Monoclonal Antibodies against Zika Virus: Therapeutics and Their Implications for Vaccine Design

THERAPEUTIC ANTIBODIES TARGETING E PROTEINS

ZIKV is an arbovirus that is transmitted to humans by Aedes mosquitoes. It belongs to the genus Flavivirus, family Flaviviridae, which also includes some important human pathogens such as dengue virus (DENV), yellow fever virus (YFV), and West Nile virus (WNV); among these, ZIKV is the species most closely related to DENV (5). In flavivirus infections, humoral immunity represents an important component of the host response, and administration of polyclonal or purified monoclonal antibodies (MAbs) helps to clear the viruses (6, 7). Accordingly, within a short period, hundreds of ZIKV MAbs have been developed from either humans or mice (8–14); among these, NS1 MAbs have been applied for diagnosis (14). Although MAbs against NS1 from other flaviviruses, such as WNV, YFV, and DENV, confer protection in vivo (15), the efficacy of NS1 MAbs in ZIKV clearance remains to be elucidated. Thus, in this minireview, we focus on the therapeutic MAbs that bind to the envelope (E) proteins (E MAbs) (summarized in Table 1).

To date, 461 MAbs that bind to E proteins have been characterized, including 70 MAbs that display moderate to high neutralizing activities, with half-maximal inhibitory concentration (IC₅₀), 50% plaque reduction neutralization titer (PRNT₅₀), or 50% focus reduction neutralization titer (FRNT₅₀) values of <1 μg/ml. Nine MAbs further confer protection against lethal ZIKV challenge in mice. Interestingly, the proportions of ZIKV-specific MAbs in the MAb groups differ. Among the 153 tested MAbs, 63 specifically bind to ZIKV E protein (E MAbs) (8–14), while 45 of 70 MAbs that convey 50% inhibition of the virus at a concentration of <1 μg/ml are specific to ZIKV (8–14). The proportion of ZIKV-specific MAbs is further increased in focusing on the MAbs with in vivo protective efficacy. Six MAbs are ZIKV specific, while three are cross-protective against other flaviviruses (8–10, 12–14). This indicates that the ZIKV-specific MAbs display higher neutralizing activities and protection efficacies against ZIKV infection than cross-binding MAbs. In addition to the protection against lethal ZIKV challenge, ZIKV-117 and convalescent-phase serum treatment markedly reduced tissue pathology, decreased vertical transmission, and prevented ZIKV-induced microcephaly in a mouse model (12, 17), emphasizing the therapeutic potential of MAbs or polyclonal antibodies in preventing ZIKV-related damage.

NEUTRALIZING MECHANISMS FOR E MAbs AND IMMUNE HOT SPOTS

Why do E MAbs exert protection against ZIKV and other flaviviruses? E protein, as a typical viral class II fusion protein, plays a pivotal role in flavivirus attachment and membrane fusion (5). The three extracellular domains (DI, DII, and DIII) of the E protein undergo major rearrangements in their relative orientations but retain most of their folded rigid-body structures in different viral life stages (Fig. 1A). On the mature virion, E proteins form dimers, but in the acidic endosome, both DII and DIII rotate clockwise with respect to DI through flexion of the interdomain linkers. Consequently, the viral envelope-anchored C terminus of DIII is close to the fusion loop (FL), which is located on top of DII and inserts into the endosomal membrane, allowing fusion to occur. ZIKV is originally assembled as immature particles, with a trimer consisting of a heterodimer of E and premembrane (prM) proteins (Fig. 1A). The prM is cleaved by furin-like protease, forming pr peptides and membranes (M), which is the hallmark for maturation. Then, peptides are released when the pH returns to neutral outside the host cells (18).

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FIG 1

Domain rearrangements in the ZIKV E protomer and the neutralizing epitopes on the E protein. In this figure, the three extracellular domains (DI, DII, and DIII) are marked in red, yellow, and blue, respectively, while the fusion loops (FLs) are colored in green. (A) The three structures displayed as cartoons represent the E protomer at the immature (PDB: 5u4w), mature (PDB: 5iz7), and postfusion stages. The ZIKV E protein in the postfusion state was generated by docking structure 5iz7 on the DENV postfusion E structure (PDB: 1ok8). (B to F) Structure-defined neutralizing epitopes are displayed with a cutoff of 4.5 Å, except for Z23. Due to the low resolution (9.6 Å) of ZIKV complexed with Z23, as determined by cryo-electron microscopy (cryo-EM), the detailed epitope could not be distinguished and is highlighted with an ellipse. Carbohydrates are indicated as spheres. In each panel, the epitope of the MAb marked in black is colored white on the surface structure. The circles in panels B, C, and E represent the epitopes of the MAbs in the same color. The PDB identifiers for the epitope analysis are as follows: 5h37 (C10), 5lcv (A11), 5lbs (C8), 5gzo (Z20), 5uhy (ZIKV-117), 5gzn (Z3L1), 5gzr (Z23), 5vig (Z006), 5kvg (ZV-67), and 5jhl (2A10G6).

Solving the structures of neutralizing MAbs can help to identify neutralizing hot spots for vaccine development. The MAbs that interfere with E protein function at any stage (e.g., blocking E-mediated viral attachment, hindering E protein rearrangement in the endosome, or blocking FL insertion into the endosomal membrane) might prevent the flavivirus infection. Structural studies have shed light on the neutralizing mechanisms of MAbs. Taking MAb C10 as an example (19), this MAb covers the region of FL on DII of one protomer of the virion and makes contact with DIII, DI, and DII on the other protomer (Fig. 1B). This MAb also binds to the interdimer interface. Thus, E dimers are locked by MAb C10, which inhibits domain reorganization. Consistently, the E protein layer of the ZIKV-C10 complex at pH 6.5 remains at a radius similar to that seen with the uncomplexed ZIKV at pH 8.0, unlike uncomplexed ZIKV at pH 6.5 (19). Similar intradimer epitopes are also observed for MAb C8 and MAb A11 (11) (Fig. 1B). Interestingly, all three MAbs were initially isolated from a DENV infection patient and then their cross-neutralization activities against ZIKV were confirmed, indicating that the epitope is a cross-neutralization hot spot (11, 20). E dimers might be also locked by MAbs interacting across DII of protomers, as exemplified by Z20 and ZIKV-117 (13, 21) (Fig. 1C). Although isolated from different patients, the two MAbs bind to similar regions, indicating that this epitope might be another neutralization hot spot. Of note, with a few varied residues in the epitope, ZIKV-117 displays ZIKV specificity, while Z20 exerts weak cross-neutralization against DENV. MAbs such as Z3L1, which was isolated along with Z20 and is specific for ZIKV, can also inhibit domain rearrangement through fixation of the interdomain linker (Fig. 1D) (13).

DIII-binding MAbs might prevent ZIKV infection by either impeding E reorganization or blocking viral attachment to host cells because DIII is hypothesized to be responsible for receptor binding (22, 23). The human MAb Z23 mainly binds to the lateral regions (LRs) on DIII and inserts into the cleft between two adjacent dimers (Fig. 1E) (13). ZV67, derived from mice, interacts with the LRs similarly to Z23 (Fig. 1E) (9). Both MAbs are specific to ZIKV, suggesting the LRs are probable ZIKV-specific hot spots. Z006, isolated from another patient, also bound to the LRs and is a strong neutralizer of both ZIKV and DENV serotype 1 virus (DENV-1) (Fig. 1E) (8).

During membrane fusion, FL-targeting MAbs might cause steric hindrance and inhibit E insertion into the endosomal membrane. The murine MAb 2A10G6, which interacts with the conserved FL (Fig. 1F) and displays broad protection against flaviviruses, is hypothesized to function this way (10, 16). Notably, the neutralizing activity of 2A10G6 against ZIKV is weaker than that seen with other flaviviruses, such as WNV and DENV (13). A majority of human and murine FL MAbs also show much weaker neutralization activities against ZIKV infection (10, 13, 14). This difference might result from the unique structural features of ZIKV. ZIKV displays higher thermal stability than other flaviviruses (24), leading to less “breathing” of the E protein and a FL that is less accessible to solvent. Instead, these MAbs might enhance the virus infection, a phenomenon called antibody-dependent enhancement (ADE). Therefore, ADE epitopes should be avoided in vaccine development.

Many reports indicate that the primary infection with DENV generates an antibody response that protects against homologous serotypes, but it might cause the ADE effect during subsequent heterologous infections and exacerbate disease (7, 25, 26). Due to the similarity between DENV and ZIKV, the MAbs stimulated by ZIKV infection also enhance DENV infection (14). The cross-reactive MAbs, especially the FL MAbs, greatly contributed to the ADE effect (27). Thus, during ZIKV vaccine design, FL MAbs should be avoided. Thus far, many platforms have been developed for ZIKV vaccines (28), among which the mRNA vaccine candidate incorporates T76R, Q77E, W101R, and L107R mutations to destroy the FL. The modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of ADE during subsequent exposure to DENV, indicating the most potential for future clinical application (29).

IMPLICATIONS FOR THERAPEUTICS AND VACCINE DESIGN

Since the latest outbreak of ZIKV infection, hundreds of E MAbs have been isolated from patients or mice. Six human MAbs, including four ZIKV-specific ones, have proven in vivo protection efficacies in mice against lethal ZIKV challenge. Moreover, ZIKV-117 treatment also protects against mother-to-fetus transmission, infection, and disease, suggesting its therapeutic potential for clinical applications. However, one obstacle for future MAb treatment is that viruses, especially those with RNA genomes, display high mutation rates and readily generate escape mutations. Combinations consisting of different MAbs targeting different epitopes and, therefore, functioning through different mechanisms might suppress escape mutations. Here, a structural analysis provides detailed epitope information and is fundamental for therapeutic MAb “cocktail” design. Moreover, more human-protective MAbs are expected to be discovered, enhancing our understanding of ZIKV immune responses and protective therapeutics.

Another antigen that is important during flavivirus infections is the NS1 protein. Previously, NS1 polyclonal antibodies or MAbs had been shown to demonstrate protection in mice challenged with YFV and WNV (30, 31). Studies of ZIKV indicate that increasing secretion of NS1 is associated with high transmission between mosquitos and humans. In support of this idea, administration of antiserum against ZIKV NS1 protein decreases transmission between the two hosts. Thus, ZIKV NS1-specific MAbs may provide additive protection in combination with E-specific MAbs, though further investigations should be conducted to support this hypothesis.

Through the dissection of MAbs targeting E protein, three neutralizing hot spots were uncovered. More E MAb complex structures are desired for identification of additional hot spots. Further, it is also proposed that FL antibodies readily stimulate the ADE effect. Although FL mutations decrease the antigenicity of this epitope (29), they reciprocally evoke lower neutralizing serum titers, possibly by destabilizing the interaction between the mutated DII on one protomer and the DIII on the other protomer. Many strategies, including but not limited to the introduction of substitution mutations on the counterpart of DIII with hydrophilic amino acids to enhance the electrostatic interactions between the two domains and the introduction of disulfide bonds between the two protomers to stabilize the dimer conformation, are worth trying to solve the problem. Recently, a hypothesis was proposed on the basis of the study of influenza vaccine that preexisting human antibody levels negatively correlate with boosting responses to the same epitope (32, 33). Whether the administration of ZIKV vaccines along with FL antibodies could suppress responses to the cognate antigenic sites remains to be elucidated, and the results will aid in ZIKV vaccine development.