Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Epidemiology and Relative Severity of Influenza Subtypes in Singapore in the Post-Pandemic Period from 2009 to 2010.

BACKGROUND: After 2009, pandemic influenza A(H1N1) [A(H1N1)pdm09] cocirculated with A(H3N2) and B in Singapore. METHODS: A cohort of 760 participants contributed demographic data and up to 4 blood samples each from October 2009 to September 2010. We compared epidemiology of the 3 subtypes and investigated evidence for heterotypic immunity through multivariable logistic regression using a generalized estimating equation. To examine age-related differences in severity between subtypes, we used LOESS (locally weighted smoothing) plots of hospitalization to infection ratios and explored birth cohort effects referencing the pandemic years (1957; 1968). RESULTS: Having more household members aged 5-19 years and frequent public transport use increased risk of infection, while preexisting antibodies against the same subtype (odds ratio [OR], 0.61; P = .002) and previous influenza infection against heterotypic infections (OR, 0.32; P = .045) were protective. A(H1N1)pdm09 severity peaked in those born around 1957, while A(H3N2) severity was least in the youngest individuals and increased until it surpassed A(H1N1)pdm09 in those born in 1952 or earlier. Further analysis showed that severity of A(H1N1)pdm09 was less than that for A(H3N2) in those born in 1956 or earlier (P = .021) and vice versa for those born in 1968 or later (P < .001), with no difference in those born between 1957 and 1967 (P = .632). CONCLUSIONS: Our findings suggest that childhood exposures had long-term impact on immune responses consistent with the theory of antigenic sin. This, plus observations on short-term cross-protection, have implications for vaccination and influenza epidemic and pandemic mitigation strategies.

Beta-actin variant is necessary for Enterovirus 71 replication.

Enterovirus 71 (EV71) is one of the main etiological agents of the Hand, Foot and Mouth Disease (HFMD) and has been known to cause fatal neurological complications such as herpangina, aseptic meningitis, poliomyelitis-like paralysis and encephalitis. EV71 is endemic in the Asia-Pacific region and causes occasional epidemics. In order to better understand EV71 infection, we compared the proteome between EV71-susceptible and EV71-resistant human Rhabdomyosarcoma (RD) cell line. We found significant differences in the ß-actin variants between the EV71-susceptible RD cells and EV71-resistant RD cells, suggesting that ß-actin, in association with other proteins such as annexin 2 is required in vesicular transport of EV71. This finding further support our previous study that actin potentially plays a role in pathogenesis and the establishment of the disease in HFMD.

Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein.

BACKGROUND: Enterovirus 71 (EV71) has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. RESULTS: We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP) of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain) did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. CONCLUSIONS: Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non-neutralizing epitope, spanning amino acids 142-146 which are located in the VP2 protein's E-F loop. The S/T(144) mutation in this epitope confers a loss of VP2 antigenicity to some newly emerged EV71-C4 strains from China. The corresponding monoclonal antibody 7C7 was used successfully in an AC-ELISA and did not cross-react to coxsackieviruses 4, 6, 10, and 16 in immunofluorescence assay and Western blots. 7C7 is the first monoclonal antibody described, that can differentiate Coxsackievirus 16 from Enterovirus 71.

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in naïve human populations.

Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia.

Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131 This cleavage triggers N-glycine-mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.

Rapid detection of enterovirus 71 by real-time TaqMan RT-PCR.

BACKGROUND: Enterovirus 71 (EV71) is the main etiological agent of Hand, Foot and Mouth Disease (HFMD) and has been associated with neurological complications which resulted in fatalities during recent outbreaks in Asia Pacific region. OBJECTIVE: Develop a real-time TaqMan RT-PCR for rapid detection of EV71. STUDY DESIGN: Specific primers and probe were designed based on highly conserved VP1 region of EV71. The sensitivity of the real-time RT-PCR was evaluated with 67 clinical specimens collected from pediatric patients with suspected HFMD. RESULTS: Our real-time TaqMan RT-PCR showed 100% specificity in detecting EV71 and showed an analytical sensitivity of 5 viral copies. High sensitivity was also achieved in detecting EV71 directly from clinical specimens. CONCLUSIONS: Real-time TaqMan RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.

Development of multiplex real-time hybridization probe reverse transcriptase polymerase chain reaction for specific detection and differentiation of Enterovirus 71 and Coxsackievirus A16.

Large outbreaks of hand, foot, and mouth disease have been reported in the Asia Pacific region over the last few years and resulted in significant fatalities. The 2 main etiologic agents are Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16). Both viruses are closely related genetically and show similar clinical symptoms. However, EV71 are associated with neurologic complications and can lead to fatalities. In this study, we developed a multiplex real-time hybridization probe reverse transcriptase polymerase chain reaction to detect and differentiate EV71 from CA16 using the LightCycler (Roche Molecular Biochemicals). Specific primers and hybridization probes were designed based on highly conserved VP1 region of EV71 or CA16. Our results showed high specificity and sensitivities in detecting EV71 or CA16 from 67 clinical specimens, and no other enterovirus serotype was detected. Rapid diagnosis to differentiate EV71 from CA16 in outbreak situations will enable pediatricians to identify and manage the patients more effectively.

Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide.

Enterovirus 71 (EV71) infections could lead to high mortalities and neither vaccine nor therapeutic treatment is available. We investigated vaccination with a synthetic peptide SP70 representing a neutralizing linear VP1 epitope of EV71 strain 41 (subgenogroup B4) and passive transfer of anti-SP70 antibodies to protect suckling Balb/c mice against EV71 infectivity. When the mouse anti-SP70 antisera with a neutralizing antibody titer of 1:32 were passively administered to one-day-old suckling mice which had been challenged with a lethal dose of 1000 TCID(50) per mouse, the neutralizing anti-SP70 antibodies were able to confer 80% in vivo protection. In contrast, suckling mice which did not receive any anti-SP70 antisera did not survive the viral challenge at day 21 postinfection. Histological examination and real-time RT-PCR assays revealed viral infiltration in small intestines of EV71-infected mice. Interestingly, anti-SP70 antibodies play a major role in the inhibition of EV71 replication in vivo and significantly reduced the viral titer. In conclusion, EV71-neutralizing antibodies elicited by the synthetic peptide SP70 were able to confer good in vivo passive protection against homologous and heterologous EV71 strains in suckling Balb/c mice.

Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides.

Enterovirus 71 (EV71) is the main causative agent of Hand, foot and mouth disease (HFMD) and has been associated with severe neurological diseases resulting in high mortalities. Currently, there is no vaccine available and treatment is limited to palliative care. In this study, antisera were raised in mice against 95 overlapping synthetic peptides spanning the VP1 capsid protein of EV71. Two peptides, SP55 and SP70, containing amino acid 163-177 and 208-222 of VP1, respectively, are capable of eliciting neutralizing antibodies against EV71 in the in vitro microneutralization assay. SP70 was identified to be particularly potent in eliciting a neutralizing antibody titer comparable to that obtained with a whole virion-immune serum. Immunization of mice with either SP55 or SP70 triggered an EV71-specific IgG response as high as that obtained with the whole virion as immunogen. The IgG sub-typing revealed that the neutralizing antibodies elicited by both synthetic peptides are likely belonging to the IgG1 sub-type. Alignment with databases showed that the amino acid residues of SP70 are highly conserved amongst the VP1 sequences of EV71 strains from various sub-genogroups. Altogether, these data indicate that SP70 represents a promising candidate for an effective synthetic peptide-based vaccine against EV71.

Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71.

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It has been associated with severe neurological complications and has caused significant mortalities in large-scale outbreaks in Asia. In this study, we demonstrated an enhanced silencing of EV71 through the use of chemically synthesized 29-mer shRNAs. The 29-mer shRNAs were designed to target three highly conserved regions of EV71 genome. Transfection of rhabdomyosarcoma (RD) cells with the 29-mer shRNAs significantly inhibited EV71 replication in a dose-dependent manner as demonstrated by reduction of viral RNA, VP1 protein and plaque forming units. The inhibitory effects were more potent and were achieved at 10-fold lower concentrations when compared to 19-mer siRNAs reported previously [Sim, A.C.N., Luhur, A., Tan, T.M.C., Chow, V.T.K., Poh, C.L., 2005. RNA interference against Enterovirus 71 infection. Virology 341, 72-79]. The viral inhibitory effects lasted 72 h post-infection and there was no adverse off-target silencing effect. Gene silencing by 29-mer shRNAs targeted at the 3D(pol) region (sh-3D) was the most effective, achieving 91% viral inhibition. Further evaluation found that no enhanced inhibitory effects were observed when sh-3D was cotransfected with each of the other two candidates. This study showed an improvement in triggering RNAi using the more potent 29-mer shRNAs, indicating its therapeutic potential against EV71.

Effective in vivo therapeutic IgG antibody against VP3 of enterovirus 71 with receptor-competing activity.

Passive immunization is an effective option for treatment against hand, foot and mouth disease caused by EV71, especially with cross-neutralizing IgG monoclonal antibodies. In this study, an EV71-specific IgG2a antibody designated 5H7 was identified and characterized. 5H7 efficiently neutralizes the major EV71 genogroups (A, B4, C2, C4). The conformational epitope of 5H7 was mapped to the highly conserved amino acid position 74 on VP3 capsid protein using escape mutants. Neutralization with 5H7 is mediated by the inhibition of viral attachment, as revealed by virus-binding and post-attachment assays. In a competitive pull-down assay with SCARB2, 5H7 blocks the receptor-binding site on EV71 for virus neutralization. Passive immunization of chimeric 5H7 protected 100% of two-week-old AG129 mice from lethal challenge with an EV71 B4 strain for both prophylactic and therapeutic treatments. In contrast, 10D3, a previously reported neutralizing antibody that takes effect after virus attachment, could only confer prophylactic protection. These results indicate that efficient interruption of viral attachment is critical for effective therapeutic activity with 5H7. This report documents a novel universal neutralizing IgG antibody for EV71 therapeutics and reveals the underlying mechanism.

Biochemical and structural characterization of the interface mediating interaction between the influenza A virus non-structural protein-1 and a monoclonal antibody.

We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.

Capsules of virulent pneumococcal serotypes enhance formation of neutrophil extracellular traps during in vivo pathogenesis of pneumonia.

Neutrophil extracellular traps (NETs) are released by activated neutrophils to ensnare and kill microorganisms. NETs have been implicated in tissue injury since they carry cytotoxic components of the activated neutrophils. We have previously demonstrated the generation of NETs in infected murine lungs during both primary pneumococcal pneumonia and secondary pneumococcal pneumonia after primary influenza. In this study, we assessed the correlation of pneumococcal capsule size with pulmonary NETs formation and disease severity. We compared NETs formation in the lungs of mice infected with three pneumococcal strains of varying virulence namely serotypes 3, 4 and 19F, as well as a capsule-deficient mutant of serotype 4. In primary pneumonia, NETs generation was strongly associated with the pneumococcal capsule thickness, and was proportional to the disease severity. Interestingly, during secondary pneumonia after primary influenza infection, intense pulmonary NETs generation together with elevated myeloperoxidase activity and cytokine dysregulation determined the disease severity. These findings highlight the crucial role played by the size of pneumococcal capsule in determining the extent of innate immune responses such as NETs formation that may contribute to the severity of pneumonia.

Human genome-wide RNAi screen reveals host factors required for enterovirus 71 replication.

Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.

Intron position conservation across eukaryotic lineages in tubulin genes.

A compilation of intron positions obtained from a large number of eukaryotic genomes across orthologous tubulins is explored for molecular evolution. Comparison of intron positions for 41 alpha, 80 beta, and 30 gamma tubulin genomic sequences indicates that the putative ancestral tubulin gene contained at least 19, 33, and 52 intron positions distributed at different sites in the coding regions for alpha, beta, and gamma tubulins, respectively. Many intron positions are old and are conserved across different eukaryotic lineages and intron distribution patterns are consistent with 'introns-early' hypothesis.

Human genome -- from pieces to patterns.

A profile of exon-intron lengths in genes shows a normal distribution. This observation suggests that different genes may have portions of their total exon and/or intron lengths in common. In order to explore the common exon-intron structural patterns that may arise due to common lengths across genes, we compared the exon-intron length patterns of annotated human genes. We discovered 1762278 conserved arrangements of exon-intron length across the otherwise unrelated and diverse genomic landscape. The existence of common exon-intron length patterns across unrelated genes suggests for their role of in gene assemblage and human genome design and architecture.

Recombinant non-structural 1 (NS1) protein of dengue-2 virus interacts with human STAT3beta protein.

A combination of yeast two-hybrid library screening, co-immunoprecipitation and immunofluorescence microscopy demonstrated that dengue-2 virus non-structural 1 (NS1) protein can interact with an N-terminally truncated form of human STAT3beta (DeltaN40-STAT3beta) protein. The NS1 protein interacted with the activated STAT3beta protein in vesicle-like structures in the cell cytoplasm. In addition, transfection of dendritic cells with plasmid expressing NS1 protein also resulted in significant induction of tumor necrosis factor-alpha (TNFalpha) and interleukin-6 (IL-6). Since the STAT3beta protein is an acute-phase response factor, its interaction with NS1 protein may influence the pathological changes observed in dengue fever, dengue hemorrhagic fever and dengue shock syndrome.

Differential expression of metallothionein isoforms in nasopharyngeal cancer and inhibition of cell growth by antisense down-regulation of metallothionein-2A.

Biological functions of metallothionein (MT) proteins which are encoded by 10 functional MT isoforms, include cell proliferation, differentiation and apoptosis. The aim of this study was to compare the relative expression levels of functional MT mRNA isoforms in three nasopharyngeal cancer (NPC) cell lines with laryngeal carcinoma and embryonic lung cell lines by quantitative real-time RT-PCR. All the NPC lines exhibited expression of the MT-2A transcript, whereas the MT-1E isoform was expressed in well differentiated HK1 and moderately differentiated TW01 but not in poorly differentiated CNE2 cells. Interestingly, TW01 and HEp-2 laryngeal cancer cells exhibited similar expression profiles with both MT-1E and MT-2A isoforms being detected at levels below those of MRC-5 embryonic lung fibroblasts. Functional studies of the MT-2A isoform by down-regulating expression of this gene with MT-2A antisense oligonucleotide in CNE2 cells, showed a reduction in cell viability and proliferation. These findings may provide valuable information in the search for novel therapeutic strategies against NPC.

Seroepidemiology of Coxsackievirus A6, Coxsackievirus A16, and Enterovirus 71 Infections among Children and Adolescents in Singapore, 2008-2010.

Coxsackieviruses A6 (CV-A6) and A16 (CV-A16) and Enterovirus 71 (EV-A71) have caused periodic epidemics of hand, foot and mouth disease (HFMD) among children in Singapore. We conducted a cross-sectional study to estimate the seroprevalence of these enteroviruses among Singapore children and adolescents. The study was conducted between August 2008 and July 2010. It involved 700 Singapore residents aged 1-17 years whose residual sera were obtained following the completion of routine biochemical investigations in two public acute-care hospitals. The levels of neutralizing antibodies (NtAb) against CV-A6, CV-A16 and EV-A71 were analyzed by the microneutralization test. The age-specific geometric mean titer (GMT) of antibodies against each of the three enteroviruses and the 95% confidence intervals (CI) were calculated. The seroprevalence of CV-A6 and CV-A16 was high at 62.7% (95% CI: 59.1-66.2%) and 60.6% (95% CI: 56.9-64.1%), respectively. However, the seroprevalence of EV-A71 was significantly lower at 29.3% (95% CI: 26.0-32.8%). About 89.7% of the children and adolescents had been infected by at least one of the three enteroviruses by 13-17 years of age. About half (52.3%) were seropositive for two or all three enteroviruses, while only 16.1% had no NtAb against any of the three enteroviruses. High NtAb levels were observed in the younger age groups. CV-A6 and CV-A16 infections are very common among Singapore children and adolescents, while EV-A71 infections are less common. Infection is continually acquired from early childhood to adolescent age.