Original antigenic sin was first described in 1953 from observations on immune responses to influenza viruses (10
). Following on from this, in his “disquisitions on original antigenic sin,” Fazekas de St. Groth made detailed observations on children vaccinated with two strains of influenza, who by virtue of their age could have been exposed to only one of these strains by natural exposure (13
). Upon heterologous vaccination, fully cross-reactive antibodies were generated against the vaccinating strain and previously encountered strain. Despite recent reports corroborating these findings (34
), there have been some studies with influenza vaccination that have questioned the significance or even the presence of original antigenic sin (21
The exact mechanisms of original antigenic sin are not fully understood. The degree of antigen similarity will clearly influence the ability of a single antibody to cross-react. It has been suggested that a difference of less than approximately 33 to 42% will result in original antigenic sin (6
). Interestingly, dengue antigens show approximately 30% amino acid difference between the serotypes, leaving them in a prime position for such a phenomenon. It is also suggested that cross-reactive memory responses are preferentially activated over naïve cells, as memory cells require a lower level of activation to proliferate (34
). Furthermore, memory cells may outnumber naïve cells meaning that, when antigen levels are low, they could outcompete naïve cells for antigen. In addition, it appears that the vaccine formulation may also have a bearing on the effect (34
Original antigenic sin in dengue was first described by Halstead in 1983 (26
) and forms the basis for serological efforts to untangle the sequence of infection in secondary cases. More recently, original antigenic sin has also been described for dengue-specific T-cell responses (49
). In this study, we have taken a close look at antibody responses to ED3 in both primary and secondary infections. Dengue seems to be an extreme example of original antigenic sin, where the secondary response is entirely constructed from antibody that cross-reacts with previously encountered virus. Furthermore, these antibodies can be competed by 20- to 10,000-fold less antigen from the putative primary infecting serotype, indicating higher avidity for these antigens than for the secondary infecting virus.
Original antigenic sin has the advantage that a response can be rapidly mobilized from memory. However, the downside is that in some cases, such as dengue, the response is dominated by inferior-quality antibody. In influenza, original antigenic sin has been shown to reduce the effectiveness of vaccination (13
). In dengue, the effect of original antigenic sin has considerable bearing on vaccine strategies. Once a response has been established, it is unlikely that repeat boosting will be able to change its scope, meaning that balanced responses against the four virus serotypes will need to be established with the first vaccine dose.
In this report, we have also shown that the primary infecting serotype, and thus antibody avidity, influences antibody-dependent enhancement in subsequent infections. The balance between neutralization and enhancement will likely determine the susceptibility of an individual to an infection and, by driving higher virus replication, may expose some individuals to the risk of developing DHF. An association between higher virus loads and DHF has been demonstrated (63
), and we believe that the mobilization of a poor-quality antibody response in the early stages of secondary dengue infection may allow further amplification of virus replication via ADE.
In mice, some antibodies generated following infection with flaviviruses react to ED3 and show potent neutralization of infection (18
). These observations have generated interest in the use of ED3 as an immunogen for subunit dengue vaccines, and several flavivirus anti-ED3 antibodies have been suggested as potential therapeutics (5
). It has recently been reported that some anti-ED3 neutralizing monoclonal antibodies do not neutralize all strains within a given serotype to equal levels (5
), meaning that therapeutic antibodies (Abs) will need to be carefully chosen.
In the experiments described here, we used prototypic dengue strains for the four serotypes and did not attempt to match to the virus causing each secondary infection. Nevertheless, the results on polyclonal human sera were clear, showing very high-level cross-reactivity during secondary infection. It remains possible that comparing a panel of ED3 strains from a given serotype in competition assays may allow a more precise description of the primary infecting virus beyond just its serotype.
ED3 has two potential attractions as an immunogen. First, antibodies directed to this domain are frequently of high avidity and show high neutralizing capacity for dengue virus compared to antibodies directed to other regions of the envelope protein (60
). Second, ED3 is a target of serotype-specific mouse monoclonal antibodies (5
). Initially, it was thought that antibodies against this domain had limited cross-reactivity between the 4 serotypes, suggesting a reduced risk of ADE following vaccination. Recent reports in macaques (30
) and humans (44
) also suggest that the primary ED3 response is relatively serotype specific. However, it has since been recognized that there are indeed cross-reactive mouse monoclonal antibodies against this domain (19
), even though neither the binding avidities nor the critical binding amino acids need be the same between serotypes (46
). Furthermore, while cross-reactive ED3 antibodies can be strong neutralizers (52
), they are generally weaker neutralizers compared to their serotype-specific counterparts (19
), meaning that the potential for ADE remains significant.
In this study of the human serological response to dengue, we demonstrate a low to moderate level of cross-reactivity in the ED3 response following primary infection, although the highest response was mostly indicative of the infecting serotype. Subsequently, however, during a heterotypic secondary infection, this low-level cross-reactivity was dramatically boosted to the extent that 100% of antibody was cross-reactive.
Cross-reactive mouse monoclonal antibodies have been shown to bind with the highest avidity to ED3 serotype 1 (19
), and following ED3 immunization in mice, anti-ED3(1) polyclonal responses are also often the strongest (37
). While these data correspond well with the high ED3(1) binding titers that we observed during secondary infections, we did not see a bias toward this serotype in our polyclonal competition avidity ELISA. It has also been reported that cross-reactive monoclonal antibodies bind poorly to ED3 from serotype 4 (19
). Consistent with this, following primary dengue infections in humans, we found reduced levels of seropositivity against ED3(4) compared to the remaining serotypes. This may be a consequence of the slight structural differences in the ED3 of this serotype (64
In humans, it has recently been reported that anti-ED3 antibodies constitute a relatively minor proportion of the response against dengue (8
), and it appears that a major part of the anti-envelope antibody response targets the fusion loop (8
). Corresponding with this, the anti-ED3 response seems to contribute little or nothing to neutralization in humans, as we demonstrate here in monocytes and as reported by Wahala et al. (66
). The limited contribution of the anti-ED3 response to neutralization in a natural infection does not however mean that inducing a high-level response by vaccination would fail, and such vaccination strategies should not be discounted.
Finally, the ELISA competition assay we describe here may well provide an alternative to neutralization assays to predict the sequence of dengue infection. The technique is fast, does not require infection-containment facilities, and yields results comparable to the results of neutralization assays. Indeed, in a number of cases, neutralization results are complex and do not allow the diagnosis of the primary infection, whereas the competition ELISA usually gives clear definition of the avidity response.