An intact humoral response is critical for the control of neuroinvasive WNV infection (16
). In this report, we evaluated the kinetics and magnitude of the antibody response against two distinct epitopes on the WNV E protein with discrete functional characteristics. One epitope, DIII-lr, is recognized by type-specific antibodies with potent neutralizing and therapeutic activity (4
). The other, DII-fl, is detected by flavivirus cross-reactive antibodies with less neutralizing and protective activity (13
). Given the differences in B-cell V-d
-J repertoire and the variation in class II major histocompatibility complex alleles among different species, it was not unexpected to see a species-related difference in the antibody response against the two epitopes. However, several interesting observations can be made, which may have implications for targeted vaccine development. In mice, DIII-lr IgM antibodies were detected soon after infection and contributed to the early neutralizing potential of serum. For unclear reasons, there was a delay in isotype switching of DIII-lr antibodies to IgG, since these were not reliably measured until day 15 after infection. Given that the vast majority of mortality in C57BL/6 mice after WNV infection occurs between days 8 and 12 (16
), DIII-lr IgG antibodies likely do not contribute to protection against primary infection. Moreover, in mice, DII-fl antibodies comprised a smaller percentage of the total antibody response compared to DIII-lr antibodies. In humans, only a subset of individuals generated a significant IgG antibody response against the DIII-lr epitope. In almost all human cases, a large fraction of the E-specific antibodies was directed against the less protective DII-fl epitope.
DIII-lr IgM in mice was observed soon after WNV infection in mice. Sequence analysis of the V-d
-J regions of ~10 different DIII-lr strongly neutralizing MAbs suggest that some mice (e.g., BALB/c and C57BL/6) have germ line gene configurations that can produce antibodies recognizing the DIII-lr epitope (S. Johnson and M. Diamond, unpublished observations). This could in part explain why neutralizing IgM were detected soon after infection. At present, it remains unclear which subset of B cells produce IgM that recognizes the DIII-lr epitope. CD5+
B-1 cells generate natural IgM, are unresponsive to B-cell receptor-mediated growth signals, and instead undergo apoptosis upon B-cell receptor cross-linking, whereas conventional CD5−
B-2 cells expand in an antigen-dependent fashion after B-cell receptor cross-linking and costimulation (2
). Of note, we did not detect DIII-lr antibodies in naive serum, although we cannot rule out that their presence was below our limit of detection. More definitive subset depletion or epitope-based ELISPOT studies are needed to define the B-cell population that produces the earliest DIII-lr antibody response in mice.
Despite an early IgM response to the DIII-lr epitope there was a delay in the isotype switch to IgG of antibodies against this epitope in mice. The lag was considerable such that DIII-lr IgG was not consistently measured until between days 10 and 15, a time after which the majority of mortality had occurred in mice after WNV infection. In contrast, there was no global delay in IgG responses, since significant WNV E protein antibody titers were measured in all infected mice by day 8. Although we cannot yet explain the epitope specific delay in isotype switching, it could be due to a requirement for germinal center formation in lymphoid tissues. Whereas some IgG antibodies can be produced in parafollicular zones, others require T-cell help, specific cytokines, or somatic hypermutation and are generated exclusively in germinal centers (7
). Experiments with signaling lymphocyte activation molecule-associated protein-deficient mice, which have impaired germinal center formation, production of class-switched IgG, and development of memory B-cell germinal center formation (15
), are in progress to directly address this question.
Although both BALB/c and C57BL/6 mice generate monoclonal (44
) and polyclonal antibodies that recognized the DIII-lr epitope, other species, including humans, showed significant variability. Accordingly, neutralization profiles with WNV RVP and mouse serum were affected more significantly by a mutation at T332K, which abolishes binding and neutralization of DIII-lr antibodies (47
). Our evaluation of convalescent human serum suggests that only a subset of infected individuals generate IgG against this DIII-lr epitope, and this accounted for a relatively small fraction of the neutralizing antibody response. One possible criticism of our analysis is that the human serum samples were obtained from individuals infected with heterogeneous WNV isolates, which might not bind to recombinant proteins derived from the New York 1999 strain of WNV. However, an alignment of all 83 published WNV isolates in North America between 1999 and 2006 showed virtually no change in amino acid sequences in the fusion loop or DIII of the E protein (G. Ebel, unpublished data): only four amino acid changes were seen in any of the residues of DIII, and each change was observed as a single mutation in a single strain and occurred at residues Q296, V364, N394, and H395, which do not engage strongly neutralizing MAbs as defined by crystallography (42
). Thus, to date, the type-specific epitope in DIII and cross-reactive epitope in DII are completely conserved in all North American WNV isolates.
The apparent lack of immunodominance of the highly protective DIII-lr epitope in humans is also consistent with two recent studies that generated human MAbs: only 2% (1 of 51) and 0% (0 of 5) of unique human single chain antibodies (scFv) generated from immune or nonimmune patient B cells by phage display reacted with the DIII-lr epitope (20
). In our sample collection, some human sera contained DIII-lr IgG, whereas others did not. Unfortunately, we could not establish a correlation between the development of an antibody response against the DIII-lr epitope and clinical outcome. Although studies with a larger patient cohort are required for confirmation, our preliminary data suggest that IgG against this epitope does not contribute significantly to protection against primary WNV infection. This is similar is to what has been observed in human immunodeficiency virus infections, where the level or type of antibodies does not predict disease progression (53
). Alternatively, serum sampling 4 to 6 months after infection may give an incomplete picture of the function of particular antibodies at a given stage of disease. Because the majority of human samples in our study were acquired at a single time point and not as part of a multiple-time-point prospective study, we cannot ascertain when in the course of the infections the DIII-lr-specific IgG developed. Finally, our results with human serum are also consistent with results obtained with sera from WNV-infected horses: the equine IgG response to the DIII-lr epitope was also variable and comprised only a small fraction of the antibodies directed against DIII (49
The fusion loop is highly conserved among flaviviruses, and antibodies against this epitope are in general highly cross-reactive (13
). Earlier studies established that DII-fl IgG MAbs behaved distinctly from DIII-lr MAbs in infection assays in cells. In general, they neutralized infection less efficiently in cells lacking Fc-γ receptors and enhanced infection across a wide range of antibody concentrations in cells expressing Fc-γ receptors (42
). Accordingly, they showed decreased efficacy in preventing or treating WNV or DENV infection in vivo (24
). The experiments in the present study are particularly intriguing since they suggest that DII-fl antibodies are produced by all humans against WNV and comprise a significant fraction of the anti-E protein response. Moreover, preliminary data with human MAbs isolated from B cells from secondarily DENV-infected patients indicate that the majority of E-specific antibodies also map to the DII-fl (C. Simmons, M. Beltramello, F. Sallusto, M. Diamond, and A. Lanzavecchia, unpublished results). Based on this, we speculate that the cross-reactive DII-fl epitope is immunodominant in humans.
The experiments with human and horse sera (49
) suggest that some animals do not make significant responses against the highly protective DIII-lr epitope. Based on passive transfer studies in rodents, it would be desirable to design vaccines that elicit high-titer DIII-lr antibodies, which block at a postattachment stage (42
). To date, WNV vaccine design has focused on eliciting a strong neutralizing response against the E protein, but the epitope specificities of the generated antibodies are largely unknown (11
). Complicating the issue, many of the initial immunization studies have been performed in mice. Because our studies show that mice generate distinct antibody responses against specific epitopes, some caution may be required in applying mouse vaccination results to humans. Although administration of live attenuated WNV vaccines to humans and nonhuman primates has induced relatively low-titer WNV-specific neutralizing antibodies that control viremia (36
), the epitope specificity of the response remains unclear. Further study into the naturally occurring and vaccine-induced antibody repertoire should enhance our understanding of the immune correlates of protection from WNV disease and promote the development of novel vaccine strategies, which focus on eliciting highly protective DIII-lr antibodies.