scholarly journals Genotypic Differences in Dengue Virus Neutralization Are Explained by a Single Amino Acid Mutation That Modulates Virus Breathing

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Kimberly A. Dowd ◽  
Christina R. DeMaso ◽  
Theodore C. Pierson
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Vaccine Development ◽  
Antibody Binding ◽  
Single Amino Acid ◽  
Genotypic Differences ◽  
Conformational Ensembles ◽  
Single Amino Acid Residue ◽  
Virus Serotype ◽  
Serotype 1

ABSTRACTFlaviviruses sample an ensemble of virion conformations resulting from the conformational flexibility of their structural proteins. To investigate how sequence variation among strains impacts virus breathing, we performed studies with the monoclonal antibody (MAb) E111, which binds an inaccessible domain III envelope (E) protein epitope of dengue virus serotype 1 (DENV1). Prior studies indicated that an observed ~200-fold difference in neutralization between the DENV1 strains Western Pacific-74 (West Pac-74) and 16007 could not be explained by differences in the affinity of MAb E111 for each strain. Through neutralization studies with wild-type and variant viruses carrying genes encoding reciprocal mutations at all 13 amino acid differences between the E proteins of West Pac-74 and 16007, we found that E111 neutralization susceptibility mapped solely to the presence of a lysine or arginine at E domain II residue 204, located distally from the E111 epitope. This same residue correlated with neutralization differences observed for MAbs specific for epitopes distinct from E111, suggesting that this amino acid dictates changes in the conformational ensembles sampled by the virus. Furthermore, an observed twofold difference in the stability of infectious West Pac-74 versus 16007 in solution also mapped to E residue 204. Our results demonstrate that neutralization susceptibility can be altered in an epitope-independent manner by natural strain variation that influences the structures sampled by DENV. That different conformational ensembles of flaviviruses may affect the landscape available for antibody binding, as well as virus stability, has important implications for functional studies of antibody potency, a critical aspect of vaccine development.IMPORTANCEThe global burden of dengue virus (DENV) is growing, with recent estimates of ~390 million human infections each year. Antibodies play a crucial role in protection from DENV infection, and vaccines that elicit a robust antibody response are being actively pursued. We report here the identification of a single amino acid residue in the envelope protein of DENV serotype 1 that results in global changes to virus structure and stability when it is changed. Our results indicate that naturally occurring variation at this particular site among virus strains impacts the ensemble of structures sampled by the virus, a process referred to as virus breathing. The finding that such limited and conservative sequence changes can modulate the landscape available for antibody binding has important implications for both vaccine development and the study of DENV-reactive antibodies.

Deuterium-Hydrogen Exchange Coupled With Mass Spectrometry Revealed A Novel Autoantibody Binding Epitope and Substrate Recognition Site In ADAMTS13 Protease,

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 455-455
Author(s):  
Veronica C. Casina ◽  
Wenbing Hu ◽  
Hayley A. Hanby ◽  
Don L. Siegel ◽  
Leland Mayne ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Substrate Recognition ◽  
Antibody Binding ◽  
Recognition Site ◽  
Single Amino Acid ◽  
Binding Epitope ◽  
Single Amino Acid Residue ◽  
Mass Spectrometry Technology ◽  
Non Linear

Acquired thrombotic thrombocytopenic purpura (aTTP), a potentially fatal syndrome, is primarily caused by autoantibodies against the metalloprotease ADAMTS13. Most patients with aTTP harbor an immunoglobulin (Ig) G isotype in blood that targets the spacer domain of ADAMTS13. The precise epitopes of the anti-ADAMTS13 IgGs and the mechanism underlying their inhibition activity are not fully understood. We hypothesized that inhibitory IgG autoantibodies from aTTP patients achieve their inhibitory function by binding to a discontinuous epitope in the spacer domain of ADAMTS13. To test this hypothesis, we determined the binding epitope of one out of >100 unique human monoclonal antibody (mAb) fragments (single-chain Fv, scFv) isolated by phage display from aTTP patients. We developed a novel hydrogen-deuterium exchange-mass spectrometry technology (HX-MS) to identify the antibody binding sites at single amino acid residue resolution. Human ADAMTS13 inhibitory scFv 4-20 was expressed in E. coli Top10 cells and purified to homogeneity by Ni-chelating affinity chromatography. In the HX-MS experiment, the mAb was coupled to affi-gel 10 resin and used to bind recombinant ADAMTS13-MDTCS fragment expressed in a stably transfected Drosophila schneider 2 (S2) cell line. After exchange with deuterium (D2O) oxide for various periods of time, the reaction was stopped, the protein was eluted, and digested to peptide fragments with pepsin, and the peptides with or without deuterium bound were resolved and identified by fast HPLC and mass spectrometry. We find that mAb scFv4-20 binds to amino acid residues Arg636, Leu637, Arg639, and Leu640 spanning from Leu632 to Leu640 (in exosite 4) in the spacer domain of ADAMTS13. This sequence is highly conserved in the ADAMTS13 spacer domains from zebrafish to mammals. In addition, mAb scFv4-20 binds Arg660, Tyr661, and Tyr665 in exosite 3, previously shown to play an important role in substrate recognition and anti-ADAMTS13 autoantibody-mediated inhibition, as well as Lys608, upstream exosites 3 and 4. Apparently, mAb scFv4-20 inhibits plasma ADAMTS13 activity (IC50 ∼0.40 nM) by binding these non-linear surface residues in the spacer domain (Fig. 1A). In agreement, site-directed mutagenesis shows that complete deletion (Δ632LTEDRLPR639) or partial deletion (Δ632LTED635 or Δ636RLPR639), or replacement of these residues with alanines (632LTED635/4A or 636RLPR639/4A) abolished or dramatically reduced mAb scFv4-20 binding. A deletion or alanine substitution of the surface residues on exosite 4 also abolished or reduced ADAMTS13 proteolytic activity toward a fluorescein-labeled VWF73 peptide and multimeric VWF (Fig. 1B), indicating that the ADAMTS13 epitope for mAb scFv4-20 is also part of ADAMTS13’s substrate recognition site. We conclude that anti-ADAMTS13 autoantibodies work by physically blocking the well-conserved VWF binding site on ADAMTS13. These results demonstrate the powerful use of HX-MS technology to determine both linear and non-linear antibody binding epitopes. The results provide valuable information concerning the mechanism of autoantibody-mediated aTTP that may be exploited to develop targeted therapy by reengineering ADAMTS13 to avoid autoantibody inhibition. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A single nine-amino acid peptide induces virus-specific, CD8+ human cytotoxic T lymphocyte clones of heterogeneous serotype specificities.

1995 ◽  
Vol 182 (3) ◽  
pp. 853-863 ◽  
Author(s):  
J Zivny ◽  
I Kurane ◽  
A M Leporati ◽  
M Ibe ◽  
M Takiguchi ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Lymphocytes ◽  
Dengue Virus ◽  
Virus Type ◽  
Nonstructural Protein ◽  
Single Amino Acid ◽  
Target Cells ◽  
Amino Acid Peptide ◽  
Hla Class I Molecules ◽  
Virus Serotype

It is generally accepted that virus-specific CD8+ cytotoxic T lymphocytes (CTLs) recognize nine-amino acid peptides in conjunction with HLA class I molecules. We recently reported that dengue virus-specific CD8+ CTLs of two different serotype specificities, which were established by stimulation with dengue virus, recognize a single nine-amino acid peptide of the nonstructural protein NS3 of dengue virus type 4 (D4V) in an HLA-B35-restricted fashion. To further analyze the relationships between the serotype specificities of T cells and the amino acid sequence of the recognized peptides, we examined the ability of this viral peptide D4.NS3.500-508 (TPEGIIPTL) to stimulate T lymphocytes of an HLA-B35-positive, dengue virus type 4-immune donor. Peptide stimulation of the PBMC generated dengue virus-specific, HLA-B-35-restricted CD8+ CTL clones. These clones lysed dengue virus-infected autologous cells, as well as autologous target cells pulsed with this peptide. Four patterns of dengue virus serotype specificities were demonstrated on target cells infected with dengue-vaccinia recombinant viruses or pulsed with synthetic peptides corresponding to amino acid sequences of four dengue virus serotypes. Two serotype-specific clones recognized only D4V. Three dengue virus subcomplex-specific clones recognized D1V, D3V, and D4V, and one subcomplex-specific clone recognized D2V and D4V. Three dengue virus serotype-cross-reactive clones recognized D1V-D4V. Thus, a single nine-amino acid peptide induces proliferation of a heterogeneous panel of dengue virus-specific CD8+ CTL clones that are all restricted by HLA-B35 but have a variety of serotype specificities. Peptides that contain a single amino acid substitution at each position of D4.NS3.500-508 were recognized differently by the T cell clones. These results indicate that a single epitope can be recognized by multiple CD8+ CTLs that have a variety of serotype specificities, but the manner of recognition by these multiple CTLs is heterogeneous.

scholarly journals Structures suggest an approach for converting weak self-peptide tumor antigens into superagonists for CD8 T cells in cancer

2021 ◽  
Vol 118 (23) ◽  
pp. e2100588118
Author(s):  
Pengcheng Wei ◽  
Kimberly R. Jordan ◽  
Jonathan D. Buhrman ◽  
Jun Lei ◽  
Hexiang Deng ◽  
...  
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
Amino Acid Substitution ◽  
Cd8 T Cells ◽  
Tumor Antigens ◽  
Vaccine Development ◽  
Single Amino Acid ◽  
Cell Repertoire ◽  
Subtle Change

Tumors frequently express unmutated self-tumor–associated antigens (self-TAAs). However, trial results using self-TAAs as vaccine targets against cancer are mixed, often attributed to deletion of T cells with high-affinity receptors (TCRs) for self-TAAs during T cell development. Mutating these weak self-TAAs to produce higher affinity, effective vaccines is challenging, since the mutations may not benefit all members of the broad self-TAA–specific T cell repertoire. We previously identified a common weak murine self-TAA that we converted to a highly effective antitumor vaccine by a single amino acid substitution. In this case the modified and natural self-TAAs still raised very similar sets of CD8 T cells. Our structural studies herein show that the modification of the self-TAA resulted in a subtle change in the major histocompatibility complex I–TAA structure. This amino acid substitution allowed a dramatic conformational change in the peptide during subsequent TCR engagement, creating a large increase in TCR affinity and accounting for the efficacy of the modified self-TAA as a vaccine. These results show that carefully selected, well-characterized modifications to a poorly immunogenic self-TAA can rescue the immune response of the large repertoire of weakly responding natural self-TAA–specific CD8 T cells, driving them to proliferate and differentiate into functional effectors. Subsequently, the unmodified self-TAA on the tumor cells, while unable to drive this response, is nevertheless a sufficient target for the CD8 cytotoxic effectors. Our results suggest a pathway for more efficiently identifying variants of common self-TAAs, which could be useful in vaccine development, complementing other current nonantigen-specific immunotherapies.

scholarly journals Genetic Stability The Protein Encoding Envelope (E) Genes Dengue Virus Serotype-4 Passaged in Vero Cell As A Candidate Chimera Vaccine Material

2020 ◽  
Vol 4 (2) ◽  
pp. 40
Author(s):  
Deya Karsari
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Genetic Stability ◽  
Rna Extraction ◽  
Vero Cells ◽  
Gene Encoding ◽  
Variable Site ◽  
Virus Serotype ◽  
Protein Encoding ◽  
Encoding Protein

This study aims to  analyze   genetic stability of  the gene encoding the envelope protein (E) dengue virus serotype-4 passaged in vero cells, Denv-4 passaged  in vero cells serially then continued with RNA extraction at passage 0, 10, 20, 30, 40 ,50 , and 60, and then continued with two step PCR and amplification, and sequencing then analyze the nucleotide stability with BLAST and MEGA 5 software. The result shows that there are many variable site in nucleotide and amino acid with high mutation rate 57.4% for nucleotide and 71.9% for amino acid ,while the similarity between passages are high ranging from 91% - 98%. The conclusion for this study is Denv-4 after analyzed shows that the gene encoding protein E has many variable site but high in similarity.

scholarly journals Tracking the polyclonal neutralizing antibody response to a dengue virus serotype 1 type-specific epitope across two populations in Asia and the Americas

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Daniela V. Andrade ◽  
Colin Warnes ◽  
Ellen Young ◽  
Leah C. Katzelnick ◽  
Angel Balmaseda ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Sri Lankan ◽  
Human Monoclonal Antibodies ◽  
Virus Serotype ◽  
Serotype 1 ◽  
Dengue Virus Serotypes ◽  
Kinetics Of ◽  
Two Populations

Abstract The four dengue virus serotypes (DENV1-4) cause major public health problems worldwide. Highly neutralizing type-specific human monoclonal antibodies (hmAbs) target conformation-dependent epitopes on the DENV envelope protein, including 1F4, a DENV1 type-specific hmAb. Using a recombinant DENV2 virus displaying the DENV1 1F4 epitope (rDENV2/1), we measured the proportion and kinetics of DENV1 neutralizing antibodies targeting the 1F4 epitope in individuals living in Asia and the Americas where different DENV1 genotypes were circulating. Samples from 20 individuals were analyzed 3 and 18 months post-primary DENV1 infection, alongside samples from 4 individuals collected annually for four years post-primary DENV1 infection, from two studies in Nicaragua. We also analyzed convalescent post-primary DENV1 plasma samples from Sri Lankan individuals. We found that neutralizing antibodies recognizing the 1F4 epitope vary in prevalence across both populations and were detected from 20 days to four years post-infection. Additionally, both populations displayed substantial variability, with a range of high to low proportions of DENV1 type-specific neutralizing antibodies recognizing the 1F4 epitope seen across individuals. Thus, the 1F4 epitope is a major but not exclusive target of type-specific neutralizing antibodies post-primary infection with different DENV1 genotypes in Asia and Latin America, and additional epitopes likely contribute to type-specific neutralization of DENV1.

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