Potential Target Antigens Research Articles

Circulating and disseminated tumor cells: diagnostic tools and therapeutic targets in motion.

Enumeration of circulating tumor cells (CTCs) in peripheral blood with the gold standard CellSearchTM has proven prognostic value for tumor recurrence and progression of metastatic disease. Therefore, the further molecular characterization of isolated CTCs might have clinical relevance as liquid biopsy for therapeutic decision-making and to monitor disease progression. The direct analysis of systemic cancer appears particularly important in view of the known disparity in expression of therapeutic targets as well as epithelial-to-mesenchymal transition (EMT)-based heterogeneity between primary and systemic tumor cells, which all substantially complicate monitoring and therapeutic targeting at present. Since CTCs are the potential precursor cells of metastasis, their in-depth molecular profiling should also provide a useful resource for target discovery. The present review will discuss the use of systemically spread cancer cells as liquid biopsy and focus on potential target antigens.

Expression pattern of subA in different tissues and blood-feeding status in Haemaphysalis flava.

Tick-borne-diseases (TBD) pose a huge threat to the health of both humans and animals worldwide. Tick vaccines constitute an attractive alternative for tick control, due to their cost-efficiency and environmental-friendliness. Subolesin, a protective antigen against ticks, is reported to be a promising candidate for the development of broad-spectrum vaccines. However, the entire length of its gene, subA, and its gene expression pattern in different tissues and blood-feeding status (or different levels of engorgement) have not been studied extensively. In our study, the full-length of subA in Haemaphysalis flava, Rhipicephalus haemaphysaloides, Rhipicephalus microplus, and Dermacentor sinicus was cloned by RACE-PCR. The subA expression pattern was analyzed further in H. flava in different tissues and blood-feeding status by RT-PCR. We found that the full-length of subA in H. flava, R. haemaphysaloides, R. microplus, and D. sinicus was 1318, 1498, 1316, and 1769bp, respectively, with encoded proteins at 180, 162, 162, and 166aa in length, respectively. The primary structure of subolesin in H. flava included three conserved regions and two hypervariable regions, with no signal peptide. SubA expression in female H. flava of different blood-feeding status was in the order of the fasted<the 1/4-engorged<the half-engorged<the fully-engorged (p<0.01). Tissue expression of subA was in the order of salivary gland>midgut>integument (p<0.01), but its expression in salivary glands was not statistically different from that in ovaries. We concluded that subolesin was a conserved antigen and that subA was expressed differentially in H. flava in different tissues and blood-feeding status. Those features made subolesin feasible as a potential target antigen for development of a universal vaccine for the control of tick infestations and a reduction in TBD.

Evaluation of a di-O-methylated glycan as a potential antigenic target for the serodiagnosis of human toxocariasis.

Serodiagnosis of human toxocariasis is based on the detection of specific IgG antibodies by the enzyme-linked immunosorbent assay (ELISA) using Toxocara larvae excretory-secretory (TES) antigens, but its production is a laborious and time-consuming process being also limited by the availability of adult females of T.canis as source for ova to obtain larvae. Chemical synthesis of the di-O-methylated (DiM) glycan structure found in the TES antigens has provided material for studying the antibody reactivity in a range of mammalian hosts, showing reactivity with human IgM and IgG. In this study, we have evaluated the performance of the DiM glycan against a panel of sera including patients with toxocariasis (n=60), patients with other helminth infections (n=75) and healthy individuals (n=94), showing that DiM is able to detect IgG antibodies with a sensitivity and specificity of 91·7% and 94·7%, respectively, with a very good agreement with the TES antigens (kappa = 0·825). However, cross-reactivity was observed in some sera from patients with ascariasis, hymenolepiasis and fascioliasis. These results show that the DiM glycan could be a promising antigenic tool for the serodiagnosis of human toxocariasis.

Enolase, a plasminogen receptor isolated from salivary gland transcriptome of the ixodid tick Haemaphysalis flava

Enolase, a multifunctional protein, is shown to act as a plasminogen receptor that contributes to fibrinolysis, which plays an important role in preventing the formation of blood clots during tick feeding. The study of enolase genes provides opportunities to develop a potential antigen target for tick control. So far, enolase has been identified in only a few species of ticks. Knowledge of the exact mechanisms of plasminogen activation and fibrinolysis by enolase as a plasminogen receptor is limited. Here, we cloned the enolase full-length complementary DNA (cDNA) from the salivary glands of Haemaphysalis flava, expressed it, and analyzed the function of the recombinant H. flava enolase. The enolase cDNA was 1988bp in length and encoded 433 amino acid residues. It contained two domains and some highly conserved functional motifs including an assumed membrane re-association region "AAVPSGASTGI." The enolase exhibited 83.3% amino acid similarity to that of the putative enolase of Ixodes ricinus, and 85% to that of Ornithodoros moubata enolase. After eukaryotic expression in insect cells, Western blot analysis showed that the mouse antiserum against the hexahistidine-tagged recombinant enolase protein recognized a band of approximately 48kDa. The recombinant enolase bound human plasminogen in a dose-dependent manner and enhanced plasminogen activation in the presence of host tissue plasminogen activator (t-PA), most probably to promote fibrinolysis and maintain blood flow at the host-tick interface. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the expression level of enolase in salivary glands was significantly higher than in other tested tissues. Although the enolase was expressed in all developmental stages, it had the highest expression in the rapid blood feeding period of ticks. These findings indicate that the enolase might play an important role in blood feeding of H. flava.

Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S.mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S.mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S.mutans NG8 strain. Therefore, our results indicate that S.mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries.

Methodology for preservation of yeast-bound single chain fragment variable antibody affinity reagents

Readily accessible affinity reagents are critical to the validation of biomarkers and to the development of new diagnostic tests. As alternatives to monoclonal antibodies, yeast-bound single chain fragment variable antibody (yeast-scFv) can be rapidly selected from yeast display libraries. An important characteristic for any diagnostic reagent is its stability or ability to store it. A lyophilization procedure that has extended the shelf life of yeast-scFv by a factor of ≥10-fold relative to previous reports is reported. Real time stability for three yeast-scFv clones to three distinct Entamoeba histolytica potential diagnostic antigen targets for one year at room temperature as well as at 37°C and 45°C. Retention of full binding activity and specificity of the yeast-scFv clones for their cognate antigens is shown by flow cytometry. Lyophilization can easily be carried out in batches and in single-use vials.

Potential utility of autoantibodies as blood-based biomarkers for early detection and diagnosis of Parkinson’s disease

IntroductionThere is a great need to identify readily accessible, blood-based biomarkers for Parkinson’s disease (PD) that are useful for accurate early detection and diagnosis. This advancement would allow early patient treatment and enrollment into clinical trials, both of which would greatly facilitate the development of new therapies for PD. MethodsSera from a total of 398 subjects, including 103 early-stage PD subjects derived from the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) study, were screened with human protein microarrays containing 9,486 potential antigen targets to identify autoantibodies potentially useful as biomarkers for PD. A panel of selected autoantibodies with a higher prevalence in early-stage PD was identified and tested using Random Forest for its ability to distinguish early-stage PD subjects from controls and from individuals with other neurodegenerative and non-neurodegenerative diseases. ResultsResults demonstrate that a panel of selected, blood-borne autoantibody biomarkers can distinguish early-stage PD subjects (90% confidence in diagnosis) from age- and sex-matched controls with an overall accuracy of 87.9%, a sensitivity of 94.1% and specificity of 85.5%. These biomarkers were also capable of differentiating patients with early-stage PD from those with more advanced (mild-moderate) PD with an overall accuracy of 97.5%, and could distinguish subjects with early-stage PD from those with other neurological (e.g., Alzheimer’s disease and multiple sclerosis) and non-neurological (e.g., breast cancer) diseases. ConclusionThese results demonstrate, for the first time, that a panel of selected autoantibodies may prove to be useful as effective blood-based biomarkers for the diagnosis of early-stage PD.

The Epstein-Barr Virus Lytic Protein BZLF1 as a Candidate Target Antigen for Vaccine Development.

The Epstein-Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. Although most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing life-threatening diseases, such as Hodgkin lymphoma and posttransplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV infection in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1 as a potential target antigen for vaccine development. Primary tumors from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) express BZLF1 protein. Pulsing human dendritic cells (DC) with recombinant BZLF1 followed by incubation with autologous mononuclear cells led to expansion of BZLF1-specific CD8(+) T cells in vitro and primed BZLF1-specific T-cell responses in vivo. In addition, vaccination of hu-PBL-SCID mice with BZLF1-transduced DCs induced specific cellular immunity and significantly prolonged survival from fatal EBV-LPD. These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide a rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases.

Role of a Novel Human Leukocyte Antigen-DQA1*01:02;DRB1*15:01 Mixed Isotype Heterodimer in the Pathogenesis of “Humanized” Multiple Sclerosis-like Disease

Gene-wide association and candidate gene studies indicate that the greatest effect on multiple sclerosis (MS) risk is driven by the HLA-DRB1*15:01 allele within the HLA-DR15 haplotype (HLA-DRB1*15:01-DQA1*01:02-DQB1*0602-DRB5*01:01). Nevertheless, linkage disequilibrium makes it difficult to define, without functional studies, whether the functionally relevant effect derives from DRB1*15:01 only, from its neighboring DQA1*01:02-DQB1*06:02 or DRB5*01:01 genes of HLA-DR15 haplotype, or from their combinations or epistatic interactions. Here, we analyzed the impact of the different HLA-DR15 haplotype alleles on disease susceptibility in a new "humanized" model of MS induced in HLA-transgenic (Tg) mice by human oligodendrocyte-specific protein (OSP)/claudin-11 (hOSP), one of the bona fide potential primary target antigens in MS. We show that the hOSP-associated MS-like disease is dominated by the DRB1*15:01 allele not only as the DRA1*01:01;DRB1*15:01 isotypic heterodimer but also, unexpectedly, as a functional DQA1*01:02;DRB1*15:01 mixed isotype heterodimer. The contribution of HLA-DQA1/DRB1 mixed isotype heterodimer to OSP pathogenesis was revealed in (DRB1*1501xDQB1*0602)F1 double-Tg mice immunized with hOSP(142-161) peptide, where the encephalitogenic potential of prevalent DRB1*1501/hOSP(142-161)-reactive Th1/Th17 cells is hindered due to a single amino acid difference in the OSP(142-161) region between humans and mice; this impedes binding of DRB1*1501 to the mouse OSP(142-161) epitope in the mouse CNS while exposing functional binding of mouse OSP(142-161) to DQA1*01:02;DRB1*15:01 mixed isotype heterodimer. This study, which shows for the first time a functional HLA-DQA1/DRB1 mixed isotype heterodimer and its potential association with disease susceptibility, provides a rationale for a potential effect on MS risk from DQA1*01:02 through functional DQA1*01:02;DRB1*15:01 antigen presentation. Furthermore, it highlights a potential contribution to MS risk also from interisotypic combination between products of neighboring HLA-DR15 haplotype alleles, in this case the DQA1/DRB1 combination.

Armed hunter killers: discerning the role of adoptive T-cell transfer for glioblastoma.

Glioblastoma (GBM) is the most malignant primary brain cancer with an incidence around 3.19 per 100,000/year in the USA [1]. Poor prognosis is due to its infiltrative nature in the brain, genetic heterogeneity and the belligerent GBM stem cells. Despite the advance in new generation alkylating agents, such as temozolomide, along with radiation and tumor vasculature-targeting monoclonal antibodies, such as bevacuzimab, the overall survival has only accented by a few months above the natural history of the untreated tumor [2]. The Cancer Genome Atlas Research Network recently unraveled a genetic alteration which pinpointed potential antigenic targets in GBM [3]. Hence, targeted gene immunotherapy could be a vital hope for finding an efficient GBM cure. Immunotherapy is an intriguing biologic which harnesses the power of the immune system to selectively kill tumor cells [4]. Active immunization with dendritic cell vaccines and immune recovery with cytokines, are encouraging approaches however, they are limited by their inability to expand tumor antigen-specific T cells, which are often anergized by the tumor hostile milieu [4]. One of the means to overcome this limitation is passive immunization with adoptively-transferred T cells [5]. Adoptivelytransferred T cells are tumor-specific T cells prepared ex vivo, either de novo or by enriching a pre-existent tumor specific T-cell population, then retransferred back to patients, often after lymphodepletion [6]. Two tactics were scrutinized to generate tumor affinitymatured T cells by transducing T cells either with high-affinity tumor reactive T-cell receptor (TCR) genes or genes encoding chimeric antigen receptors (CARs) [7]. Broader adoptive therapy concept includes natural killer cells [8], lymphokine-activated killer cells [4]; as well as neural or mesenchymal stem-cells [9,10]. This review will highlight the use of T-cell adoptive therapy potential for GBM while embarking upon some challenges that need to be overcome to pave the way to an excelled GBM cure in the near future.

Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 (LMP2) of Epstein-Barr virus as a potential vaccine and diagnostic agent.

Epstein-Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients.

Diagnosis and treatment of chronic inflammatory demyelinating polyneuropathy.

Chronic inflammatory demyelinating neuropathy (CIDP) is generally considered to be an immune-mediated neuropathy with potential target antigens, although its precise pathogenesis is unclear. Unlike its more acute counterpart, acute inflammatory demyelinating neuropathy, the diagnosis is necessarily delayed as a result of clinical definitions but also the fluctuating clinical manifestations which can evolve over months and years. In addition, as a result of its relative rarity it is not usually definitively diagnosed and treated until patients come into contact with specialist neurology services. However, even within specialist settings the diagnosis is often elusive as a result of heterogeneous clinical manifestations, the patchy nature of the demyelinating pathology and limitations of available diagnostic tests. Despite the limited understanding of disease aetiology, recent studies have underlined the important role of both cellular and humoral components of the immune system in pathogenesis which in turn has informed treatment approaches. Although there remains no overall consensus on the optimum management strategy for CIDP, it is becoming clear that individualising therapeutic interventions depending on accurate identification of the CIDP variant is important. These include the typical symmetrical motor and sensory polyneuropathy, multifocal acquired demyelinating sensory and motor neuropathy (MADSAM), pure motor, pure sensory and distal acquired demyelinating symmetric (DADs) neuropathy, all of which may demonstrate variation in response to intravenous immunoglobulins, plasma exchange (PE) and other immunosuppressive therapies possibly as a result of different underlying pathological mechanisms. In this month’s Journal Club, we review three papers focusing on different aspects of diagnosis and treatment in CIDP. The first paper explores patterns of magnetic resonance neurography and the distribution of nerve hypertrophy in CIDP variants. The second paper reviews different electrophysiological profiles and treatment response in typical and atypical forms, whilst the third investigates predictors of intravenous immunoglobulin (IVIG) responsiveness in patients with CIDP.

Identification of heat shock protein 27 as a novel autoantigen of Behçet’s disease

ObjectiveThe aim of this study was to identify candidate pathogenic autoantigens of Behçet’s disease (BD) in pathogen-stimulated target cells. MethodsFirst, three cell lines were used as target cells to screen autoantibody. Second, selected target cells were simulated with pathogens. Third, western blotting was used for detecting the auto-antigens in cell extracts. Next, immunoprecipitation was performed and the amino-acid sequences of target antigens were analyzed by LC-MALDI-TOF/TOF. Then, the potential target antigen was expressed, purified, and immunologically confirmed. And finally, an ELISA kit was developed and clinically validated through the assessments of 456 clinical samples with BD. ResultsOne antigen with a molecular weight of approximately 27-kDa was identified as heat shock protein 27 (HSP27). The reactivity of serum IgG against recombinant human HSP27 was detected in 52 of 91 BD patients (57%), 66 of 92 rheumatoid arthritis (RA) patients (72%), 32 of 90 Sjogren syndrome (SS) patients (36%), 22 of 92 systemic lupus erythematosus (SLE) patients (24%) and 0 of 91 healthy controls (HC). The reactivity of BD serum IgG antibodies against HSP27 was significantly higher than SLE (P<0.0001) SS (P<0.0001) and HC (P<0.0001). ConclusionsThis study identified HSP27 as a candidate endothelial cell autoantigen of BD, which is interesting and probably worth further exploration.

Prospects for immunocontraception in feral horse population control: exploring novel targets for an equine fertility vaccine.

Feral horses populate vast land areas and often induce significant ecological and economic damage throughout the landscape. Non-lethal population control methods are considered favourable in light of animal welfare, social and ethical considerations; however, no single effective, safe and species-specific contraceptive agent is currently available for use in free-ranging wild and feral horses. This review explores aspects of equine reproductive physiology that may provide avenues for the development of specific and long-lasting immunocontraceptive vaccines and some of the novel strategies that may be employed to facilitate appropriate antigen discovery in future research. Potential antigen targets pertaining to spermatozoa, the ovary and oocyte, as well as the early conceptus and its associated factors, are reviewed in the context of their suitability for immunocontraceptive vaccine development.

The Epstein-Barr Virus Lytic Protein BZLF1 As a Candidate Target Antigen for Vaccine Development

The Epstein-Barr virus (EBV) is an oncogenic, γ-herpesvirus associated with a broad spectrum of disease. While most immune-competent individuals can effectivley develop efficient adaptive immune responses to EBV, immunocompromised individuals are at serious risk for developing lifethreatening pathology such as Hodgkin’s lymphoma and post-transplant lymphoproliferative disorder (PTLD). Given the significant morbidity associated with EBV in high-risk populations, there is a need to develop vaccine strategies that restore or enhance EBV-specific immune responses. Here, we identify the EBV immediate-early protein BZLF1, a transcription factor that initiates latency to lytic cycle transition, as a potential target antigen for vaccine development. IHC assays on primary lymphoma samples from patients with PTLD and a chimeric human-murine model of EBV-driven lymphoproliferative disorder (EBV-LPD) revealed significant expression of BZLF1 protein (>10% tumor cells). Other EBV-positive lymphomas that exhibited staining for BZLF1 included lymphomatoid granulomatosis (1/3) and diffuse large B-cell lymphoma (5/10); whereas EBV(+) Hodgkin’s and EBV(+) unclassifiable lymphomas failed to demonstrate any BZLF1 signal. To examine the immunomodulatory properties of BZLF1, PBMCs were isolated from 24 healthy, EBV-seropositive donors of various HLA types and cultured in vitro with adenovirus encoding recombinant BZLF1 (rAd5F35/BZLF1) or null control (rAd5F35/null) for 18 hrs. Quantification of the number of IFN-γ secreting cells by ELISPOT revealed increased immune activation in response to BZLF1 exposure in the majority (20/24) of donors. These results led us to hypothesize that enhanced recognition of BZLF1 by antigen-specific cellular immunity may provide a protective effect against EBV. To address this, we next investigated whether DCs loaded with recombinant BZLF1 protein could mediate the expansion of BZLF1-specific CD8(+) T-cells in vitro. DCs were generated in vitro from healthy PBMC monocytes. Coculture of rBZLF1-loaded DCs and autologous PBMCs led to increased frequencies of BZLF1-specific CD8(+) T-cells as determined by tetramer flow assays. To determine whether BZLF1 vaccination could prime BZLF1-specific T-cell immunity in vivo, SCID mice depleted of murine NK cells were engrafted with EBV-positive PBMCs (hu-PBL-SCID) and injected simultaneously with rBZLF1 (100 µg/mouse). Four weeks post-vaccination, ex vivo splenocytes were reexposed to BZLF1-presenting DCs in vitro and assayed for IFN-γ secretion by ELISPOT. In comparison to vehicle, vaccination with rBZLF1 alone significantly enhanced BZLF1-specific IFN-γ responses (p=0.0007, n=3). In order to generate broader, more sustained immune responses against BZLF1, we next explored BZLF1 virally-transduced DC vaccination as a potential approach to prevent EBV-LPD. Monocyte-derived DCs were infected with rAd5F35/BZLF1 or rAd5F35/null (MOI=10) and cocultured with autologous PBMCs for 7 days. Exposure to BZLF1-transduced DCs induced a robust expansion of BZLF1-specific CD8(+) cells that was comparatively higher than recombinant protein presentation. Finally, we examined whether vaccination of hu-PBL-SCID mice with rAd5F35/BZLF1-transduced DCs would positively enhance cellular immunity and improve survival against fatal EBV-LPD. Hu-PBL-SCID mice were injected IP with 5 x 106 BZLF1-transduced DCs (or control). Mice in the “booster group” received a second dose of either rAd5F35/BZLF1 (or control) DCs as a booster. Five weeks post-vaccination, ex-vivo splenocytes were cultured 1:1 with autologous lymphoblastoid cell lines (LCLs) and assayed for IFN-γ secretion by ELISPOT. Mice vaccinated with rAd5F35/BZLF1-transduced DCs showed significantly higher responsiveness to LCLs relative to vector control mice (p<0.0001, n=3). Furthermore, single vaccination with BZLF1-transduced DCs showed a trend toward improved survival (p=0.085, n=10); however, vaccination followed by booster delivery at 2 weeks significantly delayed the development of EBV-LPD (p=0.014, n=10, median survival: rAd5F35/BZLF1, 62 days; rAd5F35/null, 48 days). These findings identify BZLF1 as a candidate target protein in the immunosurveillance of EBV and provide rationale for considering BZLF1 in vaccine strategies to enhance primary and recall immune responses and potentially prevent EBV-associated diseases. DisclosuresNo relevant conflicts of interest to declare.

Prediction and identification of HLA-A*0201-restricted epitopes from leukemia-associated protein MLAA-22 which elicit cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) play a critical role in the control of leukemia. However, few effective CTL epitopes have been identified to date yet. We previously reported that MLAA-22, a protein composed of 631 amino acid residues, is a novel acute myeloid leukemia (AML)-associated antigen. In the present study, ten high-score 9-mer peptides, which were selected from MLAA-22 by using ProPred1 and SYFPEITHI bioinformatics tools, were screened to identify HLA-A*0201-restricted-specific CTL epitopes. Monocyte-derived dendritic cells were generated in vitro to be used as antigen-presenting cells for the induction of CTLs. We found that peptide MLAA-22(379-387) (LLPNAIYKV) exhibited the highest binding affinity to HLA-A*0201 among all peptide candidates in the peptide-T2 binding assay. The percentage of positive T2 cells treated with MLAA-22(379-387) was about 96.3%, which is even higher than that of the positive control peptide CML28(173-181) (95.1%). MLAA-22(379-387)-induced CTLs showed the most significant cytotoxic activity and apparent killing effects on the cell lines including THP-1 (human acute monocytic leukemia), A549, T2, U937, and MCF-7, and the specific lysis ratios were 83.8, 32.6, 64.4, 64.4, and 32.6%, respectively, when the effector to target ratio (E/T) was 20:1. Specific lysis (%) of MLAA1 was significantly increased (P < 0.05, P < 0.001, respectively) in THP-1 cell than those in other cancer cell lines and were 28.5, 67.8, and 83.8% at ratio 5:1, 10:1, and 20:1, respectively. Hence, MLAA-22(379-387) is a potential tumor-associated antigen target for AML immunotherapy.

Abstract 2661: Patient-derived breast cancer targeting heavy chain antibodies

Abstract The emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab and anti-CD47 antibody has highlighted the significant role of antibodies for effective cancer therapy. The most common approach to generate tumor specific antibodies is to select an established tumor specific antigen and generate antibodies to that target. The drawback with this approach is that it does not lead to identification of novel antigens or antigenic epitopes. Therefore, we developed a strategy that will lead to the identification of both tumor specific antibodies and novel tumor antigens. The approach uses the patient's immune system to guide us to define tumor specific antigens. In order to achieve this, we identified reactive germinal centers in the tumor draining sentinel lymph nodes from breast cancer patients. Immunohistochemical analysis of reactive germinal centers defined the B-cell regions for mRNA isolation and generation of cDNA molecules for the variable heavy chain segment (VH) of antibodies. The VH cDNA molecules were cloned into a mammalian expression vector to generate bivalent heavy chain antibodies with a C-terminus mouse Fc tag. At present, we have generated about 250 antigen-driven variable heavy chain clones from 27 breast cancer patient lymph node samples. We screened 46 heavy chain antibodies against a panel of breast cancer cell lines using immunofluorescence and identified 8 antibodies that showed strong and specific staining. Of these 8 antibodies, one of them (VH6-93) showed membrane staining preferentially on MDA-MB-231 cells in comparison to MCF7 cells. VH6-93 antibody also showed strong staining on conditionally reprogrammed tumor cells derived from a breast cancer patient with only moderate staining on normal breast epithelial cells derived from the same patient. VH6-93 also revealed preferential binding to breast tumor tissues as compared to normal ductal tissues evidenced by immunofluorescence and immunohistochemistry. To identify the target antigens, a multi-tiered approach was used, which included immunoprecipitation of target antigen from cell lysates followed by peptide sequencing with mass spectrometry as well as utilizing Nucleic Acid Programmable Protein Array (NAPPA) technology. A comprehensive analysis of two patient-derived antibodies has produced a panel of potential antigen targets, which are undergoing specificity and validation analysis. In summary, this methodology of using activated B-cells from sentinel lymph nodes is a unique way to enrich for tumor-specific antibody clones. The findings from this research will not only lead to tumor specific antibodies but will also reveal novel tumor specific antigens. Identification of these novel antigens can give rise to additional therapeutic strategies such as peptide or protein vaccination. Citation Format: Charan Kumar V. Devarakonda, Daniel Kita, Kathryn N. Phoenix, Dewey M. Magee, Kevin P. Claffey. Patient-derived breast cancer targeting heavy chain antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2014-2661

Porphyromonas gingivalis antigenic determinants--potential targets for the vaccine development against periodontitis.

Periodontal disease, is the most prevalent inflammatory affliction characterized by the loss of supporting structures of the teeth. Although bacteria are identified as prime agents initiating the disease, but major part of tissue destruction is driven by the host immune response to infecting agent and is responsible for the clinical presentation of the disease. Porphyromonas gingivalis (P. gingivalis) has been identified as main microbial factor for causation and progression of periodontal disease. Three emerging concepts of periodontal disease viz wide prevalence, polymicrobial nature and their role in perpetuation of systemic diseases bear critical significance on the development of novel therapeutic approaches to eradicate or alleviate the disease burden. Therefore, development of preventive approaches such as periodontal vaccine appears as an exciting modality which can prove as a significant adjunct to current periodontal therapies. The new generation diagnostics including microbial genome sequencing and bioinformatics analysis are available now to detect and analyze the whole genomic and proteomic data regarding almost all human pathogens for exploring these as novel drug targets for vaccine development. The current review, aims to provide an update of wide-ranging virulence factors of P. gingivalis as potential antigenic targets for periodontal vaccine development.

New targets for antibody therapy of pediatric B cell lymphomas.

Antibody therapy has become standard of care for adult B cell lymphoma patients. It is a potentially less toxic and more targeted approach for lymphoma therapy and should therefore be applied to treat pediatric B cell lymphoma patients as well. In pediatric lymphoma patients, however, clinical experience with monoclonal antibodies is very limited. This is in part due to smaller patient numbers and very good outcome with conventional chemotherapy. In addition, pediatric patient and lymphoma biology differ significantly from that found in adults often precluding extrapolation of the adult experience to children. This review focuses on targeting pediatric B cell lymphoma with monoclonal antibody therapy. The special characteristics of B cell lymphomas found in children are reviewed and six potential new lymphoma target antigens are discussed.

Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.