Scientist from Friedrich-Alexander University Erlangen-Nürnberg-Germany, Ulm University Medical Center-Germany, University Hospital Tübingen-Germany and Marinomed Biotech AG-Austria have in a new study found that the safe and non-toxic natural compound iota-carrageenan has the ability to inhibit replication of all existing SARS-CoV-2 variants of concern or VOCs.
The study team 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.
The study team 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.
The study findings indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants.
The study findings were published in the peer reviewed International Journal of Molecular Sciences.
The antiviral activity of iota-carrageenan is well-established and has been demonstrated for a variety of respiratory viruses [35,36,37]. Recently, it has been shown that iota-carrageenan inhibits SARS-CoV-2 replication in various cell lines  and in primary differentiated human airway epithelial cultures . For lambda-carrageenan an antiviral activity in Vero E6 cells against SARS-CoV-2 was reported .
Moreover, treatment of respiratory viral infection with iota-carrageenan nasal spray has already been shown to be safe and effective in five randomized, double blinded, placebo-controlled clinical trials with more than 600 children and adults suffering from respiratory viral infection. Therapeutic application of an iota-carrageenan nasal spray reduced the viral load, which also manifested clinically by reducing the severity and duration of symptoms as well as the number of relapses in the verum group [41,42,43,44,45].
Very recently, it was shown in a multicenter, randomized, double-blinded, placebo-controlled clinical study, that an iota-carrageenan containing nasal spray exhibits prophylactic efficacy in preventing SARS-CoV-2 infection in healthcare workers caring for patients with COVID-19 disease with a relative risk reduction of 79.8% .
An earlier trial investigating a nasal spray containing Ivermectin and iota-carrageenan showed reduction in COVID-19 as well as of disease severity . Furthermore, there are clinical trials on COVID-19 cases running in the UK  and in Austria [49,50] investigating the prophylactic and therapeutic effect of iota-carrageenan as inhalant.
We previously published that iota-carrageenan exhibits antiviral activity against the SARS-CoV-2 Wuhan type in Vero B4 cells . In our current work, we show for the first time that carrageenans exhibits antiviral activity not only against the SARS-CoV-2 Wuhan type but also the VOCs Alpha, Beta, Gamma and Delta with comparable IC50 values.
This effect was shown for all carrageenan types with iota being the most effective, occurring in human cell lines and in the SSPL system, as shown for SARS-CoV-2 Wuhan type, and the VOCs Alpha, Beta and Gamma.
The IC50 values varied depending on the form of infection and cell lines used, ranging from 1.4–5 µg/mL iota-carrageenan in the SSPL particles system to 2.1–10.3 µg/mL in A549-ACE2/TMPRSS2 cells, and 0.04–0.15 µg/mL in Calu-3 human lung cells infected with the SARS-CoV-2 variants. Thereby, iota-carrageenan displayed an at least 10-fold higher efficacy when compared to lambda- and kappa-carrageenan.
Since the beginning of the outbreak in December 2019, the causative virus of the COVID-19 pandemic exerted a dramatic health and socioeconomic crisis worldwide. It can be assumed that, as before for SARS-CoV and MERS-CoV, in the future, coronaviruses could spread from animals to humans via zoonotic transmission, potentially causing pandemic threats.
This clearly necessitates a general need for pandemic preparedness. Regarding SARS-CoV-2, worldwide vaccination programs remain a challenge as inefficiency and a lack of supply particularly in developing countries might lead to the emergence of new variants that may be more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity.
Thus, there is a tremendous need for the development of new therapeutics that are broadly active, safe, relatively cheap and easily distributable for a wide range of patients when compared to standard antivirals. As an alternative approach to the repurposing of existing synthetic drugs, like hydroxy-chloroquine and remdesivir, which was proven ineffective , natural substances would have the advantages of a better toxicological profile with a larger therapeutic window, less side effects and a faster admission process in comparison to chemically engineered drugs.
In addition, due to their general mode of action, most naturals exhibit a broad antiviral spectrum when compared, for instance, to highly specific monoclonal antibodies or small molecule inhibitors of viral factors. In the past, the beneficial effects of natural substances were shown for many diseases, including metabolic disorders or cancer . Most importantly, they also proved promising against a variety of different viruses, including SARS-CoV, MERS-CoV and SARS-CoV-2 [31,32,33].
In this study, we demonstrate that the natural substance iota-carrageenan, exerts antiviral activity not only against the originally emerged SARS-CoV-2 Wuhan type but also against the VOCs Alpha, Beta, Gamma and Delta. Data from a SSPL system as well as from transgenic A549-ACE2/TMPRSS2 and human Calu-3 lung cells were comparable indicating that carrageenans exhibit a broad antiviral activity.
The antiviral effect of iota-carrageenan was shown against several respiratory viruses in vitro [35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59] and was also proofed in clinical trials [41,42,43,44,45,46]. Interestingly, a very recent study revealed a synergistic antiviral activity using a combination of carrageenan with another natural substance, the lectin Griffithsin, when tested against SARS-CoV-2 pseudoviruses .
Hemilä et al. performed an independent meta-analysis with publicly available data from clinical studies with an iota-carrageenan containing nasal spray . The authors attest the quality of the available clinical data as well as the broad effectiveness against influenza viruses and endemic coronaviruses .
They further conclude that carrageenan may also inhibit the new coronavirus SARS-CoV-2, an assumption that was confirmed by a recent clinical study showing that an iota-carrageenan containing nasal spray give significant protection when used as COVID-19 prophylaxis in health care workers managing patients with COVID-19 disease .
Furthermore, another recently published clinical study underlined this assumption as it demonstrated that sucking iota-carrageenan containing Lozenges inhibit viral replication of SARS-CoV-2 ex vivo . Moreover, there are regional different recommendations for the use of iota-carrageenan in prevention of COVID-19, as for example by the German Society of Hospital Hygiene and by Austrian society [62,63].
Nasal sprays, throat sprays and lozenges containing carrageenans are approved common cold prevention options and have been launched in more than 20 countries . The most commonly used carrageenan in these products is iota-carrageenan. To test whether the types of carrageenan have different antiviral properties, we compared iota-, kappa- and lambda-carrageenan in various infection experiments. Although all carrageenan types showed antiviral effects against SARS-CoV-2 Wuhan type and its VOCs Alpha, Beta and Gamma, iota-carrageenan clearly led to the strongest reduction with IC50 values that are ~1 log-stage lower than that for lambda- or kappa-carrageenan.
These results are in concert with other studies were it was shown that iota-carrageenan exhibits the most potent reduction of human rhinovirus infection and hepatitis A virus replication when compared to kappa- or lambda-carrageenan [35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65]. The molecular basis for the observed difference, however, remains widely unclear. Interpretation of data is hampered by the finding, that even purchased preparations of kappa- and lambda-carrageenan contained 16 % and 27.3 % iota-carrageenan, respectively , thus, indicating that the observed antiviral activity of kappa- and lambda-carrageenan is rather due to the presence of iota-carrageenan than an effect of the respective polymers themselves.
There are some differences between the IC50 values of iota-carrageenan obtained for the SSPL and SARS-CoV-2 systems (Table 1). Pseudo-typed virus-like particles versus replication competent viruses react different to carrageenans, as also non-human Vero as well as human lung cells exhibit differences. In addition, A549 cells are transgenic for ACE2/TMPRSS2, whereas Calu-3 express ACE2 and TMPRSS2 endogenously [31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66]. However, there was a clear antiviral activity with comparable IC50 values against the SARS-CoV-2 Wuhan type and the VOCs following treatment with iota-carrageenan for all systems.
Carrageenans not only have antiviral properties but also exert antitumor, immunomodulatory and coagulative activities, respectively [58,59,60,61,62,63,64,65,66,67,68,69,70,71]. In this context, it was published that kappa- and lambda-carrageenans exhibit both anticancer and immunostimulatory effects [58,59,60,61,62,63,64,65,66,67,68,69,70]. In contrast, such immunomodulating and antitumoral effects have not been described yet for iota-carrageenan, supporting a broad application as an otherwise biologically inert substance. Furthermore, iota-carrageenan has been approved as food safe by the EFSA  and by the FDA  for a quantum satis of 75 mg/kg b.w. per day which would translate into 10 L of plum pudding .
The sulfated polysaccharide does not enter the human cells as it is degraded before . In concert with this, a non-clinical study analyzing iota-carrageenan showed no local intolerance or toxicity when iota-carrageenan was applied intranasally or by inhalation . In addition, no immunogenicity or immunotoxicity were observed as there was no stimulation of a panel of pro-inflammatory cytokines by iota-carrageenan detectable .
In agreement with this, our data showed no toxic effect on A549-ACE2/TMPRSS2+ and Calu-3 cells when treated with different concentrations of iota-carrageenan up to 100 µg/mL. It is intriguing to hypothesize that iota-carrageenan, which shows the best antiviral activity in comparison with the other types of carrageenan, is biologically inert and, thus, can be used against SARS-CoV-2 and variants in the ongoing pandemic without any concern in the view of adverse effects.
Various carrageenans and other sulpated polysaccharides, like heparin, dextran sulfate and pentosan sulfate, exert antiviral activity against enveloped viruses [75,76,77,78,79]. In order to interact with their specific host cell receptors , it is hypothesized that viruses utilize their positive electrical charge to reach the negatively charged cell surface . Polyanionic molecules, such as iota-carrageenan may present a way of trapping the virus due to their affinity for basic viral surfaces . As iota-carrageenan non-specifically envelops viruses, thereby, preventing interaction between virus and cellular surface, the development of resistance due to the occurrence of escape mutants is unlikely.
This current knowledge is backed by a series of experiments showing that direct interaction between virus and carrageenan is needed to efficiently inhibit infection of cells [35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82]. The activity of carrageenan is based on its ability to neutralize virus particles when they first enter the nasal cavity, and additionally when newly synthesized virus particles are released from infected cells.
The long, negatively charged iota-carrageenan molecules attract and trap these newly released positively charged viruses and sterically hinder them from binding to their host cells, thereby, inhibiting the infection of adjacent epithelial cells. Finally, iota-carrageenan together with the trapped viruses will then be eliminated by mucociliary clearance .
The lack of any pharmacological, immunological or toxicological activity of large polyanionic molecules such as iota-carrageenan and their lack of absorption or metabolism makes them a safe biologically inert antiviral that can be applied topically, e.g., as lozenges or nasal spray. Since iota-carrageenan has similar IC50 values following infection with different VOCs, a covalent selective interaction of carrageenan with the spike protein of SARS-CoV-2 appears rather unlikely, while electrostatic encasement of virions mediated by the sulpated polysaccharide supports prophylactically application of this naturally occurring substance against SARS-CoV-2 regardless of newly emerging variants.