Humoral Immune Responses of Dengue Fever Patients Using Epitope-Specific Serotype-2 Virus-Like Particle Antigens

Dengue virus (DENV) is a serious mosquito-borne pathogen causing significant global disease burden, either as classic dengue fever (DF) or in its most severe manifestation dengue hemorrhagic fever (DHF). Nearly half of the world's population is at risk of dengue disease and there are estimated to be millions of infections annually; a situation which will continue to worsen with increasing expansion of the mosquito vectors and epidemic DF/DHF. Currently there are no available licensed vaccines or antivirals for dengue, although significant effort has been directed toward the development of safe and efficacious dengue vaccines for over 30 years. Promising vaccine candidates are in development and testing phases, but a better understanding of immune responses to DENV infection and vaccination is needed. Humoral immune responses to DENV infection are complex and may exacerbate pathogenicity, yet are essential for immune protection. In this report, we develop DENV-2 envelope (E) protein epitope-specific antigens and measure immunoglobulin responses to three distinct epitopes in DENV-2 infected human serum samples. Immunoglobulin responses to DENV-2 infection exhibited significant levels of individual variation. Antibody populations targeting broadly cross-reactive epitopes centered on the fusion peptide in structural domain II were large, highly variable, and greater in primary than in secondary DENV-2 infected sera. E protein domain III cross-reactive immunoglobulin populations were similarly variable and much larger in IgM than in IgG. DENV-2 specific domain III IgG formed a very small proportion of the antibody response yet was significantly correlated with DENV-2 neutralization, suggesting that the highly protective IgG recognizing this epitope in murine studies plays a role in humans as well. This report begins to tease apart complex humoral immune responses to DENV infection and is thus important for improving our understanding of dengue disease and immunological correlates of protection, relevant to DENV vaccine development and testing

Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes

The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231

Manipulation of immunodominant dengue virus E protein epitopes reduces potential antibody-dependent enhancement

<p>Abstract</p> <p>Background</p> <p>Dengue viruses (DENV) are the most important arboviruses of humans and cause significant disease. Infection with DENV elicits antibody responses to the envelope glycoprotein, predominantly against immunodominant, cross-reactive, weakly-neutralizing epitopes. These weakly-neutralizing antibodies are implicated in enhancing infection via Fcγ receptor bearing cells and can lead to increased viral loads that are associated with severe disease. Here we describe results from the development and testing of cross-reactivity reduced DENV-2 DNA vaccine candidates that contain substitutions in immunodominant B cell epitopes of the fusion peptide and domain III of the envelope protein.</p> <p>Results</p> <p>Cross-reactivity reduced and wild-type vaccine candidates were similarly immunogenic in outbred mice and elicited high levels of neutralizing antibody, however mice immunized with cross-reactivity reduced vaccines produced significantly reduced levels of immunodominant cross-reactive antibodies. Sera from mice immunized with wild-type, fusion peptide-, or domain III- substitution containing vaccines enhanced heterologous DENV infection <it>in vitro</it>, unlike sera from mice immunized with a vaccine containing a combination of both fusion peptide and domain III substitutions. Passive transfer of immune sera from mice immunized with fusion peptide and domain III substitutions also reduced the development of severe DENV disease in AG129 mice when compared to mice receiving wild type immune sera.</p> <p>Conclusions</p> <p>Reducing cross-reactivity in the envelope glycoprotein of DENV may be an approach to improve the quality of the anti-DENV immune response.</p

Differentiation of West Nile and St. Louis Encephalitis Virus Infections by Use of Noninfectious Virus-Like Particles with Reduced Cross-Reactivity▿ †

Differential diagnosis of St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) infections can be complicated due to the high degree of cross-reactivity observed in most serodiagnostic assays. In an effort to provide a more specific diagnostic test, we developed virus-like particle (VLP) antigens with reduced cross-reactivity for both SLEV and WNV by identifying and mutating envelope protein amino acids within the cross-reactive epitopes of VLP expression plasmids. To determine the serodiagnostic discriminatory ability of the antigens with reduced cross-reactivity, a panel of 134 human serum samples collected predominately from North American patients with SLEV or WNV infections was used to evaluate the performance of these novel antigens in imunoglobulin M antibody-capture enzyme-linked immunosorbent assays. Positive/negative ratios and the resulting diagnostic classifications were compared between the mutant and the wild-type (WT) VLPs. The mutant VLP antigens were more specific, with higher positive predictive values and higher likelihood ratios than the WT VLP antigens. Both the SLEV and WNV mutant VLPs greatly reduced the observed cross-reactivity, significantly increasing the specificity and sensitivity of the assay. The use of these novel VLP antigens with reduced cross-reactivity in these serodiagnostic assays and others should lead to more accurate diagnoses of current infections, thereby reducing the need for time-consuming and cumbersome confirmatory plaque-reduction neutralization tests to differentiate between SLEV and WNV infections in North America

Introduction to basic neurology

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Virus neutralization titers for primary DENV-2 infected serum samples from dengue fever patients.

1<p>days post onset of symptoms.</p>2<p>Last positive titer in 90% Focus-reduction micro-neutralization (FRμNT) assay.</p>3<p>Calculated actual 90% neutralization titers based on a nonlinear regression of the FRμNT data using a variable slope sigmoidal dose-response model.</p

Envelope protein structural domain III (EDIII) alignment of representative strains of the four dengue virus (DENV) serotypes and Japanese encephalitis virus (JEV).

<p>Single letter amino acid abbreviations are shown for EDIII of DENV-2 using DENV-2 numbering (the last digit of the residue number lies directly above the numbered residue). Amino acids conserved relative to DENV-2 in the other serotypes are shown as dots, alignment gaps are depicted with dashes, and single letter abbreviations for non-conserved amino acids are shown. Colored residues in the DENV-2 sequence depict epitope-specific determinates as determined in this report and previously published reports. DENV complex and subcomplex cross-reactive epitopes are highlighted in yellow, DENV-2 specific residues are highlighted in red, and residues from the region of overlap between these epitopes (hence affecting DENV complex and DENV-2 virus specific epitopes) are highlighted green <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004991#pone.0004991-SukupolviPetty1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004991#pone.0004991-Gromowski1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004991#pone.0004991-Gromowski2" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004991#pone.0004991-Lok1" target="_blank">[47]</a>. The substituted EDIII residues incorporated into mutant antigens in this study are marked in black for the non DENV-2 viruses: DENV EDIII complex cross-reactive knock out mutants incorporated K310D, E311R, P364R, and K388D; K305E was utilized to determine EDIII DENV-2 specific immunoglobulin responses.</p

Nucleotide sequences of mutagenic primers used and % VLP secretion from resultant plasmids relative to wild-type (100%).

1<p>Mutated nucleotides are shown in bold.</p>2<p>Average of triplicate experiments of mutant VLP secretion from transiently transformed COS-1 cells, standardized against the wild-type DENV-2 plasmid VLP secretion.</p

Epitope Specific Proportions of Envelope Protein Specific IgM and IgG from Primary and Secondary DENV-2 Infected Human Sera.

1<p>The mutant antigens used are all knock-out mutants. The symbols * and † depict significantly different mean endpoint titers between those antigens sharing the same symbol. Mean endpoint titers were considered significantly different when the 95% CI for the difference between the mean endpoints did not cross zero.</p>2<p>Epitope specific antibody populations targeted by the different knock-out antigens. WT antigen measures antibody recognizing all E-protein epitopes. EDII_FP denotes broadly cross-reactive epitopes incorporating the E-protein structural domain II fusion peptide. EDIII_CR denotes predominately complex cross-reactive epitopes incorporating residues within E-protein structural domain III. EDII_FP−EDIII_CR denotes individual or overlapping epitopes incorporating either or both the EDII fusion peptide or EDIII. EDIII_TS denotes EDIII DENV-2 type-specific epitopes and were determined by the reactivity difference between the K305E and K388D antigens (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004991#s4" target="_blank">methods</a> for details).</p>3<p>Endpoint titer determined with the knock-out antigen, thus representing immunoglobulins recognizing epitopes not targeted by the knock-out antigen. Because the EDIII DENV-2 type-specific response was calculated as the percent difference between K305E and K388D reactivities, the titers for EDIII_TS were calculated as the WT titers multiplied by the percent EDIII_TS response.</p>4<p>Because the mutant antigens knock-out antibody recognition of specific epitopes, the percent of immunoglobulin recognizing a particular epitope was determined for each individual sera by calculating the percent reactivity measured with a mutant antigen relative to that determined with the WT antigen and subtracting this value from 1.0; (1- [Endpoint_mutant/Endpoint_wt])×100; for the percent DENV-2 specific reactivity we used (1-[(Endpoint_K305E/Endpoint_WT)−(Endpoint_K388D)/Endpoint_WT)])×100.</p