Determination Of Region Research Articles

Vaccination with Altered Peptide Ligands of a Plasmodium berghei Circumsporozoite Protein CD8 T-Cell Epitope: A Model to Generate T Cells Resistant to Immune Interference by Polymorphic Epitopes.

Many pathogens, including the malaria parasite Plasmodium falciparum, display high levels of polymorphism within T-cell epitope regions of proteins associated with protective immunity. The T-cell epitope variants are often non-cross-reactive. Herein, we show in a murine model, which modifies a protective CD8 T-cell epitope from the circumsporozoite protein (CS) of Plasmodium berghei (SYIPSAEKI), that simultaneous or sequential co-stimulation with two of its putative similarly non-cross-reactive altered peptide ligand (APL) epitopes (SYIPSAEDI or SYIPSAEAI) has radically different effects on immunity. Hence, co-immunization or sequential stimulation in vivo of SYIPSAEKI with its APL antagonist SYIPSAEDI decreases immunity to both epitopes. By contrast, co-immunization with SYIPSAEAI has no apparent initial effect, but it renders the immune response to SYIPSAEKI resistant to being turned off by subsequent immunization with SYIPSAEDI. These results suggest a novel strategy for vaccines that target polymorphic epitopes potentially capable of mutual immune interference in the field, by initiating an immune response by co-immunization with the desired index epitope, together with a carefully selected “potentiator” APL peptide.

SEPIa, a knowledge-driven algorithm for predicting conformational B-cell epitopes from the amino acid sequence

BackgroundThe identification of immunogenic regions on the surface of antigens, which are able to be recognized by antibodies and to trigger an immune response, is a major challenge for the design of new and effective vaccines. The prediction of such regions through computational immunology techniques is a challenging goal, which will ultimately lead to a drastic limitation of the experimental tests required to validate their efficiency. However, current methods are far from being sufficiently reliable and/or applicable on a large scale.ResultsWe developed SEPIa, a B-cell epitope predictor from the protein sequence, which is sufficiently fast to be applicable on a large scale. The originality of SEPIa lies in the combination of two classifiers, a naïve Bayesian and a random forest classifier, through a voting algorithm that exploits the advantages of both. It is based on 13 sequence-based features, whose values in a 9-residue sequence window are compiled to predict the epitope/non-epitope state of the central residue. The features are related to the type of amino acid, its conservation in homologous proteins, and its tendency of being exposed to the solvent, soluble, flexible, and disordered. The highest signal is obtained from statistical amino acid preferences, but all 13 features contribute non-negligibly in the predictor. SEPIa’s average prediction accuracy is limited, with an AUC score (area under the receiver operating characteristic curve) that reaches 0.65 both in 10-fold cross-validation and on an independent test set. It is nevertheless slightly higher than that of other methods evaluated on the same test set.ConclusionsSEPIa was applied to a test protein whose epitopes are known, human β2 adrenergic G-protein-coupled receptor, with promising results. Although the actual AUC score is rather low, many of the predicted epitopes cluster together and overlap the experimental epitope region. The reasons underlying the limitations of SEPIa and of all other B-cell epitope predictors are discussed.

Indian Long-term Non-Progressors Show Broad ADCC Responses with Preferential Recognition of V3 Region of Envelope and a Region from Tat Protein.

HIV-specific antibody-dependent cell cytotoxicity (ADCC) is likely to be important in governing protection from human immunodeficiency virus (HIV) and slowing disease progression. Little is known about the ADCC responses to HIV-1 subtype C. We characterized ADCC responses in HIV-1 subtype C-infected Indian subjects with slow disease progression and identified the dominant antigenic regions recognized by these antibodies. ADCC responses were measured in plasma from 34 long-term non-progressors (LTNPs), who were asymptomatic and maintained CD4 count above 500 cells/mm3 for the last 7 years in the absence of antiretroviral therapy (ART), and 58 ART naïve progressors with CD4 count <500 cells/mm3 against overlapping HIV-1 peptides using a flow cytometry-based antibody-dependent natural killer (NK) cell activation assay. The assay measured CD107a expression on NK cells as a marker of antibody-dependent NK cell activation and IFN-γ secretion by NK cells upon activation. The ADCC epitopes were mapped using the matrix of overlapping peptides. Indian LTNPs showed higher and broader ADCC responses compared to the progressors. The Env-C and Tat-specific ADCC responses were associated with lower plasma viral load, whereas the Env-C responses were also associated with higher CD4 counts. Five of 10 LTNP responders targeted epitopes in the V3 region (amino acids 288–330) of Env-C. Additionally, three Tat regions were targeted by ADCC antibodies from LTNPs. ADCC responses were associated with slow HIV progression in Indian subtype C-infected cohort. The frequently recognized peptides from the V3 loop of Env and the novel epitopes from Tat by the LTNPs warrants further study to understand the role of ADCC responses to these regions in control and prevention of HIV-1 infection.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity.B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

Mapping of Surface-Exposed Epitopes of In Vitro and In Vivo Aggregated Species of Alpha-Synuclein

Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal deposits observed in Parkinson’s disease and dementia with Lewy bodies. The objective of the study was to identify surface-exposed epitopes of alpha-synuclein in vitro and in vivo formed aggregates. Polyclonal immunoglobulin Y antibodies were raised against short linear peptides of the alpha-synuclein molecule. An epitope in the N-terminal region (1–10) and all C-terminal epitopes (90–140) were found to be exposed in an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant monomeric, oligomeric, and fibrillar alpha-synuclein. In a phospholipid ELISA, the N-terminus and mid-region of alpha-synuclein (i.e., 1–90) were associated with phosphatidylserine and thus occluded from antibody binding. The antibodies that reacted most strongly with epitopes in the in vitro aggregates (i.e., 1–10 and epitopes between positions 90–140) also labeled alpha-synuclein inclusions in brains from transgenic (Thy-1)-h[A30P] alpha-synuclein mice and Lewy bodies and Lewy neurites in brains of patients with alpha-synucleinopathies. However, differences in reactivity were observed with the C-terminal antibodies when brain tissue from human and transgenic mice was compared. Taken together, the study shows that although similar epitopes are exposed in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity can be observed between different molecular species.

Evolutionary dynamic of antigenic residues on influenza B hemagglutinin

Hemagglutinin (HA) of seasonal influenza virus evolves under positive selection pressure exerted by host immunity. It was previously shown that antigenic drift in different influenza B sublineages during different time periods distributed unevenly among different epitopes, and that more recent viruses up to 2007 might have their antigenic drift more focused on certain epitope. We further analyzed whether more recent influenza B viruses up to 2016 followed that same pattern of antigenic evolution. By using Shannon entropy and relative entropy to characterize HA antigenic epitopes, the most recent viruses of both Victoria and Yamagata lineages had residues with high relative entropy located most frequently on the 120-loop region. In addition to residues in the known epitopes, possible antigenic residues were also identified outside of the known epitope regions. The data provide an insight into the antigenic evolution of current influenza B viruses and expand our knowledge on their antigenic sites.

Genetic diversity of ORF 4–6 of type 1 porcine reproductive and respiratory syndrome virus in naturally infected pigs

Genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV) has been highly prevalent throughout Korea since the virus was first detected in 2005. However, genetic analyses of genotype 1 PRRSV in Korea have been limited to ORF5 and/or ORF7. In the present study, we determined 10 representative sequence covering ORF4 to ORF6 and each individual ORFs of genotype 1 PRRSV in Korea, and performed molecular analyses. The most variable gene among the individual ORFs of field strains was ORF4, and this gene exhibited only 74.5–87.3% sequence homology compared with strains reported elsewhere. However, the strains showed analogous sequence arrangements with each other. In the phylogenetic analysis, the sequences of Korean field strains formed a distinct cluster with some Austrian and German strains compared to genotype 1 PRRSV strains available in GenBank. In the amino acid analysis, the putative antigenic region of GP4 was highly variable, whereas the predicted epitope regions of ORF5 and ORF6 were relatively conserved. The hydropathy plots of GP4 showed a highly variable pattern in the antigenic region. The non-synonymous and synonymous substitution analysis suggested that ORF4 presumably had more immunogenic pressure compare with the other ORFs. According to these findings, genotype 1 PRRSV in Korea has been diversified and indigenized in Korea, and these strains might have multifarious immunological and genetic properties. This study provides novel insights into genotype 1 PRRSV in a geographically remote area and contributes to the information for further research on the evolution of type 1 PRRSV in the Korean peninsula.

FcμR Triggering Activates BCR Signaling Pathways and Increases Survival of CLL Cells

Soluble IgM was recently shown to activate B cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) B cells and protect them from spontaneous and CpG DNA-induced apoptosis (Wagner M et al, Blood 2016, 127:436-48; Gobessi S et al, Blood 2015, 126:4131, abstract 4131). The exact mechanism behind these effects is still unknown, but interaction of the leukemic BCRs with internal epitopes in the framework regions of soluble IgM has been considered as a possible explanation. An alternative explanation for these effects is that they are mediated by binding of soluble IgM to the Fcμ receptor (FcμR), which is highly overexpressed in CLL compared to normal B cells (Li FJ et al, Blood. 2011; 118:4902-9) and was recently shown to physically interact with the BCR in normal murine B cells (Ouchida R et al, J Immunol. 2015; 194:3096-101).To evaluate the possibility that triggering of the FcμR is responsible for the activation of BCR signaling pathways and for the increased apoptosis resistance of CLL cells, we first investigated the viability of CLL cells cultured for 72 hours with or without soluble human IgM or Fcμ fragment. Both soluble human IgM and Fcμ significantly increased the viability of CLL cells with respect to unstimulated control, suggesting that triggering of the FcμR is sufficient to increase CLL cell survival (n=28, % viable IgM-stimulated: 52.6±18.3, % viable Fcμ-stimulated: 55.0±14.8, % viable unstimulated: 43.7±17.0, p<0.001 for IgM- and Fcμ-stimulated vs unstimulated, p=n.s. for IgM-stimulated vs Fcμ-stimulated). We next investigated whether triggering of FcμR can activate BCR signaling pathways. CLL cells were stimulated for 10 min with Fcμ or anti-IgM and the levels of phospho-SYK, phospho-AKT, phospho-ERK (n=7) and intracellular Ca2+ (n=5) were analyzed by immunoblotting and flow cytometry, respectively. Increased phosphorylation of SYK, AKT and ERK and increased Ca2+ flux were detected in 5 and 4 of the investigated samples, respectively. The effects of Fcμ stimulation appeared greater in samples that showed a weaker response to anti-IgM stimulation, suggesting that FcμR and BCR signaling are reciprocally regulated.Because IL-4 was recently shown to increase surface IgM expression on CLL cells (Aguilar-Hernandez MM et al, Blood. 2016, 127:3015-25; Guo B et al, Blood 2016, 128:553-562), we next investigated whether it will have an opposite effect on FcμR expression. Stimulation of CLL cells (n=7) for 48 hours with IL-4 resulted in a mean 2.4 fold reduction in surface FcμR expression and a 3.9 fold increase in surface IgM expression compared to unstimulated cells (P<0.001 and P=0.016, respectively). Since IL-4 is produced by T cells, which typically interact with CLL cells in lymph nodes (LN), we next compared surface FcμR and IgM expression in two paired LN and peripheral blood (PB) CLL samples. Interestingly, in both cases the levels of surface FcμR were lower on LN than PB CLL cells, whereas no difference was detected in the expression of surface IgM.We next compared signaling in CLL cells cultured in the presence or absence of IL-4. As previously reported, CLL cells cultured in the presence of IL-4 showed a considerably greater increase in anti-IgM induced phosphorylation of SYK, PLCγ2 and AKT compared to controls. Interestingly, greater activation of SYK, PLCγ2 and AKT was also observed when FcμR was downregulated by RNA interference in one primary CLL sample, suggesting that FcμR expression could be in part responsible for the reduced capacity of PB CLL cells to respond to anti-IgM stimulation.In summary, this study shows that stimulation of FcμR by soluble IgM increases CLL cell survival and activates BCR signaling pathways, presumably because the two receptors are physically associated on the cellular membrane. Expression of FcμR is higher on PB than LN CLL cells and is negatively regulated by IL-4, which has an opposite effect on surface IgM expression. Preliminary data suggest that FcμR expression may reduce the capacity of the BCR to respond to external ligand. The purpose of such a mechanism is currently unclear, but one possibility could be to prevent inappropriate activation of PB CLL cells by (auto)antigens in the absence of co-stimulatory signals. DisclosuresEfremov:Gilead: Honoraria.

Autoantibody recognition mechanisms of MUC1

The most cost-effective blood-based, noninvasive molecular cancer biomarkers are based on p53 epitopes and MUC1 tandem repeats. Here we use dimensionally compressed bioinformatic fractal scaling analysis to compare the two distinct and comparable probes, which examine different sections of the autoantibody population, achieving combined sensitivities of order 50%. We discover a promising MUC1 epitope in the SEA region outside the tandem repeats.

Genotypic diversity of porcine circovirus type 2 (PCV2) and genotype shift to PCV2d in Korean pig population

Porcine circovirus type 2 (PCV2) is a causative agent of PCV2-associated disease (PCVAD), which leads to enormous economic losses in the swine industry worldwide. A high nucleotide substitution rate allows for the continuous evolution of PCV2 and the emergence of novel PCV2 strains. However, the distribution of emerging PCV2 genotypes and the co-existence of multiple genotypes in Korea have not been elucidated. The objective of this study was to determine the genetic diversity of PCV2 in Korean pig herds between 2009 and 2016. The overall prevalence of PCV2, from various samples originating from commercial pigs, was 53.8% (325/604). Two cases of a genotype shift to PCV2d at the farm level showed that the genotype shift started before 2012. In addition, genotype-specific PCR, on pen-based oral fluid samples for nationwide PCV2 surveillance in 2016, indicated that the infection pattern of PCV2 genotypes at the farm level was as follows; none (6/69), PCV2a (6/69), PCV2b (2/69), PCV2d (33/69), PCV2a/b (2/69), PCV2a/d (4/69), PCV2b/d (11/69), and PCV2a/b/d (2/69), respectively. This suggests that the genotype shift to PCV2d occurred on a nationwide scale and that the co-existence of different genotypes is common in Korean pig herds. In addition, seven sites on the capsid protein of Korean PCV2 were identified as being under positive selection pressure, all of which are related to the epitope region and neutralization activity. These data provide evidence of increased genetic diversity and shifts among Korean PCV2 isolates.

Peptide mimics of a carbohydrate‑associated epitope expressed by cancer cells: Identification of vaccine candidates.

Cancer-associated antigen 215 (CA215) is an immunoglobulin molecule expressed by numerous tumor types. Membrane‑bound and soluble CA215 have been detected in the majority of cancer cells and rarely identified in normal tissues. In addition, CA215C is a carbohydrate‑associated epitope in the variable region of CA215, which is specifically recognized by the monoclonal antibody, RP215. However, CA215C is not a suitable vaccine candidate as it is a thymus‑independent antigen. In the present study, RP215 was used as a target to screen short peptide mimics of CA215C from a phage display peptide library. Following three rounds of screening, 30 positive phage clones that specifically bound to RP215 were identified and sequenced. The result of amino‑acid sequence analysis revealed five conserved sequence groups for seventeen of the positive phage clones. The sequences of phage clones 2, 13 and 42 were selected for peptide synthesis and binding analysis. The synthetic peptides R2 and R42 specifically bound RP215. Antisera from mice immunized with R2‑BSA or R42‑BSA bound purified CA215C and innate CA215C expressed on human hepatic and rectal carcinoma tissues, as demonstrated by immunohistochemistry. Furthermore, R2‑BSA and R42‑BSA antisera inhibited RP215 binding to cancer tissues. These results revealed that R2‑BSA and R42‑BSA antisera had similar characteristics to RP215 and that the synthetic peptides R2 and R42 may mimic the CA215C epitope. R2 and R42 peptides may therefore have potential for development into a tumor vaccine.

Evaluation of a synthetic peptide from the Taenia saginata 18 kDa surface/secreted oncospheral adhesion protein for serological diagnosis of bovine cysticercosis

Bovine cysticercosis is a zoonotic infection widely spread throughout Brazil, creating a burden on hygiene maintenance and the economy. Diagnosis of cysticercosis usually relies on post mortem inspection of carcasses in slaughterhouses. This detection method provides only low sensitivity. Recent advancements have improved the performance of serologic tests, such as ELISA, providing greater sensitivity and specificity. The objective of the current study was to identify and evaluate a synthetic peptide derived from the Taenia saginata 18kDa oncospheric surface protein for the diagnosis of bovine cysticercosis in ELISA. Test performance of the identified peptide was compared to an ELISA based on a heterologous crude Taenia crassiceps antigen (Tcra), widely used for the sero-diagnosis of bovine cysticercosis. Based on the primary sequence of an in silico structural model of the 18kDa protein, an epitope region designated EP1 was selected (46-WDTKDMAGYGVKKIEV-61). The peptide derived from this region yielded 91.6% (CI=80–96%) sensitivity and 90% (CI=82–95%) specificity when used in an ELISA, whereas the crude antigen yielded 70% (CI=56–8%) sensitivity and 82% (CI=73–89%) specificity. Thus, we conclude that EP1 has higher diagnostic potential for detecting bovine cysticercosis than the crude antigen Tcra.

Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits.

The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

Computational prediction of immunodominant antigenic regions & potential protective epitopes for dengue vaccination.

Background & objectives:Epitope-based vaccines (EVs) are specific, safe and easy to produce. However, vaccine failure has been frequently reported due to variation within epitopic regions. Therefore, development of vaccines based on conserved epitopes may prevent such vaccine failure. This study was undertaken to identify highly conserved antigenic regions in the four dengue serotypes to produce an epitope-based dengue vaccine.Methods:Polyprotein sequences of all four dengue serotypes were collected and aligned using MAFFT multiple sequence alignment plugin with Geneious Pro v6.1. Consensus sequences of the polyproteins for all four dengue serotypes were designed and screened against experimentally proven epitopes to predict potential antigenic regions that are conserved among all four dengue serotypes.Results:The antigenic region VDRGWGNGCGLFGKG was 100 per cent conserved in the consensus polyprotein sequences of all four dengue serotypes. Fifteen experimentally proven epitopes were identical to the immunodominant antigenic region.Interpretation & conclusions:Computationally predicted antigenic regions may be considered for use in the development of EVs for protection against dengue virus. Such vaccines would be expected to provide protection against dengue infections caused by all dengue serotypes because these would contain antigenic regions highly conserved across those serotypes. Therefore, the immunodominant antigenic region (VDRGWGNGCGLFGKG) and 15 potential epitopes may be considered for use in dengue vaccines.

Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors.

BackgroundHIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.ResultsMutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.ConclusionsMutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users.

Antibodies against HLA-DP recognize broadly expressed epitopes

HLA matching and avoidance of pre-transplant donor-specific antibodies are important in selection of donors for solid organ transplant. Solid phase testing with single antigen beads allows resolution of antibody reactivity to the level of the allele. Single antigen bead testing results at a large transplant center were reviewed to identify selective reactivity patterns of anti-HLA antibodies. Many HLA-DP antibodies were identified in the context of other HLA antibodies, but some sera had antibodies against only HLA-DP. B cell flow crossmatch testing was positive for 2 out of 9 sera with HLA-DP antibodies. Many patterns of reactivity corresponded to epitopes in hypervariable regions C and F of DPB1, but some matched epitopes in other regions or DPA1. Through analysis of single antigen bead testing from a large number of patients, we report that anti-HLA-DP antibodies predominantly recognize broadly cross-reactive epitopes. The United Network for Organ Sharing has mandated HLA-DP typing on all deceased kidney donors, and HLA-DP epitopes should be considered as the major antigens for avoidance of pre-transplant donor-specific antibodies.

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

BackgroundA few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures.ResultsSurprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer’s paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6’s efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly.ConclusionsOverall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0126-z) contains supplementary material, which is available to authorized users.

Molecular Analysis of VP7 Gene of Rotavirus G1 Strains Isolated from North India.

Rotavirus G1 strains are the predominant cause of diarrhoea in children. Universally common rotavirus vaccines (Rotarix and RotaTeq) include G1 as the immunological component. India has recently introduced rotavirus vaccine in Universal Immunization Programme. Therefore, in the present study, VP7 gene of rotavirus G1 strains circulating in Himachal Pradesh, India is analysed to study their phylogenetic characteristics, and further comparative analysis was performed for assessment of their divergence from the vaccine strains. The rotavirus strains (JU-SOL-5, JU-SOL-58, JU-SOL-77, JU-SOL-173 and JU-SHI-14) analysed in the study were isolated from the faeces of diarrhoeic children during active surveillance for rotaviruses. The Himachal strains clustered together in G1-Lineage 1 in the phylogenetic analysis. All five isolates showed 96.4-98.8% similarity with the other G1-Lineage 1 strains at amino acid level. However, none of them clustered in the pre-defined sublineages within lineage 1. Interestingly, all the strains were distantly related to the vaccine strains having 93.9-94.5 and 91.9-92.6% similarities at amino acid level with Rotarix and RotaTeq strains, respectively. The comparative sequence and structural analysis of the Himachal strains with vaccine strains revealed differences in amino acids in epitope region of the protein especially at the antibody neutralization sites. The study highlights variations between the G1 strains from Himachal Pradesh, India and Rotarix and RotaTeq vaccine strains. These differences might have an impact on the neutralization efficiency of vaccine and subsequently on vaccine efficacy. This underscores further investigation to study intragenotype antigenic variability and also impact of viral evolution on vaccine effectiveness.

Identification of a major epitope by anti-interferon-γ autoantibodies in patients with mycobacterial disease.

The binding of autoantibodies (autoAbs) to interferon (IFN)-γ in people with mycobacterial diseases has become an emerging medical concern. Many patients display specific human leukocyte antigen (HLA) class II haplotypes, which suggests that a common T cell-dependent and B cell-dependent mechanism might underlie the production of specific anti-IFN-γ autoAbs. We show here that these autoAbs target a major epitope (amino acids 121-131, designated position (P)121-131) in a region crucial for IFN-γ receptor (IFN-γR) activation to impair IFN-γ-mediated activities. The amino acid sequence of this epitope is highly homologous to a stretch in the Noc2 protein of Aspergillus spp., which was cross-reactive with autoAbs from patients. Rats immunized with Aspergillus Noc2 developed antibodies that reacted with human IFN-γ. We generated an epitope-erased variant of IFN-γ (EE-IFN-γ), in which the major neutralizing epitope region was altered. The binding affinity of anti-IFN-γ autoAbs for EE-IFN-γ was reduced by about 40%, as compared to that for IFN-γ1-131. Moreover, EE-IFN-γ activated the IFN-γR downstream signaling pathway ex vivo, irrespectively of anti-IFN-γ autoAbs. In conclusion, we identified a common, crucial B cell epitope that bound to anti-IFN-γ autoAbs in patients, and we propose a molecular-mimicry model for autoAb development. In addition, treatment with EE-IFN-γ might be worth investigating in patients producing anti-IFN-γ autoAbs.

Molecular Characterization of the ORF3 and S1 Genes of Porcine Epidemic Diarrhea Virus Non S-INDEL Strains in Seven Regions of China, 2015

In an effort to trace the evolution of porcine epidemic diarrhea virus (PEDV), S1 and ORF3 genes of viruses identified in 41 pig farms from seven regions (North, Northeast, Northwest, Central, East, South West, and South, respectively) of China in 2015 were sequenced and analyzed. Sequence analysis revealed that the 41 ORF3 genes and 29 S1 genes identified in our study exhibited nucleotide homologies of 98.2%–100% and 96.6%–100%, respectively; these two genes exhibited low nucleotide sequence similarities with classical CV777 strain and early Chinese strain LZC. Phylogenetic analysis indicated that the identified PEDV strains belonged to global non S-INDEL strains, and exhibited genetic diversity; S1 gene of the HLJ2015/DP1-1 strain harbored an unique deletion of 12 nucleotides (A1130CAACTCCACTG1141); while the Chinese PEDV S-INDEL reference strains included two types of the “CV777” S-INDEL as well as the “US” S-INDEL, and all co-circulated with Chinese non S-INDEL strains. Of 29 identified S1 genes, the SS2 epitope (Y748SNIGVCK755) was highly conserved, while the SS6 epitope (L764QDGQVKI771) and pAPN receptor-binding region (aa 490–615) exhibited amino substitutions. Nine possible recombination events were identified between the 29 identifed S1 genes and the 3 S1 reference genes from early Chinese PEDV strains. The complete S genes of selected Chinese PEDV field strains (2011–2015) showed 5.18%–6.07% nucleotide divergence, which is far higher than the divergence observed in early Chinese PEDV strains (3.1%) (P<0.05). Our data provide evidence that PEDV non S-INDEL strains with genetic diversities and potential recombination circulate in seven regions of China in 2015; Chinese PEDV S-INDEL strains exhibit genetic diversity and co-circulate with non S-INDEL strains.