A phase I study to evaluate the safety, tolerance and pharmacokinetics of anti-Shiga toxin hyperimmune equine F (ab')2 fragments in healthy volunteers.

AIMS: Shiga toxin-producing Escherichia coli-haemolytic uraemic syndrome (STEC-HUS) is considered a toxaemic disorder in which early intervention with neutralizing antibodies may have therapeutic benefits. INM004, composed of F (ab')2 fragments from equine immunoglobulins, neutralizes Stx1/Stx2, potentially preventing the onset of HUS. METHODS: A single-centre, randomized, phase 1, single-blind, placebo-controlled clinical trial to evaluate INM004 safety, tolerance and pharmacokinetics (PK) in healthy adult volunteers, was conducted; in stage I, eight subjects were divided in two cohorts (n = 4) to receive a single INM004 dose of 2 or 4 mg kg-1, or placebo (INM004:placebo ratio of 3:1). In stage II, six subjects received three INM004 doses of 4 mg kg-1 repeated every 24 h, or placebo (INM004:placebo ratio of 5:1). RESULTS: Eight subjects (57.1%) experienced mild treatment-emergent adverse events (TEAEs); most frequent were rhinitis, headache and flushing, resolved within 24 h without changes in treatment or additional intervention. No serious AEs were reported. Peak concentrations of INM004 occurred within 2 h after infusion, with median Cmax values of 45.1 and 77.7 µg mL-1 for 2 and 4 mg kg-1, respectively. The serum concentration of INM004 declined in a biphasic manner (t1/2 range 30.7-52.9 h). Systemic exposures increased with each subsequent dose in a dose-proportional manner, exhibiting accumulation. Geometric median Cmax and AUC values were 149 and 10 300 µg h mL-1, respectively, in the repeated dose regimen. Additionally, samples from subjects that received INM004 at 2 mg kg-1 showed neutralizing capacity against Stx1 and Stx2 in in vitro assays. CONCLUSIONS: The results obtained in this first-in-human study support progression into the phase 2 trial in children with HUS.

Improvement of Cellulomonas fimi endoglucanase CenA by multienzymatic display on a decameric structural scaffold.

The development of multifunctional particles using polymeric scaffolds is an emerging technology for many nanobiotechnological applications. Here we present a system for the production of multifunctional complexes, based on the high affinity non-covalent interaction of cohesin and dockerin modules complementary fused to decameric Brucella abortus lumazine synthase (BLS) subunits, and selected target proteins, respectively. The cohesin-BLS scaffold was solubly expressed in high yield in Escherichia coli, and revealed a high thermostability. The production of multienzymatic particles using this system was evaluated using the catalytic domain of Cellulomonas fimi endoglucanase CenA recombinantly fused to a dockerin module. Coupling of the enzyme to the scaffold was highly efficient and occurred with the expected stoichiometry. The decavalent enzymatic complexes obtained showed higher cellulolytic activity and association to the substrate compared to equivalent amounts of the free enzyme. This phenomenon was dependent on the multiplicity and proximity of the enzymes coupled to the scaffold, and was attributed to an avidity effect in the polyvalent enzyme interaction with the substrate. Our results highlight the usefulness of the scaffold presented in this work for the development of multifunctional particles, and the improvement of lignocellulose degradation among other applications. KEY POINTS: • New system for multifunctional particle production using the BLS scaffold • Higher cellulolytic activity of polyvalent endoglucanase compared to the free enzyme • Amount of enzyme associated to cellulose is higher for the polyvalent endoglucanase.

Non-clinical safety assessment and in vivo biodistribution of CoviFab, an RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required the urgent development of new therapies, among which passive immunotherapy is contemplated. CoviFab (INM005) is a RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies. We investigate their preclinical security and biodistribution by in vivo and ex vivo NIR imaging after intravenous administration of a dose of 4 mg/kg at time 0 and 48 h. Images were taken at 1, 12, 24, 36, 48, 49, 60, 72, 84, 96, 108, 120, 132 and 144 h after the first intravenous injection. At 96 and 144 h, mice were sacrificed for haematology, serum chemistry, clinical pathology, histopathology and ex vivo imaging. The biodistribution profile was similar in all organs studied, with the highest fluorescence at 1 h after each injection, gradually decreasing after that each one and until the end of the study (144 h). The toxicology study revealed no significant changes in the haematology and serum chemistry parameters. Further, there were no changes in the gross and histological examination of organs. Nonclinical data of the current study confirm that CoviFab is safe, without observable adverse effects in mice. Furthermore, we confirm that bioimaging studies are a useful approach in preclinical trials to determine biodistribution.

Development and Evaluation of a Novel VHH-Based Immunocapture Assay for High-Sensitivity Detection of Shiga Toxin Type 2 (Stx2) in Stool Samples.

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).

[Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome]. / Desarrollo de un producto anti-toxina Shiga para la prevención del síndrome urémico hemolítico.

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.

Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and brucella lumazine synthase confers total protection against Shiga toxins in mice.

The striking feature of enterohemorrhagic Escherichia coli (EHEC) infections is the production of Shiga toxins (Stx) implicated in the development of the life-threatening hemolytic uremic syndrome. Despite the magnitude of the social impact of EHEC infections, no licensed vaccine or effective therapy is available for human use. One of the biggest challenges is to develop an effective and safe immunogen to ensure nontoxicity, as well as a strong input to the immune system to induce long-lasting, high-affinity Abs with anti-Stx-neutralizing capacity. The enzyme lumazine synthase from Brucella spp. (BLS) is a highly stable dimer of pentamers and a scaffold with enormous plasticity on which to display foreign Ags. Taking into account the advantages of BLS and the potential capacity of the B subunit of Stx2 to induce Abs that prevent Stx2 toxicity by blocking its entrance into the host cells, we engineered a new immunogen by inserting the B subunit of Stx2 at the amino termini of BLS. The resulting chimera demonstrated a strong capacity to induce a long-lasting humoral immune response in mice. The chimera induced Abs with high neutralizing capacity for Stx2 and its variants. Moreover, immunized mice were completely protected against i.v. Stx2 challenge, and weaned mice receiving an oral challenge with EHEC were completely protected by the transference of immune sera. We conclude that this novel immunogen represents a promising candidate for vaccine or Ab development with preventive or therapeutic ends, for use in hemolytic uremic syndrome-endemic areas or during future outbreaks caused by pathogenic strains of Stx-producing E. coli.

Protection of mice against Shiga toxin 2 (Stx2)-associated damage by maternal immunization with a Brucella lumazine synthase-Stx2 B subunit chimera.

Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.

Evaluation of the efficacy of polymeric antigen BLSOmp31 formulated in a new cage-like particle adjuvant (ISPA) administered by parenteral or mucosal routes against Brucella ovis in BALB/c mice.

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.

Safety and effectiveness of RBD-specific polyclonal equine F(ab´)2 fragments for the treatment of hospitalized patients with severe Covid-19 disease: A retrospective cohort study.

BACKGROUND: Passive immunotherapy has been evaluated as a therapeutic alternative for patients with COVID-19 disease. Equine polyclonal immunotherapy for COVID-19 (EPIC) showed adequate safety and potential efficacy in a clinical trial setting and obtained emergency use authorization in Argentina. We studied its utility in a real world setting with a larger population. METHODS: We conducted a retrospective cohort study at "Hospital de Campaña Escuela-Hogar" (HCEH) in Corrientes, Argentina, to assess safety and effectiveness of EPIC in hospitalized adults with severe COVID-19 pneumonia. Primary endpoints were 28-days all-cause mortality and safety. Mortality and improvement in modified WHO clinical scale at 14 and 21 days were secondary endpoints. Potential confounder adjustment was made by logistic regression weighted by the inverse of the probability of receiving the treatment (IPTW) and doubly robust approach. FINDINGS: Subsequent clinical records of 446 non-exposed (Controls) and 395 exposed (EPIC) patients admitted between November 2020 and April 2021 were analyzed. Median age was 58 years and 56.8% were males. Mortality at 28 days was 15.7% (EPIC) vs. 21.5% (Control). After IPTW adjustment the OR was 0.66 (95% CI: 0.46-0.96) P = 0.03. The effect was more evident in the subgroup who received two EPIC doses (complete treatment, n = 379), OR 0.58 (95% CI 0.39 to 0.85) P = 0.005. Overall and serious adverse events were not significantly different between groups. CONCLUSIONS: In this retrospective cohort study, EPIC showed adequate safety and effectiveness in the treatment of hospitalized patients with severe SARS-CoV-2 disease.

Polymeric antigen BLSOmp31 formulated with class B CpG-ODN in a nanostructure (BLSOmp31/CpG-ODN/Coa-ASC16) administered by parenteral or mucosal routes confers protection against Brucella ovis in Balb/c mice.

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.

RBD-specific polyclonal F(ab´)2 fragments of equine antibodies in patients with moderate to severe COVID-19 disease: A randomized, multicenter, double-blind, placebo-controlled, adaptive phase 2/3 clinical trial.

BACKGROUND: passive immunotherapy is a therapeutic alternative for patients with COVID-19. Equine polyclonal antibodies (EpAbs) could represent a source of scalable neutralizing antibodies against SARS-CoV-2. METHODS: we conducted a double-blind, randomized, placebo-controlled trial to assess efficacy and safety of EpAbs (INM005) in hospitalized adult patients with moderate and severe COVID-19 pneumonia in 19 hospitals of Argentina. Primary endpoint was improvement in at least two categories in WHO ordinal clinical scale at day 28 or hospital discharge (ClinicalTrials.gov number NCT04494984). FINDINGS: between August 1st and October 26th, 2020, a total of 245 patients were enrolled. Enrolled patients were assigned to receive two blinded doses of INM005 (n = 118) or placebo (n = 123). Median age was 54 years old, 65•1% were male and 61% had moderate disease at baseline. Median time from symptoms onset to study treatment was 6 days (interquartile range 5 to 8). No statistically significant difference was noted between study groups on primary endpoint (risk difference [95% IC]: 5•28% [-3•95; 14•50]; p = 0•15). Rate of improvement in at least two categories was statistically significantly higher for INM005 at days 14 and 21 of follow-up. Time to improvement in two ordinal categories or hospital discharge was 14•2 (± 0•7) days in the INM005 group and 16•3 (± 0•7) days in the placebo group, hazard ratio 1•31 (95% CI 1•0 to 1•74). Subgroup analyses showed a beneficial effect of INM005 over severe patients and in those with negative baseline antibodies. Overall mortality was 6•9% the INM005 group and 11•4% in the placebo group (risk difference [95% IC]: 0•57 [0•24 to 1•37]). Adverse events of special interest were mild or moderate; no anaphylaxis was reported. INTERPRETATION: Albeit not having reached the primary endpoint, we found clinical improvement of hospitalized patients with SARS-CoV-2 pneumonia, particularly those with severe disease.

Restoration of antitumor immunity through anti-MICA antibodies elicited with a chimeric protein.

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.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina.

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.

Characterization of folding-sensitive nanobodies as tools to study the expression and quality of protein particle immunogens.

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.

Mucosal immunization with polymeric antigen BLSOmp31 using alternative delivery systems against Brucella ovis in rams.

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.

Structural and kinetic properties of lumazine synthase isoenzymes in the order Rhizobiales.

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of alpha-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs from Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.

Polymeric antigen BLSOmp31 in aluminium hydroxide induces serum bactericidal and opsonic antibodies against Brucella canis in dogs.

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.

Crystallographic studies on decameric Brucella spp. Lumazine synthase: a novel quaternary arrangement evolved for a new function?

The enzyme lumazine synthase (LS) catalyzes the penultimate step of riboflavin biosynthesis in plants, fungi and bacteria. The quaternary structure of the polypeptide differs between species, existing as pentamers or as icosahedrally arranged dodecamers of pentamers with 60 subunits. The pathogen Brucella spp. expresses two proteins that exhibit LS activity, RibH1 and RibH2. The latter enzyme belongs to a novel third category of quaternary arrangement for LS, that of a decameric structure assembled as a head-to-head oriented dimer of pentamers. In contrast, the RibH1 enzyme is assembled as a pentamer, as noted for several other LS enzymes. RibH1 appears to be the functional LS in Brucella spp., whereas RibH2, an enzyme of lower catalytic activity, is a virulence factor presumably acting in response to oxidative stress. The latter observation prompted us to further investigate the structural and catalytic properties of RibH2 in order to clarify the biological function of this enzyme. Here, we present a detailed analysis of two new crystallographic forms of RibH2 that explain the low catalytic activity of this enzyme in comparison with RibH1 and other LSs. Additionally, we analyze the effect of pH on the structure of this enzyme, and the binding of riboflavin and 6,7-dimethyl-8-ribityllumazine to its active site.

Spray dried microspheres based on chitosan: A promising new carrier for intranasal administration of polymeric antigen BLSOmp31 for prevention of ovine brucellosis.

Previous studies have demonstrated that parenteral immunization with polymeric antigen BLSOmp31 induced a strong immune response and conferred protection against Brucella ovis in rams. This work describes the development of a novel formulation strategy for the delivery of BLSOmp31 in the nasal mucosa. Chitosan microparticles were prepared by spray-drying technology processes and recombinant chimera BLSOmp31 was loaded by passive adsorption onto chitosan microspheres, which were characterized by means of the evaluation of size, zeta potential, morphology, and loading and release rate of BLSOmp31. The mucoadhesive properties of microspheres were evaluated by studying the interaction between microparticles and mucin. The antigen BLSOmp31 integrity was investigated by SDS-PAGE. The yield of production of spray-drying process was 68.95%. Microspheres had a good sphericity, 1-10 µm of particle size and had a positive charge. The loading capacity was found to be 45.19%. The initial fast release of BLSOmp31 from chitosan microparticles was 60%. The BLSOmp31 integrity was not affected by passive adsorption (ionic interaction). The amount of mucin adsorbed on the surface of CMs-BLSOmp31 was lower than on the surface of blank CMs at neutral pH. In vivo studies were carried out in rams. Intranasal immunization induced systemic and local antibodies. In conclusion, the use of BLSOmp31-loaded chitosan spray-drying microspheres offers a promising way for nasal mucosal vaccination in sheep against brucellosis.

Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS).

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.