Flavivirus group-reactive epitope A1.
The six residues identified as participating in the flavivirus cross-reactive epitopes are spatially arranged on the DENV-2 E-glycoprotein surface in two clusters (Fig. ). The most prominent grouping of these residues is the clustering of three A1 epitope residues in the highly conserved fusion peptide region of DII (4
). These residues, Gly104
, and Leu107
, are almost completely conserved among the flaviviruses (Table ). The A1 epitope is located in structural DII and is flavivirus group reactive, highly surface accessible, reduction-denaturation sensitive, and involved in virus neutralization and virus-mediated cell membrane fusion (46
). Tryptic fragment mapping with MAbs suggested that this epitope is formed from two discontinuous E-glycoprotein peptide fragments consisting of aa 1 to 120 and 158 to 142 (1
We identified substitutions at four amino acid positions, Gly104
, and Trp231
, that significantly reduced or eliminated the ability of anti-A1 MAbs to bind to the DENV-2 E glycoprotein (Table ). The first three of these residues are located within the highly conserved hydrophobic fusion peptide region of DII (Fig. and ). The fusion peptide is located at the end of the extended finger-like structure of DII in the E-glycoprotein monomer (40
). In the native dimer structure the tightly folded fusion peptide loop at the tip of one monomer is packed against the other subunit near the DI-DIII interface (Fig. and ). This close packing of the fusion peptide against its subunit partner and the glycan on the upper surface at DI residue Asn153
are believed to protect the fusion peptide from irreversible pH-induced conformational changes during maturation and secretion.
The cross-reactive MAbs that were most strongly consistently affected by substitutions in this region were 4G2 and 6B6C-1. These two MAbs are considered to be quite similar; both are flavivirus group reactive and have been grouped into the A1 epitope of the E glycoprotein (20
). We demonstrated here that although the epitopes of these two MAbs overlap spatially, they do not contain exactly the same residues. A substitution at Gly104
, or Leu107
knocked out the ability of MAb 4G2 to bind to the E glycoprotein. However, only substitutions at Gly104
interfered with the binding ability of MAb 6B6C-1. Leu107
is therefore not a component of the flavivirus group-reactive epitope that is recognized by MAb 6B6C-1 (Fig. ).
H substitution dramatically reduced the reactivities of all three of our flavivirus cross-reactive MAbs for this antigen (Table ). It is unlikely that a glycine residue, with no side chain, would directly participate in the binding energetics of an antibody-antigen (Ab-Ag) interaction. However, if a glycine residue were included in the buried surface area of this antibody epitope, the introduction of a large bulky hydrophobic side chain would likely disrupt the Ab-Ag shape complementarity and hence increase the dissociation rate constant (Kd
) of the Ab-Ag interaction (35
). In fact, a preliminary kinetic analysis of the G106
Q antigen and MAb 4G2 indicated that an increased Kd
was responsible for the affinity reductions of this Ab-Ag interaction. The G104
H mutant also reduced the recognition of the type-specific anti-A3 MAb 1A5D-1 (Table ). The A3 epitope is nonneutralizing, reduction sensitive, and moderately surface accessible (46
). All of the fusion peptide substitutions that we introduced into this region reduced the reactivity of this A3 reactive MAb, consistent with the interpretation that the buried surface area footprint of this MAb not only includes DENV-2 serotype-specific residues, but also includes these strongly conserved residues. A comparison of the DENV-2 atomic structure with flavivirus E-glycoprotein alignments identified at least two unique DII surface-accessible residues (Glu71
) and a third residue that is variable within DENV-2 but distinct from the other DENV serotypes (Thr81
). All of these residues are within 10 to 22 Å of Gly104
, a distance well within the buried surface area of a typical Ab-Ag interface (37
). Alternatively, less surface-accessible type-specific residues nearby may participate in MAb 1A5D-1 binding since this epitope itself is only moderately surface accessible (46
). Since this MAb is DENV-2 specific, these type-specific residues would be expected to provide the majority of the binding energy for this epitope.
Q substitution also knocked out all discernible reactivities for both anti-A1 reactive MAbs, 4G2 and 6B6C-1, although it did not affect the binding of the anti-A5 reactive MAb 1B7-5 (Table ; Fig. ). Type-specific anti-A3 and -C1 reactive MAbs lost all measurable reactivity to the G106
Q construct. The A3 epitope footprint appears to include conserved fusion peptide residues in addition to DENV-2 serotype-specific residues as discussed above. The reduced reactivity of the C1 reactive MAb for the G106
Q construct is difficult to explain. Because of the lack of biological activity of DI (C epitopes), epitope assignments in this domain can be problematic (46
). The apparent incorporation of Gly106
(see below) into this C1 epitope is consistent with the possibility that either the previous DI assignment was incorrect or the C1 epitope includes residues from both DI and DII. However, if this anti-C1 reactive MAb recognized such an interdomain epitope, then this high-affinity MAb would be expected to interfere with the E-glycoprotein dimer-to-trimer reorganization (2
) that occurs during virus-mediated membrane fusion, which it does not.
Leu107 is the third residue that we identified in the fusion peptide region of DII that is incorporated into the A1 epitope. Unlike the substitutions at Gly104 and Gly106, the L107K substitution knocked out the reactivity of the anti-A1 reactive MAb 4G2, but it did not interfere with the reactivity of the other anti-A1 reactive MAb, 6B6C-1 (Table ; Fig. ). Beyond this major discrepancy, the reactivity patterns of the rest of the MAbs for this construct were similar to that observed for the other fusion peptide substitutions (Table ).
Previous studies have examined the effects of mutagenesis in this fusion peptide region. Pletnev et al. (42
) performed mutagenesis of fusion peptide residues 104 and 107 in a chimeric infectious clone containing the TBEV structural genes and DENV-4 nonstructural genes. TBEV has a histidine at position 104, as do all of the tick-borne flaviviruses (Table ). Pletnev et al. constructed the opposite substitution from the one that we constructed, H104
G, replacing the tick-associated histidine with the mosquito-associated glycine, but they were unable to recover live virus from this construct. They also constructed a H104
F double mutant from which they were able to recover virus; however, they were unable to detect any effect of these mutations on mouse neurovirulence. Allison et al. (4
) also performed mutagenesis at Leu107
to examine the role of this residue in virus-mediated membrane fusion by using TBEV VLPs. They replaced Leu107
with phenylalanine, threonine, or aspartic acid. They found that all of these mutations reduced the rate of fusion. Moreover, consistent with the results presented here, they found that the L107
D substitution appeared to completely abolish the binding of their DII flavivirus group-reactive MAb, which was also assigned to the A1 epitope.
The fourth residue that we identified as having a major effect on the anti-A1 and -A5 reactive MAbs was Trp231
, an invariant residue across the flaviviruses (Table ). Both substitutions that we introduced at Trp231
dramatically reduced the reactivities of both anti-A1 MAbs and the anti-A5 MAb. This residue is structurally distant from the fusion peptide region (Fig. and ). It is somewhat surprising that substitutions at this residue affect the binding of MAbs that also recognize the distant fusion peptide residues. The strict conservation of tryptophan (Table ) and the predicted high energetic costs of substitutions at this position (Table ) suggest that this residue is important for proper DI-DII conformational structure and function. If this is the case, then the loss of reactivity of these mutants with MAbs recognizing fusion peptide residues may occur from the induction of localized structural disturbances across DII occurring at a distance from Trp231
. However, the Trp231
substitutions did not significantly affect the binding of anti-A2 and -A3 epitopes or that of 10A1D-2, whereas anti-A2, -A3, and 10A1D-2 MAb reactivities were reduced or eliminated by all three of the fusion peptide substitutions. Anti-A2 MAbs do not recognize native virus, yet they block virus-mediated cell membrane fusion, presumably by recognizing an epitope that is exposed only during or after low-pH-catalyzed conformational changes (2
). If substitutions at Trp231
induced domain-wide structural alterations, we would expect a loss of reactivity for the anti-A3 reactive MAb 1A5D-1 and a possible exposure of the non-surface-accessible A2 epitope, resulting in an increase, or at least a change, in the reactivity of the anti-A2 reactive MAb 4E5 by IFA for these constructs. Moreover, the reactivities of polyclonal MHIAF and of all of our DIII MAbs (B epitopes) were no different for these constructs than they were for the nonmutated wild-type plasmid-transfected cells (Table ). DIII, however, is reduction-denaturation stable and folds into its native IgC-like conformation even when it is expressed alone without the remainder of the E glycoprotein (6
The apparent inclusion of Trp231
in these epitopes is therefore somewhat surprising. In the context of a single E-glycoprotein dimer, it appears that the location of Trp231
is too far removed from the identified fusion peptide residues to be bound by a single immunoglobulin molecule. There is a distance of approximately 35 to 45 Å from Trp231
, and Leu107
in the same dimer subunit and of 50 to 60 Å from Trp231
to these residues in the alternate dimer subunit. However, the distance from Trp231
in one dimer to Gly104
, and Leu107
in its nearest neighbor dimer on the viral E-glycoprotein lattice surface is only 25 to 30 Å, a distance that can be spanned by a single IgG molecule. Such a potential interdimer epitope would span a relatively large gap (10 to 15 Å) between the two dimers and would therefore probably require the incorporation of many close-packed water molecules to insure complementarity and to provide the typically observed polar interactions that occur between antibody paratopes and antigen epitopes (7
). The anti-A1 reactive MAb 4G2 has a high affinity, and if epitope A1 did in fact span across dimers and include Trp231
, then we would expect that this MAb would also interfere with virus-mediated membrane fusion by disrupting the dimer-to-trimer E-glycoprotein reorganization (2
). Unfortunately, it is not known if this MAb interferes with virus-mediated membrane fusion or not. The other anti-A1 reactive MAb, 6B6C-1, does interfere with fusion (46
). Given the shared overlapping nature of the residues identified in these two A1 epitopes, it seems reasonable to assume that MAb 4G2, like 6B6C-1, does block virus-mediated membrane fusion. It is therefore possible that the fusion-blocking activity of 6B6C-1 and, by extension, that of 4G2 arise from their recognition of an interdimer epitope and thus their disruption of the dimer-to-trimer conformational change that is essential for initiating virus-mediated membrane fusion. Glu126
is a structural neighbor of Trp231
and is apparently incorporated into the anti-A1 reactive MAb 6B6C-1 epitope. Like W231
, this residue might be incorporated in an interdimer epitope. However, since substitutions at this position only reduced 6B6C-1 binding by IFA and not by Ag-ELISA, it is possible that the apparent involvement of Glu126
in this epitope is an artifact, possibly resulting from a nonnative protein conformation in acetone-fixed cells in the IFA.
Flavivirus subgroup cross-reactive epitope A5.
The A5 epitope shares all of the physical and biochemical attributes of the A1 epitope, with the exception of it being flavivirus subgroup reactive instead of flavivirus group reactive (46
). We identified at least three residues that appear to be involved in the A5 epitope, Gly104
, and Glu126
, with the possible addition of Thr226
. The high level of amino acid conservation at Gly104
(Table ) and their participation in other flavivirus cross-reactive epitopes (discussed above) are consistent with the interpretation that these residues are incorporated into distinct yet overlapping cross-reactive epitopes.
We observed reductions in the reactivity of the anti-A5 reactive MAb 1B7-5 for the E126A/T226N double mutant by both IFA and Ag-ELISA, whereas the E126A mutant alone reduced the reactivity of this MAb to similar levels in Ag-ELISA, but not in IFA, and the T226N substitution had no effect on the reactivity of this MAb in either assay (Table ). The larger decrease in the reactivity of 1B7-5 for the double mutant than for either single mutant is interesting. It is possible that the effects of substitutions at these two residues on anti-A5 MAb reactivity are additive or synergistic. Either the positions of these residues in the A5 epitope footprint or the nature of the specific substitutions that we introduced may disrupt the binding of the anti-A5 MAb to the observed magnitude only when they are present together, and the effect of either of these substitutions alone does not interfere as dramatically with anti-A5 MAb binding energetics.
L, and G104
H plasmid-transfected cells failed to secrete measurable VLP antigen into the tissue culture medium. The inability of cells transfected with these plasmids to secrete VLP antigen into the tissue culture medium may have resulted from the disruption of a variety of protein maturation processes. Interference with particle maturation may have occurred via a disruption of E-prM/M intermolecular interactions or E-glycoprotein dimer interactions or via a disruption of dimer organization into the surface lattice covering the mature particles. Although the two processes are interdependent, these substitutions may not interfere with particle formation per se, but instead may directly interfere with particle secretion itself. The IFA staining pattern of DENV-2 G104
H- and W231
F/L-transfected cells was highly punctate and appeared to be localized within inclusion bodies (data not shown). We previously observed similar IFA staining patterns with nonsecreting constructs of dengue virus and other flaviviruses (11
). Studies with TBEV VLPs have shown that interactions between prM and E are involved in the prM-mediated intracellular transport of prM-E heterodimers (3
). The location of Gly104
near the interior lateral edge of DII puts it very close to the E-dimer “hole” where the prM/M proteins are located in the heterodimer (32
) (Fig. ). Therefore, it seems likely that the G104
H mutant interferes with VLP secretion via disruption of the prM-E interactions that are necessary for intracellular transport and secretion. The mosquito-born flaviviruses have a glycine at this position, whereas the tick-borne flaviviruses have a histidine. Interestingly, Pletnev et al. (42
) introduced the reverse substitution, H104
G, into the TBEV E glycoprotein in a TBEV/DENV-4 chimeric infectious clone, and they were unable to recover virus from this mutant. The inability of G104
H mutant-transfected cells to secrete VLP antigen similarly suggests that this too is a lethal substitution in DENV-2. Taken together, these two results are consistent with the idea that vector-specific selection has produced strong epistasis between this residue and another unidentified residue(s) elsewhere in the E or prM/M glycoprotein.
Flavivirus infections elicit virus species-specific as well as flavivirus group cross-reactive immune responses. Preexisting antibodies to flaviviruses that are detectable by in vitro neutralization tests can protect laboratory animals from a challenge with a second, closely related flavivirus, reducing morbidity and mortality in the animals (21
). Dengue viruses consist of four antigenically related viruses. This concept of cross-reactive immune protection was hypothesized to explain the absence of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases during a DENV-2 outbreak among Peruvians who were previously infected with DENV-1 (31
). However, Sabin (48
) previously demonstrated that volunteers challenged with a secondary DENV serotype were fully cross-protected for only 2 months, partially protected for up to 9 months after the initial infection, and showed no protection thereafter. In contrast to this potential protective role of cross-reactive antibodies, their paradoxical role in the antibody-dependent enhancement (ADE) of secondary DENV infections, resulting in DHF/DSS, has been suggested by many prospective, population-based, cohort, and clinical studies (26
Current concepts of DENV vaccine development have focused on developing and deploying tetravalent live attenuated or chimeric attenuated vaccines in a single application. This approach theorizes that an effective tetravalent vaccine can provide long-lasting immunity against any future DENV serotype infection, thus decreasing the probability of ADE in areas where DENV is endemic. However, there are potential problems when immunogens are introduced simultaneously, as in the administration of a multivalent DENV vaccine. Although immunological interference has not been very apparent in laboratory animals, the suppression of an antibody response to one or more immunogens and/or unbalanced antibody responses have been found more frequently in humans, even when immunogen doses were adjusted in an attempt to equalize humoral immune responses (12
We have constructed eukaryotic expression plasmids that express DENV-1, −2, −3, and −4 prM/M and E glycoproteins in the form of VLPs (11
; D. Holmes, D. Purdy, and G.-J. Chang, unpublished results). The identification and initial characterization of residues that are incorporated into flavivirus cross-reactive epitopes A1 and A5 and the reduction of MAb recognition of these cross-reactive E-glycoprotein epitopes create an opportunity to investigate and understand the mechanistic basis and pathogenic ramifications of ADE. We hypothesize that the flavivirus group- and subgroup-reactive epitopes A1 and A5 and probable additional shared-antigen epitopes induce flavivirus cross-reactive antibodies that may cause ADE and increase the risk of developing DHF/DSS. We are continuing this structure-based rational mutagenesis approach to identify additional cross-reactive epitopes in the DENV-2 E glycoprotein, extending this epitope mapping to the other DENV serotypes and generalizing this epitope mapping approach to the encephalitic flaviviruses by using St. Louis encephalitis virus as a model. The E glycoproteins expressed by mutated plasmids in these VLPs with reduced cross-reactivities maintained an overall wild-type antigenic structure. Most importantly, many critical type-specific neutralizing antibody binding sites remained unaltered. Mutated plasmid DNAs and/or expressed VLPs will be used to immunize outbred mice and to assess their ability to stimulate type-specific neutralizing antibodies. These VLP antigens with reduced cross-reactivities will then be investigated for their effectiveness as DENV serotype-specific serodiagnostic reagents. Moreover, these immunogens, whether prM/E expression plasmids, VLP antigens, or live attenuated chimeric viruses, will provide both the theoretical foundation and the microbiological tools that are necessary for investigating and developing safer DENV candidate vaccines.