Antigenic Motifs Research Articles

Sequence-based epitope mapping of high pathogenicity avian influenza H5 clade 2.3.4.4b in Latin America.

High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.

Combination of oligo-fractionated irradiation with nivolumab can induce immune modulation in gastric cancer

BackgroundTumor-associated antigen (TAA)-specific CD8(+) T cells are essential for nivolumab therapy, and irradiation has been reported to have the potential to generate and activate TAA-specific CD8(+) T cells. However, mechanistic...

Synthetic Neoglycoconjugates of Hepta- and Nonamannoside Ligands for Eliciting Oligomannose-Specific HIV-1-Neutralizing Antibodies.

Oligomannose-type glycans on the spike protein of HIV-1 constitute relevant epitopes to elicit broadly neutralizing antibodies (bnAbs). Herein we describe an improved synthesis of α- and β-linked hepta- and nonamannosyl ligands that were subsequently converted into BSA and CRM197 neoglycoconjugates. We assembled the ligands from anomeric 3-azidopropyl spacer glycosides from select 3-O-protected thiocresyl mannoside donors. Chain extensions were achieved using [4+3] or [4+5] block synthesis of thiocresyl and trichloroacetimidate glycosyl donors. Subsequent global deprotection generated the 3-aminopropyl oligosaccharide ligands. ELISA binding data obtained with the β-anomeric hepta- and nonamannosyl conjugates with a selection of HIV-1 bnAbs showed comparable binding of both mannosyl ligands by Fab fragments yet lesser binding of the nonasaccharide conjugate by the corresponding IgG antibodies. These results support previous observations that a complete Man9 structure might not be the preferred antigenic binding motif for some oligomannose-specific antibodies, and have implications for glycoside designs to elicit oligomannose-targeted HIV-1-neutralizing antibodies.

Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system. Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins. Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins. Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.

Characterization of contemporary 2010.1 H3N2 swine influenza A viruses circulating in United States pigs

In 2012, swine influenza surveillance detected a novel reassorted influenza A virus (IAV) strain containing human-seasonal hemagglutinin (HA) and neuraminidase (NA). Subsequently, these viruses reassorted, maintaining only the human-origin H3, which resulted in a new lineage of viruses that became the most frequently detected H3 clade in US swine (2010.1 HA clade). Here, we assessed the antigenic phenotype, virulence, and transmission characteristics of this virus lineage following its introduction to swine. Relative to 2010.1 viruses from 2012 and 2014, recent 2010.1 contemporary strains from 2015 to 2017 resulted in equivalent macroscopic lung lesions and transmission in pigs. A single mutation at amino acid residue 145 within the previously defined HA antigenic motif was associated with a change of antigenic phenotype, potentially impairing vaccine efficacy. Contemporary 2010.1 viruses circulating in swine since 2012 were significantly different from both pre-2012H3N2 in swine and human-seasonal H3N2 viruses and demonstrated continued evolution within the lineage.

Changes in N-Glycosylation Induced By Somatic Hypermutation Modulate the Antigen Reactivity of the Immunoglobulin Receptors in CLL Stereotyped Subset #201

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Two complementary α-fucosidases from Streptococcus pneumoniae promote complete degradation of host-derived carbohydrate antigens

An important aspect of the interaction between the opportunistic bacterial pathogen Streptococcus pneumoniae and its human host is its ability to harvest host glycans. The pneumococcus can degrade a variety of complex glycans, including N- and O-linked glycans, glycosaminoglycans, and carbohydrate antigens, an ability that is tightly linked to the virulence of S. pneumoniae Although S. pneumoniae is known to use a sophisticated enzyme machinery to attack the human glycome, how it copes with fucosylated glycans, which are primarily histo-blood group antigens, is largely unknown. Here, we identified two pneumococcal enzymes, SpGH29C and SpGH95C, that target α-(1→3/4) and α-(1→2) fucosidic linkages, respectively. X-ray crystallography studies combined with functional assays revealed that SpGH29C is specific for the LewisA and LewisX antigen motifs and that SpGH95C is specific for the H(O)-antigen motif. Together, these enzymes could defucosylate LewisY and LewisB antigens in a complementary fashion. In vitro reconstruction of glycan degradation cascades disclosed that the individual or combined activities of these enzymes expose the underlying glycan structure, promoting the complete deconstruction of a glycan that would otherwise be resistant to pneumococcal enzymes. These experiments expand our understanding of the extensive capacity of S. pneumoniae to process host glycans and the likely roles of α-fucosidases in this. Overall, given the importance of enzymes that initiate glycan breakdown in pneumococcal virulence, such as the neuraminidase NanA and the mannosidase SpGH92, we anticipate that the α-fucosidases identified here will be important factors in developing more refined models of the S. pneumoniae-host interaction.

Immunoassay Platform for Casein Antigens in Hydrolyzed Casein-Based Infant Formula

Hypoallergenic formulas are the sole source of nutrition for infants that are either allergic to milk proteins or at risk of developing allergies. Strategies to provide nutritional sustenance, while preventing allergic reactions, include designing formulas based on extensively hydrolyzed casein which is presumably devoid of antigenic epitopes. Assays devoted to the assessment of antigenic protein motifs are crucial to verify the absence of relevant antigens in formulas and the raw materials used in their preparation. Evaluation of commercial immunoassay kits intended for the detection of milk proteins in foods led to the conclusion that a specific assay for extensively hydrolyzed casein-based formulas was necessary to improve allergen recoveries and assay consistency. The purpose of this investigation was to develop a reproducible path, from the generation of antibodies to the pre-validation of immunoassays optimized for the analysis of hydrolyzed casein-based infant formula. We prepared purified antisera from sheep immunized with bovine acid-precipitated casein to establish a platform consisting of a slot blot immunoassay and an enzyme-linked immunosorbent assay. Results indicate that sheep can reliably produce antibodies against epitopes in the casein fraction of bovine milk, thus providing a quantitative reagent that binds to immobilized casein in different formats. The limits of detection and quantitation for standard solutions for the enzyme-linked immunoassay were 0.8 and 2.5 ppm, respectively. The limit of detection in the extensively hydrolyzed casein-based formula was 0.5 ppm and the limit of quantitation 1.4 ppm. This account describes two reproducible immunoassays that are accessible to any laboratory or manufacturing setting and do not require proprietary ingredients or undisclosed extraction solutions. While these tests were developed to quantitate casein in hypoallergenic formula matrices, an application of the slot blot immunoassay to assess residual casein on manufacturing surfaces is also described in the present account.

CD20-Mimotope Peptides: A Model to Define the Molecular Basis of Epitope Spreading.

Antigen-mimicking peptide (mimotope)-based vaccines are one of the most promising forms of active-immunotherapy. The main drawback of this approach is that it induces antibodies that react poorly with the nominal antigen. The aim of this study was to investigate the molecular basis underlying the weak antibody response induced against the naïve protein after peptide vaccination. For this purpose, we analyzed the fine specificity of monoclonal antibodies (mAb) elicited with a 13-mer linear peptide, complementary to theantigen-combining site of the anti-CD20 mAb, Rituximab, in BALB/c mice. Anti-peptide mAb competed with Rituximab for peptide binding. Even so, they recognized a different antigenic motif from the one recognized by Rituximab. This explains their lack of reactivity with membrane (naïve) CD20. These data indicate that even on a short peptide the immunogenic and antigenic motifs may be different. These findings highlight an additional mechanism for epitope spreading and should be taken into account when designing peptides for vaccine purposes.

Engineering E. coli recombinant strains for high yield production of Burkholderia pseudomallei specific antigens

A risk of introducing into the Russian Federation exotic infections including laboratory-confirmed melioidosis regularly recorded worldwide necessitates development and improvement of express diagnostics tools. Cross reactivity between phylogenetically related species of the genus Burkholderia complicates melioidosis diagnostics by express test methods based on using monoclonal antibodies against pathogen exopolysaccharide epitopes. Searching for target antigens to create the next generation group- and species-specific immunodiagnostic reagents for identifying Burkholderia pseudomallei is still of high priority. The study was aimed at cloning complete coding sequences for cell surface proteins differentiating Burkholderia pseudomallei and optimizing recombinant antigens purification protocol. In silico comparative study allowed to select highly immunogenic B. pseudomallei outer membrane proteins Omp38 and OmpA/МotB as target biomolecules. For cloning, omp38 and ompA/motB gene-specific amplicons were obtained by PCR and ligated with the linear expression vector RIC-Ready pPAL7. Competent E. coli C-Max5 cells were transformed by a ligation mixture for producing recombinant plasmids, which were further purified to transform E. coli BL21 (DE3) cells for robust recombinant protein expression. Due to a potential multimeric protein structure, a standard protein purification protocol from native cell lysate was inefficient, which was modified to increase recombinant protein yield. However, by adding denaturing conditions at intermediate purification steps caused hydrolysis of peptide bonds in the target proteins, presumably between proline and asparagine residues. As a result, N-terminal fragments connecting recombinant proteins to the stationary phase of chromatographic column were eluted and evaluated for linear epitope detection according to their molecular weights. In silico analysis data identified highly antigenic motifs within the polypeptides studied. Thus, strains of E. coli BL21(DE3) BpsOmp39 and E. coli BL21(DE3) BpsOmpА engineered by us produce cell surface proteins Omp38 and ОmpA/МotB derived from melioidosis pathogen, which can be useful for developing diagnostic test systems.

Microarray assessment of N-glycan-specific IgE and IgG profiles associated with Schistosoma mansoni infection in rural and urban Uganda

Core β-1,2-xylose and α-1,3-fucose are antigenic motifs on schistosome N-glycans, as well as prominent IgE targets on some plant and insect glycoproteins. To map the association of schistosome infection with responses to these motifs, we assessed plasma IgE and IgG reactivity using microarray technology among Ugandans from rural Schistosoma mansoni (Sm)-endemic islands (n = 209), and from proximate urban communities with lower Sm exposure (n = 62). IgE and IgG responses to core β-1,2-xylose and α-1,3-fucose modified N-glycans were higher in rural versus urban participants. Among rural participants, IgE and IgG to core β-1,2-xylose were positively associated with Sm infection and concentration peaks coincided with the infection intensity peak in early adolescence. Responses to core α-1,3-fucose were elevated regardless of Sm infection status and peaked before the infection peak. Among urban participants, Sm infection intensity was predominantly light and positively associated with responses to both motifs. Principal component and hierarchical cluster analysis reduced the data to a set of variables that captured core β-1,2-xylose- and α-1,3-fucose-specific responses, and confirmed associations with Sm and the rural environment. Responses to core β-1,2-xylose and α-1,3-fucose have distinctive relationships with Sm infection and intensity that should further be explored for associations with protective immunity, and cross-reactivity with other exposures.

Glycan Microarray-Assisted Identification of IgG Subclass Targets in Schistosomiasis.

Infection with schistosomes is accompanied by the induction of antibodies against the parasite. Despite having IgG against both protein and glycan antigens, infected individuals remain chronically infected until treated, and re-infection is common in endemic areas as immunity does not develop effectively. Parasite specific IgG subclasses may differ in functionality and effectivity with respect to effector functions that contribute to parasite killing and immunity. In this study, we investigated if specific IgG subclasses target specific antigenic schistosome glycan motifs during human infection. Sera from 41 S. mansoni infected individuals from an endemic area in Uganda were incubated on two glycan microarrays, one consisting of a large repertoire of schistosome glycoprotein- and glycolipid- derived glycans and the other consisting of chemically synthesized core xylosylated and fucosylated N-glycans also expressed by schistosomes. Our results show that highly antigenic glycan motifs, such as multi-fucosylated terminal GalNAc(β1-4)GlcNAc (LDN) can be recognized by all IgG subclasses of infection sera, however with highly variable intensities. Detailed examination of core-modified N-glycan targets revealed individual antibody responses specific for core-xylosylated and core α3-fucosylated glycan motifs that are life stage specifically expressed by schistosomes. IgG1 and IgG3 were detected against a range of N-glycan core structures, but IgG2 and IgG4, when present, were specific for the core α3-fucose and xylose motifs that were previously found to be IgE targets in schistosomiasis, and in allergies. This study is the first to address IgG subclass responses to defined helminth glycans.

Antigenic evolution of H3N2 influenza A viruses in swine in the United States from 2012 to 2016.

BackgroundSix amino acid positions (145, 155, 156, 158, 159, and 189, referred to as the antigenic motif; H3 numbering) in the globular head region of hemagglutinin (HA1 domain) play an important role in defining the antigenic phenotype of swine Clade IV (C‐IV) H3N2 IAV, containing an H3 from a late 1990s human‐to‐swine introduction. We hypothesized that antigenicity of a swine C‐IV H3 virus could be inferred based upon the antigenic motif if it matched a previously characterized antigen with the same motif. An increasing number of C‐IV H3 genes encoding antigenic motifs that had not been previously characterized were observed in the U.S. pig population between 2012 and 2016.ObjectivesA broad panel of contemporary H3 viruses with uncharacterized antigenic motifs was selected across multiple clades within C‐IV to assess the impact of HA1 genetic diversity on the antigenic phenotype.MethodsHemagglutination inhibition (HI) assays were performed with isolates selected based on antigenic motif, tested against a panel of swine antisera, and visualized by antigenic cartography.ResultsA previously uncharacterized motif with low but sustained circulation in the swine population demonstrated a distinct phenotype from those previously characterized. Antigenic variation increased for viruses with similar antigenic motifs, likely due to amino acid substitutions outside the motif.ConclusionsAlthough antigenic motifs were largely associated with antigenic distances, substantial diversity among co‐circulating viruses poses a significant challenge for effective vaccine development. Continued surveillance and antigenic characterization of circulating strains is critical for improving vaccine efforts to control C‐IV H3 IAV in U.S. swine.

A common antigenic motif recognized by naturally occurring human VH5\u201351/VL4\u20131 anti-tau antibodies with distinct functionalities

Misfolding and aggregation of tau protein are closely associated with the onset and progression of Alzheimer’s Disease (AD). By interrogating IgG+ memory B cells from asymptomatic donors with tau peptides, we have identified two somatically mutated VH5–51/VL4–1 antibodies. One of these, CBTAU-27.1, binds to the aggregation motif in the R3 repeat domain and blocks the aggregation of tau into paired helical filaments (PHFs) by sequestering monomeric tau. The other, CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of tau seeds and mediates the uptake of tau aggregates into microglia by binding PHFs. Crystal structures revealed that the combination of VH5–51 and VL4–1 recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot interactions while the specificity and thereby functionality of the antibodies are defined by the CDR3 regions. Affinity improvement led to improvement in functionality, identifying their epitopes as new targets for therapy and prevention of AD.

Extended low-resolution structure of a Leptospira antigen offers high bactericidal antibody accessibility amenable to vaccine design.

Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection. These obligatory structures exposed to the host immune system provide important targets for rational vaccine design. Here, we use a systematically designed series of multi-domain constructs in combination with small angle X-ray scattering (SAXS) to determine the structure of the main immunoreactive region from a major antigen from Leptospira interrogans, LigB. An anti-LigB monoclonal antibody library exhibits cell binding and bactericidal activity with extensive domain coverage complementing the elongated architecture observed in the SAXS structure. Combining antigenic motifs in a single-domain chimeric immunoglobulin-like fold generated a vaccine that greatly enhances leptospiral protection over vaccination with single parent domains. Our study demonstrates how understanding an antigen's structure and antibody accessible surfaces can guide the design and engineering of improved recombinant antigen-based vaccines.

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections.

BackgroundThe Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a ‘mosaic’ structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response.Methods/Principle findingsHigh-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection.Conclusions/SignificanceIn spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection.

Prevalence of human pegivirus-1 and sequence variability of its E2 glycoprotein estimated from screening donors of fetal stem cell-containing material

BackgroundHuman pegivirus-1 (HPgV-1) is a member of the Flaviviridae family whose genomic organization and mode of cellular entry is similar to that of hepatitis C virus (HCV). The E2 glycoprotein of HPgV-1 is the principle mediator in the virus-cell interaction and as such harbors most of HPgV-1’s antigenic determinants. HPgV-1 persists in blood cell precursors which are increasingly used for cell therapy.MethodsWe studied HPgV-1 prevalence in a large cohort of females donating fetal tissues for clinical use. PCR was used for screening and estimation of viral load in viremic plasma and fetal samples. Sequence analysis was performed for portions of the 5′-untranslated and E2 regions of HPgV-1 purified from donor plasmas. Sequencing was followed by phylogenetic analysis.ResultsHPgV-1 was revealed in 13.7% of plasmas, 5.0% of fetal tissues, 5.4% of chorions, exceeding the prevalence of HCV in these types of samples. Transmission of HPgV-1 occurred in 25.8% of traceable mother-chorion-fetal tissues triads. For HPgV-1-positive donors, a high viral load in plasma appears to be a prerequisite for transmission. However, about one third of fetal samples acquired infection from non-viremic individuals. Sequencing of 5′-untranslated region placed most HPgV-1 samples to genotype 2a. At the same time, a portion of E2 sequence provided a much weaker support for this grouping apparently due to a higher variability. Polymorphisms were detected in important structural and antigenic motifs of E2.ConclusionHPgV-1 is efficiently transmitted to fetus at early embryonic stages. A high variability in E2 may pose a risk of generation of pathogenic subtypes. Although HPgV-1 is considered benign and no longer tested mandatorily in blood banks, the virus may have adversary effects at target niches if delivered with infected graft upon cell transplantation. This argues for the necessity of HPgV-1 testing of cell samples aimed for clinical use.

CD1b-mycolic acid tetramers demonstrate T-cell fine specificity for mycobacterial lipid tails.

Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T-cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αβ T cells. Most CD1-presented lipid antigens contain large hydrophilic head groups comprised of carbohydrates or peptides that dominate patterns of T-cell specificity. MA diverges from the consensus antigen motif in that it lacks a head group. Using multiple forms of natural and synthetic MA and MA-specific T-cells with different T-cell receptors, we found that, unlike antigens with larger head groups, lipid length strongly controlled T-cell responses to MA. In addition, the three forms of MA that naturally occur in M. tuberculosis that differ in modifications on the lipid tail, differ in their potency for activating MA-specific T-cell clones. Thus, naturally occurring MA forms should be considered as separate, partly cross-reactive antigens. Two of the three forms of MA could be loaded onto human CD1b proteins, creating working CD1b-MA tetramers. The creation of CD1b-MA tetramers represents a new tool for future studies that track the effector functions and kinetics of MA-specific T-cells ex vivo.

Abstract 2313: Improved killing of tumor cells by a novel flexible antibody-based modular T cell retargeting system

Abstract In recent years, bispecific antibodies (bsabs) and chimeric antigen receptors (CARs) emerged as promising candidates for an antigen-specific cancer immunotherapy. Bsabs and CARs redirect T cells in an antigen-specific manner towards tumor cells and unleash their enormous cytotoxic potential to attack the tumor. Nevertheless, several challenges have to be solved to pave the way for a more widespread application of both strategies. Both approaches are monotherapies bearing the inherent risk for development of antigen-loss tumor variants under treatment. In addition, due to their single antigen specificity each given bsab or CAR is only curative for a restricted number of tumor indications and long-lasting and demanding research is necessary to bring a new bsab- or CAR-based drug to patients in need. Moreover, current CAR T cell approaches do not allow for any control of the magnitude and duration of T cell reactivity, thus bearing the risk for overshooting anti-tumor reactions leading to severe side effects and ongoing destruction of healthy tissue carrying the target antigen after tumor clearance. To overcome these limitations, we recently introduced a novel flexible antibody-based modular platform (UniTARG) that can be rapidly and easily adapted for redirection of T cells to any TAA in both a bsAb- or CAR-setting. The UniTARG technology consists of a universal effector arm and individual targeting modules (TMs). The effector arm is either a universal CAR (UniCAR), which has specificity for a short peptide motif of 10 amino acids derived from a human nuclear protein, or a bsAb (UniMAB) with specificity for human CD3 on T cells and the respective peptide epitope. The antigen-specificity of the system is provided by TMs comprising a binding domain e.g., a tumor-antigen specific scFv, fused to the nuclear antigen motif recognized by the UniCAR/UniMAB binding domain. Here we provide first in vitro and in vivo prove of concept for these new approaches. Redirection of T cells armed with the universal effector modules was effective at picomolar concentrations of targeting modules directed against various antigens. The modular composition of both platforms prompted us to explore, if T cell retargeting against several antigens simultaneously might be feasible. Using either two single-specific TMs or single bi-specific TMs significantly enhanced specific lysis of tumor cells in vitro and durable responses in vivo were observed. Furthermore, our results proof that TMs against new targets can be developed in a couple of weeks and all TMs tested so far engaged effector module armed T cells with similar potencies. Taken together, the UniTARG platform technology represents a promising tool in the field of both bsAbs and CARs with the advantage of simultaneous or consecutive dual or even multispecific T cell retargeting. Furthermore the platform approach allows a rapid development of new therapeutic options against additional targets. Citation Format: Armin Ehninger, Marc Cartellieri, Anja Feldmann, Claudia Arndt, Stefanie Koristka, Simon Loff, Malte von Bonin, Gerhard Ehninger, Michael Bachmann. Improved killing of tumor cells by a novel flexible antibody-based modular T cell retargeting system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2313.