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Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1

View Article: PubMed Central - PubMed

ABSTRACT

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

No MeSH data available.


Related in: MedlinePlus

Labile toxin B subunit (LTB) significantly enhanced the immunogenicity of enterovirus 71 VP1 subunit (EVP1) vaccine. Balb/c mice of 3–4 weeks (male) were divided into four groups (6 mice in each group) as LTB, EVP1, LTB+EVP1, and PBS (phosphate buffer saline). After anesthetizing with chloral hydrate, the mice were vaccinated intranasally three times on day 0, 7, and 14 with 10–20 µL of LTB (10 µg/mL each mouse), EVP1 (10 µg/mL each mouse), LTB + EVP1 (20 µg/mL each mouse), and PBS, respectively. Samples were individually collected from immunized mice on day 21. Endpoint titers were determined as the dilution of each sample from groups of EVP1, LTB+EVP1, and PBS which showed a 2.1-fold higher absorbance level of 450 nm as compared to that of the negative control samples. Average OD450 values for the animals were calculated. The specific antibodies of EVP1 were significantly increased in LTB+EVP1 treatment (* p < 0.05).
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ijms-17-01419-f001: Labile toxin B subunit (LTB) significantly enhanced the immunogenicity of enterovirus 71 VP1 subunit (EVP1) vaccine. Balb/c mice of 3–4 weeks (male) were divided into four groups (6 mice in each group) as LTB, EVP1, LTB+EVP1, and PBS (phosphate buffer saline). After anesthetizing with chloral hydrate, the mice were vaccinated intranasally three times on day 0, 7, and 14 with 10–20 µL of LTB (10 µg/mL each mouse), EVP1 (10 µg/mL each mouse), LTB + EVP1 (20 µg/mL each mouse), and PBS, respectively. Samples were individually collected from immunized mice on day 21. Endpoint titers were determined as the dilution of each sample from groups of EVP1, LTB+EVP1, and PBS which showed a 2.1-fold higher absorbance level of 450 nm as compared to that of the negative control samples. Average OD450 values for the animals were calculated. The specific antibodies of EVP1 were significantly increased in LTB+EVP1 treatment (* p < 0.05).

Mentions: In this study, the adjuvanticity of LTB to non-replicating EVP1 vaccine was proved via intranasal vaccination in Balb/c mice. The EVP1 specific antibody titers had no significant change between the three tested groups after 48 h of the first vaccination (p > 0.05, data not showed). However, both serum and mucosal EVP1 specific antibody titers were boosted significantly in group LTB plus EVP1 after 21 days of the third vaccination compared with EVP1 alone vaccination (p < 0.05, Figure 1). The results indicated that LTB had vigorous mucosal adjuvant activity and elicited both systematic and mucosal immune responses.


Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1
Labile toxin B subunit (LTB) significantly enhanced the immunogenicity of enterovirus 71 VP1 subunit (EVP1) vaccine. Balb/c mice of 3–4 weeks (male) were divided into four groups (6 mice in each group) as LTB, EVP1, LTB+EVP1, and PBS (phosphate buffer saline). After anesthetizing with chloral hydrate, the mice were vaccinated intranasally three times on day 0, 7, and 14 with 10–20 µL of LTB (10 µg/mL each mouse), EVP1 (10 µg/mL each mouse), LTB + EVP1 (20 µg/mL each mouse), and PBS, respectively. Samples were individually collected from immunized mice on day 21. Endpoint titers were determined as the dilution of each sample from groups of EVP1, LTB+EVP1, and PBS which showed a 2.1-fold higher absorbance level of 450 nm as compared to that of the negative control samples. Average OD450 values for the animals were calculated. The specific antibodies of EVP1 were significantly increased in LTB+EVP1 treatment (* p < 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037698&req=5

ijms-17-01419-f001: Labile toxin B subunit (LTB) significantly enhanced the immunogenicity of enterovirus 71 VP1 subunit (EVP1) vaccine. Balb/c mice of 3–4 weeks (male) were divided into four groups (6 mice in each group) as LTB, EVP1, LTB+EVP1, and PBS (phosphate buffer saline). After anesthetizing with chloral hydrate, the mice were vaccinated intranasally three times on day 0, 7, and 14 with 10–20 µL of LTB (10 µg/mL each mouse), EVP1 (10 µg/mL each mouse), LTB + EVP1 (20 µg/mL each mouse), and PBS, respectively. Samples were individually collected from immunized mice on day 21. Endpoint titers were determined as the dilution of each sample from groups of EVP1, LTB+EVP1, and PBS which showed a 2.1-fold higher absorbance level of 450 nm as compared to that of the negative control samples. Average OD450 values for the animals were calculated. The specific antibodies of EVP1 were significantly increased in LTB+EVP1 treatment (* p < 0.05).
Mentions: In this study, the adjuvanticity of LTB to non-replicating EVP1 vaccine was proved via intranasal vaccination in Balb/c mice. The EVP1 specific antibody titers had no significant change between the three tested groups after 48 h of the first vaccination (p > 0.05, data not showed). However, both serum and mucosal EVP1 specific antibody titers were boosted significantly in group LTB plus EVP1 after 21 days of the third vaccination compared with EVP1 alone vaccination (p < 0.05, Figure 1). The results indicated that LTB had vigorous mucosal adjuvant activity and elicited both systematic and mucosal immune responses.

View Article: PubMed Central - PubMed

ABSTRACT

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

No MeSH data available.


Related in: MedlinePlus