Four Worrisome SARS-CoV-2 Variants WNY1-4 Found In American Sewers


Researchers from the City University of New York, University of Missouri-School of Medicine and The New School-New York (A private university) have in a new study alarmingly come across four new unique “cryptic” lineages of the SARS-CoV-2 coronavirus ie WNY1, 2, 3, and 4, from  wastewater sites.

Shockingly these new variants had an assortment of mutations on them, some that were already found on various variants of concern (VOCs) and some that were totally new and worrying.

The team suspects that these new variants emerged from animals that were infected with the SARS-CoV-2 virus but they were transmissible back to humans and in some cases had enhanced transmissibility potential.

It is only a matter as to when some vectors or actual infections aids in getting these variants in the wastewaters or from the animals themselves into the human population.

The study findings showed the some of these new SARS-CoV-2  variants are totally ‘super’ immune evasive and that could derail all vaccine efforts so far and all other measures in trying to contain this pandemic should these new variants start becoming dominant among the human masses.

The study team stressed that tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity.

In order to monitor New York City (NYC) for the presence of novel variants, the study team amplified regions of the SARS-CoV-2 Spike protein gene from RNA acquired from all 14 NYC wastewater treatment plants (WWTPs) and ascertained the diversity of lineages from these samples using high throughput sequencing.

The study findings alarmingly reported the detection and increasing frequencies of novel SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database.

These lineages contain mutations rarely observed in clinical samples, including Q493K, Q498Y, H519N and T572N.

Many of these mutations were found to expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor.

Importantly pseudoviruses containing the Spike amino acid sequence of these lineages were found to be resistant to many different classes of receptor binding domain (RBD) binding neutralizing monoclonal antibodies.

The study team offered several hypotheses for the anomalous presence of these mutations, including the possibility of a non-human animal reservoir.

Although wastewater sampling cannot provide direct inference of SARS-CoV-2 clinical sequences, the research revealed several lineages that could be relevant to public health and they would not have been discovered if not for wastewater surveillance.

The study findings were published on a preprint server and are currently being peer reviewed.

The study findings indirectly warn that animal reservoirs Of COVID-19 could trigger new rounds of human disease or even a new global pandemic simultaneously!

During analyzing samples from the fourteen wastewa ter treatment plants in the New York City, the study team found a number of different COVID-19 variants in their samples, including Alpha, Beta, Delta, and Gamma.

Most alarmingly, the study team found four New SARS-CoV-2 Variants  or distinct “cryptic” lineages, WNY1, 2, 3, and 4, from three of the wastewater sites. These four cryptic variants yield surprising and troubling results.

The receptor-binding domain RNA extracted from the wastewater samples contained up to 29 mutations in the four detected variants, some previously observed in the variants of concern or interest and others unique to these samples.

Importantly majority of the variants unique to the sewershed variants are extraordinarily rare among the 3.5 million sequences found in the GISAID SARS-CoV-2 database.

Most concerning is the fact that the mutations found on the receptor-binding domain, the site of attachment of the virus to the ACE2 receptor on the host cell surface and also the target for the great majority of protective antibodies, were found to enhance transmission and immune evasiveness!

It was found that most of the cryptic variant mutations occur between amino acid positions 437 and 508. This is the part of the Spike protein that is in direct contact with the ACE2 receptor.

Furthermore the extensive mutations in the receptor-binding domain were found to be functional.

The study team reported that pseudotyped viruses carrying a receptor-binding domain substituted with that of the cryptic variants are infectious. This is not entirely unexpected as polymorphisms shared with variants of interest and concern enhanced Spike protein function.

Also it was found that the shared amino acid substitutions at positions N440, L452, and N501 enhance receptor-binding affinity.

More worrying, the mutations found at positions K417, N439, K444, N460, E484, Q493, and S494 reduce virus neutralization by convalescent sera and selected monoclonal antibodies.

Also, evidently, the amino acid substitutions at the twelve novel sites are viable. This is especially notable for the deletion at position 484, a site known to interact with both ACE2 and neutralizing antibodies directly.

It is important to determine the potential role of each of these cryptic mutations in immune escape and enhanced function. They may anticipate changes yet to be seen in new variants of concern.

The study team suggests that these new variants arose not from human sources, but from nonhuman hosts.

These suggestions were based on several findings:

– Firstly, these cryptic variants are only observed in some, but not all samples. The study team said that if variants were prevalent in the human population, they would be found throughout the City and not confined to specific sewer sites.

– Secondly, the team observed that the pseudotyped viruses carrying the “cryptic” receptor-binding domain are also capable of infecting non-human ACE2 receptors, specifically those of rats and mice. 

– Thirdly, it is known that SARS-CoV-2 can infect many species other than pangolins and humans. Up to 40% of all dogs tested in the US have antibodies to SARS-CoV-2. Several variants infect feral house and field mice. Up to 30% of all white-tailed deer in the northeast test positive for COVID-19 antibodies. Reports of infection of both domestic and large cats held in zoos were reported in the early days of the pandemic. 

It is important to note that SARS-CoV-2, like the influenza virus, can engage in ‘zoonotic pingpong’.

SARS-CoV-2 infections from animals can make their way back into humans.

For instance SARS-CoV-2 strains have infected mink, and mink have returned the favor by infecting humans. As early as January 2021, farmed mink in Denmark showed signs of SARS-CoV-2 infection. These viruses were relayed back to human populations and subsequently sequenced.

Shockingly a study on these emerged SARS-CoV-2 variants that were relayed back to human populations from the mink zoonotic transmission had a unique mutation ie the Y453F in the receptor-binding domain which results in up to four-fold higher ACE2 receptor affinity, suggesting a far more transmissible virus.

The New York wasterwater study also examined the resistance of pseudotyped viruses carrying the mutant receptor-binding domain to monoclonal antibodies currently approved by the US FDA for treatment such as etesevimab, bamlanivimab, and imdevimab.

The study findings showed that WNY1 and 2 are partially neutralized by etesevimab and imdevimab.

However the WNY3 and 4 SARS-CoV-2 variants were completely resistant to all the monoclonal antibodies tested.

Furthermore the study findings showed that all four variants were also completely resistant to bamlanivimab.

The study team also tested these new mutations against naturally-developed antibodies in convalescent antisera.

It was found that convalescent sera neutralized both WNY1 and 2 but are far less effective against WNY3 and 4. The neutralizing titers of the sera from fully vaccinated individuals were also significantly reduced. 

The study team also warned that as the pseudotyped virus used to test antibody neutralization only includes receptor-binding domain mutations, whereas the full variant is far more likely to decrease antibody neutralization to a greater extent.

The study team concluded that “the properties and characteristics of these new identified variant lineages provide them the capacity to be an increased threat to public health.”

Importantly simply analyzing this small region of the virus has been insightful. The cryptic variants display the full range of variations that may occur within SARS-CoV-2 RNA. The receptor-binding domain shows here a broader degree of mutability. This epitomizes the potential for viral variation, not only in the receptor-binding domain, but in the rest of the Spike protein and viral genome as well, potentially enhancing virulence, vaccine and monoclonal antibody resistance, immune suppression, and transmission.

It should also be noted that another possibility to be aware of is that the extreme variation seen in highly localized New York City cryptic variants is not due to zoonotic variants but rather the effluent from highly regional clusters of variants replicating in human populations, perhaps wards treating immunocompromised patients. Extreme variation of the S protein is documented to occur in such patients.

The key message from this study findings are that variants documented to date represent only a subset of what the world may expect from future SARS-CoV-2 infections. We also must be aware of new variants emerging from fauna that inhabit our ecosystem.

The study team concluded, “To date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to biases, costs and throughput. Here we demonstrate the circulation of several lineages of SARSCoV-2 in the NYC metropolitan area that have not been detected by standard clinical surveillance.

While the origins of these lineages have not been determined, we have demonstrated that these lineages have expanded receptor tropism which is consistent with expansion to an animal reservoir.

Finally, we demonstrated that these lineages have gained significant resistance to some patient-derived neutralizing monoclonal antibodies. Thus, these novel lineages could be relevant to public health and necessitate further study.”

Neutralizing antibodies elicited by infection or vaccination are a central component of immunity to subsequent challenge by viruses (Plotkin, 2010) and can also confer passive immunity in prophylactic or therapeutic settings. In the case of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), an understanding of how viral variants evade antibodies and how affinity maturation could generate antibodies that maintain activity against viral variants is important to guide vaccination and treatment strategies.

The receptor-binding domains (RBDs) of the SARS-CoV-2 Spike trimer are key neutralization targets, and potent RBD-specific antibodies have been isolated from many convalescent donors (Brouwer et al., 2020; Cao et al., 2020; Chen et al., 2020; Chi et al., 2020; Hansen et al., 2020; Ju et al., 2020; Kreer et al., 2020; Robbiani et al., 2020; Rogers et al., 2020; Seydoux et al., 2020; Shi et al., 2020; Wec et al., 2020; Wu et al., 2020b; Zost et al., 2020).

Such antibodies are used for the treatment of SARS-CoV-2 infection (Chen et al., 2021; Weinreich et al., 2021). Typically, RBD-specific neutralizing antibodies isolated during early convalescence have low levels of somatic hypermutation, and nearly identical antibodies derived from specific rearranged antibody genes (e.g., VH3-53/VH3-63) (Barnes et al., 2020b; Robbiani et al., 2020; Yuan et al., 2020) are found in distinct convalescent or vaccinated individuals (Wang et al., 2021b).

Consistent with these findings, high titer neutralizing sera are generated following the administration of at least some SARS-CoV-2 vaccines (Sahin et al., 2020; Widge et al., 2021). Conversely, SARS-CoV-2 infection may sometimes fail to induce sufficient B cell stimulation and expansion to generate high neutralizing antibody titers. Neutralizing titers are low in some convalescent individuals, including those from whom commonly elicited potent antibodies can be cloned (Luchsinger et al., 2020; Robbiani et al., 2020; Wu et al., 2020a).

The RBD exhibits flexibility and binds the angiotensin-converting enzyme 2 (ACE2) receptor only in an “up” conformation, not in the “down” RBD conformation of the closed, prefusion trimer (Walls et al., 2020; Wrapp et al., 2020). Structural studies have allowed the designation of distinct RBD-binding antibody structural classes (Barnes et al., 2020b).

Class 1 antibodies are derived from VH3-53 or VH3-63 gene segments, include short CDRH3s, and recognize the ACE2 binding site on RBDs in an up conformation (Barnes et al., 2020a, 2020b; Hurlburt et al., 2020; Shi et al., 2020; Wu et al., 2020c; Yuan et al., 2020).

Class 2 antibodies are derived from a variety of VH gene segments, also target the ACE2 binding site, but can bind to RBDs in either an up or a down conformation. Some class 2 antibodies (e.g., C144, S2M11) (Barnes et al., 2020a; Tortorici et al., 2020) bridge adjacent down RBDs to lock the Spike trimer into a closed prefusion conformation.

Class 3 antibodies, which can recognize up or down RBDs, do not target the ACE2 binding site (Barnes et al., 2020a).

Despite the fact that cloned RBD-specific antibodies can select resistance mutations, such as E484K, in cell culture (Baum et al., 2020; Weisblum et al., 2020), until recently, little evidence had emerged that antibodies have imposed selective pressure on circulating SARS-CoV-2 populations. Nevertheless, variability and decay of convalescent neutralizing titers (Gaebler et al., 2021; Luchsinger et al., 2020; Muecksch et al., 2021; Robbiani et al., 2020; Seow et al., 2020) suggests that reinfection by SARS-CoV-2 may occur with some frequency.

Recent reports have documented reinfection or rapidly increasing case numbers associated with SARS-CoV-2 variants with resistance to commonly elicited antibodies (Fujino et al., 2021; Tegally et al., 2020; Volz et al., 2021; Wang et al., 2021b; West et al., 2021; Wibmer et al., 2021).

The majority of SARS-CoV-2 antibodies that have been studied in detail were cloned from individuals early in convalescence and have relatively low numbers of somatic mutations. However, recent work has shown that antibodies evolve in convalescent patients, accumulating somatic mutations that can affect function (Gaebler et al., 2021; Sakharkar et al., 2021; Sokal et al., 2021).

Here, we present a detailed functional and structural characterization of several groups of clonally related antibodies recovered from the same 5 individuals shortly after infection and then later in convalescence. We show that somatic mutations acquired in the months after infection endow some SARS-CoV-2 RBD-specific antibodies with greater neutralization potency and breadth. We further show that the acquisition of somatic mutations enables some antibodies to maintain activity in instances in which viral mutations would otherwise enable escape from neutralization.

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