Quantification of SARS-CoV-2 spike and nucleocapsid proteins using isotope dilution tandem mass spectrometry

The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.     

UspA2 is a cross-protective Moraxella catarrhalis vaccine antigen

Moraxella catarrhalis (Mcat) is a key pathogen associated with exacerbations of chronic obstructive pulmonary disease (COPD) in adults and playing a significant role in otitis media in children. A vaccine would help to reduce the morbidity and mortality associated with these diseases. UspA2 is an Mcat surface antigen considered earlier as vaccine candidate before the interest in this molecule vanished due to sequence variability. However, the observation that some conserved domains are the target of bactericidal antibodies prompted us to reconsider UspA2 as a potential vaccine antigen.We first determined its prevalence among the COPD patients from the AERIS study, as the prevalence of UspA2 in a COPD-restricted population had yet to be documented. The gene was found in all Mcat isolates either as UspA2 or UspA2H variant. The percentage of UspA2H variant was higher than in any report so far, reaching 51%. A potential link between the role of UspA2H in biofilm formation and this high prevalence is discussed.To study further UspA2 as a vaccine antigen, recombinant UspA2 molecules were designed and used in animal models and bactericidal assays. We showed that UspA2 is immunogenic and that UspA2 immunization clears Mcat pulmonary challenge in a mouse model. In a serum bactericidal assay, anti-UspA2 antibodies generated in mice, guinea pigs or rabbits were able to kill Mcat strains of various origins, including a subset of isolates from the AERIS study, cross-reacting with UspA2H and even UspA1, a closely related Mcat surface protein.In conclusion, UspA2 is a cross-reactive Mcat antigen presenting the characteristics of a vaccine candidate.     

Influenza vaccine effectiveness against influenza-associated hospitalization in children in Hong Kong, 2010–2020

BackgroundInfluenza virus infections can cause hospitalizations in children, and annual vaccination of children can provide protection against influenza.MethodsWe analyzed a test-negative design study with data spanning from 2010/11 through 2019/20 to evaluate influenza vaccine effectiveness (VE) against influenza hospitalization in children by age group, influenza type/subtype and time period within each season. We enrolled children admitted to hospital with acute febrile respiratory illnesses. Nasopharyngeal aspirates were tested by culture and/or RT-PCR to determine influenza status, and vaccination status was obtained by interviewing parents or legal guardians and was verified where possible. VE was estimated by conditional logistic regression model adjusting for sex, age and age-squared, matching on week.ResultsInfluenza seasons in Hong Kong are prolonged with influenza-associated hospitalizations occurring in almost every month of the year during the study period. Influenza vaccination was effective in preventing influenza-associated hospitalizations in children of all ages. Influenza VE was higher in younger children than in older children, and higher against hospitalization due to influenza A(H1N1)pdm09 than A(H3N2) and B.ConclusionsThe childhood influenza vaccination program in Hong Kong has prevented influenza-associated hospitalizations particularly in younger children. Our findings support the use of influenza vaccines in children as an effective approach to influenza control and prevention.     

Trends,patterns and psychological influences on COVID-19 vaccination intention: Findings from a large prospective community cohort study in England and Wales (Virus Watch)

BackgroundVaccination intention is key to the success of any vaccination programme, alongside vaccine availability and access. Public intention to take a COVID-19 vaccine is high in England and Wales compared to other countries, but vaccination rate disparities between ethnic, social and age groups has led to concern.MethodsOnline survey of prospective household community cohort study participants across England and Wales (Virus Watch). Vaccination intention was measured by individual participant responses to ‘Would you accept a COVID-19 vaccine if offered?’, collected in December 2020 and February 2021. Responses to a 13-item questionnaire collected in January 2021 were analysed using factor analysis to investigate psychological influences on vaccination intention.ResultsSurvey response rate was 56% (20,785/36,998) in December 2020 and 53% (20,590/38,727) in February 2021, with 14,880 adults reporting across both time points. In December 2020, 1,469 (10%) participants responded ‘No’ or ‘Unsure’. Of these people, 1,266 (86%) changed their mind and responded ‘Yes’ or ‘Already had a COVID-19 vaccine’ by February 2021. Vaccination intention increased across all ethnic groups and levels of social deprivation. Age was most strongly associated with vaccination intention, with 16–24-year-olds more likely to respond “Unsure” or “No” versus “Yes” than 65–74-year-olds in December 2020 (OR: 4.63, 95 %CI: 3.42, 6.27 & OR 7.17 95 %CI: 4.26, 12.07 respectively) and February 2021 (OR: 27.92 95 %CI: 13.79, 56.51 & OR 17.16 95 %CI: 4.12, 71.55). The association between ethnicity and vaccination intention weakened, but did not disappear, over time. Both vaccine- and illness-related psychological factors were shown to influence vaccination intention.ConclusionsFour in five adults (86%) who were reluctant or intending to refuse a COVID-19 vaccine in December 2020 had changed their mind in February 2021 and planned to accept, or had already accepted, a vaccine.     

Older adolescents and young adults willingness to receive the COVID-19 vaccine: Implications for informing public health strategies

ImportanceThe success in ending the COVID-19 pandemic rests partly on the mass uptake of the COVID-19 vaccine. Little work has been done to understand vaccine willingness among older adolescents and young adults. This is important since this age group may be less likely to adhere to public health guidelines.ObjectiveTo understand willingness of getting a vaccine and reasons for vaccine hesitancy among a sample of older adolescents and young adults.DesignData were from the Well-Being and Experiences study (The WE Study), a longitudinal community-based sample of older adolescents and young adults collected from Winnipeg, Manitoba, Canada from 2017 to 2020 (n = 664).SettingThe study setting was a community-based observational longitudinal study.ParticipantsParticipants for the study were aged 14 to 17 years old at baseline in 2016–17 (n = 1000). Data were also collected on one parent/caregiver. Waves 2 (n = 747) and 3 (n = 664) were collected in 2019 and 2020, respectively.ExposuresThe main exposures were sociodemographic factors, health conditions, COVID-19 knowledge, and adversity history.Main OutcomesThe main outcomes were COVID-19 vaccine willingness, hesitancy, and reasons for hesitancy.ResultsWillingness to get a COVID-19 vaccine was 65.4%. Willingness did not differ by age, sex, or mental health conditions, but did differ for other sociodemographic characteristics, physical health conditions, COVID-19 knowledge, practicing social/physical distancing, and adversity history. The most common reasons for not wanting a vaccine were related to safety, knowledge, and effectiveness. Sex differences were noted.Conclusions and RelevanceIncreasing uptake of the COVID-19 vaccine among older adolescents and young adults may rely on targeting individuals from households with lower income, financial burden, and adversity history, and generating public health messaging specifically aimed at vaccine safety, how it works to protect against illness, and why it is important to protect oneself against a COVID-19 infection.     

Older adults' vaccine hesitancy: Psychosocial factors associated with influenza,pneumococcal, and shingles vaccine uptake

Influenza, pneumococcal disease, and shingles (herpes zoster) are more prevalent in older people. These illnesses are preventable via vaccination, but uptake is low and decreasing. Little research has focused on understanding the psychosocial reasons behind older adults’ hesitancy towards different vaccines. A cross-sectional survey with 372 UK-based adults aged 65–92 years (M = 70.5) assessed awareness and uptake of the influenza, pneumococcal, and shingles vaccines. Participants provided health and socio-demographic data and completed two scales measuring the psychosocial factors associated with vaccination behaviour. Self-reported daily functioning, cognitive difficulties, and social support were also assessed. Participants were additionally given the opportunity to provide free text responses outlining up to three main reasons for their vaccination decisions. We found that considerably more participants had received the influenza vaccine in the last 12 months (83.6%), relative to having ever received the pneumococcal (60.2%) and shingles vaccines (58.9%). Participants were more aware of their eligibility for the influenza vaccine, and were more likely to have been offered it. Multivariate logistic regression analyses showed that a lower sense of collective responsibility independently predicted lack of uptake of all three vaccines. Greater calculation of disease and vaccination risk, and preference for natural immunity, also predicted not getting the influenza vaccine. For both the pneumococcal and shingles vaccines, concerns about profiteering further predicted lack of uptake. Analysis of the qualitative responses highlighted that participants vaccinated to protect their own health and that of others. Our findings suggest that interventions targeted towards older adults would benefit from being vaccine-specific and that they should emphasise disease risks and vaccine benefits for the individual, as well as the benefits of vaccination for the wider community. These findings can help inform intervention development aimed at increasing vaccination uptake in future.     

Attitudes towards influenza and uptake of the flu vaccine: A survey of pharmacy staff working in English hospitals

ObjectivesPharmacy staff working in hospitals are at risk of contracting and disseminating influenza. Previous research focuses on community pharmacists’ attitudes towards influenza and vaccination. This survey investigates the beliefs and attitudes of pharmacists and other pharmacy staff working in English Hospitals regarding influenza and the vaccine and how this relates to vaccine uptake.MethodsA self-administered survey was provided to pharmacy staff at three hospitals in the East Midlands of England. Job role, age and vaccination status (vaccinated, intended to be vaccinated, and not vaccinated) were collected alongside ratings of agreement with 20 statements regarding influenza and vaccination using a Likert scale.Results170 pharmacy staff responded; 50.6% had been vaccinated, 17.1% intended to be vaccinated and 32.4% were not vaccinated. Increasing age showed a significant (p = 0.017) positive correlation with increased vaccine uptake as did the beliefs that vaccination protects the individual from influenza (p = 0.049) and that vaccination should be mandatory for NHS staff (p = 0.006). Fear of needles and believing their immune system is strong enough to protect against influenza were negatively correlated with vaccine uptake (p = 0.016 and p = 0.010, respectively). Job role was also strongly correlated with vaccine uptake (p = 0.001), with those holding a pharmacy degree more likely to report being vaccinated or intending to be vaccinated compared to all other pharmacy staff groups.ConclusionsThis is the first survey to focus on vaccine behaviours of all pharmacy staff groups working in hospitals. Current uptake of the influenza vaccine may be increased through engagement of senior pharmacy colleagues and providing education on influenza, vaccines, and vaccination. Similar studies should be undertaken on a larger scale to fully interrogate the differences between pharmacy staff groups.     

RSV neutralization assays – Use in immune response assessment

Respiratory syncytial virus (RSV) is a leading cause of respiratory illness among children and infants worldwide, yet no licensed vaccine exists to reduce the risk of disease. At least 16 RSV vaccine candidates are currently in clinical development and many are designed to induce robust virus neutralizing immune responses. RSV neutralizing antibody (nAb)-mediated interventions such as intravenous immunoglobulin (IVIG) and palivizumab provide passive protection against serious lower respiratory tract disease due to RSV, validating nAbs as a correlate of protection. To identify correlates of protection for vaccine candidates that have demonstrated their protective efficacy, an investigator can use assays designed to measure nAb responses. However, there is no standard method of measurement; individual laboratories have developed their own methods to measure the ability of nAbs to reduce the infectivity of a defined virus dose in a variety of cell lines, leading to establishment of the broad variety of RSV neutralization assay formats currently in use.Standardizing the RSV neutralization assay is an essential step toward better assessment of nAb responses to vaccine candidates. Use of a common reference standard by all makes comparing the immunogenicity of different vaccine candidates feasible. In the context of vaccine development, the WHO 1^st International Standard for Antiserum to RSV (RSV IS) has been shown to be suitable for harmonizing results across laboratories and assay formats used to measure nAb titers to RSV/A and RSV/B in human sera.This review describes the broad variety of RSV virus neutralization assay formats currently in use and the importance of the RSV IS for harmonization of results across formats and across laboratories. It also outlines good practices for key assay components and data analysis to promote the quality and consistency of measuring RSV nAb titers in serum specimens.     

Tolerance of BNT162b2 mRNA COVI-19 vaccine in patients with a medical history of COVID-19 disease: A case control study

IntroductionStudies evaluating BNT162b2 mRNA Covid-19 vaccine safety excluded subjects with a previous history of COVID-19 infection. The aim of our study was to focus on the tolerance of this vaccine this population.MethodsAn anonymous self-reporting survey related to safety and tolerance of vaccine was completed by subjects 21 to 28 days after the first vaccine dose in two vaccination centers.ResultsSubjects with prior COVID-19 disease history (n = 61) had higher systemic reactions than subjects without any previous history (n = 1987) (45.9% vs 29.7%, p = 0.01). Asthenia, headache and fever were significantly more frequent in COVID-19 + group than negative group (25.6% vs 15.2% p = 0.045, 19.7% vs 9.3% p = 0.01, 6.5% vs 0.9% p = 0.003 respectively). Grade of severity was higher in COVID-19 + than in COVID-19 - group (p = 0.03).ConclusionOur study confirms a higher risk of side effects in patients with preexisting SARS-CoV-2 disease but with a good overall tolerance.