Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display.

Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response.

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Using crystallography tools to improve vaccine formulations.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Characterization of a virulence-modifying protein of Leptospira interrogans identified by shotgun phage display

Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Dose-sparing effect of two adjuvant formulations with a pandemic influenza A/H7N9 vaccine: a randomized, double-blind, placebo-controlled, phase 1 clinical trial

The emergence of potentially pandemic viruses has resulted in preparedness efforts to develop candidate vaccines and adjuvant formulations. We evaluated the dose-sparing effect and safety of two distinct squalene-based oil-in-water adjuvant emulsion formulations (IB160 and SE) with influenza A/H7N9 antigen. This phase I, randomized, double-blind, placebo-controlled, dose-finding trial (NCT03330899), enrolled 432 healthy volunteers aged 18 to 59. Participants were randomly allocated to 8 groups: 1A) IB160 + 15μg H7N9, 1B) IB160 + 7.5μg H7N9, 1C) IB160 + 3.75μg H7N9, 2A) SE + 15μg H7N9, 2B) SE + 7.5μg H7N9, 2C) SE + 3.75μg H7N9, 3) unadjuvanted vaccine 15μg H7N9 and 4) placebo. Immunogenicity was evaluated through haemagglutination inhibition (HI) and microneutralization (MN) tests. Safety was evaluated by monitoring local and systemic, solicited and unsolicited adverse events (AE) and reactions (AR) 7 and 28 days after each study injection, respectively, whereas serious adverse events (SAE) were monitored up to 194 days post-second dose. A greater increase in antibody geometric mean titers (GMT) was observed in groups receiving adjuvanted vaccines. Vaccinees receiving IB160-adjuvanted formulations showed the greatest response in group 1B, which induced an HI GMT increase of 4.7 times, HI titers ≥40 in 45.2% of participants (MN titers ≥40 in 80.8%). Vaccinees receiving SE-adjuvanted vaccines showed the greatest response in group 2A, with an HI GMT increase of 2.5 times, HI titers ≥40 in 22.9% of participants (MN titers ≥40 in 65.7%). Frequencies of AE and AR were similar among groups. Pain at the administration site and headache were the most frequent local and systemic solicited ARs. The vaccine candidates were safe and the adjuvanted formulations have a potential dose-sparing effect on immunogenicity against influenza A/H7N9. The magnitude of this effect could be further explored.

Using crystallography tools to improve vaccine formulations

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Off balance: Interferons in COVID-19 lung infections.

Interferons are innate and adaptive cytokines involved in many biological responses, in particular, viral infections. With the final response the result of the balance of the different types of Interferons. Cytokine storms are physiological reactions observed in humans and animals in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules. The excessive and prolonged presence of these cytokines can cause tissue damage, multisystem organ failure and death. The role of Interferons in virus clearance, tissue damage and cytokine storms are discussed, in view of COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The imbalance of Type I, Type II and Type III Interferons during a viral infection contribute to the clinical outcome, possibly together with other cytokines, in particular, TNFα, with clear implications for clinical interventions to restore their correct balance.

H7N9 pandemic preparedness: A large-scale production of a split inactivated vaccine.

In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 µg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.

ASCVac-1, a Multi-Peptide Chimeric Vaccine, Protects Mice Against Ascaris suum Infection.

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

ASCVac-1, a multi-peptide chimeric vaccine, protects mice against Ascaris suum infection / ASCVac-1, uma vacina quimérica multipeptídica, protege camundongos contra a infecção por Ascaris suum

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

Off balance: interferons in COVID-19 lung infections

Interferons are innate and adaptive cytokines involved in many biological responses, in particular, viral infections. With the final response the result of the balance of the different types of Interferons. Cytokine storms are physiological reactions observed in humans and animals in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules. The excessive and prolonged presence of these cytokines can cause tissue damage, multisystem organ failure and death. The role of Interferons in virus clearance, tissue damage and cytokine storms are discussed, in view of COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The imbalance of Type I, Type II and Type III Interferons during a viral infection contribute to the clinical outcome, possibly together with other cytokines, in particular, TNFα, with clear implications for clinical interventions to restore their correct balance.

H7N9 pandemic preparedness: a large-scale production of a split inactivated vaccine

In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 μg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.

Assessing the efficiency of SBA-15 as a nanocarrier for diphtheria anatoxin

SBA-15 ordered mesoporous silica can be considered a promising inorganic nanocarrier with emerging potential as an oral vaccine adjuvant. In this study, we investigated its application in the encapsulation of the diphtheria anatoxin (dANA). We observed a considerable preservation of dANA secondary and tertiary structures, even after the drying process by means of Circular Dichroism (CD) and fluorescence spectroscopies. Antigen loading was assessed at a number of different ratios of adjuvant-to-antigen using a combination of nitrogen adsorption porosimetry and Small Angle X-ray Scattering (SAXS). Our data showed that the mass ratio of 1:10 (dANA:SBA-15) is recommended for total encapsulation of dANA in the mesopores, considering that at this relative mass concentration antigen clustering was avoided, which is deleterious effect for immunization purposes. dANA release in mimetic intestine fluid, at a pH equal to 6.8, was followed by in-situ SAXS measurements and shown to be slow, being more pronounced after 6 h and continuous up to 35 h. Finally, the immunogenic complex was tested in isogenic Balb C mice by oral and subcutaneous immunization routes, including a comparison with the only permitted adjuvant for human use, aluminum hydroxide. A higher antibody titer was obtained by subcutaneous and oral administration routes using SBA-15 as the vehicle of dANA, compared with the conventional aluminum hydroxide, demonstrating the viability to use this ordered mesoporous silica in the formulation of oral vaccines, as already proved for the Virus Like Particles (VLP) Hepatitis B (HBsAg) case.

Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil.

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4-8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

H7N9 influenza split vaccine with SWE oil-in-water adjuvant greatly enhances cross-reactive humoral immunity and protection against severe pneumonia in ferrets.

Until universal influenza vaccines become available, pandemic preparedness should include developing classical vaccines against potential pandemic influenza subtypes. We here show that addition of SWE adjuvant, a squalene-in-water emulsion, to H7N9 split influenza vaccine clearly enhanced functional antibody responses in ferrets. These were cross-reactive against H7N9 strains from different lineages and newly emerged H7N9 variants. Both vaccine formulations protected in almost all cases against severe pneumonia induced by intratracheal infection of ferrets with H7N9 influenza; however, the SWE adjuvant enhanced protection against virus replication and disease. Correlation analysis and curve fitting showed that both VN- and NI-titers were better predictors for protection than HI-titers. Moreover, we show that novel algorithms can assist in better interpretation of large data sets generated in preclinical studies. Cluster analysis showed that the adjuvanted vaccine results in robust immunity and protection, whereas the response to the non-adjuvanted vaccine is heterogeneous, such that the protection balance may be more easily tipped toward severe disease. Finally, cluster analysis indicated that the dose-sparing capacity of the adjuvant is at least a factor six, which greatly increases vaccine availability in a pandemic situation.