Characterization of structural and immunological properties of a fusion protein between flagellin from <i>Salmonella</i> and lumazine synthase from <i>Brucella</i>

Hiriart, Yanina; Rossi, A.; Biedma, Marina Elizabeth; Errea, Agustina Juliana; Moreno, Griselda; Cayet, Delphine; Rinaldi, Jimena; Blancá, Bruno; Sirard, Jean‐Claude; Goldbaum, Fernando A.; Berguer, Paula M.; Rumbo, Martı́n

doi:10.1002/pro.3151

“…In conclusion, the results presented in this work show that BLS serves as an adjuvant in combination with ICB. This TLR4 agonist emerges as a novel immunomodulator not only due to the adjuvant properties but also due to its distinctive carrier properties 41 – 43 . As previously reported, BLS not only can induce an immune response against covalently attached peptides, but it also increases the stability of these antigens, making BLS-chimeras easy to design and produce.…”

Section: Discussionmentioning

confidence: 99%

A TLR4 agonist improves immune checkpoint blockade treatment by increasing the ratio of effector to regulatory cells within the tumor microenvironment

Farias

¹

,

Soto

²

,

Puttur

³

et al. 2021

Sci Rep

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Brucella lumazine synthase (BLS) is a homodecameric protein that activates dendritic cells via toll like receptor 4, inducing the secretion of pro-inflammatory cytokines and chemokines. We have previously shown that BLS has a therapeutic effect in B16 melanoma-bearing mice only when administered at early stages of tumor growth. In this work, we study the mechanisms underlying the therapeutic effect of BLS, by analyzing the tumor microenvironment. Administration of BLS at early stages of tumor growth induces high levels of serum IFN-γ, as well as an increment of hematopoietic immune cells within the tumor. Moreover, BLS-treatment increases the ratio of effector to regulatory cells. However, all treated mice eventually succumb to the tumors. Therefore, we combined BLS administration with anti-PD-1 treatment. Combined treatment increases the outcome of both monotherapies. In conclusion, we show that the absence of the therapeutic effect at late stages of tumor growth correlates with low levels of serum IFN-γ and lower infiltration of immune cells in the tumor, both of which are essential to delay tumor growth. Furthermore, the combined treatment of BLS and PD-1 blockade shows that BLS could be exploited as an essential immunomodulator in combination therapy with an immune checkpoint blockade to treat skin cancer.

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“…It is highly stable and resistant to the action of proteases and chaotropic agents like urea. Although it is possible to couple proteins to the structure of BLS by recombinant fusion (Craig et al 2005 ; Bellido et al 2009 ; Alvarez et al 2013 ; Mejias et al 2013 ; Rossi et al 2015 ; Hiriart et al 2017 ; Berguer et al 2022 ), these constructs are frequently expressed as inclusion bodies in Escherichia coli and require refolding steps that do not always succeed. Non-covalent coupling methods using heterodimeric partners recombinantly fused to the scaffold subunits and target proteins are useful to overcome the problems associated with the concomitant folding and association of protein modules on oligomeric particles (Jennings and Bachmann 2008 ; Craig et al 2012 ).…”

Section: Introductionmentioning

confidence: 99%

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

Rando

¹

,

Gorojovsky

²

,

Zylberman³

et al. 2023

Appl Microbiol Biotechnol

0

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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 Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12581-6.

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“…Such decamers may be decorated with any protein whose antigenicity needs to be increased. The BLS decamer has been used to significantly increase the immunogenicity of other proteins by means of the fusion between the C-terminus of the foreign protein and the N-terminus of BLS, showing that BLS is a convenient platform for antigen display 16–18 . Moreover, BLS activates dendritic cells in vitro , increasing the levels of co-stimulatory molecules and the secretion of proinflammatory cytokines, and also recruits dendritic cells in vivo , in both cases in a TLR4-dependent manner 19 .…”

Section: Introductionmentioning

confidence: 99%

“…Such decamers may be decorated with any protein whose antigenicity needs to be increased. The BLS decamer has been used to significantly increase the immunogenicity of other proteins by means of the fusion between the C-terminus of the foreign protein and the N-terminus of BLS, showing that BLS is a convenient platform for antigen display [16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

Berguer¹,

Blaustein²,

Bredeston³

et al. 2021

Preprint

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Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

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Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

Cited by 11 publications

References 45 publications

A TLR4 agonist improves immune checkpoint blockade treatment by increasing the ratio of effector to regulatory cells within the tumor microenvironment

A TLR4 agonist improves immune checkpoint blockade treatment by increasing the ratio of effector to regulatory cells within the tumor microenvironment

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

Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

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