Identification of four neutralizing antigenic sites on the enterovirus D68 capsid.

IMPORTANCE: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 ßB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.

Initial studies on the implanting sites of high and low ventricular septa using leadless cardiac pacemakers.

OBJECTIVE: To study the safety and electrical characteristics of various implanting sites of the Micra pacemaker. METHOD: A total of 15 patients from Beijing Anzhen Hospital, Capital Medical University, were included, who were implanted with Micra leadless pacemakers and allocated to either the high ventricular septum group (eight patients) or the low ventricular septum group (seven patients) based on their individual patient factors and clinical conditions. The baseline of the patients, the implanting area, the electrocardiogram change after implantation, the implantation data, the threshold, R wave, impedance, and the date of the 1-month follow-up were then analyzed. With all of the data, the characteristics of different implantation sites of the Micra pacemaker were determined. RESULTS: Overall, the thresholds were low at implantation and remained stable over the 1-, 3-, 6-month, 1-, 2-, 3-, and 4-year follow-ups. On comparing the two groups, there was no difference in QRS duration at pacing (140.00 [40.00] ms vs. 179.00 [50.00] ms), threshold at implantation (0.38 [0.22] mV vs. 0.63 [1.00] mV), R wave at implantation ([10.85 ± 4.71] V vs. [7.26 ± 2.98] V), or impedance at implantation ([906.25 ± 162.39] Ω vs. [750.00 ± 173.40] Ω). While the difference in QRS duration between the two groups was not significant, the QRS duration of the high ventricular septum group exhibited a reduced tendency compared with that of the low ventricular group. The corrected QT interval during pacing exhibited a significant difference (440.00 [80.00] ms vs. 520.00 [100.00] ms; p < .05). For the 1-, 3-, 6-month, 1-, 2-, 3-, and 4-year follow-ups, there was no difference between the threshold of the high ventricular septum group and that of the low ventricular septum group (p > .05). CONCLUSION: High ventricular septum pacing appears to be a safe site for implantation of the Micra pacemaker. It could entail a shorter QRS duration at pacing and could be more physiological than low ventricular septum pacing.

Congenitally corrected transposition of the great arteries and implantation of a leadless pacemaker: a case report.

BACKGROUND: Congenitally corrected transposition of the great arteries (ccTGA) is a rare cardiac anomaly and can lead to abnormal electrical activity of the heart. The implant of a pacemaker in such patients is more complicated than conventional operations. This case report of an adult with ccTGA who had a leadless pacemaker implant will provide a reference for diagnosing and treating such patients. CASE PRESENTATION: A 50-year-old male patient was admitted to hospital having experienced intermittent vision loss for a month. An electrocardiogram and Holter monitoring showed intermittent third-degree atrioventricular block, and echocardiography, cardiac computed tomography and cardiac magnetic resonance imaging confirmed a diagnosis of ccTGA. A leadless pacemaker was successfully implanted into the patient's anatomical left ventricle, and the postoperative parameters were stable. CONCLUSION: Implanting a leadless pacemaker into a patient with a rare anatomical and electrophysiological abnormality, such as ccTGA, is feasible and efficacious, but preoperative imaging evaluation is of considerable importance.

Occurrence of ventricular septal perforation in patients with permanent left bundle branch pacing followed up using echocardiographic and computed tomography images.

OBJECTIVE: To explore short-term changes after left bundle branch pacing (LBBP) using echocardiography and computed tomography (CT), especially for postoperative ventricular septal perforation. METHODS: Between January and September 2019, 33 patients with atrioventricular block underwent LBBP at Beijing Anzhen Hospital. All the patients were evaluated using electrocardiography, pacing, parameters and echocardiographic measurements, including for major complications, during the 1, 3, 6, 12 and 24-month follow-up. Interval perforations were examined during a 1-month follow-up echocardiogram and CT. RESULTS: Left bundle branch pacing was successfully performed in 100% (33/33) of patients. The mean seizure threshold was stable and unchanged postoperatively at the 1, 3, 6, 12 and 24-month follow-up. The paced QRS duration of the LBBP was 119.72 ± 2.53 ms and <130 ms in all patients. Unipolar impedance during the procedure was higher than 500 Ω (662.00 ± 181.50 Ω). No ventricular septal perforation occurred at the end of the procedure. At the 1-month follow-up, two patients reported transthoracic echocardiography, with CT revealing septal lead perforation. Through CT, two other patients were found to have septal lead perforation, and echocardiography indicated that the pacing lead had penetrated the interventricular septum and entered the left subendocardium. At the 1, 3, 6, 12 and 24-month follow-up, these four patients exhibited no significant increase in pacing threshold or impedance (p > .05). No ventricular thrombus or stroke was detected. CONCLUSION: Permanent LBBP is safe and feasible in patients with bradycardia. Echocardiography and/or CT can more accurately evaluate changes in cardiac structure and function after LBBP.

An AAV-based, room-temperature-stable, single-dose COVID-19 vaccine provides durable immunogenicity and protection in non-human primates.

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.

Functional and structural characterization of a two-MAb cocktail for delayed treatment of enterovirus D68 infections.

Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates.

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

Association Between Blood Glucose Variability and the Characteristics of Vulnerable Plaque in Elderly Non-ST Segment Elevation Acute Coronary Syndrome Patients.

Blood glucose variability is considered to be one of the risk factors for coronary heart disease, and there is growing evidence that blood glucose fluctuation is closely related to the characteristics of plaques. The aim of the study was to investigate the influence of blood glucose variability on the vulnerability of culprit plaques in elderly non-ST segment elevation acute coronary syndrome (NSTE-ACS) patients.Coronary angiography and VH-IVUS were applied to evaluate the components of culprit plaque in NSTE-ACS patients. CGMS monitoring was performed for 72 hours and blood glucose variability was assessed by glycemic excursions (MAGE), absolute means of daily differences (MODD), postprandial glycemic excursions (PPGE), and the largest amplitude of glycemic excursions (LAGE). An oxidative stress indicator (urinary 8-iso-PGF2α) was also tested.Eighty two elderly NSTE-ACS patients were enrolled in this study. Higher glucose variability was associated with the increased culprit plaque instability. MODD was positively correlated with urinary 8-iso-PGF2α. PPGE and urinary 8-iso-PGF2α were independent risk factors for percent fibrous and necrotic volume in culprit plaques (PPGE: ß = -0.340, P = 0.024; urinary 8-iso-PGF2α: ß = -0.294, P = 0.013).Blood glucose variability is positively related to oxidative stress. With an increase in blood glucose variability, the instability of criminal plaques in elderly NSTE-ACS patients increased.

Recombinant virus-like particle presenting a newly identified coxsackievirus A10 neutralization epitope induces protective immunity in mice.

Coxsackievirus A10 (CVA10) has emerged as one of the major pathogens of hand, foot, and mouth disease in recent years. However, there are no approved vaccines or effective drugs against CVA10. Several experimental CVA10 vaccines have been shown to elicit neutralizing antibodies that could confer protection against viral infection. However, neutralizing antigenic sites on CVA10 capsid have not been well characterized. Here, we report the characterization of linear neutralization epitopes of CVA10 and the development of a CVA10 vaccine based on the identified epitopes. We showed that peptide VP2-P28, corresponding to residues 136 to 150 of VP2, were recognized by anti-inactivated CVA10 sera and effectively inhibited anti-CVA10 sera-mediated neutralization, suggesting that this peptide contains neutralizing epitopes. Insertion of VP2-P28 into hepatitis B core antigen (HBc) resulted in a chimeric virus-like particle (VLP; designated HBc-P28) with the CVA10 epitope exposed on the particle surface. HBc-P28 VLP elicited strong antibody responses against VP2-P28 in mice. Anti-HBc-P28 sera could neutralize both CVA10 clinical isolates and prototype strain, consistent with the fact that the VP2-P28 sequence is highly conserved among CVA10 strains. In addition, anti-HBc-P28 sera failed to cross-neutralize other HFMD-causing enteroviruses, indicating that neutralizing antibodies elicited by HBc-P28 VLP were CVA10-specific. Importantly, anti-HBc-P28 sera were able to provide efficient protection against lethal CVA10 infection in recipient mice. Collectively, these data show that peptide VP2-P28 represents a CVA10-specific linear neutralizing antigenic site and chimeric VLP displaying this peptide is a promising epitope-based CVA10 vaccine candidate.

Association of Isoprostanes-Related Oxidative Stress with Vulnerability of Culprit Lesions in Diabetic Patients with Acute Coronary Syndrome.

Urinary excretion of 8-iso-prostaglandin F2α (8-iso-PGF2α), a reliable biomarker for enhanced oxidant stress in vivo, has been described in association with diabetes and coronary heart disease. The aim of this study was to evaluate the relationship between urinary 8-iso-PGF2α levels and the characteristics of coronary culprit lesion in diabetic patients with acute coronary syndrome (ACS). A total of 79 diabetic patients with ACS were included. iMAP intravascular ultrasound (iMAP-IVUS) was performed to evaluate the characteristics of culprit plaques. Fasting urinary 8-iso-PGF2α level was measured and corrected by creatinine clearance. iMAP-IVUS data showed culprit plaques in high urinary 8-iso-PGF2α level patients had a greater percentage of necrotic core and less fibrous components. High urinary 8-iso-PGF2α levels were correlated with increased necrotic plaque components (r = 0.325, P = 0.003). Meanwhile, the presence of thin-capped fibroatheroma (50.0% versus 11.5%, P = 0.003), ruptured plaques (30.8% versus 7.7%, P = 0.035), and thrombus (38.5% versus 7.7%, P = 0.008) were significantly more frequent in the upper tertile of urinary 8-iso-PGF2α levels than in the low tertile. Multivariate analysis showed high levels of urinary 8-iso-PGF2α (OR 4.240, P = 0.007) was independently associated with the presence of vulnerable culprit plaque in diabetic ACS patients. Urinary 8-iso-PGF2α also displayed a significant value in predicting vulnerable plaques in diabetic patients with ACS by constructing the receiver-operating characteristic (ROC) curve (Area under the ROC curve: 0.713, P = 0.001). Urinary 8-iso-PGF2α levels are associated with the vulnerability of the coronary culprit lesion in diabetic patients with ACS and may provide additional information for risk assessment in suspected vulnerable patients.

Urinary 8-iso-prostaglandin F2α as a risk marker for the vulnerability of culprit plaque in diabetic patients with stable coronary artery disease.

We evaluated the association of urinary excretion of 8-iso-prostaglandin F2α (8-iso-PGF2α) with the vulnerability of culprit lesions in 156 age- and sex-matched diabetic stable coronary artery disease (CAD) patients with or without thin-capped fibroatheroma (TCFA) identified by iMAP intravascular ultrasound. Fasting urinary 8-iso-PGF2α level was measured and corrected by creatinine clearance. Compared to non-TCFA group, patients with TCFA had higher urinary 8-iso-PGF2α levels [114.6 (71.1, 181.5) vs. 83.0 (63.2, 138.2) pmol/mmolCr, P = 0.012]. Urinary 8-iso-PGF2α level was positively correlated with percent necrotic volume of culprit lesion (r = 0.218, P = 0.006). High urinary 8-iso-PGF2α level (OR 2.941, P = 0.009) was independently associated with the presence of TCFA and displayed a significant value in predicting TCFA plaques in study patients. The current study indicated that urinary 8-iso-PGF2α may be an important surrogate marker for the vulnerability of culprit lesion in diabetic patients with CAD.

Admission glycemic variability correlates with in-hospital outcomes in diabetic patients with non-ST segment elevation acute coronary syndrome undergoing percutaneous coronary intervention.

OBJECTIVE: The aim of this study is to evaluate the effects of admission glycemic variability (AGV) on in-hospital outcomes in diabetic patients with non-ST segment elevation acute coronary syndrome (NSTE-ACS) undergoing percutaneous coronary intervention (PCI). METHODS: We studied 759 diabetic patients with NSTE-ACS undergoing PCI. AGV was accessed based on the mean amplitude of glycemic excursions (MAGEs) in the first 24 hours after admission. Primary outcome was a composite of in-hospital events, all-cause mortality, new-onset myocardial infarction, acute heart failure, and stroke. Secondary outcomes were each of these considered separately. Predictive effects of AGV on the in-hospital outcomes in patients were analyzed. RESULTS: Patients with high MAGE levels had significantly higher incidence of total outcomes (9.9% vs. 4.8%, p=0.009) and all-cause mortality (2.3% vs. 0.4%, p=0.023) than those with low MAGE levels during hospitalization. Multivariable analysis revealed that AGV was significantly associated with incidence of in-hospital outcomes (Odds ratio=2.024, 95% CI 1.105-3.704, p=0.022) but hemoglobin A1c (HbA1c) was not. In the receiver-operating characteristic curve analysis for MAGE and HbA1c in predicting in-hospital outcomes, the area under the curve for MAGE (0.608, p=0.012) was superior to that for HbA1c (0.556, p=0.193). CONCLUSION: High AGV levels may be closely correlated with increased in-hospital poor outcomes in diabetic patients with NSTE-ACS following PCI.

A virus-like particle-based tetravalent vaccine for hand, foot, and mouth disease elicits broad and balanced protective immunity.

Hand, foot, and mouth disease (HFMD) is an infectious disease that mainly affects infants and children, causing considerable morbidity and mortality worldwide. HFMD is commonly caused by enterovirus 71 (EV71) and coxsackieviruses A16 (CVA16), A6 (CVA6), and A10 (CVA10). Formalin-inactivated EV71 vaccines are currently available in China; however, these vaccines fail to confer cross-protection against infections by other HFMD-causing enteroviruses, highlighting the necessity of developing a multivalent HFMD vaccine. Our previous studies demonstrated that recombinant virus-like particles (VLP) of EV71, CVA16, and CVA6 are capable of inducing protective immunity against homologous virus challenges in mice. In this study, we generated CVA10-VLP using a baculovirus-insect cell expression system and then combined CVA10-VLP with EV71-VLP, CVA16-VLP, and CVA6-VLP to formulate a tetravalent VLP vaccine. Immunogenicity and protective efficacy of tetravalent VLP vaccine was compared with that of monovalent VLP vaccines. Mouse immunization studies revealed that the tetravalent vaccine elicited antigen-specific and long-lasting serum antibody responses comparable to those elicited by its corresponding monovalent vaccines. Moreover, tetravalent vaccine immune sera strongly neutralized EV71, CVA16, CVA10, and CVA6 strains with neutralization titers similar to those of their monovalent counterparts, indicating a good compatibility among the four antigens in the combination vaccine. Importantly, passively transferred tetravalent vaccine-immunized sera conferred efficient protection against single or mixed infections with EV71, CVA16, CVA10, and CVA6 viruses in mice, whereas the monovalent vaccines could only protect mice against homotypic virus infections but not heterotypic challenges. These results demonstrate that the tetravalent VLP vaccine represents a promising broad-spectrum HFMD vaccine candidate.

Insect cell-produced recombinant protein subunit vaccines protect against Zika virus infection.

Infection with Zika virus (ZIKV) may lead to severe neurologic disorders. It is of significant importance and urgency to develop safe and effective vaccines to prevent ZIKV infection. Here we report the development of ZIKV subunit vaccines based on insect cell-produced recombinant proteins. The N-terminal approximately 80% region (designated as E80) and the domain III (designated as EDIII) of ZIKV envelope (E) protein were efficiently produced as secreted proteins in a Drosophila S2 cell expression system. Both E80 and EDIII could inhibit ZIKV infection in vitro, suggesting that they may have folded properly to display native conformations. Immunization studies demonstrated that both E80 and EDIII vaccines were able to trigger antigen-specific antibody and T-cell responses in mice. The resulting anti-E80 and anti-EDIII sera could potently neutralize ZIKV infection in vitro. More importantly, passive transfer of either anti-E80 or anti-EDIII sera protected recipient mice against lethal ZIKV challenge. It is worth noting that the anti-EDIII sera possessed higher neutralizing titers and conferred more complete protection than the anti-E80 sera, indicating that the S2 cell-produced EDIII is a superior ZIKV vaccine candidate compared with the E80. These data support further preclinical and clinical development of a ZIKV subunit vaccine based on S2 cell-produced EDIII.

A Mouse Model of Enterovirus D68 Infection for Assessment of the Efficacy of Inactivated Vaccine.

In recent years, enterovirus D68 (EVD68) has been reported increasingly to be associated with severe respiratory tract infections and acute flaccid myelitis (AFM) in children all over the world. Yet, no effective vaccines or antiviral drugs are currently available for EVD68. Although several experimental animal models have been developed, immunogenicity and protective efficacy of inactivated EVD68 vaccines has not been fully evaluated. To promote the development of vaccines, we established an Institute of Cancer Research (ICR) suckling mouse model of EVD68 infection in this study. The results showed that ICR neonatal mice up to about nine days of age were susceptible to infection with EVD68 clinical strain US/MO/14-18947 by intraperitoneal injection. The infected mice exhibited progressive limb paralysis prior to death and the mortality of mice was age- and virus dose-dependent. Tissue viral load analysis showed that limb muscle and spinal cord were the major sites of viral replication. Moreover, histopathologic examination revealed the severe necrosis of the limb and juxtaspinal muscles, suggesting that US/MO/14-18947 has a strong tropism toward muscle tissues. Additionally, ß-propiolactone-inactivated EVD68 vaccine showed high purity and quality and induced robust EVD68-specific neutralizing antibody responses in adult mice. Importantly, results from both antisera transfer and maternal immunization experiments clearly showed that inactivated EVD68 vaccine was able to protect against lethal viral infection in the mouse model. In short, these results demonstrate the successful establishment of the mouse model of EVD68 infection for evaluating candidate vaccines against EVD68 and also provide important information for the development of inactivated virus-based EVD68 vaccines.

Enterovirus D68 virus-like particles expressed in Pichia pastoris potently induce neutralizing antibody responses and confer protection against lethal viral infection in mice.

Enterovirus D68 (EV-D68) has been increasingly associated with severe respiratory illness and neurological complications in children worldwide. However, no vaccine is currently available to prevent EV-D68 infection. In the present study, we investigated the possibility of developing a virus-like particle (VLP)-based EV-D68 vaccine. We found that co-expression of the P1 precursor and 3CD protease of EV-D68 in Pichia pastoris yeast resulted in the generation of EV-D68 VLPs, which were composed of processed VP0, VP1, and VP3 capsid proteins and were visualized as ~30 nm spherical particles. Mice immunized with these VLPs produced serum antibodies capable of specifically neutralizing EV-D68 infections in vitro. The in vivo protective efficacy of the EV-D68 VLP candidate vaccine was assessed in two challenge experiments. The first challenge experiment showed that neonatal mice born to the VLP-immunized dams were fully protected from lethal EV-D68 infection, whereas in the second experiment, passive transfer of anti-VLP sera was found to confer complete protection in the recipient mice. Collectively, these results demonstrate the proof-of-concept for VLP-based broadly effective EV-D68 vaccines.

A virus-like particle vaccine confers protection against enterovirus D68 lethal challenge in mice.

Enterovirus D68 (EV-D68) is increasingly associated with severe acute respiratory infection and acute flaccid myelitis (AFM) in children around the world. However, neither vaccines nor therapeutic drugs are available for EV-D68. Here we report the development of a virus-like particle (VLP) based experimental EV-D68 vaccine. We found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1 and were visualized as spherical particles of ∼30 nm in diameter. Immunization of mice with EV-D68 VLPs resulted in the production of serum antibodies that displayed potent serotype-specific neutralizing activities against EV-D68 virus in vitro. Passive transfer of anti-VLP sera completely protected neonatal recipient mice from lethal EV-D68 infection. Moreover, maternal immunization with these VLPs provided full protection against lethal EV-D68 challenge in suckling mice. Together, these results demonstrate that the recombinant EV-D68 VLP is a promising vaccine candidate against EV-D68 infection.

A bivalent virus-like particle based vaccine induces a balanced antibody response against both enterovirus 71 and norovirus in mice.

Noroviruses are the main cause of severe viral gastroenteritis, which results in estimated 200,000 deaths each year, primarily in children in the developing world. Genogroup II.4 (GII.4) strains are responsible for the majority of norovirus outbreaks. Enterovirus 71 (EV71), the leading causative agent of hand, foot and mouth disease, has recently been prevalent in Asia-Pacific regions, resulting in significant morbidity and mortality in young children. However, no vaccine is commercially available for either norovirus GII.4 or EV71. Recombinant virus-like particles (VLPs) derived from either GII.4 or EV71 have been shown to be promising monovalent vaccine candidates. In this study, we investigate the possibility to formulate a VLP-based bivalent vaccine for both norovirus GII.4 and EV71. The GII.4- and EV71-VLPs were produced in a baculovirus-insect cell expression system. A bivalent combination vaccine comprised of GII.4 and EV71 VLPs was formulated and compared with monovalent GII.4- and EV71-VLPs for their immunogenicity in mice. We found that the bivalent vaccine elicited durable antibody responses toward both GII.4 and EV71, and the antibody titers were comparable to that induced by the monovalent vaccines, indicating there is no immunological interference between the two antigens in the combination vaccine. More significantly, the bivalent vaccine-immunized mouse sera could efficiently neutralize EV71 infection and block GII.4-VLP binding to mucin. Together, our results demonstrate that the experimental combination vaccine comprised of GII.4 and EV71-VLPs is able to induce a balanced protective antibody response, and therefore strongly support further preclinical and clinical development of such a bivalent VLP vaccine targeting both norovirus GII.4 and EV71.