The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- ¿ß by TLR4-expressing cells, as well as the production of TNF- ¿ by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

Aims: The NADPH oxidases constitute a major source of superoxide anion (·O2¿) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by transforming growth factor-ß1 (TGF-ß1). We investigated whether a chronic treatment with P144, a peptide synthesized from type III TGF-ß1 receptor, inhibited NADPH oxidases in the renal cortex of spontaneously hypertensive rats (SHR). Results: Here, we show that chronic administration of P144 significantly reduced the NADPH oxidase expression and activity as well as the oxidative stress observed in control vehicle-treated SHR (V-SHR). In addition, P144 was also able to reduce the significant increase in the renal fibrosis and in mRNA expression of different components of collagen metabolism, as well as in the levels of connective tissue growth factor observed in V-SHR. Finally, TGF-ß1-stimulated NRK52E exhibited a significant increase in NADPH oxidase expression and activity as well as a TGF-ß1-dependent intracellular pathway that were inhibited in the presence of P144. Innovation: Our experimental evidence suggests that reversing oxidative stress may be therapeutically useful in preventing fibrosis-associated renal damage. We show here that (i) the TGF-ß1-NADPH oxidases axis is crucial in the development of fibrosis in an experimental hypertensive renal disease animal model, and (ii) the use of P144 reverses TGF-ß1-dependent NADPH oxidase activity; thus, P144 may be considered a novel therapeutic tool in kidney disease associated with hypertension. Conclusion: We demonstrate that P144 inhibits NADPH oxidases and prevents oxidative stress in kidneys from hypertensive rats. Our data also suggest that these effects are associated with the renal antifibrotic effect of P144.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Peptide vaccines derived from CD8(+) T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8(+) T-cell responses, even in the absence of CD4(+) T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8(+) T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8(+) T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8(+) T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8(+) T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8(+) T cells. Taken together, these results suggest that CD40L expression induced by potent CD8(+) T-cell epitopes can activate antitumor CD8(+) T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8(+) T-cell epitopes and bypassing the requirement for CD4(+) helper T cells in vaccination protocols.

NADPH oxidases constitute a major source of superoxide anion (center dot O-2(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-beta 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-beta(1) receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-beta 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-beta 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-beta 1.

Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8+ T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 57 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma.

This work shows that class II-linked humoral lack of response to an antigen can be overcome by joint immunization with the antigen and a T-helper cell determinant (TDh) well recognized by class II molecules of a non-responder individual. Thus, SJL/J mice (H-2s), which are non-responders to the S region of hepatitis B virus surface antigen (HBsAg), were rendered responders by joint immunization with a recombinant surface antigen, only composed of the S region, and a short synthetic TDh peptide well recognized by the H-2s restriction. By contrast, when this peptide is not recognized as TDh, as in B10M mice (H-2f restricted and also non-responders to the S region), no humoral response could be induced against the S region. These results have important implications for therapy and vaccination against hepatitis B virus as well as in enhancing the immunogenicity of other antigens.

An extensive outbreak characterized by the appearance of neurological symptoms in small ruminant lentivirus (SRLV) infected sheep has been identified in Spain, but the genetic characteristics of the strain involved and differential diagnostic tools for this outbreak remain unexplored. In this work, 23 Visna-affected naturally infected animals from the outbreak, 11 arthritic animals (both groups presenting anti-Visna/Maedi virus serum antibodies), and 100 seronegative animals were used. Eight of the Visna-affected animals were further studied post-mortem by immunohistochemistry. All had lesions in spinal cord, being the most affected part of the central nervous system in six of them. A representative strain of the outbreak was isolated. Together with other proviral sequences from the outbreak the virus was assigned to genotype A2/A3. In vitro culture of the isolate revealed that viral production was slow/low in fibroblast-like cells but it was high in blood monocyte-derived macrophages. The long terminal repeat (LTR) of the viral genome of this isolate lacked an U3-duplication, but its promoter activity in fibroblast-like cells was normal compared to other strains. Thus, viral production could not be inferred from the LTR promoter activity in this isolate. Analysis of the viral immunodominant epitopes among SRLV sequences of the outbreak and other known sequences allowed the design of a synthetic SU peptide ELISA that detected the Visna affected animals, representing a tool of epidemiological interest to control viral spread of this highly pathogenic strain.