Depolymerization of SUMO chains induces slender to stumpy differentiation in T. brucei bloodstream parasites.

Trypanosoma brucei are protozoan parasites that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, a quorum sensing-like mechanism coordinates its differentiation from a slender replicative form into a quiescent stumpy form, limiting growth and activating metabolic pathways that are beneficial to the parasite in the insect host. The post-translational modification of proteins with the Small Ubiquitin-like MOdifier (SUMO) enables dynamic regulation of cellular metabolism. SUMO can be conjugated to its targets as a monomer but can also form oligomeric chains. Here, we have investigated the role of SUMO chains in T. brucei by abolishing the ability of SUMO to polymerize. We have found that parasites able to conjugate only SUMO monomers are primed for differentiation. This was demonstrated for monomorphic lines that are normally unable to produce stumpy forms in response to quorum sensing signaling in mice, and also for pleomorphic cell lines in which stumpy cells were observed at unusually low parasitemia levels. SUMO chain mutants showed a stumpy compatible transcriptional profile and better competence to differentiate into procyclics. Our study indicates that SUMO depolymerization may represent a coordinated signal triggered during stumpy activation program.

Regulation of human neutrophil IL-1ß secretion induced by Escherichia coli O157:H7 responsible for hemolytic uremic syndrome.

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1ß). STEC stimulated neutrophils to release elevated levels of IL-1ß through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1ß secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1ß secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1ß secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation.

Design and characterization of chimeric Rabies-SARS-CoV-2 virus-like particles for vaccine purposes.

Due to the high number of doses required to achieve adequate coverage in the context of COVID-19 pandemics, there is a great need for novel vaccine developments. In this field, there have been research approaches that focused on the production of SARS-CoV-2 virus-like particles. These are promising vaccine candidates as their structure is similar to that of native virions but they lack the genome, constituting a biosafe alternative. In order to produce these structures using mammal cells, it has been established that all four structural proteins must be expressed. Here we report the generation and characterization of a novel chimeric virus-like particle (VLP) that can be produced by the expression of a single novel fusion protein that contains SARS-CoV-2 spike (S) ectodomain fused to rabies glycoprotein membrane anchoring region in HEK293 cells. This protein is structurally similar to native S and can autonomously bud forming enveloped VLPs that resemble native virions both in size and in morphology, displaying S ectodomain and receptor binding domain (RBD) on their surface. As a proof of concept, we analyzed the immunogenicity of this vaccine candidate in mice and confirmed the generation of anti-S, anti-RBD, and neutralizing antibodies. KEY POINTS: • A novel fusion rabies glycoprotein containing S ectodomain was designed. • Fusion protein formed cVLPs that were morphologically similar to SARS-CoV-2 virions. • cVLPs induced anti-S, anti-RBD, and neutralizing antibodies in mice.

A COVID-19 vaccine candidate based on SARS-CoV-2 spike protein and immune-stimulating complexes.

Spike protein from SARS-CoV-2, the etiologic agent of the COVID-19 pandemic disease, constitutes a structural protein that proved to be the main responsible for neutralizing antibody production. Thus, its sequence is highly considered for the design of candidate vaccines. Animal cell culture represents the best option for the production of subunit vaccines based on recombinant proteins since they introduce post-translational modifications that are important to mimic the natural antigenic epitopes. Particularly, the human cell line HEK293T has been explored and used for the production of biotherapeutics since the products derived from them present human-like post-translational modifications that are important for the protein's activity and immunogenicity. The aim of this study was to produce and characterize a potential vaccine for COVID-19 based on the spike ectodomain (S-ED) of SARS-CoV-2 and two different adjuvants: aluminum hydroxide (AH) and immune-stimulating complexes (ISCOMs). The S-ED was produced in sHEK293T cells using a 1-L stirred tank bioreactor operated in perfusion mode and purified. S-ED characterization revealed the expected size and morphology. High N-glycan content was confirmed. S-ED-specific binding with the hACE2 (human angiotensin-converting enzyme 2) receptor was verified. The immunogenicity of S-ED was evaluated using AH and ISCOMs. Both formulations demonstrated the presence of anti-RBD antibodies in the plasma of immunized mice, being significantly higher for the latter adjuvant. Also, higher levels of IFN-γ and IL-4 were detected after the ex vivo immune stimulation of spleen-derived MNCs from ISCOMs immunized mice. Further analysis confirmed that S-ED/ISCOMs elicit neutralizing antibodies against SARS-CoV-2. KEY POINTS: Trimeric SARS-CoV-2 S-ED was produced in stable recombinant sHEK cells in serum-free medium. A novel S-ED vaccine formulation induced potent humoral and cellular immunity. S-ED formulated with ISCOMs adjuvant elicited a highly neutralizing antibody titer.

Omp25-dependent engagement of SLAMF1 by Brucella abortus in dendritic cells limits acute inflammation and favours bacterial persistence in vivo.

The strategies by which intracellular pathogenic bacteria manipulate innate immunity to establish chronicity are poorly understood. Here, we show that Brucella abortus outer membrane protein Omp25 specifically binds the immune cell receptor SLAMF1 in vitro. The Omp25-dependent engagement of SLAMF1 by B. abortus limits NF-κB translocation in dendritic cells (DCs) with no impact on Brucella intracellular trafficking and replication. This in turn decreases pro-inflammatory cytokine secretion and impairs DC activation. The Omp25-SLAMF1 axis also dampens the immune response without affecting bacterial replication in vivo during the acute phase of Brucella infection in a mouse model. In contrast, at the chronic stage of infection, the Omp25/SLAMF1 engagement is essential for Brucella persistence. Interaction of a specific bacterial protein with an immune cell receptor expressed on the DC surface at the acute stage of infection is thus a powerful mechanism to support microbe settling in its replicative niche and progression to chronicity.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

RomA, A Periplasmic Protein Involved in the Synthesis of the Lipopolysaccharide, Tunes Down the Inflammatory Response Triggered by Brucella.

Brucellaceae are stealthy pathogens with the ability to survive and replicate in the host in the context of a strong immune response. This capacity relies on several virulence factors that are able to modulate the immune system and in their structural components that have low proinflammatory activities. Lipopolysaccharide (LPS), the main component of the outer membrane, is a central virulence factor of Brucella, and it has been well established that it induces a low inflammatory response. We describe here the identification and characterization of a novel periplasmic protein (RomA) conserved in alpha-proteobacteria, which is involved in the homeostasis of the outer membrane. A mutant in this gene showed several phenotypes, such as membrane defects, altered LPS composition, reduced adhesion, and increased virulence and inflammation. We show that RomA is involved in the synthesis of LPS, probably coordinating part of the biosynthetic complex in the periplasm. Its absence alters the normal synthesis of this macromolecule and affects the homeostasis of the outer membrane, resulting in a strain with a hyperinflammatory phenotype. Our results suggest that the proper synthesis of LPS is central to maximize virulence and minimize inflammation.

A Brucella spp. Protease Inhibitor Limits Antigen Lysosomal Proteolysis, Increases Cross-Presentation, and Enhances CD8+ T Cell Responses.

In this study, we demonstrate that the unlipidated (U) outer membrane protein (Omp) 19 from Brucella spp. is a competitive inhibitor of human cathepsin L. U-Omp19 inhibits lysosome cathepsins and APC-derived microsome activity in vitro and partially inhibits lysosomal cathepsin L activity within live APCs. Codelivery of U-Omp19 with the Ag can reduce intracellular Ag digestion and increases Ag half-life in dendritic cells (DCs). U-Omp19 retains the Ag in Lamp-2(+) compartments after its internalization and promotes a sustained expression of MHC class I/peptide complexes in the cell surface of DCs. Consequently, U-Omp19 enhances Ag cross-presentation by DCs to CD8(+) T cells. U-Omp19 s.c. delivery induces the recruitment of CD11c(+)CD8α(+) DCs and monocytes to lymph nodes whereas it partially limits in vivo Ag proteolysis inside DCs. Accordingly, this protein is able to induce CD8(+) T cell responses in vivo against codelivered Ag. Antitumor responses were elicited after U-Omp19 coadministration, increasing survival of mice in a murine melanoma challenge model. Collectively, these results indicate that a cysteine protease inhibitor from bacterial origin could be a suitable component of vaccine formulations against tumors.

Brucella abortus induces intracellular retention of MHC-I molecules in human macrophages down-modulating cytotoxic CD8(+) T cell responses.

Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8(+) T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN-γ-induced MHC-I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC-I down-modulation correlated with the development of diminished CD8(+) cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8(+) T lymphocytes and a diminished percentage of IFN-γ-producing CD8(+) T cells. Inhibition of MHC-I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC-I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC-I molecules whereby B. abortus would avoid CD8(+) cytotoxic T cell responses, evading their immunological surveillance.

Targeting Stat3 induces senescence in tumor cells and elicits prophylactic and therapeutic immune responses against breast cancer growth mediated by NK cells and CD4+ T cells.

Aberrant Stat3 activation and signaling contribute to malignant transformation by promoting cell cycle progression, inhibiting apoptosis, and mediating tumor immune evasion. Stat3 inhibition in tumor cells induces the expression of chemokines and proinflammatory cytokines, so we proposed to apply Stat3-inhibited breast cancer cells as a source of immunogens to induce an antitumor immune response. Studies were performed in two murine breast cancer models in which Stat3 is activated: progestin-dependent C4HD cells and 4T1 cells. We immunized BALB/c mice with irradiated cancer cells previously transfected with a dominant-negative Stat3 vector (Stat3Y705F) in either a prophylactic or a therapeutic manner. Prophylactic administration of breast cancer cells transfected with Stat3Y705F (Stat3Y705F-breast cancer cells) inhibited primary tumor growth compared with administration of empty vector-transfected cells in both models. In the 4T1 model, 50% of the challenged mice were tumor free, and the incidence of metastasis decreased by 90%. In vivo assays of C4HD tumors showed that the antitumor immune response involves the participation of CD4(+) T cells and cytotoxic NK cells. Therapeutic immunization with Stat3Y705F-breast cancer cells inhibited tumor growth, promoted tumor cell differentiation, and decreased metastasis. Furthermore, inhibition of Stat3 activation in breast cancer cells induced cellular senescence, contributing to their immunogenic phenotype. In this work, we provide preclinical proof of concept that ablating Stat3 signaling in breast cancer cells results in an effective immunotherapy against breast cancer growth and metastasis. Moreover, our findings showing that Stat3 inactivation results in induction of a cellular senescence program disclose a potential mechanism for immunotherapy research.

A Gamma-adapted subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from infection.

In the context of continuous emergence of SARS-CoV-2 variants of concern (VOCs), one strategy to prevent the severe outcomes of COVID-19 is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD vaccine derived from the Gamma SARS-CoV-2 variant adjuvanted with Alum. The Gamma-adapted RBD vaccine is more immunogenic than the Ancestral RBD vaccine in terms of inducing broader neutralizing antibodies. The Gamma RBD presents more immunogenic B-cell restricted epitopes and induces a higher proportion of specific-B cells and plasmablasts than the Ancestral RBD version. The Gamma-adapted vaccine induces antigen specific T cell immune responses and confers protection against Ancestral and Omicron BA.5 SARS-CoV-2 challenge in mice. Moreover, the Gamma RBD vaccine induces higher and broader neutralizing antibody activity than homologous booster vaccination in mice previously primed with different SARS-CoV-2 vaccine platforms. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate.

Toll-like receptor 6 plays an important role in host innate resistance to Brucella abortus infection in mice.

Brucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-κB signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.

Brucella abortus induces TNF-α-dependent astroglial MMP-9 secretion through mitogen-activated protein kinases.

BACKGROUND: Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology. METHODS: In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies. RESULTS: B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing antibody. MMP-9 activity was detected in cerebrospinal fluid (CSF) samples from patients suffering from neurobrucellosis. CONCLUSIONS: Our results indicate that the inflammatory response elicited by B. abortus in astrocytes would lead to the production of MMP-9 and that MAPK may play a role in this phenomenon. MAPK inhibition may thus be considered as a strategy to control inflammation and CNS damage in neurobrucellosis.

Bm Delta-pgm, a vaccine for the control of Brucella melitensis with cross-species protective properties.

Brucellosis remains one of the most worldwide distributed zoonosis inflicting serious economical and human health problems in many areas of the world. The disease is caused by different species of the genus Brucella that have different tropisms towards different mammals being the most relevant for human health Brucella abortus, Brucella melitensis and Brucella suis that infect cows, goats/sheep, and swine respectively. For B. melitensis, considered the species with more zoonotic potential and highly aggressive for animals, only one vaccine is available to date in the market: Rev 1. This attenuated strain has the disadvantage that is has a very high residual virulence for animals and humans and, for this reason, it is applied by ocular instillation which is technically challenging in many productive settings. For this reason, the search for new vaccines for caprine and ovine brucellosis is an active topic of research. We describe here the construction of a novel highly attenuated vaccine strain (Bm Delta-pgm) that confers excellent levels of protection against B. melitensis in the mouse model of infection. This strain is a clean deletion of the phosphoglucomutase (pgm) gene that codes for a protein that catalyzes the conversion of glucose-6-P to glucose-1-P, which is used as a precursor for the biosynthesis of many polysaccharides, including the O-antigen of the lipopolysaccharide and cyclic beta glucans. Our results indicate that vaccination with Bm Delta-pgm induces a robust memory cellular immune response but no antibody production against the O-antigen. Cross protection experiments show that this new vaccine protects against B. abortus and B. suis raising the possibility that Bm Delta-pgm could be used as a universal vaccine for the most important Brucella species.

Safety and immunogenicity of a SARS-CoV-2 Gamma variant RBD-based protein adjuvanted vaccine used as booster in healthy adults.

A Gamma Variant RBD-based aluminum hydroxide adjuvanted vaccine called ARVAC CG was selected for a first in human clinical trial. Healthy male and female participants (18-55 years old) with a complete COVID-19-primary vaccine scheme were assigned to receive two intramuscular doses of either a low-dose or a high-dose of ARVAC CG. The primary endpoint was safety. The secondary objective was humoral immunogenicity. Cellular immune responses were studied as an exploratory objective. The trial was prospectively registered in PRIISA.BA (Registration Code 6564) and ANMAT and retrospectively registered in ClinicalTrials.gov (NCT05656508). Samples from participants of a surveillance strategy implemented by the Ministry of Health of the Province of Buenos Aires that were boosted with BNT162b2 were also analyzed to compare with the booster effect of ARVAC CG. ARVAC CG exhibits a satisfactory safety profile, a robust and broad booster response of neutralizing antibodies against the Ancestral strain of SARS-CoV-2 and the Gamma, Delta, Omicron BA.1 and Omicron BA.5 variants of concern and a booster effect on T cell immunity in individuals previously immunized with different COVID-19 vaccine platforms.

The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis.

Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the first Brucella protein that without the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19. Moreover, the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route. Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells and macrophages in vitro. The latter property and its ability to induce a protective Th1 immune response against B. abortus infection have been found to be TLR4 dependent. The facts that U-Omp16 is an oral protective Ag and possesses a mucosal self-adjuvanting property led us to develop a plant-made vaccine expressing U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally, indicating that a plant-based oral vaccine expressing U-Omp16 could be a valuable approach to controlling this disease.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

Brucella abortus induces the secretion of proinflammatory mediators from glial cells leading to astrocyte apoptosis.

Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage chemoattractant protein-1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55-/- mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-alpha signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis.

Stability Studies of the Vaccine Adjuvant U-Omp19.

Unlipidated outer membrane protein 19 (U-Omp19) is a novel mucosal adjuvant in preclinical development to be used in vaccine formulations. U-Omp19 holds two main properties, it is capable of inhibiting gastrointestinal and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable U-Omp19 to enhance the adaptive immune response to co-administrated antigens. To characterize the stability of U-Omp19 we have performed an extensive analysis of its physicochemical and biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4 °C or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form aggregates. This information will play a key role in the development of a stable formulation of U-Omp19, allowing an extended shelf-life during manufacturing, storage, and shipping of a future vaccine containing this pioneering adjuvant.