Towards the Development of a Cream with Antiviral Properties Targeting Both the Influenza A Virus and SARS-CoV-2

Introduction

Respiratory viruses are responsible for the most common causes of acute respiratory infections in humans. Throughout human history, outbreaks of virally induced respiratory diseases have been reported and have caused severe death tolls. Among the many respiratory viruses, influenza, and more recently coronaviruses, are major concerns for human health and have caused millions of deaths over the last century.

The influenza type A virus pandemic (H1N1 subtype), also referred to as the ‘Spanish Flu’, was one of the most devastating viral pandemics, with an approximate death toll of about 50 million people worldwide in less than 2 years, between 1918 and 1920 [1]. More recently, the new pulmonary disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide since December 2019 and was declared a pandemic by the World Health Organization (WHO) in March 2020 [1].

Influenza viruses belong to the orthomyxovirus family of RNA viruses, which is composed of three groups (A, B and C), with type A being the most virulent [2]. Human-to-human transmission of influenza mainly occurs through the air via respiratory droplets or aerosols, but it can also be contracted via contact with contaminated surfaces. IAV infection causes a spectrum of clinical symptoms, from a benign upper respiratory tract infection to fulminant pneumonia, which mostly strikes the elderly and immunocompromised patients [3]. SARS-CoV-2—in addition to MERS-CoV and SARS-CoV-1—belongs to the beta coronavirus family and is considered to have emerged from bats and spread to humans very recently. The mode of transmission from human to human occurs through multiple routes that mainly include the emission and inhalation of droplets and aerosols, but also indirect contamination via hand contact with surfaces and objects [4]. In addition to the fact that both SARS-CoV-2 and influenza viruses are enveloped and contain a single-stranded RNA genome, they also share several common features such as the transmission route, clinical symptoms, viral shedding and the serial interval of disease. For both pathogens, vaccines are available, and although they have considerably restricted the progression of the COVID-19 pandemic and attenuated the severity of the clinical symptoms, they cannot completely eliminate the spread of and infection with viruses [2].

In order to invade and infect their hosts, both SARS-CoV-2 and influenza need to establish initial contact with the upper respiratory tract through aerosols and droplets that are inhaled through the nose and mouth. Thus, a first line of defense against these pathogens consists of wearing facial masks, and this strategy has been adopted worldwide as a simple and efficient measure for slowing down the progression of the pandemic. However, indirect contamination can also occur through contact transmission of the pathogens from various media on which they can settle. As such, virally contaminated droplets can be deposited on the surface of various objects, such as tissues, coins and bank notes, that can be touched by hand. This, in turn, can contact the mucous membranes of the mouth, nose and eyes that then mediate viral entry to and infection of the patient. As for the influenza H1N1 strain, it has been shown [5] that viruses could persist and remain infectious on stainless steel surfaces for 7 days. Recent experiments with SARS-CoV-2 [6] have also demonstrated the presence of viable virus for up to 72 h, depending on the environmental surface conditions tested. This was confirmed in several general studies [7,8] that showed that the vast majority of respiratory tract viruses, such as coronaviruses, influenza and rhinovirus, could persist on inanimate surfaces for a few days, thus concluding that fomite transmission for influenza and SARS-CoV-2 is likely to occur and form part of the infectious process.

Given that contact transmission is a substantial risk factor in the spreading of both influenza and SARS-CoV-2, this emphasizes the need to employ proper hand hygiene to prevent the transmission of these pathogens, and this was officially recommended by the WHO as one of the first preventative measures to slow down the propagation of SARS-CoV-2 [7,9,10]. Although frequent hand washing can be easily performed, this is not the case for the face, which remains exposed in the absence, or inappropriate wearing, of a facial mask. Furthermore, the frequent use of antiviral disinfectants is a major cause of skin irritations, especially in healthcare workers. As such, the prolonged use of these measures induces various skin reactions, with a prevalence ranging from 43% [11] to 97% [12] among healthcare workers exposed to infected patients.

These facts formed the rationale for investigating the design and manufacture of a cream that could be applied to both the face and hands and that would exhibit antiviral activities. Surprisingly, this approach has not been thoroughly investigated since the beginning of the SARS-CoV-2 outbreak, as to our knowledge, there has been only one study published testing the virucidal activity of a cream/lotion on human skin [13]. This is even more surprising if one considers that better face protection would certainly have a significant impact on person-to-person transmission. Therefore, this established the rationale for this work, which was to conceive and produce a daily applicable skin cream with virucidal activity.

As highly versatile vehicles, lentivectors are widely used for gene delivery purposes, since they allow for stable integration of transgenes into cell lines and differentiated cells. Interestingly, they can be engineered to incorporate envelopes from different viral origins that modulate their tropism for recipient cells [14] and authorize their use for serological investigations [15] or the identification of antiviral reagents [16]. In our attempt to compose a cream with viral protective activity, we first tested the ability of different chemical and natural compounds to inactivate lentiviral-based pseudo-particles pseudotyped with the proteins HA (hemagglutinin) and NA (neuraminidase) from influenza or the G protein from the vesicular stomatitis virus in human embryonic kidney (HEK293T) cells.

Among these compounds, cyclodextrins have been shown to possess virucidal effects against many viruses cultivated in vitro, including the herpes simplex virus (HSV), respiratory syncytial virus (RSV), dengue virus and Zika virus [17,18,19,20]. In addition, sulfated polysaccharides that can be extracted from natural plants or fungi can interfere with several steps in the virus’ life cycle without presenting adverse biological effects [21]. Dipotassium glycyrrhizinate (DG), a natural triterpene that can be isolated from the roots of licorice, has been shown to inhibit the replication of hepatitis B virus and human immunodeficiency virus [22,23]. We chose two sulfated polysaccharides from algae extracts: One was furcellaran, a sulfated polysaccharide (carrageenan), which was extracted from the cell wall matrix of red seaweed belonging to the genus Furcellaria. The second was an extracellular sulfated polysaccharide (EPS) from the genus Porphyridium (Porphyridium cruentum extract).

The typical antiviral mechanism of most sulfated polysaccharides against enveloped viruses can be explained as follows: These viruses attach to host cells through the interaction between their glycoproteic envelope and the heparan sulfate receptor on the cell surface. The formation of the virus–cell complex primarily relies on ionic interactions between the negatively charged (mostly sulfate) groups in this polysaccharide and the basic amino acids within the glycoprotein [18]. This suggests that the antiviral effect occurs by effectively neutralizing the positively charged sites on the viral envelope glycoproteins, preventing the viral adsorption process.

We also tested beta-cyclodextrin (KLEPTOSE®). Cyclodextrins (CDs) are occurring glucose derivatives with a rigid cyclic structure, consisting of (1–4)–linked glucopyranoside units. Beta-cyclodextrin has previously been reported as displaying antiviral activity against a number of enveloped viruses [19]. We also tested calcium D pantetheine (CAD) and DG. We initially tested these compounds in a model of cultured cells challenged with the pseudotyped VLPs containing HA-NA from IAV or the G protein from the vesicular stomatitis virus and expressing a reporter gene in order to assess the virucidal effects of the compounds. In the second part of this study, we used human skin explants derived from donors on which diverse versions of the cream were applied. In this experiment, in addition to HA-VPs, we added lentivectors pseudotyped with the envelope of SARS-CoV-2 into the viral inoculum, and in particular a spike variant, to assay the virucidal activity of our formula on IAV and SARS-CoV-2.

Our results show that some compounds are effective at inactivating VLPs bearing HANA and spike and significantly reduce their infectious potential. Moreover, when incorporated into a cream formulation, these compounds remain as active as in the cell culture. This suggests that they can be developed to be incorporated into a face or a hand cream formulation.

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Materials

The algae extract (AE) was sourced from the red seaweed Furcellaria lumbricalis, located in the Baltic Sea. The Baltic Sea has a salinity of 10 g·L−1, very close to physiological serum, which could well explain its affinity for the skin. This extract was obtained from Codif (Saint-Malo, France). Furcellaran is a sulfated polysaccharide that is extracted from the cell wall matrix of red seaweed belonging to the genus Furcellaria. The furcellaran was then depolymerized using a patented process. The structure of furcellaran is similar to kappa carrageenan and has been described as a hybrid of kappa–beta carrageenan complex. The essential difference is that kappa carrageenan has one sulfate ester residue per two sugars, whereas furcellaran has one sulfate ester residue per three or four sugar residues. The AE master solution (10 mg/mL) was prepared in water heated at 70 °C for complete dissolution. The working solutions were prepared rapidly through dilution of the master solution in a warm culture medium. The extracellular sulfated polysaccharide (EPS) extract was developed by Givaudan Active Beauty Marine (Toulouse, France). Red unicellular microalgae from the genus Porphyridium (Porphyridium cruentum extract) produce an extracellular sulfated polysaccharide (EPS) with acidic characteristics and with potential applications in cosmetics, as an inhibitor of hyaluronidase and having anti-allergic and antiviral properties. Betacyclodextrin (KLEPTOSE®) was developed by Roquette (La madeleine, France). The master solutions for KLEPTOSE® (10 mg/mL), dipotassium glycyrrhizinate (50 mg/mL) and calcium D pantetheine-S-sulfonate (3 mg/mL) were prepared in culture medium. All solutions were filtered through a 0.2 μm-pore-sized filter, except for AE. Hydrapatch® is a unique combination of three polysaccharides: pullulan (repeating maltotriose units), alginate (polymer of D-mannuronic and L-glucuronic units) and hyaluronic acid. Hydrapatch® can regulate the rate and kinetics of absorption of active ingredients and was developed by BASF (Levallois Perret, France). Liposkin® was developed by Lucas Meyer (Massy, France) and is a complex of C12–16 alcohols, hydrogenated lecithin, palmitic acid, phytosphingosine and cholesterol.

Mangeot, P.; Lazou, K.; Blin, A.; Gorzelanczyk, V.; Jeanneton, O.; Kurfurst, C.; Pays, K.; Bavouzet, B.; Nizard, C.; Ohlmann, T.; et al. Towards the Development of a Cream with Antiviral Properties Targeting Both the Influenza A Virus and SARS-CoV-2. Cosmetics 2024, 11, 91. https://doi.org/10.3390/cosmetics11030091

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