Seroprevalence of Avian Influenza A(H5N6) Virus Infection, Guangdong Province, China, 2022.

In 2022, we assessed avian influenza A virus subtype H5N6 seroprevalence among the general population in Guangdong Province, China, amid rising numbers of human infections. Among the tested samples, we found 1 to be seropositive, suggesting that the virus poses a low but present risk to the general population.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

L226Q Mutation on Influenza H7N9 Virus Hemagglutinin Increases Receptor-Binding Avidity and Leads to Biased Antigenicity Evaluation.

Since the first outbreak in 2013, the influenza A (H7N9) virus has continued emerging and has caused over five epidemic waves. Suspected antigenic changes of the H7N9 virus based on hemagglutination inhibition (HI) assay during the fifth outbreak have prompted the update of H7N9 candidate vaccine viruses (CVVs). In this study, we comprehensively compared the serological cross-reactivities induced by the hemagglutinins (HAs) of the earlier CVV A/Anhui/1/2013 (H7/AH13) and the updated A/Guangdong/17SF003/2016 (H7/GD16). We found that although H7/GD16 showed poor HI cross-reactivity to immune sera from mice and rhesus macaques vaccinated with either H7/AH13 or H7/GD16, the cross-reactive neutralizing antibodies between H7/AH13 and H7/GD16 were comparably high. Passive transfer of H7/AH13 immune sera also provided complete protection against the lethal challenge of H7N9/GD16 virus in mice. Analysis of amino acid mutations in the HAs between H7/AH13 and H7/GD16 revealed that L226Q substitution increases the HA binding avidity to sialic acid receptors on red blood cells, leading to decreased HI titers against viruses containing HA Q226 and thus resulting in a biased antigenic evaluation based on HI assay. These results suggest that amino acids located in the receptor-binding site could mislead the evaluation of antigenic variation by solely impacting the receptor-binding avidity to red blood cells without genuine contribution to antigenic drift. Our study highlighted that viral receptor-binding avidity and combination of multiple serological assays should be taken into consideration in evaluating and selecting a candidate vaccine virus of H7N9 and other subtypes of influenza viruses.IMPORTANCE The HI assay is a standard method for profiling the antigenic characterization of influenza viruses. Suspected antigenic changes based on HI divergency in H7N9 viruses during the 2016-2017 wave prompted the recommendation of new H7N9 candidate vaccine viruses (CVVs). In this study, we found that the L226Q substitution in HA of A/Guangdong/17SF003/2016 (H7/GD16) increased the viral receptor-binding avidity to red blood cells with no impact on the antigenicity of H7N9 virus. Although immune sera raised by an earlier vaccine strain (H7/AH13) showed poor HI titers against H7/GD16, the H7/AH13 immune sera had potent cross-neutralizing antibody titers against H7/GD16 and could provide complete passive protection against H7N9/GD16 virus challenge in mice. Our study highlights that receptor-binding avidity might lead to biased antigenic evaluation by using the HI assay. Other serological assays, such as the microneutralization (MN) assay, should be considered a complementary indicator for analysis of antigenic variation and selection of influenza CVVs.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Liu Shen capsule shows antiviral and anti-inflammatory abilities against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.

Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

The histone demethylase LSD1 has been known as a key transcriptional coactivator for DNA viruses such as herpes virus. Inhibition of LSD1 was found to block viral genome transcription and lytic replication of DNA viruses. However, RNA virus genomes do not rely on chromatin structure and histone association, and the role of demethylase activity of LSD1 in RNA virus infections is not anticipated. Here, we identify that, contrary to its role in enhancing DNA virus replication, LSD1 limits RNA virus replication by demethylating and activating IFITM3 which is a host restriction factor for many RNA viruses. We have found that LSD1 is recruited to demethylate IFITM3 at position K88 under IFNα treatment. However, infection by either Vesicular Stomatitis Virus (VSV) or Influenza A Virus (IAV) triggers methylation of IFITM3 by promoting its disassociation from LSD1. Accordingly, inhibition of the enzymatic activity of LSD1 by Trans-2-phenylcyclopropylamine hydrochloride (TCP) increases IFITM3 monomethylation which leads to more severe disease outcomes in IAV-infected mice. In summary, our findings highlight the opposite role of LSD1 in fighting RNA viruses comparing to DNA viruses infection. Our data suggest that the demethylation of IFITM3 by LSD1 is beneficial for the host to fight against RNA virus infection.

Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest.

Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.

Towards broad-spectrum protection: the development and challenges of combined respiratory virus vaccines.

In the post-COVID-19 era, the co-circulation of respiratory viruses, including influenza, SARS-CoV-2, and respiratory syncytial virus (RSV), continues to have significant health impacts and presents ongoing public health challenges. Vaccination remains the most effective measure for preventing viral infections. To address the concurrent circulation of these respiratory viruses, extensive efforts have been dedicated to the development of combined vaccines. These vaccines utilize a range of platforms, including mRNA-based vaccines, viral vector vaccines, and subunit vaccines, providing opportunities in addressing multiple pathogens at once. This review delves into the major advancements in the field of combined vaccine research, underscoring the strategic use of various platforms to tackle the simultaneous circulation of respiratory viruses effectively.

Engineering of novel hemagglutinin biosensors for rapid detection and drug screening of Influenza A H7N9 virus.

New pathogenic influenza virus strains are constantly emerging, posing a serious risk to both human health and economic growth. To effectively control the spread of this virus, there is an urgent need for early, rapid, sensitive, simple, and cost-effective detection technologies, as well as new and effective antiviral drugs. In this study, we have successfully achieved a significant milestone by successfully fusing the H7N9 influenza virus hemagglutinin (HA) protein with the nano-luciferase component, resulting in the development of a novel set of biosensors. This remarkable achievement marks the first instance of utilizing this biosensor technology for influenza antibody detection. Our biosensor technology also has the potential to facilitate the development of antiviral drugs targeting specific epitopes of the HA protein, providing a promising avenue for the treatment of H7N9 influenza virus infections. Furthermore, our biosensors have broad applications beyond H7N9 influenza virus detection, as they can be expanded for the detection of other pathogens and drug screening applications in the future. By providing a novel and effective solution to the detection and treatment of influenza viruses, our biosensors have the potential to revolutionize the field of infectious disease control.

The Impact of Medical Resources and Oral Antiviral Drugs on SARS-CoV-2 Mortality - Hong Kong SAR, China, 2022.

Introduction: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates increased transmissibility compared to earlier strains, contributing to a significant number of fatalities in Hong Kong Special Administrative Region (HKSAR), China. Adequate medical resources and medications are essential in mitigating these deaths. This study evaluates the effects of supplementary resources from the Chinese mainland during the fifth wave of the pandemic in HKSAR. Methods: Vector autoregression (VAR) was employed to analyze data from the Oxford coronavirus disease 2019 (COVID-19) Government Response Tracker to assess the effectiveness of control measures during five waves of the pandemic in HKSAR. Additionally, a transmission dynamics model was created to investigate the influence of supplementary medical resources from the Chinese mainland and oral medications on mortality. Results: In the initial four waves, workplace closures, restrictions on public events, international travel bans, and shielding the elderly significantly influenced pandemic management. Contrarily, during the fifth wave, these measures showed no notable effects. When comparing a situation without extra medical resources or COVID-19 oral medication, there was a 17.7% decrease in COVID-19 fatalities with mainland medical resources and an additional 10.2% reduction with oral medications. Together, they contributed to a 26.6% decline in fatalities. Discussion: With the rapid spread of the virus, regional reallocation of medical resources may reduce mortality even when the local healthcare system is overstretched.

Baloxavir marboxil use for critical human infection of avian influenza A H5N6 virus.

BACKGROUND: Recent outbreaks of avian influenza and ongoing virus reassortment have drawn focus on spill-over infections. The increase in human infections with highly pathogenic avian influenza H5N6 virus and its high fatality rate posed a potential threat, necessitating the search for a more effective treatment. METHODS: Longitudinal clinical data and specimens were collected from five H5N6 patients after admission. All patients received antiviral treatment of either sequential monotherapy of oseltamivir and baloxavir or the two drugs in combination. Severity of illness; viral load in sputum, urine, and blood; and cytokine levels in serum and sputum were serially analyzed. FINDINGS: All patients developed acute respiratory distress syndrome (ARDS) and viral sepsis within 1 week after disease onset. When delayed oseltamivir showed poor effects, baloxavir was administered and rapidly decreased viral load. In addition, levels of IL-18, M-CSF, IL-6, and HGF in sputum and Mig and IL-18 in serum that reflected ARDS and sepsis deterioration, respectively, were also reduced with baloxavir usage. However, three patients eventually died from exacerbation of underlying disease and secondary bacterial infection. Nonsurvivors had more severe extrapulmonary organ dysfunction and insufficient H5N6 virus-specific antibody response. CONCLUSIONS: For critical human cases of H5N6 infection, baloxavir demonstrated effects on viral load and pulmonary/extrapulmonary cytokines, even though treatment was delayed. Baloxavir could be regarded as a first-line treatment to limit continued viral propagation, with potential future application in avian influenza human infections and poultry workers exhibiting influenza-like illness. FUNDING: This work was funded by the National Natural Science Foundation of China (81761128014).

Circumventing antivector immunity by using adenovirus-infected blood cells for repeated application of adenovirus-vectored vaccines: proof of concept in rhesus macaques.

Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14(+) cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.

Active immunization with Pseudomonas aeruginosa vaccine protects mice from secondary Pseudomonas aeruginosa challenge post-influenza virus infection.

Background: Influenza virus infection complicated by secondary bacterial pneumonia contributes significantly to death during seasonal or pandemic influenza. Secondary infection of Pseudomonas aeruginosa (P. aeruginosa) in influenza virus-infected patients contributes to morbidity and mortality. Methods: Mice were first infected with PR8 influenza virus, followed by a secondary infection of P. aeruginosa. Body weights and survival rate of mice was monitored daily over 20 days. Bronchoalveolar lavage fluids (BALFs) and lung homogenates were harvested for measuring bacterial titers. Lung tissue section slides were stained with hematoxylin and eosin for microscopic observation. After vaccination with inactivated P. aeruginosa cells or recombinant PcrV protein, the mice were subjected to PR8 influenza virus infection followed by a secondary infection of a P. aeruginosa. The inhibition against P. aeruginosa of serum was evaluated by detecting the growth of P. aeruginosa in broth containing diluted sera. Results: The prior influenza infection greatly enhanced the susceptibility to secondary infection of P. aeruginosa and increased morbidity and mortality in mice. Active immunization with inactivated P. aeruginosa cells could protect mice from secondary P. aeruginosa challenge in influenza virus infected mice. Conclusions: To develop an effective P. aeruginosa vaccine might be a promising strategy to decrease the threat of secondary P. aeruginosa infection in influenza patients.

Influenza Virus Carrying a Codon-Reprogrammed Neuraminidase Gene as a Strategy for Live Attenuated Vaccine.

Live attenuated influenza vaccines offer broader and longer-lasting protection in comparison to inactivated influenza vaccines. The neuraminidase (NA) surface glycoprotein of influenza A virus is essential for the release and spread of progeny viral particles from infected cells. In this study, we de novo synthesized the NA gene, in which 62% of codons were synonymously changed based on mammalian codon bias usage. The codon-reprogrammed NA (repNA) gene failed to be packaged into the viral genome, which was achievable with partial restoration of wild-type NA sequence nucleotides at the 3' and 5' termini. Among a series of rescued recombinant viruses, we selected 20/13repNA, which contained 20 and 13 nucleotides of wild-type NA at the 3' and 5' termini of repNA, respectively, and evaluated its potential as a live attenuated influenza vaccine. The 20/13repNA is highly attenuated in mice, and the calculated LD50 was about 10,000-fold higher than that of the wild-type (WT) virus. Intranasal inoculation of the 20/13repNA virus in mice induced viral-specific humoral, cell-mediated, and mucosal immune responses. Mice vaccinated with the 20/13repNA virus were protected from the lethal challenge of both homologous and heterologous viruses. This strategy may provide a new method for the development of live, attenuated influenza vaccines for a better and more rapid response to influenza threats.

Immunization with a Prefusion SARS-CoV-2 Spike Protein Vaccine (RBMRNA-176) Protects against Viral Challenge in Mice and Nonhuman Primates.

There is an urgent need for a broad-spectrum and protective vaccine due to the emergence and rapid spreading of more contagious SARS-CoV-2 strains. We report the development of RBMRNA-176, a pseudouridine (Ψ) nucleoside-modified mRNA-LNP vaccine encoding pre-fusion stabilized trimeric SARS-CoV-2 spike protein ectodomain, and evaluate its immunogenicity and protection against virus challenge in mice and nonhuman primates. A prime-boost immunization with RBMRNA-176 at intervals of 21 days resulted in high IgG titers (over 1:819,000 endpoint dilution) and a CD4+ Th1-biased immune response in mice. RBMRNA-176 vaccination induced pseudovirus-neutralizing antibodies with IC50 ranging from 1:1020 to 1:2894 against SARS-CoV-2 spike pseudotyped wild-type and variant viruses, including Alpha, Beta, Gamma, and Kappa. Moreover, significant control of viral replication and histopathology in lungs was observed in vaccinated mice. In nonhuman primates, a boost given by RBMRNA-176 on day 21 after the prime induced a persistent and sustained IgG response. RBMRNA-176 vaccination also protected macaques against upper and lower respiratory tract infection, as well as lung injury. Altogether, these findings support RBMRNA-176 as a vaccine candidate for prevention of COVID-19.

Generation of replication-competent recombinant influenza A viruses carrying a reporter gene harbored in the neuraminidase segment.

Replication-competent influenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant influenza A viruses with a neuraminidase (NA) segment harboring enhanced green fluorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specific packaging signals for NA were largely retained for efficient packaging. An "autocleave" 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP+NA and rPR8-NA+EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent influenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.

Determination of serum neutralization antibodies against seasonal influenza A strain H3N2 and the emerging strains 2009 H1N1 and avian H5N1.

BACKGROUND: Humoral virus neutralizing activity is crucial in preventing influenza virus infection. However, the influenza neutralizing activity in the general population remains unclear. METHODS: In this study we performed a serological survey of 200 blood donors from Guangzhou, China. Using a microneutralization (MN) assay, neutralizing activities against influenza A 2009 H1N1, H3N2 and H5N1 were measured. Anti-haemagglutinin antibody was assayed by haemagglutination inhibition (HI) test. Also, antibodies against M1 and M2 matrix proteins were measured using an enzyme-linked immunosorbent assay (ELISA). RESULTS: By MN assay, 86% of the individuals showed neutralizing activity against H3N2, 11% against 2009 H1N1, and none against H5N1. The positive rate for H3N2 increased as the age of individuals increased. Interestingly, males displayed a 4 times higher positive rate against 2009 H1N1 than females. The results of ELISA revealed that 97.5% of the individuals had positive M1 titres and 21% had positive M2 titres. Furthermore, anti-haemagglutinin antibody had a much higher correlation with the neutralization activity than anti-M1 and anti-M2 antibodies. CONCLUSIONS: Neutralizing activities against H5N1 and 2009 H1N1 were low in the general population. Therefore, public health agencies should design strategies for preventing potential H5N1 and 2009 H1N1 pandemics.

Evaluation of the immune response of a H7N9 candidate vaccine virus derived from the fifth wave A/Guangdong/17SF003/2016.

Highly pathogenic influenza H7N9 viruses that emerged in the fifth wave of H7N9 outbreak pose a risk to human health. The World Health Organization has updated the candidate vaccine viruses for H7N9 viruses recently. In this study, we evaluated the immune response to an updated H7N9 candidate vaccine virus, which derived from the highly pathogenic A/Guangdong/17SF003/2016 (GD/16) in mice and rhesus macaques. GD/16 vaccination elicited robust neutralizing, virus-specific immunoglobulin G antibodies and effective protection, but poor hemagglutination inhibition antibody titers. Furthermore, mouse and rhesus macaque serum raised against the previous H7N9 CVV A/Anhui/1/2013 (AH/13) were tested for its cross-reactivity to GD/16 virus. We found that although AH/13-immune serum has poor hemagglutination inhibition reactivity against GD/16 virus, AH/13 elicit efficient cross-neutralizing antibodies and in vivo protection against GD/16. Further studies showed that the hemagglutinin of GD/16 has strong receptor binding avidity, which might be associated with the decreased hemagglutination inhibition assay sensitivity. This study underscores the point that receptor binding avidity should be taken into account when performing quantitative interpretation of hemagglutination inhibition data. A combination of multiple serological assays is required for accurate vaccine evaluation and antigenic analysis of influenza viruses.

Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E). PURPOSE: The study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro. METHODS: The antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway. RESULTS: KD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells. CONCLUSIONS: KD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.

An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques.

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.