Determination Of Polymorphism Research Articles

Systematic Determination of TCR–Antigen and Peptide–MHC Binding Kinetics among Field Variants of a Theileria parva Polymorphic CTL Epitope

CTLs are known to contribute to immunity toward Theileria parva, the causative agent of East Coast fever. The Tp967-75 CTL epitope from the Muguga strain of T. parva is polymorphic in other parasite strains. Identifying the amino acids important for MHC class I binding, as well as TCR recognition of epitopes, can allow the strategic selection of Ags to induce cellular immunity toward T. parva In this study, we characterized the amino acids important for MHC class I binding and TCR recognition in the Tp967-75 epitope using alanine scanning and a series of variant peptide sequences to probe these interactions. In a peptide-MHC class I binding assay, we found that the amino acids at positions 1, 2, and 3 were critical for binding to its restricting MHC class I molecule BoLA-1*023:01. With IFN-γ ELISPOT and peptide-MHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 5-8 in the epitope were required for TCR recognition. Only two of eight naturally occurring polymorphic Tp9 epitopes were recognized by both CTLs. Finally, using a TCR avidity assay, we found that a higher TCR avidity was associated with a stronger functional response toward one of two variants recognized by the CTL. These data add to the growing knowledge on the cross-reactivity of epitope-specific CTLs and specificities that may be required in the selection of Ags in the design of a wide-spectrum vaccine for East Coast fever.

CD4 T Cell Responses to Theileria parva in Immune Cattle Recognize a Diverse Set of Parasite Antigens Presented on the Surface of Infected Lymphoblasts.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

A polymorphic CD4 epitope related to increased susceptibility to Babesia bovis in Canchim calves

Different allelic forms of bovine CD4 were previously described in cattle and were also observed in Canchim calves examined in the present experiment. However, the functional relevance of these different CD4 phenotypes has not yet been investigated. CD4 + T helper cells are known to play a central role in immune control against Babesia bovis infection. Thus, our study aimed to compare the profiles of immune cells, specific antibody titers and blood infection levels measured by qPCR (quantitative polymerase chain reaction) in calves naturally infected with B. bovis, phenotyped as CD4- (absence of anti-CD4 staining), CD4 + (intermediate staining) or CD4 ++ (high staining). The CD4 mRNA precursor was also measured in these animals. Calves with the CD4- phenotype showed higher amounts of B. bovis DNA in blood samples, compared to the other CD4 phenotypes. It was also observed that these calves with higher levels of infection had lower amounts of natural killer cells and higher expression of the CD4 gene, which can be interpreted as a compensation for the failure of the altered CD4 receptor to recognize relevant B. bovis epitopes.

Novel insights into non-HLA alloimmunity in kidney transplantation.

SummaryRecognition of non‐self structures on donor cells represents the main immunological barrier in solid organ transplantation. The human leukocyte antigens (HLA) are considered the most important non‐self (allo)antigens in transplantation. Long‐term graft attrition is mainly caused by the formation of alloreactive antibodies that are directed against non‐self structures (i.e., epitopes) on cell surface proteins. Recently published data provided evidence for a similar importance of non‐HLA mismatches between donors and recipients in acute rejection as well as long‐term kidney allograft survival. These data suggest a broader concept of immunological non‐self that goes beyond HLA incompatibility and expands the current concept of polymorphic non‐self epitopes on cell surface molecules from HLA to non‐HLA targets. Amino acid substitutions caused by single nucleotide variants in protein‐coding genes or complete loss of gene expression represent the basis for polymorphic residues in both HLA and non‐HLA molecules. To better understand these novel insights in non‐HLA alloimmunity, we will first review basic principles of the alloimmune response with a focus on the HLA epitope concept in donor‐specific antibody formation before discussing key publications on non‐HLA antibodies.

Testing New Peptides From Toxoplasma gondii SAG1, GRA6, and GRA7 for Serotyping: Better Definition Using GRA6 in Mother/Newborns Pairs With Risk of Congenital Transmission in Mexico

Toxoplasma gondii variant influences clinical profile in human congenital and ocular toxoplasmosis. Parasite genotyping represents a challenge due to insufficient amount of genetic material of the protozoan in the host samples, and isolates are hard to obtain, especially from pediatric patients. An alternative is serotyping, which is based on the presence of specific antibodies against polymorphic proteins related to virulence; the more widely used are GRA6 and GRA7, but most works report cross reactions among the classical strains (I, II, and III). We designed new peptides of GRA6, GRA7, and SAG1 proteins, with more SNPs among the three clonal strains than those previously designed. This was done by identifying BcR and polymorphic epitopes by means of bioinformatics; then we designed peptides with linkers joining the specific regions and predicted their 3D structure. With the commercial molecules synthesized on the basis of these designs, we tested 86 serum samples from 42 mother/newborn pairs and two congenitally infected newborns, by indirect ELISA. We implemented a strategy to determine the serotype based on scatter plots and a mathematical formula, using ratios among reactivity indexes to peptides. We found low frequency of samples reactive to GRA7 and SAG1, and cross reactions between GRA6 serotypes I and III; we modified these later peptides and largely improved distinction among the three clonal strains. The chronicity of the infection negatively affected the reactivity index against the peptides. Serotyping both members of the mother/child pair improves the test, i.e., among 26% of them only one member was positive. Serotype I was the most frequent (38%), which was congruent with previous genotyping results in animals and humans of the same area. This serotype was significantly more frequent among mothers who transmitted the infection to their offspring than among those who did not (53 vs. 8%, p = 0.04) and related to disease dissemination in congenitally infected children, although non-significantly. In conclusion, serotyping using the improved GRA6 peptide triad is useful to serotype T. gondii in humans and could be implemented for clinical management and epidemiological studies, to provide information on the parasite type in specific areas.

A new polymorphic epitope of bovine CD4 antigen evidenced by flow cytometry

Cluster of differentiation 4 (CD4) is the accessory protein non-covalently bounded to T cell receptor (TCR) that recognize an invariant region of MHC class II on antigen presenting cell (APC). Its cytoplasmic tail, physically associated with a protein tyrosine kinase, is important in the activation of helper/inducer T (TH) lymphocytes. The anti-bovine CD4 monoclonal antibody (mAb) CC8 is a clone usefully used in flow cytometry assay in the last 20 years and no anomalous labelling has been described in previous reports. The aim of this study was to investigate the lack and reduced binding of clone CC8 to TH lymphocytes in three different breeds of dairy cattle: Italian Friesian (FRI), Pezzata Rossa Friulana (PRI) and their crossbreed (MET). Two unexpected flow cytometric phenotypes were found: CC8+ (reduced labelling) in 25%, 35% and 15% of the animals in the three breeds respectively, and CC8− (complete lack of labelling) in 15% of PRI cows. The complete sequencing of CD4 coding region (CDS) has allowed to identify two new missense mutations in the exon 5: Q306H and K310 N associated with the two phenotypes. A further new synonymous mutation (g.103634725 G > A) was found in exon 6. Further studies are needed to understand the effects of the news mutations, but these results demonstrate that flow cytometry is a powerful tool to discover polymorphic determinants of antigens involved in the immune responses.

Antibodies to Cryptic Epitopes in Distant Homologues Underpin a Mechanism of Heterologous Immunity between Plasmodium vivax PvDBP and Plasmodium falciparum VAR2CSA.

Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA) in vitro The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes.IMPORTANCE In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.

OR39 Potential clinical relevance of anti-HLA-DP antibodies targeting the 96K and 96R epitopes

Aim HLA-DP is conserved across the human population with the majority of polymorphism occurring at six hypervariable regions (HVR) within the β -chain. Anti-DP antibodies commonly react to specific epitopes in close proximity to AA 55, AA 84, and to a lesser extent the AA 33 region. Reactivity to these polymorphic HLA-DP epitopes can be associated with a positive flow crossmatch when single antigen bead (SAB) reactivity is high. Here we confirm another variable region of HLA-DP located at AA 96, and characterized by either a lysine (96K) or arginine (96R) residue. Methods We identified two paradoxical, robust, HLA-DP reactivity patterns by SAB, and MFI data was analyzed with HLA Matchmaker to investigate potential epitope patterns—which suggested variability at AA 96. Using patient serum samples obtained before and after the appearance of AA 96 reactivity, we performed surrogate flow crossmatch to determine the validity of antibodies targeting AA 96 epitopes. Results Using HLA Matchmaker, we have verified a variable region at AA 96, and have observed that differences at this position are capable of eliciting 96K (DP: 1, 3, 5, 6, 9, 10, 11, 13, 14, 15, 18, 19, 20) or 96R (DP: 0201, 0401, 0402, 17, 23, 28) antibody response. We identified a patient that was homozygous for 96R, received a lung from a heterozygous 96K/96R donor, and quickly developed strong anti-96K DSA determined by SAB. Flow crossmatch of this 96K reactive serum versus a surrogate donor homozygous for the 96K epitope (DP5, –) yielded a strongly positive B-cell crossmatch, while crossmatch versus a surrogate donor that was heterozygous at AA 96 (K/R: DP9, DP0201) only elicited a weakly positive B-cell crossmatch. In comparison, serum samples targeting the 96R epitope were also identified and additional surrogate testing is ongoing. Conclusions We have confirmed a HLA-DP epitope located at AA 96 with potential clinical relevance. Antibodies targeting the 96K epitope were capable of eliciting a positive B-cell crossmatch. Additional surrogate testing is ongoing to better characterize HLA-DP 96K and 96R antibodies.

Abstract 3563: Impact of the affinity of chimeric antigen receptor on immune activation profiles of T cells

Abstract Background: Chimeric antigen receptor (CAR) generally uses a single chain antibody fragment (scFv) of high affinity as an antigen-binding domain. Recent studies implied that T cells engineered with CAR (CAR T cells) of an extremely low affinity have the potential to reduce on-target off-tumor toxicity that is a serious side effect in CAR T cell therapy. Defining the relation of CAR affinity to immune activation profiles is important for understanding the nature of CAR T cells. Methods: We examined the functional variation of CAR T cells when stimulated with target antigens of various affinities. HLA-DR-specific MVR CAR T cells that were previously developed in our group were used in this study. Since MVR CAR recognizes HLA-DR, Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV LCLs) which stably express HLA-DR, stimulated MVR CAR T cells. Importantly, owing to the broad spectrum of MVR CAR affinity against polymorphic epitope of HLA-DR, six different EBV LCLs each of which express unique HLA-DRs, stimulated MVR CAR T cells with different magnitude based on the binding affinities. Using the combination of MVR CAR T cells and six different EBV LCLs, we assessed the extent of effector functions and gene expression induced by different CAR affinity. Results: The cytolytic activity of MVR CAR T cells was correlated with the affinity between MVR CAR and HLA-DR. The induced polyfunctionality was highest in intermediate MVR CAR-target affinity and was decreased in weak and strong affinities. Gene expression analysis of the stimulated MVR CAR T cells identified a panel of genes of which the expression levels were correlated with CAR-target affinity. Gene ontology analysis revealed that the genes are mainly involved in a T cell activation pathway. Interestingly, we found that the genes involved in type I interferon signaling were upregulated following the stimulation with strong affinity antigens, while those expression levels were unchanged following the stimulation with weak and intermediate affinity antigens. Conclusions: Here we describe the affinity-associate functional variation of CAR T cells defined by the use of the combination of MVR CAR T cells and EBV LCLs of various affinities. Using those effector/target cell combinations, we investigated the relation between CAR-antigen affinity and the effector functions which include cytolytic activity and polyfunctionality. Furthermore, we identified that type I interferon signaling is a distinct characteristics of strong affinity-induced activation of CAR T cells. These effector/target cell combinations and the observations will help to understand the nature of CAR T cells. Citation Format: Chungyong Han, Seon-Hee Kim, Beom K. Choi, Byoung S. Kwon. Impact of the affinity of chimeric antigen receptor on immune activation profiles of T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3563.

Combined effect of glutamine at position 70 of HLA-DRB1 and alanine at position 57 of HLA-DQB1 in type 1 diabetes: An epitope analysis.

The contribution of specific HLA Class II alleles in type 1 diabetes is determined by polymorphic amino acid epitopes that direct antigen binding therefore, along with conventional allele frequency analysis, epitope analysis can provide important insights into disease susceptibility. We analyzed the highly heterogeneous Cypriot population for the HLA class II loci of T1DM patients and controls and we report for the first time their allele frequencies. Within our patient cohort we identified a subgroup that did not carry the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04:xx-DQA1*03:01-DQB1*03:02 risk haplotypes but a novel recombinant one, DRB1*04:XX-DQA1*03:01-DQB1*02:01 designated DR4-DQ2.3. Through epitope analysis we identified established susceptibility (DQB1 A57, DRB1 H13) and resistance (DQB1 D57) residues as well as other novel susceptibility residues DRB1 Q70, DQB1 L26 and resistance residues DRB1 D70, R70 and DQB1 Y47. Prevalence of susceptibility epitopes was higher in patients and was not exclusively a result of linkage disequilibrium. Residues DRB1 Q70, DQB1 L26 and A57 and a 10 amino acid epitope of DQA1 were the most significant in discriminating risk alleles. An extended haplotype containing these epitopes was carried by 92% of our patient cohort. Sharing of susceptibility epitopes could also explain the absence of risk haplotypes in patients. Finally, many significantly associated epitopes were non-pocket residues suggesting that critical immune functions may exist spanning further from the binding pockets.

Epstein-Barr virus strain heterogeneity impairs human T-cell immunity.

The Epstein-Barr virus (EBV) establishes lifelong infections in > 90% of the human population. Although contained as asymptomatic infection by the immune system in most individuals, EBV is associated with the pathogenesis of approximately 1.5% of all cancers in humans. Some of these EBV-associated tumors have been successfully treated by the infusion of virus-specific T-cell lines. Recent sequence analyses of a large number of viral isolates suggested that distinct EBV strains have evolved in different parts of the world. Here, we assessed the impact of such sequence variations on EBV-specific T-cell immunity. With the exceptions of EBNA2 and the EBNA3 family of proteins, an overall low protein sequence disparity of about 1% was noted between Asian viral isolates, including the newly characterized M81 strain, and the prototypic EBV type 1 and type 2 strains. However, when T-cell epitopes including their flanking regions were compared, a substantial proportion was found to be polymorphic in different EBV strains. Importantly, CD4+ and CD8+ T-cell clones specific for viral epitopes from one strain often showed diminished recognition of the corresponding epitopes in other strains. In addition, T-cell recognition of a conserved epitope was affected by amino acid exchanges within the epitope flanking region. Moreover, the CD8+ T-cell response against polymorphic epitopes varied between donors and often ignored antigen variants. These results demonstrate that viral strain heterogeneity may impair antiviral T-cell immunity and suggest that immunotherapeutic approaches against EBV should preferably target broad sets of conserved epitopes including their flanking regions.

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.

Allele-specific antibodies to Plasmodium vivax merozoite surface protein-1: prevalence and inverse relationship to haemoglobin levels during infection.

BackgroundAntigenic polymorphisms are considered as one of the main strategies employed by malaria parasites to escape from the host immune responses after infections. Merozoite surface protein-1 (MSP-1) of Plasmodium vivax, a promising vaccine candidate, is a highly polymorphic protein whose immune recognition is not well understood.Methods and resultsThe IgG responses to conserved (MSP-119) and polymorphic (block 2 and block 10) epitopes of PvMSP-1 were evaluated in 141 P. vivax infected patients. Ten recombinant proteins corresponding to block 2 (variants BR07, BP29, BP39, BP30, BEL) and block 10 (BR07, BP29, BP39, BP01, BP13) often observed in Brazilian P. vivax isolates were assessed by ELISA in order to determine levels of specific antibodies and their respective seroprevalence. The magnitude and the frequency of variant-specific responses were very low, except for BR07 variant (>40%), which was the predominant haplotype as revealed by block 10 PvMSP-1 gene sequencing. By contrast, 89% of patients had IgG against the C-terminal conserved domain (PvMSP-119), confirming the high antigenicity of this protein. Using multiple linear and logistic regression models, there was evidence for a negative association between levels of haemoglobin and several IgG antibodies against block 2 variant antigens, with the strongest association being observed for BP39 allelic version. This variant was also found to increase the odds of anaemia in these patients.ConclusionsThese findings may have implications for vaccine development and represent an important step towards a better understanding of the polymorphic PvMSP-1 domain as potential targets of vaccine development. These data highlight the importance of extending the study of these polymorphic epitopes of PvMSP-1 to different epidemiological settings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1612-z) contains supplementary material, which is available to authorized users.

Implications from predicted B-cell and T-cell epitopes of Plasmodium falciparum merozoite proteins EBA175-RII and Rh5.

The leading circumsporozoite protein (CSP) based malaria vaccine, RTS,S, though promising, has shown limited efficacy in field studies. There is therefore, still a need to identify other malaria vaccine targets. Merozoite antigens are potential vaccine candidates, since naturally acquired antibodies generated against them inhibit erythrocyte invasion and in some cases result in the clinical protection from disease. We thus used in silico tools (BCPreds, NetMHCcons and NetMHCIIpan 3.0) to predict B-cell epitopes (BCEs) and T-cell epitopes (TCEs) in two merozoite invasion proteins, EBA175-RII and Rh5. Initially, we validated these tools using CSP to determine whether the algorithms could predict the epitopes in the RTS,S vaccine. In EBA175-RII, we prioritised three BCEs 15REKRKGMKWDCKKKNDRSNY34, 420SNRKLVGKINTNSNYVHRNKQ440 and 528WISKKKEEYNKQAKQYQEYQ547, a CD8+ epitope 553KMYSEFKSI561 and a CD4+ epitope 440QNDKLFRDEWWK VIKKD456. Three Rh5 epitopes were prioritised, a BCE 344SCYNNNFCNTNGIRYHYDEY363, a CD8+ epitope 198STYGKCIAV206 and a Rh5 CD4+ epitope 180TFLDYYKHLSYNSIYHKSSTY200. All these epitopes are in the region involved in the proteins’ interaction with their erythrocyte receptors, thus enabling erythrocyte invasion. Therefore, upon validation of their immunogenicity, by ELISA using serum from a malaria endemic population, antibodies to these epitopes may inhibit erythrocyte invasion. All the epitopes we predicted in EBA175-RII and Rh5 are novel. We also identified polymorphic epitopes that may escape host immunity, as some variants were not predicted as epitopes, suggesting that they may not be immunogenic regions. We present a set of epitopes that following in vitro validation provide a set of molecules to screen as potential vaccine candidates.

P053 : DISTRIBUTION OF KIR HLA LIGANDS IN TRANSPLANT PATIENTS AND THEIR CORD BLOOD UNIT (CBU) DONORS

Aim Killer cell immunoglobulin-like receptor (KIR) genes are receptors expressed on the surface of natural killer cells. KIR genes recognize polymorphic epitopes of HLA class I-A, B & C, called KIR HLA ligands (HLA-L). After binding specific ligands the KIR may transmit inhibitory or activating signals. NKC sense & kill target cells missing MHC class I molecules. Studies showed that hematopoietic stem cells (HSC) transplant patients may benefit from NK alloreactivity of the donor or HLA-C alleles (C1/x) of the recipients. Previously we reported the diversification of KIR HLA-L in CBU from MD Anderson Cord Bank. The goal of this study was to further explore the frequency (Freq) of HLA-L in selected CBU from the bank and their transplant recipients aimed at providing information for finding the best donor and improving graft outcome. Methods 640 CBU selected from MD Anderson Cord Bank and 589 recipients were included. HLA typing of class I & II loci were obtained using PCR/probe based methods & sequence-based typing as needed. Freq of Bw4, C1, and C2 group of HLA-C alleles were examined. Results KIR3DL1 recognizes allotypes with HLA-Bw4 motif; KIR2DL1 recognizes HLA-C epitope with lysine at codon 80 (group 2-C2); while KIR2DL2 and KIR2DL3 recognize C alleles with asparagine at residue 80 (group 1-C1). Among the 640 CBU and 589 recipients, 70.0% and 72.0%, respectively, carry Bw4 bearing phenotypes. HLA-C1 bearing phenotypes (C1/x) were found to be predominated in both CBU (85.2%) and their transplant recipients(81.5%). Missing HLA-C1 was lower (C2/C2, 18.5%) than missing C2 (C1/C1, 33.1%) in recipients (Table 1). Conclusion Here we show that CBU can be classified based on their KIR repertoire (licensed versus unlicensed) and the presence of KIR-ligand mismatch with the recipient. Analysis of graft outcome in these HSC transplant recipients is underway. The ultimate goal of our study is to develop algorithms based on the KIR profile of CB units to select the units with maximum antileukemic activity to improve the outcome of CBU transplant.

Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis.

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

Allelic polymorphisms of human platelets-specific alloantigens in South Tunisian population

ObjectivesHuman platelet-specific alloantigens (HPA) are polymorphic epitopes which vary among ethnic groups.BackgroundIn Tunisia, HPA frequencies were determined in North and centre; however, the pattern of HPA in South Tunisian population is not been studied yet. The aim of this work was to determine allelic frequencies of HPA-1, -3, and -5 systems in south Tunisian population, in order to estimate the risk of anti-platelet allo-immunization and to create a register of HPA-typed blood donors.MethodsOur study concerned 212 unrelated healthy, regular blood donors from southern Tunisia. Allelic polymorphisms of each system were determined using a polymerase chain reaction with sequence-specific primers.ResultsGenotype frequencies a/a, a/b, and b/b were, respectively, 0.670, 0.288, and 0.042 for HPA-1 system, 0.430, 0.462, and 0.108 for HPA-3 system, and 0.750, 0.241, and 0.009 for HPA-5 system. The allele frequencies were 0.814 and 0.186 for HPA-1a and -1b alleles; 0.660 and 0.340 for HPA-3a and -3b alleles and 0.870, and 0.130 for HPA-5a and -5b alleles.DiscussionThe reported frequencies are more similar to those of Caucasians than those of north Tunisian population.

Identification of Strain-Specific B-cell Epitopes in Trypanosoma cruzi Using Genome-Scale Epitope Prediction and High-Throughput Immunoscreening with Peptide Arrays

BackgroundThe factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.MethodologyBy performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.Findings and ConclusionsA total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

Antibodies to Polymorphic Invasion-Inhibitory and Non-Inhibitory Epitopes of Plasmodium falciparum Apical Membrane Antigen 1 in Human Malaria

BackgroundAntibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1.Methodology/FindingsWe examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies.Conclusions/SignificanceIndividuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1.

Direct binding to antigen‐coated beads refines the specificity and cross‐reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets.