Identification of a neutralizing linear epitope within the VP1 protein of coxsackievirus A10

Coxsackievirus A10 (CV-A10) is a human enterovirus belonging to the A species of the Enterovirus genus within Picornaviridae family [1]. CV-A10-associated hand, foot, and mouth diseases (HFMD) typically manifest mild and self-limiting symptoms. Rarely, severe clinical manifestations, such as herpangina, acute hemorrhagic conjunctivitis, acute respiratory tract infections, viral myocarditis, and even death, are observed in a few cases [2, 3]. Recently, the increasing incidence of CV-A10 infection has been an alarming trend [4]. In addition, the co-circulation of CV-A10 with other human enteroviruses may increase the likelihood of co-infection and the risk of EV genetic recombination. However, there is no specific medicine or vaccine registered against CV-A10. Several CV-A10 vaccines, including inactivated vaccines, virus-like particles (VLPs), and VLPs derived from non-EV-core viruses, have been developing for years [5].

CV-A10 is a non-enveloped icosahedral particle with a diameter of approximately 24–30 nm. The genome is approximately 7500 nt in length and consists of a single stranded positive sense RNA genome with two open reading frames (ORF1 and ORF2) [6]. ORF1 encodes a polyprotein. Further cleavage of polyprotein generates three precursor proteins. Four structural proteins (VP1, VP2, VP3, and VP4) derived from the precursor protein P1 are assembled to form the virus capsid [7]. P2 and P3 are subdivided into the nonstructural viral proteins, including 2A, 2B, 2C, 3A, 3B, 3C, and 3D [8].

Studies have shown that multiple protruding loops, such as B–C loop (residues 97–105), E–F loop (residues 163–177), G-H loop (residues 208–225), and proximity of C-terminus (residues 253–267) in VP1 of EV-A71 have been identified as the significant antigenic proteins exposed on the viral surface [9‐12]. The neutralizing epitopes in these loop structures have been extensively characterized by neutralizing antisera and monoclonal antibodies. The results indicate that VP1 protein of enterovirus contains potential neutralizing epitopes and is the primary target for diagnostic reagents and vaccine research [13‐15]. However, neutralizing epitopes in CV-A10 VP1 have not been experimentally confirmed.

The conventional approach for identifying immunodominant linear neutralizing epitopes on the viral capsid is made by screening a peptide library covering the entire protein sequences. These peptides are analyzed with the polyclonal or monoclonal antibody, followed by the determination of their neutralization-inhibitory effects [6, 16], which are generally considered expensive and time consuming. Bioinformatics can be utilized to rapidly predict linear epitopes and present their advanced structural characteristics [17, 18]. When combined with immunological techniques, the efficiency and precision of identifying epitopes can be improved [19]. Current linear epitope prediction includes the single parametric method and the multi-parameter synthetic prediction method utilizing online servers, such as ABCpred, BepiPred, and SVMTrip [20]. The physicochemical properties of epitopes, including secondary structure, hydrophilicity, flexibility, antigenicity, and surface accessibility, can be analyzed by PSIPRED and DNA STAR software. Furthermore, the location of epitopes can be represented visually by three-dimensional (3D) models. These techniques have been extensively applied to investigate the epitopes of influenza virus, hepatitis A virus, SARS-CoV-2, and other pathogens [21‐23].

In the current study, bioinformatics techniques were applied to predict three candidate linear neutralizing epitopes within the VP1 protein of CV-A10. The candidate epitope peptides were subsequently synthesized and conjugated with keyhole limpet hemocyanin (KLH) for the immunization of mice. The levels of specific IgG and neutralizing antibodies were evaluated in the sera of mice. EP4, a highly neutralizing epitope peptide, was identified as a potential neutralizing epitope. Notably, EP4 double-peptide demonstrated enhanced neutralizing protection, which could passively protect neonatal mice from lethal CV-A10 challenges, making it a promising candidate for CV-A10 vaccine.

Neutralizing antibodies are elicited by the neutralizing epitopes. The level of neutralization titer is related to the amount of effective viral antigen, which is suggested as a criterion for evaluating the efficacy of vaccine candidates. Therefore, neutralizing epitopes play a key role in vaccine effectiveness and can serve as biomarkers to monitor vaccine efficacy [32]. The discovery and identification of neutralizing epitopes is critical to vaccine development.

Recently, CV-A10 has become one of the most prevalent pathogens of HFMD [33]. The linear neutralizing epitopes are essential for vaccine development of CV-A10 and its diagnosis [34]. Historically, epitopes were identified and screened by chemo-synthesizing short overlapping peptides based on their sequences. Immunological testing was then used to confirm the antigenic and immunogenic properties of each peptide. This method requires a great deal of time, human resources, and materials, and it may miss some potential neutralizing epitopes despite effectively covering the entire polypeptide chain.

In this study, online servers predicted thirteen epitopes with high scores (Additional file 1: Table S1). Based on the secondary structure analysis of VP1 protein, peptides 15, 11 and 3 with the α-helical or β-strand structure were eliminated (Additional file 1: Fig. S1A). Then, based on hydrophilicity, elasticity, antigenicity, and advanced structure of VP1, five linear neutralizing epitope candidates for CV-A10 VP1 were selected. A 3D model displayed the spatial locations of these five epitopes and demonstrated that EP1 and EP2 were embedded within the capsid, whereas EP3–EP5 were located on the exterior of the capsid. Since neutralizing epitopes are immunoreactive regions on antigens and are responsible for binding to immune cell receptors or free antibodies, they are typically found on the surface of the viral capsid. EP1 and EP2, therefore, do not meet the requirement for neutralization.

Furthermore, Dai's study demonstrated that peptides in the EP1 region are antigenic but lack neutralizing properties [6]. In addition, it is demonstrated that bioinformatics is superior to overlapping peptide mapping for locating the region that corresponds to neutralizing epitope characteristics. EP3–EP5 was synthesized and then administered to mice. ELISA demonstrated that all three candidate epitopes could induce elevated levels of self-specific IgG. EP5 induced the highest IgG titer, possibly because its spatial configuration is more conducive to the recognition by immune cells. In a micro-neutralization assay, EP4 was found to have a higher neutralizing antibody titer than EP3 and EP5. It suggests that EP4 may be a potential neutralizing epitope of CV-A10 VP1. To obtain a high-titer neutralizing antibody against EP4 and demonstrate its protective efficacy in vivo, tandem arrays of EP4 double-peptides conjugated with KLH were synthesized. The geometric mean neutralizing antibody titer against CV-A10 in antiserum from mice immunized with double-peptide EP4 was 1:50.79. To verify the passive protection efficiency of double-peptide EP4 in vivo, an experimental animal model of CV-A10 infection was used [29]. The double-peptide EP4 provided 40% protection against the 50 × LD50 CV-A10 challenge in neonatal mice, whereas the PBS or the irrelevant peptide conferred no protection. After further refinement, EP4 double-peptide could be used to develop a vaccine against CV-A10 epitope based on the above results.

Studies demonstrated that EV-A71 and CV-A16 utilize human scavenger receptor class B, member 2 (hSCARB2), or human P selectin glycoprotein ligand 1 (PSGL-1) as cellular receptors [35]. The locations of epitopes in CV-A6 VP1 are comparable to those in EV-A71, including the BC loop, EF loop, GH loop, and C-terminus [9‐12]. PSGL-1 was considered the cell receptor of CV-A10 [11], so the neutralizing epitopes of CV-A10 VP1 may be located in the same region as EV-A71. Sequence analysis and 3D modeling supported the hypothesis, as mentioned earlier, EP4 was located in EF loop of CV-A10 VP1 [36], the exact location as SP55 of EV-A71 VP1 and PEP55 of CV-A16 VP1. EP4 amino acids were highly conserved in CV-A10 genotype, sharing approximately 60% and 80% sequence identity with EV-A71 SP55 and CV-A16 PEP55, respectively, indicating that EP4 can also be used as a specific target for detecting CV-A10. EP4 contains three amino acids (G164S, A167S, and Q169A) different from SP55 and PEP55 that distinguish it from EV-A71 and CV-A16, suggesting that these three amino acids determine the specificity of EP4. Using a mutagenesis strategy in EP4, the relationship between EP4 and virulence, transmissibility, or pathogenicity of CV-A10 can be further investigated.

In this study, a neutralizing epitope of CV-A10 VP1 was identified. Additional bioinformatic and immunology research is required to determine if neutralizing epitopes exist in BC loop of VP1, GH loop of VP2, and GH loop of VP3. Interestingly, the immune protective range can be expanded by replacing the original epitope with a heterologous neutralizing epitope on the capsid of an EV-A71 VLP vaccine or inactivated vaccine [36, 37], suggesting that EP4 could be used as a candidate epitope for developing the novel polyvalent chimeric vaccine.

On CV-A10 VP1, a highly conserved linear neutralizing epitope (EP4) has been identified. Neutralizing antibodies elicited by EP4 could protect mice from a lethal CV-A10 challenge. It can be used as a guide for designing CV-A10 epitope peptides or enterovirus polyvalent vaccines.