Large-scale analysis of B-cell epitopes of envelope: Implications for Zika vaccine and immunotherapeutic development

Data selection

Complete Zika polypeptide sequences for isolates identified between 1968 and 2017 were obtained from the National Center for Biotechnology Information (NCBI) Zika resource²⁸. A total of 409 complete polypeptide sequences were retrieved, and duplicate sequences were removed. Multiple sequence alignment was performed using MUSCLE in the Geneious tool, version 11.0, and the E protein region as extracted²⁹. Lastly, MUSCLE alignment was performed for E protein sequences and duplicate E sequences were subsequently removed.

Phylogenetic analysis

A phylogenetic tree for all unique Zika virus E protein sequences was constructed using the maximum-likelihood method with the PhyML tool, version 3.0³⁰, with 100 bootstrap replications. The tree applied an LG substitutional model to determine the divergence of E protein sequences. Lastly, the phylogenetic tree was edited using the Figtree tool, version 1.4.3.

Homology modeling

The SWISS model³¹ server was used to generate 3D structures of the E proteins of Zika isolates identified in 1968, 2007, 2013, 2015, and 2016. Chain A of the E protein structure (PDB: 5GZN) was used as a template for homology modeling. The best homology model was selected based on global model quality estimate (GMQE) and Qmean statistics. Each homologous 3D structure was evaluated using Ramachandran plots prepared with PROCHECK³². Hydrogen bonds were added using molprobity³³. Each model was subjected to energy minimization using the ModRefiner server described by Xu and Zhang³⁴.

Mapping of antigenic epitopes

E protein-specific antigenic epitopes of monoclonal antibodies (mAbs) isolated from Zika-virus infected humans and Zika immunized mice were retrieved from the Immune Epitope Database (IEDB)³⁵, which is a free resource funded by the National Institute of Allergy and Infectious Diseases devoted to disseminating antigenic epitope data. Linear and conformational B-cell epitopes with positive major histocompatibility complex ligands were selected. B-cell epitope regions mapped with B-cell receptor (BCR)-positive neutralizing antibodies were also selected. Epitopes that mapped with screening peptides and did not elicit an immune response were removed. A total of 7 human and 10 mouse (from mice immunized with E protein) B-cell epitopes were identified. The identified epitopes were annotated against aligned E sequences as well as the 3D structures of monomeric E proteins using the Chimera tool³⁶. Potential sites of N-glycosylation were predicted using NetNglycan 1.0 server³⁷. Potential N-glycosylation sites were defined by the sequence Asp/X/Ser/Thr, where X represents any amino acid except Pro. A threshold of >0.5 suggested an N-glycosylated residue.

Analysis of mutations on E protein stability

The effect of mutations on the stability of E protein was predicted using the mutation cutoff scanning matrix (mCSM)³⁸, site-directed mutator (SDM)³⁹, DUET⁴⁰, and I-Mutant 2.0⁴¹ tools. The mCSM is a machine-learning algorithm based on a 3D physiochemical environment, and the data are summarized as a graphical signature. The SDM is a statistical potential energy function based on the propensity of amino acids in wild-type and mutant proteins to assume folded and unfolded conformations. DUET is an integrated computational approach that utilizes both SDM and mCSM to predict the effect of non-synonymous single-nucleotide polymorphisms on protein stability. Lastly, the I-Mutant webserver is a neural network-based tool for predicting mutation-associated free energy changes. The I-Mutant2.0 tool enables prediction of free energy changes under differing conditions of pH, temperature, neighboring residues, and solvent accessibility.

Strain frequencies

Antigenic variations among Zika strains were examined by first obtaining the complete sequences of Zika polypeptides from the NCBI Zika resource¹⁹. A total of 409 Zika polypeptide sequences were retrieved. Identical polypeptide sequences were removed, resulting in a final total of 257 sequences. Sequences were aligned by MUSCLE using Geneious software, E protein sequences were extracted, and duplicate E protein sequences were removed, resulting in a total of 75 unique sequences (Table 1). Of note, the majority of the 75 unique E protein sequences were represented strains isolated in 2015 and 2016. Sequences from isolates collected in 2017 did not harbor any unique mutations in comparison to sequences from isolates collected in previous years; thus, 2017 sequences were removed after duplicate E protein sequences were removed.

Phylogeny

A phylogenetic tree of unique E protein sequences was constructed using the maximum-likelihood function in PhyML software, with 100 bootstrap replications (Figure 1). Zika strain accession numbers shown in the phylogenetic tree denote the year of isolation. Notably, E protein sequences from isolates collected in 1968, 2007, 2010, and 2012 clustered in one group, indicating that the associated strains are closely related (Figure 1). However, sequences from strains isolated in 2013 and 2014 exhibited divergence from the sequences of strains isolated in previous years (Figure 1).

Figure 1. Phylogenic tree of the unique E protein sequences between the years 1968–2017.

Year 1968 colored (purple), 2007 (yellow), 2010 (cyan), 2012 (green), 2013(red), 2014 (brown), 2015(magenta), and 2016 (blue).

Antigenic epitopes

A number of recent reports describe the isolation of mAbs specific for Zika E protein^23–28. These antibodies bind preferentially to epitopes located in DII and DIII of the E glycoprotein (Table 2). A total of 7 neutralizing mAbs have been isolated from Zika-virus infected humans, and all of these mAbs bind to discontinuous epitopes of E protein. Of note, ZIKV-117 and ZIKV-19 mAbs recognize epitopes located in DII (Table 2), whereas ZIKV-12 and ZIKV-15 mAbs recognize epitopes located in the FL region, and mAbs ZIKV-Z006, and ZIKV-116 as well as the ZKA 190 mAb recognize epitopes located in DIII. Significant overlap between antigenic epitopes specific for ZIKV-Z006 and ZIKV-116 was observed, as three residues in the epitope recognized by the ZIKV-116 mAb are shared with the epitope recognized by ZIKV-Z006 (Table 2). In addition, the epitope region recognized by the ZKA 190 mAb overlapped with that of the mAb specific for ZIKV-Z006 in two amino acids residues.

A total of 10 B-cell epitopes of Zika E protein were found to elicit humoral antibody responses in vaccinated mice (Table 2). Five of these epitopes were shown to be linear and elicited the production of neutralizing antibodies (Table 2). An additional five discontinuous epitopes have been characterized based on antibodies obtained from vaccinated mice (Table 2). The majority of those epitopes are bound to DIII domain of E.

To identify amino acid substitution mutations occurring in B-cell epitopes, we aligned the sequences of 75 unique E protein amino acids sequences among the 422 pre-epidemic and epidemic Zika strains identified. The sequence of Zika/Nigeria/9/9/1968 was used as a reference, and the E protein sequences were mapped against all of the mAbs from Zika infected humans or vaccinated mice. We then annotated the mutations in Zika E glycoprotein at predefined B-cell epitopes. Only nine Zika strains were found to carry mutations in the predefined B-cell epitopes recognized by mAbs from Zika-virus infected humans. Of note, two amino acids substitutions (R335T) and (D393E) appeared in 2007 (Figure 2A). These amino acids substitutions were retained in all subsequent strains from 2007 to 2016. Interestingly, no unique mutations were observed in predefined B-cell epitopes for isolates collected between 2008 and 2014. However, in 2015, two additional substitutions of alanine residues for threonine residues appeared at amino acid positions 309 and 333 (Figure 2A). These mutations were not retained in subsequently isolated Zika strains, with the sequences quickly reverting to those of previously isolated strains. The greatest number of amino acid substitution mutations occurred in 2016; the majority of the mutations identified in 2016 involved several deletions in specific regions of the predefined B-cell epitopes (Figure 2A). Of note, the Dominican Republic/6/6/2016 strain exhibited significant deletions in B-cell epitopes recognized by ZIKV-Z006 mAb (Figure 2A). Surprisingly, 3 B-cell epitopes were completely conserved in the pre-epidemic and epidemic strains and indeed have not changed for nearly 50 years (Figure 2A). These epitopes are recognized by the mAbs ZIKV-117, ZIKV-15, and ZIKV-19. Thus, as these mAbs could exhibit cross-reactivity against a wide range of Zika strains, they have potential for use in the development of vaccines and immunotherapeutics targeting Zika (Figure 2A).

Figure 2A. B-cell epitopes alignments mapped with human anti-E monoclonal antibodies.

A total of 9 Zika strains are variable at predefined B-cell epitopes during 1968–2017.

Conversely, none of 10 mouse B-cell epitopes were completely conserved among all Zika strains (Figure 2B, Figure 2C). Of note, the number of Zika strains exhibiting variations in the amino acid sequence at predefined antigenic epitopes of the E protein was higher in mAbs characterized in vaccinated mice than in mAbs characterized in infected humans (Figure 2B, Figure 2C). In addition, both the linear and discontinuous epitopes of vaccinated mice exhibited variations beginning in 2014 and continuing in subsequent years (Figure 2B, Figure 2C). The majority of mAbs in vaccinated mice recognized the DIII of E protein, and these epitopes exhibited higher rates of sequence variation than did the mAbs recognizing DII (Figure 2B, Figure 2C). Of note, the ZIKV-E-2A10G6 mAb binds to a highly conserved discontinuous epitope in which a single amino acid deletion was observed in Zika strain ATG29285|Homo sapiens|Mexico|17/05/2016. Remarkably, discontinuous epitopes bound the ZV-67 mAb exhibited the highest degree of variation in amino acid sequence among all the Zika B-cell epitopes examined (Figure 2B). Sequence variations were also observed in all of the Zika E protein linear epitopes (Figure 2C).

Figure 2B. Discontinuous B-cell epitopes alignments mapped with mice anti-E monoclonal antibodies.

A total of 18 Zika strains were variable at predefined B-cell epitopes during 1968–2017.

Figure 2C. Linear B-cell linear alignments mapped with mice anti-E antibodies.

A total of 20 Zika strains were variable at predefined B-cell epitopes during 1968–2017.

E protein homology modeling

The recently solved E protein structure of Zika enabled us to construct a homology model of various E protein sequences. Amino acid substitutions in B-cell epitopes were also annotated on the 3D structure of the E protein. The majority of mutations in the E protein were found to be located within DIII, whereas the B-cell epitopes in DII were highly conserved (Figure 3). Zika strain Homo sapiens/French Polynesia/11/13 did not harbor any additional mutations in B-cell epitopes compared with Zika strain Homo sapiens/Micronesia/01/06/07 (Supplementary Figure 1).

Figure 3. Comparison of B-cell epitopes of monomeric E between the years 1968, 2007, 2010, 2015, and 2016.

Homology models of E were built from PDB: 5GZN chain A from strains of following E sequences: AMR68906| Homo sapiens |Nigeria|09/09/1968, ACD75819| Homo sapiens |Micronesia|01/06/2007, ANO46307| Homo sapiens/French Polynesia/11/2013, AMX81917/Homo sapiens/Thailand/16/01/2015, and AMQ48986 | Homo sapiens |USA|02/02/2016. Color blue color represents amino acids conservation at B-cell epitopes and color pink represents amino acids substitutions. Yellow label represent N-glycosylation potential of E.

It is known that N-glycosylation can mask antigenic epitopes. However, as the glycosylation site in the Zika E protein is located remotely from the predefined B-cell epitopes, glycosylation does not mask the B-cell epitope epitopes (Figure 3). This is consistent with reports of glycosylation in E protein of WNV and JEV¹⁸. A recent report demonstrated that glycosylation at 154 is critical for Zika infection of both mammalian and mosquito hosts¹⁷. Our analyses indicate that antigenic changes occur less frequently in Zika strains (Figure 3) and suggest that highly effective neutralizing Zika vaccines and immunotherapies for treating infections with known Zika strains are possible. Consequently, monitoring antigenic changes in E proteins over time would be useful for evaluating the cross-neutralizing potential of Zika vaccines against newly mutated strains.

Mutational stability

Mutation stability was carried out to predict the effects of non-synonymous variants on the stability of E protein and antibody binding. Here, we analyzed the stability of monomeric E protein upon substitutional mutations. To investigate the effect of amino acids substitutions at antigenic epitopes on the stability of E protein and antibody binding of E, 4 prediction tools for mutational stability were selected.

We predicted the stability of B-cell epitope mutations using the 3D structure of Zika E proteins. In both the T309A and T335A substitutions, a polar threonine residue was substituted with a hydrophobic alanine residue. No change in hydrophobicity with the V391I mutation or change in charge with the D393E were observed. In the R335T mutation, a basic residue was substituted with an aromatic residue. Overall, these suggest that defined substitutions in the E glycoprotein are potentially destabilizing. However, these mutations had moderate destabilizing effect, as the ∆∆G values ranged between -0.3 and -0.7 kcal/mol (Table 3).