Type 1 diabetes is an autoimmune disease that leads to disrupted glucose metabolism. It requires lifelong insulin therapy. The Global Platform for the Prevention of Autoimmune Diabetes (GPPAD) collaborates within a European network to develop new methods to prevent this condition which is, to date incurable. AVAnT1A is GPPAD's third intervention study. It will investigate whether vaccination against SARS-CoV-2 in the first year of life can protect children who have an increased genetic risk for type 1 diabetes from developing the condition. The study is supported by funding from The Leona M. and Harry B. Helmsley Charitable Trust.
In Germany, four out of 1000 children are diagnosed with type 1 diabetes, a metabolic disorder triggered by an autoimmune reaction. In individuals with type 1 diabetes, the immune system destroys the insulin-producing cells in the Langerhans islets of the pancreas. Affected individuals require lifelong insulin treatment as insulin plays a vital role in transporting sugar from the blood into the body's cells. Approximately 90 percent of affected children and adolescents have no close relatives with type 1 diabetes, often leading to a late and unexpected diagnosis. However, researchers can identify the underlying autoimmune process long before symptoms occur, based on islet autoantibodies in the blood.
The exact causes of the underlying autoimmune reaction remain unclear. In large-scale, long-term studies, researchers at Helmholtz Munich have identified viral infections in early childhood as a crucial environmental factor for the development of type 1 diabetes. During the COVID-19 pandemic, GPPAD researchers made another important observation: after contracting a SARS-CoV-2 infection, children with an increased risk for type 1 diabetes were more likely to develop islet autoantibodies.
COVID-19 has increased the risk of the disease. We have seen that children who had COVID-19 before age 18 months were around five times more likely to develop the islet autoantibodies as those who were not infected."
Prof. Ezio Bonifacio, GPPAD researcher at the Center for Regenerative Therapies Dresden (CRTD) of TUD Dresden University of Technology
These autoantibodies are biomarkers indicating the beginning of the autoimmune process that leads to type 1 diabetes.
In a new study, GPPAD aims to further investigate this relationship. The AVAnT1A Study - short for "AntiViral Action against Type 1 Autoimmunity" - examines whether vaccination against COVID-19 at the age of six months can prevent the development of islet autoantibodies in babies at increased genetic risk of developing type 1 diabetes, thus reducing their risk of developing the condition. The vaccine used is safe and approved for children aged six months and older. The participating children are randomly assigned to two groups beforehand using a randomized, placebo-controlled study design. The intervention group will receive the COVID-19 vaccine, the control group will receive a placebo injection without active ingredients. The vaccine is provided by the Center for Pandemic Vaccines and Therapeutics (ZEPAI) at the Paul-Ehrlich-Institute, Federal Institute for Vaccines and Biomedicines. Additionally, the study is double-blinded, so the researchers, the study personnel, and the families, will not know which group a child belongs to until the study is completed.
"Since many infections in young children occur almost without symptoms, we also ask participating families to collect saliva and stool samples from their children at regular intervals," explains Prof. Anette-Gabriele Ziegler, Director of the Helmholtz Munich Institute for Diabetes Research and GPPAD, Chair of Diabetes and Gestational Diabetes at Klinikum rechts der Isar and Technical University of Munich. From these samples, researchers can identify which viruses the children had contact with. This allows the researchers to clarify further connections between type 1 diabetes and viral infections in early childhood.
"With a planned number of 2252 participants, the AVAnT1A Study is the largest intervention study to date exploring the relationship between type 1 diabetes and early childhood viral infections. Insights generated with this study will help us to move closer to our goal of a world without type 1 diabetes," says Prof. Sandra Hummel, lead scientist in the AVAnT1A study and researcher at Helmholtz Munich. Children with an increased genetic risk for developing type 1 diabetes are invited to participate in the AVAnT1A study. Their increased risk is detected within the newborn screening called "Freder1k", which for Germany is offered in Bavaria, Lower Saxony, Saxony, and Thuringia. Parents can test their newborns either directly at the birth clinic or in the pediatrician's office until six weeks of age. A tiny drop of blood from the umbilical cord or heel is sufficient for this test.
Children participating in the AVAnT1A study are invited for regular check-ups until their sixth birthday. The families benefit from participating in an early detection program for type 1 diabetes. If a child shows initial signs of the autoimmune disease, this can be detected early in the disease progression, ensuring families access to optimal support and information to support their child.
"This study holds promise to help tease out the link between viral infections and the development of autoimmunity in type 1 diabetes;" says Anne Koralova, Program Officer at the Helmsley Charitable Trust. "The investigators at GPPAD have done incredible work studying strategies to prevent the development of type 1 diabetes, and Helmsley is committed to supporting these innovative studies."
Following the POInT Study (short for: Primary Oral Insulin Trial) and the SINT1A Study (short for: Supplementation with B. INfantis for Mitigation of Type 1 Diabetes Autoimmunity), AVAnT1A is now GPPAD's third intervention study aimed at developing new preventive measures for type 1 diabetes. The POInT Study investigated whether the administration of insulin powder in the first three years of life has a protective effect on the immune system. The study will be completed in 2024. Participants in the SINT1A Study receive a probiotic in the first year of life, which aims to positively influence the microbiome and thus prevent autoimmune reactions. The SINT1A study achieved full recruitment in March 2024. The GPPAD research platform, and all studies supported within it, are financed by The Leona M. and Harry B. Helmsley Charitable Trust in the U.S.
In a recent review published in the journal Nature Reviews Immunology, researchers discussed the limitations of current influenza vaccines and the potential for future vaccines to induce both T-cell responses and antibodies for enhanced protection. They examined the strategies to develop influenza vaccines with broad strain specificity and long-term efficacy, covering protection requirements, immune response evaluation, expected outcomes, and financial considerations.
Study: Opportunities and challenges for T cell-based influenza vaccines. Image Credit:CI Photos/ Shutterstock
Influenza, a longstanding public health challenge, has caused significant morbidity and mortality worldwide, with annual death tolls up to 650,000. The coronavirus disease 2019 (COVID-19) pandemic temporarily lowered influenza activity, but as restrictions ease, cases are resurging. Vaccination remains pivotal in mitigating influenza's impact, yet existing vaccines have limitations, including variable effectiveness. Prioritizing vaccination for high-risk groups is crucial. The present review highlights the importance of ongoing influenza prevention efforts amid evolving public health landscapes. It explores enhancing traditional influenza vaccines by eliciting broader and more durable immune responses across multiple influenza strains and seasons.
The past decade has seen advancements in influenza vaccine technologies, including quadrivalent formulations and non-egg-based production methods, aiming to improve strain specificity and durability. Antibody-focused strategies dominate, but recent research suggests inducing broadly reactive antibodies targeting hemagglutinin stem regions and neuraminidase for universal influenza vaccines. Effectiveness varies yearly, with recent high-dose, adjuvanted, and novel cell-based vaccines showing modest improvements. However, even the best influenza vaccines fall short compared to highly efficacious vaccines for other pathogens, underscoring the need for further research and innovation.
Influenza vaccine performance is challenged by antigenic variation, original antigenic sin, high levels of pre-existing immunity in the population, and a focus on antibody responses rather than broader immune activation. Antigenic shifts in circulating viruses, coupled with pre-existing immunity, reduce vaccine effectiveness. Original antigenic sin may limit vaccine efficacy by preferentially boosting cross-reactive antibodies. Moreover, the predominance of antibody-based vaccines may not fully replicate natural immune responses, suggesting a need for innovative strategies to induce more comprehensive immunity, including CD4+ (cluster of differentiation 4) and CD8+ T-cell responses.
T-cells play a crucial role in influenza virus immunity, contributing to protection through cytotoxicity, inflammatory cytokine release, and support for antibody responses. CD4+ T-cells, particularly TH1 cells, are essential for promoting antibody responses and clearing infected cells, while CD8+ cytotoxic T lymphocytes (CTLs) target and kill infected cells. Additionally, CD4+ T helper cells support B-cell responses, and resident memory T-cells (TRM) provide rapid resistance to local infection, potentially intercepting early infectious events. Despite challenges such as antigenic variation and immune memory, T-cell responses remain vital for influenza vaccine efficacy and could be harnessed for improved protection strategies.
Multiple studies in both mouse models and humans demonstrate the protective role of T-cells, particularly CD8+ and CD4+ T cells, against influenza virus infection. While T-cell responses may not prevent infection, they reduce symptomatic illness and contribute to viral clearance. Studies involving human challenge with influenza viruses show correlations between pre-existing T-cell responses and reduced virus shedding and symptom severity. Observational studies during the 2009 H1N1 pandemic and subsequent seasons further support the protective effects of T-cell immunity against symptomatic influenza illness. However, the potential role of non-neutralizing antibodies in mediating protection alongside T-cell responses warrants further investigation. Overall, these findings underscore the importance of T-cell-mediated immunity in influenza virus defense.
Hemagglutinin in vaccines generates neutralizing antibodies, and neuraminidase induces non-neutralizing antibodies. Adjuvants enhance immune function. Vaccine delivery routes influence systemic or local responses. Messenger ribonucleic acid (mRNA) vaccines induce CD4+ and CD8+ T cells. Viral vectors elicit CD8+ T-cell responses. Nanoparticles promote tissue-resident memory T-cell priming. Optimizing these factors can enhance influenza vaccine efficacy by stimulating appropriate immune responses.
Recent trials of T-cell-inducing influenza vaccines yielded mixed results. While OVX836 showed 84% protection, FLU-v demonstrated efficacy with a single dose, and M-001 showed no efficacy. Concerns include narrow T-cell responses. Safety was good, highlighting the need for more extensive phase IIb or III trials. Criteria for success of T-cell vaccines require careful consideration.
Combining T-cell-inducing vaccines with antibody-inducing ones enhances protection, replicating the synergistic response observed in natural infections. This approach is currently under investigation in vaccine development against COVID-19 and acquired immunodeficiency syndrome (AIDS).
Measuring immune responses in vaccine development poses challenges due to the complexity of correlating protective mechanisms. Evaluating T-cell responses is particularly intricate, requiring sophisticated assays and considerations of compartmentalization, while assessing success relies on endpoints that may not fully capture T-cell-mediated protection, emphasizing the need for comprehensive evaluation methods in clinical trials.
In conclusion, efforts to improve influenza vaccines, including T-cell-inducing strategies, are crucial given their modest efficacy compared to other vaccines. Innovative approaches, bolstered by lessons from COVID-19, hold promise for addressing the ongoing burden of influenza, necessitating increased research, investment, and a redefined approach to vaccine development and evaluation.
MITCHELL South Dakota saw a flu season that was stronger than normal this year.
According to a recent report issued by the South Dakota Department of Health, the 2023-24 flu season is the worst in the past five years. The state has set five-year highs for the number of confirmed flu cases along with the most hospitalizations and deaths since 2018-19.
This flu season has included 19,911 confirmed cases. Davison County accounts for 529 of that total. The 831 hospitalizations statewide were the most since 2017-18 when there were 878 hospitalizations. The number of deaths was also the highest at 44, one more than 2018-19 when there were 43 deaths.
Those between the ages of 25 and 49 are the most likely to catch the flu, followed by younger people ages 5 to 24. The highest affected age group this season however was those between the ages of 5 to 24.
While numbers were higher than they have been in previous seasons, the data does not represent the severity of illness, according to State Epidemiologist Dr. Joshua Clayton. Clayton said the severity of the illness was similar to influenza seasons prior to the Covid-19 pandemic.
Though it is tough to pinpoint any single reason for this seasons bustling flu activity, medical professionals shed light on what they saw and why this flu season is setting records.
Dr. Darren Manthey, an emergency medicine physician with Avera Medical Group in Mitchell, agreed that flu activity seemed widespread this year.
We had high rates of emergency department visits and outpatient visits, Manthey said. I would say it was a moderately severe season.
Manthey also says the season ran later this year. Flu season typically peaks in the first week of February in South Dakota. However, the DOH stated the peak for the 2023-24 flu season wasnt until the second week of March.
Manthey suggests that one of the reasons the flu hit the state harder this year is because fewer people chose to get their flu shots this year.
The vaccination rate is slightly down this year, Manthey said. Lower vaccination rates mean more people arent protected and there are greater opportunities for the virus to spread.
The downward trend in Influenza immunization has Avera Vice President of Clinical Quality Dr. David Basel concerned.
The lower herd immunity will likely lead to increased numbers and severity of the disease especially in the very young, the very old and the immunosuppressed, Basel said.
While those getting the flu shot were down this year, according to Megan Jensen, a spokesperson for Sanford Health. System officials for Sanford said the flu shot this year was very protective against the influenza strains that were in existence this season.
Vaccine makers have to make educated guesses as they create flu shots well in advance of the respiratory virus season. Sometimes, the guesses are only so-so. But the ingredients in this years vaccine appeared to be right on target.
The influenza and Covid vaccines were extremely effective, lowering the number of severe cases and hospitalizations, Jensen said. Vaccines continue to be the best protection against respiratory illnesses.
Clayton also attributes the higher number to improvements in testing and reporting. There have been improvements in lab testing, so more clinics were able to do confirmatory influenza testing in clinic rather than having to send specimens to outside laboratories.
There have also been advancements in electronic laboratory reporting that have resulted in more timely and more accurate reporting that is automatic and not submitter based, according to Clayton.
Manthey points out that people often misuse the term flu. Influenza is different than having whats often called the stomach flu. Sometimes people mistake symptoms of the stomach flu for the viral infection commonly called flu. But theyre not the same.
Lots of people talk about getting the stomach flu, so theyll get nausea, vomiting and diarrhea. They will say they have the flu, Manthey said. Its important to recognize that influenza is a different disease.
The flu, or influenza, comes with symptoms like fever, congestion, muscle aches and fatigue.
While the severity of influenza affecting people was not abnormal, some people report feeling sick or longer or struggling to recover from seasonal illnesses.
The Influenza A I had was terrible, said Karli Kiner, of Mitchell. I had it three weeks ago and still have a lingering cough."
While people seem to be feeling a lot more rotten than usual, the good news it's probably not Covid or even Covid-related. While there are some theories out there that say immune changes may play a role in those with long-Covid syndrome, the linkage remains unclear at this time.
Basel said while there have been some studies suggesting that those who had severe (hospitalized) Covid have detectable changes in the immune system for many months, there is no clear evidence of immune changes in a more generalized population that had milder cases.
We are still learning about what, if any, effect Covid will have on long-term immunity, Basel said.
A small study done last year by the National Institute of Allergy and Infectious Disease found that people who recovered from severe Covid infections had long-lasting changes to their immune system for up to one year. It found stem cells from people with severe Covid produced more white blood cells which then produced more inflammatory signals in the body.
That study was done with cells in a laboratory Petri dish. Experts dont really know what it all means clinically in a human body.
Theres no evidence to support that Covid has damaged immune systems, Manthey said.
Manthey suggests people might just be more aware of how we feel.
A logical explanation as to why people seem more susceptible to illness than ever before is because we went through a respiratory virus pandemic and we are now more attuned to looking for illness than we were before the pandemic, Manthey said.
Jennifer Leither joined the Mitchell Republic in April 2024. She was raised in Sioux Falls, S.D. where she attended Lincoln High School. She continued her education at South Dakota State University, graduating in December 2000 with a bachelor's degree in Journalism. During her time in college, Leither worked as a reporter for the campus newpaper, The Collegian. She also interned for Anderson Publications in Canistota, SD the summer of 2000. Upon graduation, Leither continued to reside in the Sioux Falls area and worked as a freelance writer for the Argus Leader for a number of years. /jennifer-leither
KOLKATA: Worried Covid survivors in Kolkata have been calling up doctors and hospitals following pharmaceutical company AstraZenecas admission in a UK court earlier this week that its Covid-19 vaccine has the potential to cause thrombosis with thrombocytopenia syndrome (TTS). The vaccine was administered in India under the name of Covishield. More than 175 crore doses of Covishield were administered across the country. A section of physicians doesnt rule out the possibility of some of their patients suffering or succumbing to TTS but added that there was no need to press the panic button now since the period for side effects to strike is long over. The time window for side effects of a vaccine is usually a month and that period is long over, pointed out cardiac surgeon Kunal Sarkar. While there is no need to worry about it now, we had a few patients suffering from side-effects during the initial stages of vaccine administration. I operated upon two who had suffered serious cardiac clots soon after taking the vaccine. While symptoms suggested it was a side-effect of Covishield, we have no test in India to confirm that. But the advantages of the vaccine far outweighed the side-effects, said Sarkar. AstraZenecas vaccine formula was licensed to the Pune-based Serum Institute of India (SII) for the production of Covishield. Sarkar rued that the Indian population was not made aware of the possibility of side effects. On the other hand, any vaccine is bound to have some side effects since it replicates the virus and its ill-effects. So, this vaccine, too, in some cases triggered thrombocytopenia or clots in the brain and elsewhere, explained Sarkar. A private hospital said they had received numerous calls from vaccine recipients over the last three days. We have spoken to our doctors and assured them that its too late for side effects now and that they were all safe, said a hospital spokesperson. We knew about the side effects and some did get them. A few are predisposed to such side effects but their number has been very few. Covishield recipients are worried but its too late for side effects, said AMRI infectious diseases physician Sayan Chakrabarty. A section of experts, however, pointed out that though its too late for thrombocytopenia or platelet count drop, clots in the brain may still occur as a long-term side effect of a vaccine. Covid vaccines like Covishield were prepared in a hurry and their long-term implications were not studied. We did receive several cases of thrombosis after the introduction of vaccines though it cant be concluded that they were triggered by Covishield since there was no clinical evidence. Cases of platelet drop were very rare though some patients did suffer from it, said Peerless Hospital microbiologist Bhaskar Narayan Chaudhury. Less than one in 1 lakh had TTS that can happen only within the first month of taking the vaccine and certainly not after years, said M S Purkait, medical superintendent, Techno India DAMA Hospital.
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The Department of Beaches and Harbors is pleased to announce a free COVID-19 Vaccine Clinic at Burton Chace Park in Marina del Rey. Tuesday, May 7, 2024 from 10 AM 3 PM at Burton Chace Park Community Room, 13650 Mindanao Way, Marina del Rey, CA 90292
Open to the public 6 months and older regardless of insurance or immigration status. Minors must be accompanied by parent/guardian.
Updated Moderna COVID Vaccine (while supplies last) and/or Flu Shot Visit https://bit.ly/dbhvaccine to make your appointment. Walk-ups are also welcome.
COVID-19 has killed two more New Brunswickers, while a child under four and a youth aged five to 19 are among the 19 people hospitalized for or with the virus, data released by the province Tuesday shows.
COVIDactivity remains moderate, andmost indicators remained stable during the reporting period,April 21 to April 27, theRespiratory Watch report says.
The report comes just days after theNational Advisory Committee on Immunization, known as NACI, issued its latest guidance on fall COVID-19 vaccines.
A fall dose is "strongly recommended"for people at higher risk of infection or severe disease, including seniors, people with underlying medical conditions, and those who provide essential community services, NACI said.
People aged six months or older who are not at increased risk may receive a fall dose,NACI said.
CBC News has asked the Department of Health for comment about its fall vaccine plans.
NACI said there isn't sufficient data yet to determine the best time to start fall COVID-19 vaccinations, but preliminary observations from previous seasons suggest COVID activity began to increase before fall campaigns were launched. Last year, the national percent positivity began to increase in mid-August, it said. (Jonathan Hayward/The Canadian Press)
NACI "emphasizes the benefits of available vaccines for COVID-19 protection, and particularly for those most at risk of severe illness, as we know that protection against severe illness due to COVID-19 can wane over time," chair Dr. Robyn Harrison said in a statement Friday.
"An updated COVID-19 vaccine formulation may also be available by the fall that would better target the currently circulating strains," she said.
Spring boosters have been available since April 2to New Brunswickers considered most at risk of severe illness.
Nearly 2,000 COVID vaccines were administered in the past week, raising the total to 155,620 since Oct. 4, according to figures fromthe department.
The spring doses will be available until June 15, the department has said.
4 outbreaks, 29 new cases
The two people who died from COVIDduring the reporting week wereaged 45 to 64 and 65 or older.
Their deaths raise the provincial pandemic total to at least 1,034. The actual number is unclear because the Department of Healthcounts only people who die in hospital as COVID deaths.
The19 people who were hospitalized either because of COVID or for something else and later tested positive for the virusis up from 17 in the previous report. One person was admitted to intensive care, the same number as a week ago.
In addition to the child and youth, those admitted to hospital included one person aged 20 to 44, two people aged 45 to 64 and 14 aged 65 or older, one of whom required intensive care.
PCR lab tests are restricted in New Brunswick to those with symptoms who have a referral from a primary care provider and for whom the outcome will 'directly influence treatment or care.' (Kamran Jebreili/The Associated Press)
The number of lab-confirmed COVID outbreaks doubledto four since the last report. One was in a nursing home and the other three were in facilities listed only as "other."
Twenty-ninenew cases of COVIDwere confirmed through polymerase chain reaction (PCR) lab tests,down from31.
The positivity rate or the percentage of lab tests performed that produced a positive result is four per cent, unchanged.
Flu sends child andyouth to hospital
No influenzadeaths were reported betweenApril 21 and April 27,but the flu sent seven people to hospital, up from six the previous week.
Among those admitted to hospital were a child under four,a youth aged five to 19, one person aged 20 to 44, oneaged 45 to 64 and three aged 65 or older.
Influenza activity remainsrelatively stable, thereport says.
Forty-two new flu cases were confirmed by lab tests,down from59, with a positivity rate of five per cent, down from seven.
Eleven of the new cases were influenza A and 31 were influenza B.
Since the respiratory season began on Aug. 27, there have been3,630 confirmed flu cases confirmed.
A total of 224,109 New Brunswickers have been vaccinated against the flu, as of Tuesday, an increase of 146 from a week ago, figures from the department show.
Horizon andVitalit
Horizon Health Network has eightactive COVID admissions, as of Saturday, down from 10 last week, according to its latest COVID report.
None are in intensive care, down from one.
Two health-care workers are off the job, after they tested positive for COVID-19, compared to one a week ago, the report says.
Horizon'sCOVID-19 outbreakpage shows one outbreak, as of Monday, at the Moncton Hospital's cardiology unit.
VitalitHealth Network updates its COVID report only monthly, with the next update expected on May 28.
American depositary receipts (ADRs) of BioNTech(BNTX) were down less than 1% in intraday trading Monday after the COVID-19 vaccine maker shrugged off losses and tumbling sales in the first quarter, giving a positive outlook for the year.
The German biotech firm, which collaborated with Pfizer (PFE) on a COVID-19 vaccine, reported a loss per share of 1.31 euros ($1.41), with revenue plunging 85% year-over-year to EUR187.6 million ($202.3 million). Both were worse than expected as demand for the shot slumped since the end of the pandemic.
However, CFO Jens Holstein said that first-quarter revenues reflect the seasonal demand for COVID-19 vaccines, and we expect to recognize approximately 90% of our full year revenues in the last months of 2024, mostly in Q4 of 2024.
Holstein added that the company has a strong cash position of EUR16.9 billion ($18.2 billion), which he argued makes it well positioned to expand its business into treatments for cancer.
The company affirmed its full-year guidance of revenue between EUR2.5 billion and EUR3.1 billion ($2.7 billion and $3.3 billion).
BioNTech ADRs have lost about 13% of their value in 2024.
Vaccines and immunisation
Researchers say experimental shot is step towards goal of creating vaccines before a pandemic has started
Scientists have created a vaccine that has the potential to protect against a broad range of coronaviruses, including varieties that are not yet even known about.
The experimental shot, which has been tested in mice, marks a change in strategy towards proactive vaccinology, where vaccines are designed and readied for manufacture before a potentially pandemic virus emerges.
The vaccine is made by attaching harmless proteins from different coronaviruses to minuscule nanoparticles that are then injected to prime the bodys defences to fight the viruses should they ever invade.
Because the vaccine trains the immune system to target proteins that are shared across many different types of coronavirus, the protection it induces is extremely broad, making it effective against known and unknown viruses in the same family.
Weve shown that a relatively simple vaccine can still provide a scattershot response across a range of different viruses, said Rory Hills, a graduate researcher at the University of Cambridge and first author of the report. It takes us one step forward towards our goal of creating vaccines before a pandemic has even started.
Tests in mice showed that the vaccine induced a broad immune response to coronaviruses, including Sars-Cov-1, the pathogen that caused the 2003 Sars outbreak, even though proteins from that virus were not added to the vaccine nanoparticles. Details of the work, a collaboration between the universities of Cambridge and Oxford and the California Institute of Technology, are published in Nature Nanotechnology.
The universal coronavirus vaccine can be made in existing facilities for microbial fermentation, Hills said, adding that the researchers were working with industrial partners on ways to scale up the process. The nanoparticles and viral proteins can be made at different times in different places and mixed together to produce the vaccine.
Medical regulators do not have procedures for proactive vaccinology and the researchers say these would have to be worked out with the relevant bodies. If the vaccine were found to be safe and effective in humans, one option would be to use it as a Covid booster with the added benefit of it protecting against other coronaviruses.
More likely is that countries would hold stocks of the vaccine, and others designed to target separate pathogens, once they have been manufactured and approved. In the event that a coronavirus or other pathogen crosses over you could have pre-existing vaccine stocks ready and a clear plan to quickly scale up production if needed, Hills said.
Prof Mark Howarth, a senior author of the study, said: Scientists did a great job in quickly producing an extremely effective Covid vaccine during the last pandemic, but the world still had a massive crisis with a huge number of deaths. We need to work out how we can do even better than that in the future, and a powerful component of that is starting to build the vaccines in advance.
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Plasmids and cloning
Cloning was performed using standard PCR methods with Q5 High-Fidelity 2 Master Mix (New England Biolabs) and Gibson assembly. All open-reading frames were validated by Sanger sequencing (Source Bioscience).
pET28a-SpyCatcher003-mi3 (GenBank MT945417, Addgene 159995) was previously described58. pET28a-SpyTag-MBP (GenBank MQ038699, Addgene 35050) has been published59. pDEST14-SpySwitch (GenBank ON131074, Addgene plasmid ID 184225) was previously described19. Monomeric sarbecovirus RBD expression vectors contained a C-terminal SpyTag003 (RGVPHIVMVDAYKRYK)16 and His8-tag (ref. 12) in the plasmid p3BNC-RBD-His8-SpyTag003 and were previously described19: SARS2 (GenBank ON131086), SARS1 (GenBank ON131087), RaTG13-CoV (GenBank ON131088), SHC014-CoV (GenBank ON131089), Rs4081-CoV (GenBank ON131090), pangolin17 (pang17)-CoV (GenBank ON131091), RmYN02-CoV (GenBank ON131092), Rf1-CoV (GenBank ON131093), WIV1-CoV (GenBank ON131094), Yunnan2011 (Yun11)-CoV (GenBank ON131095), BM-4831-CoV (GenBank ON131096) and BtKY72-CoV (GenBank ON131097). The origins of the sarbecovirus RBDs are SARS1 (GenBank AAP13441.1; residues 318510), WIV1 (GenBank KF367457; residues 307528), SHC014 (GenBank KC881005; residues 307524), BM-4831 (GenBank NC014470; residues 310530), BtKY72 (GenBank KY352407; residues 309530), pang17 (GenBank QIA48632; residues 317539), SARS2 (GenBank NC045512; S protein residues 331529), RaTG13 (GenBank QHR63300; S protein residues 319541), Rs4081 (GenBank KY417143; S protein residues 310515), RmYN02 (GSAID EPI_ISL_412977; residues 298503) and Rf1 (GenBank DQ412042; residues 310515). The monomeric SARS2 VOC RBDs for Supplementary Fig. 18 ELISAs were cloned into pcDNA3.1 with the influenza H7 hemagglutinin (A/HongKong/125/2017) signal peptide followed by a SpyTag followed by the RBD: Wuhan (GenBank MT945427.1, Addgene 159999), Delta (GenBank PP136028, Addgene plasmid ID 214723), BQ.1.1 (GenBank PP136030, Addgene plasmid ID 214725) and XBB.1.5 (GenBank PP136029, Addgene plasmid ID 214724). The SARS2 Wuhan Spike protein was the HexaPro variant (a gift from Jason McLellan, Addgene plasmid ID 154754) that contains six proline substitutions (F817P, A892P, A899P, A942P, K986P, V987P) which confer greater stability60. The SARS2 Beta variant Spike protein was cloned from HexaPro to match the B.1.351 variant (L18F, D80A, D215G, 242-244, R246I, K417N, E484K, N501Y, D614G, A701V) in addition to the previously outlined six proline mutations. The SARS2 Delta variant Spike protein was cloned from HexaPro to match the B.1.617.2 variant (T19R, T95I, G142D, 156-157, R158G, L452R, T478K, D614G, P681R, D950N) in addition to the previously outlined six proline mutations.
Quartet RBD constructs were cloned using Gibson assembly in competent Escherichia coli DH5 cells and began with the influenza H7 hemagglutinin (A/HongKong/125/2017) signal-peptide sequence. Each RBD was separated with an eight or nine residue Gly-Ser linker. Each linker was distinct from all others in the construct to reduce potential recombination and facilitate sequence analysis. pcDNA3.1-Quartet-SpyTag was created by cloning from the N terminus to C-terminal SHC014 RBD, Rs4081 RBD, RaTG13 RBD and SARS2 RBD with a C-terminal SpyTag into pcDNA3.1 (Fig. 1c; GenBank PP136033, Addgene plasmid ID 214726). This is the construct used for Figs. 1 and 2. For subsequent figures, pcDNA3.1-SpyTag-Quartet was cloned with a SpyTag after the signal sequence and then the same order of RBDs (SpyTag-SHC014-Rs4081-RaTG13-SARS2) (Supplementary Fig. 8; GenBank PP136031, Addgene Plasmid ID 214727). pcDNA3.1-Quartet [SARS1] was cloned with SpyTag after the signal sequence, with SARS1 in the position of SARS2 (SpyTag-SHC014-Rs4081-RaTG13-SARS1) (Supplementary Fig. 8; GenBank PP136034, Addgene plasmid ID 214729). pcDNA3.1-Alternate Quartet was cloned with SpyTag after the signal sequence, followed by pang17 RBD, RmYN02 RBD, Rf1 RBD and WIV1 RBD (Supplementary Fig. 8; GenBank PP136032, Addgene plasmid ID 214728). pcDNA3.1-SpyTag-Quartet_NoLinker was cloned with the same order of RBDs as SpyTag-Quartet (SpyTag-SHC014-Rs4081-RaTG13-SARS2) but did not have any Gly-Ser linker between RBDs (Supplementary Fig. 8; GenBank PP136036, Addgene plasmid ID 214731). pcDNA3.1-Kraken Quartet was identical to SpyTag-Quartet with the SARS2 XBB.1.5 RBD in place of SARS2 Wuhan RBD (Supplementary Fig. 8; GenBank PP136035, Addgene plasmid ID 214730).
pET28a-SpyCatcher003-mi3 or pET28a-SpyTag-MBP was transformed into E. coli BL21(DE3) cells (Agilent) and grown on LB-Agar plates with 50gml1 kanamycin for 16h at 37C. A single colony was added in 10ml of LB medium containing 50gml1 kanamycin and grown for 16h at 37C with shaking at 200rpm. This starter culture was then added to 1l of LB containing 50gml1 kanamycin and incubated at 37C and with 200rpm shaking until optical density (OD)600 0.6. Cultures were induced with 0.5mM isopropyl -d-1-thiogalactopyranoside. For SpyCatcher003-mi3, cells were grown at 22C with shaking at 200rpm for 16h. For SpyTag-MBP, cells were grown at 30C with shaking at 200rpm for 4h. Cultures were pelleted by centrifugation at 4,000g.
Cell pellets were resuspended in 20ml of 20mM Tris-HCl, 300mM NaCl, pH 8.5, supplemented with 0.1mgml1 lysozyme, 1mgml1 cOmplete mini EDTA-free protease inhibitor (Roche) and 1mM phenylmethanesulfonyl fluoride. The lysate was incubated at 4C for 45min with end-over-end mixing. An Ultrasonic Processor equipped with a microtip (Cole-Parmer) was used to perform sonication on ice (four times for 60s, 50% duty-cycle). Centrifugation at 35,000g for 45min at 4C was used to clear cell debris. Then, 170mg of ammonium sulfate was added per ml of lysate and incubated at 4C for 1h, while mixing at 120rpm, to precipitate the particles. The solution was centrifuged for 30min at 30,000g at 4C. The pellet was resuspended in 10ml of mi3 buffer (25mM Tris-HCl, 150mM NaCl, pH 8.0) at 4C and filtered sequentially through 0.45m and 0.22m syringe filters (Starlab). The filtrate was dialysed for 16h against 1,000-fold excess mi3 buffer. The dialysed particles were centrifuged at 17,000g for 30min at 4C and filtered through a 0.22-m syringe filter. The purified SpyCatcher003-mi3 was loaded onto a HiPrep Sephacryl S-400 HR 16-60 column (GE Healthcare), which was equilibrated with mi3 buffer using an KTA Pure 25 system (GE Healthcare). The proteins were separated at 0.1mlmin1 while collecting 1ml of elution factions. The fractions containing the purified particles were pooled and concentrated using a Vivaspin 20 100kDa molecular weight cut-off centrifugal concentrator (GE Healthcare) and stored at 80C.
Mammalian expression of all RBD and Spike constructs was performed in Expi293F cells (Thermo Fisher, A14635). Expi293F cells were grown under humidified conditions at 37C and 8% (v/v) CO2 in Expi293 Expression Medium (Thermo Fisher) with 50Uml1 penicillin and 50gml1 streptomycin. Transfections were performed using the ExpiFectamine 293 Transfection Kit (Thermo Fisher). Expi293F cells were brought to 3106cells per ml and then 1g of plasmid DNA per ml of culture was incubated with ExpiFectamine 293 reagent for 20min, before being added dropwise to the Expi293F culture. After approximately 20h, ExpiFectamine 293 Transfection Enhancers 1 and 2 were added. Cell supernatants were collected after 5d by centrifuging for 4,000g at 4C for 5min and were passed through a 0.45m filter and then a 0.22m filter (Starlab).
RBDs, Quartets and SpyTag-MBP were purified by SpySwitch19. Purifications were performed at 4C. For SpyTag-MBP, cells were lysed according to the same procedure as SpyCatcher003-mi3 and supplemented with 10SpySwitch buffer (500mM Tris-HCl, pH 7.5, +3M NaCl) 10% (v/v). For mammalian proteins, 10SpySwitch buffer was added to mammalian culture supernatant at 10% (v/v). SpySwitch resin19, packed in an Econo-Pac Chromatography Column (Bio-Rad), was pre-equilibrated with 210 column volumes (CV) of SpySwitch buffer (50mM Tris-HCl, pH 7.5, +300mM NaCl). The supernatant was incubated with SpySwitch resin for 1h at 4C on an end-over-end rotator. The column was washed twice with 15CV of SpySwitch buffer. Proteins were eluted using a weakly acidic pH switch. The protein was incubated with 1.5CV of SpySwitch Elution Buffer (50mM acetic acid/sodium acetate, pH 5.0, +150mM NaCl) at 4C with the column capped. The cap was removed and the elution flow-through was collected into a microcentrifuge tube containing 0.3CV of 1M Tris-HCl pH 8.0. The microcentrifuge tube was mixed by inversion to minimize the time spent at an acidic pH. This elution step was repeated for a total of six times. Purification was assessed by SDSPAGE with Coomassie staining. Briefly, 10l volumes of fractions were mixed with 2l of 6 SDS loading buffer (234mM Tris-HCl pH 6.8, 24% (v/v) glycerol, 120M bromophenol blue, 234mM SDS), before heating at 95C for 5min in a C1000 Touch Thermal Cycler (Bio-Rad) and loading onto 12% SDSPAGE, then staining with Coomassie. Typical yields for the RBD Quartets were 50100mg per litre of culture. Typical yields for RBD monomers were 80160mg per litre of culture, as measured by bicinchoninic acid. Elution fractions were dialysed for 16h against 1,000-fold excess Tris-buffered saline (TBS) (50mM Tris-HCl, 150mM NaCl, pH 7.4 at 25C). Proteins were stored in aliquots at 80C.
SARS2 Spike proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Mammalian supernatants were supplemented with 10 Ni-NTA buffer (500mM Tris-HCl, 3M NaCl, pH 7.8) at 10% (v/v). Ni-NTA agarose (Qiagen) was packed in an Econo-Pac Chromatography Column (Bio-Rad) and washed with 210CV of Ni-NTA buffer (50mM Tris-HCl, 300mM NaCl, pH 7.8). Mammalian supernatant was incubated in the Ni-NTA column for 1h at 4C with rolling. The supernatant was allowed to flow through by gravity, before being washed with 210CV of Ni-NTA wash buffer (10mM imidazole in Ni-NTA buffer). Elutions were performed by incubating resin with Ni-NTA elution buffer (200mM imidazole in Ni-NTA buffer) for 5min, before eluting by gravity. A total of six 1-CV elutions were performed. Elution fractions were assessed by SDSPAGE with Coomassie staining, pooled and dialysed for 16h against 1,000-fold excess TBS.
Quartets were loaded onto a HiPrep Sephacryl S-200 HR 16-600 column (GE Healthcare), which was equilibrated with PBS pH 7.4 and run with an KTA Pure 25 system (GE Healthcare). The proteins were separated at 0.5mlmin1 while collecting 1ml of elution factions. A Gel Filtration Standard (Bio-Rad) was run over the column under the same conditions for comparison. All size-exclusion chromatography was performed at 4C.
Quartet protein (2g) was incubated with 1l of Glycoprotein Denaturing Buffer (10) (New England Biolabs) at 100C for 10min with a C1000 Touch Thermal Cycler (Bio-Rad). The denatured protein was then chilled on ice for 1min and centrifuged for 10s at 2,000g with a MiniStar Silverline (VWR). Then 2l of GlycoBuffer 2 (10) (New England Biolabs), 2l of 10% (v/v) NP-40, 6l of MilliQ water and 1l of PNGase F (New England Biolabs) at 500,000 units per ml were added and incubated at 37C for 1h. Proteins were resolved on 12% SDSPAGE, stained with Coomassie and imaged using a ChemiDoc XRS imager.
First, 2M SpyTag antigens were conjugated with 2M SpyCatcher003-mi3 for 48h at 4C. Proteins were centrifuged for 30min at 16,900g at 4C and 30l of the supernatant was loaded into a quartz cuvette. Samples were measured at 20C using a Viscotek 802 (Viscotek) with 20 scans of 10s each, using 50% laser intensity, 15% maximum baseline drift and 20% spike tolerance. Before collecting data, the cuvette was incubated in the instrument for 5min to allow the sample temperature to stabilize. The intensity of the size distribution was normalized to the peak value using OmniSIZE v.3.0 software, calculating the mean and standard deviation from the multiple scans (Viscotek).
First, 2M SpyCatcher003-mi3 was incubated for 48h at 4C with 2M of the appropriate antigens to make Homotypic SARS2 Nanocage, Mosaic-8 and Quartet Nanocage or without any antigen present in 25mM Tris-HCl, 150mM NaCl, pH 8.0. Samples were applied to a freshly glow-discharged TEM grid, blotted twice with water and stained with 2% (w/v) uranyl acetate for 30s. Samples were imaged using a Tecnai G2 80200-keV transmission electron microscope at the Cambridge Advanced Imaging Centre. For size analysis, the particle diameter for each group was measured manually (n=75) and plotted with 2-nm bin size in Excel (Microsoft).
Endotoxin was removed from all vaccine components using Triton X-114 phase separation61,62. Triton X-114 at a final 1% (v/v) was added to the protein on ice and incubated for 5min. The solution was incubated at 37C for 5min and centrifuged for 1min at 16,000g at 37C. The top phase was transferred to a fresh tube. This procedure was repeated for a total of three times. A final repetition without the addition of Triton X-114 was performed, to account for residual Triton X-114. A Pierce Chromogenic Endotoxin Quant Kit (Thermo Fisher) was used according to manufacturer instructions to quantify the final endotoxin concentration. All vaccine components were below the accepted endotoxin levels for vaccine products of 20endotoxin units per ml (ref. 63).
The concentration of vaccine components was measured using bicinchoninic acid assay (Pierce). Where multiple antigens were coupled to the nanocage, the antigens were first mixed in equimolar amounts in TBS. Doses were normalized by the number of SpyTags, to facilitate an equimolar amount of SpyCatcher003-mi3 nanocages with similar occupancy in each condition. For high-dose immunizations (Supplementary Figs. 1416), SpyCatcher003-mi3 at 8M was incubated with 8M SpyTagged antigen for 48h at 4C in TBS, pH 8.0. For other immunizations, SpyCatcher003-mi3 at 0.8M was incubated with 0.8M total SpyTagged antigen for 48h at 4C in TBS, pH 8.0. Uncoupled RBD and Uncoupled Quartet were incubated at 0.8M for 48h at 4C in TBS, pH 8.0, without the addition of SpyCatcher003-mi3. Before immunization, samples were analysed by SDSPAGE/Coomassie and DLS. For Fig. 5, SARS2 Spike prime and boost doses were performed with 10g of SARS2 Wuhan Spike (HexaPro) protein in TBS pH 8.0 at 4C.
Animal experiments were performed according to the UK Animals (Scientific Procedures) Act 1986, under Project License (PBA43A2E4 and PP9362617) and approved by the University of Oxford Animal Welfare and Ethical Review Body. Mice that were 6weeks old (at the time of the first immunization) were obtained from Envigo. For high-dose immunizations (Supplementary Figs. 1416), we used BALB/c female mice, and for all other immunizations we used C57BL/6 female mice. Mice were housed in accordance with the UK Home Office ethical and welfare guidelines and fed on standard chow and water ad libitum. Before immunization, immunogens were mixed 1:1 with VAC 20 adjuvant (SPI Pharma) (25l+25l), except for the high-dose immunizations (Supplementary Figs. 1416) where immunogens were mixed 1:1 with AddaVax (Invivogen). This procedure gave final doses of 0.2nmol of total SpyTagged antigen for high-dose immunizations and 0.02nmol of total SpyTagged antigen for normal-dose immunization. For normal-dose immunization, this relates to 0.6g of Uncoupled RBD. Isoflurane (Abbott)-anaesthetised mice were immunized on day 0 and day 14 intramuscularly in the gastrocnemius muscle with the specified antigenadjuvant mix. Post-prime blood samples were obtained on day 13 via tail vein using Microvette (CB300, Sarstedt) capillary tubes. Post-boost samples were obtained on days 32 to 41 (exact day for each set of immunizations is indicated in the figure) via cardiac puncture of humanely killed mice. The collected whole blood in microtainer SST tubes (Becton Dickinson) was allowed to clot at 25C for 12h, before spinning down at 10,000g for 5min at 25C. The sera were heat-inactivated at 56C for 30min, before storing at 20C.
Nunc MaxiSorp plates (Thermo Fisher) were coated with 80nM purified SpyTag-RBD, SpyTag-MBP or SpyCatcher003-mi3 in PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.7mM KH2PO4, pH 7.4) at 4C for 16h. Where SARS2 was analysed, this refers to the Wuhan variant, unless indicated. In Supplementary Fig. 5b, the response to different SARS2 variants was measured by coating 1gml1 of the indicated HexaPro Spike protein in PBS and incubating at 4C for 16h. Plates were washed three times with PBS supplemented with 1% (v/v) Tween 20 (PBST). Plates were blocked by 2h of incubation at 25C with 5% (w/v) skimmed milk in PBS. Plates were then washed three times with PBST. Sera were serially diluted into the blocking buffer using eight-point, fourfold series starting at 1:100. Plates were incubated with sera for 1h at 25C and then washed three times with PBST. Plates were incubated at 25C for 1h with a 1:1,600 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich, A9044). Plates were washed three times with PBST. Plates were then incubated at 25C for 5min with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific) before the reaction was stopped with 1M H2SO4. Absorbance measurements at 405nm (A405) were taken with a FLUOstar Omega plate reader (BMG Labtech) using Omega MARS software (BMG Labtech). A sigmoidal doseresponse curve was fit to the absorbance data using the optimize.curve_fit() function from the Python SciPy library64. The sigmoidal doseresponse function was:
$${{y}}={rm{Bottom}}+frac{{rm{Top}}-{rm{Bottom}}}{1+{10}^{{log }_{10}left({{rm{IC}}}_{50}right)-{{x}}}}$$
IC50 is the serum concentration that gives a 50% signal between the maximum and minimum of the curve. The area under the fitted curve was determined using the trapz function from the Python NumPy library65. Area under the curve was used instead of endpoint titre to account better for data across the entire range of values66. For calculation of midpoint titre, sigmoidal doseresponse curve absorbance data and the midpoint were calculated using GraphPad Prism (GraphPad Software v.9.4.1). Results were plotted using GraphPad Prism (GraphPad Software v.9.4.1).
These assays were performed in the James & Lillian Martin Centre, University of Oxford, operating under license from the Health and Safety Authority, UK, on the basis of an agreed Code of Practice, Risk Assessments (under the Advisory Committee on Dangerous Pathogens) and standard operating procedures. The microneutralization assay determines the serum concentration that induces a 50% reduction in focus-forming units of SARS2 in Vero cells (American Type Culture Collection, CCL-81). A serial dilution of immunization sera (seven steps from 1/40 to 1/40,000 diluted into DMEM) was pre-incubated for 30min at 25C with a fixed dose of 100200 focus-forming units (20l) of different authentic SARS2 variants. This procedure was performed in triplicate for samples from high-dose immunizations outlined in Supplementary Figs. 1416 and in quadruplicate for all other samples. DMEM on its own was used for serum-free control wells, which were used to define 100% infectivity. The Victoria 01/2020 isolate (Pango B) was used for Wuhan neutralization67. The Beta variant (Pango B.1.351) used for neutralizations is the HV001 isolate, sequenced and kindly supplied by CAPRISA, Durban, South Africa68. The isolates for Delta (Pango B.1.617.2), Omicron BA.1 (Pango B.1.1.529.1) and Omicron BQ.1.1 (Pango B.1.1.529.5.3.1.1.1.1.1.1) were kindly supplied by Gavin Screaton (University of Oxford). This mixture was incubated with 100l of Vero cells (4.5104) at 37C with 5% (v/v) CO2. At 2h into this incubation, a 1.5% (w/v) carboxymethyl cellulose-containing overlay was applied to prevent satellite focus formation. At 18h post-infection, the monolayers were fixed with 4% (w/v) paraformaldehyde in PBS and then permeabilized with 2% (v/v) Triton X-100. The cells were stained using the FB9B monoclonal antibody at 1gml1 (ref. 69). These samples were developed using an anti-human IgG (Fc-specific) peroxidase-conjugated antibody (1:5,000 dilution, cat. no. A0170-1ML, Sigma-Aldrich) and True Blue peroxidase substrate. The infectious foci were enumerated by Classic ELISpot Reader (AID GmbH). Data were analysed using four-parameter logistic regression (Hill equation) using GraphPad Prism (GraphPad Software v.8.3). Statistical significance of differences between groups was determined using a one-way analysis of variance (ANOVA) test, followed by Tukeys multiple comparison post hoc test of half-maximal inhibitory dilution (ID50) values converted to log10 scale using GraphPad Prism (GraphPad Software v.9.4.1).
SARS2 BQ.1.1, SARS1, WIV1, SHC014 and BtKY72 K493Y/T498W pseudotyped viruses were prepared as described70,71. The double mutation BtKY72 K493Y/T498W in the BtKY72 Spike protein has previously been shown to enable entry to human cells via ACE2 (ref. 72). This technique for producing pseudoviruses employs HIV-based lentiviral particles with genes encoding the appropriate Spike protein lacking the cytoplasmic tail. A threefold serial dilution of sera was incubated with pseudotyped virus for 1h at 37C. The mixture was incubated with 293TACE2 target cells for 48h at 37C (ref. 12). Cells were washed twice with PBS, before being lysed with Luciferase Cell Culture Lysis 5 reagent (Promega). NanoLuc Luciferase activity in the lysates was measured using the Nano-Glo Luciferase Assay System (Promega). The relative luminescence units were normalized to values derived from cells infected with pseudotyped virus in the absence of serum. ID50 was determined using four-parameter nonlinear regression in AntibodyDatabase73 and plotted using GraphPad Prism (GraphPad Software v.9.4.1). Statistical significance of differences between groups was determined using an ANOVA test, followed by Tukeys multiple comparison post hoc test of ID50 values converted to log10 scale using GraphPad Prism (GraphPad Software v.9.4.1).
We incubated 2M SpyTag-Quartet or 2M Quartet-SpyTag with or without 2M SpyCatcher003-mi3 in 25mM Tris-HCl, 150mM NaCl, pH 8.0, for 16h at 4C to allow for coupling. We added the protein samples at 50nM to Nunc MaxiSorp plates (Thermo Fisher) and incubated for 16h at 4C in PBS pH 7.4. We then washed three times with PBST and blocked with 5% (w/v) skim milk for 2h at 25C. We washed three times more and incubated with 50nM of the specified antibody for 1h at 25C. The monoclonal antibodies used in this study, namely C121 71, EY-6A, FI-3A, FP-12A, IY-2A (ref. 45), LCA60 (ref. 74), FP-8A and FD-5D (refs. 19,69), have all been previously described. Heavy and light chain expression vectors for these antibodies were co-transfected into ExpiCHO cells (Thermo Fisher Scientific, A29133) using the ExpiCHO expression system kit, and the monoclonal antibodies were purified from the supernatant by Protein A Sepharose (GE Healthcare). After three washes, we incubated with a 1/2,500 dilution of anti-human IgG horseradish peroxidase (Sigma-Aldrich, A8667) for 1h at 25C. After three washes, we incubated with TMB for 30s (for comparison of coupled and uncoupled Quartet) or 2min (for comparison of SpyTag-Quartet and Quartet-SpyTag), before stopping with 1M HCl. A405 measurements of triplicate wells per condition were taken at 25C with a FLUOstar Omega plate reader (BMG Labtech) using Omega MARS software (BMG Labtech).
Serum mapping studies were performed following the previously established approach26: 25l of each serum sample was heat-inactivated for 30min at 56C, before depleting twice by incubation with 50 OD units of AWY101 yeast containing an empty vector, to deplete serum of non-specific yeast-binding antibodies. Yeasts that were generously provided by Tyler Starr (University of Utah) were induced to express the SARS2 RBD library in galactose-containing synthetic defined medium with casamino acids: 6.7gl1 Yeast Nitrogen Base, 5.0gl1 casamino acids, 1.065gl1 2-(N-morpholino)ethanesulfonic acid (MES), 2% (w/v) galactose and 0.1% (w/v) dextrose26. After a 1618-h induction, cells were washed and incubated with serum at a range of dilutions for 1h at 25C with gentle agitation. For each serum sample, a subsaturating dilution enabled the fluorescent signal from antibody binding to be equivalent across samples. The libraries were washed and labelled for 1h with 1:100 fluorescein-conjugated anti-myc tag antibody (Immunology Consultants Lab, CYMC-45F) to quantify RBD expression and 1:200 Alexa Fluor-647-goat anti-mouse-IgG Fc-gamma (Jackson ImmunoResearch, 115-605-008) to detect mouse antibodies from serum. Approximately 5106 RBD-positive cells were processed on a Sony SH800 cell sorter. A flow cytometric gate was drawn to capture RBD mutants with reduced antibody binding compared with their level of RBD expression26. These cells were grown overnight, before plasmid extraction in a synthetic defined medium with casamino acids: 6.7gl1 Yeast Nitrogen Base, 5.0gl1 casamino acids, 1.065gl1 MES, 2% (w/v) dextrose, 100Uml1 penicillin and 100gml1 streptomycin. Plasmid samples were then prepared from 30 OD units (1.6108colony forming units; cfu) of preselection yeast populations and 5 OD units (~3.2107cfu) of overnight cultures of serum-escaped cells (Zymoprep Yeast Plasmid Miniprep II)26. The 16-nucleotide barcodes identifying each RBD variant were amplified by PCR and sequenced on an Illumina HiSeq 2500 with 50-base pair single-end reads26. We computationally filtered out variants with >1 amino acid mutation, low sequencing counts or highly deleterious mutations that might escape antibody binding because of poor RBD expression or folding26. The escape fraction represents the proportion of cells expressing that specific variant that falls in the escape bin: a value of 0 means that the variant is always bound by serum antibody and a value of 1 means that the variant always escapes serum antibody binding. The height of each letter indicates the escape fraction for that amino acid mutation, calculated as described above. The static logo plots feature any site where, for at least one serum sample, the site-total antibody escape was >10 the median across all sites and at least 10% the maximum of any site. RBD sites are categorized based on antibody epitope region28. Class 1 epitopes are defined as residues 403, 405, 406, 417, 420, 421, 453, 455460, 473478, 486, 487, 489, 503 and 504. Class 2 epitopes are defined as residues 472, 479, 483485 and 490495. Class 3 epitopes are defined as residues 341, 345, 346, 354357, 396, 437452, 466468, 496, 498501 and 462. Class 4 epitopes are defined as residues 365390 and 408.
The phylogenetic tree of sarbecovirus RBD sequences was constructed using MEGA X v.11.0.13 software75. Multiple sequence alignment and calculation of amino acid identity were performed using Clustal Omega v.1.2.4 (ref. 76). The structure of SARS2 RBD was based on PDB ID: 6ZER (ref. 77) and analysed using PyMOL v.2.5.2.
No statistical method was used to predetermine sample size. Significance for ELISAs was calculated with an ANOVA test using Tukeys post hoc test in GraphPad Prism (GraphPad Software v.9.4.1). Comparisons for neutralizations were calculated with an ANOVA test, followed by Tukeys multiple comparison post hoc test of ID50 values converted to log10 scale using GraphPad Prism (GraphPad Software v.9.4.1). For ELISAs and neutralizations, Tukeys test was used to correct for the multiple comparisons between the responses raised to each individual antigen within each set of immunizations. Significance was assigned according to: *P<0.05, **P<0.01, ***P<0.001. On graphs where some conditions are compared, where no test is marked then the difference was non-significant. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
