ABSTRACT
Brucella ovis is an economically important cause of epididymitis in rams worldwide. Polymeric BLSOmp31 was previously identified as a protective immunogen against this pathogen. In this study, BLSOmp31 was formulated with a modified version of ISCOMATRIX adjuvant called ISPA (BLSOmp31/ISPA) and was administered in BALB/C by the subcutaneous and ocular route. The systemic and mucosal immune responses, the opsonic activity of antibodies and the protection conferred against B. ovis were evaluated. BLSOmp31+ISPA injected subcutaneously or by ocular route induced significantly higher IgG antibody levels with a mixed Th1/Th2 profile compared to non-immunized mice. IgA and IgG were detected in sera and nasal, tracheobronchial, vaginal secretions, tears and faeces, from SC immunized mice while in the group immunized by the ocular route a slight increase in both isotypes was mainly observed in all secretions, except in vaginal fluid. Opsonic antibodies stimulated binding and increased uptake of PHrodo™ Green-labelled B. ovis by neutrophils and monocytes. BLSOmp31 administered subcutaneously induced the highest levels of IFN-É£. The ocular immunization not only produced significant levels of this cytokine but also IL-4 compared to non-immunized mice. Both, subcutaneous and ocular routes of immunization, significantly protected against B. ovis infection. These results indicate that BLSOmp31/ISPA administered parenterally or by ocular route is a safe and effective vaccine against B. ovis in the murine model.
Subject(s)
Brucella ovis , Brucellosis , Rodent Diseases , Animals , Antibodies, Bacterial , Antigens, Bacterial , Brucellosis/prevention & control , Brucellosis/veterinary , Female , Male , Mice , Mice, Inbred BALB C , SheepABSTRACT
Previously, we demonstrated that the chimera BLSOmp31 formulated in chitosan microspheres or Poloxamer407-Chitosan administered via the nasal and the ocular mucosa conferred partial protection in sheep against B. ovis. In this work, we tested a new delivery system for mucosal immunization with BLSOmp31 in the murine model to improve the efficacy of previously used formulations. First, we evaluated the protective efficacy against B. ovis induced by BLSOmp31 administered by the subcutaneous route using either BLSOmp31 alone, co-administered with immunostimulatory synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG-ODN) or with CpG-ODN in a nanostructure called Coa-ASC16 compared with BLSOmp31 emulsified in Incomplete Freund Adjuvant. Then, we evaluated the protection conferred by the best performing formulation (BLSOmp31/CpG-ODN/Coa-ASC16) administered by both subcutaneous and ocular routes. BLSOmp31/CpG-ODN/Coa-ASC16 injected subcutaneously did not induce higher IgG antibody levels compared to BLSOmp31 alone or BLSOmp31/CpG-ODN but it did stimulate a mixed immune Th1-Th2 response with the highest levels of IFN-É£ and conferred significant protection against the B. ovis challenge. Although ocular instillation of BLSOmp31/CpG-ODN/Coa-ASC16 showed a similar degree of protection compared to the parenteral route (3.66 and 3.60 logs of protection, respectively), it induced lower levels in serum of specific IgG (with mixed IgG1/IgG2a) and IgA antibodies and, less IFN-É£ and IL-4 than the subcutaneous route. No antibodies were detected in vaginal lavages or saliva. Fecal antigen-specific IgA was slightly higher in mice immunized with BLSOmp31/CpG-ODN/Coa-ASC16 subcutaneously compared with the ocular route. These results indicate that BLSOmp31/CpG-ODN/Coa-ASC16 was a safe and effective vaccine against B. ovis in mice.
Subject(s)
Antigens, Bacterial/immunology , Brucella ovis/immunology , Nanostructures/chemistry , Oligodeoxyribonucleotides/chemistry , Adjuvants, Immunologic , Animals , Antibodies, Bacterial/blood , Antigens, Bacterial/chemistry , Drug Administration Routes , Female , Immunization/veterinary , Immunoglobulin A/blood , Immunoglobulin G/blood , Mice , Mice, Inbred BALB C , Vaccination/veterinaryABSTRACT
The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.
La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab')2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.
Subject(s)
Antibodies, Viral , Coronavirus Infections/therapy , Immune Sera/immunology , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin G/isolation & purification , Pandemics , Pneumonia, Viral , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Argentina , Betacoronavirus , COVID-19 , Horses , Humans , Immunization, Passive , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Neutralization Tests , SARS-CoV-2 , COVID-19 SerotherapyABSTRACT
BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/immunology , Histocompatibility Antigens Class I/immunology , Lymphoma/immunology , Recombinant Fusion Proteins/immunology , Urinary Bladder Neoplasms/immunology , Animals , Brucella/enzymology , Female , Histocompatibility Antigens Class I/genetics , Lymphoma/pathology , Lymphoma/therapy , Male , Mice , Mice, Inbred C57BL , Multienzyme Complexes/genetics , Multienzyme Complexes/immunology , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/immunology , Tumor Cells, Cultured , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/therapyABSTRACT
The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.
La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.
Subject(s)
Humans , Animals , Immunoglobulin Fab Fragments/isolation & purification , Coronavirus Infections/therapy , Immune Sera/immunology , Antibodies, Viral/isolation & purification , Antibodies, Viral/immunology , Antibodies, Viral/chemistry , Argentina , Immunoglobulin G/isolation & purification , Immunoglobulin G/chemistry , Immunoglobulin Fab Fragments/chemistry , Neutralization Tests , Pandemics , Betacoronavirus , SARS-CoV-2 , COVID-19 , HorsesABSTRACT
Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti-Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotin-streptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).
Subject(s)
Enterohemorrhagic Escherichia coli/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Hemolytic-Uremic Syndrome/diagnosis , Shiga Toxin 1/isolation & purification , Shiga Toxin 2/isolation & purification , Shiga-Toxigenic Escherichia coli/isolation & purification , Single-Domain Antibodies/chemistry , Animals , Argentina , Child, Preschool , Chlorocebus aethiops , Early Diagnosis , Feces/microbiology , Humans , Sensitivity and Specificity , Vero CellsABSTRACT
Subcellular vaccines against ovine contagious epididymitis due Brucella ovis can solve some shortcomings associated with the use of Brucella melitensis Rev 1. We have demonstrated that the parenteral immunization with polymeric antigen BLSOmp31 emulsified in oil adjuvant conferred significant protection against B. ovis in rams. In our previous studies, we have characterized chitosan microspheres (ChMs) and a thermoresponsive and mucoadhesive in situ gel (Poloxamer 407-Ch) as two novel formulation strategies for the delivery of BLSOmp31 in nasal as well as conjunctival mucosa. In the present work, we evaluated the immunogenicity and protection conferred by the intranasal and conjunctival immunization with these two mucosal delivery systems against B. ovis in rams. BLSOmp31-ChM administered by intranasal route and BLSOmp31-P407-Ch applied by intranasal or conjunctival routes induced systemic, local and preputial IgG and IgA antibody response. Neither formulation showed interference in the serological diagnosis. Thus, mucosal immunization using either formulation induced significant specific cellular immune responses (in vitro and in vivo) and it prevented the excretion of B. ovis in semen. Although these vaccines did not prevent infection in immunized rams, colonization reduction of infected organs and bacterial distribution differed significantly between vaccinated and unvaccinated rams.
Subject(s)
Brucella Vaccine/immunology , Brucella ovis , Brucellosis/veterinary , Sheep Diseases/prevention & control , Administration, Intranasal/veterinary , Administration, Ophthalmic , Animals , Bacterial Outer Membrane Proteins/immunology , Brucella Vaccine/administration & dosage , Brucella Vaccine/chemistry , Brucellosis/prevention & control , Immunogenicity, Vaccine , Immunoglobulin A/blood , Interferon-gamma/metabolism , Male , Microspheres , Semen/microbiology , Sheep , Sheep Diseases/microbiology , Vaccines, Synthetic/immunologyABSTRACT
Vaccination is as one of the most beneficial biopharmaceutical interventions against pathogens due to its ability to induce adaptive immunity through targeted activation of the immune system. Each vaccine needs a tailor-made set of tests in order to monitor its quality throughout the development and manufacturing. The analysis of the conformational state of protein nanoparticles is one of the key steps in vaccine quality control. The enzyme lumazine synthase from Brucella spp. (BLS) acts as a potent oral and systemic immunogen. BLS has been used as a carrier of foreign peptides, protein domains and whole proteins, serving as a versatile platform for vaccine engineering purposes. Here, we show the generation and characterization of four families of nanobodies (Nbs) which only recognize BLS in its native conformational state and that bind to its active site. The present results support the use of conformation-sensitive Nbs as molecular probes during the development and production of vaccines based on the BLS platform. Finally, we propose Nbs as useful molecular tools targeting other protein scaffolds with potential applications in nano-and biotechnology.
Subject(s)
Multienzyme Complexes , Single-Domain Antibodies , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Brucella/enzymology , Escherichia coli/genetics , HEK293 Cells , Humans , Multienzyme Complexes/chemistry , Multienzyme Complexes/physiology , Protein Conformation , Protein Folding , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/physiology , Vaccines, SubunitABSTRACT
The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.
Subject(s)
Drugs, Investigational , Escherichia coli Infections/drug therapy , Hemolytic-Uremic Syndrome/prevention & control , Immunoglobulin Fab Fragments/therapeutic use , Shiga Toxin 1/antagonists & inhibitors , Shiga Toxin 2/antagonists & inhibitors , Antibodies/immunology , Argentina , Clinical Trials, Phase II as Topic , Escherichia coli/immunology , Escherichia coli/isolation & purification , Escherichia coli Infections/complications , Hemolytic-Uremic Syndrome/immunology , Humans , Shiga Toxin 1/immunology , Shiga Toxin 2/immunologyABSTRACT
El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.
The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.
Subject(s)
Humans , Immunoglobulin Fab Fragments/therapeutic use , Drugs, Investigational , Shiga Toxin 1/antagonists & inhibitors , Shiga Toxin 2/antagonists & inhibitors , Escherichia coli Infections/drug therapy , Hemolytic-Uremic Syndrome/prevention & control , Argentina , Clinical Trials, Phase II as Topic , Shiga Toxin 1/immunology , Shiga Toxin 2/immunology , Escherichia coli/isolation & purification , Escherichia coli/immunology , Escherichia coli Infections/complications , Hemolytic-Uremic Syndrome/immunology , Antibodies/immunologyABSTRACT
Polymeric antigen BLSOmp31 is an immunogenic vaccine candidate that confers protection against Brucella canis in mice. In this preliminary study, the immunogenicity and safety of BLSOmp31 adsorbed to aluminum hydroxide gel (BLSOmp31-AH) were evaluated in Beagle dogs. In addition, the potential to elicit serum antibodies with complement-dependent bactericidal activity and/or to enhance phagocytosis by neutrophils were analyzed. Dogs were immunized three times with BLSOmp31-AH by subcutaneous route, followed by an annual booster. The vaccine elicited specific antibodies 3 weeks after the first immunization. Annual booster induced comparable antibody response as the primary series. Humoral immune response stimulated by BLSOmp31-AH did not interfere with routine agglutination test for canine brucellosis. Antibodies demonstrated a high complement-dependent bactericidal activity against B. canis. Moreover, opsonization by immune serum not only stimulated binding and uptake of the bacteria by neutrophils but effectively enhanced the destruction of B. canis. Specific IgG was detected in 3/4 immunized dogs in preputial secretions. The antibody profile corresponded to a marked Th2 response, since IgG1 prevailed over IgG2 and cellular immune response was not detected in vitro or in vivo. These results require further evaluation in larger field studies to establish the full prophylactic activity of BLSOmp31 against canine brucellosis.
Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Brucella canis/immunology , Brucellosis/veterinary , Dog Diseases/immunology , Aluminum Hydroxide , Animals , Brucellosis/immunology , Brucellosis/microbiology , Dog Diseases/microbiology , Dogs/immunology , Dogs/microbiology , Immunity, Humoral/immunology , Immunoglobulin G/immunology , MaleABSTRACT
Control of ovine brucellosis with subcellular vaccines can solve some drawbacks associated with the use of Brucella melitensis Rev.1. Previous studies have demonstrated that the polymeric antigen BLSOmp31 administered by parenteral route was immunogenic and conferred significant protection against B. ovis in rams. Immunization with BLSOmp31 by conjunctival route could be efficient for the induction of mucosal and systemic immune responses. In this work, we evaluated the conjunctival immunization using a thermoresponsive and mucoadhesive in situ gel composed of Poloxamer 407 (P407) and chitosan (Ch) as vaccine delivery system for BLSOmp31 in rams. Serum samples, saliva, lacrimal, preputial and nasal secretions were analyzed to measure specific IgG and IgA antibodies. Cellular immune response was evaluated in vivo and in vitro. Immunization with BLSOmp31-P407-Ch induced high IgG antibody levels in serum and preputial secretions which remained at similar levels until the end of the experiment. Levels of IgG in saliva, lacrimal and nasal secretions were also higher compared to unvaccinated control group but decreased more rapidly. IgA antibodies were only detected in nasal and preputial secretions. BLSOmp31-P407-Ch stimulated a significant cellular immune response in vivo and in vitro. The induction of systemic and local immune responses indicates a promising potential of P407-Ch for the delivery of BLSOmp31 by conjunctival route.
Subject(s)
Brucellosis/veterinary , Sheep Diseases/prevention & control , Animals , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/immunology , Brucella Vaccine/administration & dosage , Brucella ovis/immunology , Brucellosis/immunology , Brucellosis/prevention & control , Conjunctiva/immunology , Drug Delivery Systems/methods , Drug Delivery Systems/veterinary , Gels , Immunity, Cellular , Immunity, Mucosal , Male , Sheep , Sheep Diseases/immunology , Vaccination/methods , Vaccination/veterinary , Vaccines, Synthetic/administration & dosageABSTRACT
Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.
Subject(s)
Antitoxins/isolation & purification , Hemolytic-Uremic Syndrome/therapy , Immunotherapy/methods , Shiga Toxin 2/immunology , Single-Chain Antibodies/isolation & purification , Animals , Antitoxins/therapeutic use , Camelus , Disease Models, Animal , Hemolytic-Uremic Syndrome/diagnosis , Mice , Serologic Tests/methods , Single-Chain Antibodies/therapeutic use , Therapeutics , Treatment OutcomeABSTRACT
Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1) h(-1)) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data establish cannabinoid receptors as an interesting target for the treatment of implantation deficiencies.
Subject(s)
Arachidonic Acids/pharmacology , Blastocyst/cytology , Blastocyst/physiology , Nitric Oxide Synthase/metabolism , Polyunsaturated Alkamides/pharmacology , Uterus/drug effects , Uterus/enzymology , Animals , Benzamides/pharmacology , Cannabinoid Receptor Modulators/pharmacology , Carbamates/pharmacology , Embryo Implantation , Endocannabinoids , Female , Immunohistochemistry/methods , Polymerase Chain Reaction , Rats , Rats, Wistar , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolismABSTRACT
BACKGROUND/AIM: Autophagy is a degradation process of cytoplasmic cellular constituents. We have described the vacuole membrane protein-1 (VMP1) whose expression triggers autophagy in mammalian cells. The aim of this study was to analyze the role of autophagy in human pancreatic cancer cell death. METHODS/RESULTS: Here we show that gemcitabine, the standard chemotherapy for pancreatic cancer, induced autophagy in PANC-1 and MIAPaCa-2 cells, as evidenced by the accumulation of acidic vesicular organelles, the recruitment of microtubule-associated protein-1 light chain-3, and electron microscopy. In addition, gemcitabine treatment induced early expression of VMP1 in cancer cells. Gemcitabine also induced apoptosis detected by morphology, annexin V-positive cells, and cleavage of caspase-3. Surprisingly, 3-methyladenine, an autophagy inhibitor, decreased apoptosis in gemcitabine-treated cells, showing that autophagy leads to cancer cell apoptotic death. Finally, VMP1 knockdown decreased autophagy and apoptosis in gemcitabine-treated cancer cells. CONCLUSIONS: The VMP1-autophagy pathway promotes apoptosis in pancreatic cancer cells and mediates gemcitabine-induced cytotoxicity. and IAP.
Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Deoxycytidine/analogs & derivatives , Membrane Proteins/physiology , Pancreatic Neoplasms/pathology , Adenine/analogs & derivatives , Adenine/pharmacology , Caspase 3/metabolism , Cell Line, Tumor , Deoxycytidine/pharmacology , Gene Knockdown Techniques , Humans , Membrane Proteins/genetics , Pancreatic Neoplasms/metabolism , Vacuoles/metabolism , GemcitabineABSTRACT
Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.
