Impaired arginine/ornithine metabolism drives severe HFMD by promoting cytokine storm.

Introduction: The Hand, Foot and Mouth Disease (HFMD), caused by enterovirus 71 infection, is a global public health emergency. Severe HFMD poses a significant threat to the life and well-being of children. Numerous studies have indicated that the occurrence of severe HFMD is associated with cytokine storm. However, the precise molecular mechanism underlying cytokine storm development remains elusive, and there are currently no safe and effective treatments available for severe HFMD in children. Methods: In this study, we established a mouse model of severe HFMD to investigate the molecular mechanisms driving cytokine storm. We specifically analyzed metabolic disturbances, focusing on arginine/ornithine metabolism, and assessed the potential therapeutic effects of spermine, an ornithine metabolite. Results: Our results identified disturbances in arginine/ornithine metabolism as a pivotal factor driving cytokine storm onset in severe HFMD cases. Additionally, we discovered that spermine effectively mitigated the inflammatory injury phenotype observed in mice with severe HFMD. Discussion: In conclusion, our findings provide novel insights into the molecular mechanisms underlying severe HFMD from a metabolic perspective while offering a promising new strategy for its safe and effective treatment.

Recent Progress in Innate Immune Responses to Enterovirus A71 and Viral Evasion Strategies.

Enterovirus A71 (EV-A71) is a major pathogen causing hand, foot, and mouth disease (HFMD) in children worldwide. It can lead to severe gastrointestinal, pulmonary, and neurological complications. The innate immune system, which rapidly detects pathogens via pathogen-associated molecular patterns or pathogen-encoded effectors, serves as the first defensive line against EV-A71 infection. Concurrently, the virus has developed various sophisticated strategies to evade host antiviral responses and establish productive infection. Thus, the virus-host interactions and conflicts, as well as the ability to govern biological events at this first line of defense, contribute significantly to the pathogenesis and outcomes of EV-A71 infection. In this review, we update recent progress on host innate immune responses to EV-A71 infection. In addition, we discuss the underlying strategies employed by EV-A71 to escape host innate immune responses. A better understanding of the interplay between EV-A71 and host innate immunity may unravel potential antiviral targets, as well as strategies that can improve patient outcomes.

TLR4 signalling: the key to controlling EV71 replication and inflammatory response.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity.

Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9-23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

Low-frequency, exhausted immune status of CD56dim NK cells and disordered inflammatory cytokine secretion of CD56bright NK cells associated with progression of severe HFMD, especially in EV71-infected patients.

BACKGROUND: The roles of CD56bright and CD56dim natural killer (NK) subsets in the viral clearance and inflammatory processes of hand, foot, and mouth disease (HFMD) remain undefined. METHODS: A total of 39 HCs and 55 patients were enrolled to analyze peripheral CD56bright and CD56dim NK cells according to cell number, surface receptors, cytotoxic activities, and cytokine production. The plasma concentrations of IL-2, IL-6, IL-10, IFN-γ, TNF-α,and MCP-1 were detected using ELSA. RESULTS: Peripheral blood NK cells was significantly lower in severe patients than in HCs due to the dramatic loss of CD56dim NK cells with no changes in the cell count of CD56bright NK cells. For mild patients, decreased NKp46 expression coincided with enhanced cytolysis (CD107a, GNLY, and GrB) in CD56dim NK cells and decreased NKG2A expression with enhanced IL-10 production in CD56bright NK cells. In contrast, severe patients showed the dominant expression of NKG2A and decreased expression of NKG2D accompanied by cytotoxic dysfunction in CD56dim NK cells. Imbalanced receptor expression coincided with the increased concentrations of TNF-α in CD56bright NK cells. Moreover, EV71+ patients showed significantly decreased counts of CD56dim NK cells with cytolysis dysfunction, displayed cytokine hypersecretion in CD56bright NK cells, while the EV71- patients displayed significantly higher plasma cytokine concentrations. The changes in the immune function of NK subsets and their subpopulations were closely related to clinical inflammatory parameters. CONCLUSIONS: Low-frequency, exhausted immune status of CD56dim NK cells and disordered inflammatory cytokine secretion of CD56bright NK cells were associated with the progression of severe HFMD, especially in EV71-infected patients. This promoted the severity of inflammatory disorders, leading to enhanced disease pathogenesis.

Proliferation characteristics of coxsackievirus A10 in mice and immune protection ability of experimental inactivated vaccine.

Coxsackievirus A10 (CVA10) is the main pathogen of hand, foot, and mouth disease in China. However, there are no CVA10-specific drugs and vaccines, and the pathogenesis and effects of this virus in the body are unknown. We investigated the effect of a clinically isolated CVA10 virus strain (CVA10-25) to investigate its effect in suckling mice through different infection routes. We observed the dynamic distribution and proliferation of the virus in mouse tissues by infecting suckling mice with different doses of the virus and mice of different ages with the same dose of the virus. We also analysed the pathological characteristics after infection. A formaldehyde-inactivated experimental vaccine was prepared to immunise 5-week-old BALB/c female mice three times, and newborn suckling mice were tested for the presence of maternally transmitted antibodies. The viral load in each organ after intracerebral administration was higher than that after intraperitoneal administration; the peroral administration route did not cause disease in mice. Mouse paralysis and death after infection were related to age. The skeletal muscles, heart, and lung showed histopathological changes after infection. We established a 2-day-old BALB/c suckling mouse model that could be infected intracranially to study the pathogenesis and pathology of CVA10. Maternally transmitted antibodies protected the mice against the virus. This study provides a reference for CVA10-related pathogenesis and vaccine research.

A 10-Day-Old Murine Model of Coxsackievirus A6 Infection for the Evaluation of Vaccines and Antiviral Drugs.

Coxsackievirus A6 (CVA6) is recognized as a major enterovirus type that can cause severe hand, foot, and mouth disease and spread widely among children. Vaccines and antiviral drugs may be developed more effectively based on a stable and easy-to-operate CVA6 mouse infection model. In this study, a wild CVA6-W strain was sub-cultured in newborn mice of different ages (in days), for adaptation. Therefore, a CVA6-A mouse-adapted strain capable of stably infecting the mice was generated, and a fatal model was built. As the result indicated, CVA6-A could infect the 10-day-old mice to generate higher levels of IFN-γ, IL-6, and IL-10. The mice infected with CVA6-A were treated with IFN-α1b at a higher dose, with complete protection. Based on this strain, an animal model with active immunization was built to evaluate antiviral protection by active immunization. The three-day-old mice were pre-immunized with inactivated CVA6 thereby generating IgM and IgG antibodies within 7 days that enabled complete protection of the pre-immunized mice following the CVA6 virus challenge. There were eight mutations in the genome of CVA6-A than in that of CVA6-W, possibly attributed to the virulence of CVA6 in mice. Briefly, the CVA6 infection model of the 10-day-old mice built herein, may serve as an applicable preclinical evaluation model for CVA6 antiviral drugs and vaccine study.

A practical method for evaluating the in vivo efficacy of EVA-71 vaccine using a hSCARB2 knock-in mouse model.

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy in vivo. Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5-6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week-old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titre of antibodies was measured after 1 week. Mice at 4 weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain, and muscles, accompanied by clear pathological changes. The expression of IL-1ß, IL-13, IL-17, and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the in vivo efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.

Kinetics of the neutralising antibody response in patients with hand, foot, and mouth disease caused by EV-A71: A longitudinal cohort study in Zhengzhou during 2017-2019.

BACKGROUND: Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) poses a serious threat to children's health. Kinetics of the neutralising antibody (NAb) response in EV-A71 infected HFMD patients remains unclear. The ideal sampling time of paired serum samples for serological diagnosis of EV-A71 infection is not well defined. METHODS: HFMD inpatients admitted to Henan Children's Hospital between February 15, 2017 and February 15, 2018 were enrolled. Serial serum samples collected during hospitalisation and up to 1.5 years after discharge were tested for NAb against EV-A71. Random intercept modelling with B-spline was conducted to characterize the kinetics of the EV-A71 NAb response over time after illness onset. FINDINGS: A total of 524 serum samples from 264 EV-A71 RNA positive HFMD inpatients were collected. NAb titres of EV-A71 infected patients were estimated to increase from 40 (95% CI: 9-180) at the day of onset to the peak of 2417 (95% CI: 1859-3143) at day 13, then remained above 1240 until 26 months. For serological diagnosis of EV-A71 infection, if at least a 4-fold rise in titre was used as the criteria, the acute phase serum should be collected at 0-4 days, the corresponding convalescent serum should be collected 14.9 days (95% CI: 9.1-23.8) after illness onset. INTERPRETATION: EV-A71 infection induced a strong and persistent humoral immune response in HFMD patients. The findings provide a scientific support for determining the collection time of paired serum samples for serological diagnosis of EV-A71 infected HFMD patients. FUNDING: National Science Fund for Distinguished Young Scholars.

The epidemiological characteristics of enterovirus infection before and after the use of enterovirus 71 inactivated vaccine in Kunming, China.

Enterovirus A71 (EV-A71) inactivated vaccines have been widely inoculated among children in Kunming City after it was approved. However, there was a large-scale outbreak of Enteroviruses (EVs) infection in Kunming, 2018. The epidemiological characteristics of HFMD and EVs were analysed during 2008-2018, which are before and three years after EV-A71 vaccine starting to use. The changes in infection spectrum were also investigated, especially for severe HFMD in 2018. The incidence of EV-A71 decreased dramatically after the EV-A71 vaccine starting use. The proportion of non-CV-A16/EV-A71 EVs positive patients raised to 77.17-85.82%, while, EV-A71 and CV-A16 only accounted for 3.41-7.24% and 6.94-19.42% in 2017 and 2018, respectively. CV-A6 was the most important causative agent in all clinical symptoms (severe HFMD, HFMD, Herpangina and fever), accounting from 42.13% to 62.33%. EV-A71 only account for 0.36-2.05%. In severe HFMD, CV-A6 (62.33%), CV-A10 (11.64%), and CV-A16 (10.96%) were the major causative agent in 2018. EV-A71 inactivated vaccine has a good protective effect against EV-A71 and induced EVs infection spectrum changefully. EV-A71 vaccine has no or insignificant cross-protection effect on CV-A6, CV-A10, and CV-A16. Herein, developing 4-valent combined vaccines is urgently needed.

Regulation of RDN on Th1/ILC1 cell imbalance in HFMD patients caused by EV71 infection.

Enterovirus 71 (EV71) infection is more likely to cause hand, foot and mouth disease (HFMD) in children, which can lead to neurogenic complications and higher mortality. As a commonly used clinical medicine, Reduning injection (RDN) helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children. However, the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed. This study collected detailed treatment data of 56 children with HFMD who entered the affiliated Children's Hospital of Nanjing Medical University during 2019. Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group. To explore its mechanism, the relevant detection indicators were detected by flow cytometry, enzyme-linked immunosorbent assay and real-time quantitative PCR. It was found that the number and function of innate immune (ILCs) and adaptive immunity (Th1, Th2 and secreted cytokines) were reduced, suggesting that RDN plays a role by regulating cellular immunity. The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

Immunomodulatory effects of platelets on the severity of hand, foot, and mouth disease infected with enterovirus 71.

BACKGROUND: Enterovirus 71 (EV71) infection contributes to hand, foot, and mouth disease (HFMD) with severe neurogenic complications, leading to higher morbidity. In addition to their typical roles in coagulation, platelets could serve as essential immune regulatory cells to play a key role in the pathogenesis of this viral infection. METHODS: Platelet parameters were measured using an automatic hematology analyzer. T-helper type 1 (Th1) and Th2 cells were analyzed by flow cytometry. The levels of cytokines and key transcription factors were determined. RESULTS: The levels of platelet count and plateletcrit were positively associated with the severity of HFMD. Th1 and Th2 cells as well as their corresponding cytokines were increased in the severe group compared to the healthy volunteers. Moreover, the levels of platelets were negatively correlated with the level of interferon-γ (IFN-γ), but positively correlated with the frequency of Th1 cells. Coculture of platelets and naive CD4+ T cells showed that platelets from mild patients promote Th1 cell differentiation and IFN-γ secretion. CONCLUSIONS: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4+ T cell differentiation and function in the pathogenesis of HFMD caused by EV71. IMPACT: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4+ T cell differentiation and function in the pathogenesis of HFMD caused by EV71. For the first time, we have discovered the role of platelets in children's HFMD caused by EV71 infection, which may provide a better treatment for HFMD in the future. This article describes new discoveries in platelet immunity.

Abnormalities of ILC1 in children with hand, foot and mouth disease during enterovirus 71 infection.

Children with HFMD due to EV71 infection are more likely to suffer from neurogenic complications, leading to higher morbidity and mortality. ILCs play crucial roles in the initiation of host immunity. However, the contribution of ILCs to the occurrence and development of HFMD due to EV71 infection remains to be explored. The results of our study showed that the levels of peripheral ILC1s and Th1 cells were increased in children with severe HFMD compared to healthy children, as were ILC1- and Th1-related cytokines and transcription factors. Furthermore, HFMD children with a higher frequency of circulating ILC1s exhibited a 2.9-fold greater risk of severity when HFMD was accompanied by VEM. Our study is the first to show that ILC1 abnormalities contribute to the pathogenesis of the severity of HFMD, in which ILC1s are aberrant increased and affect the cellular and humoral immunity. ILC1s could be used in the diagnosis of HFMD.

Immune responses of a CV-A16 live attenuated candidate strain and its protective effects in rhesus monkeys.

Coxsackievirus A16 (CV-A16) is a major causative pathogen of hand, foot, and mouth diseases (HFMDs). The licensed HFMD vaccine targets EV-A71 without cross-protection against CV-A16. Thus, a CV-A16 vaccine is needed. In this study, the immunogenicity and protective efficacy of a live attenuated CV-A16 candidate, K168-8Ac, were evaluated in a rhesus monkey model. Four passages of this strain (P35, P50, P60, and P70) were administered to monkeys, and its protective effect was identified. The immunized monkeys were clinically asymptomatic, except for slight fever. Weak viraemia was observed, and two doses of vaccination were found to significantly reduce virus shedding. High levels of antibody responses were observed (1:1024-1:2048), along with a significant increase in plasma IL-8. The I.M. group showed a much stronger humoural immunity. Pathological damage was detected mainly in lung tissues, although thalamus, spinal cord, lymph nodes, and livers were involved. After the viral challenge, it was found that two doses of vaccine reduced virus shedding, and the degree of lung damage and the number of organs involved decreased as the passage number increased. Overall, a robust immune response and partial protection against CV-A16, triggered by the K168-8Ac strain, were demonstrated. This study provides valuable data for CV-A16 vaccine development.

Emergence of genotype C1 Enterovirus A71 and its link with antigenic variation of virus in Taiwan.

An outbreak of the hand-foot-mouth disease with severe neurological cases, mainly caused by the genotype C1 enterovirus A71 (EV-A71), occurred in Taiwan between 2018 and early 2019. In the recent decade, the most dominant EV-A71 genotypes in Taiwan were B5 and C4 but changed to C1 in 2018. Antibody-mediated immunity plays a key role in limiting the EV-A71 illness in humans. However, the level of neutralizing activities against genotype C1 virus by human polyclonal and monoclonal antibodies (MAbs) remains largely unclear. In the study, we demonstrated that that 39% (9 in 23) of post-infection sera from the genotype B5- or C4-infected patients in 2014-2017 exhibit reduced titers with the 2018-2019 genotype C1 viruses than with the earlier B5 and C4 viruses tested. This finding with polyclonal sera is confirmed with human MAbs derived from genotype B5 virus-infected individuals. The 2018-2019 genotype C1 virus is resistant to the majority of canyon-targeting human MAbs, which may be associated with the residue change near or at the bottom of the canyon region on the viral capsid. The remaining three antibodies (16-2-11B, 16-3-4D, and 17-1-12A), which target VP1 S241 on the 5-fold vertex, VP3 E81 on the 3-fold plateau and VP2 D84 on the 2-fold plateau of genotype C1 viral capsid, respectively, retained neutralizing activities with variable potencies. These neutralizing antibodies were also found to be protective against a lethal challenge of the 2018-2019 genotype C1 virus in an hSCARB2-transgenic mice model. These results indicate that the EV-A71-specific antibody response may consist of a fraction of poorly neutralizing antibodies against 2018-2019 genotype C1 viruses among a subset of previously infected individuals. Epitope mapping of protective antibodies that recognize the emerging genotype C1 virus has implications for anti-EV-A71 MAbs and the vaccine field.

BNIP3 deletion ameliorated enterovirus 71 infection-induced hand, foot and mouth disease via inhibiting apoptosis, autophagy, and inflammation in mice.

Bcl2/adenovirus E1B protein-interacting protein 3 (BNIP3) plays a key role in cellular response to stress by regulating apoptosis and selective autophagy. The present study aimed to determine the effects of BNIP3 on enterovirus (EV) 71 infection-induced hand, foot and mouth disease (HFMD), and the apoptosis, autophagy and inflammatory in mice and SH-SY5Y human neuroblastoma cell line. Neonatal BALB/c mice were injected with EV 71 strain to induce the HFMD. Western blotting and ELISA were used to measure the protein expression and cytokine levels. The BNIP3 mRNA and protein levels in the brain were increased in EV 71-infected mice. By contrast, the BNIP3-knockout (KO) mice with EV 71 infection had higher health score and survival rate. BNIP3 deletion reversed the increase of cleaved-caspase 3, cleaved-caspase 8, Bax, LC3 II and LC3 II/LC3 I levels, and the decrease of Bcl2 and Bcl2/Bax and LC3 I levels in the brain of mice with EV 71 infection. The EV 71 infection-induced increase of tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß, IL-6, interferon (IFN)-α and IFN-γ levels were inhibited in BNIP3-KO mice. BNIP3 knockdown with small interfering RNA (siRNA) inhibited the EV 71 infection-induced the increases of apoptosis, autophagy and inflammatory factors in SH-SY5Y cells. BNIP3 overexpression further facilitated the EV 71 infection-induced increase of these inflammatory factors in SH-SY5Y cells. These results demonstrated that BNIP3 deletion ameliorated EV 71 infection-induced HFMD via inhibiting apoptosis, autophagy and inflammation in mice. BNIP3 may be a therapeutic target for HFMD.

Seroprevalence of EV-A71 neutralizing antibodies following the 2011 epidemic in HCMC, Vietnam.

Enterovirus-A71 (EV-A71) cyclically causes hand-foot-mouth disease (HFMD) epidemics in Asian children. An EV-A71 epidemic occurred in Southern Vietnam in 2011, but its scale is not clear. We collected residual sera from non-HFMD Vietnamese inpatients in 2012-2013 to determine seroprevalence of EV-A71 neutralizing antibodies, and measured cross-reactive neutralizing antibody titers against three EV-A71 genogroups. About 23.5% of 1-year-old children in Southern Vietnam has been infected by EV-A71, and the median age of infection was estimated to be 3 years. No significant antigenic variation could be detected among the three EV-A71 genogroups. The high seroprevalence of EV-A71 neutralizing antibody in children living in southern Vietnam indicates the necessity of introducing EV-A71 vaccines in southern Vietnam, particularly for children under 6 months of age. Moreover, it is critical to understand EV-A71 disease burden for formulating national vaccination policy.

Identification of Antibodies with Non-overlapping Neutralization Sites that Target Coxsackievirus A16.

Hand, foot, and mouth disease is a common childhood illness primarily caused by coxsackievirus A16 (CVA16), for which there are no current vaccines or treatments. We identify three CVA16-specific neutralizing monoclonal antibodies (nAbs) with therapeutic potential: 18A7, 14B10, and NA9D7. We present atomic structures of these nAbs bound to all three viral particle forms-the mature virion, A-particle, and empty particle-and show that each Fab can simultaneously occupy the mature virion. Additionally, 14B10 or NA9D7 provide 100% protection against lethal CVA16 infection in a neonatal mouse model. 18A7 binds to a non-conserved epitope present in all three particles, whereas 14B10 and NA9D7 recognize broad protective epitopes but only bind the mature virion. NA9D7 targets an immunodominant site, which may overlap the receptor-binding site. These findings indicate that CVA16 vaccines should be based on mature virions and that these antibodies could be used to discriminate optimal virion-based immunogens.

Development of an Enterovirus 71 Vaccine Efficacy Test Using Human Scavenger Receptor B2 Transgenic Mice.

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing.IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines.