key: cord-0269466-wis4pmna
authors: Mukherjee, Sayandip; Vincent, Carol K.; Jayasekera, Harshinie W.; Yekhe, Ashish Shrikant
title: Personal care formulations demonstrate virucidal efficacy against multiple SARS-CoV-2 variants of concern: implications for hand hygiene
date: 2021-09-23
journal: bioRxiv
DOI: 10.1101/2021.09.20.461073
sha: fa0b599c1e43007a3a501d22335626b26b0e5ddb
doc_id: 269466
cord_uid: wis4pmna

The second and third waves of COVID-19 pandemic have largely been driven by the surge of successive SARS-CoV-2 variants of concern (VOC). These VOC have rapidly spread through multiple geographies being enabled by high transmission rates and/or high viral load compared to the original parent strain. Consequently, the altered phenotypes of these VOC have posed greater challenges to diagnostic and clinical management of COVID-19. Despite considerable progress being made on vaccine roll out, practicing proper hand hygiene has been advocated as a consistent precautionary intervention as more virulent VOC continue to emerge and spread across geographies. Two variants of concern, namely beta and delta, have recently been shown to escape antibody-mediated neutralization by virtue of acquired mutations in the receptor-binding domain of the viral spike protein which binds to the human ACE2 receptor for cellular entry. In this report we have empirically determined the efficacy of a range of personal care formulations in inactivating the beta and delta variants of SARS-CoV-2. High titres of these variants were exposed to marketed personal care formulations from Unilever under standard in-vitro suspension test-based conditions relevant to end-user habits. All the formulations demonstrated greater than 99.9% reduction in viral infective titres. The rate of inactivation by these products were comparable to that of the original strain of SARS-CoV-2 virus tested under the same conditions. Therefore, it can be concluded that well-designed personal care formulations when tested under consumer-centric conditions, and with proven efficacy against the parent strain of SARS-CoV-2 will continue to be effective against extant and emerging variants of SARS-CoV-2. This is through their broad-spectrum mode of action (disruption of lipid bilayer of the host-derived viral envelope, denaturation of envelop and nucleocapsid proteins, and disruption of genome) which is independent of the escape mutations that facilitate immune evasion or enhanced transmissibility.

2 Abstract 22

The second and third waves of COVID-19 pandemic have largely been driven by the surge of 23 successive SARS-CoV-2 variants of concern (VOC). These VOC have rapidly spread through 24 multiple geographies being enabled by high transmission rates and/or high viral load 25 compared to the original parent strain. Consequently, the altered phenotypes of these VOC 26 have posed greater challenges to diagnostic and clinical management of COVID-19. Despite 27 considerable progress being made on vaccine roll out, practicing proper hand hygiene has 28 been advocated as a consistent precautionary intervention as more virulent VOC continue to 29 emerge and spread across geographies. 30

Two variants of concern, namely beta and delta, have recently been shown to escape 31 antibody-mediated neutralization by virtue of acquired mutations in the receptor-binding 32 domain of the viral spike protein which binds to the human ACE2 receptor for cellular entry. 33

In this report we have empirically determined the efficacy of a range of personal care 34 formulations in inactivating the beta and delta variants of SARS-CoV-2. High titres of these 35 variants were exposed to marketed personal care formulations from Unilever under standard 36

in-vitro suspension test-based conditions relevant to end-user habits. All the formulations 37 demonstrated greater than 99.9% reduction in viral infective titres. The rate of inactivation by 38 these products were comparable to that of the original strain of SARS-CoV-2 virus tested 39 under the same conditions. Therefore, it can be concluded that well-designed personal care 40 Introduction 48 Generation of successive variants is an expected consequence of viral evolution and results 49 primarily from extended viral persistence and propagation in the human population. While 50 majority of genetic variants are of insignificant consequence to the human host, certain traits 51 of a selected number of variants could prolong the pandemic and reintroduce risk of COVID-52 19 even among vaccinated individuals [1, 2] . The World Health Organization (WHO) defines 53 classes of SARS-CoV-2 variants based on severity of illness, transmissibility, and their impact 54 on public health and social countermeasures such as lockdowns and masking, and medical 55 countermeasures such as diagnostics, therapeutics, and vaccination. Variants of Concern 56 (VOC) are those with evidence of inducing more severe disease, increasing transmissibility, 57 or having negative impact on countermeasures. There are currently four VOC designated by 58 the WHO as Alpha, Beta, Gamma, and Delta (https://www.who.int/en/activities/tracking-59 SARS-CoV-2-variants/). In an analysis of the global spread of SARS-CoV-2 variants and 60 estimated changes in the effective reproduction numbers (R0) at country-specific level, it was 61 clearly demonstrated that WHO-designated VOC have rapidly replaced previously circulating 62 lineages with estimated transmissibility increases of 29%, 25%, 38% and 97% respectively for 63

Alpha, Beta, Gamma, and Delta [3] . In addition to enhanced transmissibility, many of these 64 for 20-and 10-seconds contact time respectively. Each test product was tested in triplicate. 110

All controls (virus recovery, neutralization, and cytotoxicity) were performed according to 111 ASTM standard. Host cells were examined microscopically for virus-specific cytopathic effects 112 and product-specific cytotoxic effects. 113

The 50% tissue culture infective dose per mL (TCID50/mL) was determined using the 115 Spearman-Karber method, and in cases where no virus was detectable, a statistical analysis 116 using the Poisson distribution was performed to determine the theoretical maximum possible 6 titre for the product. The average log10 reduction was calculated as the difference in TCID50 118 between the test product and virus recovery control. In the absence of soap and water, the CDC recommends the use of alcohol-based hand rub 136 containing at least 60% alcohol (w/w) for hand disinfection. Therefore, we evaluated alcohol-137 based hand sanitizer containing 65% alcohol (w/w) for virucidal efficacy against the SARS-138

CoV-2 VOC. As noted previously with hand washing, we believe that even for alcohol-based 139 hand sanitizers it is critical to look at lower contact time-points which are more consumer 140 relevant. In an observation of 50 episodes of hand hygiene with an alcohol-based hand rub of 141 health care personnel working in an intensive care unit, it was recorded that the mean time of 142 application (i.e., rubbing product onto hands) was 11.6 seconds with a median time of 10 143 seconds [14] . As shown in table 1, the 65 % alcohol-based sanitizer gave greater than equal 144 to log10 4.4 (≥ 99.99%) reduction of SARS-CoV-2 Beta titre in 10 seconds while the same 145 sanitizer achieved greater than equal to log10 3.2 (≥ 99.9%) reduction for Delta over the same 146 contact duration. It is worthwhile to mention here that this discrepancy can be accounted for 147 by the lower input titre that was employed for the Delta variant. All the test products reduced 148 the infective titre of SARS-CoV-2 VOC below detectable limits by greater than 99.9% within 149 10 to 20 seconds of exposure and at dilutions relevant to end-user usage. CoV-2 strain. To the best of our knowledge, this is the first study to report inactivation of Beta 158 and Delta VOC by commercially formulated personal care products for cleansing. Our results 159 re-affirm the importance of hand-hygiene products with adequate levels and composition of 160 surfactants (for soap bars and liquid cleansers) and alcohol (for sanitizers) to deliver efficacy 161 in consumer habit-centric conditions to prevent further spread of VOC. We believe that these 162 results will lend confidence to members of the public and encourage them to continue to 163 practice good hand hygiene. However, we should not lose sight of the fact that the world at 164 this point is closely monitoring the spread and behaviour of multiple variants of interest (VOI) 165 namely Eta, Iota, Kappa, Lambda and Mu, and numerous variants of high consequence, all of 166 which have the potential to become future VOC [15] . Therefore, it is important to consider 167 whether daily hygiene measures involving handwashing and hand-sanitation with soaps and 168 alcohol-based hand-rubs should be expected to be effective against emerging VOC. To 169 answer this, we must look at the mechanism of action as related to viral mutations and their 170 effects on fundamental virus particle structure and function. 171

Coronaviruses including SARS-CoV-2 are multimolecular assemblies of virally derived nucleic 172 acids (RNA), proteins (spike, membrane, envelope and nucleocapsid), and host derived lipids 173 (viral envelope). The nucleocapsid which encloses the viral genomic RNA is surrounded by 174 the lipid bilayer in which the spike proteins, membrane glycoproteins, and envelope proteins 175 are anchored. The different constituents of a mature virion are held together by non-covalent 176 interactions. These non-covalent interactions are weak in nature and the virus relies on the 177 host-derived envelope for its shape, integrity, and infectivity. 178

Surfactants present in soaps, liquid handwashes and detergents, are amphipathic molecules 179 with a hydrophobic fatty acid tail and a hydrophilic head group. When the surfactants 180 encounter the virus, they insert themselves into the lipid bilayer aided by the hydrophobic tails 181 while the hydrophilic head groups remain outside in contact with water. The key tendency of 182 a micelle-forming amphiphile inserting into a lipid bilayer is its preference for a locally curved 183 interface (its spontaneous curvature) that conflicts with the planar topology of a bilayer. This 184 misfit causes a curvature stress. As more and more surfactant molecules wedge themselves 185 into the ordered lipid bilayer, the viral envelope is ruptured thereby destroying the 186 multimolecular assembly and rendering the virus inactive. When the surfactant concentration 187 reaches the critical micellar concentration (CMC), micelles form around the disintegrated viral 188 particles and remove them during rinsing [16, 17] . Many other broad-spectrum virucidal agents such as organic acids, quaternary ammonium 205 compounds, phenolics, and oxidizers are also known to disrupt the viral envelope, the viral 206 genome, and the viral envelope proteins through physico-chemical modes of action. The 207

representative everyday hand hygiene products studied here do not contain such agents, but 208 it would be expected that effective formulations containing the above-mentioned agents would 209 also be minimally impacted by viral mutations given similar mechanisms of action to those 210 ingredients discussed here. 211

Majority of the lineage-defining mutations occur in the spike protein for both VOC and VOI. 212

These mutations are chiefly single-nucleotide substitutions which alter the affinity of the spike 213 protein for the ACE2 receptor and/or for neutralizing antibodies. While these mutations affect 214 binding of the RBD to the receptor or antibodies at the molecular level, they do not alter the 215 macromolecular property of spike protein which therefore remains susceptible to denaturation 216 by soaps, surfactants, alcohols, organic acids, phenolic compounds and oxidizers. Similarly, 217 the lipid components of the host-derived viral envelope remain completely permissive to 218 disruption by surfactants and alcohol as they are unaffected by the mutations and remain 219 unchanged across the variants. 220

In conclusion, we have provided empirical proof that marketed personal care formulations from 221

Unilever have high virucidal efficacy against the SARS-CoV-2 variants Beta and Delta. This 222 direct experimental evidence fully supports the broader argument that well-designed personal 223 care hand hygiene products capable of delivering efficacy in user centric conditions which act 224 through physico-chemical mechanism against the basic structure of the virus particle should 225 be efficacious regardless of mutations [21] . Furthermore, we argue that due to broad-spectrum 226 mode of action of these tested formulations, the continued practice of good hand hygiene 227 practices with everyday used products such as those described herein holds significant 228 promise as an easily accessible, economic and effective non-therapeutic intervention towards 229 reducing the transmission of present and future variants of SARS-CoV-2 across communities 230 and populations. 231 Table 1 . Virucidal efficacy of a range of commercially available proprietary personal care formulations tested against selected SARS-CoV-2 232 variants of concern (VOC) at user relevant dilutions and contact duration. TCID50, 50% tissue culture infective dose. 

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We would like to thank Microbac Laboratories, Sterling, Virginia, USA for providing technical 240 assistance. The authors would also like to thank Dr. Timothy Tobery and Dr. Naresh Ghatlia 241 for critical review of the manuscript. 242 243