Association between inflammatory cytokines/chemokines, clinical laboratory parameters, disease severity and in-hospital mortality in critical and mild COVID-19 patients without comorbidities or immune-mediated diseases.

There are limited data on inflammatory cytokines and chemokines; the humoral immune response; and main clinical laboratory parameters as indicators for disease severity and mortality in patients with critical and mild COVID-19 without comorbidities or immune-mediated diseases in Saudi Arabia. We determined the expression levels of major proinflammatory cytokines and chemokines; C-reactive protein (CRP); procalcitonin; SARS-CoV-2 IgM antibody and twenty-two clinical laboratory parameters and assessed their usefulness as indicators of disease severity and in-hospital death. Our results showed a significant increase in the expression levels of SARS-CoV-2 IgM antibody; IL1-ß; IL-6; IL-8; TNF-α and CRP in critical COVID-19 patients; neutrophil count; urea; creatinine and troponin were also increased. The elevation of these biomarkers was significantly associated and positively correlated with in-hospital death in critical COVID-19 patients. Our results suggest that the levels of IL1-ß; IL-6; IL-8; TNF-α; and CRP; neutrophil count; urea; creatinine; and troponin could be used to predict disease severity in COVID-19 patients without comorbidities or immune-mediated diseases. These inflammatory mediators could be used as predictive early biomarkers of COVID-19 disease deterioration; shock and death among COVID-19 patients without comorbidities or immune-mediated diseases.

Similarities between the effect of SARS-CoV-2 and HCV on the cellular level, and the possible role of ion channels in COVID19 progression: a review of potential targets for diagnosis and treatment.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted an urgent need to identify effective medicines for the prevention and treatment of the disease. A comparative analysis between SARS-CoV-2 and Hepatitis C Virus (HCV) can expand the available knowledge regarding the virology and potential drug targets against these viruses. Interestingly, comparing HCV with SARS-CoV-2 reveals major similarities between them, ranging from the ion channels that are utilized, to the symptoms that are exhibited by patients. Via this comparative analysis, and from what is known about HCV, the most promising treatments for COVID-19 can focus on the reduction of viral load, treatment of pulmonary system damages, and reduction of inflammation. In particular, the drugs that show most potential in this regard include ritonavir, a combination of peg-IFN, and lumacaftor-ivacaftor. This review anaylses SARS-CoV-2 from the perspective of the role of ion homeostasis and channels in viral pathomechanism. We also highlight other novel treatment approaches that can be used for both treatment and prevention of COVID-19. The relevance of this review is to offer high-quality evidence that can be used as the basis for the identification of potential solutions to the COVID-19 pandemic.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

A novel non-toxic combined CTA1-DD and ISCOMS adjuvant vector for effective mucosal immunization against influenza virus.

Here we demonstrate that by using non-toxic fractions of saponin combined with CTA1-DD we can achieve a safe and above all highly efficacious mucosal adjuvant vector. We optimized the construction, tested the requirements for function and evaluated proof-of-concept in an influenza A virus challenge model. We demonstrated that the CTA1-3M2e-DD/ISCOMS vector provided 100% protection against mortality and greatly reduced morbidity in the mouse model. The immunogenicity of the vector was superior to other vaccine formulations using the ISCOM or CTA1-DD adjuvants alone. The versatility of the vector was best exemplified by the many options to insert, incorporate or admix vaccine antigens with the vector. Furthermore, the CTA1-3M2e-DD/ISCOMS could be kept 1 year at 4°C or as a freeze-dried powder without affecting immunogenicity or adjuvanticity of the vector. Strong serum IgG and mucosal IgA responses were elicited and CD4 T cell responses were greatly enhanced after intranasal administration of the combined vector. Together these findings hold promise for the combined vector as a mucosal vaccine against influenza virus infections including pandemic influenza. The CTA1-DD/ISCOMS technology represents a breakthrough in mucosal vaccine vector design which successfully combines immunomodulation and targeting in a safe and stable particulate formation.

The proton translocation domain of cellular vacuolar ATPase provides a target for the treatment of influenza A virus infections.

BACKGROUND AND PURPOSE: Cellular vacuolar ATPases (v-ATPase) play an important role in endosomal acidification, a critical step in influenza A virus (IAV) host cell infection. We investigated the antiviral activity of the v-ATPase inhibitor saliphenylhalamide (SaliPhe) and compared it with several older v-ATPase inhibitors concanamycin A, bafilomycin A1, (BafA) and archazolid B targeting the subunit c of the V(0) sector. EXPERIMENTAL APPROACH: An in vitro assay was devised to quantify the anti-influenza effect of v-ATPase inhibitors by measuring green fluorescent protein fluorescence of a reporter IAV. These data were combined with cytotoxicity testing to calculate selectivity indices. Data were validated by testing v-ATPase inhibitors against wild-type IAV in vitro and in vivo in mice. KEY RESULTS: In vitro SaliPhe blocked the proliferation of pandemic and multidrug resistant viruses at concentrations up to 51-fold below its cytotoxic concentrations. At essentially non-toxic concentrations, SaliPhe protected 62.5% of mice against a lethal challenge of a mouse-adapted influenza strain, while BafA at cytotoxic concentrations showed essentially no protection against infection with IAV (SaliPhe vs. BafA P < 0.001). CONCLUSIONS AND IMPLICATIONS: Our results show that a distinct binding site of the proton translocation domain of cellular v-ATPase can be selectively targeted by a new generation v-ATPase inhibitor with reduced toxicity to treat influenza virus infections, including multi-resistant strains. Treatment strategies against influenza that target host cellular proteins are expected to be more resistant to virus mutations than drugs blocking viral proteins.

Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection.

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

M2e-based universal influenza A vaccine.

Human influenza causes substantial morbidity and mortality. Currently, licensed influenza vaccines offer satisfactory protection if they match the infecting strain, but they come with significant drawbacks. These vaccines are derived from prototype viruses, containing the hemagglutinin of influenza viruses that are likely to cause the next epidemic. Their usefulness against a future pandemic, however, remains problematic. A vaccine based on the ectodomain of influenza matrix protein 2 (M2e) could overcome these drawbacks. M2e is highly conserved in both human and avian influenza A viruses. The low immunogenicity against natural M2e can be overcome by fusing M2e to an appropriate carrier such as Hepatitis B virus-derived virus-like particles. Such chimeric particles can be produced in a simple and safe bacterial expression system, requiring minimal biocontainment, and can be obtained in a pure form. Experiments in animal models have demonstrated that M2e-based vaccines induce protection against a lethal challenge with various influenza A virus subtypes. Furthermore, the production and use of an effective M2e-vaccine could be implemented at any time regardless of seasonality, both in an epidemic as well as in a pandemic preparedness program. In animal models, M2e-vaccines administered parenterally or intranasally protect against disease and mortality following challenge with various influenza A strains. Adjuvants suitable for human use improve protection, which correlates with higher anti-M2e antibody responses of defined subtypes. Recently, Phase I clinical studies with M2e-vaccines have been completed, indicating their safety and immunogenicity. Further clinical development of this universal influenza A vaccine candidate is being pursued in order to validate its protective efficacy in humans.

CTA1-M2e-DD: a novel mucosal adjuvant targeted influenza vaccine.

At present few vaccine candidates exists against potentially pandemic influenza virus infections. We provide compelling evidence that a targeted fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the matrix protein 2 (M2e) ectodomain epitope, CTA1-3M2e-DD, confers strong protective immunity against a potentially lethal challenge infection with influenza virus in mice. The formulation was highly effective for mucosal immunizations and promoted high M2e-specific serum IgG and mucosal IgA antibody titers and an hitherto unknown anti-M2e CD4 T cell immunity. This novel CTA1-3M2e-DD fusion protein combines adjuvant and a conserved influenza A antigen in a promising candidate for a universal anti-influenza vaccine.

LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation.

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-kappaB (NF-kappaB). Although LPS-induced activation of NF-kappaB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-kappaB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-kappaB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-kappaB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-kappaB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKbeta. NF-kappaB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-kappaB activity in the liver, thereby partially protecting mice against LPS/D-(+)-galactosamine-induced mortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-kappaB activation.

Adenoviral gene transfer of ABIN-1 protects mice from TNF/galactosamine-induced acute liver failure and lethality.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a central role in acute and chronic hepatitis B and C infection and alcoholic liver disease as well as fulminant liver failure. TNF-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. The transcription factor NF-kappaB is believed to mediate at least part of the proinflammatory effects of TNF, and is therefore a favorite drug target. However, NF-kappaB also suppresses TNF-mediated hepatocyte apoptosis, implicating a potential cytotoxic effect of NF-kappaB inhibitors in the liver. This dual function of NF-kappaB emphasizes the need for therapeutics that can inhibit both TNF-induced NF-kappaB activation and cell death. Here we describe that adenoviral expression of the NF-kappaB inhibitory protein ABIN-1, but not an IkappaBalpha superrepressor (IkappaBalpha(s)), completely prevents lethality in the TNF/D-(+)-galactosamine-induced model of liver failure. Protection was associated with a significant decrease in TNF-induced leukocyte infiltration as well as hepatocyte apoptosis. The differential effects of ABIN-1 and IkappaBalpha(s) suggest a role for an NF-kappaB independent function of ABIN-1. Indeed, ABIN-1 was found to prevent not only NF-kappaB activation, but also apoptosis of cultured hepatocytes in response to TNF, explaining its protective effect against TNF-induced liver failure. In conclusion, ABIN-1 has a dual NF-kappaB inhibitory and anti-apoptotic activity in the liver, which might be of considerable interest for the treatment of inflammatory liver diseases.

2',5'-Oligoadenylate size is critical to protect RNase L against proteolytic cleavage in chronic fatigue syndrome.

A dysregulation in the 2',5'-oligoadenylate (2-5A)-dependent RNase L antiviral pathway has been detected in peripheral blood mononuclear cells (PBMC) of chronic fatigue syndrome (CFS) patients, which is characterized by upregulated 2-5A synthetase and RNase L activities, as well as by the presence of a low molecular weight (LMW) 2-5A-binding protein of 37-kDa related to RNase L. This truncated protein has been shown to originate from proteolytic cleavage of the native 83-kDa RNase L by m-calpain and human leukocyte elastase (HLE). We investigated the possible role of 2-5A oligomers in the proteolytic action toward the endonuclease and show that incubation of CFS PBMC extracts with 2-5A trimer and tetramer, but not with the dimer, results in a significant protection of the native 83-kDa RNase L against cleavage by endogenous and purified proteases. Similar results are obtained with a purified recombinant RNase L. An analysis of the size of 2-5A oligomers produced by the catalytic activity of the 2-5A synthetase present in PBMC extracts further shows that samples containing the 37-kDa RNase L preferentially produce 2-5A dimers instead of higher oligomers. Taken together, our results indicate that homodimerization of RNase L by 2-5A oligomers higher than the dimer prevents its cleavage by proteolytic enzymes. The presence of the truncated 37-kDa RNase L in PBMC extracts is therefore likely to result, not only from the abnormal activation of inflammatory proteases, but also from a dysregulation in 2-5A synthetase induction or activation towards the preferential production of 2-5A dimers.

Adenoviral gene transfer of the NF-kappa B inhibitory protein ABIN-1 decreases allergic airway inflammation in a murine asthma model.

Airway inflammation is a characteristic of many lung disorders, including asthma and chronic obstructive pulmonary disease. Using a murine model of allergen-induced asthma, we have demonstrated that adenovirus-mediated delivery of the nuclear factor-kappaB (NF-kappaB) inhibitory protein ABIN-1 to the lung epithelium results in a considerable reduction of allergen-induced eosinophil infiltration into the lungs. This is associated with an ABIN-1-induced decrease in allergen-specific immunoglobulin E levels in serum, as well as a significant reduction of eotaxin, interleukin-4, and interleukin-1beta in bronchoalveolar lavage fluid. These findings not only prove that NF-kappaB plays a critical role in the pathogenesis of allergic inflammation but also illustrate that inhibiting NF-kappaB could have therapeutic value in the treatment of asthma and potentially other chronic inflammatory lung diseases.

In vivo anti-tumour activity of recombinant MVM parvoviral vectors carrying the human interleukin-2 cDNA.

BACKGROUND: The natural oncotropism and oncotoxicity of vectors derived from the autonomous parvovirus, minute virus of mice (prototype strain) [MVM(p)], combined with the immunotherapeutic properties of cytokine transgenes, make them interesting candidates for cancer gene therapy. METHODS: The in vivo anti-tumour activity of a recombinant parvoviral vector, MVM-IL2, was evaluated in a syngeneic mouse melanoma model that is relatively resistant in vitro to the intrinsic cytotoxicity of wild-type MVM(p). RESULTS: In vitro infection of the K1735 melanoma cells prior to their injection resulted in loss of tumorigenicity in 70% of mice (7/10). Tumour-free mice were protected against a challenge with non-infected parental cells. In addition, MVM-IL2-infected tumour cells induced an anti-tumour activity on parental cells injected at a distant location. These non-infected tumour cells were injected either at the same time or 7 days before the injection of MVM-IL2-infected cells. In the latter setting, which mimics a therapeutic model for small tumours, 4/10 mice were still tumour-free after 4 months. CONCLUSIONS: Our results show that (i) the MVM-IL2 parvoviral vector efficiently transduces tumour cells; and (ii) the low multiplicity of infection (MOI = 1) used in our experiments was sufficient to elicit an anti-tumour effect on distant cells, which supports further studies on this vector as a new tool for cancer gene therapy.