Though recent developments in the management of the coronavirus disease 2019 (COVID-19) pandemic have resulted in significant progress, its continued persistence demands continued consideration both of larger scale public health factors as well as individual patient management. We present a case that provides a broad perspective across several issues within both categories, of a morbidly obese 34-year-old unvaccinated female presenting with respiratory distress secondary to COVID-19 pneumonia, managed through remdesivir therapy. Though this case presents an example of successful management, it nonetheless emphasizes the demand for a renewed focus on vaccine hesitancy and obesity as public health issues, particularly within the context of the pandemic. Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the highly contagious infectious disease coronavirus disease 2019 (COVID-19) has resulted in more than 6 million deaths worldwide as of March 2022. As the most consequential global health crisis since the influenza pandemic of 1918, COVID-19 was designated as a global pandemic by the World Health Organization (WHO) on March 11, 2020. The past 2.5 years have resulted in the development of novel therapeutics and vaccine development at unprecedented speed. The most important measure to prevent and decrease the transmission of SARS-CoV-2 is shown to be through vaccination [Advanced age and comorbidities such as obesity, chronic lung disease, and cardiovascular disease are risk factors for the development of severe COVID-19 and its associated complications. A common complication of severe COVID-19 illness is progressive or sudden clinical deterioration leading to acute respiratory failure and acute respiratory distress syndrome (ARDS). Currently, therapeutic options for COVID-19 include antiviral drugs, anti-SARS-CoV-2 monoclonal antibodies, anti-inflammatory drugs, and immunomodulatory agents available under FDA-issued Emergency Use Authorization (EUA). Remdesivir  was approved by the FDA for clinical use to treat hospitalized patients with COVID-19 and was recently shown to produce an 87% lower risk of hospitalization or death than placebo when at-risk nonhospitalized patients with COVID-19 were treated with a three-day course of remdesivir in a randomized double-blind placebo-controlled trial .Although advances in treatment and vaccine development have improved, lagging vaccination rates have contributed to the lack of transition from pandemic to endemic. As cases continue to persist, we must globally utilize effective vaccines, anti-viral medications, and diagnostic tests, and develop vaccines with protection against a broader range of variants. To mitigate continued severe COVID-19 cases with associated complications, vaccination must remain a continued public health effort. We present a case of a morbidly obese 34-year-old unvaccinated female presenting with respiratory distress secondary to COVID-19 pneumonia, managed through remdesivir therapy.A 34-year-old morbidly obese female presented to the ED by ambulance with a chief complaint of shortness of breath, with a recent history of gastrointestinal symptoms including diarrhea over the previous five days. She had previously visited the ED during the course of these symptoms suspecting possible COVID-19 but had tested negative at the time. She denied a history of alcoholism, recreational drug use, or asthma. She was unvaccinated against COVID-19 and stated she had no intention of ever receiving it.Prehospital vital signs taken by emergency medical services (EMS) were significant for a low pulse oximetry oxygen saturation of 90% on room air. Four milligrams of ondansetron and albuterol nebulizer treatment were administered in the field by EMS.Upon presentation, the patient was febrile with a temperature of 100.3ºF. Additional vitals at this time include a blood pressure of 139/78 mmHg , pulse rate of 105, and respiratory rate of 24 breaths per minute. A focused physical examination confirmed a patent airway and moderate respiratory distress with accompanying stridor on auscultation. Physical examination did not otherwise reveal additional signs or symptoms, and the patient remained fully alert and oriented. She was given 125 mg methylprednisolone intravenously and started on bilevel positive airway pressure with a positive end-expiratory pressure of 10 cmH20 and pressure support of 5. She received acetaminophen for her fever and a sepsis protocol was initiated.D-dimer elevated at 527 ng/mL FEU. The patient tested positive for COVID-19 through nucleic acid amplification and was diagnosed with acute respiratory distress secondary to COVID-19 pneumonia. The radiological examination included a chest radiograph that demonstrated bilateral infiltrates and pneumonia  many unvaccinated individuals. At the time of the case, those getting very sick from COVID-19 were all mostly unvaccinated. Patients were asked about their vaccine reservations and time was spent by the involved clinicians dispelling vaccine myths. The only barrier to vaccines in this and other similar cases at the time was the misinformed disbelief of vaccine safety and efficacy.Though the current state of the COVID-19 pandemic remains far superior compared with the pre-vaccine era, emerging research on acute complications, chronic long-term adverse events, and “brain fog” associated with contracting the disease reveals the need for continued disease vigilance ,5. In thIndeed, vaccine hesitancy is not a particularly novel concept, but rather one that has become ubiquitous in the realms of public health, epidemiology, and infectious disease management summarizing various positions on vaccine administration ranging from directly opposed to cautiously in acquiescence. Its societal spread, especially in recent years, has brought about a drop in vaccination rates leading to a resurgence of vaccine-preventable disease (VPD) over the past half-decade to a degree previously unseen .In the context of the COVID-19 pandemic, vaccine hesitancy has unsurprisingly manifested in a significant population of unvaccinated individuals. Despite all available vaccines in the US having achieved demonstrable and remarkable benchmarks in both individual and societal risk reduction, with a CDC-led report published earlier this year finding 14- and 53-fold higher risk of COVID-19 infection and death, respectively, in unvaccinated versus fully vaccinated persons, prevalent beliefs ranging from the skepticism of the pandemic severity (perhaps driven by case examples of relatively low-virulence or low acute severity COVID-19 infection) to mistrust of the vaccine itself persist -9. UnforInterestingly, vaccine hesitancy presents even among healthcare workers despite their unique roles through the past years in managing the pandemic . VaccineAs shown through the case we report, such vaccine hesitancy can result in emergent symptoms. Though management of patient condition was ultimately successful in this case, a more directed effort to address the problem of vaccine hesitancy both overall and within the context of the COVID-19 pandemic is needed.The patient at hand additionally presented with a second risk factor in her morbid obesity. Patients suffering from obesity are more likely to suffer from respiratory infections due to mechanical and inflammatory issues . CompareThe use of remdesivir therapy was indicated in this case and played a role in the successful management of the patient. Remdesivir is a nucleoside analog that became the first approved treatment for severe COVID-19 cases due to its broad anti-viral properties . This trIn summary, we present a case that highlights several important aspects of the modern management of the COVID-19 pandemic and acute COVID-19 pneumonia and secondary respiratory distress. Firstly, continued overall progress against the disease demands a renewed emphasis on vaccine hesitancy and vaccine-hesitant individuals, with a growing need for further research into the motivations behind such viewpoints and holistic solutions. Secondly, obesity as a risk factor for COVID-19 presents another public health avenue into pandemic management. Finally, the use of remdesivir presents a promising emerging therapeutic approach for the management of COVID-19. Continued awareness of current topics in COVID-19 management remains critical, particularly as pandemic restrictions continue to loosen or expire.


There are still no definite curative or preventive strategies for COVID-19 disease. It is crucial to fully comprehend the pathogenesis of COVID-19 infection so that we can develop expedient pharmacological protocols. While the impact of cytokine storm on COVID-19 severity has been one of the most tested hypotheses, the role of bradykinin and various other oxidative stress markers has been relatively under-researched. Their levels can be determined immediately after a hospital admission so they could be used as early predictors of the further development of the disease. Material and methods. The study was conducted as a prospective cross-sectional study. It included 47 consecutive adult patients with confirmed SARS-CoV-2 infection and COVID-19 pneumonia. All study subjects were admitted for a hospital treatment to the tertiary Clinical Hospital Center Bezanijska kosa, Belgrade, Serbia on June 2021. The blood samples were collected at the patients' admission. The analyses of demographic, radiological, and clinical data were later conducted for both groups (the deceased patients and those who survived). In addition, we analyzed the potential relations between the outcome and the levels of bradykinin and galectin-3 measured immediately after the patients were admitted to the hospital.  The study aims at evaluating the possibility of using bradykinin and galectin-3 levels as early predictors that COVID-19 disease will progress into a severe case.  The patients who passed away were predominantly older men with comorbidities. We recorded higher CT scores in the deceased patients and the significantly higher levels of urea, creatinine, CK, troponine, CRP, and other laboratory markers. They stayed at the ICU unit longer and required mechanical ventilation more frequently than the patients who survived. On the other hand, no differences were recorded in the time periods passing from the onset of the systems to the hospital admissions. Finally, we can highlight several independent predictors of mortality in patients with COVID-19 pneumonia, including the following: (1) patients who are 50 or more years old, (2) with in-hospital stays are longer that 4 days, (3) bradykinin levels surpass 220000 pg/ml, (4) D-dimer, creatinine, and CRP are elevated, and (5) comorbidities were present (such as hypertension and diabetes).  The present study strongly supports the bradykinin storm hypothesis. Since elevated bradykinin levels have been found in most COVID-19 cases with fatal outcomes, the future therapeutical strategies for COVID-19 have to be focused on reducing bradykinin serum concentrations. Coronavirus disease COVID-19), caused by SARS-CoV-2 viral infection, has resulted in more than 583 million infections and more than six million deaths globally [9, causedThe pathophysiological processes of SARS-CoV-2 infection manifest as disturbances of laboratory markers and deterioration of respiratory functions, but also as venous and arterial thromboembolism . AngioteBradykinin storm is an imbalance of bradykinin caused by a dysregulation in kallikrein-kinin system (KKS). Since its discovery in snake venom, bradykinin has been used as an explanation for physiopathological phenomena in inflammatory conditions. BK is a regulator of tissue blood flow and a regulator of vasomotor activity. It is an additional component of the renin-angiotensin system (RAS). On the other hand, in very high concentrations, BK has a prominent role in inflammatory and oxidative process [Bradykinin (BK) is a hypotensive cytokine. It has been mentioned in the previous studies as a potential contributing factor in the pathophysiology of severe COVID-19 disease manifestations . Bradyki process .Galectin process . It play process , 9. Gale process –9.On the other hand, a host's response is essential by the virtue of immune response and antibody release , 11. SARThe connection between oxidative stress and inflammatory response during COVID-19 disease results from the concurrent activity of endothelial cells and immune response. The authors have suggested that the regulation of endothelial function could prevent the cytokine storm .Still, there are no curative or preventive strategies for COVID-19. The deeper understanding of the pathogenesis of a COVID-19 infection is essential for the rational development of pharmacological protocols. Due to their proinflamatory and oxidative effects, it is crucial to investigate the clinical repercussions of BK and Galectin 3 in survived and deceased patients with confirmed COVID-19 pneumonia. Several studies have revealed that the hyperinflammatory response, induced by SARS-CoV-2 infection and acute pneumonia, is a major risk factor for developing the severe forms of the disease that can result in death. Still, there is a lack of evidence on how bradykinin and galectin-3 are involved in the pathogenesis and fatal clinical outcomes of COVID-19 disease. These findings can indicate promising therapeutical targets and independent predictors of patients' chances of survival. The present study aims at investigating the potential usefulness of bradykinin and galectin-3 levels, obtained at hospital admission, as predictors of COVID-19 progression into severe or deadly forms of the disease.This study was approved by the Local Institutional Committee of the University Clinical Hospital Center Bežanijska kosa, Belgrade, Serbia (reference number 6609). All procedures were conducted in accordance with the Declaration of Helsinki (revision 2013) and Good Clinical Practice. The written informed consents were obtained from all participants before their inclusion in the study. A preprint of this manuscript has been previously published .The study was conducted as a prospective cross-sectional study. It included 47 adult patients with confirmed SARS-CoV-2 infection and radiologically-confirmed pneumonia who were admitted to University Clinical Hospital Center Bežanijska kosa, Belgrade, Serbia, in June 2021. The main criteria for the participation in the study in this study included the following: (1) that a patient was 18 or more years old, (2) that COVID-like pneumonia was confirmed radiographically, and (3) that the patients consented to participate. All patients were given adequate supportive care and were administered the proper therapy according to the pharmacological protocols .COVID-19 infection was confirmed with reverse transcriptase-polymerase chain reaction (RT-PCR) from nasopharyngeal swab sample. COVID-19 pneumonia was confirmed radiographically by chest X-ray or chest CT. Chest CT was performed in all participants so that we could evaluate the stage and extensiveness of interstitial pneumonia with more precision. The typical COVID-19 pneumonia changes were evaluated by examining the bilateral lung involvement (with ground-glass opacities), the lung consolidations, the interlobular septal thickenings, the crazy pavings, the pleural effusions, and the lymphadenopathy of our patients. According to the severity of the observed changes, every lobe was given 0–5 points  forming the maximum possible score of 25 points. Except scoring, the disease was also classified into four stages . The main clinical criteria for the ICU admission was the detected progression of the disease observed through radiographic or chest CT examinations, the peripheral oxygen saturation (Sp02) below 93% , and the further impairment of the results obtained through arterial blood gas tests and laboratory tests (mainly the increases of inflammatory parameters).The demographic data, comorbidities, and laboratory parameters were collected from all patients at their admission. The laboratory parameters included a complete hemogram, neutrophile-lymphocyte ratio, blood sugar, serum ferritin, coagulation status and D-dimer, serum lactate dehydrogenase, and troponin; renal function test, liver function test, and electrolyte balance. The treatment details were also recorded, such as the use of steroids, anticoagulants, high-flow nasal cannula, noninvasive, and invasive ventilation.2−), hydrogen peroxide (H2O2), nitrite (NO2−), index of lipid peroxidation , and activity enzymes , superoxide-dismutase (SOD), and reduced glutathione (GSH)). All these biochemical parameters of oxidative stress were measured spectrophotometrically .The following biomarkers of oxidative stress were measured in plasma samples: superoxide anion radical  reagent was added to TRIS buffer (assay mixture) and the measurements were performed at the wavelength of 530 nm .2O2 was based on the oxidation of phenol red by H2O2 in the presence of horseradish peroxidase as a catalyst. The levels of H2O2 were then measured at the wavelength of 610 nm [The protocol for the quantification of Hf 610 nm .The TBARS method was used for evaluating the lipid peroxidation indices. The biological specimen (with 1% thiobarbituric acid (TBA) and 0.05 M sodium hydroxide (NaOH)) were heated at 100° C for 15 minutes. Finally, the measurements were performed at 530 nm .The nitric oxide levels were evaluated indirectly, i.e., via measuring the nitrite levels. Green's method, with the Griess reagent  was applied here . The sam2O2. The CAT activity was measured spectrophotometrically at the wavelength of 360 nm and was expressed in nmol/ml plasma.The antioxidant enzyme CAT quantification was carried out according to McCord's method . We usedThe SOD activity was evaluated by applying Beutler's epinephrine method . The samThe GSH levels were measured by following Beutler's method . The metThe serum bradykinin levels were measured with a bradykinin ELISA kit  by following the manufacturer's instructions. All samples were tested in duplicate. The optical densities (OD) were measured at the wavelength of 450 nm on a microtiter plate reader .Ninety-six-well microplates were precoated with capture polyclonal antibody (goat) to human galectin-3  and washed three times with wash buffer 1% Tween 20  in PBS. The samples (50 ll) were added in duplicate to each well, which contained 50 ll of the sample diluent. Detection antibody was diluted in reagent diluent and was then added to each well and incubated at room temperature (RT) for 2 hours on a microplate shaker set at 200 rpm. After washing and incubation, the reaction was stopped by a stop solution. The absorbance of each sample was measured at 450 nm in a microtiter plate reader . A standard curve ranging from 0.156 to 10 ng/ml of galectin-3 was generated for each ELISA.These data were collected retrospectively from the hospital's electronic medical records by trained staff. An electronic data capture tool was used for these purposes. We recorded the time periods passing from the first symptoms to the patients' admission, the durations of the ICU stays, and the durations of mechanical ventilation for both groups (the survived and deceased patients).U test as appropriate, while the patients' data were tested with the Chi Square Test. In addition, the Cox regression model was used to test the relations between cytokine values and patient demographics and comorbidities. The Cox proportional hazards model was employed to estimate the death hazards. It includes covariance adjustment. The covariates  were determined by the backward elimination method. The point estimates (HRs), along with the corresponding 95% CIs, predicted probabilities for survival. We obtained the cumulative incidence curves. The analyses used two-sided tests and were performed in the SPSS version 26.0 for Macintosh.The patients' data were summarized by using standard descriptive statistics, i.e., means for continuous variables and count/percent for categorical variables. The correlation analyses were used to test the potential impact of cytokines and oxidative biomarkers. The laboratory test results were evaluated with the Mann–Whitney Between the August and September of 2021, 47 consecutive patients with confirmed COVID-19 pneumonia were recruited for the study. 31.2% of them passed away. The mean age of the survivors was 46.50 years and the mean age of the deceased patients was 72.53 .. The maRegarding the presence of comorbidities, a higher percentage of patients with hypertension (93.3%), diabetes (20.0%), and obesity (6.67%) was recorded among the group of the deceased patients. Other comorbidities  were also more frequent in the deceased patients. Chronic obstructive pulmonary disease and asthma were not detected in any group .The laboratory blood tests were significantly impacted in both groups . The levThe significant differences between the survived and deceased patients were detected in the total blood counts and coagulation statuses . The decThe secondary outcomes were also different . The decThe Figures 11The correlation analysis among all COVID-19 patients confirmed the statistically significant correlation between some markers of oxidative stress and cytokine bradykinin and peptide galectin-3 . Serum bSince bradykinin showed a good linear correlation with the detected oxidative stress levels, we evaluated the potential cut-off values of bradykinin, which could be taken as the borderline between the positive and fatal outcomes. Figures 55The Cox regression analysis reveals the following independent predictors of mortality in the patients suffering from COVID-19 pneumonia: (1) age above 50, (2) in-hospital stays surpassing four days, (3) the bradykinin levels above 220000 pg/ml, (4) the elevated D-dimer, creatinine, and/or CRP, and (5) the presence of comorbidities (hypertension and diabetes) .This was conducted as a prospective cross-sectional study. It included 47 consecutive adult patients with confirmed SARS-CoV-2 infection and COVID-19 pneumonia. All patients fulfilled the criteria for hospital treatment and were admitted to the University Clinical Hospital Center Bežanijska kosa, Belgrade, Serbia, in June 2021. The main criteria for the participation in the study included the following: (1) that a patient was 18 or more years old, (2) that COVID-like pneumonia was confirmed radiographically, and (3) that patients consented to participate. All patients were given adequate supportive care and were administered the proper therapy according to the pharmacological protocols . This study aimed at detecting the major risk factors for developing severe conditions of COVID-19 disease, with special reference to bradykinin and galectin-3. Their levels can be measured immediately after the admission and they might be used as indicators that the disease will progress in undesirable directions. The data regarding the role and mechanisms of bradykinin and galectin-3 in severe COVID-19 cases have not been fully explored. Thus, our findings can provide a valuable insight. They can indicate the therapeutical targets and can be used as independent predictors of patients' survival.The first part of the study focused on the basic demographic characteristics of the patients. The deceased patients were older than the patients who survived. In both groups, male patients were dominant.We recorded the higher shares of the patients with hypertension (93.3%), diabetes (20.0%), and obesity (6.67%) among the deceased patients. Other comorbidities, such as coronary disease, cardiomyopathy, and chronic kidney disease were also more frequent in this group. Chronic obstructive pulmonary disease and asthma were not detected in any group . The preThe second part of the study focused on the routine and specific laboratory blood tests. The laboratory blood test results were significantly different in the two groups . The decRegarding the total blood count and coagulation status, there were significant differences between the groups . The higIt has been recognized that one algorithm of laboratory markers could be a potential guide for making prognosis and developing useful therapeutical protocols. As a standard of care, the baseline blood tests and inflammatory markers are obtained when patients are admitted to a hospital. The proper approach to risk assessment should allow physicians to forecast a patient's future worsening based on these initial results. As it has been mentioned in the literature on numerous occasions, D-dimer is associated with a bad prognosis. Our findings confirm this. The seconds early diagnostic markers of hyper-coagulopathy are the disturbances in CRP, fibrinogen, and white blood cells. They can indicate the necessity to include direct factor Xa inhibitors in a treatment –31.Elevated D-dimer level suggests extensive thrombin generation and fibrinolysis. It is associated with poor prognosis in COVID-19. This has prompted clinicians to hypothesize that increased D-dimer concentrations are indicative of coexisting venous thromboembolisms that may lead to ventilation-perfusion mismatch. Moreover, there is a lot of evidence about the high risk for thromboembolism in patients with COVID-19 disease. The activation of KKS in plasma leads to BK overproduction .Moreover, it is essential to obtain the cut-off values for the severity of the disease for each laboratory marker in order to reduce the number of severe and fatal cases.The secondary outcome for the two groups also differed . The durIn conclusion, we tried to find the molecular mechanisms responsible for fatal outcomes of COVID-19 pneumonia. We detected the elevated oxidative stress and very high levels of serum bradykinin and galectin-3 at the patients' admission in those who passed away. Their levels of nitric oxide and the activity of superoxide dismutase were lower while the levels of superoxide anion radical and the indices of lipid peroxidation were higher Figures 1. BradykiUsing a correlation analysis, we confirmed there are certain relations between oxidative stress markers and cytokine bradykinin, but also peptide galectin-3 . The ser6Definitely, bradykinin storm was present in the deceased patients. Unfortunately, the ongoing storm can probably result in increased microvascular permeability, edema, and further inflammation, which worsen prognoses. Our study strongly supports the bradykinin storm hypothesis. In that sense, future therapeutical strategies must be focused on reducing a bradykinin serum concentration in COVID-19 patients. Studies conducted by Ghahestani et al. suggested that blocking the B2 receptor with icatibant may be a good strategy for large BK degradation in COVID-19 patients. As they concluded, this drug could be able to also reduce angioedema, improve oxygenation in severe forms of the disease, and prevent poor clinical outcomes in patients with COVID-19 pneumonia . This phBradykinin can be a prognostic marker of mortality due to its linear dynamics and high sensitivity. At the heart of the pandemic, there was a growing concern about the pathophysiology of COVID-positive patients and their prognoses. Moreover, since there are no unique and effective pharmacological protocols for the patients infected with SARS-CoV-2, the importance of knowing the pathophysiological molecular mechanisms has been increasingly emphasized. The proinflammatory cytokines and peptides, such as bradykinin and galectin-3, are dominant in the deceased patients and may contribute to leaky vasculature and cell necrosis, which results in microvascular endotheliopathy. Moreover, a bradykinin course follows free radicals, which are identified in high bioavailability in the systemic circulation of patients with COVID-19 pneumonia. The cytokine and bradykinin storm theory could be a possible explanation for the severe forms of COVID-19 disease. As such, they offer many potential therapeutic targets for the prevention of multiorgan failure, such as inhibitors of bradykinin production –42.The limitation of this study is its relatively small sample size. However, we included matched patients with similar clinical features and the duration of the disease prior to admission. Moreover, given the different pathophysiology of COVID-19 disease, we plan to include the selected patients with 7 or 28 day follow-ups who had a mild type of disease to test all these assumptions further.Bradykinin storm and oxidative stress in patients with the fatal outcomes are probably responsible for the overactive inflammatory response and the resulting symptoms in COVID-19 patients. Understanding the mechanisms by which the two storms, bradykinin and cytokine, affect the body separately or jointly is crucial because it may help us develop more effective treatments, save lives, and mitigate the effects of the pandemic.


Chinese-Americans are one of the largest groups of Asian-Americans in the US with distinctive behavioral and cultural characteristics that influence health service use. Although Chinese-Americans have significantly higher COVID-19-related mortality rates, relative to other racial and ethnic groups, limited literature is available examining their willingness to accept the COVID-19 vaccine. With recent development of the combination influenza-COVID-19 vaccine by biotechnology companies to mitigate COVID-19 infection, we examined factors associated with Chinese-Americans’ acceptance of hypothetical annual doses of COVID-19 vaccination before the vaccine rollout. A total of 241 Chinese-Americans who received at least one dose of the COVID-19 vaccine completed an online questionnaire developed and based on health behavior theories. Our results indicated that Chinese-American participants who were satisfied with their prior COVID-19 vaccination experience, who had more accurate knowledge and perceived higher susceptibility of getting COVID-19, were more willing to receive the annual COVID-19 vaccine in the future. The findings of our current study may be used to guide the development of strategic messages to promote uptake of the annual COVID-19 vaccine by Chinese-Americans in the U.S. The COVID-19 vaccine is a safe and effective way to build immunity against COVID-19 infection and variants . The vir“Booster shots” is a term used as by health authorities , which suggests that the vaccine may need to be regularly repeated to maintain protection against the infection . The emeChinese-Americans make up the largest subgroup of Asian-Americans who often have limited English language proficiency and significant cultural barriers . Up untiTo the best of our knowledge, however, evidence concerning Chinese-Americans’ perspectives toward COVID-19 vaccine is limited. To address this gap in the literature, the purpose of the present study is to (1) assess intention to receive an additional hypothetical annual does of COVID-19 vaccination among Chinese-Americans who have received at least one dose of the COVID-19 vaccine; and (2) identify factors associated with their annual vaccination intention. This study was approved by the Towson University Institutional Review Board. Participation criteria included (1) Chinese-Americans who were living in the U.S. when filling out the survey, (2) were 18 years of age or older, and (3) had received at least one shot of COVID-19 vaccine. Qualtrics was used as the platform to collect survey data. Participants accessed the survey via a link or QR code from 5 June 2021 to 13 June 2021, when Delta variant became the dominant variant in the U.S. and a third wave of infections began. Responses were anonymous and participation was voluntary. Participant consent was obtained via information sheets. To de-identify participants when collecting their names and emails for respondent incentive (a $10 electronic gift card) distribution, all participants were provided with a link to a separate survey to enter their name and email. We used convenience sampling and promoted our study through social media, fliers posted in Asian stores and restaurants, and by networking with Asian-American churches and community organizations. A total of 374 participants filled out the survey. After excluding incomplete surveys, non-Chinese-American participants, and those who did not receive any shot of COVID-19 vaccine, a total of 241 respondents was included in the analytic models. We conducted a power analysis to calculate the estimated sample size. Our calculation indicates a sample size of 245 is needed for estimating the binary outcome variable with 95% confidence interval, an estimated 80% of respondents who would be willing to receive annual vaccination, and a 5% margin of error . Our finWe developed a 20-min online survey adapted from the questionnaires published in the literature ,25,26,27Besides cognitive constructs adapted from theories, appropriate knowledge is the primary foundation of an individual’s behavior. Previous studies documented that knowledge plays an important role in protection behaviors against the pandemic, such as wearing facemasks, using hand sanitizer, and receiving the COVID-19 vaccine ,38. SpecTo ensure the face validity, the survey was reviewed by experts on health behavior, healthcare management, and health disparities. The survey was then revised based upon their comments. The survey was originally developed in English and then translated into Chinese for the participants who preferred to use the Chinese language to fill out the survey. Translation and back translation procedures were utilized to ensure face and conceptual equivalence and cultural relevance . SpecifiIntention to receive boosters was measured by a single item , with responses provided on a 5-point scale (1 = very unlikely to 5 = very likely). Satisfaction with previous shots of COVID-19 vaccination was measured by one item, “To what extent are you satisfied with your experience of getting the COVID-19 vaccine? 1 = not at all satisfied; 5 = extremely satisfied”.Perceived susceptibility of getting COVID-19 was measured with 4 items  adapted from previous literature ,25. The Perceived severity of getting COVID-19 was measured with 6 items  adapted from previous literature ,25. The Perceived benefits of getting the COVID-19 vaccine were measured with 6 items adapted from previous literature  ,24. The Perceived barriers to getting the COVID-19 vaccine were measured with 4 items adapted from previous literature  ,24. The p < 0.001).Self-efficacy for getting the COVID-19 vaccine was measured with 3 items adapted from previous studies  . Cronbacp < 0.001).Subjective norms for getting the COVID-19 vaccine refers to perceived social pressure from important person or group of people for an individual’s decision on vaccination. This construct was measured with 3 items adapted from previous studies  . CronbacTen items adapted from previous literature were used to measure cue to actions . If participants opted for any of these choices, they were coded as “yes” for cue to actions of COVID-19 vaccine. Knowledge of COVID-19 vaccine was measured by 7 false/true questions developed by the research team based on educational materials from the CDC website   was measured using two questions: “1. What is the manufacturer/developer of the COVID-19 vaccine you actually received or will potentially receive? ” and “2. What is your preferred manufacturer/developer of the COVID-19 vaccine? ”. Participants were categorized as “failure to receive the preferred type of COVID-19 vaccine in previous shot(s)” if their responses for the first question were not consistent with those for the second one. For the participants who picked the same types of vaccine for both questions or chose “no preference” for the second question, they were not considered as “failure to receive the preferred type of COVID-19 vaccine”.Sociodemographic information  was also collected in the survey because it may also influence Chinese-Americans’ decision to receive the booster vaccines.The frequency, percentages, means, and standard deviations of variables were examined with descriptive statistics. Psychometric testing was conducted for cognitive constructs with multiple items. Specifically, Cronbach’s alpha was used to check construct reliability, and confirmatory factor analysis (CFA) was used to check the unidimensional validity of each construct with 3 items or more based on three fit indices: the root mean square error of approximation (RMSEA), comparative fit index (CFI), and standardized root mean residual (SRMR). An RMSEA < 0.08, a CFI > 0.90, and an SRMR < 0.06, were adopted as the cut-off point for an adequate model fit . CFA wasp < 0.05) were included in a multivariable modified Poisson regression model to assess factors associated with Chinese-Americans’ willingness to receive an annual COVID-19 vaccine. Constructs adopted from theories were all included in the model no matter whether significant or unsignificant results in bivariate analyses. The choice of survey language was also included in the final model because it is considered as an important factor which may influence immigrants’ decision making in healthcare. STATA 17 was used to conduct all analyses with the alpha level of p < 0.05. Bivariate analyses were conducted to examine the relationship between outcome variables  and predictors. Specifically, we used modified Poisson regressions to examine the associations. Modified Poisson regression is considered as a better approach compared to logistic regression when using common binary outcomes with more than 10% of occurrence ,46. Demon = 220, 93.2%) were born outside of the U.S. More than half of the participants had a graduate degree . More than half of our participants were employed  and had an annual household income above $75,000 . Less than half of the participants  affiliated with a particular religion . Most participants  chose English as the language to fill out the survey, while the rest of the participants  chose the Chinese version of the survey.The majority of the participants (86.3%) reported that they were willing to receive the boosters, while the rest of the participants (13.7%) were not willing to receive annual boosters even if they had previously received shots of the COVID-19 vaccine. The mean of satisfaction is 4.1 with a range from 1 to 5. Most participants (83.0%) felt either very satisfied or extremely satisfied with their experience with the COVID-19 vaccination. However, more than fifty-one participants (16.2%) reported that they did not receive their preferred type of COVID-19 vaccine. As shown in The current survey study examined Chinese-Americans’ intention to receive an additional hypothetical annual COVID-19 vaccine dose and explored the factors associated with this intention. The findings of our study indicated that while most Chinese-American participants were willing to accept an annual COVID-19 vaccine, nearly 14% of the participants were hesitant to receive an annual vaccine. The factors significantly associated with acceptance of the hypothetical annual COVID-19 vaccine included satisfaction with prior COVID-19 vaccination experience, knowledge regarding COVID-19 vaccine, and perceived risk of getting COVID-19. The majority of our participants (86.3%) were willing to get vaccinated annually against COVID-19. Our findings are similar with a cross-sectional survey study conducted in Europe, which indicated that 82.4% of participants from a German-speaking region were willing to receive annual vaccinations. However, the percentage from our study is higher than that of a survey study to examine Canadian adults’ willingness to accept annual vaccines, which indicated that 64.7% of the respondents reported the acceptance of an annual COVID-19 vaccine dose . The higNotably, our results indicated that Chinese-American participants who were satisfied with prior COVID-19 vaccination were more likely to accept the annual vaccine than those who were not satisfied. If the annual COVID-19 vaccine is recommended in the future, it will be important to ensure that those who plan to receive it act on their intentions and efforts must be taken to encourage those who are hesitant to get it to change their minds. It is critically important to provide Chinese-Americans with high-quality and accessible vaccine services, including providing easy scheduling of vaccination appointments through patient/community navigation, convenient access to vaccination sites (such as mobile or community-based vaccination programs), translation services, culturally and linguistically appropriate vaccine educational materials and services, good vaccination management, and efficient pre-vaccination communication with pharmacists, nurses, or doctors . Along with satisfaction, a high level of knowledge of the COVID-19 vaccine was positively and significantly associated with participants’ intention to accept an annual vaccine. This finding is consistent with previous studies exploring other population members’ intentions to receive the COVID-19 vaccine. A nationally representative survey of 1027 adults in the U.S. found that many respondents, especially racial/ethnic minorities and people with lower education levels, overestimated the side effects of the COVID-19 vaccine, significantly underestimated the size/scale of the clinical trials for the COVID-19 vaccine, and perceived that the vaccine contained live coronavirus . Our resCompared to other populations outside of the U.S., our sample perceived low-level knowledge of COVID-19 and vaccines. For example, a survey study conducted among elderly people in Southern Italy indicated a satisfactory level of knowledge about COVID-19 and related control measures (with a correct answer rate ≥ 81.5%) . AnotherThus, delivering accurate vaccine information to Chinese-Americans is critically important to counter the myths and encourage vaccination. Educational efforts are needed to help racial/ethnic minorities (including Chinese-Americans) to better understand the benefits of COVID-19 vaccination and to eliminate misconceptions about the vaccine. Informing members of this population about the safety of vaccination and addressing their concerns about the vaccine may decrease hesitancy about their receiving of continuous annual vaccine doses. For example, future public health outreaches for Chinese-American communities are needed to educate the uninformed regarding the potential benefits and immunization process of annual doses of the COVID-19 vaccine. Our results also showed that Chinese-American participants’ intention to accept an annual COVID-19 vaccination was significantly associated with greater perceived susceptibility. This finding was consistent with previous literature regarding COVID-19 vaccine hesitancy and annual influenza vaccinations ,49. The The previous literature indicates that English proficiency is an important factor associated with Asian-Americans’ perceptions of COVID-19 vaccination . The AsiThis study had some limitations. First, our sample was not recruited using random selection methods. More than half of our participants had received a high level of education and had a high income. However, these sample factors reinforce that vaccine hesitancy issues are not just limited to those with lower education and income levels. The findings of this study may not be generalized to all Chinese-Americans living in the U.S. Our relatively small sample size tempers our ability to generalize our findings to the entirety of the U.S. or other countries. Second, this study did not explore the reasons behind the unwillingness or willingness to receive the annual COVID-19 vaccine. Future research should identify specific motivators and barriers to continuing vaccination. In addition, as the annual COVID-19 vaccine is a hypothetical situation, participants’ responses were based on a hypothetical scenario. Their opinions may change if and when the actual situation may occur, where epidemiological data and vaccination policy may play an important role in their decision making. Fourth, this study did not assess the association between annual vaccination intention and other cognitive constructs from TBP . Future research is needed to test the application of the full theory. Fifth, although construct items were adopted from a previously validated instrument, the questionnaire was not piloted before full implementation. However, the psychometric testing showed that our instrument is valid and reliable. Fifth, due to the nature of cross-sectional design, the causal relationship of the effect cannot be concluded . Future To the best of our knowledge, this is a first-of-its-kind study in the assessment of Chinese-Americans’ willingness to accept a hypothetical annual COVID-19 vaccine. Our findings indicated that most Chinese-American participants were willing to get an annual COVID-19 vaccine. In addition, our findings highlight the importance of enhancing the positive experience of the previous vaccination, accurate knowledge regarding COVID-19 vaccination, and perceived risk of COVID-19 infections among Chinese-Americans to promote annual COVID-19 vaccination. If annual COVID-19 vaccination is required in the future, a clear guideline from health authorities, advocation by governments, public health educational efforts, and recommendations from healthcare providers will play important roles in improving vaccine acceptance rates among Chinese-Americans.


Clinical results demonstrate the potential of this immune process to trigger a systemic effect. We conclude that the properly applied amRF promotes antitumor HSP activity, and in situ, it could support the tumor-specific immune effects produced locally but acting systemically for disseminated cells and metastatic lesions.The role of Heat Shock Proteins (HSPs) is a “double-edged sword” with regards to tumors. The location and interactions of HSPs determine their pro- or antitumor activity. The present review includes an overview of the relevant functions of HSPs, which could improve their antitumor activity. Promoting the antitumor processes could assist in the local and systemic management of cancer. We explore the possibility of achieving this by manipulating the electromagnetic interactions within the tumor microenvironment. An appropriate electric field may select and affect the cancer cells using the electric heterogeneity of the tumor tissue. This review describes the method proposed to effect such changes: amplitude-modulated radiofrequency (amRF) applied with a 13.56 MHz carrier frequency. We summarize the preclinical investigations of the amRF on the HSPs in malignant cells. The preclinical studies show the promotion of the expression of HSP70 on the plasma membrane, participating in the immunogenic cell death (ICD) pathway. The sequence of guided molecular changes triggers innate and adaptive immune reactions. The amRF promotes the secretion of HSP70 also in the extracellular matrix. The extracellular HSP70 accompanied by free HMGB1 and membrane-expressed calreticulin (CRT) form damage-associated molecular patterns encouraging the dendritic cells’ maturing for antigen presentation. The process promotes CD8 The stress- or heat shock proteins (HSPs) represent a large family of highly conserved molecules vital in almost every living cell, on their surfaces, and in their extracellular microenvironments, throughout the cells’ lifetime regardless of their evolutionary level . Their rAny change in the dynamic equilibrium of the cell’s life, such as environmental stresses , pathogeThe roles and impacts of HSPs are highly complex. The complexity determines their role in immune modulation and in cancer development. For example, increased expressions of the members of the HSP70 family HSPa1a, HSPa1b, and HSPa7 are associated with a poor prognosis in human colorectal cancer observed in the comparison of the pretreatment tumor samples with the clinical data. However, the increased expression of the same family member, HSPa9, in tumors was associated with a favorable prognosis . Heat shWhile the primary function of most HSPs is the protection of cells against stresses, their function is not limited to protection on a cellular basis. Under certain conditions, they participate in the collective protection of multicellular structures and tissues and may even play a role in systemic processes ,17.HSPs are able to transport intracellular antigenic peptides to antigen-presenting cells (APCs), which produce antigen-specific cytotoxic T-lymphocytes , and the cytosol . The mHSIntracellularly, the HSPs as molecular chaperones assist in maintaining the balance between the intracellular proteins, participate in the regulation of apoptosis, and protect the cells from stress  caused bThe different families of HSPs represent the further functional division at numerous locations. Therefore, each has specific activities and functions in a cooperative network of homeostatic control.The multifunctional behavior of HSPs results in opposing actions. They may either support cellular defense  or, in sThe distinction of functions in malignancy is rather complex. The individually well-functioning cells with unicellular preference renounce multicellularity, causing fundamental challenges in malignancy decisions . The malSensing the immunosurveillance of the system could serve as the reversing factor from a foe to a friend. The effects of the stress proteins were recognized early on in immunology  and haveMost oncological treatment methods cause extra stress, which induces HSPs. The increased HSP synthesis is seen after conventional hyperthermia , chemothStress also induces the translocation of iHSPs to the malignant cell’s membrane , formingElectromagnetism is one of the active factors in biological processes, and it has a broad therapeutic application in oncology. One of the widely used treatments is the local-regional heating of the tumor and its environment. The absorption of electromagnetic energy heats the tissues in these therapies utilizing radiofrequency (RF) and microwaves ,58. It iThe heat stress-induced iHSPs present a significant challenge for oncological hyperthermia treatment because these chaperones’ elevated level develops treatment resistance and promotes the malignant processes . The HSPParadoxically, hyperthermia has shown profound success in inhibiting cancerous growth. Hyperthermia combined with conventional chemo- and radiotherapies, surgery, and the emerging immunotherapies  achievedThe apparent controversy between supporting or inhibiting tumor growth indicates, again, the complex behavior of heating interventions, which appears in the “double-edged sword” phenomena Cooperation with the natural homeostatic regulations could be a decisional factor to push the balance to the favorite side. One of the overall regulators of the homeostatic control is the immune-surveillance, which may have an excellent partner to win .The bioelectromagnetic interactions potentially manipulate the locations and functions of the HSPs, driving the complex challenge of cooperating with the immune effects and re-establishing the healthy homeostatic balance.One way is to heat the whole tumor volume isothermally. The applied focusing techniques intend to maximize the temperature of the tumor volume and minimize it in healthy surroundings . The ori–The method injects artificial  into the tumors. These invasively placed exclusive energy absorbers ,73 distr–The tumor’s heterogenic structure offers natural targets. The optimally chosen electromagnetic properties such as the frequency, the intensity, the phase, and the delivered time–information (modulation) allow for tuning to select the chosen particle (molecular cluster). For example, the membrane rafts (lipid micro-domains found in the plasma membranes of cells ) offer tAnother way heats small selected targets, either artificially or naturally available centers in the tumor volume:Bioelectromagnetic energy absorption heats the targets. Heating has two fundamental concepts in oncological practice:All heating methods induce the expression of various HSPs . While hThe lipid raft micro-domains respond to electromagnetic fields . MembranThe heating of the molecular clusters on the tumor cells causes extreme stress on the cells, which can trigger programmed cell death . The eleThis review focuses on hyperthermia driving HSP activity in a “friendly way”. Our goal is to convert the HSPs’ role from “foe” to “friend”, promoting ICD of the tumor cells. This could shift the approach to treating malignancy with hyperthermia from targeting local disease to producing systemic activity.The heterogenic selection principle proposes a solution to the challenge of tissue selection in hyperthermia  by applyThe selection uses the relatively high number of membrane rafts on the surface of malignant cells , which hThe mEHT technology applies capacitive-coupled energy, using precise impedance matching  in orderrthermia . This sorthermia   and Bag3. The transcription of HSP90 alpha (HSP90AA1) and HSP60 (HSPD1) was also up-regulated in HT29 xenografts. An increase in a broad spectrum of HSP families at an mRNA level was observed in HT29 xenografts four hours post-treatment [The heat-map of gene expression showed that mEHT induced significant up- or down-regulation of 48 transcripts of 39 genes compared to controls in the study on HT29 xenografts. The relative mRNA expression of HSP70 reached maximum expression four hours post-treatment . Gene coreatment .HSPb1, HSPa1a, HSPa1b, and HSPh1, was also seen in triple-negative 4T1 breast cancer isografts treated with mEHT. This observation was noted 24 h post-treatment and was associated with inhibiting tumor growth and proliferation. HSPa1a and HSPa1b are the most common isoforms of the HSP70 family. The RNA sequencing showed significant HSP70-1 (HSP72), HSP70-2, HSPB1, and chaperone HSP105 development 24 h after treatment, along with the up-regulation of the associated genes [HSP70 concentrations around the peripheral margin of the tumors 24 h post-treatment was observed.The up-regulation of several genes, including ed genes . An incrHSP105 and HSP90A, have been shown to block apoptosis by interfering with caspase activation and directly and indirectly inhibit apoptosis. In this study, the HSPs appeared to play an antiapoptotic role as they prevented apoptotic cell death in the cells heated with wHT, but not in the cells heated with mEHT [EGR1, JUN, and CDKN1A genes. Despite the same thermal condition, the mEHT treatment does not appear to activate the cytoprotective network while the water-bath treatment activates it. The activation of the ERK-JUN pathway was present only in the mEHT-treated samples. The FAS, c-JUN N-terminal kinases (JNK), and ERK signaling pathways dominate the apoptotic pathway. The ingenuity pathway study uncovered the HSP network  cell line showed an activation of the cytoprotective gene network in samples heated with water-bath homogeneous heating (wHT). The up-regulation of genes, such as  network a–c, whicThe level of mRNA-associated iHSP70 peaks at four hours post-treatment in 4T1 isograft models , while tIt is clearly shown that the up-regulation of the HSP70 in the HT29 xenograft model treated with mEHT increases, peaking at 14 h post-treatment, followed by a decline and a return to baseline levels at 48 h. The distribution of HSP70 shows a large increase in iHSPs during this first peak. The second peak in HSP expression occurs between 72 and 120 h post-treatment, followed by a return to baseline levels after 168 to 216 h post-treatment . The secThe levels of mHSP70 and eHSP70 also show an increase in B16F10 melanoma allograft models treated with mEHT, compared to the control, at 24 h post-treatment .Yang et al. showed that heat stress in the HepG2 cell line produces massive increases in iHSPs following all heating methods. The secretion of eHSP70 also appears post-treatment between 24 and 48 h in all heating methods. However, the levels of eHSPs are significantly higher following mEHT treatments  Figure . The datThe inoculation of tumors into two different regions in vivo, e.g., the left and right femoral region , or the In a follow-up study by Andocs et al., mice were inoculated with HT29 xenograft cells in both hind legs. One of the resulting tumors in each model was treated with mEHT and the other was left untreated. A consistent increase in HSP90 was observed in the tumors treated with mEHT. In these tumors, two peaks in the expression of iHSP90 were observed; the first at ~24 h post-treatment and the second between 168 and 216 h post-treatment  Figure . An incrThe results of the preclinical apoptosis studies suggest that despite the increased expression of iHSPs, the mEHT has the potential to inhibit tumor growth and support the development of an apoptotic process . When HSIn the event that the protective mechanisms from the iHSPs are unable to restore normal cell functions after exposure to stress, the stress on the cell results in cell cycle arrest, which is typically by caspase-dependent apoptosis . During The tumor destruction ratio  also peaked at approximately 24 h after the final mEHT treatment, suggesting that the protective mechanisms from the HSPs had been exhausted by this time. Furthermore, the proportion of the cCas3-positive regions overlapped extensively with the damaged regions of the tumor seen on consecutive sampling . The proApoptosis-inducing factor (AIF), a flavoprotein that resides in the mitochondrial intermembrane space, may also have a role in apoptosis caused by mEHT . FollowiThe mEHT may induce other apoptotic pathways detected in vitro  Figure . VariousThe comparison of the wHT and mEHT under the same thermal conditions also shows significant differences in the intercellular calcium  ), increa1: WAF1) .Another preclinical study on a melanoma model treated with mEHT  showed NMyeloperoxidase  positive neutrophil granulocytes (and monocytes) were significantly elevated in xenografts treated with mEHT compared to control tumors from 48 h to approximately 216 h post-treatment  in HT29 Modulated electro-hyperthermia may improve the immunological tumor microenvironment, followed by dendritic cell (DC) immune support . This adactivity   in the CT26 allografts  Figure .The eHSP70 appears together with a set of other molecules, forming a damage-associated molecular pattern (DAMP), which includes the release of calreticulin (CRT), high-mobility group Box 1 (HMGB1), and adenosine triphosphate (ATP). The cytoplasmic CRT translocates to the plasma membrane during the early stages (four hours) after treatment with mEHT in HT29 xenografts . The DAMThe addition of other stimuli supporting mEHT has enhanced the success of tumor-specific immune activation. The combination with fluorescently labeled primary NK cells (NK92MI) increased the NK cell penetration to the tumor , and theThe radiation-induced abscopal effect does not frequently appear in clinical practice. Radiation therapy demonstrated the first observable abscopal effect  as a lesMinnaar et al. (2019), in a Phase III mEHT study, described the possible observation of the abscopal effect in 24% of women with locally advanced cervical cancer and extra-pelvic nodal disease . CisplatThe high biological heterogeneity of the tumor, and its structural and functional differences from the healthy host tissue, are apparent from the electromagnetic and thermal variability of the energy transmission and absorption . These dImmunogenic cell death describes the cellular death pathway triggered by the chronic exposure of DAMPs to the immune system. This immune-stimulatory form of apoptosis promotes an adaptive immune response to the dying cell. In oncology, this process appears to promote the immune recognition of malignant cells and anti-tumor immunity .The electromagnetic interaction resulting from the electric heterogeneities in the tumor tissue offers a selection opportunity for malignant cells, causing stress, which allows for the manipulation of HSP production. Therefore, the applied electric field causes stress via both thermal and nonthermal processes.The malignant cells have a higher concentration of membrane microdomains (rafts) than the nontumorigenic cells . These rThe initial peak of HSPs between 12 and 24 h post-treatment likely represents a coexistence of iHSPs and mHSPs. The second peak of HSPs seen several days after treatment with mEHT  is likelThe gradual temperature increase applied in the step-up heating protocol of mEHT induces more HSPs in healthy cells than in the heavily stressed malignant cells . The difIn the pre-clinical studies, temperature measurements were taken during mEHT and classical heating techniques, such as water bath heating and capacitive heating. Most of the studies evaluated the samples from the different heating methods at the same temperature. However, Andocs et al. also compared mEHT heating at 38 degrees Celsius and 42 degrees Celsius to other heating methods at 42 degrees Celsius. This study revealed the nonthermal effects (field effects) as well as the thermal effects of mEHT. During mEHT, the thermal and nonthermal effects occur together, working synergistically ,145. TheFurthermore, high nonthermal doses can stop, or even decrease, the expression of iHSPs ,148. TheIn the case of high nonthermal doses, the strong apoptotic forces overcome the protection facility of the iHSPs, destroying the cellular integrity . The iHSThe tumor-specific mHSP70 enables the recognition of the malignant cells by NK cells ,160. TheThe therapeutic goal in the case of mEHT is to both induce the local (treated tumor) and systemic (distant tumors and tumor cells) effect, and the local treatment could produce tumor-specific immune reactions which tackle the systemic targets. We propose using electromagnetically triggered apoptosis to target the local tumor to achieve this. While the local excitation triggers the increased expression of iHSPs, the electric field triggers the transition of iHSPs to the ECM and promotes apoptotic cell death. The overexpression of the iHSPs in the malignant cells is quickly exhausted, paving the way for apoptosis . AdditioThe extrinsic excitation of rafts triggers the cooperation of and FADD . The eleand FADD . The diroptosis) . The RF optosis) , potenti pathway . The Cas pathway , resulti pathway . Measure pathway . Eventua pathway . At eigh pathway , which aThe set of DAMPs triggered by mEHT-induced apoptosis further paves the way toward promoting the immune targeting of systemic disease. DAMPs have variants of molecular sets expressed in response to different stimuli, and these variants are all hallmarks of various types of cell death . The mEHThe key to eliciting systemic immunogenic effects against malignant lesions with mEHT is the promotion of the molecular sequences leading ICD . The memThe extensive heating in conventional homogeneous hyperthermia processes causes necrosis. The heat triggers the expression of iHSPs, which are mostly antiapoptotic. The necrotic cellular rupture also produces eHSP70 as the iHSP70 is released into the extracellular matrix ,169. Howeg cells . The TreDuring hyperthermia, the arteries deliver blood, which acts as a cooling media. However, the distribution of the vasculature throughout the tumor is not even, resulting in hot spots in poorly perfused regions. There is a risk that the control of the eHSP action is lost in these overheated micro-regions. Furthermore, the relatively high temperature in these heated regions (>40 °C) inactivates the immune cells , whose aThe mEHT method attempts to bypass the necrotic process, avoid inflammation, and suppress immunity . The mEHPreclinical studies confirm that the DAMP-inducing apoptosis dominates in mEHT, and the necrotic cell destruction is relatively low ,164. TheFurthermore, the apoptosis-inducing stress from mEHT does not degrade the various DAMP molecules, which can deliver the necessary information and sensitizing actions for both the native and adaptive immune processes, offering a way to develop tumor-specific immune effects. The eHSP presents the “info signal” as the carrier  of tumorCalreticulin acts as the “eat me” signal for phagocytosis ,180, andCa2+ . Calretiows mEHT .The HMGB1, another member of DAMP, represents a “danger signal” . The HMGThe DAMP, therefore, also has a “double-edged sword” effect , acting The nonthermal effects of mEHT influence the homeostatic control using the time-fractal modulation . The appIn this proposal, the electric field effectively participates in limiting the malignant growth , harnessThe tumor-specific immune activity of mEHT promotes the response of untreated distant metastases to the local irradiation of the primary tumor, known as the abscopal effect. The DAMPs with the delivered genetic information by eHSP70 activate DCs, generating antigen-presenting cells (APCs) . This primmunity ,203. A nas 38 °C . The γδT systems , particint cells .The various HSPs have crucial roles in forming immune-specific antitumor processes all over the body, including targeting micro- and macro-metastases . The infHeat shock proteins have a complex “double-edged sword” effect, promoting and suppressing the malignant processes. Our goal is to apply an external stimulus, such as an electric field, to induce immune effects and tip the balance towards the side of tumor suppression, promoting the systemic immune recognition of tumor cells. Despite the plethora of literature on heat shock proteins, the precise roles and mechanisms of each HSP in adaptive and innate immunity are still not known. There is still much to learn about these molecules about the precise effects of mEHT and electric fields on the immune modulation of malignant cells. Following a review of the literature, we have proposed a potential mechanism for the outcomes seen in the preclinical and clinical data on mEHT, in which HSPs play a crucial role. The electric field’s synergistic action of the thermal and nonthermal effects selectively heats the tumor cells and targets its membrane components . The non


Over 2021, COVID-19 vaccination programs worldwide focused on raising population immunity through the primary COVID-19 vaccine series. In Singapore, two mRNA vaccines (BNT162b2 and mRNA-1273) and the inactivated vaccine CoronaVac are currently authorized under the National Vaccination Programme for use as the primary vaccination series. More than 90% of the Singapore population has received at least one dose of a COVID-19 vaccine as of December 2021. With the demonstration that vaccine effectiveness wanes in the months after vaccination, and the emergence of Omicron which evades host immunity from prior infection and/or vaccination, attention in many countries has shifted to how best to maintain immunity through booster vaccinations.The objectives of this phase 3, randomized, subject-blinded, controlled clinical trial are to assess the safety and immunogenicity of heterologous boost COVID-19 vaccine regimens (intervention groups 1–4) compared with a homologous boost regimen (control arm) in up to 600 adult volunteers. As non-mRNA vaccine candidates may enter the study at different time points depending on vaccine availability and local regulatory approval, participants will be randomized at equal probability to the available intervention arms at the time of randomization. Eligible participants will have received two doses of a homologous mRNA vaccine series with BNT162b2 or mRNA-1273 at least 6 months prior to enrolment. Participants will be excluded if they have a history of confirmed SARS or SARS-CoV-2 infection, are immunocompromised, or are pregnant. Participants will be monitored for adverse events and serious adverse events by physical examinations, laboratory tests and self-reporting. Blood samples will be collected at serial time points  for assessment of antibody and cellular immune parameters. Primary endpoint is the level of anti-SARS-CoV-2 spike immunoglobulins at day 28 post-booster and will be measured against wildtype SARS-CoV-2 and variants of concern. Comprehensive immune profiling of the humoral and cellular immune response to vaccination will be performed.This study will provide necessary data to understand the quantity, quality, and persistence of the immune response to a homologous and heterologous third booster dose of COVID-19 vaccines. This is an important step in developing COVID-19 vaccination programs beyond the primary series.ClinicalTrials.govNCT05142319. Registered on 2 Dec 2021. The order of the items has been modified to group similar items  declined in the months following vaccinations. For example, 6-month follow-up data for the mRNA-1273 vaccine estimated the half-life for binding antibodies for all participants was 52 days (95% CI 46 to 58), with significantly lower titers with increasing age [Some of the explanations for waning vaccine effectiveness is the emergence of SARS-CoV-2 variants of concern (VOC) which partially evade host immunity from prior infection/vaccination. This includes B.1.617.2 (Delta) and B.1.1.529 (Omicron) . StudiesSeveral studies have demonstrated that a COVID-19 vaccine “booster” dose significantly increases viral neutralization titers against wildtype, Omicron –9, and oIn Singapore, the mRNA vaccines produced by Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273) received interim authorization under the Pandemic Special Access Route (PSAR). Sinovac-CoronaVac has also been added to the National Vaccination Program, primarily for those who are medically ineligible for mRNA vaccines. By 17 January 2022 more than 9 million COVID-19 vaccine doses had been administered, with 88% of Singapore’s resident population fully vaccinated, 90% having received at least one dose, and 53% having received booster shots.Clinical trials of vaccine candidates which target VOCs are ongoing, though even if successful these vaccines are not expected to be available till late in 2022. Pending the availability of these VOC vaccines, the need for booster vaccinations, particularly in vulnerable populations needs to be assessed. A third dose of the vaccines developed by Pfizer–BioNTech, Moderna, Oxford–AstraZeneca, and Sinovac prompted significant increases in neutralizing antibodies titers when administered several months after the second dose .Preclinical and clinical data have also suggested heterologous prime-boost vaccination may enhance the immune response against SARS-CoV-2 or HIV –20. At dPRIBIVAC will assess a heterologous prime-boost-boost-boost strategy in comparison with a homologous regimen in order to compare short and long-term immunogenicity of different COVID-19 vaccine combinations against the ancestral SARS-CoV-2 as well as different VOCs.The primary objective is to determine whether heterologous prime-boost-boost COVID-19 vaccine regimens lead to non-inferior humoral immunity compared with homologous prime-boost-boost vaccine regimen against wildtype SARS-CoV-2 and/or 1≥ VOC at day 28 post-vaccination.The secondary objectives are to (1) determine whether heterologous prime-boost-boost COVID-19 vaccine regimens lead to non-inferior humoral and cellular immunity compared with homologous prime-boost-boost vaccine regimen against wildtype SARS-CoV-2 and/or 1≥ VOC at any other time points  post-vaccination, and (2) assess the reactogenicity and safety of heterologous and homologous prime-boost-boost COVID-19 vaccine schedules.PRIBIVAC is an adaptive, randomized, subject-blinded, controlled, non-inferiority phase 3 trial to assess the immunogenicity and safety of heterologous boost COVID-19 vaccination (intervention groups) compared with a homologous boost regimen (control group). This is an adaptive trial as new intervention groups may also be added depending on what COVID-19 vaccines are authorized for use in Singapore and may include new vaccines or vaccines targeting SARS-CoV-2 variants.Participants will be enrolled at the National Centre for Infectious Diseases (NCID), Singapore.The key eligibility criterion is individuals who had received a homologous primary vaccines series with BNT162b2 or mRNA-1272 at least 6 months prior to study enrolment.Willing and able to provide informed consent for participation in this study;Aged ≥21years at the time of study enrolment;Received the second dose of BNT162b2 or mRNA-1273 vaccines at least 6 months prior to enrolment;Willing and able to comply with all scheduled visits, vaccination plan, laboratory tests, and other study procedures.Inclusion criteria:Known history of SARS-CoV-2 or SARS-CoV-1 infection;Previously received an investigational coronavirus vaccine;Previously received a SARS-CoV-2 monoclonal antibody;Current or planned simultaneous participation in another interventional study;A history of anaphylaxis, urticaria, or other significant adverse reaction requiring medical intervention after receipt of a COVID-19 vaccine, or otherwise have a contraindication to one of the available study vaccines per the approved label;Individuals who are immunocompromised ;Received systemic immunosuppressants or immune-modifying drugs for >14 days in total within 6 months prior to screening . Topical tacrolimus is allowed if not used within 14 days prior to day 1;Individuals who are pregnant or breastfeeding;Chronic illness that, in the opinion of the study team, is at a stage where it might interfere with trial conduct or completion;Deprived of freedom by an administrative or court order, or in an emergency setting, or hospitalized involuntarily;Current alcohol abuse or drug addiction that might interfere with the ability to comply with trial procedures in the opinion of the study team;Moderate or severe acute illness/infection (according to study team’s judgment) on the day of vaccination, or febrile illness (temperature ≥ 37.5°C). A prospective participant should not be included in the study until the condition has resolved or the febrile event has subsided.Exclusion criteria:The principal investigator (PI) or medically qualified co-investigators are responsible for ensuring freely given consent is obtained from each potential participant prior to the conduct of any protocol-specific procedures.Leftover biological specimen and/or data may be used for future research, subject to participant consent.The PRIBIVAC study aims to assess the immunogenicity and safety of heterologous boost COVID-19 vaccine regimens compared with a homologous boost regimen.n = 164) retained >30% inhibition level of neutralizing antibodies against SARS-CoV-2 at 6 months post infection [Circulating antibody levels wane following vaccination and COVID-19 infection. In our previous study of COVID-19 recovered patients, ~60% of the cohort  was approved by WHO under the EUL procedure on 3 Nov 2021 for use in two doses for primary vaccination and is available in Singapore via the Special Access Route (SAR). A randomized, double-blind, placebo-controlled, multicenter, phase 3 clinical trial in India has confirmed the safety and efficacy of COVAXIN® against laboratory-confirmed symptomatic COVID-19 disease in adults [The booster vaccine for the control arm will be a homologous mRNA vaccine , while for individuals randomized to intervention group 1 the mRNA booster vaccine administered will be heterologous to the primary series . The booster vaccine candidates for intervention groups 2–4 will be an alternative non-mRNA COVID-19 vaccine. At the time of writing , vaccine candidates for intervention groups 3 and 4 have not been confirmed.Control group: Homologous mRNA booster vaccineIntervention group 1: Heterologous mRNA booster vaccineIntervention group 2: COVAXIN®Intervention group 3: Non-mRNA booster vaccine BIntervention group 4: Non-mRNA booster vaccine CThe participant may be withdrawn prematurely from the trial if he/she withdraws consent to participate in the study. In such instances, the withdrawal will be applicable to both data collection as well as vaccination.Participants may voluntarily decline the booster vaccination, or they may be withdrawn by their attending physician and/or by the study investigator(s) on the basis of the participant’s best interest. Even if the participant did not receive the booster vaccination, collected samples will still be analyzed up until the point the participant withdraws consent.All participants, including those who declined the booster vaccination, will be followed up until 12 months, unless the subject withdraws informed consent or is lost to follow-up or has died.Safety and futility will be reviewed by an independent Data and Safety Monitoring Board (DSMB) after the first 10 participants in each of the intervention arms have completed assessments at study day 28. DSMB may recommend discontinuation of a study arm on the basis of safety concerns or futility in immunogenicity.Booster dose will be given once only; patient adherence is not applicable.There will be no restrictions on diet, exercise, or concomitant medication imposed on participants, except for the restriction of receiving any other COVID-19 vaccines outside of this study.Unless there are any serious adverse events that need to be followed up closely, no post study follow-up and procedures need to be performed after the final study visit. If the participant follows the directions of the study team and is physically injured due to the procedure given under the plan for this study, NCID/TTSH will pay the medical expenses for the treatment of that injury.The outcomes are shown in Table The participant timeline is shown in Table Based on data from our ongoing COVID-19 vaccine immune-monitoring study (SCOPE) , the meaSince recruitment of subjects to intervention arms may be activated in a staggered way depending on availability of the vaccines, and recruitment to the control arm will continue until the target sample size of 100 is achieved in each intervention arm, we prepare to enroll a double sample size in the control arm for the purpose of study planning. This gives a total sample size of up to 600 subjects.Recruitment will occur over 6–9 months with 1-year follow-up period.Age Time from 2nd vaccine dose administered Primary vaccine series (BNT162b2 or mRNA-1273)Randomization will be performed using randomized permuted blocks and stratified by the following criteria:Eligible participants will be randomized in equal proportions to each of the study arms that are open for randomization. Enrolment to a study arm will be discontinued if recommended by DSMB or if the target sample size of 100 in the study arm has been reached. In the scenario where recruitment is discontinued in any of the intervention arms from 1 to 4 at the interim analysis, randomization to the control arm may continue beyond 100 participants until the target sample size of 100 participants has been reached in the remaining intervention arms.Randomization will be performed using a web-based randomization system hosted by Singapore Clinical Research Institute (SCRI). Password secured accounts will be assigned to the site personnel responsible for the randomization, where he/she can log into the randomization system using the Internet. Allocation concealment will be maintained until the registration and randomization process has been completed.The study team from SCRN in charge of participant enrolment will perform the randomization using the web-based randomization system.Vaccine allocation will be single-blind, i.e. only participants will be blinded. This is to reduce the risk of bias in participant-reported adverse events. Data analysts are not blinded.Blinding will be maintained for the participants until their day 28 visit (visit 3).All physical examinations will be performed by the investigator or co-investigator. It includes cardiovascular, respiratory, abdomen, neurological, musculoskeletal, and skin assessments. Vital signs including pulse rate, systolic and diastolic blood pressure, respiratory rate, and body temperature will also be recorded.The participant will be given a diary to record all the local and general symptoms experienced after receiving the vaccination. Local symptoms may include pain, redness, swelling, bruising, itchiness, muscle ache or movement limitation at the injection site. General symptoms may include headache, fever, nausea, vomiting, sore throat, cough, runny nose, tiredness, and red (sore) eyes, as well as symptoms suggestive of COVID-19 infections . This diary will cover 7 days post-vaccination and must be returned to the study team when the participant comes for the next visit.Safety will be assessed via physical examinations, vital sign measurements, medical reviews, AE and SAE evaluation, safety laboratory tests, and participant self-recorded diary. Blood samples will be taken during screening or day 0 and at the follow-up visits at days 7, 28, 180, and 360 post-enrollment for evaluation of immunogenicity and/or safety. All adverse events will be entered in the appropriate eCRF  and in the source documents.The study team will remind participants prior to their follow-up visits to minimize any missed appointments. Participants will be reimbursed for their time, inconvenience, and transportation costs for every study visit they complete.Paper Case Report Forms (CRFs) will be used for initial data collection. These will be transcribed to an eCRF using Research Electronic Data Capture (REDCap). The trial database will include information on demographics, medical history, vaccine arm allocation, vital signs, laboratory investigation tests, as well as AEs and SAEs. Paper documents will be maintained and stored in a locked office, with access restricted to study personnel. All electronic documents with participant information will be password-protected.Study monitors will visit the study site periodically to assess data quality and study integrity. Study monitors will review the study records on site to directly compare them with source documents, discuss the conduct of the study with the Investigator, and verify that the facilities and workflow are compliant with Good Clinical Practice (GCP). Such visits will be scheduled in advance to allow for logistical arrangements to be made. In addition, the study may be evaluated by government inspectors who must be allowed access to CRFs, source documents, and other study files.All study findings and documents will be regarded as confidential. The investigators and other study personnel must not disclose such information without prior written approval from the PI. Participant confidentiality will be strictly maintained to the extent possible under the law and local hospital policy. Identifiable information will be removed from any published data.Blood samples will be collected at NCID research clinic, de-identified/coded before dispatch to collaborating research laboratories such as A*STAR ID Labs and Singapore Immunology Network, Duke-NUS, and NCID’s NPHL for immunogenic studies as described below:5 cells per well in 96 well V-bottom plates. The cells will be first incubated with human serum (diluted 1:100 in 10% FBS) before a secondary incubation with a double stain, consisting of Alexa Fluor 647-conjugated anti-human IgG (diluted 1:500) and propidium iodide . Cells were read on BD Biosciences LSR4 laser and analyzed using FlowJo (Tree Star). A standard ELISA will also be used with the whole spike-protein, RBD fragment, or peptides encompassing dominant epitopes of the S and N proteins immobilized in the microplate wells. To identify the repertoire of epitopes induced by the vaccines, a peptide library ELISA will be used. This will provide fine resolution of the antibody responses against the S-proteins at different time points and help to determine whether there is a fixation of the antibody response to certain epitopes (antigenic sin).To determine the presence and levels of anti-SARS-COV-2 in human sera using S-protein flow cytometry-based (SFB) assay. Cells expressing the full S protein in its native configuration will be seeded at 1.5 × 10To examine the neutralizing capacity of the antibodies in the human sera, two different assays will be performed: the sVNT and the pseudovirus or live-virus assay inhibition.TM Aurora cytometer running SpectroFlo® Version 2.2.0.3 with automated unmixing. Compensation and analysis of flow cytometry data was performed with FlowJo Version 10.6.1.Quantitative T-cell responses to the vaccines will be measured using SARS-CoV-2 peptides from spike protein to stimulate the PBMCs isolated from the donor’s blood by ELISPOT or flow cytometry. To detect CD8 T-cells, PBMCs will be stimulated with a peptide pool consisting of peptides 8–10 amino acids in length. Negative controls using media and DMSO (~0.5%) will be used as reference. Assays will be performed in duplicates. The cell and peptides will be incubated in ELISpot 96-well plates. After 24 h, the plates will be washed and assayed for IFNγ. To detect CD4 Th1/2 cells, PBMCs will be stimulated with a peptide pool consisting of peptides that are 15 amino acids in length. After 24 h, the plates will be washed and assayed for IFNγ (Th1) or IL4/IL5/IL13 (Th2). Spot forming units (SFU) will be measured using IRIS reader (MabTech). SARS-CoV-2-specific T effector subsets will be tested after stimulation with pooled SARS-CoV-2 PepTivator® S, S1, peptides (0.6 nmol/mL each) (Miltenyi Biotec) for 6 h. Brefeldin A and Monesin (ThermoFisher Scientific) was added at 2 h post-stimulation. Cells were stained with surface stain markers in the dark at room temperature for 20 min, followed by fixation and permeabilization for 20 min with Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scientific). Permeabilized cells were then stained for intracellular cytokines for 20 min. Cells were then acquired with the CytekThe study team based at the NCID research clinic will maintain the codes linking the blood samples to its donor. Subjected to participant consent, any de-identified leftover blood samples may also be analyzed for exploratory research to find new scientific information about coronaviruses and related diseases, which may occur locally or overseas.Data of AEs, SAEs, grade 3 and 4 clinical or laboratory AEs will be summarized by the study arm with frequency and proportion of participants having the event, as well as the number of events. For SAEs and grade 3 and 4 AEs, the proportion of participants with the events will be provided together with its 95% confidence interval (CI).Graphical plots will be produced for the various immunity endpoints to depict the change of immunity levels over time. Levels of SAS-CoV-2 anti-spike immunoglobulins will be summarized by study arm and by randomization stratification factors. Mean difference and its 95% confidence interval in levels of SAS-CoV-2 anti-spike immunoglobulins between an intervention arm and the control arm will be estimated from a general linear regression model which adjusts for level of SAS-CoV-2 anti-spike immunoglobulins at baseline, and randomization stratification factors. If the lower bound of the 95% CI falls above − 10% (the pre-specified non-inferiority margin), non-inferiority of the intervention arm will be concluded. Analysis of repeated measurements of immunity endpoints will be performed with the use of mixed effects models that adjust for baseline values and randomization stratification factors. Log-transformation of the data may be applied as needed before the analyses.Proportion of participants with SAE is at least 25% greater (in absolute difference) in the intervention arm compared with the control armProportion of participants with Grade 3 and 4 AEs is at least 25% greater (in absolute difference) in the intervention arm compared with the control armGeometric mean ratio of anti-SARS-CoV-2 between the intervention arm and the control arm falls below 0.60.Interim analyses will be performed for DSMB review after 10 participants from each of the intervention arms from 1 to 4 have completed assessments at study day 28. DSMB may recommend discontinuation of participant enrolment to a study arm if any of the following criteria is met:The above guidelines may be revised in the DSMB charter, which implies the stopping guidelines will take precedence should there be any difference in the guidelines between the protocol and the DSMB charter.Analysis will be stratified by age , time from 2nd vaccine dose administered , and primary vaccine series (BNT162b2 or mRNA-1273).Even if the participant did not receive the booster vaccination, collected samples will still be analyzed up until the point the participant withdraws consent. Participants who drop out of the study will not be replaced.Datasets analyzed during the study will be available from the corresponding author on reasonable request.This study is led by NCID’s Singapore Infectious Disease Clinical Research Network (SCRN) and supported by Singapore Clinical Research Institute (SCRI). SCRN is responsible for project management, subject recruitment and data entry. SCRI is responsible for project and data management, as well as regulatory compliance/ study monitoring.An independent DSMB has been established to monitor the study. The DSMB will evaluate the study design and protocol, and review the accumulative data of safety and immunogenicity, as well as subject enrolment, protocol deviations, and data quality. The DSMB will meet after 10 participants in each intervention group have completed assessments at study day 28. Since participant recruitment may start at different time points for intervention groups 1 to 4 depending on vaccine availability and local regulatory approval, separate DSMB meetings may be needed for data review of intervention groups 1 to 4.A DSMB Charter will be developed describing the scope of data to be reviewed, memberships of DSMB, terms of reference, decision-making process, and timing and frequency of interim analyses (with specification of stopping guidelines). An interim analysis report will be prepared for each DSMB data review, which will include a summary data on participant enrolment, data quality, demographic and baseline characteristics, protocol deviation, safety data, and immunogenicity data. Following a review of interim data, DSMB may recommend discontinuation of participant enrolment to a study arm on the basis of safety concern and/or futility in immunogenicity compared with the control arm. The DSMB may request additional data review if necessary.Advent event reporting and harms are shown in Table Clinical site monitoring is conducted to ensure that the rights and well-being of trial subjects are protected and that the reported trial data are accurate, complete, and verifiable. Clinical monitoring also ensures the conduct of the trial is in compliance with the currently approved protocol/ amendment(s), ICH, GCP, and with applicable regulatory requirement(s) and sponsor requirement(s). Clinical monitoring will also verify that any critical study procedures are completed following specific instructions in the protocol-specific manual of procedures. Details of clinical site monitoring are documented in a clinical monitoring plan (CMP). The CMP describes in detail who will conduct the monitoring, at what frequency monitoring will be done, at what level of detail monitoring will be performed, and the distribution of monitoring reports. Monitoring visits will include, but are not limited to, review of regulatory files, accountability records, CRFs, ICFs, medical and laboratory reports, site study intervention storage records, training records, and protocol and GCP compliance.During the trial, any amendments to the protocol or consent materials will be approved by the Ethics Committee (NHG DSRB) before implementation. Participants will be informed in a timely manner of any new information that becomes available during the course of the study that may affect their willingness to continue study participation.Following completion of the study, results will be published in a medical/scientific journal. Preliminary results may be released by the sponsor to help inform policy-making decisions pertaining to COVID-19 vaccination booster regimens.PRIBIVAC is an adaptive, randomized, subject-blinded, controlled trial to assess the immunogenicity and safety of heterologous boost COVID-19 vaccination (intervention groups) compared with a homologous boost regimen (control group). The booster vaccine for the control arm will be the homologous mRNA vaccine , while for individuals randomized to intervention group 1 the mRNA booster vaccine administered will be heterologous to the primary series . The booster vaccine candidates for intervention groups 2–4 will be an alternative COVID-19 vaccine. New intervention groups may also be added depending on what COVID-19 vaccines are authorized for use in Singapore, and may include new vaccines or vaccines targeting SARS-CoV-2 variants.Since access to non-mRNA vaccine candidates is dependent on various factors outside the control of the study team availability of all vaccines at the outset of the study may be limited. Participants will be randomized at equal probability to the available intervention groups at the time of randomization. This will reduce the risk of bias  compared with a non-randomized design. While the ideal scenario is to have all the selected vaccines approved prior to the start of recruitment, due to the unknown time of availability of vaccine candidates A , B and C, and the urgency of the current situation , the study was initiated with the control and intervention group 1.rd or 4th booster dose.With the emergence of increasingly infectious VOCs such as the Omicron variant and the increasing frequency of vaccine breakthrough infections, an effective long-term COVID-19 immunization strategy is urgently needed. Currently, there are no COVID-19 vaccine booster trials to evaluate the efficacy of newly approved vaccines such as COVAXIN® against the primary mRNA series (BNT162b2 and mRNA-1273) as a booster dose to induce protective immunity against emerging VOCs. In addition, the effects of aging on the immune system result in elderly individuals not responding to immune challenges  as robustly as the young , thus loThe trial began recruiting participants in Oct 2021. Recruitment is expected to conclude by mid-2022, with the final participant visit by mid-2023.