Broad-spectrum pan-genus and pan-family virus vaccines.

Although the development and clinical application of SARS-CoV-2 vaccines during the COVID-19 pandemic demonstrated unprecedented vaccine success in a short time frame, it also revealed a limitation of current vaccines in their inability to provide broad-spectrum or universal protection against emerging variants. Broad-spectrum vaccines, therefore, remain a dream and challenge for vaccinology. This review will focus on current and future efforts in developing universal vaccines targeting different viruses at the genus and/or family levels, with a special focus on henipaviruses, influenza viruses, and coronaviruses. It is evident that strategies for developing broad-spectrum vaccines will be virus-genus or family specific, and it is almost impossible to adopt a universal approach for different viruses. On the other hand, efforts in developing broad-spectrum neutralizing monoclonal antibodies have been more successful and it is worth considering broad-spectrum antibody-mediated immunization, or "universal antibody vaccine," as an alternative approach for early intervention for future disease X outbreaks.

Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

Antibody response of heterologous vs homologous mRNA vaccine boosters against the SARS-CoV-2 Omicron variant: interim results from the PRIBIVAC study, A Randomized Clinical Trial.

BACKGROUND: Waning antibody levels post-vaccination and the emergence of variants of concern (VOCs) capable of evading protective immunity has raised the need for booster vaccinations. However, which combination of COVID-19 vaccines offers the strongest immune response against Omicron variant is unknown. METHODS: This randomized, subject-blinded, controlled trial assessed the reactogenicity and immunogenicity of different COVID-19 vaccine booster combinations. 100 BNT162b2-vaccinated individuals were enrolled and randomized 1: 1 to either homologous (BNT162b2 + BNT162b2 + BNT162b2; 'BBB') or heterologous mRNA booster vaccine (BNT162b2 + BNT162b2 + mRNA-1273; 'BBM'). Primary endpoint was the level of neutralizing antibodies against SARS-CoV-2 wild-type and VOCs at Day 28. RESULTS: 51 participants were allocated to BBB and 49 to BBM; 50 and 48 respectively were analyzed for safety and immunogenicity outcomes. At Day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBB (22,382  IU/mL 95% CI, 18,210 to 27,517) vs BBM (29,751  IU/mL 95% CI, 25,281 to 35,011, p = 0.034) as was the median level of neutralizing antibodies: BBB 99.0% (IQR 97.9 to 99.3%) vs BBM 99.3% (IQR 98.8 to 99.5%, p = 0.021). On sub-group analysis, significant differences in mean spike antibody titer and live Omicron neutralization titer was only observed in older adults. Median surrogate neutralizing antibody level against all VOCs was also significantly higher with BBM in older adults, and against Omicron was BBB 72.8% (IQR 54.0 to 84.7%) vs BBM 84.3% (IQR 78.1 to 88.7%, p = 0.0073). Both vaccines were well tolerated. CONCLUSIONS: Heterologous mRNA-1273 booster vaccination induced a stronger neutralizing response against the Omicron variant in older individuals compared with homologous BNT123b2.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

The Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination and Infection on Neutralizing Antibodies: A Nation-wide Cross-sectional Analysis.

BACKGROUND: Neutralising antibodies (nAbs) play a critical role in the protection against severe COVID-19. In the era of vaccine boosters and repeated SARS-CoV-2 outbreaks, identifying individuals at risk represents a public health priority. METHODS: Relying on the Monaco COVID Public Health Programme, we evaluated nAbs from July 2021-June 2022 in 8,080 SARS-CoV-2 vaccinated and/or infected children and adults, at their inclusion visit. We stratified by infection status and investigated variables associated with nAbs using a generalised additive model. RESULTS: Infected and vaccinated participants had high and consistent nAbs (>800 IU/mL), which remained stable over time since injection, regardless of the number of vaccine doses, body mass index, sex, or age. By contrast, uninfected participants showed larger variability (two doses [V2] median 157.6; interquartile range [IQR] 43.3-439.1 IU/mL) versus three doses [V3] median 882.5; [829.5-914.8] IU/mL). NAbs decreased by 20% per month after V2 (adjusted ratio 0.80; 95%CI [0.79-0.82]), but remained stable after V3 (adjusted ratio 0.98; 95%CI [0.92-1.05]). CONCLUSIONS: Hybrid immunity provided stable, high and consistent nAbs over time. The benefit of boosters was marked to restore decaying nAbs in uninfected participants. NAbs could identify individuals at risk of severe COVID-19 and provide more targeted vaccine boosters' campaigns.

Genomic Characterization of a Relative of Mumps Virus in Lesser Dawn Bats of Southeast Asia.

The importance of genomic surveillance on emerging diseases continues to be highlighted with the ongoing SARS-CoV-2 pandemic. Here, we present an analysis of a new bat-borne mumps virus (MuV) in a captive colony of lesser dawn bats (Eonycteris spelaea). This report describes an investigation of MuV-specific data originally collected as part of a longitudinal virome study of apparently healthy, captive lesser dawn bats in Southeast Asia (BioProject ID PRJNA561193) which was the first report of a MuV-like virus, named dawn bat paramyxovirus (DbPV), in bats outside of Africa. More in-depth analysis of these original RNA sequences in the current report reveals that the new DbPV genome shares only 86% amino acid identity with the RNA-dependent RNA polymerase of its closest relative, the African bat-borne mumps virus (AbMuV). While there is no obvious immediate cause for concern, it is important to continue investigating and monitoring bat-borne MuVs to determine the risk of human infection.

HIV viremia is associated with compromised SARS-CoV-2 Beta variant neutralization.

BACKGROUND: SARS-CoV-2 infection may be associated with worse clinical outcomes in people with HIV (PWH). We report anti-SARS-CoV-2 antibody responses in COVID-19 hospitalized patients in Durban, South Africa during the second SARS-CoV-2 infection wave dominated by the Beta (B.1.351) variant. METHODS: Thirty-four participants with confirmed SARS-CoV-2 infection were followed up with weekly blood sampling to examine antibody levels and neutralization potency against SARS-CoV-2 variants. Participants included 18 PWH, of whom 11 were HIV viremic. RESULTS: SARS-CoV-2 specific antibody concentrations were generally lower in viremic PWH relative to virologically suppressed PWH and HIV-negative participants and neutralization of the Beta variant was 4.9-fold lower in viremic PWH. Most HIV-negative participants and ART-suppressed PWH also neutralized the Delta (B.1.617.2) variant, whereas the majority of viremic PWH did not. CD4 counts <500 cells/µL were associated with lower frequencies of IgG and IgA seroconversion. In addition, there was a high correlation between a surrogate virus neutralization test and live virus neutralization against ancestral SARS-CoV-2 virus in both PWH and HIV-negative individuals, but correlation decreased for the Beta variant neutralization in PWH. CONCLUSIONS: HIV viremia was associated with reduced Beta variant neutralization. This highlights the importance of HIV suppression in maintaining an effective SARS-CoV-2 neutralization response.

Generation of self-replicating airway organoids from the cave nectar bat Eonycteris spelaea as a model system for studying host-pathogen interactions in the bat airway epithelium.

Bats are reservoir hosts for various zoonotic viruses with pandemic potential in humans and livestock. In vitro systems for studying bat host-pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air-liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species specific virus susceptibility and the reservoir likelihood for emerging infectious agents.

COVID-19 mRNA vaccine immunogenicity decay and breakthrough illness in adolescents and young adults with childhood-onset rheumatic diseases.

OBJECTIVES: To evaluate the humoral immunogenicity for 6 months after the two-dose COVID-19 mRNA vaccination in adolescents and young adults (AYAs) with childhood-onset rheumatic diseases (cRDs). METHODS: This monocentric observational study was conducted between August 2020 to March 2022. Humoral immunogenicity was assessed at 2-3 weeks after first vaccine dose and 1, 3, and 6 months after the second dose by the cPass™ SARS-CoV-2 Neutralisation Antibody (nAb) Assay. An inhibition signal of ≥ 30% defined seroconversion threshold and the readings calibrated against the World Health Organisation (WHO) International Standard for SARS-CoV-2 antibodies. RESULTS: 169 AYAs with cRDs were recruited (median age 16·8 years (IQR : 14·7-19·5), 52% female, 72% Chinese). Juvenile Idiopathic Arthritis (JIA) (58%) and Systemic Lupus Erythematosus (18%) comprised the major diagnoses. After second vaccine dose, 99% seroconverted with a median nAb titre of 1779·8 IU/ml (IQR : 882·8-2541·9), declining to 935·6 IU/ml (IQR : 261·0-1514·9) and 683·2 IU/ml (IQR : 163·5-1400·5) at the 3- and 6-month timepoints respectively. The diagnosis of JIA (OR 10·1, 95%CI 1·8-58·4, p= 0·010) and treatment with anti-Tumour Necrosis Factor-α (aTNF) (OR 10·1, 95%CI 1·5-70·0, p= 0·019) were independently associated with a > 50% drop of nAb titres at 6 months. Withholding methotrexate or mycophenolate mofetil did not affect the vaccine response or decay rate. The COVID-19 breakthrough infection was estimated at 18·2 cases/1000 patient-months with no clinical risk factors identified. CONCLUSION: Over half of AYAs with cRDs had a significant drop in SARS-CoV-2 nAb at 6-month despite an initial robust humoral response. JIA and aTNF usage are predictors of a faster decay rate.

Engineering antiviral immune-like systems for autonomous virus detection and inhibition in mice.

The ongoing COVID-19 pandemic has demonstrated that viral diseases represent an enormous public health and economic threat to mankind and that individuals with compromised immune systems are at greater risk of complications and death from viral diseases. The development of broad-spectrum antivirals is an important part of pandemic preparedness. Here, we have engineer a series of designer cells which we term autonomous, intelligent, virus-inducible immune-like (ALICE) cells as sense-and-destroy antiviral system. After developing a destabilized STING-based sensor to detect viruses from seven different genera, we have used a synthetic signal transduction system to link viral detection to the expression of multiple antiviral effector molecules, including antiviral cytokines, a CRISPR-Cas9 module for viral degradation and the secretion of a neutralizing antibody. We perform a proof-of-concept study using multiple iterations of our ALICE system in vitro, followed by in vivo functionality testing in mice. We show that dual output ALICESaCas9+Ab system delivered by an AAV-vector inhibited viral infection in herpetic simplex keratitis (HSK) mouse model. Our work demonstrates that viral detection and antiviral countermeasures can be paired for intelligent sense-and-destroy applications as a flexible and innovative method against virus infection.

The establishment of COPD organoids to study host-pathogen interaction reveals enhanced viral fitness of SARS-CoV-2 in bronchi.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

Immunogenicity of COVID-19 vaccines and levels of SARS-CoV-2 neutralising antibody in the Bruneian population: Protocol for a national longitudinal study.

INTRODUCTION: Neutralising antibodies (NAbs) have been shown to be correlative of immune protection against SARS-CoV-2. We report the protocol for a national longitudinal study to assess and compare the level of NAbs generated in response to COVID-19 vaccines in Brunei Darussalam in adults 2-6 weeks post primary series (BBIBP-CorV, AZD1222, or mRNA-1273 vaccines) and their subsequent follow-up after administration of a third (booster-1) dose (BBIBP-CorV, mRNA-1273, or BNT162b2). METHODS AND ANALYSIS: Participant data will be extracted and processed from the national electronic health record system (Bru-HIMS) and the national mobile health application (BruHealth) into a research data platform. Eligible adults who have received their primary or booster vaccine will be invited using a stratified random sampling strategy based on age, gender and vaccine type (baseline target population, n=3000; 2-6 weeks post last dose). Blood serum will be isolated, and NAb levels assessed using the cPass surrogate virus neutralisation test. Baseline participants will then be screened for eligibility for subsequent longitudinal analysis. Those who have received a third dose will be followed up at 1, 3, 6, 9 and up to 12 months. NAb levels will be evaluated across the participant population according to vaccine platform/booster type, time since the last dose and correlated with demographic data. The study period is from December 2021 to January 2023 and aims to evaluate how NAb levels wane following a third vaccine dose across different vaccine platforms and determine the impact and rate of breakthrough infections. ETHICS AND DISSEMINATION: This study has been approved by the Medical and Ethical Research Committee of Ministry of Health, Brunei Darussalam. Individual NAb test results will be shared with each participant by text message. The findings from this study will help policy-makers in Brunei develop future vaccination strategies and establish regulations across multiple agencies.

Biparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2.

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.

Absence of SARS-CoV-2 antibodies in pre-pandemic plasma from children and adults in Vietnam.

We tested pre-pandemic (2015--2019) plasma samples from 148 Vietnamese children and 100 Vietnamese adults at high risk of zoonotic infections for antibodies against SARS-CoV-2 nucleocapsid and spike proteins. None was positive. The data thus demonstrated no evidence of prior serological cross-reactivity with SARS-CoV-2 that might explain the low numbers of COVID-19 in Vietnam. No pre-existing cross-reactivity might explain Vietnam success of COVID-19 control.

Unique Evolution of Antiviral Tetherin in Bats.

Bats are recognized as important reservoirs of viruses deadly to other mammals, including humans. These infections are typically nonpathogenic in bats, raising questions about host response differences that might exist between bats and other mammals. Tetherin is a restriction factor which inhibits the release of a diverse range of viruses from host cells, including retroviruses, coronaviruses, filoviruses, and paramyxoviruses, some of which are deadly to humans and transmitted by bats. Here, we characterize the tetherin genes from 27 bat species, revealing that they have evolved under strong selective pressure, and that fruit bats and vesper bats express unique structural variants of the tetherin protein. Tetherin was widely and variably expressed across fruit bat tissue types and upregulated in spleen tissue when stimulated with Toll-like receptor agonists. The expression of two computationally predicted splice isoforms of fruit bat tetherin was verified. We identified an additional third unique splice isoform which includes a C-terminal region that is not homologous to known mammalian tetherin variants but was functionally capable of restricting the release of filoviral virus-like particles. We also report that vesper bats possess and express at least five tetherin genes, including structural variants, more than any other mammal reported to date. These findings support the hypothesis of differential antiviral gene evolution in bats relative to other mammals. IMPORTANCE Bats are an important host of various viruses which are deadly to humans and other mammals but do not cause outward signs of illness in bats. Furthering our understanding of the unique features of the immune system of bats will shed light on how they tolerate viral infections, potentially informing novel antiviral strategies in humans and other animals. This study examines the antiviral protein tetherin, which prevents viral particles from escaping their host cell. Analysis of tetherin from 27 bat species reveals that it is under strong evolutionary pressure, and we show that multiple bat species have evolved to possess more tetherin genes than other mammals, some of which encode structurally unique tetherins capable of activity against different viral particles. These data suggest that bat tetherin plays a potentially broad and important role in the management of viral infections in bats.

Lessons from the pandemic: Responding to emerging zoonotic viral diseases-a Keystone Symposia report.

The COVID-19 pandemic caught the world largely unprepared, including scientific and policy communities. On April 10-13, 2022, researchers across academia, industry, government, and nonprofit organizations met at the Keystone symposium "Lessons from the Pandemic: Responding to Emerging Zoonotic Viral Diseases" to discuss the successes and challenges of the COVID-19 pandemic and what lessons can be applied moving forward. Speakers focused on experiences not only from the COVID-19 pandemic but also from outbreaks of other pathogens, including the Ebola virus, Lassa virus, and Nipah virus. A general consensus was that investments made during the COVID-19 pandemic in infrastructure, collaborations, laboratory and manufacturing capacity, diagnostics, clinical trial networks, and regulatory enhancements-notably, in low-to-middle income countries-must be maintained and strengthened to enable quick, concerted responses to future threats, especially to zoonotic pathogens.

SARS-CoV-2 Omicron variant emerged under immune selection.

The SARS-CoV-2 Omicron variant (B.1.1.529 lineage) escapes antibodies that neutralize the ancestral virus. We tested human serum panels from participants with differing infection and vaccination status using a multiplex surrogate virus neutralization assay targeting 20 sarbecoviruses. We found that bat and pangolin sarbecoviruses showed significantly less neutralization escape than the Omicron variant. We propose that SARS-CoV-2 variants have emerged under immune selection pressure and are evolving differently from animal sarbecoviruses.

Biparatopic nanobodies targeting the receptor binding domain efficiently neutralise SARS-CoV-2

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralise virus, have potential for treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralise the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralising capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralisers with enhanced ability to control viral escape mutants. Graphical

Technology-assisted adaptive recruitment strategy for a large nation-wide COVID-19 vaccine immunogenicity study in Brunei.

A national study was conducted in Brunei to assess and compare the immunogenicity of the various brands of COVID-19 vaccines administered to the population as part of the National COVID-19 Vaccination Programme. Most of the population have had received at least 2 doses of BBIBP-CorV, AZD1222 or MRNA-1273 vaccines. Neutralising antibodies against SARS-CoV-2 induced by these vaccines will be analysed to infer population-level immune protection against COVID-19. During the 5-week recruitment period, 24,260 eligible individuals were invited to the study via SMS, out of which 2,712 participants were enrolled into the study. This paper describes the novel adaptive strategy used to recruit the study participants. Digital technology was leveraged to perform targeted online recruitment to circumvent the limitations of traditional recruitment methods. Technology also enabled stratified random selection of these eligible individuals who were stratified based on age, gender and vaccine brand. Data was extracted from the electronic health records, the national mobile health application and a third-party survey platform and integrated into a dedicated research platform called EVYDResearch. The instant availability and access to up-to-date data on EVYDResearch enabled the study team to meet weekly and adopt an adaptive recruitment strategy informed by behavioural science, where interventions could be quickly implemented to improve response rates. Some examples of these include incorporating nudge messaging into SMS invitations, involving the Minister of Health to make press announcements on this study, media coverage, setting up an enquiries hotline and reaching out to foreign language speaking expatriates of a local multinational company to participate in this study. Data integration from various data sources, real time information sharing and a strong teamwork led to good outcomes adaptable to the progress of recruitment, compared to the more time-consuming and static traditional recruitment methods.