Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor-binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are unique in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Engineering Innovative Interfaces for Point-of-Care Diagnostics.

The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas --in place of traditional tests-- and outside of traditional clinical settings --to enable new testing paradigms. Hallmark examples of POCTs are the pregnancy test lateral flow assay and the blood glucose meter. Other uses for POCTs include diagnostic assays for diseases like COVID-19, HIV, and malaria but despite some successes, there are still unsolved challenges for fully translating these lower cost and more versatile solutions. To overcome these challenges, researchers have exploited innovations in colloid and interface science to develop various designs of POCTs for clinical applications. Herein, we provide a review of recent advancements in lateral flow assays, other paper based POCTs, protein microarray assays, microbead flow assays, and nucleic acid amplification assays. Features that are desirable to integrate into future POCTs, including simplified sample collection, end-to-end connectivity, and machine learning, are also discussed in this review.

IoT-Based Home Automation System with Covid-19 Detector

As the advancement in technology continues to increase, systems also need to be upgraded to meet new trends and solve developing problems. Existing systems of home automation generally focus on energy management, security, and comfort These smart homes would he more efficient if they can also perform health diagnoses. In this paper, a cost-effective IoT-based home automation system that performs the function of energy conservation, smart security, and disease detection is designed. The system uses the NodeMCU ESP8266 IoT board with its in-built Wi-Fi shield, eliminating the need to acquire a separate Wi-Fi module. The NodeMCU is interfaced with the user's android device where the connected loads on the relay modules are controlled thus enabling the system to control home appliances via an android application. The ESP8266 is also connected to a PIR sensor that checks for motion and triggers an alarm when motion is detected and connected to a contactless temperature sensor that measures the body temperature of anyone and sends the readings to the display while also triggering an alarm if the temperature is above normal indicating the sign of illness or diseases such as coronavirus, high fever, influenza. This paper presents a design of a simple system that not only assists the user to control his home appliances, conserve energy, and improve home security but also helps to protect the user from allowing covid-19-infected persons into their home.

Oxygenated right ventricular assist device as part of veno-venopulmonary extracorporeal membrane oxygenation to support the right ventricle and pulmonary vasculature.

COVID-19 infection can lead to severe acute respiratory distress syndrome (ARDS), right ventricular (RV) failure and pulmonary hypertension. Venovenous extracorporeal membrane oxygenation (V-V ECMO) has been used for patients with refractory hypoxemia. More recently dual-lumen right atrium to pulmonary artery oxygenated right ventricular assist devices (Oxy-RVAD) have been utilized in the severe medical refractory COVID ARDS setting. Historically, animal data has demonstrated that high continuous non-pulsatile RVAD flows, leading to unregulated and unprotected circulation through the pulmonary vessels is associated with an increased risk of pulmonary hemorrhage and increased amount of extravascular lung water. These risks are heightened in the setting of ARDS with fragile capillaries, left ventricular (LV) diastolic failure, COVID cardiomyopathy, and anticoagulation. Concurrently, due to infection, tachycardia, and refractory hypoxemia, high V-V ECMO flows to match high cardiac output are often necessary to maintain systemic oxygenation. Increase in cardiac output without a concurrent increase in VV ECMO flow will result in a higher fraction of deoxygenated blood returning to the right heart and therefore resulting in hypoxemia. Several groups have suggested using a RVAD only strategy in COVID ARDS; however, this exposes the patients to the risk of pulmonary hemorrhage. We present one of the first known cases using an RV mechanical support, partial flow pulmonary circulation, oxygenated Veno-venopulmonary (V-VP) strategy resulting in RV recovery, total renal recovery, awake rehabilitation, and recovery.

Private equity in ophthalmology and optometry: a time series analysis from 2012 to 2021.

Purpose: To identify temporal and geographic trends in private equity (PE)-backed acquisitions of ophthalmology and optometry practices in the United States from 2012 to 2021. Methods: In this cross-sectional time series, acquisition data from 10/21/2019 to 9/1/2021 and previously published data from 1/1/2012 to 10/20/2019 were analyzed. Acquisition data were compiled from 6 financial databases, 5 industry news outlets, and publicly available press releases. Linear regression models were used to compare rates of acquisition. Outcomes included number of total acquisitions, practice type, locations, provider details, and geographic footprint. Results: A total of 245 practices associated with 614 clinical locations and 948 ophthalmologists or optometrists were acquired by 30 PE-backed platform companies between 10/21/2019 and 9/1/2021. Of 30 platform companies, 18 were new vis-à-vis our prior study. Of these acquisitions, 127 were comprehensive practices, 29 were retina practices, and 89 were optometry practices. From 2012 to 2021, monthly acquisitions increased by 0.947 acquisitions per year (P < 0.001*). Texas, Florida, Michigan, and New Jersey were the states with the greatest number of PE acquisitions, with 55, 48, 29, and 28 clinic acquisitions, respectively. Average monthly PE acquisitions were 5.71 per month from 1/1/2019 to 2/29/2020 (pre-COVID), 5.30 per month from 3/1/2020 to 12/31/2020 (COVID pre-vaccine [P = 0.81]), and 8.78 per month from 1/1/2021 to 9/1/2021 (COVID post-vaccine [P = 0.20]). Conclusions: PE acquisitions increased during the period 2012-2021 as companies continue to utilize regionally focused strategies for acquisitions.

COVID-19 Diagnosis and SARS-CoV-2 Strain Identification by a Rapid, Multiplexed, Point-of-Care Antibody Microarray.

Antigen tests to detect SARS-CoV-2 have emerged as a promising rapid diagnostic method for COVID-19, but they are unable to differentiate between variants of concern (VOCs). Here, we report a rapid point-of-care test (POC-T), termed CoVariant-SPOT, that uses a set of antibodies that are either tolerant or intolerant to spike protein mutations to identify the likely SARS-CoV-2 strain concurrent with COVID-19 diagnosis using antibodies targeting the nucleocapsid protein. All reagents are incorporated into a portable, multiplexed, and sensitive diagnostic platform built upon a nonfouling polymer brush. To validate CoVariant-SPOT, we tested recombinant SARS-CoV-2 proteins, inactivated viruses, and nasopharyngeal swab samples from COVID-19 positive and negative individuals and showed that CoVariant-SPOT can readily distinguish between two VOCs: Delta and Omicron. We believe that CoVariant-SPOT can serve as a valuable adjunct to next-generation sequencing to rapidly identify variants using a scalable and deployable POC-T, thereby enhancing community surveillance efforts worldwide and informing treatment selection.

Intracannula Thrombus Formation Associated With Dual Lumen ProtekDuo Cannula in Extracorporeal Membrane Oxygenation (ECMO).

Extracorporeal membrane oxygenation (ECMO) is used in cases of severe respiratory failure refractory to medical management. Use of ECMO is increasing, along with new cannulation strategies including oxygenated right ventricular assist devices (oxy-RVADs). Multiple dual lumen cannulas are now available, which increase the potential for patient mobility and decrease the number of vascular access sites. However, dual lumen, single cannula flow can be limited by adequate inflow, requiring the need for an additional inflow cannula to meet patient demands. This cannula configuration may result in differential flows in the inflow and outflow limbs and altered flow dynamics, increasing the risk of intracannula thrombus. We describe a series of four patients treated with oxy-RVAD for COVID-19-associated respiratory failure complicated by dual lumen ProtekDuo intracannula thrombus.

The Role of Nutrition in Mitigating the Effects of COVID-19 from Infection through PASC.

The expansive and rapid spread of the SARS-CoV-2 virus has resulted in a global pandemic of COVID-19 infection and disease. Though initially perceived to be acute in nature, many patients report persistent and recurrent symptoms beyond the infectious period. Emerging as a new epidemic, "long-COVID", or post-acute sequelae of coronavirus disease (PASC), has substantially altered the lives of millions of people globally. Symptoms of both COVID-19 and PASC are individual, but share commonality to established respiratory viruses, which include but are not limited to chest pain, shortness of breath, fatigue, along with adverse metabolic and pulmonary health effects. Nutrition plays a critical role in immune function and metabolic health and thus is implicated in reducing risk or severity of symptoms for both COVID-19 and PASC. However, despite the impact of nutrition on these key physiological functions related to COVID-19 and PASC, the precise role of nutrition in COVID-19 infection and PASC onset or severity remains to be elucidated. This narrative review will discuss established and emerging nutrition approaches that may play a role in COVID-19 and PASC, with references to the established nutrition and clinical practice guidelines that should remain the primary resources for patients and practitioners.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection (preprint)

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs, including recently circulating BA.4/BA.5, in both pseudovirus-based and live virus assays, and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are novel in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Morning SARS-CoV-2 Testing Yields Better Detection of Infection Due to Higher Viral Loads in Saliva and Nasal Swabs upon Waking.

Optimizing specimen collection methods to achieve the most reliable SARS-CoV-2 detection for a given diagnostic sensitivity would improve testing and minimize COVID-19 outbreaks. From September 2020 to April 2021, we performed a household-transmission study in which participants self-collected specimens every morning and evening throughout acute SARS-CoV-2 infection. Seventy mildly symptomatic participants collected saliva, and of those, 29 also collected nasal swab specimens. Viral load was quantified in 1,194 saliva and 661 nasal swab specimens using a high-analytical-sensitivity reverse transcription-quantitative PCR (RT-qPCR) assay. Viral loads in both saliva and nasal swab specimens were significantly higher in morning-collected specimens than in evening-collected specimens after symptom onset. This aspect of the biology of SARS-CoV-2 infection has implications for diagnostic testing. We infer that morning collection would have resulted in significantly improved detection and that this advantage would be most pronounced for tests with low to moderate analytical sensitivity. Collecting specimens for COVID-19 testing in the morning offers a simple and low-cost improvement to clinical diagnostic sensitivity of low- to moderate-analytical-sensitivity tests. IMPORTANCE Our findings suggest that collecting saliva and nasal swab specimens in the morning immediately after waking yields higher SARS-CoV-2 viral loads than collection later in the day. The higher viral loads from morning specimen collection are predicted to significantly improve detection of SARS-CoV-2 in symptomatic individuals, particularly when using moderate- to low-analytical-sensitivity COVID-19 diagnostic tests, such as rapid antigen tests.

Data Analysis on Detection and Prediction of Covid-19

Covid-19 has created a huge problem throughout the world. Due to the sudden increase in spread of Covid-19 many of them has lost their lives. Currently there is no proper solution for prevention of virus. Many research are working on to find out the spread of the disease as well as the preventive measures that can be taken to control. We are going to survey various set of research work proposed by different researchers. Based on the information that are collected from different research work. The paper analyses the spread of covid across particular location and to generate the graph based on the data. The details of the particular location such as the no of persons affected, recovered and other details. © 2022 IEEE.

Well-Being Among Respiratory Therapists in an Academic Medical Center During the COVID-19 Pandemic.

BACKGROUND: Recent studies have revealed high rates of burnout among respiratory therapists (RTs), which has implications for patient care and outcomes as well as for the health care workforce. We sought to better understand RT well-being during the COVID-19 pandemic. The purpose of this study was to determine rates and identify determinants of well-being, including burnout and professional fulfillment, among RTs in ICUs. METHODS: We conducted a mixed-methods study comprised of a survey administered quarterly from July 2020-May 2021 to critical-care health care professionals and semi-structured interviews from April-May 2021 with 10 ICU RTs within a single health center. We performed multivariable analyses to compare RT well-being to other professional groups and to evaluate changes in well-being over time. We analyzed qualitative interview data using thematic analysis, followed by mapping themes to the Maslow needs hierarchy. RESULTS: One hundred eight RTs responded to at least one quarterly survey. Eighty-two (75%) experienced burnout; 39 (36%) experienced professional fulfillment, and 62 (58%) reported symptoms of depression. Compared to clinicians of other professions in multivariable analyses, RTs were significantly more likely to experience burnout (odds ratio 2.32 [95% CI 1.41-3.81]) and depression (odds ratio 2.73 [95% CI 1.65-4.51]) and less likely to experience fulfillment (odds ratio 0.51 [95% CI 0.31-0.85]). We found that staffing challenges, safety concerns, workplace conflict, and lack of work-life balance led to burnout. Patient care, use of specialized skills, appreciation and a sense of community at work, and purpose fostered professional fulfillment. Themes identified were mapped to Maslow's hierarchy of needs; met needs led to professional fulfillment, and unmet needs led to burnout. CONCLUSIONS: ICU RTs experienced burnout during the pandemic at rates higher than other professions. To address RT needs, institutions should design and implement strategies to reduce burnout across all levels.

IoT-Based Home Automation System with Covid-19 Detector

As the advancement in technology continues to increase, systems also need to be upgraded to meet new trends and solve developing problems. Existing systems of home automation generally focus on energy management, security, and comfort. These smart homes would be more efficient if they can also perform health diagnoses. In this paper, a cost-effective IoT-based home automation system that performs the function of energy conservation, smart security, and disease detection is designed. The system uses the NodeMCU ESP8266 IoT board with its in-built Wi-Fi shield, eliminating the need to acquire a separate Wi-Fi module. The NodeMCU is interfaced with the user's android device where the connected loads on the relay modules are controlled thus enabling the system to control home appliances via an android application. The ESP8266 is also connected to a PIR sensor that checks for motion and triggers an alarm when motion is detected and connected to a contactless temperature sensor that measures the body temperature of anyone and sends the readings to the display while also triggering an alarm if the temperature is above normal indicating the sign of illness or diseases such as coronavirus, high fever, influenza. This paper presents a design of a simple system that not only assists the user to control his home appliances, conserve energy, and improve home security but also helps to protect the user from allowing covid-19-infected persons into their home. © 2022 IEEE.

SPIKE MUTATION T403R ALLOWS BAT CORONAVIRUS RaTG13 to USE HUMAN ACE2

Background: The bat coronavirus RaTG13 shares 96% sequence identity to SARS-CoV-2, the causative agent of the COVID-19 pandemic. However, the RaTG13 Spike (S) protein interacts only weakly with the human SCoV-2 receptor Angiotensin-converting Enzyme 2 (ACE2) and does not mediate efficient infection of human cells. Here, we examined which alterations are required to allow the RaTG13 S protein to use human ACE2 for efficient entry into human cells. Methods: Sequence alignments showed that SARS-CoV-2 almost invariantly encodes a positively charged amino acid at position 403 of its S protein, while RaTG13 has a neutral Threonine (T). REAX based computational modeling suggested that S R403 contributes to binding of human ACE2. Wild-type and T403R mutant RaTG13 S proteins were investigated for their ability to bind ACE2 and to mediate infection of pseudotyped VSV particles in human lung-and intestine-derived cell lines as well as hPSC-derived gut organoids. Replication-competent recombinant SCoV2 S R403T was produced and replication monitored. In addition, we mutated human ACE2 to map the interacting residue of S R403. Finally, sera of vaccinated individuals were analyzed for their neutralizing potential against various WT CoV and RaTG13 S as well as mutant S containing pseudoparticles. Results: Our results show that a single amino acid change of T403R allows the RaTG13 S to utilize human ACE2 for viral entry. Spike T403R enhanced infection of VSV-based RatG13 S pseudotypes in human lung and colon cells as well as gut-derived organoids. Vice versa R403T mutation reduced infectivity of SCoV2 S pseudotypes and recombinant SCoV2 replication. The enhancing effect of T403R in RaTG13 S depends on E37 in ACE2. RaTG13 T403R S-mediated infection was blocked by the fusion inhibitor EK-1 but not by the SCoV-2 antibody Casirivimab. SARS-CoV-2 and the T403R RaTG13 S were equally susceptible to neutralization by sera from individuals vaccinated against COVID-19. Conclusion: A positively charged amino acid at position 403 in the S protein of bat coronaviruses is critical for efficient utilization of human ACE2. Our results help to better assess the zoonotic potential of bat sarbecoviruses and suggest that COVID-19 vaccination will also protect against closely bat relatives of SARS-CoV-2 that may emerge in the future.

Reduced survival in patients requiring chest tubes with COVID-19 acute respiratory distress syndrome.

Background: Numerous complications requiring tube thoracostomy have been reported among critically ill patients with COVID-19; however, there has been a lack of evidence regarding outcomes following chest tube placement. Methods: We developed a retrospective observational cohort of all patients admitted to an intensive care unit (ICU) with confirmed COVID-19 to describe the incidence of tube thoracostomy and factors associated with mortality following chest tube placement. Results: In total, 1705 patients with laboratory confirmed COVID-19 patients were admitted to our ICUs from March 7, 2020, to March 1, 2021, with 69 out of 1705 patients (4.0%) receiving 130 chest tubes. Of these, 89 out of 130 (68%) chest tubes were indicated for pneumothorax. Patients receiving tube thoracostomy were much less likely to be alive 90 days post-ICU admission (52% vs 69%; P < .01), and had longer ICU (30 vs 5 days; P < .01) and hospital (37 vs 10 days; P < .01) lengths of stay compared with those without tube thoracostomy. Patients who received tube thoracostomy and survived at least 90 days post-ICU admission had shorter times to first chest tube insertion (8.5 vs 17.0 days; P = .01) and a nonsignificantly higher static compliance (20.0 vs 17.5 mL/cm H2O; P = .052) at the time of chest tube placement than those who had expired. Logistic regression analysis demonstrated an association between time to first chest tube and decreased survival when adjusted for covariates. Conclusions: Requiring a chest tube in COVID-19 is a negative prognostic end point. Delayed development of chest tube requirement was associated with a decreased survival and could reflect a poor healing phenotype.

Harmonizing units and values of quantitative data elements in a very large nationally pooled electronic health record (EHR) dataset.

OBJECTIVE: The goals of this study were to harmonize data from electronic health records (EHRs) into common units, and impute units that were missing. MATERIALS AND METHODS: The National COVID Cohort Collaborative (N3C) table of laboratory measurement data-over 3.1 billion patient records and over 19 000 unique measurement concepts in the Observational Medical Outcomes Partnership (OMOP) common-data-model format from 55 data partners. We grouped ontologically similar OMOP concepts together for 52 variables relevant to COVID-19 research, and developed a unit-harmonization pipeline comprised of (1) selecting a canonical unit for each measurement variable, (2) arriving at a formula for conversion, (3) obtaining clinical review of each formula, (4) applying the formula to convert data values in each unit into the target canonical unit, and (5) removing any harmonized value that fell outside of accepted value ranges for the variable. For data with missing units for all the results within a lab test for a data partner, we compared values with pooled values of all data partners, using the Kolmogorov-Smirnov test. RESULTS: Of the concepts without missing values, we harmonized 88.1% of the values, and imputed units for 78.2% of records where units were absent (41% of contributors' records lacked units). DISCUSSION: The harmonization and inference methods developed herein can serve as a resource for initiatives aiming to extract insight from heterogeneous EHR collections. Unique properties of centralized data are harnessed to enable unit inference. CONCLUSION: The pipeline we developed for the pooled N3C data enables use of measurements that would otherwise be unavailable for analysis.

Timeout? The Epidemiology of Pediatric Sports Injuries During the COVID-19 Pandemic.

BACKGROUND: The COVID-19 pandemic resulted in closure of schools and playgrounds while requiring social distancing, changes that likely affected youth sports participation. The purpose of this study was to identify changes in the epidemiology of pediatric sports injuries during the COVID-19 pandemic. METHODS: This retrospective cohort study included patients between the ages of 4 and 18 years who presented to orthopaedic clinics within a single children's hospital network with an acute injury sustained during athletic activity between March 20, 2020, and June 3, 2020 (the strictest period of state-level shelter-in-place orders). These patients were compared with those within the same dates in 2018 and 2019. Chi square and Mann-Whitney U tests were used, as appropriate. RESULTS: Significantly less sports injuries were seen during the pandemic (n = 257) compared with the same dates in 2018 (n = 483) and 2019 (n = 444) despite more providers available in 2020 (P < 0.001). During the pandemic, patients with sports injuries were younger (median age 11 versus 13 years, P < 0.001) and had less delay in presentation (median 5 versus 11 days, P < 0.001). A higher proportion were White (66.9% versus 47.7%, P < 0.001), privately insured (63.4% versus 48.3%, P < 0.001), and seen at a nonurban location (63.4% versus 50.2%, P < 0.001). Most sports injuries during the pandemic were fractures (83.7%). Although 71.4% of all injuries in the prepandemic period occurred in the context of formal sports, only 15.2% were sustained in a formal athletic context in 2020 (P < 0.001). The frequency of surgical treatment was higher during the pandemic (14.8% versus 7.8%, P = 0.001), mainly because most of these injuries were fractures requiring surgical intervention. CONCLUSIONS: Fewer sports injuries were seen in the outpatient setting during the COVID-19 pandemic, and most of these injuries were fractures and occurred outside of organized sports settings. Patients were more likely to be White, privately insured, and seen at a nonurban location.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine.

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%-50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.