Christian Setz

Background/Objectives: Seasonal waves of respiratory viruses—including SARS-CoV-2, influenza A virus (IAV), and respiratory syncytial virus (RSV)—continue to pose a global health burden and highlight the need for antiviral agents that are effective, safe, broadly active, affordable, and widely accessible. Current interventions are limited by the need for their early administration, the risk of resistance, their costs, and the restricted availability in large parts of the world. For certain natural products, such as European black elderberry (Sambucus nigra L.) fruit extract (ElderCraft^®; EC) and the seaweed-derived sulfated polymer iota-carrageenan (IC), antiviral activities against respiratory viruses, particularly IAV and SARS-CoV-2, have previously been shown. Here, we assessed the antiviral activity of IC and an anthocyanin-standardized EC extract against SARS-CoV-2, IAV, and RSV, either as monotherapy or in multiple-dose combinations. Methods: MDCKII cells were infected with IAV_PR8, human Calu-3 lung epithelial cells with the SARS-CoV-2 Omicron variant, and HEp-2 cells with RSV (A2 strain). Inhibitors were administered either by pre-incubation of cell-free virions prior to infection or, in separate time-of-addition experiments, during or post-infection. Viral replication was quantified by qRT-PCR or intracellular immunostaining. Cytotoxicity was evaluated using a neutral red uptake assay. Results: Most intriguingly, both EC and IC are able to neutralize virions derived from SARS-CoV-2, IAV, or RSV extracellularly in a dose-dependent manner. Notably, EC and IC alone exhibited strong anti-RSV activity, which was not reported previously. Most importantly, combined treatment with IC and EC caused a pronounced synergistic antiviral effect against the tested viruses, as confirmed by the Bliss independence model, without any detectable impact on cell viability. Finally, solutions prepared from matrix-standardized mono- or combi-lozenges, containing IC and/or EC in high or low doses, reproduced the antiviral and synergistic combination effects observed with the pure compounds. Conclusions: In summary, these findings support further development of EC and IC as a topically accessible, virion-neutralizing combination (e.g., lozenges) to provide additional protection against major respiratory viruses and potentially strengthen pandemic preparedness.

Background/Objectives: The persistent threat of emerging respiratory RNA viruses like SARS-CoV-2 and Influenza A virus (IAV) necessitates the continuous development of effective, safe, broadly acting, and generally accessible antiviral agents. Current treatments often face limitations such as early administration requirements, resistance development, and limited global access. Natural products, like European black elderberry (Sambucus nigra L.; S. nigra) fruit extract and quinine, have been used historically against viral infections. In this study, we investigated the antiviral efficacy of a standardized black elderberry fruit extract containing 3.2% anthocyanins (EC 3.2) and, as a second natural antiviral product, quinine, against IAV and SARS-CoV-2 in vitro. Methods: Madin–Darby Canine Kidney II (MDCKII) cells were infected with IAV PR-8, while human Calu-3 lung epithelial cells were infected with Wuhan-type SARS-CoV-2. Cells were treated with varying concentrations of EC 3.2 and quinine either as mono- or combinational therapy. Viral replication was assessed using quantitative RT-PCR, and cell viability was evaluated using WST-1 assays. Results: Our results demonstrate, for the first time, that both EC 3.2 and quinine individually inhibited IAV replication in a dose-dependent manner, with IC₅₀ values of approximately 1:400 for EC 3.2 and 250 nM for quinine. Most importantly, the combinational treatment exhibited a strong synergistic antiviral effect, as confirmed by the Bliss independence model (synergy scores of 14.7 for IAV, and 27.8 for SARS-CoV-2), without affecting cell viability. Conclusions: These findings suggest that the combined use of black elderberry extract and quinine might serve as an effective antiviral strategy against IAV and SARS-CoV-2, particularly since the synergistic effect allows for lower doses of each product while retaining therapeutic efficacy. In summary, this combinational in vitro approach, when expanded to other respiratory RNA viruses and confirmed in clinical studies, has the potential to open a promising avenue for pandemic preparedness.

The persistent threat of emerging respiratory RNA viruses like SARS-CoV-2 and Influenza A virus (IAV) necessitates the continuous development of effective, safe, broadly acting and gener-ally accessible antiviral agents. Current treatments often face limitations such as early admin-istration requirements, resistance development, and limited global access. Natural products like European black elderberry (Sambucus nigra L.) fruit extract and quinine have been used histori-cally against viral infections. In this study, we investigated the antiviral efficacy of a standard-ized black elderberry fruit extract containing 3.2 % anthocyanins (EC 3.2) and, as a second natu-ral antiviral product, quinine, against IAV and SARS-CoV-2 in vitro. Madin-Darby Canine Kid-ney II (MDCKII) cells were infected with IAV PR-8, while human Calu-3 lung epithelial cells were infected with SARS-CoV-2 Wuhan type. Cells were treated with varying concentrations of EC 3.2 and quinine as either mono– or combinational therapy. Viral replication was assessed using quantitative RT-PCR, and cell viability was evaluated using WST-1 assays. Our results demonstrate for the first time that both EC 3.2 and quinine individually inhibited IAV replica-tion in a dose-dependent manner, with IC₅₀ values of approximately 1:400 for EC 3.2 and 250 nM for quinine. Most importantly, the combinational treatment exhibited a strong synergistic anti-viral effect, as confirmed by the Bliss independence model (synergy scores of 14.7 for IAV and 27.8 for SARS-CoV-2) without affecting cell viability. These findings suggest that the combined use of black elderberry extract and quinine may serve as an effective antiviral strategy against IAV and SARS-CoV-2. The synergistic effect allows for lower doses of each product while keep-ing therapeutic efficacy. In summary, this combinational approach should be expanded to other respiratory RNA viruses, and thus might offer a promising avenue for pandemic preparedness. Moreover, the in vitro data obtained in this study have to be confirmed in clinical studies.

Even with its endemic transition, the COVID-19 pandemic remains a public health threat, particularly in the light of emerging variants of concern (VoCs) and the need for pandemic preparedness in the future. In November 2021, the SARS-CoV-2 VoC Omicron emerged and its subvariants BA.1, BA.2 and BA.5 became predominant. Although the protease inhibitor Paxlovid^® and the polymerase inhibitors Molnupiravir and Remdesivir were approved as specific antiviral treatment options for COVID-19 patients in the early stages after infection, effective prophylactically acting substances without adverse effects are not available yet. In a recent study, we demonstrated that iota-carrageenan, a sulfated polysaccharide extracted from red seaweed, efficiently inhibits the replication of the SARS-CoV-2 Wuhan Type and the VoCs Alpha, Beta, Gamma and Delta. Now, we extended this study by investigating the antiviral effects of iota-, lambda- and kappa-carrageenans on the VoC Omicron subvariants BA.1, BA.2 and BA.5. Using a VoC Omicron BA.1 spike pseudotyped murine leukemia virus (BA.1 MLV_OMVLP) as well as patient-derived SARS-CoV-2 Omicron isolates BA.1, BA.2 and BA.5 (SARS-CoV-2_{OM BA.1}, SARS-CoV-2_{OM BA.2} and SARS-CoV-2_{OM BA.5}), we demonstrate that iota-carrageenan exhibits similar antiviral activity against all analyzed Omicron subvariants. As with other VoCs shown before, the biologically inert iota-carrageenan was more efficient than kappa- and lambda-carrageenan. Altogether, these results confirm that, independent of the current and potential future variants, the physical barrier provided by iota-carrageenan might be applicable for prophylaxis and early treatment of SARS-CoV-2 infections.

Coronavirus disease-19 (COVID-19) is still affecting the lives of people around the globe and remains a major public health threat. Lipid levels in the host cells have been shown to promote SARS-CoV-2 replication, and since the start of COVID-19 pandemic, several studies have linked obesity and other components of the metabolic syndrome with severity of illness, as well as mortality in patients with COVID-19. The aim of this study was to obtain insights into the pathophysiological mechanisms of these associations. First, we established an in vitro model simulating high fatty acid levels and showed that this situation induced the uptake of fatty acids and triglyceride accumulation in human Calu-3 lung cells. Importantly, we found that lipid accumulation significantly enhanced the replication of SARS-CoV-2 Wuhan type or the variant of concern, Delta, in Calu-3 cells. In summary, these findings indicate that hyperlipidemia as found in patients with obesity promotes viral replication and herewith the disease course of COVID-19.

Coronavirus disease-19 (COVID-19) is still affecting the lives of people round the globe and remains a major public health threat. The emergence of new variants more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity creates a long-term negative outlook for the management of the pandemic. The development of effective and viable prevention and treatment options to reduce viral transmission is of the utmost importance. The fruits of the European black elderberry and extracts thereof have been traditionally used to treat viral infections such as coughs, cold and flu. Specifically, its efficacy against the Influenza A virus has been shown in vitro as well as in human clinical trials. In the current project, we investigated the antiviral activity of a black elderberry extract, mainly containing anthocyanins and phenolic compounds, against SARS-CoV-2 and its variants of concern and explored the possible mode of action by performing time of addition experiments. The results revealed that the extract displayed a strong anti-SARS-CoV-2 activity against the Wuhan type as well as the variants of concern Alpha, Beta, Gamma, Delta and Omicron with a comparable antiviral activity. Based on cytotoxicity data, a 2-log theoretical therapeutic window was established. The data accumulated so far suggest that the viral replication cycle is inhibited at later stages, inasmuch as the replication process was affected after virus entry. Therefore, it would be legitimate to assume that black elderberry extract might have the potential to be an effective treatment option for SARS-CoV-2 infections.

The ubiquitin proteasome system (UPS), particularly its deubiquitinating enzymes (DUBs), play a key role in the replication cycle of coronaviruses. The SARS-CoV-2 papain-like protease (Plpro) is known to process the viral polyproteins to form the replicase transcriptase complex and to counteract the host viral response. Recently, it was shown that this viral protease can also act as a deubiquitinating enzyme. In this study, we demonstrate that certain DUB-Inhibitors (DIs) interfere with SARS-CoV-2 replication. The DIs PR-619 and HBX41108 restrict SARS-CoV-2 in both Vero B4 and human Calu-3 lung cells where cells were infected with a Multiplicity of Infection (MOI) of 0.02. An in vitro protease assay using recombinant Plpro and Amido-4-methylcoumarin (AMC)-conjugated substrate revealed that PR-619 and HBX41108 are able to block the protease at concentrations where the interventions restricted virus replication. In contrast, DIs that do not inhibit Plpro had no influence on virus replication, which indicated that the protease might be at least one major target. Future vertical studies that would gain more insights into the mechanisms of how DUBs effect the replication of SARS-CoV-2 will further validate them as a potential therapeutic target.

The SARS-CoV-2 pandemic remains a major public health threat, especially due to newly emerging SARS-CoV-2 Variants of Concern (VoCs), which are more efficiently transmitted, more virulent, and more able to escape naturally acquired and vaccine-induced immunity. Recently, the protease inhibitor Paxlovid^® and the polymerase inhibitor molnupiravir, both targeting mutant-prone viral components, were approved for high-risk COVID-19 patients. Nevertheless, effective therapeutics to treat COVID-19 are urgently needed, especially small molecules acting independently of VoCs and targeting genetically stable cellular pathways which are crucial for viral replication. Pamapimod is a selective inhibitor of p38 Mitogen-Activated Protein Kinase alpha (p38 MAPKα) that has been extensively clinically evaluated for the treatment of rheumatoid arthritis. Signaling via p38 has recently been described as a key pathway for the replication of SARS-CoV-2. Here, we reveal that the combination of pamapimod with pioglitazone, an anti-inflammatory and approved drug for the treatment of type 2 diabetes, possesses potent and synergistic activity to inhibit SARS-CoV-2 replication in vitro. Both drugs showed similar antiviral potency across several cultured cell types and similar antiviral activity against SARS-CoV-2 Wuhan type, and the VoCs Alpha, Beta, Gamma, Delta, and Omicron. These data support the combination of pamapimod and pioglitazone as a potential therapy to reduce duration and severity of disease in COVID-19 patients, an assumption currently evaluated in an ongoing phase II clinical study.

The COVID-19 pandemic continues to spread around the world and remains a major public health threat. Vaccine inefficiency, vaccination breakthroughs and lack of supply, especially in developing countries, as well as the fact that a non-negligible part of the population either refuse vaccination or cannot be vaccinated due to age, pre-existing illness or non-response to existing vaccines intensify this issue. This might also contribute to the emergence of new variants, being more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity. Hence, the need of effective and viable prevention options to reduce viral transmission is of outmost importance. In this study, we investigated the antiviral effect of iota-, lambda- and kappa-carrageenan, sulfated polysaccharides extracted from red seaweed, on SARS-CoV-2 Wuhan type and the spreading variants of concern (VOCs) Alpha, Beta, Gamma and Delta. Carrageenans as part of broadly used nasal and mouth sprays as well as lozenges have the potential of first line defense to inhibit the infection and transmission of SARS-CoV-2. Here, we demonstrate by using a SARS-CoV-2 spike pseudotyped lentivirus particles (SSPL) system and patient-isolated SARS-CoV-2 VOCs to infect transgenic A549ACE2/TMPRSS2 and Calu-3 human lung cells that all three carrageenan types exert antiviral activity. Iota-carrageenan exhibits antiviral activity with comparable IC₅₀ values against the SARS-CoV-2 Wuhan type and the VOCs. Altogether, these results indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants.

Even in the face of global vaccination campaigns, there is still an urgent need for effective antivirals against SARS-CoV-2 and its rapidly spreading variants. Several natural compounds show potential as antiviral substances and have the advantages of broad availabilities and large therapeutic windows. Here, we report that lectin from Triticum vulgaris (Wheat Germ Agglutinin) displays antiviral activity against SARS-CoV-2 and its major Variants of Concern (VoC), Alpha and Beta. In Vero B4 cells, WGA potently inhibits SARS-CoV-2 infection with an IC₅₀ of 50 values. Altogether, our data indicate that topical administration of WGA might be effective for prophylaxis or treatment of SARS-CoV-2 infections.