Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

Visualized RNA detection of SARS-CoV-2 in a closed tube by coupling RT-PCR with nested invasive reaction.

A simple, cost-effective and reliable diagnosis of pathogen nucleic acids assay is much required for controlling a pandemic of a virus disease, such as COVID-19. Our previously developed visualized detection of pathogen DNA in a single closed tube is very suitable for POCT. However, virus RNA could not be detected directly and should be reverse-transcribed into cDNA in advance. To enable this visualized assay to detect virus RNA directly, various types of reverse transcriptase were investigated, and finally we found that HiScript II reverse transcriptase could keep active and be well adapted to the one-pot visualized assay in optimized conditions. Reverse transcription, template amplification and amplicon identification by PCR coupled with invasive reaction, as well as visualization by self-assembling of AuNP probes could be automatically and sequentially performed in a closed tube under different temperature conditions, achieving "sample (RNA)-in-result (red color)-out" only by a simple PCR engine plus the naked eye. The visualized RT-PCR is sensitive to unambiguous detection of 5 copies of the N and ORFlab genes of SARS-CoV-2 RNA comparing favourably with qPCR methods (commercialized kit), is specific to genotype 3 variants (Alpha, Beta and Omicron) of SARS-CoV-2, and is very accurate for picking up 0.01% Omicron variant from a large amount of sequence-similar backgrounds. The method is employed in detecting 50 clinical samples, and 10 of them were detected as SARS-CoV-2 positive samples, identical to those by conventional RT-PCR, indicating that the method is cost-effective and labor-saving for pathogen RNA screening in resource-limited regions.

HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the serum cytokine and chemokine levels in asymptomatic, mild, moderate, severe, and convalescent SARS-CoV-2-infected cases. Proinflammatory cytokine and chemokine production induced by SARS-CoV-2 were observed not only in symptomatic patients but also in asymptomatic cases, and returned to normal after recovery. IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG, and MIP-1α were found to be associated with the severity of COVID-19. Moreover, a set of cytokine and chemokine profiles were significantly higher in SARS-CoV-2-infected male than female patients. The serum levels of MCP-1, G-CSF, and VEGF were weakly and positively correlated with viral titers. We suggest that combinatorial analysis of serum cytokines and chemokines with clinical classification may contribute to evaluation of the severity of COVID-19 and optimize the therapeutic strategies.

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae.

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.

Outbreak of Vaccinia Virus Infection from Occupational Exposure, China, 2017.

Human infections with vaccinia virus (VACV), mostly from laboratory accidents or contact with infected animals, have occurred since smallpox was eradicated in 1980. No recent cases have been reported in China. We report on an outbreak of VACV from occupational exposure to rabbit skins inoculated with VACV.

Rapid detection of human mastadenovirus species B by recombinase polymerase amplification assay.

BACKGROUND: As an important component of the causative agent of respiratory tract infections, enteric and eye infections, Human mastadenoviruses (HAdVs) species B spread easily in the crowd. In this study, we developed a recombinase polymerase amplification (RPA) assay for rapidly detecting HAdVs species B which was comprised of two different formats (real-time and lateral-flow device). RESULTS: This assay was confirmed to be able to detect 5 different HAdVs species B subtypes (HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 and HAdV-B55) without cross-reactions with other subtypes and other respiratory tract pathogens. This RPA assay has not only highly sensitivity with low detection limit of 50 copies per reaction but also short reaction time (< 15 min per detection). Furthermore, the real-time RPA assay has excellent correlation with real-time PCR assay for detection of HAdVs species B presented in clinical samples. CONCLUSIONS: Thus, the RPA assay developed in this study provides an effective and portable approach for the rapid detection of HAdVs species B.

Co-circulation of multiple genotypes of influenza A (H7N9) viruses in eastern China, 2016-2017.

Five epidemic waves of human infection with influenza A (H7N9) virus have emerged in China since spring 2013. We previously described the epidemiological characterization of the fifth wave in Jiangsu province. In this study, 41 H7N9 viruses from patients and live-poultry markets were isolated and sequenced to further elucidate the genetic features of viruses of the fifth wave in Jiangsu province. Phylogenetic analysis revealed substantial genetic diversity in the internal genes, and 18 genotypes were identified from the 41 H7N9 virus strains. Furthermore, our data revealed that 41 isolates from Jiangsu contained the G186V and Q226L/I mutations in their haemagglutinin (HA) protein, which may increase the ability of these viruses to bind the human receptor. Four basic amino acid insertions were not observed in the HA cleavage sites of 167 H7N9 viruses from Jiangsu, which revealed that highly pathogenic avian influenza (HPAI) H7N9 viruses did not spread to Jiangsu province in the fifth wave. These findings revealed that multiple genotypes of H7N9 viruses co-circulated in the fifth wave in Jiangsu province, which indicated that the viruses have undergone ongoing evolution with genetic mutation and reassortment. Our study highlights the need to constantly monitor the evolution of H7N9 viruses and reinforce systematic influenza surveillance of humans, birds, and pigs in China.

Identification and genetic characterization of a novel circular single-stranded DNA virus in a human upper respiratory tract sample.

Metagenomic analysis through high-throughput sequencing is a tool for detecting both known and novel viruses. Using this technique, a novel circular single-stranded DNA (ssDNA) virus genome was discovered in respiratory secretions from a febrile traveler. The virus, named human respiratory-associated PSCV-5-like virus (HRAPLV), has a genome comprising 3,018 bases, with two major putative ORFs inversely encoding capsid (Cap) and replicase (Rep) protein and separated by two intergenic regions. One stem-loop structure was predicted in the larger intergenic region (LIR). The predicted amino acid sequences of the Cap and Rep proteins of HRAPLV showed highest identity to those of porcine stool-associated circular virus 5 isolate CP3 (PoSCV 5) (53.0% and 48.9%, respectively). The host tropism of the virus is unknown, and further study is warranted to determine whether this novel virus is associated with human disease.

Pyrosequencing with di-base addition for single nucleotide polymorphism genotyping.

We develop color code-based pyrosequencing with di-base addition for analysis of single nucleotide polymorphisms (SNPs). When a di-base is added into the polymerization, one or several two-color code(s) containing the type and the number of incorporated nucleotides will be produced. The code information obtained in a single run is useful to genotype SNPs as each allelic variant will give a specific pattern compared to the two other variants. Special care has to be taken while designing the di-base dispensation order. Here, we present a detailed protocol for establishing sequence-specific di-base addition to avoid nonsynchronous extension at the SNP sites. By using this technology, as few as 50 copies of DNA templates were accurately sequenced. Higher signals were produced and thus a relatively lower sample amount was required. Furthermore, the read length of per flow was increased, making simultaneous identification of multiple SNPs in a single sequencing run possible. Validation of the method was performed by using templates with two SNPs covering 37 bp and with three SNPs covering 58 bp as well as 82 bp. These SNPs were successfully genotyped by using only a sequencing primer in a single PCR/sequencing run. Our results demonstrated that this technology could be potentially developed into a powerful methodology to accurately determine SNPs so as to diagnose clinical settings.

Cytokine and chemokine levels in patients infected with the novel avian influenza A (H7N9) virus in China.

H7N9 avian influenza is an emerging viral disease in China caused by avian influenza A (H7N9) virus. We investigated host cytokine and chemokine profiles in serum samples of H7N9 patients by multiplex-microbead immunoassays. Statistical analysis showed that IP-10, IL-6, IL-17, and IL-2 were increased in H7N9 infected patients. Furthermore, IL-6 and the chemokine IP-10 were significantly higher in severe H7N9 patients compared to nonsevere H7N9 cases. We suggest that proinflammatory cytokine responses, characterized by a combined Th1/Th17 cytokine induction, are partially responsible for the disease progression of patients with H7N9 infection.

Portable all-in-one microfluidic system for CRISPR-Cas13a-based fully integrated multiplexed nucleic acid detection.

Point-of-care testing of "sample in, answer out" is urgently needed for communicable diseases. Recently, rapid nucleic acid tests for infectious diseases have been developed for use in resource-limited areas, but they require types of equipment in central laboratories and are poorly integrated. In this work, a portable centrifugal microfluidic testing system is developed, integrated with magnetic bead-based nucleic acid extraction, recombinase-assisted amplification and CRISPR-Cas13a detection. The system, with the advantage of its power-supplied active rotating chip and highly programable flow control through integrated addressable active thermally-triggered wax valves, has a rapid turnaround time within 45 min, requiring only one user step. All reagents are preloaded into the chip and can be automatically released. By exploiting a multichannel chip, it is capable of simultaneously detecting 10 infectious viruses with limits of detection of 1 copy per reaction and 5 copies per reaction in plasmid samples and mock plasma samples, respectively. The system was used to analyse clinical plasma samples with good consistency compared to laboratory-based molecular testing. Moreover, the generalizability of our device is reported by successfully testing nasopharyngeal swabs and whole blood samples. The portable device does not require the operation of professional technicians, making it an excellent assay for on-site testing.

A novel fast hybrid capture sequencing method for high-efficiency common human coronavirus whole-genome acquisition.

Rapid and accurate sequencing of the entire viral genome, coupled with continuous monitoring of genetic changes, is crucial for understanding the epidemiology of coronaviruses. We designed a novel method called micro target hybrid capture system (MT-Capture) to enable whole-genome sequencing in a timely manner. The novel design of probes used in target binding exhibits a unique and synergistic "hand-in-hand" conjugation effect. The entire hybrid capture process is within 2.5 hours, overcoming the time-consuming and complex operation characteristics of the traditional liquid-phase hybrid capture (T-Capture) system. By designing specific probes for these coronaviruses, MT-Capture effectively enriched isolated strains and 112 clinical samples of coronaviruses with cycle threshold values below 37. Compared to multiplex PCR sequencing, it does not require frequent primer updates and has higher compatibility. MT-Capture is highly sensitive and capable of tracking variants.IMPORTANCEMT-Capture is meticulously designed to enable the efficient acquisition of the target genome of the common human coronavirus. Coronavirus is a kind of virus that people are generally susceptible to and is epidemic and infectious, and it is the virus with the longest genome among known RNA viruses. Therefore, common human coronavirus samples are selected to evaluate the accuracy and sensitivity of MT-Capture. This method utilizes innovative probe designs optimized through probe conjugation techniques, greatly shortening the time and simplifying the handwork compared with traditional hybridization capture processes. Our results demonstrate that MT-Capture surpasses multiplex PCR in terms of sensitivity, exhibiting a thousandfold increase. Moreover, MT-Capture excels in the identification of mutation sites. This method not only is used to target the coronaviruses but also may be used to diagnose other diseases, including various infectious diseases, genetic diseases, or tumors.

Development and application of a one-step real-time RT-PCR using a minor-groove-binding probe for the detection of a novel bunyavirus in clinical specimens.

A highly sensitive one-step real-time RT-PCR method using a minor-groove-binding (MGB) probe was developed for detection and quantitation of severe febrile with thrombocytopenia syndrome virus (SFTSV). The assay could discriminate SFTSV infection from other related viral diseases in human with a minimum detection limit of 10 viral RNA copies/µl and was 1,000 times more sensitive than the conventional PCR. Strong linear correlations (r(2) > 0.99) between the C(t) values and viral RNA standards over a linear range were obtained. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The RT-PCR was also shown to be highly specific, as no positive signals were detected for other related viruses. Evaluation of this assay with serum samples from laboratory confirmed cases and healthy donors showed 100% clinical diagnostic sensitivity and over 99% specificity. Clinical application with samples from 287 patients admitted to the hospital with suspected SFTSV infection showed that 15% were infected by SFTSV. This assay was rapid, requiring just over 2 hr, including the nucleic acid extraction step.

Antigenic and genetic characterization of a European avian-like H1N1 swine influenza virus from a boy in China in 2011.

Cross-species transmission of influenza A viruses from swine to human occurs occasionally. In 2011, an influenza A H1N1 virus, A/Jiangsu/ALS1/2011 (JS/ALS1/2011), was isolated from a boy who suffered from severe pneumonia in China. The virus is closely related antigenically and genetically to avian-like swine H1N1 viruses that have recently been circulating in pigs in China and that were initially detected in European pig populations in 1979. The isolation of JS/ALS1/2011 provides additional evidence that swine influenza viruses can occasionally infect humans and emphasizes the importance of reinforcing influenza virus surveillance in both pigs and humans.

Serum microRNA expression profile as a biomarker for the diagnosis of pertussis.

Pertussis is a highly contagious, respiratory disease associated with substantial morbidity and mortality. A rapid and reliable diagnostic method is essential for appropriate treatment and prevention. Expression profiles of circulating microRNAs (miRNAs) have been proven as new non-invasive biomarkers for infectious diseases. We aimed to investigated the serum miRNA profile in pertussis patients and explored its potential as a novel diagnostic biomarker for pertussis. Among 664 different miRNAs analyzed using a miRNA array, 50 were overexpressed and 81 were underexpressed in the serum of pertussis patients. Expression levels of seven candidate miRNAs were further evaluated by real-time qRT-PCR. A panel of five miRNAs (miR-202, miR-342-5p, miR-206, miR-487b, miR-576-5p) was confirmed overexpressed in pertussis patients (p < 0.05). Risk score and receiver-operating characteristic (ROC) analysis showed that the area under the curve of the five-member miRNA profile was 0.980. At an optimal cutoff value (0.707), this panel of miRNAs yielded a sensitivity of 97.4 % and a specificity of 94.3 %. These data suggest that the five-member serum miRNA profile may serve as a new biomarker for pertussis diagnosis with high specificity and sensitivity.

Case report: Clinical characteristics of two cases of pneumonia caused with different strains of Chlamydia psittaci.

Background: With the development of metagenomic sequencing technologies, more and more cases of pneumonia caused with Chlamydia psittaci (C. psittaci) have been reported. However, it remains unknown about the characteristics of patients with pneumonia caused by different strains of C. psittaci. Here, we shared the clinical characteristics of two cases of pneumonia caused with C. psittaci strains SZ18-2 and SZ15 which were rarely identified in humans. Case presentation: Case 1: A 69-year-old male farmer who fed ducks presented to hospital for cough, diarrhea and lethargy with the temperature of 39.8°C. Case 2: A 48-year-old male worker who slaughtered ducks was transferred to hospital for high fever, cough, myalgia, diarrhea and loss of appetite. Both patients did not take any protective measures (wearing face masks or gloves) while processing ducks. C. psittaci pneumonia was diagnosed by metagenomic next-generation sequencing and polymerase chain reaction. After treatment with doxycycline and azithromycin individually, they recovered well and discharged from hospital. Through OmpA sequencing, two different strains of SZ18-2 and SZ15 were identified in case 1 and case 2, respectively. Conclusions: Patients infected with different strains of C. psittaci may own different clinical manifestations. C. psittaci infection should be suspected when pneumonia appears, accompanied by digestive symptoms and multiple organ dysfunction, especially under the exposure of specific birds.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Antibody persistence and safety after heterologous boosting with orally aerosolised Ad5-nCoV in individuals primed with two-dose CoronaVac previously: 12-month analyses of a randomized controlled trial.

Antibody persistence and safety up to 12 months of heterologous orally administered adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in individuals who were primed with two-dose inactivated SARS-CoV-2 vaccine (CoronaVac) previously, has not been reported yet. This randomized, open-label, single-centre trial included Chinese adults who have received two-dose CoronaVac randomized to low-dose or high-dose aerosolised Ad5-nCoV group, or CoronaVac group. In this report, we mainly evaluated the geometric mean titres (GMTs) of neutralizing antibodies (NAbs) against live wild-type SARS-CoV-2 virus and omicron BA.4/5 pseudovirus at 12 months after the booster dose and the incidence of serious adverse events (SAEs) till month 12. Of 419 participants, all were included in the safety analysis and 120 (28.64%) were included in the immunogenicity analysis. Serum NAb GMT against live wild-type SARS-CoV-2 was 204.36 (95% CI 152.91, 273.14) in the low-dose group and 171.38 (95% CI 121.27, 242.19) in the high-dose group at month 12, significantly higher than the GMT in the CoronaVac group (8.00 [95% CI 4.22, 15.17], p < 0.0001). Serum NAb GMT against omicron BA.4/5 pseudovirus was 40.97 (95% CI 30.15, 55.67) in the low-dose group and 35.08 (95% CI 26.31, 46.77) in the high-dose group at month 12, whereas the GMT in the CoronaVac group was below the lower limit of detection. No vaccine-related SAEs were observed. Orally administered aerosolised Ad5-nCoV following two-dose CoronaVac priming has a good safety profile and is persistently more immunogenic than three-dose CoronaVac within 12 months after the booster dose.Trial registration: ClinicalTrials.gov identifier: NCT05043259..

Safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster following three doses of CoronaVac: a multicentre, open-label, phase 4, randomised trial.

BACKGROUND: Aerosolised Ad5-nCoV is the first approved mucosal respiratory COVID-19 vaccine to be used as a booster after the primary immunisation with COVID-19 vaccines. This study aimed to evaluate the safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster. METHODS: This is an open-label, parallel-controlled, phase 4 randomised trial enrolling healthy adult participants (≥18 years) who had completed a two-dose primary immunisation and a booster immunisation with inactivated COVID-19 vaccines (CoronaVac only) at least 6 months before, in Lianshui and Donghai counties, Jiangsu Province, China. We recruited eligible participants from previous trials in China (NCT04892459, NCT04952727, and NCT05043259) as cohort 1 (with the serum before and after the first booster dose available), and from eligible volunteers in Lianshui and Donghai counties, Jiangsu Province, as cohort 2. Participants were randomly assigned at a ratio of 1:1:1, using a web-based interactive response randomisation system, to receive the fourth dose (second booster) of aerosolised Ad5-nCoV (0·1 mL of 1·0 × 1011 viral particles per mL), intramuscular Ad5-nCoV (0·5 mL of 1·0 × 1011 viral particles per mL), or inactivated COVID-19 vaccine CoronaVac (0·5 mL), respectively. The co-primary outcomes were safety and immunogenicity of geometric mean titres (GMTs) of serum neutralising antibodies against prototype live SARS-CoV-2 virus 28 days after the vaccination, assessed on a per-protocol basis. Non-inferiority or superiority was achieved when the lower limit of the 95% CI of the GMT ratio (heterologous group vs homologous group) exceeded 0·67 or 1·0, respectively. This study was registered with ClinicalTrials.gov, NCT05303584 and is ongoing. FINDINGS: Between April 23 and May 23, 2022, from 367 volunteers screened for eligibility, 356 participants met eligibility criteria and received a dose of aerosolised Ad5-nCoV (n=117), intramuscular Ad5-nCoV (n=120), or CoronaVac (n=119). Within 28 days of booster vaccination, participants in the intramuscular Ad5-nCoV group reported a significantly higher frequency of adverse reactions than those in the aerosolised Ad5-nCoV and intramuscular CoronaVac groups (30% vs 9% and 14%, respectively; p<0·0001). No serious adverse events related to the vaccination were reported. The heterologous boosting with aerosolised Ad5-nCoV triggered a GMT of 672·4 (95% CI 539·7-837·7) and intramuscular Ad5-nCoV triggered a serum neutralising antibody GMT of 582·6 (505·0-672·2) 28 days after the booster dose, both of which were significantly higher than the GMT in the CoronaVac group (58·5 [48·0-71·4]; p<0·0001). INTERPRETATION: A heterologous fourth dose (second booster) with either aerosolised Ad5-nCoV or intramuscular Ad5-nCoV was safe and highly immunogenic in healthy adults who had been immunised with three doses of CoronaVac. FUNDING: National Natural Science Foundation of China, Jiangsu Provincial Science Fund for Distinguished Young Scholars, and Jiangsu Provincial Key Project of Science and Technology Plan.