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Alanine scanning mutagenesis of SP70 epitope in characterizing species?specific antibodies induced by enterovirus 71?based antigens.

ABSTRACT: The enterovirus 71 (EV71) SP70 epitope, derived from amino acids 208?222 of VP1, is a neutralizing epitope. The present study aimed to assess the inter?species differences of the antibodies induced by EV71?based antigens in responses to SP70 mutant peptides. BALB/c mice and Lou/C rats were immunized with EV71 SP70. Monoclonal antibodies (Mabs) were produced by hybridoma clones. Serum polyclonal antibodies (Pabs) were produced from BALB/c mice and New Zealand white rabbits immunized with recombinant EV71 VP1 (rEV71?VP1) protein or inactivated EV71. Micro?neutralization and immunofluorescence assays were used to evaluate the capacity of the antibodies to bind to EV71. Reactivity of Mabs and Pabs to mutated SP70 were determined by alanine scanning mutagenesis. Furthermore, serums from EV71?infected patients were collected to examine the affinity of SP70 antibody in the serum to mutated SP70, using competitive ELISA. The binding affinity of mouse Mabs to the SP70 epitope was increased by alanine substitution at sites of 210, 212, 213, 214, and 221. The binding affinity of rat Mabs to the SP70 epitope was increased by alanine substitution at sites 210, 217, 219, and 221. Mouse serum Pabs elicited by inactivated EV71 bound wild?type SP70, but lost affinity for mutated peptides. Conversely, rabbit serum Pabs elicited by inactivated EV71 robustly recognized SP70 mutants. Mouse serum Pabs elicited by rEV71?VP1 presented the same trend as mouse Mabs. Mutations at sites 214, 215, and 217 led to loss of recognition by rabbit Pabs elicited by rEV71?VP1, while most mutations did not influence antibody binding. Compared with the wild?type, mutations at the sites 209, 219 and 221 of SP70 lead to increased affinity with the serum antibodies produced by the EV71?infected patients. Antibody responses triggered by inactivated EV71, rEV71?VP1 and EV71 SP70 differed among species in neutralizing capacity and affinity for SP70 mutant peptides.

SUBMITTER: Gao M

PROVIDER: S-EPMC5780067 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Alanine scanning mutagenesis of SP70 epitope in characterizing species‑specific antibodies induced by enterovirus 71‑based antigens.

Gao Meng M Zhang Feng F Zhu Yun Y Gao Limei L Jiang Yunshui Y Luo Yongneng Y Zhuang Fangchang F Mao Zian Z Mao Jiangsen J

Molecular medicine reports 20171106 1

The enterovirus 71 (EV71) SP70 epitope, derived from amino acids 208‑222 of VP1, is a neutralizing epitope. The present study aimed to assess the inter‑species differences of the antibodies induced by EV71‑based antigens in responses to SP70 mutant peptides. BALB/c mice and Lou/C rats were immunized with EV71 SP70. Monoclonal antibodies (Mabs) were produced by hybridoma clones. Serum polyclonal antibodies (Pabs) were produced from BALB/c mice and New Zealand white rabbits immunized with recombin ...[more]

PMID: 29115505

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Project description:Despite significant advances in health care, outbreaks of infections by enteroviruses (EVs) continue to plague the Asia-Pacific region every year. Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), for which there are currently no therapeutics. Here, we report two new antibodies, A9 and D6, that potently neutralize EV71. A9 exhibited a 50% neutralizing concentration (neut50) value of 0.1 nM against EV71, which was 10-fold lower than that observed for D6. Investigation into the mechanisms of neutralization revealed that binding of A9 to EV71 blocks receptor binding but also destabilizes and damages the virus capsid structure. In contrast, D6 destabilizes the capsid only slightly but interferes more potently with the attachment of the virus to the host cells. Cryo-electron microscopy (cryo-EM) structures of A9 and D6 bound with EV71 shed light on the locations and nature of the epitopes recognized by the two antibodies. Although some regions of the epitopes recognized by the two antibodies overlap, there are differences that give rise to dissimilarities in potency as well as in the mechanisms of neutralization. Interestingly, the overlapping regions of the epitopes encompass the site that the virus uses to bind SCARB2, explaining the reason for the observed blocking of the virus-receptor interaction by the two antibodies. We also identified structural elements that might play roles in modulating the stability of the EV71 particles, including particle integrity. The molecular features of the A9 and D6 epitopes unveiled in this study open up new avenues for rationally designing antiviral drugs.IMPORTANCE During the course of viral infections, the human body produces neutralizing antibodies which play a defining role in clearing the virus. From this study, we report two new, highly potent neutralizing antibodies, A9 and D6, against enterovirus 71 (EV71), the causative agent of HFMD. Both antibodies prevent the virus from entering the host cell, a step that is important for establishing a successful infection. A9 destabilizes and damages the virus capsid that forms an outer protective covering around the genome of the virus, while also interfering with virus attachment to the host cells. In contrast, D6 only prevents binding of the virus to its receptor(s). The mechanism of neutralization of A9 is unique and has not been observed before for neutralizing antibodies targeting EVs. The two antibodies that we are reporting in this study have potential to be developed into much-needed therapeutic interventions for treatment of HFMD, outbreaks of which are reported every year in the Asia-Pacific region.

Project description: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.

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Alanine scanning mutagenesis of SP70 epitope in characterizing species?specific antibodies induced by enterovirus 71?based antigens.

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Alanine scanning mutagenesis of SP70 epitope in characterizing species‑specific antibodies induced by enterovirus 71‑based antigens.

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