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17 p.-7 fig., Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin‐mediated metastatic progression. CDH6 preferentially bound to αIIbβ3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin RGD motif. Furthermore, CDH6 and CDH17 were found to interact with α2β1 in αIIbβ3low cells. Transient silencing of CDH6, ITGA2B or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion and lung colonization through the downregulation of SRC, FAK, AKT and ERK signaling. Interaction of αIIbβ3 with CDH6, and subsequent αIIbβ3 activation, promoted activation of α2β1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically‐approved αIIbβ3 inhibitors could block pro‐metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbβ3 regulate α2β1‐mediated adhesion and invasion of ovarian and renal cancer metastatic cells, and constitutes a therapeutic target of broad potential for treating metastatic progression., This research was supported by grants from the Ministerio de Ciencia e Innovación (Spain) (RTC2017-2017-6260-1 and RTI2018-095055-B-100).

15 p.-7 fig.-2 tab., C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome., This study was supported by the Spanish Ministryof Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Develop-ment Fund (ERDF); and the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [ED431C2018/21; predoctoral grant Plan I2C 2014; and CentroSingular de investigación de Galicia accreditation 2016-2019, ED431G/05], co-funded by the European Regional Development Fund (ERDF). The study also received funding from the European Union’s Horizon 2020 research andinnovation programme under the Marie Skłodowska-Curiegrant agreement No 766118. J.A.E. is supported by DeutscheForschungsgemeinschaft [DFG grant: EB177/13-1]

1 p. JSMO2021 Virtual Congress. 2021 the Japanese Society of Medical Oncology Annual Meeting. February 18 - 21, 2021, Background: Metallothionein-3 (M-3) has poorly characterized functions in hepatocellular carcinoma (HCC). HCC is a significant health problem. Globally is the second most common cause of cancer-associated death. Sorafenib was originally identified as an inhibitor of multiple oncogenic kinases and remains the only approved systemic therapy for advanced HCC. However, acquired resistance to sorafenib has been found in HCC patients, which results in poor prognosis. Overexpression of MT-3 decreased the sensitivity of HCC cells to sorefenib. Here, we investigated the impact of MT-3 up-regulation in HCC cells and the mechanisms underlying the sorafenib-resistance., Methods: To increase the expression of MT-3 HCC cells were transiently transfected with a plasmid containing MT-3 gene or with empty vector. The cDNA microarrays were accomplished using the ElectraSenseTM Reader. MS analysis was performed using a Q-Exactive MS. We used chick chorioallantoic membrane assay as in vivo model., Results: A cDNA profiling revealed that sorafenib resistance has a specific transcriptomic signature involving genes responsible for ion transport, trafficking and DNA repair. Also, The MS analysis data strongly suggest that resistance HCC cells acquired a complex regulatory network that significantly affects the ability of HCC cells to remove the ROS and activation of glycolysis. We provide the first evidence that up-regulation of MT3 resulted in increased dissociation, invasion, and intravasation from the primary tumours to the veins. In addition, MT3 profoundly impacted blood migration of Nbl cells and their extravasation to chicken organs., Conclusion: From a perspective of future utilization of our data, we anticipate that several identified genes and proteins could serve as prognostic biomarkers of outcome of sorafenib therapy. The increased expression of MT-3 within tumour mass should inform about worse prognosis and also decreased efficiency of sorafenib-based chemotherapy in HCC.

16 p.-4 fig., Low-grade, early-stage endometrial carcinoma (EC) is the most frequent malignant tumor of the uterine corpus. However, the molecular alterations that underlie these tumors are far from being fully understood. The purpose of this study is to describe dysregulated molecular pathways from EC patients. Sixteen samples of tumor tissue and paired healthy controls were collected and both were subjected to mass spectrometry (MS)/MS proteomic analysis. Gene ontology and pathway analysis was performed to discover dysregulated pathways and/or proteins using different databases and bioinformatic tools. Dysregulated pathways were cross-validated in an independent external cohort. Cell signaling, immune response, and cell death-associated pathways were robustly identified. The SLIT/ROBO signaling pathway demonstrated dysregulation at the proteomic and transcriptomic level. Necroptosis and ferroptosis were cell death-associated processes aberrantly regulated, in addition to apoptosis. Immune response-associated pathways showed a dominance of innate immune responses. Tumor immune infiltrates measured by immunofluorescence demonstrated diverse lymphoid and myeloid populations. Our results suggest a role of SLIT/ROBO, necroptosis, and ferroptosis, as well as a prominent role of innate immune response in low-grade, early-stage EC. These results could guide future research in this group of tumors, This research was funded by the Instituto de Salud Carlos III (ISCIII) (PI17/01723), co-financed by the European Development Regional Fund “A way to achieve Europe” (FEDER).

28 p.-6 fig., Cisplatin (CDDP) is a widely used agent in the treatment of neuroblastoma. Unfortunately, the development of acquired chemoresistance limits its clinical use. To gain a detailed understanding of the mechanisms underlying the development of such chemoresistance, we comparatively analysed established cisplatin-resistant neuroblastoma cell line (UKF-NB-4CDDP) and its sensitive counterpart (UKF-NB-4). First, using viability screenings, we confirmed the decreased sensitivity of tested cells to cisplatin and identified a cross-resistance to carboplatin and oxaliplatin. Then, the proteomic signatures were analysed using nanoLC MS/MS. Among the proteins responsible for UKF-NB-4CDDP chemoresistance, ion channels transport family proteins, ABC superfamily proteins, SLC-mediated trans-membrane transporters, proteasome complex subunits and V-ATPases were identified. Moreover, we detected markedly higher proteasome activity in UKF-NB-4CDDP cells and a remarkable lysosomal enrichment that can be inhibited by bafilomycin A to sensitize UKF-NB-4CDDP to CDDP. Our results indicate that lysosomal sequestration and proteasome activity may be one of key mechanisms responsible for intrinsic chemoresistance of neuroblastoma to CDDP., This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No.759585). The authors also gratefully acknowledge financial support from AZV project 15-28334A, ERASMUS + Staff Mobility from European Commission and from the Ministry of Health of the Czech Republic for conceptual development of research organization 00064203 17(University Hospital Motol, Prague, Czech Republic).

The transcription factor Twist1 is involved in the epithelial–mesenchymal transition and contributes to cancer metastasis through mostly unknown mechanisms. In colorectal cancer, Twist1 expression is mainly restricted to the tumor stroma. We found that human fibroblast cell lines stably transfected with Twist1 acquired characteristics of activated cancer-associated fibroblasts (CAFs), such as hyperproliferation, an increased ability to migrate and an alignment of the actin cytoskeleton. Further, Twist1-activated fibroblasts promoted increased matrix stiffness. Using quantitative proteomics, we identified palladin and collagen α1(VI) as two major mediators of the Twist1 effects in fibroblast cell lines. Co-immunoprecipitation studies indicated that palladin and Twist1 interact within the nucleus, suggesting that palladin could act as a transcription regulator. Palladin was found to be more relevant for the cellular biomechanical properties, orientation and polarity, and collagen α1(VI) for the migration and invasion capacity, of Twist1-activated fibroblasts. Both palladin and collagen α1(VI) were observed to be overexpressed in colorectal CAFs and to be associated with poor colorectal cancer patient survival and relapse prediction. Our results demonstrate that Twist1-expressing fibroblasts mimic the properties of CAFs present at the tumor invasive front, which likely explains the prometastatic activities of Twist1. Twist1 appears to require both palladin and collagen α1(VI) as downstream effectors for its prometastatic effects, which could be future therapeutic targets in cancer metastasis., IG-P was supported by a contract S2010/BMD-2344/ Colomics2 from the Comunidad de Madrid. ST was a recipient of a Juan de la Cierva programme. RAB was supported by a grant to established research groups of the Asociación Española Contra el Cáncer (AECC). ML-L was a recipient of a ProteoRed contract. AP-G and BE-P were FPI fellows from the Ministry of Economy and Competitiveness (MINECO). This research was supported by grants to established research groups of the S2010/BMD-2344/Colomics2 from the Comunidad de Madrid, ‘Asociación Española Contra el Cancer (AECC)’, BIO2012-31023 from the MINECO, PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER), RD12/0036/0041 and RD12/0036/0021 from the Instituto de Salud Carlos III-FEDER.

Large bowel adenocarcinoma is one of the most frequent human neoplasms and despite recent insights into the pathophysiology and molecular basis of this disease, mortality remains high in advanced and metastatic cases. Most guidelines recommend adjuvant chemotherapy for tumours involving lymph nodes, but not for patients with localized stage I or II disease. However, it is well known that approximately 20% of stage II colorectal carcinoma patients eventually recur, mainly with distant or peritoneal involvement and show bad prognosis. It would be important to predict which patients are at increased risk of recurrence to guide potential adjuvant therapy use in this controversial setting. In this sense, only microsatellite stability has been proposed as a predictive tool in some guidelines. The tripartite motif family protein 72 (TRIM72) is a ubiquitin ligase, involved in the cell membrane repair machinery and known to be associated to insulin resistance. Its potential role in colon cancer has recently been proposed. The aim of this study is to determine the potential predictive value of TRIM72 immunohistochemical expression in stage II colon carcinoma. We have retrospectively reviewed a series of 95 patients with stage II colon microsatellite stable carcinomas operated with a curative intent at a single large tertiary hospital in Madrid (Spain) between 2006 and 2012. None of the patients received adjuvant chemotherapy. We reviewed the histopathological slides and constructed a tissue microarray (TMA) of three representative areas to perform immunohistochemical staining for TRIM72. In our series 30 patients (31.7%) recurred after a median follow-up of 17.5 months. Lack of immunohistochemical expression of TRIM72 in the tumor was significantly and independently associated to recurrence. A recent report by Chen et al. has shown that TRIM72 can be measured in plasma for colon carcinoma detection as an alternative to CEA or CA19.9, with lower levels in patients with carcinoma. Our report is the first one to show that lower immunohistochemical expression of TRIM72 predicts recurrence in colon stage II carcinoma. We feel this predictive influence can be related to its crucial role as a central regulator in many signaling pathways (PI3K-AKT, ERK). As an ubiquitin ligase, the lack of TRIM72 could increase the levels of several potential oncogenic molecules and therefore lead to a more aggressive phenotype. It remains to be shown whether chemotherapy could change the clinical behaviour of this bad prognosis group. We propose TRIM72 immunohistochemical analysis as a potential tool to predict recurrence risk in stage II colon carcinoma patients. Our results should be confirmed in larger series, but could open the way to management strategies refinement in this early stage group of patients.

8 p.-2 fig., The transcription factor (TF) Snail1 is a major inducer of the epithelial–mesenchymal transition (EMT) during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC) abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC) for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT) for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157 (Vizcaino et al., 2014) [1]. Data are associated with a research article published in Molecular and Cellular Proteomics (Pelaez-Garcia et al., 2015), This research was supported by grants to established research groups (AECC), BIO2012-31023 from the Spanish Ministry of Economy and Competitiveness, S2011/BMD-2344/ (Colomics2) from Comunidad de Madrid and Grant PRB2 (IPT13/0001 – ISCIII-SGEFI/FEDER) from ProteoRed-ISCIII.

10 páginas, 6 figuras, 2 tablas -- PAGS nros. 559-568, We present data that reveal crucial differences between the binding mode of anti-gastrin17 (G17, pyroEGPWLEEEEEAYGWMDF-NH2) monoclonal antibodies (mAbs) and their CDR-derived synthetic binders (SBs) with G17. The mAbs recognize the N-terminal sequence of G17 (pyroEGPWL) with nanomolar affinity and high sequence selectivity. Molecular simulations suggest that G17 recognition is based primarily on a multitude of weak antibody–ligand interactions (H-bonding, van der Waals, etc.) inside a structurally well-defined cleft-like binding pocket. Relatively small structural changes (e.g. G-2 to A for G17) have a drastic impact on affinity, which is characteristic for antibody-like binding. In contrast, SBs recognize various sequences, including G17-unrelated targets with affinities of 1:1 complexes estimated in the 0.1–1.0 mM range. In most cases however, the G17/SB complex stoichiometries are not well-defined, giving rise to multimer aggregate formation with high apparent complex stabilities. Mutational studies on both G17 and SBs reveal the importance of positively charged (K/R) and aromatic residues (W/Y/F) for G17/SB complex formation. We propose that the synthetic binders use combinations of electrostatic, hydrophobic, and/or cation–π interactions in a variety of ways due to their intrinsic flexibility. This may also be the reason for their relatively low target specificity. We speculate that our findings are of general relevance, in showing that high-affinity mAbs do not necessarily provide the optimal basis for functional mimics design, This work was financially supported by a European Commission FP6 cooperative research project (COOP-CT-2004-512691). RB was recipient of a contract from the Fondo de Investigaciones Sanitarias of the Spanish Ministry of Health. DA and SGS acknowledge support from the CNRS (Centre National de la Recherche Scientifique) and UdS (University of Strasbourg). PT and RHM acknowledge also financial support from the Dutch Ministry of Economic Affairs (grant TSGE2017). RB and IC acknowledge financial support from the Spanish Ministry of Science (grant BIO2006-07689)

p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. We describe here that p120-catenin is required for Wnt pathway signaling. p120-catenin binds and is phosphorylated by CK1ε in response to Wnt3a. p120-catenin also associates to the Wnt co-receptor LRP5/6, an interaction mediated by E-cadherin, showing an unexpected physical link between adherens junctions and a Wnt receptor. Depletion of p120-catenin abolishes CK1ε binding to LRP5/6 and prevents CK1ε activation upon Wnt3a stimulation. Elimination of p120-catenin also inhibits early responses to Wnt, such as LRP5/6 and Dvl-2 phosphorylation and axin recruitment to the signalosome, as well as later effects, such as β-catenin stabilization. Moreover, since CK1ε is also required for E-cadherin phosphorylation, a modification that decreases the affinity for β-catenin, p120-catenin depletion prevents the increase in β-catenin transcriptional activity even in the absence of β-catenin degradation. Therefore, these results demonstrate a novel and crucial function of p120-catenin in Wnt signaling and unveil additional points of regulation by this factor of β-catenin transcriptional activity different of β-catenin stability.

20 p.-1 fig.-2 tab., Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2-D DIGE, MS and protein microarray technologies, in their different formats, to identify up- or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low-abundant proteins, which may play a key role in CRC diagnostics and progression., This study was supported partially by the BIO2006-07689 grant from the Spanish Ministry of Education and Science.Additional funding was provided by grants from the Fundación Médica Mutua Madrileña and the Comunidad Autónoma de Madrid to the ‘‘Colomics’’ research program.

Gastrin and its derivatives are becoming important targets for immunotherapy of pancreatic, gastric and colorectal tumors. This study was conducted to design antibodies able to block gastrin binding to the gastrin/cholecystokinin-2 (CCK-2) receptor in order to delay tumor growth. The authors have used different gastrin molecules, combined with the diphtheria toxoid, to generate and select human single chain variable fragments (scFvs) as well as mouse monoclonal antibodies and scFvs against different regions of gastrin. There was a remarkable conservation in the antibody repertoire against gastrin, independently of the approach and the species. The germlines most frequently used in gastrin antibody formation were identified. Three different epitopes were identified in the gastrin molecule. The resulting mouse monoclonal antibodies and scFvs were analyzed for gastrin neutralization using Colo 320 WT cells, which overexpress the CCK-2 receptor. The gastrin neutralizing activity assay showed that N-terminal specific mouse monoclonal antibodies were more efficient to inhibit proliferation of Colo 320 WT cells than the anti-C terminal antibodies. Moreover, the human antigastrin scFvs obtained in this study inhibited significantly the proliferation of Colo 320 tumoral cells. These findings should contribute to a more rational design of antibody-based antigastrin therapies in cancer, including passive administration of human antibodies with blocking activity. (c) 2008 Wiley-Liss, Inc.

Objective Understand the role of ZEB1 in the tumour initiation and progression beyond inducing an epithelial-to-mesenchymal transition. Design Expression of the transcription factor ZEB1 associates with a worse prognosis in most cancers, including colorectal carcinomas (CRCs). The study uses survival analysis, in vivo mouse transgenic and xenograft models, gene expression arrays, immunostaining and gene and protein regulation assays. Results The poorer survival determined by ZEB1 in CRCs depended on simultaneous high levels of the Wnt antagonist DKK1 , whose expression was transcriptionally activated by ZEB1. In cancer cells with mutant TP53 , ZEB1 blocked the formation of senescence-associated heterochromatin foci at the onset of senescence by triggering a new regulatory cascade that involves the subsequent activation of DKK1, mutant p53, Mdm2 and CtBP to ultimately repress macroH2A1 ( H2AFY ). In a transgenic mouse model of colon cancer, partial downregulation of Zeb1 was sufficient to induce H2afy and to trigger in vivo tumour senescence, thus resulting in reduced tumour load and improved survival. The capacity of ZEB1 to induce tumourigenesis in a xenograft mouse model requires the repression of H2AFY by ZEB1. Lastly, the worst survival effect of ZEB1 in patients with CRC ultimately depends on low expression of H2AFY and of senescence-associated genes. Conclusions The tumourigenic capacity of ZEB1 depends on its inhibition of cancer cell senescence through the activation of a herein identified new molecular pathway. These results set ZEB1 as a potential target in therapeutic strategies aimed at inducing senescence.

The Spanish Chromosome 16 consortium is integrated in the global initiative Human Proteome Project, which aims to develop an entire map of the proteins encoded following a gene-centric strategy (C-HPP) in order to make progress in the understanding of human biology in health and disease (B/D-HPP). Chromosome 16 contains many genes encoding proteins involved in the development of a broad range of diseases, which have a significant impact on the health care system. The Spanish HPP consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. Proteomics strategies have enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. BIOLOGICAL SIGNIFICANCE: In this manuscript we describe how the Spanish HPP-16 consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. We show how the Proteomic strategy has enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. This article is part of a Special Issue entitled: HUPO 2014.

A wide array of systems have been developed to improve “classic” vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project.

African horse sickness virus (AHSV) causes a fatal disease in horses. The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5. VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes. The biological function of VP5, the other component of the capsid, is unknown. In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies. Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated. To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system. The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed. The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151–200) and II (residues 83–120). The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule. Neutralizing epitopes were defined at positions 85–92 (PDPLSPGE) for MAb 10AE12 and at 179–185 (EEDLRTR) for MAb 10AC6. Epitope 10AE12 is highly conserved between the different orbiviruses. MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques. These data will be especially useful for vaccine development and diagnostic purposes.

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal antibodies (MAbs) and 12 overlapping fragments of the protein expressed in E. coli. Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid. This domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appears to be the most immunodominant region.

12 p.-7 fig., Liver metastasis is the major cause of death associated to colorectal cancer. Cadherin-17 (CDH17) is a non-classical, seven domain, cadherin lacking the conserved cytoplasmic domain of classical cadherins. CDH17 was overexpressed in highly metastatic human KM12SM and present in many other colorectal cancer cells. Using tissue microarrays, we observed a significant association between high expression of CDH17 with liver metastasis and poor survival of the patients. On the basis of these findings, we decided to study cellular functions and signaling mechanisms mediated by CDH17 in cancer cells. In this report, loss-of-function experiments demonstrated that CDH17 caused a significant increase in KM12SM cell adhesion and proliferation. Coimmunoprecipitation experiments demonstrated an interaction between CDH17 and α2β1 integrin with a direct effect on β1 integrin activation and talin recruitment. The formation of this complex, together with other proteins, was confirmed by mass spectrometry analysis. CDH17 modulated integrin activation and signaling to induce specific focal adhesion kinase and Ras activation, which led to the activation of extracellular signal-regulated kinase and Jun N-terminal kinase and the increase in cyclin D1 and proliferation. In vivo experiments showed that CDH17 silencing in KM12 cells suppressed tumor growth and liver metastasis after subcutaneous or intrasplenic inoculation in nude mice. Collectively, our data reveal a new function for CDH17, which is to regulate α2β1 integrin signaling in cell adhesion and proliferation in colon cancer cells for liver metastasis., This research was supported by grant BIO2009-08818 from the Spanish Ministry of Science and Innovation, grant to established research groups (AECC) and grant S2010/BMD-2344/ Colomics2 from Comunidad de Madrid.

The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species-MEV, canine parvovirus, and feline panleukopenia virus- and thus the same vaccine could be used in three economically important viral hosts-mink, dogs, and cats, respectively.

All participating laboratories are members of ProteoRed-ISCIII.-- et al., The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC–MS/MS and gel/LC–MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study., This work was supported by: ProteoRed and the Carlos III National Health Institute Agreement, ProteoRed-ISCIII; the agreement between FIMA and the “UTE project CIMA”; grants SAF2011-29312 from Ministerio de Ciencia e Innovación and ISCIII-RETIC RD06/0020 to FJC and EU FP7 grant ProteomeXchange [grant number 260558]. APM and DTM have been funded by Spanish grants from Ministerio de Ciencia e Innovación BIO2010-17527 and the Government of Madrid (P2010/BMD-2305). BBVA Foundation for its support to HUPO initiatives.

Two recently developed vaccine--one based on synthetic peptide and one based on recombinant capsid protein--fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology ( > 98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 micrograms of conjugated peptide or 3 micrograms of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale.

7 p.-4 fig.

12 p.-7 fig., Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.Oncogene advance online publication, This study was supported by grants awarded by the Ministerio de Ciencia e Innovación (BFU2006-03203 and BFU2009-07578 to MD and SAF2006-00339 and SAF2010-16089 to AGH) and Fundació La Marató de TV3 (to AGH). The partial support from the Instituto Carlos III-Fondos FEDER (RTICCC, C03710, RD06/0020/0040) and the Generalitat de Catalunya (2009SGR867) is also appreciated. PV and RV-C were supported by predoctoral fellowships from the Ministerio de Ciencia y Tecnología and Instituto Carlos III, respectively

11 páginas, 6 figuras, 2 tablas.-- et al., SNAI1, ZEB1, E-cadherin (CDH1), and vitamin D receptor (VDR) genes regulate the epithelial–mesenchymal transition (EMT) that initiates the invasion process of many tumor cells. We hypothesized that this process could also affect the behavior of normal cells adjacent to the tumor. To verify this hypothesis, the expression level of these genes was determined by quantitative RT–PCR in tumor, normal adjacent, and normal distant tissues from 32 colorectal cancer (CC) patients. In addition, we extended the study to human HaCaT normal keratinocytes and SW480-ADH colon cancer cells co-cultured with SW480-ADH cells overexpressing the mouse Snai1 gene. Of 18 CC cases with SNAI1 expression in tumor tissue, five also had SNAI1 in normal adjacent tissue (NAT). Expression of SNAI1 in tumor tissue correlated with downregulation of CDH1 and VDR genes in both tumor (P=0.047 and P=0.014, respectively) and NAT lacking SNAI1 expression (P=0.054 and P=0.003). ZEB1 expression was directly related to VDR expression in tumor tissue (r=0.39; P=0.027) and inversely to CDH1 in NAT (r=−0.46; P=0.010). CDH1 and VDR were also downregulated in SW480-ADH and MaCaT cells, respectively, when they were co-cultured with Snai1-expressing cells. Furthermore, cytokine analysis showed differences in the conditioned media obtained from the two cell types. These results indicate that histologically normal tissue adjacent to tumor tissue expressing the EMT-inducing gene SNAI1 shows alterations in the expression of epithelial differentiation genes such as CDH1 and VD, This study was supported by the grants SAF2007-60431, CAM: S-GEN/0266/2006, ISCIII-RETIC RD06/0020 and /0009, and a grant from the Accion Transversal del Cancer (ISCIII)

Aplidin (plitidepsin) is an antitumoral agent that induces apoptosis via Rac1-JNK activation. A proteomic approach using 2D-DIGE technology found 52 cytosolic and 39 membrane proteins differentially expressed in wild-type and Aplidin-resistant HeLa cells, of which 39 and 27 were identified by MALDITOF mass spectrometry and database interrogation. A number of proteins involved in apoptosis pathways were found to be deregulated. Alterations in Rab geranylgeranyltransferase, protein disulfide isomerase (PDI), cystathionine γ-lyase, ezrin, and cyclophilin A (CypA) were confirmed by immunoblotting. Moreover, the role of PDI and CypA in Aplidin resistance was functionally confirmed by using the inhibitor bacitracin and overexpression, respectively. These deregulated proteins are candidates to mediate, at least partially, Aplidin action and might provide a route to the cells to escape the induction of apoptosis by this drug. © 2007 American Chemical Society., This work has been partially supported by Grant SAF2004-01015 from the “Ministerio de Educación y Ciencia” of Spain. The CNIO and the Instituto de Investigaciones Biomédicas were partially supported by the RTICC (Red de Centros de Cáncer, FIS RD06/0020/009 and RD06/0020/0083) and the Comunidad Autónoma de Madrid (S-GEN-0266/2006)

8 p.-4 fig., The selection and production of scFvs from phage display synthetic antibody libraries are frequently delayed by the presence of amber (TAG) stop codons within the sequences corresponding to the variable CDRs. This is due to the use of randomised oligonucleotides for library design and amber mutations for joining the scFv to the phage protein pIII. The screening of such libraries may lead to the selection of scFvs containing stop codons. Then, multiple site-directed mutagenesis is required for their removal or, alternatively, the proteins must be expressed as scFv-pIII fusions, which are not suitable for many functional assays. We describe here an alternative procedure to express soluble scFvs, despite the presence of TAG stop codons, in the currently used Escherichia coli suppressor strain TG1. It is based on a simple mutagenesis protocol that replaces the amber codon between the scFv and the pIII gene by a different stop codon (TAA), functional in E. coli TG1. The expression of soluble scFvs in the suppressor strain TG1 permits their fully functional characterization including the determination of affinity constants, which are critical for selecting the right scFvs for further studies., This project was partially supported by an EU grant COOP-CT-2004-512691 and the grant B102003-01481 from the Ministry of Science and Technology (MCYT) of Spain.

10 páginas, 6 figuras -- PAGS nros. 23-34, Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization. It might constitute an important target in cancer therapy. In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells. Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E. coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form. Further studies demonstrated that this protein was not functional. Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also. However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained. Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated. The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase

8 página, 5 figuras, 1 tabla -- PAGS nros. 2727-2734, NIH mice were vaccinated subcutaneously or intranasally with chimaeric cow pea mosaic virus (CPMV) constructs expressing a 17-mer peptide sequence from canine parvovirus (CPV) as monomers or dimers on the small or large protein surface subunits. Responses to the chimaeric virus particles (CVPs) were compared with those of mice immunized with the native virus or with parvovirus peptide conjugated to keyhole limpet haemocyanin (KLH). The characteristics of the immune response to vaccination were examined by measuring serum and mucosal antibody responses in ELISA, in vitro antigen-induced spleen cell proliferation and cytokine responses. Mice made strong antibody responses to the native plant virus and peptide-specific responses to two of the four CVP constructs tested which were approximately 10-fold lower than responses to native plant virus. The immune response generated by the CVP constructs showed a marked TH1 bias, as determined by a predominantly IgG2a isotype peptide-specific antibody response and the release of IFN-γ but not IL-4 or IL-5 from lymphocytes exposed to antigen in vitro. In comparison, parvovirus peptide conjugated to KLH generated an IgG1-biased (TH2) response. These data indicate that the presentation of peptides on viral particles could be used to bias the immune response in favor of a TH1 response. Anti-viral and anti-peptide IgA were detected in intestinal and bronchial lavage fluid of immunized mice, demonstrating that a mucosal immune response to CPV can be generated by systemic and mucosal immunization with CVP vaccines. Serum antibody from both subcutaneously-vaccinated and intranasally-vaccinated mice showed neutralizing activity against CPV in vitro

Expression of the VP2 gene of autonomous parvoviruses in insect cells with the use of the baculovirus system has led to the production of virus-like particles (VLPs) formed by the self-assembly of VP2. These VLPs are expressed at high levels and can easily be purified by salt fractionation. They are highly immunogenic in the corresponding host, being fully protective at doses as low as 1-2 microg of purified material per animal. No special adjuvants are required. An interesting property of these particles is their usefulness as a diagnostic reagent for ELISA kits, which have successfully replaced conventional methods for parvovirus diagnostics based on haemagglutination. Another application of the hybrid recombinant parvovirus-like particles of pig parvovirus (PPV) and canine parvovirus (CPV) is its use as an antigen delivery system. PPV:VLPs containing a CD8(+) epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein are able to evoke a potent cytolytic T-lymphocyte response and to protect mice against a lethal infection with LCMV. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a T helper response in mice. These T-cell epitopes were inserted into the N-terminus of the VP2 protein. Unfortunately, the N-terminus is not adequate for antibody responses because it is inside the particle. Recent findings have shown that fine tailoring of the point of insertion around the tip of loop 2 of the surface of CPV allowed the elicitation of a potent antibody response. Thus mice immunized with chimaeric C3:B CPV:VLPs were able to elicit a strong neutralizing antibody response (3 log10 units) against poliovirus. We now have the possibility of using these particles to elicit different immune responses against single or multiple pathogens in a simple and economic way.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N protein genes from two PRRSV isolates Olot/91 (European) and Quebec 807/94 (North American) were cloned and expressed in Escherichia coli using the pET3x system. The antigenic structure of the PRRSV N protein was dissected using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E.coli. Three antigenic sites were found. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein or at least a large fragment comprising the first 78 residues, respectively. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50 to 66). This hydrophillic region is well conserved among different isolates of European and North American origin. However, since this epitope is not recognized by many pig sera, it is not adequate for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire N protein.

The ultimate vaccine is an oral vaccine which given once protects against a multitude of diseases. Furthermore this ultimate vaccine needs to be very stable and inexpensive to produce. Probably this latter condition can be met only if the vaccines are produced in plants. Such vaccines are called 'edible vaccines'. Edible vaccines can be produced in plants in many ways. Using recombinant plantvirus, CPMV, it was shown that plants can produce massive amounts of chimaeric virus particles which protect after a single injection the target animal against disease. The final step, oral administration, is being addressed at present. Preliminary experiments by others suggest that this step may be solved sooner than expected.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N protein genes from two PRRSV isolates Olot/91 (European) and Quebec 807/94 (North American) were cloned and expressed in Escherichia coli using the pET3x system. The antigenic structure of the PRRSV N protein was dissected using seven monoclonal antibodies (MAbs) and overlapping fragments of the protein expressed in E.coli. Three antigenic sites were found. Four MAbs recognized two discontinuous epitopes that were present in the partially folded protein or at least a large fragment comprising the first 78 residues, respectively. The other three MAbs revealed the presence of a common antigenic site localized in the central region of the protein (amino acids 50 to 66). This hydrophillic region is well conserved among different isolates of European and North American origin. However, since this epitope is not recognized by many pig sera, it is not adequate for diagnostic purposes. Moreover, none of the N protein fragments were able to mimic the antigenicity of the entire N protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tübingen isolates and between 61-64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.

African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors.

Overlapping fragments of the outermost capsid protein VP2 of African horse sickness virus serotype 4 (AHSV-4) have been expressed in Escherichia coli. Horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for AHSV were used to screen these fragments for antigenic regions. The screening revealed that the major antigenic domain of the AHSV-4 VP2 is localized in a central region (amino acids 200 to 413) and that both the N-terminal region (aa 1-159) and the half C-terminal region (aa 414-1060) are not immunogenic. All the fragments containing a region between amino acids 253 and 413 (fragment H) were able to elicit consistently high titers of neutralizing antibodies. The ability of several subfragments of this region to evoke neutralizing antibodies indicates the presence of several sites inside this domain. However, neutralizing antibodies in sera of horse infected or vaccinated with attenuated viruses were not absorbed by fragment H, indicating that this domain is not immunodominant in AHSV. This information might be useful in designing a subunit vaccine against AHSV infection.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.

Ten antigenic sites on canine parvovirus (CPV) were mapped with a complete set of overlapping nonapeptides of the capsid proteins VP1 and VP2: five of these sites were recognized by sera from CPV-infected dogs, three were recognized by a rabbit anti-CPV antiserum, and two were recognized by murine monoclonal anti-CPV antibodies. A region covering the first 21 amino-terminal amino acid residues of VP2 was recognized by three sera from infected dogs, one neutralizing rabbit antiserum, and one neutralizing murine monoclonal antibody. Immunoabsorption experiments with full virions indicated that at least 6 of the 10 antigenic sites are located on the surface. Of these six, three sites occur in the amino terminus of VP2. When superimposed on the three-dimensional structure of canine parvovirus (J. Tsao, M. S. Chapman, M. Agbandje, W. Keller, K. Smith, H. Wu, M. Luo, T. J. Smith, M. G. Rossmann, R. W. Compans, and C. R. Parrish, Science 251:1456-1464, 1991), the other three epitopes are located on two loops of VP2 which form the highly exposed "spike" around the threefold-symmetry axis of the virus. Thus, these regions (amino terminus and loops 1 and 3) are of interest as major target sites for induction of neutralizing antibodies.

Peptide vaccines would form the ideal ultimate vaccine because they are safe both in production as well as application, easy to handle, store and transport. Peptide vaccines have the advantage that they can be produced in a completely reproducible manner, they are cheap compared to subunit or whole protein vaccines and potentially they can be precisely targeted to meet specific demands. For instance, peptide vaccines may be used to break maternal immunity or raise very specific antibodies for research or diagnostics. Furthermore synthetic peptide vaccines offer the potential possibility for multivalent vaccines and to incorporate adjuvanticity. Notwithsanding numerous attempts only one peptide vaccine has found its way to the market. This peptide vaccine is however not targeted to a pathogen but to a self-protein, Gonadotropin Releasing Hormone (GnRH), and used to immunocastrate production, animals (Hoskinson et al, 1990). In this contribution we shall discuss why peptide vaccines for pathogens, notably viruses, have failed so far, by focusing on Foot-and-Mouth Disease Virus (FMDV). For FMDV substantial attempts have been made to develop a synthetic peptide vaccine. These attempts have failed. However an attempt to develop a peptide vaccine against CPV was successful and may help to learn how failures with other attempts can be overcome.

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log10 units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses.

A synthetic peptide vaccine which protects dogs against challenge with virulent canine parvovirus is described. The amino acid sequence used was discovered in previous studies on the immunogenic properties of previously mapped antigenic sites and represents the amino-terminal region of viral protein VP2. As with marker vaccines, it is possible to discriminate between vaccinated dogs that have not been exposed to the virus and dogs that have been infected with the virus. The protective mechanism can be explained by a humoral response against the peptide aided by T-cell epitopes contained in the carrier protein used for peptide coupling. This is the first example of a synthetic peptide vaccine that induces protection in target animals.

Mucosal surfaces are frequently the first site of contact between the host and environmental hazards such as infectious agents or carcinogens. Therefore, the mucosa-associated lymphoid tissues are particularly important for protection against diseases for which entry and pathogenesis involve the mucosal system (i.e., the respiratory, gastrointestinal, and genital tracts), such as salmonellosis, tuberculosis, and AIDS. The mucosal immune system contains defense mechanisms, including secretory immunoglobulin A (IgA) antibodies and cytotoxic-T-cell (CTL) responses (6, 11), against foreign aggressions. Therefore, antigen carrier vectors would have to elicit mucosal, as well as systemic, immune responses in order to develop fully efficient prophylactic or therapeutic vaccines against various pathogens such as, for instance, the human papillomaviruses, which cause the development of cervical cancer, or Helicobacter pylori, which is responsible for the development of gastric ulcer. However, vaccines administered by parenteral routes generally fail to stimulate mucosal immune responses. Therefore, it is necessary to develop efficient and safe antigen vectors which will be able to trigger systemic and mucosal immune responses when administered by mucosal routes. Virus-like particle (VLP) vectors represent promising vaccine candidates. Indeed, they can stimulate immune responses (i) against the VLP proteins themselves, such as parvovirus-like particles (12, 14), human immunodeficiency virus type 1 (HIV-1) Gag particles (4), rubella virus VLP (20), and human papillomavirus-like particles (3), or (ii) against foreign peptides or proteins expressed by chimeric VLP, such as yeast retrotransposon Ty:VLP (7, 9) hepatitis B surface-antigen VLP (15), Semliki Forest virus VLP (23), or potyvirus-like particles (5). We previously studied the immunogenicity of virus-like particles produced in insect cells by self-assembly of the recombinant VP2 capsid protein from porcine parvovirus (PPV:VLP) carrying various heterologous peptides corresponding to CD4+ or CD8+ T-cell epitopes. These PPV:VLP administered in mice by the intraperitoneal (i.p.) route, in the absence of adjuvant, were shown to induce a whole range of immune responses, including Th1 CD4+ T lymphocytes (10, 22) and protective antiviral cytotoxic CD8+ T lymphocytes (21). These results prompted us to investigate the immune responses induced by these pseudoparticles after mucosal administration. This study demonstrates that VLP prepared with a single PPV protein and administered by the intranasal route, in the absence of adjuvant, induced specific IgG and/or IgA antibodies in the serum and in bronchoalveolar lavages and intestinal fluids. Chimeric PPV:VLP carrying a lymphocytic choriomeningitis virus (LCMV) CD8+ CTL epitope inserted into the N terminus of PPV VP2 and administered by the intranasal (i.n.) route also stimulated an LCMV-specific CTL response showing the strong potential of this antigen carrier system in developing safe and efficient vaccines that are able to stimulate systemic and mucosal immune responses.

Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. To learn more about the time course of production and specificity of AHSV-specific antibodies, sera were analyzed by immunoblot analysis. Only animals that survived for more than 9 days were able to develop a humoral immune response detectable by immunoblotting. The earliest serological markers corresponded mainly to VP5, VP6, and NS2 and to a lesser extent to VP3, NS1, and NS3. Neutralizing antibodies to VP2 were not detected by immunoblotting, suggesting that they are mostly conformation dependent. VP7-specific antibodies were detected later in infection. These results make NS2 and VP6 the most attractive candidates for the rapid diagnosis of the infection.

In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.