Immunization with Immune Complexes Modulates the Fine Specificity of Antibody Responses to a Flavivirus Antigen.

UNLABELLED: The antibody response to proteins may be modulated by the presence of preexisting antigen-specific antibodies and the formation of immune complexes (ICs). Effects such as a general increase or decrease of the response as well as epitope-specific phenomena have been described. In this study, we investigated influences of IC immunization on the fine specificity of antibody responses in a structurally well-defined system, using the envelope (E) protein of tick-borne encephalitis (TBE) virus as an immunogen. TBE virus occurs in Europe and Asia and-together with the yellow fever, dengue, West Nile, and Japanese encephalitis viruses-represents one of the major human-pathogenic flaviviruses. Mice were immunized with a dimeric soluble form of E (sE) alone or in complex with monoclonal antibodies specific for each of the three domains of E, and the antibody response induced by these ICs was compared to that seen after immunization with sE alone. Immunoassays using recombinant domains and domain combinations of TBE virus sE as well as the distantly related West Nile virus sE allowed the dissection and quantification of antibody subsets present in postimmunization sera, thus generating fine-specificity patterns of the polyclonal responses. There were substantially different responses with two of the ICs, and the differences could be mechanistically related to (i) epitope shielding and (ii) antibody-mediated structural changes leading to dissociation of the sE dimer. The phenomena described may also be relevant for polyclonal responses upon secondary infections and/or booster immunizations and may affect antibody responses in an individual-specific way. IMPORTANCE: Infections with flaviviruses such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses pose substantial public health problems in different parts of the world. Antibodies to viral envelope protein E induced by natural infection or vaccination were shown to confer protection from disease. Such antibodies can target different epitopes in E protein, and the fine specificities of polyclonal responses can differ between individuals. We conducted a mouse immunization study with TBE E protein alone or complexed to monoclonal antibodies specific for each of the three protein domains. We demonstrated that phenomena such as epitope shielding and antibody-induced structural changes can profoundly influence the fine specificity of antibody responses to the same immunogen. The study thus provided important new information on the potential immunomodulatory role of preexisting antibodies in a flavivirus system that can be relevant for understanding individual-specific factors influencing antibody responses in sequential flavivirus infections and/or immunizations.

Aluminum hydroxide influences not only the extent but also the fine specificity and functional activity of antibody responses to tick-borne encephalitis virus in mice.

Aluminum hydroxide is the most widely used adjuvant in human vaccines and serves as a potent enhancer of antibody production. Its stimulatory effect strongly depends on the adsorption of the antigen to the adjuvant, which may influence antigen presentation and, as a consequence, the fine specificity of antibody responses. Such variations can have functional consequences and can modulate the effectiveness of humoral immunity. Therefore, we investigated the influence of aluminum hydroxide on the fine specificity of antibody responses in a model study in mice using an inactivated purified virus particle, the flavivirus tick-borne encephalitis (TBE) virus, as an immunogen. To dissect and quantify the specificities of polyclonal antibodies in postimmunization sera, we established a platform of immunoassays using recombinant forms of the major target of neutralizing antibodies (protein E) as well as individual domains of E (DIII and the combination of DI and DII [DI+DII]). Our analyses revealed a higher proportion of neutralizing than virion binding (as detected by enzyme-linked immunosorbent assay) antibodies after immunization with aluminum hydroxide. Furthermore, the induction of antibodies to DIII, a known target of potently neutralizing antibodies, as well as their contributions to virus neutralization were significantly greater in mice immunized with adjuvant and correlated with a higher avidity of these antibodies. Thus, our data provide evidence that aluminum hydroxide can lead to functionally relevant modulations of antibody fine specificities in addition to its known overall immune enhancement effect.

Immunodominance and functional activities of antibody responses to inactivated West Nile virus and recombinant subunit vaccines in mice.

Factors controlling the dominance of antibody responses to specific sites in viruses and/or protein antigens are ill defined but can be of great importance for the induction of potent immune responses to vaccines. West Nile virus and other related important human-pathogenic flaviviruses display the major target of neutralizing antibodies, the E protein, in an icosahedral shell at the virion surface. Potent neutralizing antibodies were shown to react with the upper surface of domain III (DIII) of this protein. Using the West Nile virus system, we conducted a study on the immunodominance and functional quality of E-specific antibody responses after immunization of mice with soluble protein E (sE) and isolated DIII in comparison to those after immunization with inactivated whole virions. With both virion and sE, the neutralizing response was dominated by DIII-specific antibodies, but the functionality of these antibodies was almost four times higher after virion immunization. Antibodies induced by the isolated DIII had an at least 15-fold lower specific neutralizing activity than those induced by the virion, and only 50% of these antibodies were able to bind to virus particles. Our results suggest that immunization with the tightly packed E in virions focuses the DIII antibody response to the externally exposed sites of this domain which are the primary targets for virus neutralization, different from sE and isolated DIII, which also display protein surfaces that are cryptic in the virion. Despite its low potency for priming, DIII was an excellent boosting antigen, suggesting novel vaccination strategies that strengthen and focus the antibody response to critical neutralizing sites in DIII.