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Virus-Like Particles Produced in Pichia Pastoris Induce Protective Immune Responses Against Coxsackievirus A16 in Mice

View Article: PubMed Central - PubMed

ABSTRACT

Background: Coxsackievirus A16 (CA16) is one of the main causative agents of hand, foot, and mouth disease (HFMD), and the development of a safe and effective vaccine has been a top priority among CA16 researchers.

Material/methods: In this study, we developed a Pichia pastoris yeast system for secretory expression of the virus-like particles (VLPs) for CA16 by co-expression of the P1 and 3CD proteins of CA16. SDS-PAGE, Western blot, and transmission electron microscopy (TEM) were performed to identify the formation of VLPs. Immunogenicity and vaccine efficacy of the CA16 VLPs were assessed in BABL/c mouse models.

Results: Biochemical and biophysical analysis showed that the yeast-expressed CA16 VLPs were composed of VP0, VP1, and VP3 capsid subunit proteins, and present spherical particles with a diameter of 30 nm, similar to the parental infectious CA16 virus. Furthermore, CA16 VLPs elicited potent humoral and cellular immune responses, and VLPs-immunized sera conferred efficient protection to neonatal mice against lethal CA16 challenge.

Conclusions: Our results demonstrate that VLPs produced in Pichia pastoris represent a safe and effective vaccine strategy for CA16.

No MeSH data available.


Related in: MedlinePlus

Proliferation of splenocyte derived from immunized mice. The mice were immunized as in Figure 3, and splenocytes were harvested and stimulated with purified ConA or VLPs. The SI values as described in the Material and Methods section were calculated as an indicator of cell proliferation. The data represent the mean ± standard deviation (S.D.) of two independent immunization experiments (n=8 for each group). Statistical significance was indicated as follows: n.s. p>0.05; * p<0.05; and ** p<0.01.
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f4-medscimonit-22-3370: Proliferation of splenocyte derived from immunized mice. The mice were immunized as in Figure 3, and splenocytes were harvested and stimulated with purified ConA or VLPs. The SI values as described in the Material and Methods section were calculated as an indicator of cell proliferation. The data represent the mean ± standard deviation (S.D.) of two independent immunization experiments (n=8 for each group). Statistical significance was indicated as follows: n.s. p>0.05; * p<0.05; and ** p<0.01.

Mentions: Cellular immune response elicited by VLPs immunization was evaluated by splenocyte proliferation assay and cytokine production analysis. The splenocytes were isolated from immunized mice at week 2 after the booster immunization and subjected to the above assays. The VLPs-specific splenocyte proliferation assay was performed using CCK-8 methods. Figure 4 shows that the splenocytes collected from VLPs-immunized or VLPs+FA-immunized mice could be significantly induced to proliferate by VLPs compared with splenocytes of negative control groups (p<0.05). Compared to the inactivated CA16 group, the SI values for the VLPs group were statistically higher with ConA or VLPs stimulation (p<0.05). Profiles of cytokines secreted by VLPs-stimulated splenocytes were evaluated by measuring the levels of INF-γ and IL-2 (Th1 cytokines) and IL-4, IL-6, and IL-10 (Th2 cytokines). As shown in Figure 5, splenocytes of VLPs-immunized or VLPs+FA-immunized mice produced significantly higher levels of INF-γ, IL-2, IL-4, IL-6, and IL-10 compared with those from the negative controls (p<0.05). Under the same sample treatment, cytokine profiles of inactivated CA16-immunized mice were similar to those of the VLPs group, except for lower levels of IL-4 (p<0.05). The results suggested that VLPs immunization induced both Th1-type and Th2-type cellular immune response.


Virus-Like Particles Produced in Pichia Pastoris Induce Protective Immune Responses Against Coxsackievirus A16 in Mice
Proliferation of splenocyte derived from immunized mice. The mice were immunized as in Figure 3, and splenocytes were harvested and stimulated with purified ConA or VLPs. The SI values as described in the Material and Methods section were calculated as an indicator of cell proliferation. The data represent the mean ± standard deviation (S.D.) of two independent immunization experiments (n=8 for each group). Statistical significance was indicated as follows: n.s. p>0.05; * p<0.05; and ** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036384&req=5

f4-medscimonit-22-3370: Proliferation of splenocyte derived from immunized mice. The mice were immunized as in Figure 3, and splenocytes were harvested and stimulated with purified ConA or VLPs. The SI values as described in the Material and Methods section were calculated as an indicator of cell proliferation. The data represent the mean ± standard deviation (S.D.) of two independent immunization experiments (n=8 for each group). Statistical significance was indicated as follows: n.s. p>0.05; * p<0.05; and ** p<0.01.
Mentions: Cellular immune response elicited by VLPs immunization was evaluated by splenocyte proliferation assay and cytokine production analysis. The splenocytes were isolated from immunized mice at week 2 after the booster immunization and subjected to the above assays. The VLPs-specific splenocyte proliferation assay was performed using CCK-8 methods. Figure 4 shows that the splenocytes collected from VLPs-immunized or VLPs+FA-immunized mice could be significantly induced to proliferate by VLPs compared with splenocytes of negative control groups (p<0.05). Compared to the inactivated CA16 group, the SI values for the VLPs group were statistically higher with ConA or VLPs stimulation (p<0.05). Profiles of cytokines secreted by VLPs-stimulated splenocytes were evaluated by measuring the levels of INF-γ and IL-2 (Th1 cytokines) and IL-4, IL-6, and IL-10 (Th2 cytokines). As shown in Figure 5, splenocytes of VLPs-immunized or VLPs+FA-immunized mice produced significantly higher levels of INF-γ, IL-2, IL-4, IL-6, and IL-10 compared with those from the negative controls (p<0.05). Under the same sample treatment, cytokine profiles of inactivated CA16-immunized mice were similar to those of the VLPs group, except for lower levels of IL-4 (p<0.05). The results suggested that VLPs immunization induced both Th1-type and Th2-type cellular immune response.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Coxsackievirus A16 (CA16) is one of the main causative agents of hand, foot, and mouth disease (HFMD), and the development of a safe and effective vaccine has been a top priority among CA16 researchers.

Material/methods: In this study, we developed a Pichia pastoris yeast system for secretory expression of the virus-like particles (VLPs) for CA16 by co-expression of the P1 and 3CD proteins of CA16. SDS-PAGE, Western blot, and transmission electron microscopy (TEM) were performed to identify the formation of VLPs. Immunogenicity and vaccine efficacy of the CA16 VLPs were assessed in BABL/c mouse models.

Results: Biochemical and biophysical analysis showed that the yeast-expressed CA16 VLPs were composed of VP0, VP1, and VP3 capsid subunit proteins, and present spherical particles with a diameter of 30 nm, similar to the parental infectious CA16 virus. Furthermore, CA16 VLPs elicited potent humoral and cellular immune responses, and VLPs-immunized sera conferred efficient protection to neonatal mice against lethal CA16 challenge.

Conclusions: Our results demonstrate that VLPs produced in Pichia pastoris represent a safe and effective vaccine strategy for CA16.

No MeSH data available.


Related in: MedlinePlus