VANCOUVER — A Quebec man who fell and broke his jaw, cheekbone and a bone around his left eye while visiting British Columbia says his surgery was cancelled after he was told his home province “won’t pay” for the procedure.
Patrick Bélanger, 23, said his experience is a warning for residents of Quebec and all Canadians who take pride in a universal health-care system because doctors in other provinces could deny treatment to Quebecers by maintaining they won’t be compensated.
Bélanger’s ordeal began when he and his girlfriend were walking along a trail in the resort town of Sun Peaks, B.C., on the evening of June 10. He tripped andstumbled backwards in the dark, hitting his face on a boulder.
He was taken by ambulance to Royal Inland Hospital in Kamloops just before midnight and was told he needed surgery for a “broken face.” But a surgeon was not available on Saturday, so he was discharged with a prescription for the opioid-containing drug Percocet to manage his pain, Bélanger said.
The following morning, he and his girlfriend, Beth Cooper, returned to the hospital for surgery. But Bélanger said that just as he was being prepared for the operating room, the surgeon cancelled the procedure.
“He said that the hospital would not let him do the surgery because I was from Quebec,” Bélanger said, adding he’d presented his provincial health card when he arrived at Royal Inland.
“I was kind of in shock. As I thought about it more, I thought that doesn’t make sense. Normally, you’d do the surgery and figure out billing afterwards, or at least I thought that’s what was going to happen,” he said.
“I was pretty scared. I was still pretty out of it because I was in quite a bit of pain and on pain medications. And I was calling my parents trying to figure out what to do.”
Bélanger said he offered to pay for the surgery through his family’s private insurance, but the surgeon rejected that option, saying he first needed to speak with a hospital administrator who was not available on the weekend.
“When he told me that the surgery couldn’t be done today, he suggested that I fly back to Quebec City to go get the surgery done,” Bélanger said.
He was given a window of 10 days before his facial bones would start to fuse.
“We thought it was just completely absurd that I, with a broken face, was to take a commercial airline to go get a surgery done in my own country.”
Bélanger’s father and mother arrived in Kamloops later that week and tried unsuccessfully to speak with an administrator at the Interior Health authority about the best options for their son, Richard Bélanger said.
“We were baffled about his basic rights as a Canadian,” he said, calling his son’s experience a “nightmare.”
Richard Bélanger said he went to the surgeon’s private clinic to provide information on the family’s insurance plan as well his credit card in case the surgery could be done there. But staff told him the serious facial fractures his son suffered meant the surgery had to be performed in a hospital, he added.
Four days of anguish since the surgery was cancelled had Bélanger managing “excruciating pain” with prescription opioids and morphine before his case was passed on to another surgeon, he said.
“I would wake up in the middle of the night crying and screaming in pain.”
The second doctor said he needed quick intervention, and the surgery was done seven days after he fell, said Bélanger, an economics student at Bishop’s University in Sherbrooke, Que.
His family said they are still puzzled about why the original surgeon did not do the surgery.
“It’s gross incompetence on the part of the hospital and a failure on the part of the Canadian health-care system,” said Bélanger’s mother, Martha Ferris.
Both Patrick and Richard Bélanger say the end result was “discrimination” against a patient from Quebec, which pays hospital costs but does not participate in a reciprocal billing agreement for physician fees involving all other provinces and territories.
Doctors of BC, an association representing physicians, said an agreement allows its members to bill their own provincial Medical Services Plan for patients from outside the province and the plan is then reimbursed by the patients’ home jurisdiction.
“The doctors get paid as if the patient is a B.C. resident,” it said in a written statement.
“When a Quebec resident needs medical care in B.C., doctors are not able to bill MSP for it and get paid,” the association said.
However, physicians providing services to Quebecers can either bill that province or bill the patient, who would seek reimbursement from their government.
The Health Department in Quebec said doctors elsewhere are paid at rates as if the patient received the same treatment in their home province. Patients must pay any difference in cost and could apply to have the money reimbursed through private insurance, if they have it, the department said in a written response.
The department recommended Quebecers get private insurance before travelling outside the province.
Richard Bélanger said the family’s private insurance company refused to pay any costs after the first surgeon wrote in his son’s medical chart that he could return to Quebec and have the surgery there within 10 days.
The insurer had also initially refused to pay costs when an emergency room doctor noted possible intoxication in his son’s chart, Bélanger said. But that was later ruled out when the family asked why a test to determine the presence of any substances, including alcohol, was not done, he added.
Dr. Peter Stefanuto, the original surgeon, declined requests for an interview.
He said in an email that he could not speak about any specific case but that “care is provided to all patients regardless of their province or country of origin on an emergency basis.”
Issues involving compensation for services would be best addressed through the B.C. and Quebec governments, Stefanuto added.
Dr. Bob Rishiraj, who ended up doing the operation, said he was not concerned about any “politics” surrounding billing, especially after learning the patient had been taking opioids and methadone for days, and a longer wait for surgery carried the risk of infection.
“It became very concerning for me that he was using a lot of morphine and his pain was not well controlled. If we don’t do it, we have a problem of possibly having somebody with narcotics abuse potential down the road,” he said.
“I think a patient is a patient and it doesn’t matter if they’re from Quebec or Ontario or wherever. I think they should just be treated,” Rishiraj said.
The risk of a patient who is billed not paying a physician is low, and cost did not appear to be an issue for Bélanger’s family, he said.
Interior Health did not respond to a request for an interview but said in an email that physicians are not employees of the health authority.
Ferris said the family paid Rishiraj $2,563 and will apply for reimbursement from Quebec.
The irony is that the family has used its private health insurance while travelling outside the country but did not expect they would have to rely on it in Canada, she said.
“It’s mind-blowing to me, kind of shocking.”
British Columbia’s Health Ministry did not respond to questions about patients from Quebec being denied surgery.
Dr. Katharine Smart, president of the Canadian Medical Association, said Canada’s universal health-care system is intended to provide care to all Canadian citizens and permanent residents.
“We strongly encourage provincial and territorial governments to work together to ensure Canadians receive the care they need, when and where they need it, and the federal government to enforce the principles of the Canada Health Act uniformly across the country,” she said in a written statement.
Health Canada said the reciprocal billing agreements are administrative arrangements between provinces and territories to help facilitate the portability criterion of the act while people are temporarily away in another part of the country and need care.
“These agreements are voluntary and not a requirement of the Canada Health Act,” it said in a written response.
Bélanger, who had trouble speaking because his jaw was wired shut for six weeks following surgery, said the emotional toll he has suffered is “incalculable,” on top of the physical pain, which still includes migraines.
Damien Contandriopoulos, a University of Victoria nursing professor and health policy researcher, said that regardless of Quebec’s billing scheme, the province pays, on average, higher physician rates than other jurisdictions for the same care, a reversal from its practice years ago.
It’s common for thousands of patients from Quebec to get care from family doctors in Ontario border towns and for their province to reimburse the cost, he said, adding he is “shocked” that services would be denied to a patient based on billing issues.
However, doctors in British Columbia, where relatively few Quebecers get care, may be deterred from seeking information on rates paid by that province because they’re listed on about 3,000 pages in some complicated categories, and in French, said Contandriopoulos, a former resident of Quebec.
In Bélanger’s case, the surgeon could have contacted his insurance company’s 24-7 phone line to get information from a representative, rather than saying administrators were not available on the weekend, Contandriopoulos said.
He called Bélanger’s weeklong wait for surgery “insane.”
This report by The Canadian Press was first published July 7, 2022.
Camille Bains, The Canadian Press
Quebec COVID-19 booster rates stay low as province launches new vaccination campaign
MONTREAL — As Quebec prepares to launch a provincewide COVID-19 vaccination campaign ahead of a potential new fall wave, it’s unclear whether it will be enough to prompt a pandemic-weary public to roll up their sleeves for another booster.
As of Wednesday, only 56 per cent of Quebecers aged five and older had received a third vaccine dose — a number that has hardly budged in months. Government officials have said that the low booster uptake is due to the fact that millions of Quebecers have caught the novel coronavirus and consider themselves adequately protected.
Health experts, meanwhile, say pandemic fatigue and government communication have also played a role.
Dr. Don Vinh, an infectious diseases specialist at the McGill University Health Centre, points the finger at the provincial government’s messaging. Vinh says the government should have more strongly promoted boosters over the past six to eight months instead of making a big push at a time when the current wave of COVID-19 is waning.
If there had been a “consistent and clear” advisory on boosters, “more people would have been protected and all the consequences of the infection over the last few months would have been avoided,” he said in an interview.
Jason Harley, an associate professor in the department of surgery at McGill University, believes many people have shifted to a “post-COVID mentality” that leads them to stop listening to public health advice. After two years of the pandemic, worry has changed to overconfidence.
“A lot of this has to do with exhaustion with the pandemic,” they said in an interview.
“It has been over two years, and there was a tremendous amount of anxiety that a lot of people were living with on a day-to-day, sometimes hour-to hour-basis, so it’s it’s normal and reflexive that people would look for ways to feel better,” said Harley, who is also has a PhD in educational psychology.
The challenge, Harley said, is to convince people to continue to protect themselves and listen to guidelines, which requires connecting to people through clear, easy-to-follow public health messaging.
That’s something that isn’t always easy when people are on “different timelines” because they’ve become eligible for boosters at different stages of the pandemic, they said. There are also differences between how provinces communicate on COVID-19, and even on who is eligible for boosters.
Health Minister Christian Dubé on Tuesday acknowledged that booster rates had “stalled,” but he attributed the phenomenon in large part to the fact that so many people have been infected with COVID-19 in recent months. He told a news conference that 88 per cent of the most vulnerable age cohort — those over 60 — have received their third dose.
Nevertheless, he said all adults should get a booster if it’s been five months or more since their last shot, or three months since they were infected with the novel coronavirus. Dubé said vaccine appointments will open for people 60 and older next week and for everyone 18 and up on Aug. 29. Quebec started offering fifth doses of COVID-19 vaccine to residents of long-term care homes and private seniors residences on Monday.
Caroline Quach-Thanh, a microbiologist and the president of Quebec’s immunization committee, told the same news conference there was a chance that new vaccines under development will provide longer immunity. The current offerings provide good protection, “but in terms of durability, the long-term response isn’t there,” she said.
Vinh suggested Quebecers need to get used to the idea of getting regular COVID-19 shots, at least for now. Vaccines still protect very well against serious disease and death, he said, but it’s now clear that people will need periodic boosters to maintain immunity.
While some people might be tired or say “we’re vaccinating too much,” people should instead think of vaccines like any other drug, Vinh said.
“There are drugs you give every day or two, or three times a day, and you know why?
“Because the effect of that drug wears off and it’s time for the next dose.”
Another microbiologist praised the Quebec government’s booster campaign, and especially the decision to drop the terms “fourth” or “fifth” dose of vaccine in favour of calculating the amount of time that has elapsed since the last shot.
Anne Gatinol believes people will get boosters but need a nudge.
“I found out that if people are not encouraged to do it, they just don’t do it,” the McGill professor in experimental medicine said in an interview.
Both she and Vinh agree that the government is doing the right thing in offering boosters to everyone 18 and older, even though some jurisdictions such as the United States are limiting them to older and vulnerable people.
The health experts note that young people can still get very ill from COVID-19 even if they are less likely to die from it, and they say vaccines can help protect both individuals and the health system.
This report by The Canadian Press was first published Aug. 18, 2022.
Morgan Lowrie, The Canadian Press
SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization – Nature.com
Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (VH ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.
Genomic surveillance of SARS-CoV-2 during the first year of the COVID-19 pandemic revealed that the D614G mutation in the spike glycoprotein (S protein) was the sole widespread consensus mutation, with the G614 genotype largely replacing the D614 genotype in February 20201,2. In November 2020 however, the emergence of the Alpha (B.1.1.7) variant began capturing global headlines and coincided with a surge in COVID-19 cases in the United Kingdom. Within 4 months, the Alpha variant became the globally dominant SARS-CoV-2 lineage1. The emergence of the Alpha lineage was quickly followed by the emergence of the Beta (B.1.351), Gamma (P.1), and Epsilon (B.1.427/429) variants in early 2021, with the Kappa and Delta variants emerging shortly thereafter. The Delta variant achieved global dominance until it was replaced by the Omicron BA.1 sub-lineage in early 2022, which was swiftly replaced by the BA.2 sub-lineage of Omicron, followed by increasing prevalence of the BA.5 sub-lineage.
SARS-CoV-2 utilizes a trimeric spike glycoprotein for attachment to the host cell receptor angiotensin-converting enzyme 2 (ACE2) and for the subsequent cell entry step which involves the fusion of host cell and viral membranes. Given its crucial role in the viral replicative cycle, the spike protein represents an important therapeutic target and is a critical antigen in host immune responses. All emerging variants contain defining mutations within their spike proteins, with multiple mutations clustering within the receptor-binding domain (RBD) impacting both ACE2 binding and antibody neutralization escape3,4,5, while mutations within the highly antigenic loops in the N-terminal domain (NTD) across these variants reduce antibody neutralization6. Given the rapidly changing mutational and antigenic landscape of the SARS-CoV-2 spike protein, a structural understanding of spike protein mutational effects and the discovery of broadly neutralizing epitopes is of importance.
Here, we present an antibody fragment (ab6) with neutralization activity against multiple variants (Alpha, Beta, Gamma, Delta, Kappa, Epsilon, and Omicron) and report its epitope within the RBD using cryogenic electron microscopy (cryo-EM). This antibody epitope is remote from most VoC mutations, explaining its ability to confer pan-variant neutralization. Given the enhanced antibody escape of circulating variant spikes, the epitope we define here provides opportunities for rational therapeutic targeting of variant SARS-CoV-2 S proteins. We also report studies of spike structure, ACE2 affinity, and evasion of antibodies afforded by previously emerged variant spikes, providing a general structural rationale for enhanced viral fitness of the variants.
Broad neutralization of the SARS-CoV-2 spike protein by an unconventional antibody fragment
VH ab6 is a phage-display-derived antibody with the unusual biochemical property of exhibiting enhanced RBD affinity as a monomeric fragment as compared to a bivalent fusion7 and was recently shown to exhibit tolerance to several circulating RBD mutations8. We first confirmed this anomalous property of ab6, showing that the bivalent VH-Fc fusion has lower neutralization potency relative to the monovalent VH construct in both pseudotyped and live virus neutralization assays (Supplementary Fig. 1a–c). We next assessed VH ab6 binding and neutralization of variant spikes, (Fig. 1a and Supplementary Fig. 1d). Ab6 neutralized all variant spike pseudotyped viruses but exhibited 9–26-fold decreased potency for Epsilon, Kappa, and Delta and 4- and 3-fold lower potency for the BA.1 and BA.2 Omicron spikes respectively. We additionally assessed ab6 neutralization of Alpha, Beta, and Delta live viruses via authentic virus neutralization assays, confirming the lower potency of ab6 for the Delta variant (Supplementary Fig. 1b, c).
We next determined the cryo-EM structure of VH ab6 bound to the WT spike at 2.57 Å (Supplementary Fig. 2), showing that ab6 binds to the RBD in both the up and down positions, via a unique binding mode (Fig. 1b). Local refinement of the down RBD bound by VH ab6 enabled visualization of the ab6-RBD interface at 3.21 Å (Supplementary Fig. 2g, h) and revealed that the ab6-RBD interaction is dominated by contacts with the ab6 beta-sheet scaffold, which wraps around the RBD, extending this large interface to include its CDR2 and CDR3 loops but leaving the CDR1 loop free (Fig. 1c). This scaffold-mediated interaction necessitates a near perpendicular angle of approach for ab6 relative to the RBD, which likely can only be accommodated by a single VH within a bivalent fusion construct. Furthermore, accessibility to the VH scaffold may be limited within a bivalent fusion construct. Thus, the unusual angle of approach and dominance of scaffold-mediated contacts may account for the lower potency of the VH-Fc ab6 construct relative to VH ab6.
The ab6 footprint involves multiple RBD residues (Fig. 1d) and overlaps that of ACE2, consistent with a mechanism of neutralization via ACE2 competition7 (Fig. 1e). The CDR1 and CDR3 loops of ab6 occupy positions that result in clashes with ACE2 upon superposition with an ACE2-bound RBD, with the CDR3 region directly competing with the amino terminal helix of ACE2 for RBD binding contacts, while the CDR1 loop poses a steric clash with the second helix of ACE2 without making RBD contacts (Fig. 1f). To confirm the ACE2 competitive nature of ab6 we employed competition ELISA (Supplementary Fig. 3) and competitive SPR experiments (Fig. 1g). Both experiments demonstrate the ability of ab6 to compete with ACE2 for spike protein binding.
Analysis of the ab6 footprint reveals the inclusion of L452 and Q493, consistent with the reduced potencies against the Epsilon, Delta, and Kappa spikes, which harbor the L452R mutation, along with the BA.1 and BA.2 Omicron sub-lineages which harbor the Q493R mutation (Fig. 1d, h). Genomic sequences from the GISAID database confirms the conserved nature of the ab6 epitope, highlighting the Q493R mutation to be the only significantly occurring variation in circulating variants as of May 1st, 2022. (Fig. 1h). Analysis of the relative neutralization potencies of L452R and Q493R containing variants suggests that ab6 exhibits greater sensitivity to the L452R mutation (Fig. 1h). To uncover the structural basis for the attenuation of ab6 potency by the L452R mutation, we obtained the cryo-EM structure of the Epsilon spike bound to ab6. A global 3D reconstruction was obtained at 2.45 Å (Supplementary Fig. 4), in which ab6 bound both up and down RBDs, as seen in the WT-ab6 complex. Focused refinement enabled visualization of the ab6-Epsilon spike interface at 3 Å (Supplementary Fig. 4g, h), revealing R452 to extend towards the ab6 scaffold (Fig. 1i and Supplementary Fig. 4i). This orientation places the positively charged R452 sidechain in close proximity to a hydrophobic portion of ab6, centered around F58. Thus, the reduced potency observed for R452-containing spikes is likely a result of unfavorable charge and steric effects. The Q493R mutation places R493 in close proximity to the ab6 CDR3 loop, and accommodation of this mutation may involve similar charge and steric penalties which give rise to the attenuation in ab6 potency against the BA.1 and BA.2 variant spike proteins.
To contextualize the broad-spectrum activity of ab6 we sought to investigate variant spike mutational effects on antibody evasion, receptor engagement, and spike structure. As we have previously reported these analyses on the Kappa and Delta variant spikes9, along with the BA.1 Omicron spike10 we proceed here with our analysis of the Alpha, Beta, Gamma, and Epsilon variant spike proteins.
RBD- and NTD-directed antibodies are escaped by variant spike proteins
Having demonstrated the broad neutralization of variant spikes by ab6, we next aimed to provide a comparative analysis of spike mutational effects and antibody breadth using a representative panel of previously reported monoclonal antibodies. We selected RBD-directed antibodies11,12,13,14 which cover the four distinct anti-RBD antibody classes15 and an ultrapotent antibody, S2M1116, which uniquely binds two neighboring RBDs simultaneously (Fig. 2a). We additionally included the NTD-directed antibodies 4–8 and 4A8 to investigate the impact of NTD mutations within these variant spikes. (Fig. 2a). Antibody binding was quantified via enzyme-linked immunosorbent assay (ELISA), and compared with neutralization, which was measured via a pseudoviral entry assay (Fig. 2b). S309 and CR3022 are cross-reactive SARS-CoV-1 directed antibodies whose footprints do not span VoC mutations, and accordingly exhibited relatively unchanged binding across all variant spikes. We have previously characterized the mutational sensitivity of ab1, ab8, and S2M11 to spikes bearing only RBD mutations, and the current analysis of antibody evasion using spikes bearing all VoC mutations is consistent with our previous report3: (1) The N501Y mutation within the Alpha variant reduces but does not abolish the potency of ab1, while dramatic loss of ab1 activity is seen in Beta and Gamma variants due to mutation of K417 to N or T, respectively; (2) The E484K mutation abrogates ab8 activity in the Beta and Gamma variants; and (3) the L452R mutation reduces but does not abrogate activity of S2M11 in the Epsilon variant spike, drawing similarity to the mutational sensitivity of ab6. To compare the structural basis for the effect of L452R on S2M11 and ab6, we performed cryo-EM studies on the Epsilon-S2M11 complex (Supplementary Fig. 6). We obtained a global 3D reconstruction of the Epsilon spike bound to three S2M11 Fabs at 2.16 Å. In contrast to the Epsilon-ab6 structure wherein R452 protrudes into the antibody interface (Fig. 1i), R452 extends away from the S2M11 interface (Supplementary Fig. 7). As the L452 sidechain is accommodated within the footprint of S2M11, this positioning of R452 is likely due to steric clashing and charge repulsion effects which may increase the interaction energy and underlie the observed attenuation of potency.
Evasion of NTD-directed antibodies was observed in cases when mutations were either within, or adjacent to, antibody footprints (Fig. 2b), corroborating the recently described remodeling of antigenic loops in the Alpha, Beta, and Epsilon spikes17,18. The W152C substitution within the Epsilon NTD is inside the footprints of 4A8 and 4–8, and both antibodies escaped by this variant spike. The Beta NTD contains a deletion (Δ242–245) which spans the 4–8 footprint, along with the R261I substitution spanning both 4A8 and 4–8 footprints, leading to escape from both antibodies. The footprint of 4A8 and 4–8 spans a deleted site within the Alpha NTD (Δ144–145) leading to escape. These direct and allosteric mutational effects are consistent with previous findings on NTD rearrangement within these variants and demonstrate their antibody evasive properties.
Having characterized monoclonal antibody evasion by variant spikes, we extended our analysis to include polyclonal antibody escape from human sera. Sera were collected from a spectrum of patients with varying COVID-19 infection histories and vaccination statuses (Supplementary Fig. 8a) and subjected to neutralization and binding assays (Supplementary Figs. 8b, c and 9). Potent neutralization of WT spike pseudovirus was observed in all COVID-19 positive or vaccinated samples but not with pre-pandemic sera from uninfected patients, suggesting limited pre-existing immunity (Supplementary Fig. 8b, c). While serum levels of spike ectodomain binding antibodies correlated poorly with wild-type spike neutralization, strong correlations were observed between NTD and RBD binding antibody levels and neutralization (Supplementary Fig. 10), corroborating the dominance of neutralizing epitopes within the NTD and RBD15,19,20. We observed various effects on neutralization escape when sera samples were assayed using variant spike pseudotyped viruses, obtaining statistically significant decreases in neutralization efficacy for both Beta and Gamma variants relative to WT (Fig. 2c). Interestingly, the high correlation between serum NTD and RBD binding antibodies and pseudovirus neutralization for wild-type spikes was markedly reduced for all variant spikes (Fig. 2d). Taken together, these results highlight the role of mutations within the NTD and RBD of variant spikes in driving evasion of SARS-CoV-2 directed monoclonal and polyclonal antibodies, providing a basis to evaluate the broad mutational tolerance exhibited by S309 and ab6.
Enhanced receptor binding by variant spike proteins
In addition to driving antibody escape, variant spike mutations can enhance receptor engagement, which may underlie increases in infectivity. To investigate the ACE2 binding potential of SARS-CoV-2 variant spikes, recombinant S protein ectodomains bearing variant spike mutations were used in biolayer interferometry (BLI) experiments. All mutant spikes exhibited slightly higher affinities for immobilized dimeric Fc-ACE2 when compared to wild-type (Supplementary Fig. 11a, b). Additionally, we used flow cytometry to evaluate the ability of recombinant dimeric Fc-ACE2 to bind wild-type or variant full-length spikes which we transiently expressed in Expi-293 cells. We did not observe major differences in spike protein expression across the variants (Supplementary Fig. 12a), and all mutant spikes tested demonstrated marginally enhanced ACE-2 binding potencies relative to wild-type (Supplementary Fig. 11c). These complementary assays demonstrate that the totality of mutations within each variant S protein enable slightly enhanced ACE2 binding, suggesting a contributing factor for the increased infectivity observed for these SARS-CoV-2 variants.
Structural effects of variant spike protein mutations
Having demonstrated mutational effects on antibody evasion and receptor engagement, we next sought to characterize the structural impacts of variant S protein mutations. To this aim, ectodomains bearing variant spike mutations were used for cryo-EM structural studies. Global 3D reconstructions were obtained at resolutions ranging from (2.25–2.5 Å) (Supplementary Figs. 13–16), yielding open trimers with one RBD in the up conformation and 2 RBDs in the down conformation for all spikes (Fig. 3). The resolution within the NTD and RBD was insufficient for accurate visualization of mutational impacts within these domains, due to high degrees of conformational heterogeneity. In contrast, we were able to confidently model sidechains close to, and within the S2 domain, owing to its limited flexibility. We therefore first focused our analysis on mutational effects within this region, which predominantly localized to inter-protomer interfaces.
Inspection of the structure of the Alpha variant shows that the A570D and S982A mutations appear to contribute protomer-specific structural effects with implications for RBD positioning (Fig. 4b). Within both “RBD down” protomers, D570 either occupies a position within hydrogen bonding distance of N960 within the adjacent “RBD down” protomer or sits within intra-protomer hydrogen bonding distance with T572 when the RBD of the adjacent protomer is in the up conformation (“RBD up”). D570 within the “RBD up” protomer uniquely forms a salt bridge with K854 in the adjacent “down RBD” protomer. S982 sits within the hydrogen bonding distance of residues G545 and T547 within adjacent “RBD down” protomers only, and such interactions are not possible with the S982A mutation. Thus, possibilities for the effects of the A570D and S982A mutations include allosteric modulation of RBD conformation through (1) addition of the K852–D570 salt bridge, and (2) loss of stabilizing interactions afforded by S982, in agreement with recent reports4,21,22.
The D1118H mutation within the Alpha variant enables local side chain rearrangements, giving rise to additional interprotomer contacts, via pi-cation interactions between R1091 and H1118 of adjacent protomers, and electrostatic interactions between R1091 and adjacent E1092 residues (Fig. 4c). Superposition of wild-type and Alpha spike models clearly demonstrates the differential positioning of H1118 compared to D1118, and the resulting movement of adjacent R1091 towards E1092 (Fig. 4d). These additional interprotomer contacts enabled by the D1118H mutation may aid in the stabilization of the Alpha spike protein in its trimeric form.
Additional mutations were visualized within the Beta and Gamma variant spike proteins, with implications for inter-protomer and intra-protomer contacts. The A701V mutation in the Beta variant S protein lies at the protein’s surface and the larger sidechain conferred by V701 may enable tighter interprotomer packing with Q787 on adjacent protomers (Supplementary Fig. 17a). The T1027I mutation lies within the Gamma spike S2 core and the bulky hydrophobic I1027 sidechain faces inwards, increasing the hydrophobicity of the local buried environment (Supplementary Fig. 17b). This may decrease the local dielectric constant, enhancing the strength of the nearby network of interprotomer salt bridges between E1031 and R1039 on adjacent protomers (Supplementary Fig. 17b) and thus stabilize the Gamma variant trimer.
To identify structural mutational impacts on ACE2 binding we next determined the cryo-EM structures of variant spike-ACE2 complexes. The resulting maps were obtained at average resolutions of ~2.6–3 Å (Supplementary Figs. 18–21). Focus-refined structures of the ACE2-RBD interface enabled visualization of RBD mutations, revealing local structures which are identical to our previously reported structures using spikes harboring variant RBD mutations alone3. Superposition of local RBD-ACE2 complex models revealed no significant structural changes (Supplementary Fig. 22). These structural findings confirm that mutations outside of the RBD do not modulate the positioning of ACE2-contacting residues at the receptor interface via allosteric mechanisms.
Structure of the gamma variant NTD
The structure of NTD region in the Alpha, Beta, and Epsilon variants has been previously reported17,18. Here, we report structural analysis of the NTD region in the Gamma variant, stabilized using Fab fragments of the NTD-directed antibodies 4A823 and 4–824. We obtained Cryo-EM reconstructions for Gamma spike protein–Fab complexes with all 3 NTDs bound for both 4A8 (Fig. 5a) and 4–8 (Supplementary Figs. 23 and 24). The bound antibody fragments improve the resolution of this flexible domain upon focused refinement, enabling the determination of structures at resolutions of ~2.6 Å both for the overall spike and the NTD-antibody interface (Supplementary Figs. 23 and 24).
Superposition of 4A8-bound wild-type and Gamma NTDs reveals remodeling of the antigenic supersite N1 loop25 within the Gamma variant but not the N3 and N5 loops, which comprise the majority of the 4A8 binding site (Fig. 5b). Analysis of nearby mutational effects provides additional reasoning for conformational remodeling of the N1 loop. The mutations L18F, D138Y, and T20N cluster close together, forming multiple interactions which stabilize the alternate N1 conformation (Fig. 5c). Namely, F18 and Y138 form an interconnected network of T-shaped pi stacking interactions with each other and the adjacent F140 residue. Additionally, Y138 and N20 sit within hydrogen bonding distance of the main chain carbonyl of F79 and the sidechain of D80, respectively. Comparison of sidechain positioning between wild-type and gamma structures in this region reveals steric clashes between Gamma residue Y138 and wild-type residues F79 and L18, and between Gamma residue N20 and the main chain of wild-type residue L18, resulting in differential positioning of D80 and F79 in the Gamma NTD (Fig. 5d). Identical positioning of the N1 loop is observed in the Gamma NTD-4-8 structure, further confirming these mutational effects (Supplementary Fig. 25). Thus, the unique interactions conferred by mutations within the Gamma NTD stabilize local conformations which are sterically incompatible with wild-type N1 positioning, causing N1 loop rearrangement.
Mutational enhancement of SARS-CoV-2 viral fitness can arise from effects on receptor engagement and evasion of neutralizing antibodies, with structural origins in the spike glycoprotein. Here we have examined these effects, demonstrating domain-specific differences in the roles and structural mechanisms of S protein mutations. Although such mutational changes can pose threats to natural and vaccine-induced immunity, the existence of preserved epitopes within functional domains holds great potential for future antigenic focus. This is highlighted in our analysis of variant SARS-CoV-2 spikes, which despite exhibiting effects on antibody evasion and ACE2 binding, shared a conserved epitope within the RBD which conferred broad neutralization.
The structural impacts of VoC S protein mutations offer insight regarding the differing mutational heterogeneity observed for the NTD and RBD. While VoC mutations within the RBD are limited to only substitutions, the NTD hosts a large array of deletions and substitutions, along with one documented insertion as seen in the BA.1 subvariant. These NTD mutations predominantly localize to the three loops constituting the “NTD neutralization supersite” (N1: residues 14–26, N3: residues 141–156, N5: residues 246–260)25. Our structures of VoC S proteins in complex with ACE2 demonstrate minimal structural changes in the RBD, reflecting its functional constraints in cell attachment, only permitting mutations that preserve the ACE2 binding interface. In contrast, our structure of the Gamma NTD confirms the role of mutations within this domain as enabling structural rearrangement of antigenic loops, a feature common to all variant spike NTDs (Alpha, Beta, Delta, Epsilon, BA.1, BA.2)17,18,26,27,28,29. These rearrangements are likely directed primarily by immune-evasive pressures. Taken together, these contrasting structural effects between variant NTD and RBD mutations likely arise due to different functional requirements and selective pressures between these domains.
Despite these domain-specific mutational pressures, several lines of evidence have emerged from the present study demonstrating the existence of pan-variant epitopes. The high correlation between RBD + NTD binding antibody levels and viral neutralization potency reflects the dominance of neutralizing epitopes within these domains of the WT spike (Fig. 2d). The diminished correlation between these parameters when assessing variant spikes demonstrates mutational escape within these domains. However, the fact that neutralization of variant spikes is attenuated, but not abolished, suggests the preservation of neutralizing epitopes. The existence of such epitopes within the RBD is corroborated by the unaltered potency of the SARS-CoV-1 directed antibody S309 across Alpha, Beta, Gamma, and Epsilon spikes. Most importantly, we reveal an epitope within the RBD conferring broad neutralization of all variants of concern spike proteins, including the rapidly spreading BA.2 subvariant. This epitope has largely survived viral evolution thus far, with the only prominent mutational changes being L452R and Q493R. These mutations are accommodated by ab6, albeit yielding decreased neutralization potencies. Therefore, we highlight this epitope for focus in the design of broadly protecting therapeutic antibodies and immunogens.
A comparison of ab6 to several other reported RBD-directed VH domains highlights the unique epitope and mechanism of binding exhibited by ab6 (Supplementary Fig. 26). While VH fragments ab814, H330, and C530 approach the RBD with more acute angles relative to ab6, C130, and n311331 both exhibit near perpendicular angles of approach involving some scaffold interactions, similar to ab6. Although the H3 footprint overlaps significantly with that of ab6, it is completely escaped by mutations within the Beta variant spike protein, unlike ab6. C1 binds an epitope distal to that of ab6 and the ACE2 binding site, yet is able to compete for ACE2 binding due to steric effects, whereas n3113 binds an epitope which overlaps with that of ab6 but is noncompetitive with regards to ACE2 binding. The neutralization breadth of n3113 extends to multiple variants31, consistent with its epitope overlap to that of ab6. Of note, n3113 was shown to bind exclusively to the RBD in the up conformation31 in contrast to ab6 which can recognize both down and up RBD conformations. Thus, distinguishing features of ab6 include its ability to adopt a near perpendicular angle of approach relative to the RBD in both up and down conformations via scaffold-mediated interactions, while also utilizing its CDR regions to compete with ACE2 via steric effects (CDR1) and direct binding of ACE2 interacting residues on the RBD (CDR3).
Analysis of the ab6 sequence using IMGT32 indicates the most closely aligning VH gene to be VH3-30 (Supplementary Fig. 27A). Comparison of the ab6-RBD structure to that of several other VH3-30 encoded RBD-directed antibodies demonstrates heterogeneity in the RBD epitopes recognized by VH3-30 encoded fragments (Supplementary Fig. 27B). Of these antibodies, the footprint of the heavy chain of antibody P17 shares the highest extent of overlap with that of ab6. In contrast to ab6, P17 potency was shown to be sensitive to mutations at position 484 within the RBD and was significantly escaped by the BA.1 Omicron subvariant33.
Several RBD mutation-resistant antibodies against SARS-CoV-2 have been reported during the preparation of this manuscript34,35,36,37,38, providing additional context regarding the conserved epitope we report here. Antibodies DH104739 and STE90-C1136 were isolated from convalescent patients and SARS2-3834 from immunized mice. All three antibodies are RBD directed and bind epitopes distal to that of ab6 (Fig. 6). While STE90-C11 tolerated most circulating RBD mutations, it exhibited loss of activity against the K417T, K417N, and N501Y mutations36, which are present in many VOC/VOI spike proteins. In contrast, SARS2-38 and DH1047 bind highly conserved epitopes, retaining potency across all VOC/VOI spikes, with DH1407 exhibiting cross-reactivity with additional sarbecoviruses34,38. VH ab6 is distinguished from these previously reported antibodies by its unique angle of approach and binding mode involving multiple VH scaffold – RBD contacts (Fig. 1e), along with its small (15 kDa) size. Small antibody fragments are attractive therapeutic modalities given their enhanced tissue penetration compared to conventional monoclonal antibodies40,41.
A recent study by a global consortium defined seven RBD binding antibody communities and showed broadly neutralizing antibodies either bind cryptic epitopes within the inner RBD face (communities RBD-6, RBD-7), or are non-ACE2 competing antibodies that bind the outer RBD face (community RBD-5)37. Ab6 binds the inner RBD face and contacts the RBM, enabling ACE2 competition, drawing similarity to the RBD-4 antibody community, which interestingly was not shown to contain any broadly neutralizing antibodies. Structural comparison of the ab6 footprint with a representative RBD-4 antibody (C002)15 reveals an overlapping footprint shared by the C002 heavy chain and ab6 despite differences in binding modes (Fig. 6). C002 is derived from a convalescent patient, suggesting the potential for such an epitope to be recognized by natural antibodies. This evidence further supports the potential value of focus on the ab6 binding epitope for future therapeutic design.
Plasmids and cloning
SARS-CoV-2 S protein Alpha, Beta, Gamma, and Epsilon genes were synthesized and inserted into pcDNA3.1 (GeneArt Gene Synthesis, Thermo Fisher Scientific). Alpha, Beta, Gamma, Epsilon, Delta, and Kappa mutated S protein ectodomain genes were synthesized incorporating the hexa-proline stabilizing mutations, the transmembrane domain replaced with a fibrin trimerization motif, and a C-terminal hexa-histidine tag (GeneArt Gene Synthesis, Thermo Fisher Scientific). The HexaPro expression plasmid was a gift from Jason McLellan (Addgene plasmid #154754; http://n2t.net/addgene:154754; RRID: Addgene_154754) which we used to construct the D614G mutant via site-directed mutagenesis (Q5 Site-Directed Mutagenesis Kit, New England Biolabs).
C-terminal 7x his tagged NTD (amino acids 1–305) and RBD (amino acids 319–541) constructs were PCR amplified from D614G, Alpha, Beta, Gamma, and Epsilon full-length spike ORFs. NTD constructs were cloned into pcDNA 3.1 using NheI and MssI restriction enzyme cloning, while the RBD constructs were introduced in frame to the mu phosphatase signal sequence and incorporated within pcDNA3.1 via Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit, New England Biolabs).
Human ACE2 (residues 1–615) with a C terminal 7x his tag was amplified from “hACE2”, a kind gift from Hyeryun Choe (Addgene plasmid # 1786) and cloned into pcDNA3.1 via BstXI and XbaI restriction enzyme cloning. Successful cloning was confirmed by Sanger sequencing (Genewiz, Inc.) for all constructs.
Patient-derived sera samples were collected according to the CARE COVID Study (http://www.bccdc.ca/health-professionals/clinical-resources/covid-19-care/covid-19-serology-care-covid-study) with ethics approval from the UBC Clinical Research Ethics Board. Informed consent was received from all study participants.
Pseudovirus neutralization assay
Variant and D614G spike pseudotyped retroviral particles were produced in HEK293T cells as described previously42. Briefly, a third-generation lentiviral packaging system was utilized in combination with plasmids encoding the full-length SARS-CoV-2 spike, along with a transfer plasmid encoding luciferase and GFP as a dual reporter gene. Pseudoviruses were harvested 60 h after transfection, filtered with a 0.45 µm PES filter, and frozen. For neutralization assays, HEK293T-ACE2-TMPRSS2 cells43 (BEI Resources cat# NR-55293) were seeded in 96-well plates at 50 000 cells. The next day, pseudovirus preparations normalized for viral capsid p24 levels (Lenti-X™ GoStix™ Plus) were incubated with dilutions of the indicated antibodies or sera, or media alone for 1 h at 37 °C prior to addition to cells and incubation for 48 h. Cells were then lysed and luciferase activity was assessed using the ONE-Glo™ EX Luciferase Assay System (Promega) according to the manufacturer’s specifications. Detection of relative luciferase units was carried out using a Varioskan Lux plate reader (Thermo Fisher). Percent neutralization was calculated relative to signals obtained in the presence of the virus alone for each experiment.
Expression and purification of recombinant proteins
Expi293F cells (Thermo Fisher, Cat# A14527) were grown in suspension culture using Expi293 Expression Medium (Thermo Fisher, Cat# A1435102) at 37 °C, 8% CO2. Cells were transiently transfected at a density of 3 × 106 cells/mL using linear polyethylenimine (Polysciences Cat# 23966-1). The media was supplemented 24 h after transfection with 2.2 mM valproic acid, and expression was carried out for 3–7 days at 37 °C, 8% CO2.
For ectodomain trimer and monomeric human ACE2 (residues 1–615) purification, the supernatant was harvested by centrifugation and filtered through a 0.22-μM filter prior to loading onto a 5 mL HisTrap excel column (Cytiva). The column was washed for 20 CVs with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl), 5 CVs of wash buffer supplemented with 20 mM imidazole, and the protein eluted with elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions containing the protein were pooled and concentrated (Amicon Ultra 100 kDa cut off for ectodomain, 10 kDa for monomeric ACE2) before gel filtration. Gel filtration was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer (20 mM Tris pH 8.0, 150 mM NaCl). Peak fractions corresponding to soluble protein were pooled and concentrated to 4.5–5.5 mg/mL (Amicon Ultra 100 kDa cut off for ectodomain, 10 kDa for monomeric ACE2). Protein samples were flash-frozen in liquid nitrogen and stored at −80 °C.
For purification of the RBD and NTD constructs, the supernatant was harvested after 7 days of expression and incubated with 300 µL of Ni-NTA resin (Qiagen) at 4 °C overnight. The resin was washed three times with 5 mL of PBS, then three times with 5 mL of PBS supplemented with 20 mM of imidazole. Proteins were eluted in PBS containing 300 mM of imidazole and then buffer exchanged into PBS and concentrated to 5–10 mg/mL (Amicon Ultra 10 kDa cut off, Millipore Sigma) before flash freezing and storage at −80 °C.
For purification of dimeric human ACE2-FC, the supernatant was harvested after 6 days of expression and flowed through a gravity column containing over 400 µL of Protein A Plus Agarose (Thermo Fisher Cat# 22812) once. The column was washed once with 5 mL of PBS before elution with 0.1 M glycine pH3.5 and immediate neutralization with 1 M Tris pH 8.0. Elutions were pooled and concentrated using an Amicon Ultra 50 kDa cut-off concentrator before gel filtration. Gel filtration was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer (20 mM Tris pH 8.0, 150 mM NaCl). Peak fractions corresponding to soluble protein were pooled and concentrated to 2–5 mg/mL (Amicon Ultra 50 kDa cut-off). Protein samples were flash-frozen in liquid nitrogen and stored at −80 °C.
VH-FC ab8, IgG ab1, Fab S309, and Fab S2M11 were produced as previously described3,13,14. Plasmids encoding light and heavy chains for Fab 4A8 and Fab 4–8 were synthesized (GeneArt Gene Synthesis, Thermo Fischer Scientific). Heavy chains were designed to incorporate a C terminal 6x histidine tag. Expi293 cells were transfected at a density of 3 × 106 cells/mL using linear polyethylenimine (Polysciences Cat# 23966-1). Twenty-four hours following transfection, media was supplemented with 2.2 mM valproic acid, and expression was carried out for 3–5 days at 37 °C, 8% CO2. The supernatant was harvested by centrifugation and filtered through a 0.22 μM filter prior to loading onto a 5 mL HisTrap excel column (Cytiva). The column was washed for 20 CVs with wash buffer (20 mM Tris pH 8.0, 500 mM NaCl), 5 CVs of wash buffer supplemented with 20 mM imidazole. The protein was eluted with elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 500 mM imidazole). Elution fractions containing the protein were pooled and concentrated (Amicon Ultra 10 kDa cut-off, Millipore Sigma) for gel filtration. Gel filtration was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer (20 mM Tris pH 8.0, 150 mM NaCl). Peak fractions corresponding to soluble protein were pooled and concentrated to 8–20 mg/mL (Amicon Ultra 10 kDa cut-off, Millipore Sigma). Protein samples were stored at 4 °C until use.
Electron microscopy sample preparation and data collection
For cryo-EM, S protein samples were prepared at 2.25 mg/mL, with and without the addition of ACE2 or antibody (1:1.25 S protein trimer:ACE2 molar ratio, 1:9 S protein trimer:VHab6 molar ratio, 1:8 S protein trimer:S2M11 fab molar ratio, 1:4 S protein trimer:4A8/4-8 fab molar ratio). Mixtures were incubated on ice for 20 min prior to centrifugation at 14,000 × g for 10 min. Vitrified samples of all S protein samples were prepared by first glow discharging Quantifoil R1.2/1.3 Cu mesh 200 holey carbon grids for 30 seconds using a Pelco easiGlow glow discharge unit (Ted Pella) and then applying 1.8 µL of protein suspension to the surface of the grid at a temperature of 10 °C and a humidity level of >98%. Grids were blotted (12 s, blot force −10) and plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). All cryo-EM samples were imaged using a 300 kV Titan Krios G4 transmission electron microscope (Thermo Fisher Scientific) equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at ×155,000 magnification (calibrated pixel size of 0.5 Å per physical pixel) over a defocus range of −0.5 µm to −3 μm with a total dose of 40 e−/Å2 using the EPU automated acquisition software.
The detailed workflow for the data processing is summarized in Supplementary Figs. 1–4, 6–7, 11–14, 17, 23, and 25. In general, all data processing was performed in cryoSPARC v.3.244 unless stated otherwise. Patch mode motion correction (EER upsampling factor 1, EER number of fractions 40), patch mode CTF estimation, reference free particle picking, and particle extraction were carried out on-the-fly in cryoSPARC live. After preprocessing, particles were subjected to 2D classification and/or 3D heterogeneous classification. The final 3D refinement was performed with an estimation of per particle CTF and correction for high-order aberrations.
For the complexes of spike protein ectodomain and human ACE2, focused refinements were performed with a soft mask covering a single RBD and its bound ACE2. For the complexes of spike protein ectodomain and VH ab6, a soft mask covering VH-ab6 and its bound RBD was used in focused refinement. For the complexes of Gamma spike protein ectodomain and fab 4-8/4-A8, another round of 3D refinement with C3 symmetry was carried out, followed by symmetry expansion. The derived particles were then focused-refined with a soft mask covering single NTD and its bound fab.
Global resolution and focused resolution were determined according to the gold-standard FSC45.
Model building and refinement
Initial models either from published coordinates (PDB code 7MJG, 7MJM, 7MJN, 7LXY, 7K43, and 7MJI) or from homology modeling (for 4–8, 4-A8, and VH-ab6) were docked into the focused refinement maps or global refinement maps using UCSF Chimera v.1.1546. Then, mutation and manual adjustment were performed with COOT v.0.9.347, followed by iterative rounds of refinement in COOT and Phenix v.1.1948. Model validation was performed using MolProbity49. Figures were prepared using UCSF Chimera, UCSF ChimeraX v.1.1.150, and PyMOL (v.2.2 Schrodinger, LLC).
Biolayer interferometry (BLI) S protein—ACE2 binding assay
The kinetics of SARS-CoV-2 trimers and human ACE2 binding were analyzed with the biolayer interferometer BLItz (ForteBio, Menlo Park, CA). Protein-A biosensors (ForteBio: 18–5010) were coated with ACE2-mFc (40 µg/mL) for 2 min and incubated in DPBS (pH = 7.4) to establish baselines. Concentrations of 125, 250, 500, and 1000 nM spike trimers were used for the association for 2 min followed by dissociation in DPBS for 5 min. The association (kon) and dissociation (koff) rates were derived from the fitting of sensorgrams and used to calculate the binding equilibrium constant (KD).
Flow cytometry S protein—ACE2 binding assay
Expi293 cells were transfected with plasmids encoding the various full-length variant S proteins using linear PEI as performed during recombinant spike production. After 3 days of expression, cells were washed with PBS then pelleted and resuspended in incubation buffer (PBS pH 7.4 with 0.02% Tween-20 and 4% BSA) at a concentration of 5 × 106 cells/mL. In all, 100 µl of this mixture was deposited into each well within a 96-well plate followed by a 5 min incubation on ice. Cells were pelleted and resuspended in an incubation buffer containing increasing concentrations of recombinant FC-ACE2 (Sino Biological Cat# 10108-H05H-100) followed by incubation for 20 min on ice. The cells were pelleted and washed with 200 µl of wash buffer (PBS pH 7.4 with 0.02%Tween-20). Next, the cells were incubated in 100 µL of a 1:300 dilution of secondary antibody (Thermo Fisher Cat# A-21235) in an incubation buffer for 10 min. Cells were pelleted and washed twice in 100 µL of wash buffer prior to staining with propidium iodide (Biolegend). Cells were analyzed using a Cytoflex LX at the ubcFLOW cytometry facility. Data were analyzed using FlowJo. The gating strategy employed along with results for un-transfected (negative control) Expi293 cells are available in Supplementary Fig. 10B.
Enzyme-linked immunosorbent assay (ELISA)
For ELISA, 100 µL of wild-type (D614G), or variant SARS-CoV-2 S proteins, NTDs, and RBDs were coated onto 96-well MaxiSorp™ plates at 2 µg/mL in PBS + 1% casein overnight at 4 °C. All washing steps were performed 3 times with PBS + 0.05% Tween 20 (PBS-T). After washing, wells were incubated with blocking buffer (PBS-T + 1% casein) for 1 h at room temperature. After washing, wells were incubated with dilutions of primary antibodies in PBS-T + 0.5% BSA buffer for 1 h at room temperature. For fab and IgG primary antibodies, wells were incubated with goat anti-human IgG (Jackson ImmunoResearch) at a 1:8000 dilution in PBS-T + 1% casein buffer for 1 h at room temperature. For VH ab6, wells were incubated with mouse anti-FLAG M2 antibody (Sigma Aldrich) at a 1:50 dilution in PBS-T + 1% casein buffer for 1 h at room temperature, washed, and incubated with goat anti-mouse IgG (Thermo Fisher Scientific Cat# 31439) at a 1:5000 dilution in PBS-T + 1% casein buffer for 1 h at room temperature. For ACE2 competition experiments, serial dilutions of VH ab6 were incubated in wells in PBS-T + 1% casein buffer for 1 h at room temperature and washed before incubation with either 0.2 or 1 ng/mL of human ACE2-FC in PBS-T + 0.5% casein for 1 h at room temperature. After washing, wells were incubated with goat anti-human IgG (Jackson ImmunoResearch) at a 1:8000 dilution in PBS-T + 1% casein buffer for 1 h at room temperature. After washing, the substrate solution (Pierce™ 1-Step™) was used for color development according to the manufacturer’s specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific).
Surface plasmon resonance (SPR)
SPR experiments were performed on a Biacore T200 instrument. CM5 chips were functionalized with monoclonal anti-Strep-Tag antibody (BIO-RAD Cat# MCA2489) at a concentration of 50 µg/mL for the capture of spike protein ectodomains. After the capture of D614G hexapro ectodomain, either buffer, 175 nM, or 440 nM of VH ab6 was injected onto the chip, followed by 500 nM human ACE2-FC. The surface was regenerated in 10 mM glycine, pH 1 between experiments. The experiments were performed at 25 degrees Celsius, using 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v Surfactant P20 as running buffer. Reference-subtracted curves were utilized for the qualitative assessment of ACE2 competition. Relative response units (RUs) were normalized to a value of 0 immediately before ACE2-FC injection for ease of ACE2-FC binding assessment.
Authentic SARS-CoV-2 plaque reduction neutralization assay
Neutralization assays were performed using Vero E6 cells (ATCC CRL-1586) that were seeded 24 h prior to the assay in 24-well tissue culture plates at a density of 3 × 105 cells per well. Antibodies were serially diluted twofold (starting concentration of 4, 10, or 40 µg/mL, depending on the antibody being tested) and mixed with an equal volume of 30–50 plaque-forming units of SARS-CoV-2. This results in a final antibody concentration of 2, 5, or 20 µg/mL in the antibody–virus mixture. The following SARS-CoV-2 variants were used: isolate USA-WA1/2020 (NR-52281, BEI Resources); isolate hCoV-19/South Africa/KRISP-EC-K005321/2020 (NR-54008, BEI Resources); isolate hCoV-19/USA/CA_UCSD_5574/2020 (NR-54008, BEI Resources); and isolate hCoV-19/USA/PHC658/2021 (NR-55611, BEI Resources). The antibody-virus mixture was then incubated at 37 °C in a 5% CO2 incubator for 1 h and added to the Vero E6 cell seeded monolayers, in duplicate. Plates were then incubated for 1 h at 37 °C in a 5% CO2 incubator. Following incubation, an overlay media with 1% agarose-containing media (2× Minimal Essential Medium, 7.5% bovine albumin serum, 10 mM HEPES, 100 µg/mL penicillin G, and 100 U/mL streptomycin) was added to the monolayers. The plates were incubated for 48–72 h (depending on the SARS-CoV2 variant) and then cells were fixed with formaldehyde for 2 h. Following fixation, agar plugs were removed, and cells were stained with crystal violet. In order to assess the input virus, a viral back-titration was performed using a culture medium as a replacement for the antibodies. To estimate the neutralizing capability of each antibody, IC50s were calculated by non-linear regression using the sigmoidal dose–response equation in GraphPad Prism 9. All assays were performed in the University of Pittsburgh Regional Biocontainment Laboratory BSL-3 facility.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
The data that support this study are available from the corresponding author upon request. The atomic models and cryo-EM density maps have been deposited into the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) as follows: Alpha (B.1.1.7) apo spike protein: PDB 8DLI and EMD-27502, Alpha (B.1.1.7) spike protein-ACE2 complex (global): PDB 8DLJ and EMD-27503, Alpha (B.1.1.7) spike protein-ACE2 complex (focused): PDB 8DLK and EMD-27504, Beta (B.1.351) apo spike protein: PDB 8DLL and EMD-27505, Beta (B.1.351) spike protein-ACE2 complex (global): PDB 8DLM and EMD-27506, Beta (B.1.351) spike protein-ACE2 complex (focused): PDB 8DLN and EMD-27507, Gamma (P.1) apo spike protein: PDB 8DLO and EMD-27508, Gamma (P.1) spike protein-ACE2 complex (global): PDB 8DLP and EMD-27509, Gamma (P.1) spike protein-ACE2 complex (local): PDB 8DLQ and EMD-27510, Gamma (P.1) spike protein-4-8 complex (global): EMD-27511, Gamma (P.1) spike protein-4-8 complex (focused): PDB 8DLR and EMD-27512, Gamma (P.1) spike protein-4A8 complex (global): EMD-27513, Gamma (P.1) spike protein-4A8 complex (focused): PDB 8DLS and EMD-27514,Epsilon (B.1.429) apo spike protein: PDB 8DLT and EMD-27515, Epsilon (B.1.429) spike protein-ACE2 complex (global): PDB 8DLU and EMD-27516, Epsilon (B.1.429) spike protein-ACE2 complex (focused): PDB 8DLV and EMD-27517, Epsilon (B.1.429) spike protein-S2M11 complex: PDB 8DLW and EMD-27518, Epsilon (B.1.429) spike protein-VH ab6 complex (global): PDB 8DLX and EMD-27519, Epsilon (B.1.429) spike protein-VH ab6 complex (focused): PDB 8DLY and EMD-27520, D614G spike protein-VH ab6 complex (global): PDB 8DLZ and EMD-27521, D614G spike protein-VH ab6 complex (focused): PDB 8DM0 and EMD-27522. Source data are provided with this paper.
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We thank the study participants, The BC Centre for Disease Control Public Health Laboratory, along with Dr. Inna Sekirov and Dr. Ana Citlali Marquez for providing human sera and patient demographic information. This work was supported by awards to S.S. from a Canada Excellence Research Chair Award; the VGH Foundation; Genome BC, Canada; and the Tai Hung Fai Charitable Foundation. D.M. is supported by a CIHR Vanier Canada Graduate Scholarship. J.W.S. is supported by a CIHR Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award (CGS-D) and a University of British Columbia President’s Academic Excellence Initiative PhD Award. S.M.B.-B. is funded by NIH R21AI47017. This work was supported by the University of Pittsburgh Medical Center.
Z.S., W.L., J.W.M., and D.S.D. are coinventors of a patent, filed by the University of Pittsburgh, related to ab6, ab1, and ab8 that were assessed in this manuscript. S.S. is the Founder and CEO of Gandeeva Therapeutics Inc. The remaining authors declare no competing interests.
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Cite this article
Mannar, D., Saville, J.W., Sun, Z. et al. SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization.
Nat Commun 13, 4696 (2022). https://doi.org/10.1038/s41467-022-32262-8
Received: 07 December 2021
Accepted: 21 July 2022
Published: 18 August 2022
Polio Is Back. Trust the Vaccines. – The Bulwark
A polio outbreak is upon us. How our government, public health professionals, and the media respond could lead to further politicization of vaccines, or a step toward restoring faith in public health.
First, we need to get the facts straight about the current outbreak: While just one man in Rockland Country, New York has thus far been paralyzed by the virus, this case is likely the tip of the iceberg. The reemergence of this virus, last spread in the United States in 1979, is certainly reason for worry—the virus is 2-3 times as infectious as the original strain of COVID-19 and paralyzes 1 in 200 people that it infects (about 1 in 4 will show “flu-like” symptoms). As recently as the 1940s, it disabled 35,000 Americans a year, and there is no way to cure the paralysis once someone has been infected. In especially severe cases, polio can cause death as the muscles that control breathing become paralyzed and the patient suffocates. Since this case was uncovered, wastewater testing in adjoining Orange County and New York City has demonstrated spread. (London and Jerusalem have also found evidence of the virus.)
Humankind has been dealing with poliovirus for millennia, yet the strain of this ancient virus making the rounds in New York is relatively new. The virus at issue is a mutated vaccine-derived poliovirus, meaning its ancestor is used to vaccinate people across the globe (outside the U.S.) in the form of a drink. Oral vaccination with an “attenuated” or nearly harmless virus has rare side effects (including paralysis, but at a rate of less than 1 percent of the lifetime likelihood of being struck by lightning). This vaccination technique is still used in other countries because it is cheap, easy to store and distribute, and can immunize others in the community as the attenuated virus infects and delivers immunity to unvaccinated individuals. We ceased using this very inexpensive, but effective, method in the U.S. in 2000, mostly due to these rare side effects. All U.S. polio vaccinations are now a four-shot series given to children 2 months to 6 years of age that contain only the dead (inactivated) virus with no chance of replicating or causing paralysis. It is especially important to vaccinate children, as they have the greatest risk of paralysis from the virus.
In the Rockland County case, it appears that the unlucky patient was an unvaccinated individual with recent contacts from foreign countries where the oral vaccination technique is still used. The attenuated vaccine-derived virus had spread and replicated among unvaccinated people in a country with less robust polio vaccination rates than in the United States. Somewhere in this replication, the vaccine-derived virus mutated and re-developed the ability to paralyze. The fact that its genetic material has been found on three continents suggests that it has retained its ancestors’ infectiousness.
Since the virus came about from a vaccine, why should people get vaccinated? For one, the current U.S. standard inactivated poliovirus (IPV) shot series cannot cause infection or paralysis, and is given routinely to infants across the country. Secondly, the IPV shot series protects against paralysis should an individual ever come in contact with this new virus at rates of over 99 percent. Finally, high rates of vaccination in a community can lead to herd immunity, where a virus is unable to propagate an outbreak because most people are already immune. Herd immunity is a hyper-local phenomenon and requires more than 80 percent of people in a community to be immune to prevent an outbreak. Less than half of New York counties with available data have children vaccinated at that 80 percent rate. And we have communities all across America with much lower vaccination rates—New York State’s overall polio vaccination rate is actually higher than the national average.
Somewhat understandably, it would be easy for those on either side of the political aisle to pervert the story of a vaccine-derived virus to further their own agenda. A narrative about a virus originating from a vaccine and causing harm has the potential to further mistrust, and if spun incorrectly could damage vaccination rates in this country even further. The public health community in this country must take action against this possibility with a unified, truthful, and straightforward explanation about how this infection came about and how to prevent its spread. Our vaccination rates among children have been on a downtrend since before COVID. Misinformation and disinformation during this pandemic, in no small part promoted by licensed medical professionals, may contribute to even lower rates in the future. The fields of science, public health, and medicine must act in unison and not allow this type of malicious “skepticism” about vaccines to proliferate into mainstream discourse if we wish to keep preventable infectious diseases at bay in this country.
The usefulness of childhood vaccines is a settled matter among public health experts. Vaccines save lives, and they do it at rates that easily justify their comparatively minimal costs. Even as we face this new threat, there are state legislatures that want to offer parents yet more freedom not to vaccinate their children. It is time to get our message together in a way that reflects the inestimable health benefits vaccines provide for our country every year.
Polio does not look at political affiliation before it ravages a host. It has infected Republicans as high ranking as Senate Minority Leader Mitch McConnell, and Democrats as powerful as President Franklin Delano Roosevelt. This outbreak has yet to receive the news coverage it deserves, which is a shame, but also an opportunity to get the story right: Just as with COVID, the American vaccines work superbly well, and they can save untold numbers of people from suffering and even death.
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