Potential use of endemic human coronaviruses to stimulate immunity against pathogenic SARS-CoV-2 and its variants

ABSTRACT

While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes significant morbidity and mortality in humans, there is a wide range of disease outcomes following virus exposures. Some individuals are asymptomatic while others develop complications within a few days after infection that can lead to fatalities in a smaller portion of the population. In the present study, we have analyzed the factors that may influence the outcome of post-SARS-CoV-2 infection. One factor that may influence virus control is pre-existing immunity conferred by an individual’s past exposures to endemic coronaviruses (eCOVIDs) which cause the common cold in humans and generally, most children are exposed to one of the four eCOVIDs before 2 years of age. Here, we have carried out protein sequence analyses to show the amino acid homologies between the four eCOVIDs (i.e. OC43, HKU1, 229E, and NL63) as well as examining the cross-reactive immune responses between SARS-CoV-2 and eCOVIDs by epidemiologic analyses. Our results show that the nations where continuous exposures to eCOVIDs are very high due to religious and traditional causes showed significantly lower cases and low mortality rates per 100,000. We hypothesize that in the areas of the globe where Muslims are in majority and due to religious practices are regularly exposed to eCOVIDs they show a significantly lower infection, as well as mortality rate, and that is due to pre-existing cross-immunity against SARS-CoV-2. This is due to cross-reactive antibodies and T-cells that recognize SARS-CoV-2 antigens. We also have reviewed the current literature that has also proposed that human infections with eCOVIDs impart protection against disease caused by subsequent exposure to SARS-CoV-2. We propose that a nasal spray vaccine consisting of selected genes of eCOVIDs would be beneficial against SARS-CoV-2 and other pathogenic coronaviruses.

KEYWORDS:

• eCovids
• religious practice
• acquired immunity
• COVID-19
• vaccines
• children

1. Introduction

Coronaviruses (CoVs) are a large group of positive-sense single-stranded RNA viruses belonging to the Coronaviridae family [1,2]. Two species of coronavirus- namely, severe acute respiratory syndrome coronavirus 1 (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), have caused global outbreaks in 2003 and 2012, respectively [1–4]. In late December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in patients with viral pneumonia in Wuhan City, China [1] and is now commonly known as COVID-19 or SARS-CoV-2. As of September 2022, there have been over 600 million confirmed cases worldwide and an estimated 6.5 million deaths due to SARS-CoV-2. In the US, there are over one million confirmed deaths due to the virus. The mortality rate of SARS-CoV-2 is estimated to be about 1% which dwarfs the impact of the related SARS coronavirus (SARS-CoV-1), which caused about 8,000 infections and 800 deaths (~10% mortality) and MERS-CoV which carries a mortality rate of 40–50% [3,5].

In addition to the three known pathogenic viruses, there are four species of COVID namely, OC43, HKU1, NL63, and 229E, referred to as endemic human Coronaviruses or eCOVIDs, causing the common cold in immunocompromised patients [2–5]. The symptoms of infection with eCOVIDs are similar to that of other ‘common colds’ caused by other seasonal human respiratory viruses like human rhinoviruses (HRVs), adenoviruses (AV), respiratory syncytial virus (HRSV), and human parainfluenza viruses. All eCOVIDs have originated by zoonotic transmission from animals to humans, and their possible ancestors share similar natural animal reservoirs and intermediate hosts (Table 1) [2–8]. For example, it appears that human coronavirus (HCoV) −229E, HCoV-NL63 SARS- CoV-1 and 2 as well as MERS-CoV primarily originated from bats, while intermediate hosts for H229E and MERS appear to be Arabian camels/Alpaca. The primary reserve for HKU1 and OC43 appears to be rodents. The intermediate host for HCoV-OC43 is most likely domestic animals such as cattle or swine [2–5,7,8].

Table 1. Comparison of four Endemic Coronaviruses with SARS-CoV-2.

	SARS-CoV-2	HCoV-229E	HC0V-OC43	HCoV-HKU1	HCoV-NL63
Year of Isolation	2019 Betacoronavirus	1962 Alphacoronavirus	1965 Betacoronavirus	2004 Betacoronavirus	2004 Alphacoronavirus
Receptor	Angiotensin-Converting Enzyme 2 (ACE-2)	Aminopeptidase N (ANP)	Sialic Acid	Sialic Acid	Angiotensin-Converting Enzyme 2 (ACE-2)
Additional receptor	transmembrane serine protease 2 (TRMPSS2)	Unknown	Unknown	Unknown	Unknown
Transmission routes	Respiratory droplets, contaminated fomites, airborne, oral-fecal	Respiratory droplets, contaminated fomites	Respiratory droplets, contaminated fomites	Respiratory droplets, contaminated fomites	Respiratory droplets, contaminated fomites
Incubation Period	2–14 days	2–5 days	2–5 days	2–4 days	2–4 days
Reservoir (primary hosts)	Horseshoe Bats (Rhinolophus)	Bats	Rodents	Rodents	Bats
Intermediate hosts	Malayan Pangolin? Cavet cats?	Alpaca	Cattle	Unknown	Unknown
Mortality Rate	~3%*	Very low	Very low	Very low	Very low
Classification	Betacoronaviruses	Alphacoronavirus	Betacoronaviruses	Betacoronaviruses	Alphacoronavirus
Symptoms	Fever, dry cough, malaise, myalgia, dyspnea, diarrhea, conjunctivitis, severe respiratory distress*, loss of taste and smell, acute kidney failure^$,	Malaise, headache, runny nose, sore throat@.	Malaise, headache, runny nose, sore throat.	Fever, dyspnea, cough, Malaise, headache, runny nose, sore throat.	Cough, croup, runny nose, sore, hypoxia, and obstructive laryngitis in children.
Epidemiology	Pandemic	Common in winter, cases occur globally.	Common in winter, cases occur globally.	Common in winter, cases occurs globally.	Common in winter, cases occur globally.

Note: *Depend on the variant and vaccination status..

$Age related, more severe in elderly and nursing home individuals..

@Can be severe in elderly and immune-compromised patients.

Accessed from Coronavirus – AccessScience from McGraw-Hill Education.

The wide variability in transmissibility and clinical manifestations of infections by SARS-COV-1 and 2, MERS-COV, and eCOVID among humans remains poorly understood. For example, the case fatality rate for MERS is between 40%– 50%, which is the highest among all pathogenic coronaviruses. There are several risk factors associated with the progression to acute respiratory distress syndrome (ARDS) in all three highly pathogenic viruses, i.e. MERS, SARS-COV-1, and 2. The most common risk factors appear to be advanced age (i.e. people aged 65 years or over), cancer, diabetes mellitus, hypertension, renal and lung diseases, smoking, and coinfections with other pathogenic microorganisms that can increase the risk [6]. Surprisingly, in children and young adults with a robust immune system, infections with MERS-CoV and SARS-COV-1 and 2 can be completely asymptomatic or exhibit mild symptoms with loss of smell or taste and low-grade fever, for a few days to one or two weeks [6,9]. There is ample evidence that children are generally less susceptible to infection with SARS-CoV-2, and it appears that they had prior exposures to eCOVID [10–19]. There is mounting evidence that prior infections with eCOVIDs can provide a certain degree of protection against SARS-CoV-2 [10]. One clear evidence of this preexisting immunity is that during the last two years of the SARS-CoV-2 pandemic a very small percentage of children have died, most with other risk factors like cancer, and other serious preexisting health diseases, and immunocompromised immunity due to autoimmune disorders [11–21]. There is also strong evidence that in the areas of the globe with low income and high population density, where there is a high probability of eCOVID exposure, the mortality and morbidity from SARS-CoV-2 have been low (discussed in a later section; Tables 4–6). Our understanding of the cross-immunity induced by eCOVID infection is limited and the only evidence is that children are significantly less susceptible to SARS-CoV-2 and in low-income, high-density regions of the world where continuous exposure to eCOVIDs is evident, the serious incidence of SARS-CoV-2 associated hospitalization and death are significantly low [22–27]. Several recent studies have shown that a considerable proportion of individuals without a history of SARS-CoV-2 infection possess antibodies to SARS-CoV-2 and SARS-CoV-2-reactive T cells, indicating that cross-reactive T cell subsets originating from past infections by eCOVID may play a role in the clinical outcome of infection with the phylogenetically related eCOVID [3–8].

In our research, we aim to analyze the potential source of anti-SARS-CoV-2 pre-existing immunity and determine if the degree of protein sequence homologies between eCOVIDs and SARS-CoV-2 plays a significant role in imparting cross-reacting immunity against SARS-CoV-2. It is important to note that amongst the four eCOVIDs, only HCoV-NL63 utilizes the ACE-2 receptor [28]. Since an important part of all three pathogenic COVIDs (i.e. SARS-COV-1 and 2, MERS-COV) is the entry through the ACE-2 receptor, it is pertinent to explore the degree of sequence homologies between these viruses. In this study, we have carried out the comparisons of protein sequences of all four eCOVIDs with SARS-Cov-2 at the Spike, Envelope, and M proteins levels [1–5] [7,8,29–32]. Our eventual goal is to determine if the degree of homologies between eCOVIDs and SARS-CoV-2 can be used to determine if any of the four eCOVIDs or parts of their combinations or selected protein subunits can be used to develop a nasal vaccine to induce sufficient cross-immunity against SARS-CoV-2. We hypothesized that a nasal spray containing selective subunits with the appropriate mixture may serve as an inexpensive preventive vaccine for the general population, especially the vulnerable groups and in developing nations where SARS-CoV-2 vaccines are still scarce or unavailable. Since all four viruses can be cultured easily in various cell lines, the development of an attenuated eCOVID-based nasal vaccine will be much simpler and significantly inexpensive as compared to the synthetic mRNA vaccine. Such a vaccine could generate enough antibodies that would cross-react with proteins of the pathogenic coronaviruses. A recent report by Hue has shown that nasal priming by murine coronavirus provides immunity against the lethal heterologous virus [33]. Currently the most dominant mRNA vaccine requires frequent booster shots and may require new mRNA synthetic sequences to stimulate immunity against new variants. In our opinion, the mRNA vaccine is far short of the formalin-fixed whole virus or subunit, or attenuated vaccines. It is also too expensive to produce and is not suitable for global immunization against the SARS-CoV-2 pandemic, since most developing nations cannot afford to purchase mRNA-based vaccines.

1.1. Short description of endemic and pathogenic coronaviruses

The name coronavirus is derived from the Latin word corona because its spike (S) proteins, which protrude outside of the mature virion, resemble the Sun’s corona under an electron microscope. Coronaviruses are enveloped viruses and contain a single-stranded ribonucleic acid (RNA) genome. The spike protein contains two subunits: S1 contains the receptor-binding domain (RBD), which binds the host cell receptors, and S2 mediates viral and host cell membrane fusion. The S2 fusion peptide is highly conserved amongst endemic and pathogenic coronaviruses, whereas S1 is more variable [2–5] [7,8,29–32]. The SARS-CoV-2 genome encodes for four major proteins, i.e. spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The S-protein of SARS-CoV-1 and 2 as well as MERS uses angiotensin-converting enzyme 2 (ACE2) expressed in human lung and olfactory neurons [6,9]. Upon binding, the viral membrane fuses with host cells. After the fusion, transmembrane serine protease 2 (TMPRSS2) present on the host cell surface activates the ACE2 S-protein complex followed by the conformational changes that allow the virus to enter the host cells [1–3,5–8,28]. Of note, only~10% of cells in the lung express both ACE2 and TMPRSS2 [1,28–32]. Those cells include goblet cells in the nasal cavity that secrete mucus; type II pneumocytes that help maintain the alveoli, the sacs where oxygen/CO2 exchange occurs; and one type of so-called enterocytes that line the small intestine and are involved in nutrient absorption. There is emerging evidence that SARS-CoV-2 also infects certain parts of the brain, especially the progenitor olfactory neurons and about 30% of patients report loss of smell and some patients permanently lose their sense of smell [20,34]. Coronaviruses’ genomes range in length from 26 to 32 kilobases and are the longest in length among all known RNA viruses. The viral genome is complexed with nucleocapsid (N) proteins to form a helical nucleocapsid. In addition to the genes that code for the structural proteins that are the building blocks of the coronavirus particle, the genome codes for 16 or more additional genes that function in replication, modulating the activities of the host cell by downregulating the cell’s protein synthesis machinery, allowing the adaptation to different tissues or cell types of hosts, and evading an affected body’s immune system [2,3,5–8,28–35].

Before the SARS-COV-1 pandemic in 2003, HCoV 229E and HCoV OC43 were the only two known coronavirus strains, causing 15–29% of all common colds. Epidemiological data have shown that over 30% of children test positive for antibodies with one of the four eCOVIDs strains within the first 12 months of life. Therefore, prior to the SARS-COV-1 pandemic, Coronaviruses were known as mildly pathogenic viruses to humans causing the common cold, especially HCoV 229E and OC43 strains.

2. Classification

Coronaviruses are classified in the family Coronaviridae and the subfamily Orthocoronavirinae which is divided into four genera, based on differences in protein sequences: Alpha, Beta, Gamma, and Delta. Alphacoronaviruses and beta coronaviruses circulate in mammals, including, horses, cows, rodents, and bats, especially causing respiratory illnesses in humans and gastroenteritis in animals [3,4,6,9]. These viruses also take a heavy burden on livestock. Delta coronaviruses mostly infect birds, whereas Gamma coronaviruses infect birds and mammals. Table 1 lists various characteristics of the eCOVIDs.

3. Method

1. Sequence blast Analyses: The length of the surface, envelope, and membrane proteins of SARS-CoV-2 are 1273, 75, and 222 amino acids (AA), respectively. The AA lengths of the four eCOVIDs are similar surface proteins ranging from 1353 to 1173 AA. The envelope proteins range from,77 to 84 AA: and the membrane proteins range from 223 to 230 AA, as shown in Table 2. To compare the relative lengths of SARS-CoV-2 surface, envelope, and membrane proteins, amino acid (AA) length with the eCOVIDs surface, envelope, and membrane proteins we performed the protein blast for SARS-CoV-2 (NC_045512.2) to four eCOVIDs species (NC_002645, NC_005831.2, NC_006213.1, and NC_006577.2) to identify the sequence similarities between the 5 viruses (Table 2). The length of the surface, envelope, and membrane proteins of SARS-CoV-2 are 1273, 75, and 222 AA, respectively. The length AA lengths of the four eCOVIDs are similar; therefore, surface protein lengths range from 1353 to 1173 AA. The envelope proteins range from, 77 to 84 AA: and the membrane proteins range from 223 to 230 AA, as shown in Table 2.

Table 2. Protein Sequences of the four endemic Coronaviruses were compared with SARS-CoV-2.

	Protein
	S			E			M
Accession	Protein AC.	Preferred Name	Protein length	Protein AC.	Preferred Name	Protein length	Protein AC.	Preferred Name	Protein length
SARS-CoV-2	YP_009724390.1	Surface glycoprotein	1273	YP_009724392.1	envelope protein	75	YP_009724393.1	membrane glycoprotein	222
HCoV- 229E	NP_073551.1	Surface glycoprotein	1173	NP_073554.1	envelope protein	77	NP_073555.1	membrane protein	225
HCoV-NL63	YP_003767.1	Surface glycoprotein	1356	YP_003769.1	envelope protein	77	YP_003770.1	membrane protein	226
HCoV-OC43	YP_009555241.1	spike glycoprotein	1353	YP_009555243.1	envelope protein	84	YP_009555244.1	membrane protein	230
HC0V-HKU1	YP_173238.1	spike glycoprotein	1356	YP_173240.1	envelope protein	82	YP_173241.1	membrane glycoprotein	223

Query sequence.

NC_045512.2 (SARS-CoV-2).

Subject sequences.

NC_002645.1 (HCoV- 229E).

NC_005831.2 (HCoV-NL63).

NC_006577.2 (HC0V-HKU1).

NC_006213.1(HCoV-OC43).

2. Percent Query Coverage is the percent of the query length that hits the aligned segments. The more query coverage means the more similarity and homology. E value refers to the number of hits expected to be observed by chance. In other words, an E-value of 0.05 obtained from a sequence alignment imply that there is a 5 out of 100 chance of taking place by chance only. However, the E value in the range of 10e-10 and 10e-50 are closely related sequences and could be a domain match or similar. So, the lower the E-value, the better the hit and the higher homology.

Percent identity refers to the percent of amino acids that are identical to the query. It is essential to take the e value into account and look for homology between conserved regions at the protein level.

3. Epidemiology Analyses: Data on the current SARS-COV-2 cases and death records were downloaded from BBC online sources (Covid map: Coronavirus cases, deaths, vaccinations by country – BBC News). The data from these sites are changing daily so we fixed February 19^th, 2022, as the last collection date for our analyses. The Muslim population by country was downloaded from Muslim Population by Country 2021 (worldpopulationreview.com) site. The analyses were carried out by entering the percentage of the Muslim population and then linking the highest Muslim population with the majority and minority of Muslims and the numbers of cases and deaths per 100,000 individuals. We eliminated the data which has high tourist trafficking regardless of the Muslim population since it tends to skew the data. These nations included the Caribbean Islands and giant transient airport hubs like Qatar, Istanbul, and UAE. We also did not include the major active War Zones, where international armies are entering and leaving in large numbers, potentially contaminating the data.

4. Results

Sequence Blast Analysis: In the sequence blast analyses the maximum score states the highest bit score from matched and mismatched amino acids and gaps according to Blossom 62 algorithm for the aligned region to the query and the total score referees to the sum of all aligned regions. The equality of maximum score and total score means there is no gap in an alignment. Ultimately, the higher the score, the better the alignment [36].

The max and total score of surface proteins showed that HCOV-OC43 has the highest max and total score to SARS-CoV-2 (Max score: 467, Total score: 363) and HCOV-HKU1 (Max score: 452, Total score: 540), HCOV-NL63 (Max score: 394, Total score: 394) and HCOV-229E (Max score: 364, Total score: 364), respectively.

The max and total score of envelope proteins showed that HCOV-HKU1 (Max score: 32.7, Total score: 32.7) and HCOV-229E (Max score: 27.7, Total score: 27.7) exhibited the highest max and total scores for SARS-CoV-2. Whereas HCOV-OC43 (Max score: 29.3, Total score: 50), HCOV-229E (Max score: 27.7, Total score: 27.7) and HCOV-NL63 (Max score: 25.8, Total score: 25.8), respectively.

The max and total scores of membrane protein showed that all four eCOVIDs: HCOV-OC43 (Max score: 165, Total score: 165), HCOV-HKU1 (Max score: 153, Total score: 153), HCOV-NL63 (Max score: 115, Total score: 115) and HCOV-229E (Max score: 86.7, Total score: 86.7), exhibited the highest max and total score to SARS-CoV-2. (Table 3).

Table 3. Protein Homology Analyses of the four endemic Coronaviruses as compared with SARS-CoV-2.

	Proteins: of Homology
	S					E					M
Accession	Max Score	Total Score	Query Cover %	E value	Percent Identity	Max Score	Total Score	Query Cover %	E value	Percent Identity	Max Score	Total Score	Query Cover %	E value	Percent Identity
HCoV- 229E	364	364	57	1e-110	31.07%	27.7	27.7	86	2e-06	27.14	86.7	86.7	99	2e-25	30.67
HCoV-NL63	394	394	60	3e-104	30.78	25.8	25.8	85	8e-06	18.64	115	115	90	2e-36	31.10
HCoV-OC43	467	363	73	2e-146	37.63	29.3	29.3	50	6e-07	31.58	165	165	95	6e-56	40.85
HC0V-HKU1	452	540	70	3e-141	35.43	32.7	32.7	81	3e-08	31.15	153	153	93	3e-51	36.36

In this study, 3 surface proteins of four eCOVID species were aligned to SARS-CoV-2 using blastp (https://blast.ncbi.nlm.nih.gov). We used Blosume62 matrix and threshold and word size of 0.05 and 3 were considered.

Our findings showed that the query coverages for surface protein in HCOV-229E, HCOV-NL63, HCOV-OC43 and HCOV-HKU1are 57% (E value = 1e-110), 60% (E value = 3e-104), 73% (E value = 2e-146) and 70% (E value = 3e-141), respectively, whereas, the percent identities are 31.07%, 30.78%, 37.63% and 35.43%, respectively (Table 1). However, the query coverages for Envelope protein in HCOV-229E, HCOV-NL63, HCOV-OC43 and HCOV-HKU1 were 86% (E value = 2e-06), 85% (E value = 8e-06), 50% (E value = 6e-07) and 81% (E value = 3e-08) respectively, and the percent identities are 27.14%, 18.64%, 31.58% and 31.15%, respectively (Table 1).

Moreover, the query coverages for Membrane protein in HCOV-229E, HCOV-NL63, HCOV-OC43 and HCOV-HKU were 99% (E value = 2e-25), 90% (E value = 2e-36), 95% (E value = 40.85) and 93% (E value = 3e-51) respectively, and the percent identities are 30.67%, 31.10%, 40.85% and 36.36%, respectively (Table 1).

The result of protein alignment showed that membrane protein is more conserved than the surface glycoprotein and the envelope proteins. Of all species, the membrane protein of HCoV-OC43 exhibited the highest similarity, and SARS-CoV-2 and HC0V-HKU1, HCoV- 229E, and HCoV-OC43 showed less similarity (Table 1B). Moreover, the surface glycoprotein proteins showed the lowest level of query coverage percent than the envelope and the membrane proteins. The HCoV- 229E, HCoV-OC43, HC0V-HKU1, and HCoV-OC43 had the highest to lowest variability respectively (Table 1B). The membrane protein of HCoV-OC43 has the highest similarity to the HCoV-NL63 and three other species are in the next level. In short, the surface glycoprotein protein has the highest, and membrane protein has the lowest variability (Table 1).

4.1. Structural homologies between SARS-CoV-2 and the four eCovids

Despite the variable degree of sequence conservation among different the four eCOVIDs species, the results of our structural protein sequence of the virions showed a remarkable degree of homologous regions. Tables 2 and 3 depict the regions of SARS-CoV-2 and the important regions of the structural eCOVIDs proteins (Figure 1). The regions for receptor binding and the proteolytic cleavage sites of the S protein, as well as the N-terminal RNA-binding and C-terminal dimerization domains of the N protein, which have been shown to be critical for virus attachment and entry, cell-to-cell fusion, and virus replication showed shared homologies [37] (41). The region of the S1 subunit responsible for receptor binding differs considerably among each of the four eCOVIDs species, which utilize different domains and host cell receptors and consequently differ in their tissue tropism [20,33]. The S2 subunit resembling the fusion system, both structurally and in the amino acid sequence was conserved [38]. The high degree of amino acid sequence and conformational conservation of the α-helical region immediately adjacent to the S2′ cleavage site likely explains why antibodies targeting this region also cross-reacted with orthologous peptides of related COVIDs, including those of MERS-CoV, SARS-CoV-1 and 2 Figure 1 [25,37–39]. Therefore, it is not surprising that individuals with past infections with eCOVIDs have elicited cross-reactive antibodies and/or led to the generation of longer-lived memory B cells with specific reactivity to this linear epitope, which may provide cross-protection against SARS-CoV-2. And this may explain why individuals who are regularly exposed to eCOVIDs exhibit cross-immunity against COVID-19, among children, who generally experience much less severe disease infection with SARS-CoV-2, as we showed in Tables 4–6 particularly [9–19,40–49]. In this context, it is important to highlight that antiviral antibodies can have a variety of protective effector functions that operate through different mechanisms. These include antibody-dependent neutralization of enveloped viruses, such as inhibition of virus replication by blocking viral entry into the host cell, the competitive binding of high-affinity antibodies where the variable fragment antigen-binding site to a specific epitope within the viral attachment and fusion protein(s) may interfere at the critical site on host cell receptor(s), and interference in the activating host proteases [6,10,50]. Neutralizing antibodies may block the fusion machinery, which is known to undergo intense conformational changes upon viral attachment to overcome the repulsive electrostatic force between the viral envelope and host cell membrane bilayers [42]. Additional antibody effector functions include complement-dependent cytotoxicity and enhancement of antibody-dependent cell-mediated CD8+ T cell cytotoxicity and/or phagocytosis by macrophages [51].

Figure 1. Illustration of incidence and mortality rates of COVID-19 in Muslim Majority versus non-Muslim majority nations.

Table 4. COVID-19 Related Mortality, Incident and Doubling rates in Muslim Majority Nations.

Country	Percentage of Muslim population	Cases/100K	Doubling Time (days)	Death/100K	Muslim population
Afghanistan	99.60%	176	323	7	28,072,000
Somalia	99.80%	95	85	5	89,95,000
Yemen	99.10%	523	65	4	23,363,000
Egypt	95.1%	254	153	15	83,870,000
Pakistan	96.4%	418	158	9	184,000,000
Bangladesh	90.6%	485	214	8	145,312,000
Iraq	99.0%	2,994	154	41	36,200,000
Algeria	97.9%	295	195	8	37,210,000
Morocco	99.00%	1,418	197	25	31,993,000
Mauritania	100%	424	169	10	32,610,000
Kyrgyzstan	86.6%	1,595	220	27	4,734,000
South Sudan	99.2%	96	100	1	30,121,000

Table 5. COVID-19 Related Mortality, Incidence and Doubling rates in Muslim Minority Countries.

Country	Percentage of Muslim population	Cases/100K	Doubling time (days)	Death/100K	Total Muslim Population
US	0.8%	10,098	162	180	2,454,000
Czech Republic	<0.02	15,547	135	282	20,000
Montenegro	19%	15,960	140	253	111,000
Israel	16.7%	9,272	145	71	1,194,000
Slovenia	2.4%	12,074	140	209	49,000
Portugal	<1%	8,233	143	166	15000
Spain	~1%	7,748	154	170	650,000
Belgium	~3%	9,142	193	216	281,000
Switzerland	4.3%	8,048	172	126	323,000
Sweden	~2%	10,339	126	140	149,000
United Kingdom	0.8%	6,681	150	191	1,647,000
Netherland	5.7%	9398	142	101	946,000

Table 6. The GDPs of countries related to the number of Cases and Death rates

Country	Percentage of Muslim population	GDP ($Million))	Cases/100K	Death/100K
Indonesia	88%	1,119,190	660	18
Mexico	<1%	1,256,440	1879	174
Saudi Arabia	97%	792,966	1294	21
Netherland	5.7%	907,050	9412	101
Turkey	95%	761,425	6237	56
Switzerland	4.3%	731,425	8048	126
UAE	76%	421,142	5724	17
Austria	4.2%	445,142	7234	119
Malaysia	60%	364,684	1646	7
S. Africa	1.5%	351,430	2797	95
Algeria	98%	171,157	296	8
Hungary	0.2%	163,469	8214	303
Kuwait	95%	134,623	7142	41
Ukraine	1%	153,781	5056	116
Morocco	99%	119,700	1419	25
Ecuador	<0.1%	107,435	2413	116
Uzbekistan	96%	57,921	294	2
Lithuania	0.1%	54,174	9749	151
Nigeria	51%	474,516	83	1
Argentina	1.9%	449,663	7927	166

Table 2 summarizes structure identity parameters for aligned residues. We found several interesting and important residues conserved amongst 4 strains (Figure 2). Some like I54, L55, and S59 (from 5zhy) have been observed to be conserved amongst HCoV-229E, NL-63, mouse hepatitis virus (MHV), severe acute respiratory syndrome (SARS) and middle east respiratory syndrome coronavirus (MERS) strains [36]. Some of these residues are essential to forming an interface of strong hydrophobicity. Overall structures and conserved fusion cores can help with the possibility of broad-spectrum inhibitor design targeting proteins of major HCoV strains.

Figure 2. Illustration of incident mortality rates in Muslims versus non-Muslim nations.

4.2. Epidemiological analyses

The specific effector functions and mechanisms that are primarily responsible for the generally milder clinical outcome among children when infected with eCOVID and in adults who are exposed to eCOVIDs on a regular basis due to cultural and religious traditions were explored. We analyzed the mortality, incidence, and case-doubling rates of COVID-19 in Muslim majority nations and compared the same rates with Muslim minority nations. We did not include nations which are war zones like Syria and Iraq and did not include the countries where there is large human traffic like Qatar, UAE, and Istanbul. We believe this would skew the data. As shown in Table 4, the COVID-19 cases/100,000 in the Muslim majority nations ranged from 95–485 cases/100,000. There is a clear correlation between the degree of in-person daily prayers practice and a lower number of cases/100K. It also appears Kyrgyzstan has a relatively less percentage of the Muslim population (i.e. 99.6 vs 86.6), there was a relatively higher number of cases/100K, but it was not statistically significant. Also, human-to-human transmission of eCOVIDs may be less since over 80% of the country is mountainous with the remainder made up of valleys and basins. In addition, about 60% of the Muslim population appears to be non-practicing Muslims, according to the 2021 census data. We compared the cases/100K of COVID-19 in developed nations where the Muslim population is a minority, in these nations the cases/100K were significantly higher ranging from 6,681–15,960 (P < 0.001: Table 5). We also analyzed the case-doubling time in days, in Muslim majority vs Muslim minority nations. As shown in Tables 4 and Table 5, the case doubling time in the Muslim majority nations ranged from 65 days to 323 days. Whereas, in the nations with a Muslim minority the doubling time ranged from 135–193 days. There was no statistical difference between the case doubling time (P < 0.39) suggesting that infection rates with COVID-19 are comparable. However, the most startling data was the death/100,000 analyses. In Muslim majority nations, it varied from 1 death/100K to 25, whereas, in the developed nations with a Muslim minority, it varied from 71–216 (P < 0.001). This set of data indicates that constant exposure to eCOVIDs in the general population may be playing an important role in reducing mortality in Muslim-majority nations. Therefore, the role of pre-existing immunity due to eCOVIDs appears to play an important role as has been shown by numerous scientific studies [6,9–19,21–23,26,37,43,44,46,47,52–57].

Next, we analyzed if there is a correlation between the nations with a Muslim majority and minority with comparative GDP, and COVID-19 cases/100K (Table 6). Therefore, cases/100 in the Muslim majority COVID-19 cases per 100K showed a significant difference (P <  0.0164). We also analyzed death/100K between the Muslim majority vs Muslim minority. Our analyses showed a significantly lower death in Muslim-majority nations with comparable GDPs (P < 0.002). We also noticed a trend that suggests that if the Muslim population is more than 50% it appears to significantly reduce the death due to COVID-19. An example is Nigeria vs Argentina, where 1 death/100K vs 116 deaths/100K.

5. Discussion

While SARS-CoV-2 causes serious morbidity and mortality in humans [1,5,7] there is a significant difference between mortality and morbidity [6,9,10]. For example, some individuals remain completely asymptomatic whereas others exhibit severe morbidities and die after short- or long-term hospitalization post-infection. There is also a great degree of variation between different variants of SARS-CoV-2 [6,9,10]. The delta variant is much more lethal than the omicron variant, which is highly contagious [58,59]. Presently, the scientific reasons why the disease is severe in some individuals and very mild in others are not clear. Most notably, infection with SARS-CoV-2 is generally very mild to asymptomatic in children, and the morbidity and mortality rates are remarkably low in low-income, high-dense populations. The latter point is significant since we believe that in this population widespread infection with the endemic human coronaviruses (eCOVIDs) may be contributing to acquired immunity against SARS-CoV-2. We hypothesized that one of the important factors that provide immunity against SARS-CoV-2 is pre-existing immunity in individuals who were infected in the recent past with eCOVIDs. As we presented in Tables 4–6, in the population of the globe where transmission of COVID-19 is most likely to be widespread due to the religious traditions where people of all ages gather multiple times a day and even more individuals gather on Fridays, the opportunities to SARS-CoV-2 increases many-fold and hence the probability of exposure to eCOVIDs become very high.

Acquired Immunity against the eCOVIDs Can Have Protected Effects Against SARS-CoV-2: In developed nations eCOVID infections are also common and usually acquired during early childhood [44,47–49], and many of children aged 7 to 15 years have already been exposed to the eCOVIDs multiple times in their lifetime in the middle and high school years. Therefore, it is no surprise that age is negatively associated with seroprevalence in many studies, suggesting that immunity against eCOVIDs plays a positive role in protection against SARS-CoV-2 [6,9–19,21–23,26,37,43,44,46,47,52–57].

Despite the structural variations in the degree of sequences among different four eCOVIDs species, the results of our systematic antibody screen highlight that the structural proteins of the virions share common antigenic sites. Indeed, several of the immunodominant regions we have identified the structural proteins of eCOVID are orthologous to the regions thought to be immunodominant targets for immune responses to SARS-CoV-2 [38] including 2 linear epitopes on the SARS-CoV-2 S protein that elicit potent neutralizing antibodies in SARS-CoV-2 patients [54]. Importantly, antigenic regions that were found to be immunodominant in a study by Sealy et al [9]. These included regions for receptor binding and the proteolytic cleavage sites of the S protein, as well as the N-terminal RNA-binding and C-terminal dimerization domains of the N protein, which have been shown to be critical for virus attachment and entry, cell-to-cell fusion, and virus replication [9,37,56–59]. The region of the S1 subunit responsible for receptor binding differs considerably among CoV species, which utilize different domains and host cell receptors and consequently differ in their tissue tropism (Table 1 [36,60,61], [39,47,62,63]). However, the S2 subunit resembling the fusion machinery is more conserved, both structurally and in amino acid sequence [60,64]. The high degree of amino acid sequence and conformational conservation of the α-helical region immediately adjacent to the S2′ cleavage site likely explains why antibodies targeting this region also cross-reacted with orthologous peptides of SARS-CoV, further supporting our hypothesis that regular exposure to eCOVIDs plays an important role in protection against pathogenic variants of SARS-CoV-2 [39,54]. It is logical to speculate that at least in some individuals, past infections with eCOVID have elicited cross-reactive antibodies and/or led to the generation of longer-lived memory B cells with specific reactivity to this linear epitope, which may provide cross-protection against SARS-CoV-2. This may be the case, particularly among children, who are generally less likely to experience severe disease outcomes from infection with eCOVID [11,12,16,23,63], and in adults and elderly who are regularly being exposed to eCOVIDs due to their religious practices, as we have shown in Tables 4–6. In this context, it is important to highlight that antiviral antibodies can have a variety of protective effector functions that operate through a variety of different mechanisms, including antibody-dependent neutralization of enveloped viruses, the competitive binding of high-affinity antibodies via variable fragment antigen-binding regions to specific regions within the viral attachment and fusion protein(s) that are also critical for the interaction with the host cell receptor(s) or activating host proteases [54]. Neutralizing antibodies can interfere with the fusion machinery, which undergoes profound activating conformational changes upon viral attachment to overcome the repulsive force between the viral envelope and host cell membrane bilayers [51].

Cellular Responses to SARS-CoV-2: In several studies, cross-reactive T cell responses and epitopes have been mapped between SARS-CoV-2 and the eCOVIDs [9,12,14,19,21,54,56–59,65–71]. Bert et al. [70], reported frequent T cell responses to SARS-CoV-2 non-structural and N proteins among SARSCoV-2 naïve donors. Both CD8+ and CD4+ T cell cross-reactive populations have been identified, and for each population, cross-reactive peptide epitopes have been described [9,12,14,19,21,54,56–59,65–71]. Of note, the cell-mediated immune responses primed by eCOVIDs can exhibit a variety of effector functions toward SARS-CoV-2 including the cytotoxic effects by killing of SARS-CoV-2 infected cells via CD8+ killer cells (CTL), by secretion of cytokines/chemokines, via T helper (TH) or T follicular helper (TFH) to activate epitope-specific B lymphocytes, and via Regulatory T cell or Treg. In some studies, more than half of humans with no known previous exposures to SARS-CoV-2 have exhibited T cell reactivity toward SARS-CoV-2 [72]. Cross-reactive T cells, like B cells, were upregulated upon exposure to SARS-CoV-2. Of note, a T cell that recognizes an N peptide of SARS-CoV-2 would be sufficient to activate a B cell that produces S-specific, neutralizing antibodies.

Acquired Immunity against the eCOVIDs Can Have Protected Effects Against SARS-CoV-2: Analyses of Developmental Nations with Islamic Traditions. In developed nations eCOVID infections are also common and usually acquired during early childhood [3–8], and many of children aged 7 to 15 years have already been exposed to eCOVIDs multiple times in their lifetime in the middle and high school years. Therefore, it is no surprise that age is negatively associated with seroprevalence in many studies, suggesting that immunity against eCOVIDs plays a positive role in protection against SARS-CoV-2 as well as against MERS and SARS-CoV-1 [9–19,21,23]. Also, numerous studies have found a significant positive association between the seroprevalence of eCOVID and the male sex [73].

Despite the structural variations in the degree of sequences among different four eCOVIDs species, the results of our systematic antibody screen highlight that the structural proteins of the virions share common antigenic sites. Indeed, several of the immunodominant regions we have identified the structural proteins of eCOVID are orthologous to the regions thought to be immunodominant targets for immune responses to SARS-CoV-2 [38] including 2 linear epitopes on the SARS-CoV-2 S protein that elicit potent neutralizing antibodies in SARS-CoV-2 patients [54]. Importantly, antigenic regions that were found to be immunodominant in a study by Tajuelo et al (59). These included regions for receptor binding and the proteolytic cleavage sites of the S protein, as well as the N-terminal RNA-binding and C-terminal dimerization domains of the N protein, which have been shown to be critical for virus attachment and entry, cell-to-cell fusion, and virus replication [37,53,56–59]. The region of the S1 subunit responsible for receptor binding differs considerably among CoV species, which utilize different domains and host cell receptors and consequently differ in their tissue tropism (Table 1 [36,39,47,60,62,63]). However, the S2 subunit resembling the fusion machinery is more conserved, both structurally and in amino acid sequence [60,64]. The high degree of amino acid sequence and conformational conservation of the α-helical region immediately adjacent to the S2′ cleavage site likely explains why antibodies targeting this region also cross-reacted with orthologous peptides of related CoVs in our study, including those of MERS-CoV, SARS-CoV, and nonhuman isolates, further supporting our overall hypothesis and the important role of this particular region as a pan-CoV target site [39,54]. It is, therefore, logical to speculate that at least in some individuals, past infections with eCOVID have elicited cross-reactive antibodies and/or led to the generation of longer-lived memory B cells with specific reactivity to this linear epitope, which may provide cross-protection against SARS-CoV-2. This may be the case, particularly among children, who are generally less likely to experience severe disease outcomes from infection with eCOVID [11,12,16,23], and in adults and elderly who are regularly being exposed to eCOVIDs due to their religious practices, as we have shown in Tables 4–6. In this context, it is important to highlight that antiviral antibodies can have a variety of protective effector functions that operate through a variety of different mechanisms, including antibody-dependent neutralization of enveloped viruses, the competitive binding of high-affinity antibodies via variable fragment antigen-binding regions to specific regions within the viral attachment and fusion protein(s) that are also critical for the interaction with the host cell receptor(s) or activating host proteases [54]. Neutralizing antibodies can interfere with the fusion machinery, which undergoes profound activating conformational changes upon viral attachment to overcome the repulsive force between the viral envelope and host cell membrane bilayers [60].

5.1. ACE2 and TMPRSS2

The main receptor for the entry of SAR-CoV-2 is the Angiotensin-converting enzyme 2 (ACE2) receptor. This receptor is on multiple different cell types throughout the body into human cells [60,62]. SARS0CoV-2 gains entry into the target cells via Transmembrane serine protease 2 (TMPRSS2), which subsequently cleaves and activates the SARS-CoV-2 S protein, thus greatly facilitating the entry of the virus into cells [62].

The degree of expression and affinity of ACE2 and TRPMSS2 between adults and children is debated. Some studies suggest higher levels of these two receptors in the elderly as compared to children [45,74]. Other studies suggest no difference in the affinity of these receptors between adult and children’s nasal epithelia [45,74]. Conflicting results have also been reported for the expression of ACE2 and TMPRSS2 in the lungs. Some studies report that the expression of ACE2 and TMPRSS2 in lungs increases with age [45,74], whereas others report a higher expression of ACE2 in lungs in children compared with elderly adults [45,74], and no difference between the age groups [45,74].

The contradictory reports on the expression of ACE2 and TMPRSS2 are due to the fact that the ACE2-angiotensin system is complex. besides age, there are multiple other factors that can affect the ACE2 expression levels and affinity including, smoking, diet, vitamin D, BMI, drugs, genetics, sex, and comorbidities including diabetes mellitus, chronic obstructive pulmonary disease, and hypertension [45,74]. ACE2 serves as the entry receptor for SARS-CoV-2 and also plays an important role in regulating immune responses, especially in the lungs. Therefore, after SARS-CoV-2 enters cells, ACE2 receptor expression is down-regulated, resulting in preventing them from converting angiotensin II to angiotensin [45,74]. The high levels of angiotensin II may be partly responsible for the organ injury in COVID-19, as serum levels of angiotensin II are significantly elevated in SARS-CoV-2-infected patients and there is a positive correlation between viral load and lung damage [45,74].

5.2. Viral load

There is no clear evidence to support the notion that viral load is responsible for age-related differences in COVID-19 severity since viral load in the respiratory tract is similar [74]. One study found significantly greater viral loads in nasopharyngeal samples from children less than 5 years of age compared with older children or adults [74].

Why Low-Income, High-Density Nations Have Fared So Well Against SARS-COV-2: An epidemiology Overview? Another indication that the eCOVIDs may provide a degree of protection against SARS-CoV-2 is that in low-income, high-density regions, where eCOVID exposures are presumed to be high, there are relatively high rates of SARS-CoV-2 infections, but low rates of serious disease or deaths (Tables 4 and Table 6). As we show in our analyses that the nations where exposures to eCOVIDs are common due to religious practices and when the mixing of children and adults takes place on continuous bases the death rate from COVID-19 are significantly lower as compared to death rates in the developed nations. Of course, there are ample evidence that clearly shows that pre-existing immunity against SARS-CoV-2 exits and it is due to prior exposure to eCOVIDs. We also show that there is a significant protein sequence homologies between eCOVIDs and SARS-CoV-2 (Figure 1, Table 2).

Sagar et al [18] utilized a comprehensive respiratory panel PCR (CRP-PCR) test and analyzed 1812 patients who were SARS-COV-2+ by PCR. They observed that the eCOVID⁺ as compared with the eCOVID^– the group had lower rates of ICU admission and death after SARS-COV-2 diagnosis. They proposed that even without neutralizing immunity, patients with prior eCOVID infections may have lung-localized primed immune responses that prevent severe disease from a heterologous virus [18]. Heterotypic lung-localized resident memory T and B cells prevent severe infections from respiratory pathogens [18].

Another example is the result of a study in Mumbai, India SARS-CoV-2 infection rates were higher in slums compared to non-slums, but fatality rates due to SARS-CoV-2 were lower in slums compared to non-slums [75]. It has also been observed that poor, highly populated countries have suffered fewer deaths from SARS-CoV-2 per million individuals compared to Western nations. In Tables 4–6 we present the results of our epidemiology analyses and how the normal population is repeatedly exposed to eCOVIDs on a daily and weekly basis due to the cultural and religious practices have fared well as opposed to many other nations, even densely populated. In Tables 4–6 we have summarized the results of our epidemiology survey. What are the causes of relatively low infection and mortality rates in developing nations and especially the Muslim majority nations? We have looked at the data that includes doubling rates, the total number of SARS-CoV-2 cases per 100,000 individuals, and the mortality rates of many countries. We noticed striking natural protection in some nations that have been overlooked by the pandemic experts. Based on the data presented here we hypothesized that there are five major factors that have prevented the potential devastating sequences of the SARS-CoV-2 pandemic in some developing nations. Usually, science and religious practices are looked at with suspicion. However, many times traditions and science meet in an amazing fashion. Here we have merged scientific analyses with the 14,00 years old Islamic traditions that we show have significantly quelled the major devastating outcomes from SARS-CoV-2. Some of the major factors that we have analyzed are the percentage of Muslim populations vs SARS-CoV-2-related death as well as SARS-CoV-2 cases/100,000. Our findings may surprise many, but the answers are scientific, and clues have been right in front of our eyes, known for a long time but overlooked due to panic over the SARS-CoV-2 pandemic. We believe that these ‘resistant’ populations have pre-existing immunity mediated by continuous exposures to non-pathogenic coronaviruses (i.e. 229E, NL63, OC43, and HKU1) that provides partial immunity against SARS-CoV-2. However, to impart a cross-reacting immunity regular exposure to these coronaviruses enhances humoral immunity (IgG and more specifically IgA). Since a significant percentage of Muslims gather in Mosques for daily 5-times daily prayers and for Friday prayers which are held in large Mosques and the way the prayers are performed increases the opportunity for the exchange of many microbes but also unknown variants of coronaviruses. The process of acquiring immunity against a large array of microbes including coronaviruses is documented in several publications, including books [76]. William McNeal in his book ‘Plagues and People’, offered a radical interpretation of the extraordinary impact of infectious diseases on culture and vice versa. He described in detail how travel to Mecca for the annual pilgrimages might have protected Muslim populations around the globe from many plagues in the past including Black Plague. In addition to daily prayers, there are several other rituals that are linked to the prayers including ablution where before each prayer the individuals must clean themselves. The cleaning ritual (Wadu or Ablution) is more thorough and robust than simple hand washing advocated in the media. This ritualistic washing requires washing hands, face, arm, mouth, nose, and feet, at least three times, 5-times daily. All Muslims are required to cleanse their private parts with water after urination and defecation. It may not look that significant, but the feces stuck on one’s underwear can play an important role in spreading microbes and we know that COVID-19 is found in human faces. Of note, acquiring immunity against potentially non-pathogenic coronaviruses and other viruses begins during early childhood when Muslim children crowd the mosques and the Qur’anic schools, madrasas, and the crowded pre-schools. We believe that traveling to Mecca expands the immunity among the Muslim community on the global scale even further when between 3–5 million pilgrims gather in Mecca and Medina around the globe and exchange microbes, acquire and transmit germs that eventually help spread the immunity by the cross-national exchange at a global scale. One more factor is the practice of Hijab. Pre-COVID period the Hijab was banned in many European countries and France is still insisting on banning Hijab [77,78], but the benefit of covering the face is obvious in preventing the spread of infectious diseases has become clearer during SARS-CoV-2 and undoubtedly other respiratory germs [79,80]. This year the common flu has almost disappeared in many countries due to the mask mandate. In this study, we compared the mortality and infection rates as well as the doubling time of SARS-CoV-2 in the Muslim majority with the nations where Muslims are a small minority. It should be noted that not all Muslim-majority nations equally practice 5-times daily prayers in mosques, and we have analyzed the impact of this factor and other related factors. For example, Shia in Iran do not practice in-person prayers in mosques and a very low percentage of Muslims go to mosques in Turkey.

After reviewing a large set of data sometimes we noted conflicting results. Therefore, some studies have shown no difference in cross-reactivity between SARS-CoV-2 and eCOVIDs. Others have detected no or few cross-reactive antibodies, whereas many have identified antibodies and T cells with strong cross-reactivity between SARS-CoV-2 and the eCOVIDs epitope targets. Some authors have reported neutralization capacities and others have reported negative results. These discrepancies are a consequence of differences in research subjects, methodologies, the small size of samples, and the regions of the area where population densities differ significantly. As we mentioned earlier that low-income, religious, and cultural background and population densities impart a remarkable and statistically significant impact on the degree of cross-reactive between SARS-CoV-2 and the eCOVIDs morbidities and mortalities. Most importantly, the methodologies used in many studies are not standardized, and can significantly differ between laboratories regarding target antigens, developing reagents, protocols, and interpretations. Some obvious flaws that we noted are that the antibodies often respond to 3 or 4-dimensional structures that cannot be accurately replicated by truncated peptide fragments. It should also be noted that T cell responses of antigens are an evolutionary marvel and is evolving for more than 400 million years and replicating a complex system in a test tube would be very difficult to duplicate. It should be noted that antigen presentation requires precise positioning of a target peptide within its viral protein and antigen presentation events and even when a target peptide is known to exist within a viral protein, the success of antigen processing for T cell recognition will depend on peptide context. Knowing these short comings in the in vitro assays the investigators must view published scientific data with an understanding that assessments of cross-reactive antibodies, T cells, and effector potentials are extremely complex, and the results must be viewed with caution.

6. Conclusions

In a significant fraction of SARS-CoV-2-unexposed humans, cross-reactive T cells and antibodies that recognized both eCOVIDs and SARS-CoV-2 were found. Additionally, when humans were naturally exposed to SARS-CoV2, there were increases in immune responses toward the eCOVIDs. Epidemiological analyses confirmed that there is a significant reduction in morbidity and mortality with SARS-CoV-2 in the regions of the globe where exposure to eCOVIDs is significant due to cultural and religious traditions. We propose that a genetically engineered nasal spray vaccine composed of various proteins from eCOVIDS that produce protective immune responses to SARS-CoV-2 would be highly beneficial as an inexpensive vaccine (s). Such a vaccine would protect against a wide variety of pathogenic coronaviruses, will be affordable for low-income nations, and will be easy to deliver [81,82].

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.