Immunodominance and Functional Activities of Antibody Responses to Inactivated West Nile Virus and Recombinant Subunit Vaccines in Mice

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Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. IMPORTANCETick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.

Section: Methodsmentioning

confidence: 99%

Variation of the Specificity of the Human Antibody Responses after Tick-Borne Encephalitis Virus Infection and Vaccination

Jarmer

Zlatkovic

Tsouchnikas

et al. 2014

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“…(a) Wild-type (wt) DIII-TR-His. DIII proteins of TBE virus strain Neudörfl (amino acids 302 to 398 of E protein) were expressed in Escherichia coli strain BL21 as a fusion protein with thioredoxin (TR) carrying a C-terminal His tag using the pET 32a Xa/LIC vector (Novagen) as described previously (35). After clarification of E. coli cell lysates, the DIII-TR-His protein was purified by Ni 2ϩ affinity chromatography (GE-Healthcare Life Sciences).…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression was induced by the addition of 500 M CuSO 4 to the cell culture medium, and the supernatants were harvested 7 to 11 days postinduction. His-tagged TBE and WN virus sE proteins were purified by immunoaffinity chromatography using the monoclonal antibody (MAb) 4G2 as described previously (35). The bound sE proteins were eluted from the column by 20 mM glycine, pH 2.7, and immediately back neutralized to pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

“…Studies using monoclonal antibodies (MAbs) demonstrated that binding to each of the three domains of E can lead to virus neutralization, and highly potent antibodies were shown to be directed at a surface-exposed epitope within DIII, the so-called DIII lateral ridge (DIII-lr) epitope (34). In mice (35,36), in contrast to humans (36)(37)(38)(39)(40) or horses (41), DIII-specific antibodies comprise a significant fraction of the polyclonal antibody response following flavivirus infection and vaccination.…”

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confidence: 99%

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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

Zlatkovic

Tsouchnikas

Jarmer

et al. 2013

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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 tickborne 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.

“…The S2 cells stably expressing the proteins were generated by cotransfection with a blasticidin selection vector as previously described (19). Seven to 11 days after induction of expression by CuSO 4 , the proteins were purified by Streptactin affinity chromatography (IBA BioTAGnology, Germany).…”

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confidence: 99%

The Membrane-Proximal “Stem” Region Increases the Stability of the Flavivirus E Protein Postfusion Trimer and Modulates Its Structure

Stiasny

Kiermayr

Bernhart

et al. 2013

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The flavivirus fusion protein E contains a "stem" region which is hypothesized to be crucial for driving fusion. This sequence element connects the ectodomain to the membrane anchor, and its structure in the trimeric postfusion conformation is still poorly defined. Using E trimers of tick-borne encephalitis virus with stem truncations of different lengths, we show that the Nterminal part of the stem increases trimer stability and also modulates the trimer structure outside the stem interaction site. Flaviviruses, members of a genus in the family Flaviviridae, comprise a number of medically relevant viruses, such as dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus (TBEV) (1). These small enveloped viruses infect cells by receptor-mediated endocytosis and acidic-pH-induced membrane fusion in endosomes (2, 3). The fusion reaction is mediated by the major envelope protein E, a class II fusion protein (4), which contains three domains (DI, DII, and DIII), a membrane-proximal region (the so-called "stem"), and the carboxy-terminal transmembrane anchor (Fig. 1A).The current flavivirus fusion model ( Fig. 1A to C) is based on atomic structures of truncated dimeric prefusion and trimeric postfusion structures of E that lack the stem-anchor region (soluble E [sE]). According to this model, the E dimers dissociate into monomers at acidic pH, leading to the exposure of the fusion peptide (FP) and its insertion into the outer leaflet of the target membrane ( Fig. 1B) (5). Membrane merger has been proposed to be driven by the relocation of DIII and a subsequent "zippering" reaction of the stem along DII, thus yielding a hairpin-like postfusion trimer with the FPs and transmembrane domains at the same end of the molecule ( Fig. 1C; reviewed in references 4 and 6). In accord with this model, soluble forms of DIII containing part of the stem and stem-derived peptides inhibited fusion (7-10).As shown by cryo-electron microscopy (cryo-EM) of dengue virions, the prefusion stem consists of two amphipathic helices (H1 and H3) that lie flat on the viral membrane and flank an additional short perimembrane helix (H2) (11, 12) ( Fig. 1A and D, lower panels). H2 has been resolved in the 3.5-Å structure of dengue virus only recently (12). With the exception of the first few amino acids following the C terminus of the E ectodomain (13,14), details of the stem in the context of the postfusion trimer are presently unknown. In X-ray crystallographic analyses of truncated dengue virus sE trimers that included N-terminal portions of the stem, most of the stem parts were disordered (14). The objective of our work was to generate experimental evidence for interactions of the stem with the core of the postfusion E trimer. For this purpose, we analyzed E trimers of TBEV with different C-terminal truncations using two approaches: (i) we assessed the impact of the presence of the stem as well as parts thereof on the thermostability of E trimers, and (ii) we examined possible influences ...