Adoption of non-pharmaceutical interventions (NPIs) remains critical to curtail the spread of COVID-19. Using self-reported adherence to NPIs in Canada, assessed through a national cross-sectional survey of 4498 respondents, we aimed to identify and characterize non-adopters of NPIs, evaluating their attitudes and behaviours to understand barriers and facilitators of adoption. A cluster analysis was used to group adopters separately from non-adopters of NPIs. Associations with sociodemographic factors, attitudes towards COVID-19 and the public health response were assessed using logistic regression models comparing non-adopters to adopters. Of the 4498 respondents, 994 (22%) were clustered as non-adopters. Sociodemographic factors significantly associated with the non-adoption cluster were: (1) being male, (2) age 18–34 years, (3) Albertans, (4) lower education level and (5) higher conservative political leaning. Participants who expressed low concern for COVID-19 and distrust towards several institutions had greater odds of being non-adopters. This information characterizes individuals at greatest odds for non-adoption of NPIs to inform targeted marketing interventions. The negative impacts of the COVID-19 pandemic have been extensive and broad reaching impacting healthcare, the economy and the general population globally6. In an effort to curtail the spread of SARS-CoV-2, medical and public health communities have worked together with governments to provide recommendations and develop policies for promoting uptake of public health behaviors ), including limiting social gatherings, physical distancing, using face masks, avoiding public spaces and quarantining when sick9.Over 1 year following the discovery of SARS-CoV-2 and with over 194 million cases and 4.1 million deaths globally as of July 202112. Using modelling, the number of cases in mainland China at the end of February 2020 were estimated to be 67-fold higher without NPIs than with NPIs8. A model was also used to estimate the effect of NPIs across 11 European countries between February and May 2020. This model estimated that the interventions were sufficient to drive the time-varying reproduction number below one and demonstrated that NPIs had a large effect on reducing transmission of COVID-1910. Unfortunately, these NPIs have also led to high economic, health and social costs globally16.The implementation of NPIs has coincided with a decline in the number of new COVID-19 cases among many different countries20. Prior studies have demonstrated higher adoption for NPIs among individuals who have higher concern about the harms of SARS-CoV-2, a greater knowledge of the pandemic, are older  has been typically associated with greater adherence and less conspiracy beliefs compared to people who use social media as an information source23.Distinct attitudes towards NPIs for COVID-19 have emerged with variation in the public’s agreement with and willingness to adopt these recommendations25, and the initiation of a mass roll-out strategy, many individuals are under false assumptions that NPIs are no longer needed or important in controlling the spread of COVID-19. However, experts are warning that as vaccinations are distributed, the public must continue to adhere to NPIs27. Public health messaging will be critical during vaccine roll-out to encourage adoption and continued adherence to NPIs26. Lack of information transparency and failure to customize information to different subpopulations have been cited as major factors contributing to communication ineffectiveness regarding emerging infectious diseases in the past and the COVID-19 pandemic thus far30. Therefore, identifying the characteristics of individuals who are most likely to be non-adopters is crucial to the development of effective communication strategies including; knowledge translation tools, targeted marketing programs, and community engagement.With the recent approval of several COVID-19 vaccinations across the globeWe aimed to characterize self-reported non-adopters of NPIs compared to people who reported following these behaviors. We wanted to understand the differences in sociodemographic factors, attitudes towards COVID-19 and risk and trust measures across these 2 groups. We also wanted to identify the informational and social media platforms they use to obtain their COVID-19 data. This information will help identify people at highest risk for non-adoption of NPIs to inform targeted marketing interventions, including optimal communication strategies and platforms, to encourage behaviour change among non-adopters and reduce spread of COVID-19.31 as they distribute broadly to their panel to promote quick responses. The proportion of individuals who completed the survey were similar in age, sex, ethnicity, highest level of education, and province of residence to those who did not complete the survey. Overall, 4498 participants completed the survey  clicked on the survey link, with 1395 (24% of respondents) excluded due to not completing and submitting the survey. A response rate of 40% is typical for the Angus Reid ForumUsing a cluster analysis, two distinct non-overlapping clusters were identified: 3504 (78%) were clustered as adopters and 994 (22%) were non-adopters , liberal  or very liberal  political leaning had lower odds of non-adoption compared to people reporting moderate/middle of the road political leaning. Conversely, compared with moderate/middle of the road, people reporting slightly conservative , conservative (aOR 4.12 95% CI 3.26–5.21) or very conservative  political views had greater odds of non-adoption  higher than respondents expressing concern about their friends or family becoming sick ; however, 36% of people who disagreed with this statement were of the age 18–34 years compared to 27% of people Adoption of NPIs were positively correlated with adoption of contact tracing and exposure notification apps, with respondents who had downloaded an app having lower odds of being non-adopters  than those who had not downloaded an app , mask in indoor public spaces , avoid public spaces  and stay home when sick  had significantly higher odds of non-adoption compared to people reporting no difference in their behaviors with messaging or advertising. Across all NPIs evaluated, people who reported not seeing any messaging or advertising had significantly higher odds of non-adoption . Whereas the greatest proportion of adopters reported that they had intended to follow the guideline but simply forgot 78/3504, 2%) as the reason for having not followed public health guidelines , health media briefings , television/radio  and physician/healthcare provider . People who reported using or trusting Google or other internet search engines and information from friends and family had higher odds of non-adoption. Whereas those that reported using or trusting public health websites, health media briefings, television/radio or their physician for COVID-19 information were negatively associated with non-adoption Fig. . People %, healthBased on self-reported adoption of recommended NPIs intended on slowing transmission of COVID-19 including physical distancing, masking in public spaces, avoiding crowded public spaces and staying home when feeling sick, we segmented the population into adopters and non-adopters of NPIs through cluster analysis. Sociodemographic factors associated with non-adoption were: (1) being male, (2) age 18–34 years, (3) Albertans, (4) lower education level, and 5) a higher conservative political leaning. Participants who expressed low concern for COVID-19 had greater odds of being non-adopters. Non-adoption was associated with greater distrust among several institutions including technology, professional services, healthcare and government. Respondents who reported that public health messaging has been unclear and inconsistent and those where messaging has made them less likely to adopt NPIs had greater odds of non-adoption  followed by Saskatchewan and Manitoba (40%). Other studies have also demonstrated that the higher the degree of trust in the health system, the greater the compliance with guidelines47. We identified a high degree of trust in healthcare among our respondents with 75% reporting they either trusted or completely trusted healthcare; however, there were greater odds of non-adoption in people expressing distrust in the healthcare system.A previous survey conducted in Canada at the end of April 2020 reported respondents from Ontario and Quebec as having the least amount of trust in the Canadian government48. The greatest proportion of people in the non-adopter cluster reported that they did not believe that the recommendations work as the reason for having not followed the public health guidelines. People who felt that COVID-19 restrictions were harming the economy had lower odds of adoption of NPIs. Prior research has found that there is a positive association between risk perception and economic threat perception, meaning that people that perceive a personal economic threat may as a result be less likely to adhere to guidelines49.Belief in the efficacy of NPIs has been found to be critical for compliance with NPI recommendations50. Social media misinformation and lack of well-designed education programs without community engagement can impact on compliance and acceptability of NPIs17. Mobilizing an effective public health response during a pandemic requires clear communication and trust51. Non-adopters were more likely to report that public health messaging has been unclear and inconsistent. As well, they were more likely to report that messaging has actually made them less likely to adopt NPIs. Our assessment of the trust people have in institutions . Informed consent was obtained, and participation was voluntary. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist was used to report our findings59.A cross-sectional survey  aged 18 years or older, (2) live in a Canadian province, (3) speak either English or French, and (4) have access to the internet. The survey was administered between October 27 and November 2, 2020, in both French and English by the Angus Reid Institute, a national, not-for-profit, research foundation. The survey was programed using Askia60 and launched using Platform One61. The survey was distributed to 14,887 potential participants who were randomly selected from the Angus Reid Forum of 70,000 individuals who are representative of the Canadian population31, in order to obtain a sample size of 4500. Sample size calculated for a 95% confidence interval with a margin of error of 3% for the adult population of Canada was 1068. However, in order to be able to stratify for equal representation of Alberta residents and residents of the other Canadian provinces combined we used a sample size of 4500 respondents. This sampling strategy was used to allow for comparison of two Canadian applications used to facilitate contact tracing, ABTraceTogether (a contact tracing application which is only available in Alberta) and COVID Alert (an exposure notification application available in eight provinces and the Northwest Territories).The Angus Reid Forum was used for selection of participantsThe main outcome measure was adoption of NPIs assessed by respondents answering the question; over the last few weeks, how often have you been performing each of the behaviors: (1) social/physical distancing-keeping at least 2 m from other people who are not in your social bubble, (2) wearing a mask in public indoor spaces when physical distancing is not possible, (3) avoiding places & activities where you would interact with a large number of people outside your household, and (4) staying home if you were sick with any symptoms, even mild ones. Adoption of these NPIs was assessed on a Likert scale of; all the time, most of the time, sometimes, rarely and never.Sociodemographic factors including sex, age, province of residence, household income, highest level of education, ethnicity, and political leaning were collected and categorized. Questions identifying attitudes towards COVID-19, public heath recommendations and reasons for non-adoption of recommendations were asked. Likert scales were used to assess agreement with statements on public health messaging and restrictions, how effective people believe public health recommendations are at reducing spread of COVID-19, effectiveness of public messaging thus far, and trust in specific institutions. Respondents’ usage and trust of information platforms and social media platforms were collected.Descriptive statistics (percentage frequencies) were calculated for all sociodemographic characteristics, attitudes toward COVID-19 and towards NPIs and adoption of NPIs. Categorical variables were compared using chi-squared tests. Respondents who had not completed all survey responses were excluded, therefore there were no missing data.Cluster analysis was used as a data-driven method to identify the most important and meaningful patterns in the survey. This analysis highlighted how different or similar individuals are in their attitudes toward NPIs such that empirical patterns can be summarized in an insightful and concise manner. The goal of cluster analysis is to estimate a limited number of clusters with the most similarity within clusters but most dissimilarity between clusters. Clustering was based on individuals’ attitudes and behaviors toward NPIs , this includes reported actions and opinions on effectiveness of NPIs and on clarity of public messaging about NPIs.A series of statistical tests were conducted to determine the clustering method and the optimal number of clusters to explain the empirical variations in the data. The Kmeans algorithm was used for cluster analysis to partition the dataset into two distinct non-overlapping clusters labeled as adopter and non-adopter clusters. Kmeans is an iterative algorithm that assigns observations or data points to a cluster with the objective to minimize the sum of squared distance between the data points and the cluster’s arithmetic mean of all the data points that belong to that cluster. The output of analysis in this part is a data-driven and detailed way to allocate each observation to their appropriate cluster and generate a clustering indicator variable (adopter and non-adopter cluster) to be used for further investigation.Logistic regression was used to calculate the odds ratio (OR) with 95% confidence interval (CI) for the non-adoption cluster compared to the adoption cluster as a reference. Logistic regression was used to identify risk factors for non-adoption of NPIs by sociodemographic factors, attitudes towards COVID-19, attitudes towards NPIs and towards public health messaging, and trust in specific institutions. Logistic regression was also used to identify communication channels and social media platforms at higher odds of being used and trusted by non-adopters. Backwards stepwise regression identified sociodemographic factors significantly associated with adoption clusters. These sociodemographic factors were included in multivariable regression models estimating the adjusted odds ratio (aOR) comparing adoption clusters with (1) attitudes towards COVID-19, (2) attitudes towards NPIs and (3) attitudes toward public health messaging. All P-values were two-tailed tests, and the statistical significance level was set at P < 0.05. All statistical analyses were performed using STATA version 15.1 .All experiments were performed in accordance with relevant guidelines and regulations.The study was approved by the University of Calgary Conjoint Health Research Ethics Board (REB20-1228).All authors give their consent to publication of this work.Supplementary Information.


Buteo buteo, a raptor with a history of human-induced setbacks. Using Resource-Area-Dependence Analysis (RADA), with which we estimated individuals' resource needs using 10-day radio-tracking sessions and the 1990s Land Cover Map of GB, and agent-based modelling, we predict that buzzards in our study area in lowland UK had fully recovered (to 2.2 ind km−2) by 1995. We also anticipate that the conversion of 30%, 60% and 90% of economically viable meadow into woodland would reduce buzzard abundance nonlinearly by 15%, 38% and 74%, respectively. The same approach used here could allow for cost-effective anticipation of other animals' population patterns in changing landscapes, thus helping to harmonize economy, landscape change and biodiversity.Harmonious coexistence between humans, other animals and ecosystem services they support is a complex issue, typically impacted by landscape change, which affects animal distribution and abundance. In the last 30 years, afforestation on grasslands across Great Britain has been increasing, motivated by socio-economic reasons and climate change mitigation. Beyond expected benefits, an obvious question is what are the consequences for wider biodiversity of this scale of landscape change. Here, we explore the impact of such change on the expanding population of common buzzards  The imp2, over the 25 years from 1990 to 2015, of woodland in the UK. This increase has come largely at the expense of grassland [One example of land use change that is both important in itself and an insightful model from which wider lessons can be drawn is the increase by 5236 kmrassland , motivatrassland . Recentlrassland . Howeverrassland ,14. As eButeo buteo is a medium-sized generalist raptor that in the UK has suffered periodic declines due to human intervention. Over 500 years ago, they were regarded as vermin and payments were made for their corpses. Then gamebird rearing and shooting at the end of the eighteenth century eradicated buzzards from much of eastern Britain. This decline was further exacerbated in the 1950s by the depletion of a key resource, rabbits Oryctolagus cuniculus, infected with the myxomatosis virus [The common buzzard is virus —this lasis virus . The buzis virus . The expWe built an agent-based model (ABM) in which virtual buzzards populated a land-cover map of the 1990s lowland UK. In this landscape, we also explored the consequences for the buzzard population of the gradual replacement of meadow fields of economically viable size  by woodland, for timber extraction and climate change mitigation ,18. Mode. 2 (a)T) and mark-resighting (M-R) buzzard counts were conducted [Individual buzzard home-ranges, resource use and reproduction were quantified in Dorset, southern UK. Between 1990 and 1995, 114 home-ranges were recorded from 72 radio-tagged buzzards. Nests were counted and transect (onducted . Each raonducted . Home-raonducted . Cores ionducted . In the onducted . After donducted . If one onducted . No sex-onducted . The lanonducted . (b)We followed a pattern-oriented modelling approach (POM) . The mod (i)Our primary purpose was to predict the distribution and abundance of common buzzards in the geographical space of the real landscape in lowland UK (i) in the 1990s, and (ii) in landscape change scenarios depicting a gradual replacement of meadow for woodland, motivated by socio-economic reasons and climate change mitigation. Our secondary purpose was to predict buzzard home-ranges and territories. (ii)Entities and state variables are presented in 2 of land-cover and 4 km2 of seawater) with grain size of 25 m × 25 m (the pixel size of LCMGB of 1990). Most of the land-covers occurring in lowland UK were present in the study area [The study area was 22 km × 6 km (128 km (iii) 1.The run starts with the unpopulated buzzard resource map, which is based on a translation of the LCMGB of 1990 using RADA. 2.my-roost in A virtual buzzard settles on an undefended woodland patch, which becomes its roosting site. It will defend this patch ( 3.rgr-dist). In finding one, it will fly to it. This will be the seed from where it will search the entire landscape patch.The virtual buzzard will then randomly choose a free rough-ground pixel within its rough-ground foraging distance ( 4.my-rgr).From the seed, the virtual buzzard begins an iterative search for its free neighbouring pixels of rough-ground, incorporating each into the territory (as  5.rgr-area) and moved back to the roosting site, or (ii) it has not met these requirements and hence left the area.Then, when no free rough-ground neighbouring pixels are left, the virtual buzzard will search for rough-ground pixels at the edges of the raster patch it is incorporating into its territory and check whether these have free rough-ground neighbours. If any of them has, the virtual buzzard will iteratively move to each edge pixel with a free rough-ground neighbour, set it as a new base and apply the iterative neighbour search described in 4. When the landscape patch under scrutiny has been fully incorporated into the territory, the virtual buzzard will move back to its roosting site and continue the search for additional free rough-ground patches (by repeating steps 3–5). This search will only stop when (i) the virtual buzzard has met its area requirements for rough-ground ( 6.mead-area and to mead-dist). It stops when (i) the meadow resource needs (mead-area) have been met or (ii) they have not been met and hence the virtual buzzard left the area. When the meadow resource needs have been met, the territory will have been fully formed. Another virtual buzzard will then arrive at a random woodland patch which is as yet undefended and try to establish a territory by following the exact same rules (steps 2–6).The search for meadow begins from the roosting site and happens in exactly the same way as that for rough-ground The main assumptions of our model were that: (i) the random selection of the woodland patch for roosting does not lead to different maximum distribution and abundance than some other form of colonization; (ii) the wild buzzard does not actively avoid any land-cover that does not contain a key resource; (iii) the key resource-containing categories of LCMGB of 1990 were perfectly mapped (an assumption of RADA); (iv) there is a proportional relationship between patch area and amount of accessible resource (an assumption of RADA); (v) wild buzzards defend only the patches that provide them with the minimum amount of the needed resources. (v)Parameter values, calibration intervals, steps and references are shown in  (vi)Maps of landscape change scenarios represented the rural economy gradually shifting from meadow to woodland, motivated by, inter alia, woodland's role in flood management, timber's value for construction and vegetarianism/veganism reducing the need for grassland to feed livestock –11. The  (vii)The model was assessed qualitatively and quantitatively with regard to producing virtual buzzards with home-ranges with size, shape and pattern of overlap similar to wild buzzards. Virtual and wild buzzard home-range cores and territories were plotted in a GIS to visually assess these three characteristics of home-range structure relevant to defence of resource patches. Virtual buzzard pairwise home-ranges overlaps, a proxy for their territorial behaviour and an emergent pattern in the model, were compared with those of wild buzzards using two-tailed tests . (viii)Local sensitivity analysis (LSA) and global sensitivity analysis (GSA) were performed. LSA used a modified version of the Morris screening method, which makes no assumptions about the model and uses individually randomized one-factor-at-a-time designs to assess the effects of changes in parameter values on outputs ,34. The  (ix)Predictions for maximum abundance were compared with estimates obtained via mark-resighting and radio-corrected distance-transects carried out in 1995–1996 . The fin. 3 (a)Home-ranges of virtual buzzards were similar to those of wild buzzards visually  and quanU-test, two-tailed, pairwise, involving overlaps between 114 wild and 276 virtual buzzards and null hypothesis that there was no difference between the two samples yielded: W = 24 548, p = 0.219). Thus, in addition to roosting on a woodland patch and defending the same key resources, virtual buzzards also shared space with neighbours similar to wild buzzards.The collective territorial pattern of virtual buzzards, which was emergent in the model, was also similar to that of wild buzzards ( (b)The mean maximum abundance over 100 runs was about 7% and 10% larger than the mean abundance based on transect-counting or mark-resighting, respectively ( (c)Predictions for 30%, 60% and 90% of meadow fields (of greater than 20 ha) being converted into woodland were that the number of buzzard territories within the study region would decline nonlinearly by 15%, 38% and 74%, respectively . The rea (d)LSA showed the main parameter influencing abundance, and in an inverse way, was the individual's meadow area requirement . Rough-ground area also inversely influenced abundance, but much less strongly. Pairwise home-range core overlaps depended mainly and inversely on how far a buzzard could search for meadow, though overall variability was less than 7%. Home-range area and perimeter lengths of the inner and outer cores depended more strongly on how much meadow the individual required, and how far it would go to find it, the latter prevailing with regard to outermost core areas and perimeter lengths. We note that despite the small effect on outputs of rough-ground area and forage search distance, these parameters were kept because they refined results and made the model applicable to landscapes with greater variation in structure.. 4In the study area, the mean virtual buzzard abundance (100 runs) was between 7% and 10% larger than field estimates based on Transect-counting and Mark-resighting, respectively . A few iWhen the landscape change scenarios were considered, buzzard abundance in the study area decreased steeply and nonlinearly with the increased conversion of meadow patches of economically viable size (greater than 20 ha) into woodland . As expl (a)Accipiter gentilis [Vulpes cana [Crocuta crocuta [Panthera leo [Ursus americanus [. Naturally, animals following this strategy are particularly affected by changes to landscape structure, as exemplified here For distribution and abundance, uncertainty in prediction was associated mainly with (i) the estimation of meadow area requirement and (ii) how well the virtual buzzard's territorial behaviour represented that of the wild buzzard. The precision of the meadow area requirement depended on the applicability of the assumptions that the LCMGB of 1990 was fully accurate and of there being a proportional relationship between patch area and resource accessibility, both of which depended to a large extent on the quality of home-range estimation and mapping . In pracPredictions for the change scenarios did not consider possible functional relationships with other species . For exa (i)Resource selection functions (RSFs) seem to be the most popular method to predict animal distribution and abundance in the geographical space of a real landscape. An RSF can predict these population patterns from correlations between a wide variety of data about animal presence (e.g. spoor or GPS tracking) and for resources or conditions  ,49. ImpoSciurus vulgaris and S. carolinensis [Haematopus ostralegus, UK [Alauda arvensis in Denmark [Canis lupus in the Italian alps [Loxodonta africana in the Kenya–Tanzania border [Panthera tigris in Nepal's Chitwan National Park [ABM offers greater flexibility by allowing for the explicit representation of an ecological mechanism that connects animal space use with landscape structure—the procurement by individual organisms of the resources needed to survive and breed ,44,50,51linensis , or fourlinensis . Alternaegus, UK , skylark Denmark  and rive Denmark , sometimian alps , Africana border  and tigenal Park .Caprimulgus europaeus that weighs around 60 g [The strength of the ABM approach we applied to buzzards comes from having estimated the virtual animals' resource needs from wild animals using RADA, which relies on remote animal tracking and mapping of the resources. Such use of remote sensing to assess individuals' resource requirements has the potential to allow for more efficient estimation of the ABM parameters, and to considerably increase the range of species that can be simulated –62. For und 60 g . In turnund 60 g ,65, whilund 60 g ,66–70. Iund 60 g . Additiound 60 g , thus faund 60 g . Indeed,und 60 g .Our results have shown that replacing meadow with trees is likely to reduce the buzzard density in the area we studied. We can apply the model to other areas of the UK if land managers want to consider the wider effects of planting trees on a prominent and protected species, or develop similar models of other species tracked in appropriately mapped areas. Anticipating that a crucial population will prosper, or an agricultural pest collapse, can help harmonize economic development with animal conservation and ecosystem service provision. We thus hope that the breadth, efficiency and simplicity of the modelling approach used here may contribute to addressing the more general conservation paradox that ‘we are not limited by lack of knowledge but failure to synthesize and distribute what we know’ .Click here for additional data file.


We assessed the performance of CoronaCHEK lateral flow assay on samples from Uganda and Baltimore to determine the impact of geographic origin on assay performance.Serum samples from SARS-CoV-2 PCR+ individuals  and from pre-pandemic individuals  were evaluated. Prevalence ratios (PR) were calculated to identify factors associated with a false-positive test.After first positive PCR in Ugandan samples the sensitivity was: 45%  at 0-7 days; 79%  8-14 days; and 76%  >15 days. In samples from Baltimore, sensitivity was: 39%  0-7 days; 86%  8-14 days; and 100%  15 days post positive PCR. The specificity of 96.5%  in Ugandan samples was significantly lower than samples from Baltimore 99.3% , p<0.01. In Ugandan samples, individuals with a false positive result were more likely to be male  or individuals who had a fever more than a month prior to sample acquisition .Sensitivity of the CoronaCHEK was similar in samples from Uganda and Baltimore. The specificity was significantly lower in Ugandan samples than in Baltimore samples. False positive results in Ugandan samples appear to correlate with a recent history of a febrile illness, potentially indicative of a cross-reactive immune response in individuals from East Africa. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID-19) . Many stSerologic assays used for the detection of antibodies to different viral infections can vary in performance based on the origin of the samples being tested, as has been seen in HIV , HCV 1010, and HThe use of samples from Baltimore was approved by The Johns Hopkins University School of Medicine Institutional Review Board . The use of samples from Uganda was approved by the Uganda Virus Research Institute’s Research Ethics Committee , Western Institutional Review Board, protocol 200313317 and the Uganda National Council for Science and Technology (HS637ES). The parent studies were conducted according to the ethical standards of the Helsinki Declaration of the World Medical Association, where all subjects provided written informed consent. All samples were de-identified prior to testing.To assess sensitivity, samples from subjects known to be SARS-CoV-2 PCR+ from Uganda and the United States with known duration from first PCR+ date were evaluated. Samples from 78 PCR+ individuals at different time intervals were identified at the Uganda Virus Research Institute in Entebbe, and Makerere University in Kampala, Uganda. None of the Ugandan individuals were hospitalized and all had mild disease. Samples (n=266) from the United States were from 38 hospitalized COVID-19 patients, attending the Johns Hopkins Hospital in Baltimore, Maryland in the United States .To assess the specificity of the assay, pre-pandemic samples were tested. This included 1077 stored samples from the Rakai Community Cohort Study, collected between 2011 and 2013 . The UgaAll samples were analyzed with the CoronaCHEK LFA (Hangzhou Biotest Biotech Co Ltd) according to the manufacturer’s protocol. Sensitivity by duration of infection and specificity among pre-pandemic samples were assessed for the presence of either IgM or IgG bands for any reactivity. Statistical analysis was performed with STATA 14.2 , and 95% confidence intervals (95% CI) for sensitivity and specificity were calculated with the Clopper-Pearson exact method. Bivariate Poisson regression models were used to calculate prevalence ratios (PR) for factors associated with a false-positive test among pre-pandemic samples.There were significant differences in the performance for the CoronaCHEK LFA between samples from Uganda and Baltimore . When coThere were four and 38 false positive results in Baltimore pre-pandemic samples and Ugandan samples, respectively. All four from Baltimore were all faint IgM bands while 82% (31/38) of the false positive samples from Uganda had only reactive IgM bands. Of the seven pre-pandemic Ugandan samples that were IgG reactive, two were also reactive for IgM. Ugandan samples were significantly more likely to misclassify if they came from men  or the individual had reported fever more than a month prior to sample collection . There was a trend to test positive if they had reported pneumonia-like symptoms . Other factors not associated with a false positive result included age, community type, and HIV status . There wThis study demonstrates differential performance of the CoronaCHEK LFA on samples collected from Uganda compared to those collected from Baltimore. Though sensitivity for both IgG and IgM in samples from Baltimore was 100% by 14 days after the subjects first PCR+ date, unlike samples from Uganda, this difference was not significantly different. Specificity was significantly lower in the Ugandan pre-pandemic samples compared to those from Baltimore, though this difference was all associated with the IgM band. False positive results in Ugandan samples were higher among men and those who had reported a febrile episode more than a month prior to sample acquisition. Of the false positive results detected, the vast majority were IgM reactivity.These results demonstrate that the performance characteristics of serological assays for SARS-CoV-2 antibody detection cannot be extrapolated to different populations without adequate validation studies. This study supports the need for validation studies on SARS-CoV-2 serologic assays in Africa, an area where little data exists . Though There are a number of limitations of our study. First, the samples from Uganda of SARS-CoV-2 infected patients were limited, with only six samples within the first week post first PCR positive test and no serial samples for a given individual. Additionally, these samples from known infected Ugandan individuals had limited symptoms, while the Baltimore samples from known SARS-CoV-2 positive individuals were all hospitalized subjects. The pre-pandemic samples from Baltimore were not matched to those from Uganda based on symptomology, though historically, individuals attending the ED in the United States have a high prevalence of fever and viral infections . SamplesIn summary, the geographical origin of the samples appeared to impact the performance of the CoronaCHEK LFA. IgM reactivity was the main cause for the false positive results. Given that IgM responses generally appear a couple days before IgG, it may be useful not to measure IgM at all in serological studies given the improvement in specificity. Further evaluations of serologic assays are needed to find appropriate tools for sero-surveillance in an African setting.


The concerning increase in HIV-1 resistance argues for prioritizing the development of host-targeting antiviral drugs because such drugs can offer high genetic barriers to the selection of drug-resistant viral variants. Targeting host proteins could also yield drugs that act on viral life cycle events that have proven elusive to inhibition, such as intracellular events of HIV-1 immature capsid assembly. Here, we review small molecule inhibitors identified primarily through HIV-1 self-assembly screens and describe how all act either narrowly post-entry or broadly on early and late events of the HIV-1 life cycle. We propose that a different screening approach could identify compounds that specifically inhibit HIV-1 Gag assembly, as was observed when a potent rabies virus inhibitor was identified using a host-catalyzed rabies assembly screen. As an example of this possibility, we discuss an antiretroviral small molecule recently identified using a screen that recapitulates the host-catalyzed HIV-1 capsid assembly pathway. This chemotype potently blocks HIV-1 replication in T cells by specifically inhibiting immature HIV-1 capsid assembly but fails to select for resistant viral variants over 37 passages, suggesting a host protein target. Development of such small molecules could yield novel host-targeting antiretroviral drugs and provide insight into chronic diseases resulting from dysregulation of host machinery targeted by these drugs. Currently, over 25 million of the 38 million people living with AIDS worldwide are being treated with antiretroviral therapy (ART), with the result being a 60% reduction in AIDS-related deaths relative to the peak of the AIDS pandemic in 2004 . HoweverTo date, 28 medicines have been FDA-approved for the treatment of HIV disease, of which 20 are ARV small molecules that act by seven different mechanisms to inhibit specific steps in the viral life cycle . NineteOf the 20 ARV small molecules only one is a host-targeting antiviral agent (HTA)—maraviroc, which targets the entry coreceptor CCR5 (reviewed in ). While Notably, while the high genetic barrier to resistance provides a strong argument in favor of development of HTAs, there are also potential disadvantages to HTAs. Most importantly, host targeting raises concerns of side effects since the targeted host protein could be important for host functions. However, depending on their exact mechanisms of action, host-targeting drugs can be very safe, for example if the host is able to use other proteins or pathways in place of the drug target, if the HTA binds to its host target in a manner that allows the target protein to be used by the host but not by the virus, or if the virus alters the target so that it cannot be used by the host until rescued by the drug and returned to its functional state. The latter mechanism has been reported for a host-targeting drug that inhibits multiple respiratory viruses in a recent bioRxiv preprint , althougExamples of HTAs that have serious side effects and those that are very well tolerated are found among drugs that inhibit the chemokine co-receptors (CXCR4 and CCR5) required for HIV-1 entry into cells (reviewed in ). The CXHost-targeting antiretroviral compounds would be of particular interest for stages of the life cycle that have not yet been successfully targeted. The HIV-1 life cycle can be broadly divided into the early events, which encompass initial infection of a target cell through proviral DNA integration, and late events, which encompass viral transcription through production of infectious virus particles . The 20 pol gene) to yield four component proteins—matrix (MA), capsid (CA), nucleocapsid (NC) and p6  are widely accepted. HIV-1 Gag assembly is initiated in the cytoplasm, when a dimer or oligomer of Gag associates with unspliced HIV-1 genomic RNA ,24. Thisiewed in ). Rearraiewed in ).specifically inhibit assembly of immature capsids, leading to reduced virus production, but such compounds had not been identified as of 2019. One reason for the lack of potent and specific inhibitors of immature capsid assembly could be that assembly is able to proceed when a drug is bound to a few Gag subunits, while the later maturation and post-entry events are less tolerant of drug binding. Alternatively, it is possible that screens have missed promising small molecule inhibitors of immature capsid assembly, perhaps because the screens that were used did not recapitulate events of immature capsid assembly and focused instead either too narrowly on one aspect of assembly or too broadly. Support for this latter hypothesis is provided by the identification by our group in 2020 of a potent and specific inhibitor of HIV-1 immature capsid assembly using a novel screening approach that reconstituted the entire pathway of HIV-1 immature capsid assembly, including both Gag-Gag interactions and Gag-host interactions [Given the underlying molecular biology, one might expect HIV-1 immature capsid assembly to be a particularly good target for inhibition by small molecules. Structures composed of multimers, like capsids, should be highly susceptible to inhibition, since an inhibitor need bind only a few of the thousands of Gag subunits in the immature capsid to disrupt capsid formation and/or function . Consistractions , as discThe working model one uses to study a stage of the viral life cycle influences the design of drugs screens used to identify inhibitors, which in turn results in some inhibitors being identified while other promising inhibitors are missed—thus the starting model matters. For decades the dominant model for understanding Gag multimerization has been the self-assembly model, which proposes that Gag polypeptides multimerize spontaneously in the presence of nucleic acids due to intrinsic properties that promote Gag-RNA and Gag-Gag interactions . Data in support of host-catalyzed Gag assembly was first generated when, in an attempt to better approximate HIV-1 infection in vivo, assembly of nascent Gag polypeptides was studied in the context of host proteins, using cell extracts (a cell-free system). Unlike self-assembly studies, which examine recombinant Gag at high concentrations in the complete absence of host proteins ,36, the The mechanisms by which ABCE1 and DDX6 promote Gag assembly have not been fully elucidated. However, in the case of DDX6, clues come from the known role of RNA helicases in RNP remodeling, a process by which proteins that are associated with RNAs and dictate RNA fates are replaced by other proteins —thus, DDDespite fundamental differences between the host-catalyzed and spontaneous models of Gag assembly, there are many ways in which the host-catalyzed model complements the self-assembly model. For example, the self-assembly studies show that RNA plays a key role in promoting Gag-Gag interactions . HoweverSimilarly, data showing that HIV-1 utilizes host ATPases to facilitate virus assembly ,46, therIn a recent paper Deng et al. argue against the existence of the stepwise pathway for HIV-1 assembly , but forA second reason why Deng et al. may have failed to detect assembly intermediates relates to their use of antibody-mediated detection of Gag epitopes to assess the multimerization state of Gag-containing complexes. This technique is problematic since it is well known that Gag epitopes become inaccessible as Gag multimerizes . Given tFinally, because Deng et al. find that the assembly-defective G2A Gag mutant forms a complex that is similar in size to our ~500S assembly intermediate, they argue that the ~500S complex formed by WT Gag must not be a late assembly intermediate. However, multiple groups have shown that non-myristoylated G2A Gag can overcome the assembly block, multimerize, and produce virus like particles in the cytoplasm ,59,60, mThus, while many aspects of the assembly pathway we have proposed remain to be understood, the study by Deng et al. did not employ approaches that would have allowed them to make conclusions about HIV-1 assembly intermediates and therefore sheds little light on the stepwise pathway of HIV-1 capsid assembly. Those concerns having been noted, probably the best approach to validating any viral life cycle model is to use that model to identify novel classes of inhibitors that are predicted by the model and can also be used to further understand the model.Until now, two screening approaches have been used to identify small molecules that inhibit viral late events and/or bind to CA: (1) hypothesis-neutral cell-based replication assays; and (2) in vitro reactions that assay self-assembly of recombinant CA peptides or CA-NC peptides (the latter encompasses CA and the adjacent NC domain of Gag). As HIV-1 capsid inhibitors have been reviewed relatively recently , here weA full viral replication screen yielded the CA-binding small molecule PF74 . PF74 dMost other screens for assembly inhibitors have involved self-assembly assays, which largely focus on self-assembly of recombinant CA, in part because of the critical role played by CA during immature and mature capsid assembly as well as during capsid disassembly. The CA domain of the Gag polyprotein contains residues critical for Gag-Gag interactions that mediate multimerization during assembly of the immature capsid. Subsequently, after Gag cleavage, CA is a key component in assembly of the mature capsid. After target cell entry, CA disassembly is required for reverse transcription, trafficking of the pre-integration complex to the nucleus, and integration (reviewed in ). CA conA CA self-assembly assay was used to validate the first CA-targeting small molecule, CAP-1, which was initially identified in silico as a potential CA-binding compound . CAP-1 rOther CA-binding small molecules have also been identified using variations of the self-assembly assay. For example, ebselen, an organoselenium compound, was identified using a high throughput screen that monitored recombinant CA-CTD peptide dimerization (the first step of self-assembly) using time-resolved fluorescence resonance energy transfer . EbselenMore recently, self-assembly screens yielded the most promising CA-binding small molecule identified to date, GS-6207 . Both GSWhile numerous experimental approaches have validated the host-catalyzed assembly model in cells, aspects of the model have been difficult to completely prove using currently available approaches. For example, since the assembly intermediates are highly transient and labile multiprotein complexes that are present in minute quantities, purifying them and cataloguing their components by mass spectrometry has been challenging. Likewise, the host components in these complexes have not been visualized during live imaging of virus assembly in cells since tagging multiple host proteins with fluorophores is much more difficult and problematic than tagging multiple viral proteins. Similarly, knockdowns that produce log-fold effects are unlikely when numerous host enzymes are involved in facilitating assembly, each contributing only modestly and likely with redundancy. However, ultimately the goal of understanding steps in the viral life cycle is to find novel approaches to antiviral therapeutics; thus, one way to bypass the technical issues that have limited acceptance of the host-catalyzed assembly model is to simply ask if screens based on this model yield successful assembly inhibitors.The first evidence that establishing screens based on host-catalyzed capsid assembly could be effective came from a study that identified the first potent small molecule inhibitor of rabies virus (RABV) replication in cell culture . In thisThe cell-free system in which the host-catalyzed HIV-1 assembly pathway was first identified was also used to develop an HIV-1 capsid assembly plate screen , analogoAnalysis of compounds from the master hit collection in cell culture assays identified a chemotype that was used for analog development, resulting in PAV-206, a small molecule that inhibits replication in an HIV-1 infected MT-2 T cell line and in HIV-1 infected human peripheral blood PBMCs with EC50s of 34 nM and 75 nM, respectively  found that the PAV-206 analog displays a dose-dependent colocalization with Gag in HIV-1-expressing cells, while a biotinylated control compound that lacks antiretroviral activity does not colocalize with Gag  develop during that time . This coThe direct-binding target of PAV-206 and its analogs has not yet been identified, and much remains to be done before an analog of PAV-206 can be advanced to a preclinical stage. Nevertheless, there are important lessons in the discovery of this potent and specific small molecule inhibitor of Gag assembly. First, the findings show that a screen recapitulating the host-catalyzed HIV-1 assembly pathway led to the first potent and selective inhibitor of Gag assembly, after 15 years of screens based on CA and Gag self-assembly, indicating that screens that more closely recapitulate the cell biology of HIV-1 assembly can identify molecules missed by other screens. Second, the conclusion that the PAV-206 appears to target a host protein argues that host-targeting inhibitors of HIV-1 assembly could be highly successful. Third, identification of PAV-206 suggests that development of ARV drugs that will not select for resistant viral variants is an achievable goal—although it may require a shift in the paradigm that is used for target identification and antiviral drug development as discussed below. Finally, the success of this screen in discovering a potent assembly inhibitor is an important validation of the host-catalyzed model of HIV-1 capsid assembly model, since an inhibitor of this pathway would only be expected to block virus production if the pathway is indeed critical for virus assembly as has been proposed ,45,46,49Antiviral drugs that bind to viral proteins constitute the low-hanging fruit of antiviral drug development, attractive on many fronts. However, the best viral targets have been the subject of numerous screens over the past three decades and could be nearly fully tapped from a small molecule perspective. This leaves host-targeted therapies as the approach that is most likely to yield big gains in the future. While more complex and requiring novel approaches, host-targeting drugs are likely to yield the exciting reward of high genetic barriers that will greatly extend drug utility . The ideGiven the increasing dependence on ARV drugs not just for treatment but also for prevention ,85, the Assembly pathway screens have two added advantages. First, they might identify small molecules that inhibit replication of unrelated viruses if those viruses use common host machinery during assembly . Secondl


Introduction: The COVID-19 pandemic has greatly impacted people's life across the globe. In a public health crisis, rural adolescents are more prone to mental health problems. The current study aimed to investigate the prevalence of depressive symptoms among Chinese rural adolescents during the COVID-19 outbreak, and examine the association between perceived social support and depressive symptoms and its underlying mechanisms.Method: Perceived Social Support Scale, UCLA Loneliness Scale, Patient Health Questionnaire-9 were administrated to 826 rural adolescents from Anhui Province, China, amid the COVID-19 crisis. Mackinnon's four-step procedure was employed to examine the mediating effect, while Hayes PROCESS macro was utilized to test the moderated mediation model.Results: The results showed the rate of depressive symptoms among rural adolescents in China was 77.6% during the outbreak of COVID-19. Female left-behind students and non-left-behind students from disrupted families experienced more depressive symptoms . Loneliness mediated the association between perceive social support and depressive symptoms and the indirect effect was stronger in left-behind adolescents in comparison to non-left-behind adolescents during the COVID-19 pandemic.Conclusion: Depressive symptoms are extremely prevalent among Chinese rural adolescents during the COVID-19 outbreak, and perceived social support plays a protective role against depressive symptoms. Chinese rural adolescents, especially left-behind students, could benefit from the interventions aimed at enhancing the perceived social support and reducing loneliness during the COVID-19 pandemic. The outbreak of COVID-19 has brought great challenges for both physical and mental healthcare around the world and disrupted daily life for everyone . As of JPerceived social support refers to an individual's cognitive appraisal of the quality and quantity of social connections. Evidence suggested that social and community ties played a critical role in the etiology of disease through the underlying psychological and physiologic mechanisms . There iLoneliness, also termed perceived social isolation , is a coAlthough perceived social support could influence loneliness among adolescents, not all adolescents with lower levels of perceived social support report higher levels of loneliness. Therefore, it is of great importance to explore the influential factor that might moderate the relationship between social support and loneliness. Over the last three decades, the rapid urbanization of China has led to a large scale of domestic migration . Due to To date, there is an absence of the studies on the potential mechanisms underlying the association between social support and depressive symptoms among rural Chinese adolescents during COVID-19 pandemic. Therefore, the present study aimed to investigate (a) the prevalence of depressive symptoms among rural adolescents during COVID-19 pandemic, (b) whether perceived social support is negatively associated with depressive symptoms, (c) whether loneliness mediates the association between perceived social support and depressive symptoms, and (d) whether the path between perceived social support and loneliness differs between left-behind students and non-left-behind students in rural China during COVID-19 epidemic. A moderated mediation model see  is constThe cross-sectional study was conducted from 28 April to 1 May, 2021 in rural regions of Anhui province, China, which is a one of the principal migrant-sending areas of China . A randoThis research was approved by the Research Ethics Commission of Navy Medical University. The participants aged 18 years or the parent or legal guardian of the participants aged under 18 years provided written informed consent before the participants anonymously completed the survey in the classroom. All participants were free to withdraw from the study at any time.The socio-demographic variables included age (≤ 16 or >16), sex , parental highest educational attainment (junior middle school or below and high school or above), family structure (intact family and disrupted family), perceived socioeconomic status (blow average and average or above) , 27, onlThe Chinese version of Perceived Social Support Scale (PSSS) is a 12-item self-report measure assessing the perception of social support from three domains : family The 20-item UCLA Loneliness Scale was used to evaluate the feelings of loneliness. Subjects were asked to rate each item on a 4-point Likert-type scale from “0” (I never feel this way) to “3” (I often feel this way). All 20 items were summed up to create a total score ranging from 0 to 60, with higher scores indicating higher levels of loneliness. The measure has been widely used among Chinese adolescents with demonstrated psychometric properties . In the Depressive symptoms were measured by Patient Health Questionnaire-9 (PHQ-9). Each item is answered on a 4-point Likert scale ranging from “0” (none) to “3” . The range for the scale is between 0 and 27, with a higher score denoting greater depressive symptoms. The cutoff score for detecting depressive symptoms was 5 , 36. TheFirstly, common method bias was examined using Harman single factor test and descriptive analyses were calculated to describe the sociodemographic characteristics stratified by left-behind status. Secondly, linear regressions were performed to calculate the univariate association between demographic variables and greater depressive symptoms stratified by left-behind status and Pearson's correlation analyses were conducted to investigate the bivariate correlations between the variables of interest. Thirdly, the mediation effect was tested by MacKinnon's four-step procedure . Four spp < 0.05 (two-tails).Age, sex, parental highest educational attainment, perceived family economic status, family structure and only-child status were included in all models as potential confounders. We standardized all variables before modeling. Statistical analyses were conducted using SPSS version 26. Statistical significance was considered as p < 0.001), which indicated the research data were suitable for factor analysis. There were seven values with eigenvalue more than 1 and the first factor presented a variance of 23.786%, which did not reach the criterion of 40%. Therefore, the results suggested common method bias was not a serious problem in the study.Given that the research data were obtained by self-report using an online questionnaire, Harman single factor was employed to examine whether common method bias would be a potential validity threat , 41. TheThe prevalence of depressive symptoms was 77.6% among Chinese rural students during COVID-19 pandemic. The prevalence of depressive symptoms was 77.7% among left-behind students, while the rate of depressive symptoms was 77.4% among non-left-behind students. The results of the univariate logistic regression see  showed tP < 0.001). Loneliness was significantly and negatively related to depression (P < 0.001).P < 0.001) (see Model 1 in P < 0.001) (see Model 2 in P < 0.001) , indicating the effect of perceived social support on loneliness was moderated by left-behind status , while for non-left-behind students, the association between perceived social support and loneliness was still significant, but much weaker . For descriptive purposes, the current study plotted the association of perceived social support with loneliness, separately for left-behind and non-left-behind adolescents  during COVID-19 pandemic , 43. In Our results presented that females reported higher levels of depressive symptoms only among left-behind students. According to ABC model, the sex differences of depression could be attributed to the affective , biological  and cognitive  factors , 48. EviAs hypothesized, loneliness partially mediated the association between perceived social support and depressive symptoms among rural adolescents during COVID-19 pandemic, which was consistent with the previous literature , 18, 55.The current study has profound implications both theoretically and practically. In theory, our findings shed insight into the underlying mechanisms linking perceived social support to depressive symptoms among left-behind adolescents during the COVID-19 outbreak. In practice, our results could inform healthcare professionals to develop more targeted interventions for preventing depressive symptoms in rural adolescents amid the COVID-19 crisis. Programs to enhance perceived social support may be an important aspect of depression prevention for rural students. Moreover, interventions for alleviating loneliness, such as mediation, mindfulness and social cognitive skills training , should Several limitations need to be noted when evaluating the findings of our study. Firstly, the study design of the current research is cross-sectional, indicating the causality could not be established since the data only represent a given point in time. Hence, further longitudinal studies should be conducted to validate the results. Secondly, self-reported measurements were employed in the study, which might result in self-report biases. Further research could collect data from multi-informants . Thirdly, students in our study sample were only from Anhui province in China, which might limit the generalization of our results. Currently, our results could generalize to rural students in Anhui province. In the future studies, multicenter approaches are recommended to recruit participants. Finally, depressive symptoms of students in rural China could be influenced by many other variables. The model in the present study could only explain a part of the variance. Therefore, further research is suggested to incorporate more factors for a more comprehensive understanding of depressive symptoms among rural students.As far as we know, this is the first study reporting the prevalence of depressive symptoms among rural adolescents during COVID-19 pandemic and investigating the potential mechanisms underlying the association between perceived social support and depression among this specific group. Depressive symptoms are prevalent in 77.6% of rural adolescent during the COVID-19 pandemic. Healthcare professionals and the government should pay special attention to rural adolescent, especially female left-behind students and non-left-behind students from disrupted families. Rural adolescents with lower levels of perceived social support, especially left-behind students, could benefit from the interventions aimed at enhancing the perceived social support and reducing loneliness.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Second Military Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.TH, YX, and XM contributed to the writing of this article and the statistical analysis and are the co-first authors. WC organized the whole study, including carrying out this study, putting forward the study, and were the corresponding authors. YL, JW, YC, and ZL contributed to the design and statistical analysis. All authors contributed for editing the article and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The unprecedented progress in addressing unmet needs in haemophilia care to date includes developing several novel therapies that rebalance haemostasis by restoring thrombin generation in patients with haemophilia A or B with and without inhibitors. These novel therapies are FVIII mimetics, antithrombin interference RNA therapy and several monoclonal antibodies directed against the tissue factor pathway inhibitor (anti-TFPI). In this review, we provide an update on the progress made in developing anti-TFPI therapie. Phase 1 data from the three anti-TFPI studies showed acceptable safety profiles, and currently, available phase 2 data are encouraging. While these data support these molecules' further development progression, there is uncertainty on several aspects of their evolution. Two of the three anti-TFPIs have shown drug-related thrombosis, with one study consequently terminated. None of the thrombotic events is predictable with current monitoring tools, and none correlate with known coagulation parameters. All three anti-TFPIs undergo target mediated drug disposition, which impacts the formulation of dosing regimen fo these therapies. They would require more frequent dosing than some of the extended half-life clotting factor products and antithrombin RNAi therapy. There is no assay to measure the TFPI as the physiological levels are very low, which makes monitoring the impact of the anti-TFPI a challenge. The anti-TFPIs have several advantages, including their bioavailability when administered subcutaneously, their stable pharmacokinetics and their ability to prevent bleeds in haemophilia A or B patients with and without inhibitors. Whether these advantages can be realized will depend on the outcome of the currently ongoing studies. Haemophilia is a rare X-linked inherited bleeding disorder characterised by remarkable management progress in the past several decades. Advances in novel haemophilia therapy development have primarily been driven by the efforts to address the many currently unmet needs in haemophilia care, which include the high disease and treatment burden –3, the nThe unprecedented progress in the development of NFRT in the last decade includes developing factor VIII (FVIII) mimetics, antithrombin RNA interference and several tissue factor pathway inhibitors (TFPI). Potential inhibitors of TFPI have included an aptamer and several monoclonal antibodies directed against this natural inhibitor of coagulation. While the anti-TFPI aptamer showed promise in the preclinical evaluations , 14, it The aptamer termination disappointment was followed by a switch to the development of monoclonal antibodies, of which three with anti-TFPI activities have been engineered. These are the befovacimab, concizumab and marstacimab. All three anti-TFPI molecules target the Kunitz-2 (K2) domain of the TFPI, with the befovacimab also targeting the Kunitz 1 (K1) TFPI domain. Under physiological conditions, the TFPI molecule binds activated Factor X (FXa) via its K2 domain to form the TFPI-FXa complex which inhibits FXa. The TF-FVIIa complex then binds the TFPI-FXa through the TFPI K1 domain to form the TFPI-FXa-TF-FVIIa quartenary complex with resultant inhibition of FVIIa. Therefore, binding of TFPI to the K2 is a rate limiting steps in this haemostasis inhibition process making development of anti-TFPI therapies directed against the TFPI K2 domain an attractive target for anti-TFPI therapy development.Studies on the evaluation of these novel molecules have been ongoing for the last 5 years. This review will summarise currently available clinical data on the development of anti-TFPI monoclonal antibodies. The pertinent characteristics of the anti-TFPI monoclonal antibodies and their progress in human studies are summarised in in vivo studies, befovacimab showed a pro-coagulant effect, reducing blood loss in mouse and non-human primate bleed models. These were associated with the absence of thrombosis  and Kunitz 2 (K2) domains of the TFPI . In the rombosis . The phao models .In the first-in-human studies, befovacimab was evaluated in a multicentre, open-label study in patients with severe HA or HB, with or without inhibitors . Safety,Clinicaltrial.org identifier NCT03597022) was a multiple-dose, dose-escalating study evaluating the safety of befovacimab in patients with haemophilia A or B, with or without inhibitors, in three dose cohorts  (The phase II befovacimab study ( 400 mg) . Eight pThree participants developed thromboses which were all in the central nervous system. Two of the thromboses were in the intermediate dose cohort  and one in the high dose cohort . None of the participants was having a bleeding event at the time of thrombosis development, and none received additional haemostatic agents. There was no evidence of haemolysis, platelet consumption, and other monitored laboratory parameters within normal limits. Further analysis revealed no correlation between the befovacimab dose, TFPI inhibition, efficacy and safety outcomes. The absence of specific laboratory findings or any differentiating PK/PD characteristics in patients experiencing serious adverse events raised concerns on the predictability of thrombosis during treatment and led to the sponsor's termination of the study.In vitro, concizumab demonstrated an affinity for both soluble and cell surface-bound TFPI  or subcutaneous dose  were administered to 28 healthy volunteers or 24 people with haemophilia . When giExplorer3 (NCT02490787) was a multinational, multicentre, randomised, double-blind, placebo-controlled, multiple-dose, dose-escalation, phase 1b trial, conducted at 18 sites in 13 countries . It evalTwo phase 2 studies were conducted to evaluate the safety of concizumab in haemophilia patients. Explorer 4 was a multicentre, randomised, open-label, controlled trial evaluating the efficacy and safety of prophylactic administration of concizumab in haemophilia A and B patients with inhibitors (NCT03196284), and Explorer 5 was conducted in haemophilia patients without inhibitors (NCT03196297) . The 53 The acceptable efficacy and safety profile from phase 2 studies led Novo Nordisk to initiate the explorer7 phase 3 clinical trial in October 2019 with concizumab in patients with haemophilia A or B with and without inhibitors. A parallel phase 3 trial in haemophilia A or B patients without inhibitors, explorer8, was initiated in November 2019. The two trials' objective was to establish the safety and efficacy of once-daily prophylactic subcutaneous concizumab delivered in a pen device to prevent bleeds. The recruitment target was 293 patients from 32 countries. The trials were paused in March 2020 due to non-fatal thrombotic events in three patients enrolled in the ongoing phase 3 programme. Novo Nordisk worked with relevant regulatory authorities and identified a new path forward for concizumab. When new mitigating safety measures and guidelines were put in place, the study hold was lifted, and there has been no new incidence since measures were in place.in vitro and in vivo in a concentration-dependent manner (In vitro thrombin generation assays (TGAs) and dilute prothrombin time (dPT) assays evaluating Marstacimab added to donor plasma from healthy normal volunteers and patients with haemophilia A, haemophilia B, and haemophilia A with inhibitors have all shown the ability of marstacimab to increase coagulation activity and promote haemostasis. Bleed control was demonstrated in severe injury mice models of haemophilia A and B  investigated the safety, efficacy, pharmacokinetics, and pharmacodynamics of marstacimab, in haemophilia A or B patients, with or without inhibitors. This was a multicentre study conducted at ten sites in three countries. Twenty-six participants were exposed to Marstacimab in four dose cohorts  . The posPreliminary efficacy was evaluated by comparing the study participant bleed rates in the 6 months before the study and then 6 months on study. The ABR was zero for most of the dose cohorts, and the reduction in the bleed rate was 80–96% across the four-dose cohorts. These data supported the progression of this study to phase 3 of marstacimab clinical development.in vitro to restore thrombin generation in animals deficient in FVIII or FIX with and without inhibitors  of TFPI. It has been shown hibitors . In the 10 mg/kg . This isThe phase 1 and phase 2 data reviewed indicates that progress in developing anti-TFPI therapies is promising, with two of these molecules progressing to phase 3 evaluation. The occurrence of thrombotic events should remind us that the safety of these novel therapies should come first, and the drugs should be thoroughly evaluated before they are accepted as a standard of care. Recent experience with development of FVIII mimetics indicate that thrombotic events can be mitigated by better pharmacokinetic understanding and appropriate dose adjustment.This gives optimism that an anti-TFPI with an acceptable profile may be identified in the currently ongoing clinical studies. If the currently evolving safety and efficacy data remain positive and acceptable; the anti-TFPI therapies may represent a significant advance in the care of patients with haemophilia A or B with and without inhibitors.JM was responsible for the conception, content research, data collection, analysis, and approval of this manuscript's final version.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mass vaccination is effective in reducing SARS-CoV-2 infections among vaccinated individuals. However, it remains unclear how effectively COVID-19 vaccines prevent people from spreading the virus to their close contacts. Using nationwide administrative datasets on SARS-CoV-2 infections, vaccination records, demographics, and unique household IDs, we conducted an observational cohort study to estimate the direct and indirect effectiveness of mRNA-based COVID-19 vaccines in reducing infections among vaccinated healthcare workers and their unvaccinated household members. Our estimates for adults imply indirect effectiveness of 39.1% (95% CI: −7.1% to 65.3%) two weeks and 39.0% (95% CI: 18.9% to 54.0%) eight weeks after the second dose. We find that the indirect effect of mRNA-based COVID-19 vaccines within households is smaller for unvaccinated children than for adults and statistically insignificant. Here, we show that mRNA-based COVID-19 vaccines are associated with a reduction in SARS-CoV-2 infections not only among vaccinated individuals but also among unvaccinated adult household members in a real-world setting. COVID-19 vaccines are effective in preventing disease, but the extent of protection against transmission is unclear. Here, the authors use data from a cohort of healthcare workers in Finland and show that vaccination is associated with reduced infection in their unvaccinated adult household contacts. Governments around the world are hoping to lift COVID-19 restrictions as vaccination coverage increases. However, with the limited supply of vaccines and the emergence of new virus variants, decision makers in public health are facing a constant need to update their vaccination and contact restriction strategies. There is currently no consensus about the potential benefits of including all children in mass vaccination programs and much uncertainty about the prospect of achieving herd immunity through vaccination.4. Furthermore, there is evidence that vaccinated individuals infected with SARS-CoV-2 have lower viral loads than unvaccinated infected individuals, even though this might have changed after the emergence of the Delta variant8. However, it remains unclear how effectively COVID-19 vaccines prevent people from becoming infected and spreading the virus to their close contacts, most notably to children and other household members, in real-world circumstances.COVID-19 vaccines have been shown to be effective in preventing symptomatic and asymptomatic disease among vaccinated adults, both in clinical trials and in nationwide mass vaccination settingsThis paper provides evidence about the direct and indirect effectiveness of COVID-19 vaccines in reducing infections among vaccinated individuals and their unvaccinated household members. Our analysis exploits the rollout of the mass vaccine program in a large cohort of healthcare workers in Finland, allowing us to estimate the indirect effects of vaccines in a large sample of household members with discordant vaccination status.We used national databases that record all polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infections and mRNA-based (BNT162b2 by Pfizer-BioNTech or mRNA-1273 by Moderna) vaccine doses administered in Finland up to the beginning of the mass vaccination program among the general working-age population  on April 25, 2021. These data were merged with administrative full-population datasets that include information on each person’s occupation and unique identifiers for partners and children living in the same household.Here, we show that mRNA-based COVID-19 vaccines are associated with a reduction in SARS-CoV-2 infections not only among vaccinated individuals but also among unvaccinated adult household members in a real-world setting. We do not find a decrease in the cumulative incidence of SARS-CoV-2 infections for unvaccinated children in households with at least one vaccinated adult. Taken together, our results suggest that mRNA-based vaccines do not only prevent SARS-CoV-2 infections among vaccinated individuals but also lead to a substantial reduction in infections among unvaccinated adult household members.To estimate the direct and indirect effectiveness of mRNA-based vaccines, we compared the cumulative incidence of SARS-CoV-2 infections between vaccinated and unvaccinated healthcare workers as well as between their unvaccinated partners and children living in the same household , we ensure that the estimates are not biased by the changing nature of the epidemic during the follow-up period. Moreover, to make individuals in the treatment and control groups comparable in terms of their SARS-CoV-2 history, we focused only on persons who had not been infected before their follow-up period. We report vaccine effectiveness estimates from follow-up week 2 onwards because the cumulative incidence of infections is very small in the first 2 follow-up weeks in our sample , 782 (0.61%), and 820 (0.48%) for healthcare workers, partners, and children, respectively. The mean ages of the healthcare workers, their partners, and children were 44 (SD 14), 45 (SD 12), and 11 (SD 5) years, respectively. Most healthcare workers (86.6%) in our sample were women. A total of 112,496 healthcare workers (42.4%) obtained at least one dose of mRNA-based vaccine during the period from December 27, 2020, to April 25, 2021. The number of double vaccinated healthcare workers during the same time period was 63,986 .We observe that the number of infections in different subgroups largely follows the general demographic and occupational profile of our sample. The group of practical nurses is the only occupational group in which the share of infections is somewhat larger than the relative share of the occupational group. Overall, 73.3% of infections are observed among the practical nurses. The most notably overrepresented demographic subgroup in terms of infections relative to the sample share of the subgroup is the group of individuals with a foreign background. While the relative share of individuals with a foreign background is 6.3% in our sample of healthcare workers, their relative share of all infections in our sample is 23.7%. A corresponding overrepresentation of infected foreign individuals is observed among the partners and children of healthcare workers. Supplementary Tables 10. This tendency might be explained by several factors. First, and most importantly, our direct effectiveness estimates  4 weeks and 23.0% (95% CI: 6.2% to 36.9%) 12 weeks after the first dose Fig. . The indFigure The indirect effectiveness estimates for 3–12-year-old children are not statistically significantly different from zero Fig. : –37.1% Our estimates for direct vaccine effectiveness are consistent with the results from clinical trials and previous observational studies assessing first-dose mRNA-vaccine effectiveness. Our results document vaccine effectiveness in a real-world setting, in which it is difficult to separate the effectiveness of the first dose and the additional protection conferred by a second dose of the vaccine , mimicking the RCT design. Thus, our research design complements existing observational studies that have relied on comparisons of individuals at different time periods before and after administration of a vaccine to their close contacts12, we are not able to explicitly study relative infectiousness among vaccinated and infected individuals.Our study has several important limitations. First, our study is limited by the fact that healthcare workers may differ from the general population, for example, in terms of their SARS-CoV-2 virus exposure. Second, virus exposure could have differed between vaccinated and unvaccinated healthcare workers as persons directly involved with COVID-19 patients were more likely to be vaccinated. These differences in virus exposure are likely to bias our vaccine effectiveness estimates towards zero. However, our results for direct effectiveness after the first and second dose are very close to the results from randomized controlled clinical trials and prior observational studies, suggesting that the sample selection and the non-random assignment of vaccines to healthcare workers do not introduce substantial bias into our direct vaccine effectiveness estimates. Third, our study is limited by the fact that we lack data on the potential biological and behavioral mechanisms that could clearly pinpoint the mechanism leading to the observed reduction in confirmed SARS-CoV-2 infections among unvaccinated adult household members. Finally, our study does not provide evidence about the risk of secondary infection conditional on a positive SARS-CoV-2 test for the index case. Thus, in contrast to a recent study in EnglandTaken together, our results suggest that mRNA-based COVID-19 vaccines do not only prevent SARS-CoV-2 infections among vaccinated individuals but also lead to a substantial reduction in infections among unvaccinated adults living in the same household. However, we do not find a statistically significant decrease in the cumulative incidence of SARS-CoV-2 infections for unvaccinated children living in households with vaccinated adults. The statistical power to detect statistically significant effects in children is weakened in our study due to a smaller risk of SARS-CoV-2 infections among children than adults.17.This paper provides indirect evidence that mRNA-based vaccines affect susceptibility in vaccinated individuals and may prevent transmission from vaccinated to unvaccinated adults within households. The observed reduction in household transmission from vaccinated to unvaccinated individuals is expected to curb the current COVID-19 pandemic as the household transmission of SARS-CoV-2 is believed to have a significant role in the overall spread of infections in the community18. Further studies are required to understand whether booster vaccinations do not only improve direct vaccine effectiveness but also strengthen vaccine-associated reduction in transmission. Overall, there is a need for new evidence to understand how the indirect effects of COVID-19 vaccines on unvaccinated adults and children support the prospect of herd immunity and to inform questions related to vaccine booster strategies and the possible mass vaccination of children.After our study period, the SARS-CoV-2 Delta (B.1.617.2) variant has become the dominant strain in many parts of the world. While mRNA-based COVID-19 vaccines are still expected to lower the risk of infection and remain highly effective against severe disease, the emergence of the Delta variant has likely eroded direct and indirect vaccine effectiveness by increasing the likelihood of breakthrough infections and viral transmission from vaccinated individuals who become infectedThis research complies with all relevant ethical regulations. The final data used in the study were de-identified and therefore research does not constitute human subject research. Ethical approval was waived by the Institutional Review Board of the Finnish Institute for Health and Welfare (IRB: 00007085).The analyses were conducted using multiple population-wide administrative datasets linked at the individual level. All PCR-confirmed SARS-CoV-2 infections and their dates up to April 25, 2021, were recorded in the Finnish National Infectious Diseases Register. The Finnish National Vaccination Register provided information about the type of vaccine and the date of vaccine administration for all COVID-19 vaccines administered up to April 25, 2021. These two registers are maintained by the Finnish Institute for Health and Welfare.We used the Finnish Incomes Register for the year 2020 from the Finnish Tax Authority to identify healthcare workers following Statistics Finland’s classification of occupations. These occupations are presented in Supplementary Table N = 2987; 1.08%) of healthcare workers and  of their partners are dropped from the linked dataset and therefore, from the estimation samples.Our linked dataset is based on nationwide administrative data sources that cover the entire Finnish population and are developed to record all SARS-CoV-2 infections and mRNA-based vaccine doses administered in Finland. However, some individuals and observations are missing in our linked dataset. The most notable missing groups of individuals are children born after 2019 and persons who immigrated to Finland after 2019, because the most recent total population data (FOLK-data) are available for the year 2019. Furthermore, our linked data do not include foreign individuals who do not have permanent residency in Finland but could have been infected during their stay in Finland. Finally, even though we use comprehensive data on the Finnish population, the household size (one of our covariates) had 1.1% and 0.2% of the total number of observations as missing for healthcare workers and their partners, respectively. Other characteristics or covariates  had even smaller shares of missing observations. The children’s sample does not have any missing observations. Individuals with missing observations in any of the covariates . This sample consisted of working-age healthcare workers aged 15–74 years). The number of healthcare workers and their vaccination status by detailed (3-digit) occupation code is shown in Supplementary Table  years. T19. RRR measures how much vaccination reduced the cumulative risk of SARS-CoV-2 infection in the treatment group relative to the control group, who did not receive a vaccination. We separately investigated healthcare workers and their unvaccinated family members (partners and children).Following the current standard practice in COVID-19 vaccine effectiveness reporting, we estimate vaccine effectiveness as relative risk reduction (RRR)l, defined as the number of periods in calendar week t from obtaining the first (second) dose of vaccination:Pit = P (yit = 1) is the probability of the cumulative SARS-CoV-2 infection of individual i recorded in calendar week t (yit = 1). Moreover, Ti is an indicator variable for treatment group status , and λtl contains week fixed effects that capture the state of the epidemic in each time-to-event week l. Xit contains controls at the individual level: age, age squared, sex, occupational group, household size , ethnicity (birth of origin: Finnish or foreign) and geographic area . These covariates include the characteristics of the individuals  themselves, except that occupation always refers to the healthcare worker’s occupation. αl refers to the log risk in the control group and βl refers to differences in the log risks between the treatment and control groups in time-to-event week l. The key advantage of a log-binomial model is that it allows easy access to an estimate of the RRR: the RRR is simply 1 – exp(βl). The corresponding 95% confidence interval estimates were calculated using the standard errors of the βl coefficients. Moreover, we assessed the sensitivity of our results to non-linear  vs. linear estimation (linear probability model)  for these unvaccinated individuals. The random assignment of a follow-up period for each unvaccinated healthcare worker and their family member parallels an assumption that vaccine coverage among healthcare workers increases linearly over time. In practice, the weekly number of vaccines administered to healthcare workers varied substantially during the study period depending on the supply of vaccines and timing of vaccine administration. Importantly, our regression models control for time-varying factors using week fixed effects, and thereby changes in the supply of vaccines and state of the epidemic over time.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary