ZKV is a small enveloped plus strand RNA virus belonging to the genus Flavivirus, which includes many human pathogenic viruses, such as Dengue virus (DNV), yellow fever virus (YFV), West Nile Virus (WNV), and hepatitis C virus (HCV). ZKV has a 10.8 kb RNA genome, containing a single open reading frame flanked by a 5′-UTR (106 nt long) and a 3′-UTR (428 nt long). The open reading frame encodes a polyprotein precursor, which is processed into three structural proteins [capsid (C), premembrane (prM), and envelope (E)] and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The viral E protein is the major surface glycoprotein of flavivirus, and the non-structural NS3 and NS5 encode essential enzyme activities for viral reproduction. The E protein is divided into three discernible domains (Domain I, Domain II, and Domain III). Domain I is involved in the envelope structure organization, and Domain II and Domain III are related to the monomers interaction and receptor binding, respectively (4).

Significant information is available about the protective role of T cell responses against other flaviviruses of clinical importance. Prevention of infection is achieved primarily by neutralizing antibodies but T cell responses (both CD4+ and CD8+) are of utmost importance for virus clearance. Cytotoxic CD8+ T cells are critical to eliminate virus-infected cells while CD4+ T cells provide help to cytotoxic CD8+ T cells and antibody production (5, 6). DNV-specific CD8+ T cells play a protective role in natural DNV infection both in humans and in animal models (7) and polyfunctional CD8+ responses are associated with protection against disease (8). CD8+ T cell immunity has been shown to be protective against WNV infection (9). Vaccination with a tetravalent DNV vaccine elicits CD8+ T cell responses against highly conserved epitopes (10). Similar, the live-attenuated 17D-based YFV vaccine elicits potent and long-lasting CD8+ T cell responses (11–13). Progress toward understanding the role of CD4+ T cell immunity in flavivirus infection is recent. YFV 17D-204 vaccination and adoptive transfer experiments demonstrate that CD4+ T cells contributed to protection against virulent YFV (14). Similar CD4+ responses have been found to be critical for protection against DNV challenge (15) and for the prevention of encephalitis during WNV infection (16). More recently, the CTL response in a murine ZKV model has shown to be crucial for protection against ZKV infection, both in CD8 depletion experiments in mice and passive transfer of memory CD8+ T cells to naive mice exposed to infection. Furthermore, deletion of the CD8a−/− gene leads to 100% death after infection. This CD8+ T cell response is cytotoxic, polyfunctional, and targeted to several H-2D-restricted epitopes (17).

Following the acute phase infection of ZKV (with or without clinical symptoms), the persistence of biomarkers of ZKV infection (e.g., viral RNA in semen) suggest that some cells may be chronically infected. The wide distribution of types and anatomical locations of cells permissive for ZKV infection, sometimes beyond the easy reach of antibodies (e.g., blood–brain barrier), suggest that a cell mediated immune response will be critical for immune surveillance of chronically infected cells. While there can be little doubt that a ZKV vaccine stimulating a neutralizing antibody response will be a key resource in limiting viremia during the acute phase of ZKV infection, there are some concerns regarding the exact nature of the antibody response provoked. The exact pathological mechanism which drives Guillain–Barré syndrome (GBS) remains unknown although there seems to a general consensus that antiglycolipid antibodies play an important role, although not every GBS patient develop this type of antibody. As discussed earlier, there is an increased incidence of GBS associated with ZKV infection (18, 19), but it is not known whether antiganglioside antibodies have a role in this specific comorbidity of ZKV infection. Each of the four different DNV serotypes (DNV 1–4) provoke cross-reactive antibody responses that may contribute to the increased disease severity observed following subsequent infection with a different serotype. The first DNV infection is either subclinical or result in a mild disease, and results in long lasting immunity to the serotype. The next DNV infection, if initiated by a different serotype, can induce severe, potentially lethal disease termed Dengue hemorrhagic fever/Dengue shock syndrome (20, 21). The immunopathogenesis of severe disease is not completely understood. One model, termed antibody-dependent enhancement (ADE), works as follows: anti-DNV antibodies evoked by the primary infection, which were once neutralizing but are not with the current serotype, bind the second serotype viral particles and promote antibody mediated phagocytosis by myeloid antigen-presenting cells which in turn become infected serving as a future reservoir for infectious virions with impaired functional activity (22). Of note are recent reports demonstrating that preexisting anti DNV abs can enhance ZKV infection (23, 24). Conversely, preexisting serum anti-ZKV antibodies were able to enhance DNV infection in vitro (25). This is due to the high serological crossreactivity between both flaviviruses which may not be cross-neutralizing. This crossreactivity is so relevant that it has delayed the development of highly specific, non-DNV crossreactive serodiagnostic tests for ZKV infection. An additional concern for flavivirus vaccination-induced pathogenic antibodies in humans came from the recent reports of severe DNV breakthrough infections requiring hospitalization, after vaccination of seronegative volunteers with an antibody-inducing DNV attenuated virus tetravalent vaccine (Dengvaxia^®), a phenomenon possibly related to ADE (26). This is a special concern since epidemics of both flaviviruses occur simultaneously in the same regions (27). Their research using a mouse model exhibiting much of the same symptoms/pathology of Dengue fever in humans, concluded “a sub-protective humoral response may, under some circumstances, have pathological consequences.” This group has since shifted their focus to inducing CD8+ T cell-mediated immunity to DNV (7, 28–31). Furthermore, the possibility that preexisting non-neutralizing anti-ZKV antibody-dependent enhancement could facilitate infection of fetal–mother interface tissues and contribute to fetal ZKV infection has not been excluded yet. Of note, currently studied ZKV candidate vaccines currently in the pipeline, either in the preclinical or phase I trial (one ongoing trial) phases, aim to elicit antibodies and are all based on whole envelope proteins, or whole inactivated or live attenuated virus (32). Preclinical studies using vaccines encoding whole ZKV preM/E proteins in DNA form, using adenovirus vectors, or whole inactivated ZKV in non-human primate models have been able to elicit neutralizing antibodies and protection after ZKV challenge (33, 34).

Taken together, these findings suggest caution in needed in the development of whole protein ZKV vaccines where evoked antibody responses that are not neutralizing may possibly enhance infection or be pathogenic (i.e., autoimmune) or could facilitate infection of maternal–fetal interface tissue.

Given the concerns with antibody-inducing flavivirus vaccines, one possible alternative would be to harness the power of the T cell immune response in protecting against flavivirus infection, as mentioned above. A recent report has shown that CD8+ T cell prevent antigen induced antibody-dependent enhancement of Dengue disease in a murine model and several studies have identified DNV T cell epitopes appropriate for inclusion in a T cell-based vaccine (31, 35–38). Another recent study shows the critical role of CTL response for protection against ZKV infection in a mouse model; this article identifies ZKV H-2D restricted epitopes recognized by CD8+ T cells from infected mice (17). Recent clinical trials have demonstrated the efficacy of T-cell-inducing vaccines against a number of diseases (39), but immunization with whole proteins may favor responses to regions subject to antigenic drift and immune escape. A way to counteract this is to focus the response into specific desirable epitopes. The T cell epitope-based vaccine approach may target the immune response only to desirable and relevant epitopes, instead of the whole protein. Relevant epitopes include those that come from conserved viral protein regions, and/or where mutations could lead to reduced viral fitness, and those that bind to multiple MHC variant molecules—thus potentially recognized by the majority of the target population—while avoiding regions that are poorly immunogenic, variable and subject to antigenic drift, or that could cause a harmful response (40). These targeted immune responses could lead to increased potency, as well as increasing safety (41, 42). There are several ongoing clinical trials of T cell epitope-based Influenza vaccines aiming to be universal vaccines (43). Mapping and selection of potential immunogenic T cell epitopes is a crucial step that may be performed either with the aid of bioinformatics tools and experimental confirmation or by empirical approach using peptide library spanning the antigen full sequence.

In chronic HIV infection there exists a reservoir of latent, transcriptionally silent viral infection within the resting memory CD4+ T cell compartment and specific myeloid lineage cells (e.g., CD14+/CD16+ monocytes) [reviewed in Ref. (44, 45)]. The resting CD4+ memory cells have long life spans, can remain quiescent, and similar to some of the ZKV tissue targets such placental, neuronal, and gonadal tissues as recently described in mice (46), may reside in immune-privileged sites such as the B cell follicle of lymph nodes, allowing escape from existing immune surveillance mechanisms (47). While the mechanism that triggers active replication in HIV+ CD4+ memory cells is poorly understood, interruption of antiretroviral therapy is associated with the resumption of viral replication. Unfortunately, preexisting HIV-1-specific CD8+ T cell responses have shown to be ineffective [reviewed in Ref. (48)] due to viral evolution of CTL epitopes, resulting in a limited repertoire of effective of cytotoxic T cell-mediated immune responses (49) and progression to AIDS. In HIV infection there are selection pressures exerted by the cellular immune system which result in antigenic drift in new virons (50).

A recent murine model study has demonstrated the potential importance of the CTL response to ZKV infection where H-2D restricted CTL epitopes were identified (17). Studies of HIV specific CTL responses in a subset of HIV+ individuals may also prove informative. HIV controllers (i.e., individuals who are HIV+ yet maintain low viral loads and do not progress to AIDS) have been carefully studied (51). HIV controller status is associated with the ability to develop CTL responses to regions of HIV proteins critical for maintenance of their structure–function (and viral fitness). Pereyra et al. (51) demonstrated that it may be possible to predict CTL class I epitopes favored by HIV controllers and suggested that CTL vaccines designed to evoke cellular immune responses to MHC class I restricted epitopes found within viral protein regions resistant to antigenic drift could lead to improved efficacy of HIV vaccines perhaps mimicking what happens naturally in HIV controllers. Our group has been inspired by these studies and has selected this general approach in the development of a CTL vaccine for ZKV.

Flaviviruses mutate in response to immune system pressure, both by antibodies and T cells. It has been reported that HLA class I-binding residues of a CD8+ T cell epitope encompassing the conserved catalytic site of DNV NS3 protease suffer variation that can abrogate HLA class I binding, suggesting evasion of DNV from a specific CD8+ T cell response by antigenic drift (52). Antigenic drift in ZKV has not been thoroughly studied, but a phylogenetic analysis of contemporary human isolates show a common ancestor and as many as 34 amino acid substitutions relative to the common ancestors with most of the variation contained within the prM protein (53, 54), suggesting that ZKV does not undergo viral evolution as fast as HIV does. However, a recent phylogenetic study on 17 whole ZKV genomes from human isolates in the present epidemic has shown the mutation rate varies between 12 and 25 bases (0.12–0.25% of the polyprotein) per year since the 2013 Polynesia outbreak. The latest sequence shows 64 mutations; and overall, 62 non-synonymous amino acid changes were observed among all sequences analyzed, demonstrating that the ZKV continues to mutate at a rapid rate during the current epidemic (55). The rationale of focusing CTL attack to ZKV protein regions that are “intolerant” to amino acid substitutions thus remains sound.

Human class I epitopes have not yet been formally identified for ZKV. Some authors have published ZKV MHC class II epitope prediction based on MHC binding search engines alone (35, 56, 57). In order to generate a realistic list of MHC-1 binding peptides on ZKV E and M proteins, not only did we use a binding prediction tool, but we also performed matching known DNV class I epitopes to peptides on ZKV. This is warranted due to the antigenic similarity of DNV and ZKV, which display 44–68% sequence identity, as well as the reported crossreactivity to ZKV of DNV envelope-specific antibodies (58). An additional layer of identification was the structural entropy analysis described in the next section.

We generated the predicted ZKV epitope list using the sequence of ZKV Strain H/PF/2013 (GenBank Accession number: KJ776791.2) (59). This strain was isolated from an infected patient during the French Polynesia epidemic in 2013–2014. The E and M protein amino acid sequences were run through the MHC-I Binding Predictions tool available on IEDB (60). This tool combines data from multiple prediction methods, which include artificial neural networks stabilized matrices. Choosing only those alleles that occur in at least 1% of the human population, we generated a list of predicted epitopes for MHC-A and B alleles. Percentile rank is calculated by comparing a given predicted peptide’s IC50 (concentration of the query peptide which inhibits 50% of a reference peptide binding) against those of a random set drawn from (61) where smaller rank indicates higher affinity. The highest ranking MHC-A and -B alleles are presented in Tables 1 and 2.

In order to maximize matching known DNV class I epitopes against the ZKV sequences, we were indiscriminate with respect to the DNV strain sequences. We used epitope sequence data from all of DNV strains 1–4, as downloaded from IEDB. Alignments between predicted ZKV epitopes and DNV were calculated using MAFFT (62) and webPRANK (63).

A recent study by Stettler (58) indicated ZKV/DNV cross-recognition observed for antibodies may not also be present for T-cell epitopes. Because more work is needed on this topic, and in order to analyze a larger set of potential ZKV epitopes, the class I epitopes listed in Tables 1 and 2 are initially predicted, and only afterward aligned to DNV. Allowing for sequence divergence between DNV and ZKV, as well as keeping in mind the antigenic divergence between strains of ZKV, we did not require strict conservation between the predicted ZKV epitopes and the DNV epitopes they were compared to. As such, non-homologous but predicted epitopes were included in these tables. There are no table entries for epitopes matched to DNV but not predicted. Reported HLA specificities refer specifically to ZKV epitope predictions.

The rational selection of CD4+ T cell epitopes in vaccine formulation is crucial for successful application of vaccination strategies that focus on induction of CD8+ T cell immunity, given the role of CD4+ T cell response in long-term maintenance of CD8+ T cell-dependent protective immunity. Recently, CD4+ T cells with cytotoxic features have been identified in PBMC from patients with chronic viral infections (67–70). Bioinformatics tools for identification of HLA class II epitopes have been reviewed by Ref. (71). The TEPITOPE HLA-DR binding prediction algorithm (72) and the derived ProPred algorithm (73) use the concept that each HLA-DR pocket in the antigen-binding groove can be characterized by “pocket profiles,” a quantitative representation of the interaction of all natural amino-acid residues with a given pocket, creating a matrix incorporated in the TEPITOPE and ProPred softwares. For each HLA-DR specificity, the algorithms generated a binding score corresponding to the algebraic sum of the strength of interaction between each residue and pocket, which correlated with binding affinity. Peptide scores along a scanned protein sequence are normalized for each HLA-DR as the proportion of the best binder peptides (74). Since the software predicts binding to a significant number of HLA-DR specificities (25 in the case of TEPITOPE, 51 for PROPRED), it is also capable of predicting promiscuous peptide ligands each capable of binding to multiple HLA class II variant molecules (58). The TEPITOPE prediction algorithm has been successfully applied to the identification of dozens of promiscuous T cell epitopes frequently recognized in 59 antigenic proteins from several human pathogens including viruses, bacteria, protozoa, fungi, and helminths (HIV, SIV, CMV. M. tuberculosis, P. vivax, P. brasiliensis, S. mansoni), and in silico prediction correlated with promiscuity in HLA-binding assays and frequency of T cell recognition by exposed individuals (75). This has led to several epitope-based vaccines which were shown to be immunogenic using conventional or and HLA class II-transgenic mice (71, 76) and protective (77) in mice. The incorporation of a promiscuous CD4+ T cell epitope in a recombinant protein-based P. vivax vaccine led to significant increase in its immunogenicity (41). A recent study from our group in non-human primates showed that a HIV CD4+ T cell epitope-based DNA vaccine was highly immunogenic and induced significant responses to most encoded epitopes in all animals tested (unpublished observations). Vaccines encoding promiscuous peptides able to bind to multiple HLA-DR molecules may thus allow wide population coverage. Here, we used the TEPITOPE and ProPred algorithms to identify potential “promiscuous” CD4+ T cell epitopes—predicted to bind to multiple HLA-DR molecules—derived from conserved regions of ZKV majority/consensus E and M protein sequences from circulating strains in the recent epidemic in Brazil and Polynesia.

The amino acid sequences derived from the ZKV strains BeH818995 (Genbank accession number KU365777.1), BeH819015 (Genbank accession number KU365778.1), BeH815744 (Genbank accession number KU365780.1), BeH819966 (Genbank accession number KU365779.1), SPH2015 (Genbank accession number KU321639.1), and SSABR1 (Genbank accession number KU707826.1), isolated in Brazil; and the H/PF/2013 strain (Genbank accession number KJ776791.2) isolated in French Polynesia were assembled and aligned with Clustal W (MegAlign, DNASTAR, Madison, WI, USA, Figure 2). We scanned the generated consensus sequence with the TEPITOPE and ProPred algorithms. We selected ZKV M and E peptides (Table 3) whose sequences were predicted to bind to at least 2/3 out of the 25 or 51 HLA-DR molecules in the TEPITOPE or ProPred matrixes, respectively, corresponding to an inner nonamer core selected as the HLA binding motif with flanking amino acids added when possible at either or both N- and C-terminal ends, to increase the efficiency of in vitro peptide presentation to CD4+ T cells.

H-2D-restricted class I epitopes, when injected intradermally without adjuvants, produce a weak immune response in C57BL/6 mice. Methods have been described for eliciting immune responses to class I. For example, the target epitopes are linked together as a “string of beads” (78). In another example, the DNA corresponding to the desired string of epitopes is inserted in a modified vaccinia Ankara (MVA) vector. Immune responses have been elicited in mice using this technique (79). A DNA string has also been administered with electroporation (80). Immune responses in Macaques have been elicited in this manner (81). In order to add and subtract epitopes from the formulation used in these types of vaccines, new linker elements must be identified and proper presentation of the desired epitopes after “string-of-beads” processing by antigen-presenting cells confirmed (82).

The use of a biodegradable, PLGA microsphere-based vaccine delivery platform allows one or more unmodified peptides to easily be incorporated into the vaccine formulation (83). The limitations of PLGA microsphere-based vaccines have been described in the literature. For example, double-emulsion sphere fabricating technologies may degrade the tertiary structure of the delivered antigen due to exposure to solvents or high temperatures used during spray drying processes (84). In a previous report, we manufactured our microspheres avoiding double emulsion sphere manufacturing technology using a precision spray drying process that operates at room temperature (85).

In contrast to previous studies which incorporated only a single peptide epitope in spheres (86), we showed that it was possible to elicit an immune response from each of two epitopes delivered simultaneously, when the two epitopes were loaded into the same spheres or different spheres.

This is an important consideration, especially because the HLA restricted nature of the class I epitopes being delivered will require the development of a “master vaccine” containing enough different peptide epitopes to cover a target population.

The fact that the majority of the epitopes listed in the first four rows of Tables 1 and 2 have the best predicted HLA match as HLA*02 suggests that a vaccine directed against these class I epitopes could readily tested in Brazil where the frequency of HLA A*02 frequency varies from 21.7 to 47.5% between states (87).