AEC from asthmatic donors. LNR125 treatment reduced IL-25 mRNA and significantly increased RV induced IFN-β a and IFN-λ protein expression and this was confirmed by Nanostring transcriptomic analyses which also identified down-regulated type-2 immune genes CCL26 (eotaxin 3) and IL1RL1(IL-33 receptor). LN125 treatment also increased IFN-λ expression by 229E-infected differentiated AECs. IL-25 treatment increased viral load associated with 50% reduced expression of IFN-β and CXCL10 and 75% reduced IFN-λ. Allergen challenged, RV-infected mice treated with LNR125 had significantly increased BAL IFN-β protein and 60% reduction in lung viral load associated with reduced IL-25, IL-4, IL-5 and IL-13 BAL proteins compared to controls. Conclusion IL-25-induced inflammation combined with suppression of AEC anti-viral immunity identify IL-25 as a central mediator of viral asthma exacerbations and therefore a target for mAb-based treatment.

TYPE 2 DIABETES IMPAIRS ANTIVIRAL IMMUNITY BY PREVENTING THE INDUCTION OF FASTING METABOLISM

Background and Aims: Type 2 diabetes (T2D) is a major risk factor for developing severe infectious disease, such as COVID-19. The endocrine and immune system closely interact following viral infection, which is deregulated in T2D. Previously, we showed in humans and mice that viral infection causes transient insulin resistance, which can lead to permanent loss of glycemic control in subjects with pre-diabetes. How changes in systemic glycemia benefit the antiviral response, and how this derails in T2D is mostly unknown. Methods: Mice were infected with virulent strains of cytomegalovirus or lymphocytic choriomeningitis virus. Glucose-, insulin- and pyruvate-tolerance tests and hyperinsulinemic euglycemic clamping were used to determine the metabolic state of animals. Conditional knock-out models were used to measure the impact of cytokines on metabolism of specific organs. Dietinduced obesity models were used to determine the impact of hyperglycemia on the antiviral response. Results: Severe viral infection causes pancreatic β-cell hyperfunctionality following their stimulation with the cytokine IFNγ by local T cells. Virus-induced hyperinsulinemia impaired glucose release by the liver and promoted induction of fasting metabolism, because of reduced hepatic glycogenolysis, causing relative, transient hypoglycemia (RHG). RHG was beneficial to the antiviral response by promoting the release of antiviral cytokines by endothelial cells, which impaired viral replication. Obese mice failed to induce fastng metabolsim, resulting in lower antiviral cytokines, higher viral titers and increased pathology. Conclusions: Metabolic adaptations following infection are of major importance for optimal control of viral replication. In context of T2D, these changes cannot be accomplished, thus leading to more frequent and severe infections.

DEEP MUTATIONAL SCANNING to INTERPRET VIRAL EVOLUTION

Advances in genome sequence have enabled rapid identification of new SARS-CoV-2 variant lineages, but a major challenge is understanding how mutations in these variants will affect anti-viral immunity. Here I describe new high-throughout assays that provide data that can be used to rapidly interpret the consequences of SARS-CoV-2 mutations.

Intra tumoral electroporation of IL-12 and SARS-Cov-2 spike plasmids drives a coordinated vaccine response and elicits robust anti-tumor immunity

Despite extensive clinical evidence on the efficacy and safety of SARS-CoV-2 vaccines, there remains a paucity of data on their effectiveness in cancer patients who are actively receiving antineoplastic therapeutics. A recent study demonstrated only ∼30% of cancer patients had positive serologic test following 2 doses of FDA-authorized SARS-CoV-2 vaccines, in contrast to ∼80% positivity rate in healthy individuals, regardless of the age. Therefore, furtherinvestigation into novel approaches to boost immune response to SARS-CoV-2 vaccines in cancer patients isrequired. Our previous preclinical and clinical studies have established intratumoral IL-12 plasmid (TAVO)electroporation (EP) induces localized expression of IL-12p70, converting immune-excluded tumors into inflamedimmunogenic lesions, thereby generating objective responses in both treated and untreated, distant tumors. Basedon the enhancement of immunotherapy efficacy by IL-12, we leveraged the flexibility of our DNA plasmid-EPplatform to express SARS-CoV-2 spike protein in addition to IL-12 (CORVax12) as an intratumoral vaccine candidate which we hypothesized could not only drive anti-SARS-CoV-2 immune responses but also generate aproductive anti-tumor response. Naïve mice were vaccinated via intradermal injection of SARS-CoV-2 spike plasmidfollowed immediately by EP with or without plasmid-encoded mIL-12 on days 1 and 21. Longitudinal serum samples were collected to interrogate virus-specific cellular responses as well anti-spike IgG antibody. A surrogate viralneutralization test (sVNT) assessed serum blockade of soluble human ACE2 binding to immobilized SARS-CoV-2spike. Our data demonstrated that intradermally electroporated CORVax12 elicits significantly higher anti-SARS-CoV-2 spike IgG antibodies and neutralization when compared with EP of SARS-CoV-2 spike alone. Next, we askedif improved SARS-CoV-2 immune response may be observed when CORVax12 is incorporated into intratumoral EPin single-tumor bearing mice. CORVax12 robustly inhibited tumor growth, induced high percentages of germinal-center B cells and class switched B cells in tumor draining lymph nodes, and generated high of anti-spike IgG and neutralization antibodies. To further investigate systemic effects of this combination, we continued with contralateraltumor mice models. In both CT26 and B16-F10 tumor models, CORVax12 intratumoral EP induced strong systemicanti-tumor responses similar to IL-12 EP alone while also producing high serum levels of anti-SARS-CoV-2 spikeIgG and neutralization antibodies. Critically, this anti-viral immunity did not limit this IL-12-based intratumoral anti-tumor therapy. In summary, our preclinical data indicates that intratumoral EP of CORVax12 can induce IgGresponses to SARS-CoV-2 spike as well as apparent viral neutralizing activity all while maintaining local and systemic anti-tumor effects expected from TAVO Treatment. This combined intratumoral therapy represents a novelstrategy to address both tumor burden and anti-SARS-CoV-2 immunity in patients with cancer.

The safety of mRNA vaccine in patients with common variable immunodeficiency

Background: Common variable immunodeficiency (CVID) is a heterogeneous group of disorders characterized by the disturbed production of immunoglobulin and specific antibodies. CVID usually manifests with recurrent bacterial respiratory tract infections, although broad spectrum of non-infectious complications affecting lungs and many other vital organ systems may develop. Therefore, CVID patients may be recognized as a risk group for Covid-19 infection and the vaccination should be considered despite of the terrain of humoral immunodeficiency. However, only limited data are available on the safety of mRNA vaccine in CVID patients Method: A single-center prospective observational study focused on the safety of mRNA vaccine Comirnaty administration in a cohort of CVID patients. Only patients who had met inclusion and exclusion criteria were enrolled upon signed written informed consent. Total blood count with differential, coagulation, biochemistry and immunologic parameters were assessed before a first dose of the vaccine (Day 0), then at Day 21 (before a second dose) and at Day 51. The study was approved by Motol University Hospital Ethic Committee. Results: Together 25 CVID patients were included into the study. The most reported adverse events were local pain, fatigue, myalgia, arthralgia or increased body temperature. Neither severe adverse events nor anaphylaxis were reported. No safety concern were also revealed in the evaluated laboratory parameters. Conclusion: The administration of mRNA vaccine Comirnaty in the terrain of CVID-severely impaired humoral immunity is safe. However, there is an open question of its efficacy, particularly from the point of view of T cell response that plays crucial role in antiviral immunity.

Viroporins and inflammasomes: A key to understand virus-induced inflammation.

Viroporins are virus encoded proteins that alter membrane permeability and can trigger subsequent cellular signals. Oligomerization of viroporin subunits results in formation of a hydrophilic pore which facilitates ion transport across host cell membranes. These viral channel proteins may be involved in different stages of the virus infection cycle. Inflammasomes are large multimolecular complexes best recognized for their ability to control activation of caspase-1, which in turn regulates the maturation of interleukin-1 ß (IL-1ß) and interleukin 18 (IL-18). IL-1ß was originally identified as a pro-inflammatory cytokine able to induce both local and systemic inflammation and a febrile reaction in response to infection or injury. Excessive production of IL-1ß is associated with autoimmune and inflammatory diseases. Microbial derivatives, bacterial pore-forming toxins, extracellular ATP and other pathogen-associated molecular patterns trigger activation of NLRP3 inflammasomes. Recent studies have reported that viroporin activity is capable of inducing inflammasome activity and production of IL-1ß, where NLRP3 is shown to be regulated by fluxes of K+, H+ and Ca2+ in addition to reactive oxygen species, autophagy and endoplasmic reticulum stress. The aim of this review is to present an overview of the key findings on viroporin activity with special emphasis on their role in virus immunity and as possible activators of inflammasomes.

The Impact of Pre-existing Comorbidities and Therapeutic Interventions on COVID-19.

Evidence from the global outbreak of SARS-CoV-2 has clearly demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from COVID-19. This is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. Not all comorbidities confer the same risk, however, many affect the function of the immune system, which in turn directly impacts the response to COVID-19. Furthermore, the myriad of drugs prescribed for these comorbidities can also influence the progression of COVID-19 and limit additional treatment options available for COVID-19. Here, we review immune dysfunction in response to SARS-CoV-2 infection and the impact of pre-existing comorbidities on the development of COVID-19. We explore how underlying disease etiologies and common therapies used to treat these conditions exacerbate COVID-19 progression. Moreover, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of COVID-19 in patients with pre-existing comorbidities.