Enterovirus virus-like-particle and inactivated poliovirus vaccines do not elicit substantive cross-reactive antibody responses.

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.

Seroprevalence of enterovirus D68 in Yamagata, Japan, between 1976 and 2019.

To clarify the epidemiology of enterovirus D68 (EV-D68), an enterovirus rarely identified in the 20th century, we performed seroepidemiological analysis against EV-D68 using sera collected in 1976, 1985, 1990, 1999, 2009, and 2019, as well as Yamagata isolate (EVD68/Yamagata.JPN/2023-89), in Yamagata, Japan. The neutralizing antibody (Ab)-positive rates for those under 20 years old were 61.0%, 82.5%, 84.3%, 46.7%, 50.5%, and 67.9%, in each year, whereas the rates for those above 20 years old were between 93.4% and 99.1%. Generally, geometric mean titers (GMTs）increased with age among children and the total GMT in each year was 25.4, 49.2, 37.2, 30.8, 29.5, and 33.9, from 1976 to 2019, respectively. The findings in this Yamagata-based study showed that the seroprevalence of EV-D68 over the last four decades has increased with age among children, as a susceptible group, and then reaches a plateau of over approximately 80% among adults. This study clearly revealed that EV-D68 was stably transmitted among children in the 20th century, when EV-D68 detection was quite rare.

Seroepidemiology of enterovirus D68 in a healthy population in Beijing, China, between 2012 and 2017: A retrospective study.

To investigate the seroepidemiological features of enterovirus D68 (EV-D68) in the healthy population from 2012 to 2017 in Beijing, China. A retrospective cross-sectional investigation was conducted using serum specimens collected from healthy individuals in Beijing from 2012 to 2017. These samples were tested for neutralization antibodies (NtAbs) against EV-D68. The sera from six EV-D68 infected patients in the acute or convalescent phase were used to determine the protection level of NtAbs against EV-D68. The geometric means of the titers (GMT) of EV-D68 NtAbs in 2012 and 2017 were 92.82 and 242.91, respectively; the seroprevalences of EV-D68 were 89.43% and 98.43%, respectively. The GMT reached its peak in the 11 to 15 age group in 2012, while in 16 to 20 age group in 2017. We also observed that EV-D68 NtAbs titers of six sera from the acute phase were all less than equal to 1:64 and that of three sera from the convalescent phase were all more than 1:64. Anti-EV-D68 NtAbs in the population remained low from 2012 to 2016 but increased significantly in 2017. Although most of the EV-D68 infections remain undetected in Beijing, the risk of a large outbreak of EV-D68 exists and should be taken seriously.

Neutralizing Antibody against Enterovirus D68 in Children and Adults before 2014 Outbreak, Kansas City, Missouri, USA1.

We evaluated enterovirus D68 seroprevalence in Kansas City, Missouri, USA, from samples obtained during 2012-2013. Neutralizing antibodies against Fermon and the dominant 2014 Missouri isolate were universally detected. Titers increased with age. Widespread circulation of enterovirus D68 occurred before the 2014 outbreak. Research is needed to determine a surrogate of protection.

Enterovirus D68 serosurvey: evidence for endemic circulation in the Netherlands, 2006 to 2016.

BackgroundEnterovirus D68 (EV-D68) has caused major outbreaks of severe respiratory illness worldwide since 2010.AimOur aim was to evaluate EV-D68 circulation in the Netherlands by conducting a serosurvey of EV-D68 neutralising antibodies (nAb) among the Dutch general population.MethodsWe screened 280 sera from children and adults in the Netherlands and used two independent sets of samples collected in the years 2006 and 2007 and in the years 2015 and 2016, time points before and after the first EV-D68 upsurge in 2010. Neutralisation capacity of the sera was tested against the prototype Fermon EV-D68 strain isolated in 1962 and against a recent EV-D68 strain (genotype B3) isolated in France in 2016.ResultsRegardless of the time of serum collection, we found remarkably high overall seropositivity (94.3-98.3%) for nAb against both EV-D68 strains. Geometric mean titres increased in an age-dependent manner.ConclusionsOur data suggest that EV-D68 has been circulating in the Netherlands for decades and that the enterovirus surveillance does not accurately capture the prevalence of this clinically relevant pathogen.

Non-polio Enterovirus detection with acute flaccid paralysis: A systematic review.

Acute flaccid paralysis (AFP), as defined by the World Health Organization (WHO), is characterized by an acute onset of limb weakness. In the post-polio era, other enterovirus (EV) serotypes associated with AFP may become more prominent. This study aims to collate the data on the non-polio enteroviruses (NPEV) associated with AFP. A systematic review of published case reports, case series, and surveillance studies of AFP from 1960 through 2017 was undertaken. Data were collected including the country of the study, number of specimens positive for NPEV and available clinical data. The majority of studies originated from Asia. In surveillance studies, EV 71 (a serotype of Enterovirus A) was the most commonly detected serotype with AFP, followed by Enterovirus B serotype echovirus 11 and then Enterovirus B serotype echovirus 11. In case studies and case reports, EV 71 and EV 68 (a serotype of Enterovirus D), were the most commonly detected NPEV. As poliovirus eradication continues, there is a need to ensure that AFP surveillance will also detect other potentially vaccine preventable viruses.

Evaluating Treatment Efficacy in a Mouse Model of Enterovirus D68-Associated Paralytic Myelitis.

Background: Enterovirus D68 (EV-D68)-associated acute flaccid myelitis (AFM) is a devastating neurological disease for which there are no treatments of proven efficacy. The unpredictable temporal and geographic distribution of cases and the rarity of the disease make it unlikely that data from randomized controlled trials will be available to guide therapeutic decisions. We evaluated the following 3 widely used empirical therapies for the ability to reduce the severity of paralysis in a mouse model of EV-D68 infection: (1) human intravenous immunoglobulin (hIVIG), (2) fluoxetine, and (3) dexamethasone. Methods: Neonatal mice were injected intramuscularly with a human 2014 EV-D68 isolate that reliably induces paralysis in mice due to infection and loss of spinal cord motor neurons. Mice receiving treatments were evaluated for motor impairment, mortality, and spinal cord viral load. Results: hIVIG, which contained neutralizing antibodies to EV-D68, reduced paralysis in infected mice and decreased spinal cord viral loads. Fluoxetine had no effect on motor impairment or viral loads. Dexamethasone treatment worsened motor impairment, increased mortality, and increased viral loads. Conclusion: Results in this model of EV-D68-associated AFM provide a rational basis for selecting empirical therapy in humans and establish this model as a useful system for evaluating other potential therapies.

3C Protease of Enterovirus D68 Inhibits Cellular Defense Mediated by Interferon Regulatory Factor 7.

UNLABELLED: Human enterovirus 68 (EV-D68) is a member of the EV-D species, which belongs to the EV genus of the Picornaviridae family. Over the past several years, clusters of EV-D68 infections have occurred worldwide. A recent outbreak in the United States is the largest one associated with severe respiratory illness and neurological complication. Although clinical symptoms are recognized, the virus remains poorly understood. Here we report that EV-D68 inhibits innate antiviral immunity by downregulation of interferon regulatory factor 7 (IRF7), an immune factor with a pivotal role in viral pathogenesis. This process depends on 3C(pro), an EV-D68-encoded protease, to mediate IRF7 cleavage. When expressed in host cells, 3C(pro) targets Q167 and Q189 within the constitutive activation domain, resulting in cleavage of IRF7. Accordingly, wild-type IRF7 is fully active. However, IRF7 cleavage abrogated its capacity to activate type I interferon expression and limit replication of EV-D68. Notably, IRF7 cleavage strictly requires the protease activity of 3C(pro). Together, these results suggest that a dynamic interplay between 3C(pro) and IRF7 may determine the outcome of EV-D68 infection. IMPORTANCE: EV-D68 is a globally emerging pathogen, but the molecular basis of EV-D68 pathogenesis is unclear. Here we report that EV-D68 inhibits innate immune responses by targeting an immune factor, IRF7. This involves the 3C protease encoded by EV-D68, which mediates the cleavage of IRF7. These observations suggest that the 3C(pro)-IRF7 interaction may represent an interface that dictates EV-D68 infection.

Full genome analysis of enterovirus D-68 strains circulating in Alberta, Canada.

A widespread outbreak of enterovirus (EV)-D68 that started in the summer of 2014 has been reported in the USA and Canada. During the course of this outbreak, EV-D68 was identified as a possible cause of acute, unexplained severe respiratory illness and a temporal association was observed between acute flaccid paralysis with anterior myelitis and EV-D68 detection in the upper respiratory tract. In this study, four nasopharyngeal samples collected from patients in Alberta, Canada with a laboratory diagnosis of EV-D68 were used to determine the near full-length genome sequence directly from the specimens. Phylogenetic analysis was performed to study the genotypes and pathogenesis of the circulating strains. Our results support the contention that mutations in the VP1 gene and other regions of the genome causing altered antigenicity, as well as lack of immunity in the younger population, may be responsible for the increased severe respiratory disease outbreaks of EV-D68 worldwide.

A self-amplifying RNA vaccine prevents enterovirus D68 infection and disease in preclinical models.

The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68-neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.

Intrafamilial spread of enterovirus infections at the clinical onset of type 1 diabetes.

BACKGROUND: At the clinical onset of type 1 diabetes mellitus (T1D), enterovirus (EV) infections are suspected to play a role. EVs in blood are seen as a possible biomarker of T1D. EV infections may occur in temporal and geographic clusters and may spread within families. OBJECTIVE: We checked whether EVs were present in the blood of newly diagnosed diabetic probands and of their consenting siblings and parents. We aimed at evaluating the frequency of EV infection, whether infections were spreading within families, and which EV species were involved. SUBJECTS AND METHODS: Blood was drawn from 24 newly diagnosed diabetic children/adolescents and their family members (20 siblings and 41 parents). Blood donors and non-diabetic children/adolescents diagnosed with overweight/short stature were used as controls. RNA was extracted from plasma/leukocytes. Reverse-transcription polymerase chain reaction assays capable of detecting virtually all EV types and of giving preliminary species identification were used. RESULTS AND CONCLUSIONS: EV genomes were found in the blood of 19 of 24 (79%) diabetics, 12 of 20 (60%) non-diabetic siblings, 26 of 41 (63%) parents, and 1 of 29 (3%) pediatric controls. EVs of the A, B, C, and D species were detected, with the B and C species more prevalent. Probands and virus-positive members of each family consistently shared the same EV species. During follow-up, 4 of 20 (20%) siblings of diabetic probands developed T1D with a latency of 3-25 months. In conclusion, infection by different EV species is highly prevalent at the clinical onset and extends to family members. EV may represent a precipitating factor of T1D. However, the disease only develops in a subset of infected individuals.

A peptide vaccine based on a B-cell epitope on the VP1 protein of enterovirus 70 induces a strong antibody response.

Enterovirus 70 (EV70) is the causative agent of acute hemorrhagic conjunctivitis (AHC), for which no effective vaccine is available. This study revealed a high reactivity of the N-terminal region of EV70 VP1 (VP1-1) with an anti-EV70 mouse serum. The analysis of overlapping synthetic peptides of VP1-1 identified a B-cell epitope in this region. The E-peptide (14-ANTVESEIKAELGVI-28) showing the highest reactivity with the anti-EV70 serum induced neutralizing antibodies in mice and reduced the virus titer in the eyes, suggesting that it is a candidate vaccine against AHC caused by EV70.

Global prevalence and case fatality rate of Enterovirus D68 infections, a systematic review and meta-analysis.

A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1-5.0) and 66.3% (95% CI = 40.0-88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1ß, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

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.

Human antibodies neutralize enterovirus D68 and protect against infection and paralytic disease.

Enterovirus D68 (EV-D68) causes outbreaks of respiratory illness, and there is increasing evidence that it causes outbreaks of acute flaccid myelitis (AFM). There are no licensed therapies to prevent or treat EV-D68 infection or AFM disease. We isolated a panel of EV-D68-reactive human monoclonal antibodies that recognize diverse antigenic variants from participants with prior infection. One potently neutralizing cross-reactive antibody, EV68-228, protected mice from respiratory and neurologic disease when given either before or after infection. Cryo-electron microscopy studies revealed that EV68-228 and another potently neutralizing antibody (EV68-159) bound around the fivefold or threefold axes of symmetry on virion particles, respectively. The structures suggest diverse mechanisms of action by these antibodies. The high potency and effectiveness observed in vivo suggest that antibodies are a mechanistic correlate of protection against AFM disease and are candidates for clinical use in humans with EV-D68 infection.

Fluctuations in Antibody Titers against Enterovirus D68 in Pediatric Sera Collected in a Community before, during, and after a Possible Outbreak.

We previously reported a hospital-based epidemiological study on enterovirus (EV)-D68 infection among children during the autumn of 2015, which indirectly inferred an outbreak in Sendai, Japan. In this study, stocked sera of children (aged 0-6 years; without symptoms of infectious diseases) in the Sendai community collected during 4 periods (1 year before, 6 months before, immediately after, and 1 year after the possible outbreak period) were analyzed using the neutralization antibody titer assay to determine community children's immunity levels against EV-D68 infection. The immunity levels were confirmed to have increased during the possible outbreak period and to have gradually waned over 1 year without another outbreak. These results provide background information supporting the results of our previous hospital-based surveillance study.

Antibodies to Enteroviruses in Cerebrospinal Fluid of Patients with Acute Flaccid Myelitis.

Acute flaccid myelitis (AFM) has caused motor paralysis in >560 children in the United States since 2014. The temporal association of enterovirus (EV) outbreaks with increases in AFM cases and reports of fever, respiratory, or gastrointestinal illness prior to AFM in >90% of cases suggest a role for infectious agents. Cerebrospinal fluid (CSF) from 14 AFM and 5 non-AFM patients with central nervous system (CNS) diseases in 2018 were investigated by viral-capture high-throughput sequencing (VirCapSeq-VERT system). These CSF and serum samples, as well as multiple controls, were tested for antibodies to human EVs using peptide microarrays. EV RNA was confirmed in CSF from only 1 adult AFM case and 1 non-AFM case. In contrast, antibodies to EV peptides were present in CSF of 11 of 14 AFM patients (79%), significantly higher than controls, including non-AFM patients (1/5 [20%]), children with Kawasaki disease (0/10), and adults with non-AFM CNS diseases (2/11 [18%]) (P = 0.023, 0.0001, and 0.0028, respectively). Six of 14 CSF samples (43%) and 8 of 11 sera (73%) from AFM patients were immunoreactive to an EV-D68-specific peptide, whereas the three control groups were not immunoreactive in either CSF (0/5, 0/10, and 0/11; P = 0.008, 0.0003, and 0.035, respectively) or sera (0/2, 0/8, and 0/5; P = 0.139, 0.002, and 0.009, respectively).IMPORTANCE The presence in cerebrospinal fluid of antibodies to EV peptides at higher levels than non-AFM controls supports the plausibility of a link between EV infection and AFM that warrants further investigation and has the potential to lead to strategies for diagnosis and prevention of disease.

Molecular epidemiological study of enterovirus D68 in hospitalised children in Hong Kong in 2014-2015 and their complete coding sequences.

Background: Human enterovirus D68 (EV-D68) was first isolated in 1962 and has aroused public concern recently because of a nationwide outbreak among children in 2014-2015 in the USA. The symptoms include fever, runny nose, sneezing, cough and muscle pains. It might be associated with severe respiratory illness in individuals with pre-existing respiratory conditions and its potential association with acute flaccid myelitis is under investigation. In Asia, EV-D68 cases have been reported in several countries. The study: We aimed to understand the EV-D68 prevalence and their genetic diversity in Hong Kong children. Methods: A total of 10 695 nasopharyngeal aspirate (NPA) samples from hospitalised patients aged <18 years were collected from September 2014 to December 2015 in two regional hospitals. NPAs tested positive for enterovirus/rhinovirus (EV/RV) were selected for genotyping. For those identified as EV-D68, their complete coding sequences (CDSs) were obtained by Sanger sequencing. A maximum-likelihood phylogeny was constructed using all EV-D68 complete coding sequences available in GenBank (n=482). Results: 2662/10 695 (24.9%) were tested positive with EV/RV and 882/2662 (33.1%) were selected randomly and subjected to molecular classification. EV-D68 was detected in 15 (1.70%) samples from patients with clinical presentations ranging from wheezing to pneumonia and belonged to subclade B3. Eight CDSs were successfully obtained. A total of 10 amino acid residue polymorphisms were detected in the viral capsid proteins, proteases, ATPase and RNA polymerase. Conclusion: B3 subclade was the only subclade found locally. Surveillance of EV-D68 raises public awareness and provides the information to determine the most relevant genotypes for vaccine development.