Development of an Antigen Delivery Platform Using Lactobacillus acidophilus Decorated With Heterologous Proteins: A Sheep in Wolf’s Clothing Story

Uriza, Paula J.; Trautman, Cynthia; Palomino, María Mercedes; Martin, Joaquina Fina; Ruzal, Sandra M.; Roset, Mara S.; Briones, Gabriel

doi:10.3389/fmicb.2020.509380

Cited by 10 publications

(17 citation statements)

References 40 publications

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“…A variety of fusion protein constructs of inactivated vtx proteins fused to adhesins and other antigens have stimulated strong serological responses and increased survival in mice, as recently reviewed [ 15 ], but none of these developments have been approved to date. Moreover, recent alternative platforms have been examined, including Lactobacillus acidophilus strains decorated with EspA, intimin, Tir and H7 Flagellin, and they showed a reduction in fecal shedding [ 30 ] at day 11 post-challenge. A salmonella-based oral vaccine bearing the same four antigens stimulated mucosal antibody responses and showed a reduction in bacterial shedding at day 6 [ 31 ].…”

Section: Introductionmentioning

confidence: 99%

GlnH, a Novel Antigen That Offers Partial Protection against Verocytotoxigenic Escherichia coli Infection

et al. 2023

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Verotoxin-producing Escherichia coli (VTEC) causes zoonotic infections, with potentially devastating complications, and children under 5 years old are particularly susceptible. Antibiotic treatment is contraindicated, and due to the high proportion of infected children that suffer from severe and life-changing complications, there is an unmet need for a vaccine to prevent VTEC infections. Bacterial adhesins represent promising candidates for the successful development of a vaccine against VTEC. Using a proteomic approach to identify bacterial proteins interacting with human gastrointestinal epithelial Caco-2 and HT-29 cells, we identified eleven proteins by mass spectrometry. These included a glutamine-binding periplasmic protein, GlnH, a member of the ABC transporter family. The glnH gene was identified in 13 of the 15 bovine and all 5 human patient samples tested, suggesting that it is prevalent. We confirmed that GlnH is involved in the host cell attachment of an O157:H7 prototype E. coli strain to gastrointestinal cells in vitro. Recombinant GlnH was expressed and purified prior to the immunisation of mice. When alum was used as an adjuvant, GlnH was highly immunogenic, stimulating strong serological responses in immunised mice, and it resulted in a modest reduction in faecal shedding but did not reduce colonisation. GlnH immunisation with a T-cell-inducing adjuvant (SAS) also showed comparable antibody responses and an IgG1/IgG2a ratio suggestive of a mixed Th1/Th2 response but was partially protective, with a 1.5-log reduction in colonisation of the colon and caecum at 7 days relative to the adjuvant only (p = 0.0280). It is clear that future VTEC vaccine developments should consider the contribution of adjuvants in addition to antigens. Moreover, it is likely that a combined cellular and humoral response may prove more beneficial in providing protective interventions against VTEC.

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Section: Introductionmentioning

confidence: 99%

GlnH, a Novel Antigen That Offers Partial Protection against Verocytotoxigenic Escherichia coli Infection

et al. 2023

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“…As a working chromatographic matrix, a culture of Bacillus subtilis natto was processed as described in Materials and Methods to generate a bacterial-derived affinity chromatography matrix, named here Bio-Matrix (BM). Interestingly, although B. subtilis has no S-layer, we have demonstrated previously that this bacterium can be externally covered with SLAP-tagged proteins to generate a recombinant S-layer on its bacterial cell wall, a process that we called decoration 15 . Decoration of B. subtilis with SLAP-tagged proteins is possible because teichoic acid and lipoteichoic acid (which are the molecules responsible for SLAP TAG association) have the same chemical composition than Lactobacillus acidophilus .…”

Section: Resultsmentioning

confidence: 99%

“…The resulting chimeric antigen (EIT-SLAP TAG ) recombinantly expressed and purified was able to associate to the bacterial cell-wall of L. acidophilus , a process that we named decoration. Thus, EIT-decorated L. acidophilus after oral administration was able to deliver the EIT antigen to the intestinal mucosa eliciting a protective immune response that controls an experimental STEC infection in mice 15 . Remarkably, the decoration process does not modify genetically the Lactobacillus genome preserving its GRAS (Generally Recognized as Safe) status, a trait that is important for vaccine purposes.…”

Section: Mainmentioning

confidence: 98%

“…Here, this dual SLP-A domain was named SLAP TAG , and the adaptation as a molecular tag was evaluated. Recently, we have characterized the binding properties of the SLAP TAG and characterized its association to live Lactobacillus wall for vaccine purposes 15 . Thus, the SLAP TAG was fused in frame to a chimeric antigen derived from the Shiga toxin-producing Escherichia coli (STEC) formed by the peptides EspA 36–192 , Intimin 653–953 and Tir 240–378 (or EIT).…”

Section: Mainmentioning

confidence: 99%

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Adaptation of the binding domain ofLactobacillus acidophilusS-layer protein as a molecular tag for affinity chromatography development

Muruaga

Uriza

Eckert

et al. 2022

Preprint

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The SLAPTAG is a novel molecular TAG derived from a protein domain present in the sequence of Lactobacillus acidophilus SlpA (SlpA284-444). Proteins from different biological sources, with different molecular weights or biochemical functions, can be fused in frame to the SLAPTAG and efficiently purified by the specific binding to a bacterial-derived chromatographic matrix named here Bio-Matrix (BM). To set an optimized protocol for the SLAPTAG-based affinity chromatography (SAC) different binding and elution conditions were evaluated. The binding equilibrium between SLAPTAG and BM was reached after a few minutes at 4oC, being the apparent dissociation constant (KD) of 4.3 uM, a value similar to the one determined for other S-layer proteins and their respective bacterial cell-walls. A reporter protein was generated (H6-GFP-SLAPTAG ) to compare the efficiency of SAC against a commercial system based on a Ni2+-charged agarose matrix, observing no differences in the H6-GFP-SLAPTAG purification performance. The stability and reusability of the BM were evaluated, and it was determined that the matrix was stable for more than a year, being possible to reuse it five times without a significant loss in the efficiency for protein purification. Alternatively, we explored the recovery of bound SLAP-tagged proteins by proteolysis using the SLAPASE (a SLAP-tagged version of the HRV-3c protease) that released a tag-less GFP (SLAPTAG-less). Additionally, iron nanoparticles were linked to the BM and the resulting BMmag was successfully adapted for a magnetic SAC, a technique that can be potentially applied for high-throughput-out protein production and purification.

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“…Enzyme-linked immunosorbent assay was performed as published ( Uriza et al, 2020 ). Briefly, wells were coated with 125 ng of recombinant proteins in 50 μl of buffer (0.5 M carbonate–bicarbonate pH 9.6).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different

Muruaga

Briones²,

Roset³

2022

Front. Microbiol.

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Brucella spp. are the etiological agent of animal and human brucellosis. We have reported previously that cyclophilins of Brucella (CypA and CypB) are upregulated within the intraphagosomal replicative niche and required for stress adaptation and host intracellular survival and virulence. Here, we characterize B. abortus cyclophilins, CypA, and CypB from a biochemical standpoint by studying their PPIase activity, chaperone activity, and oligomer formation. Even though CypA and CypB are very similar in sequence and share identical chaperone and PPIase activities, we were able to identify outstanding differential features between them. A series of differential peptide loops were predicted when comparing CypA and CypB, differences that might explain why specific antibodies (anti-CypA or anti-CypB) were able to discriminate between both cyclophilins without cross-reactivity. In addition, we identified the presence of critical amino acids in CypB, such as the Trp134 which is responsible for the cyclosporin A inhibition, and the Cys128 that leads to CypB homodimer formation by establishing a disulfide bond. Here, we demonstrated that CypB dimer formation was fully required for stress adaptation, survival within HeLa cells, and mouse infection in B. abortus. The presence of Trp134 and the Cys128 in CypB, which are not present in CypA, suggested that two different kinds of cyclophilins have evolved in Brucella, one with eukaryotic features (CypB), another (CypA) with similar features to Gram-negative cyclophilins.

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Development of an Antigen Delivery Platform Using Lactobacillus acidophilus Decorated With Heterologous Proteins: A Sheep in Wolf’s Clothing Story

Cited by 10 publications

References 40 publications

GlnH, a Novel Antigen That Offers Partial Protection against Verocytotoxigenic Escherichia coli Infection

GlnH, a Novel Antigen That Offers Partial Protection against Verocytotoxigenic Escherichia coli Infection

Adaptation of the binding domain ofLactobacillus acidophilusS-layer protein as a molecular tag for affinity chromatography development

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different

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