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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.


Titer profiles of total IgG and neutralizing antibody. (A) Titers of total anti-CA16 IgG. (B) Neutralization titers. Eight groups of adult female BABL/c mice received priming and boosting injection at two-week intervals with PBS, FA, pGAPZaA, P1, VLPs, VLPs+FA, CA16, and CA16+FA, respectively. The serum samples were collected at the indicated times and measured by ELISA as described in the Material and Methods section. 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 comparing between each vaccine group and the PBS control group.
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f3-medscimonit-22-3370: Titer profiles of total IgG and neutralizing antibody. (A) Titers of total anti-CA16 IgG. (B) Neutralization titers. Eight groups of adult female BABL/c mice received priming and boosting injection at two-week intervals with PBS, FA, pGAPZaA, P1, VLPs, VLPs+FA, CA16, and CA16+FA, respectively. The serum samples were collected at the indicated times and measured by ELISA as described in the Material and Methods section. 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 comparing between each vaccine group and the PBS control group.

Mentions: Humoral immune response elicited by VLPs immunization was evaluated by levels of anti-CA16 IgG and neutralizing antibodies. Groups of female BALB/c mice were i.p. immunized twice at two-week intervals with PBS, Freund’s adjuvant (FA), empty pGAPZαA vector antigen (pGAPZαA), P1 antigen (P1), purified VLPs (VLPs) or purified VLPs with Freund’s adjuvant (VLPs+FA), purified heat-inactivated CA16 virus (CA16), and purified CA16 virus with FA (CA16+FA), respectively. The serum samples were collected and pooled at different time points after priming and boosting. Levels of specific IgG against CA16 in sera were measured by ELISA as described in the Material and Methods section. Figure 3 shows that the FA, pGAPZαA, and P1 groups induced negligible anti-CA16 IgG titers, flatting against the background level induced by negative control PBS. Conversely, the VLPs or VLPs+FA triggered significantly higher anti-CA16 IgG antibodies than those provoked by negative controls since week 1. The titers gradually increased and peaked at 2020 or 7132 at week 2 after booster immunization (p<0.05), respectively. Despite the slight drop, the anti-CA16 IgG antibodies elicited by the VLPs or VLPs+FA were sustained at high levels at week 4 after booster immunization (the last time point tested). Compared with the VLPs or VLPs+FA, the inactivated CA16 or CA16+FA induced statistically similar IgG antibodies with the same changing trends (p>0.05). Furthermore, the profiles of specific IgG subclasses were measured in the sera harvested from VLPs-immunized and inactivated CA16-immunized mice at week 2 after booster immunization. The results showed that IgG1 and IgG2b were dominantly induced by the VLPs or inactivated CA16, followed by IgG2a and IgG3; the ratio of IgG1 to IgG2a was 1.54 in VLPs and 1.16 in inactivated CA16 (Table 1).


Virus-Like Particles Produced in Pichia Pastoris Induce Protective Immune Responses Against Coxsackievirus A16 in Mice
Titer profiles of total IgG and neutralizing antibody. (A) Titers of total anti-CA16 IgG. (B) Neutralization titers. Eight groups of adult female BABL/c mice received priming and boosting injection at two-week intervals with PBS, FA, pGAPZaA, P1, VLPs, VLPs+FA, CA16, and CA16+FA, respectively. The serum samples were collected at the indicated times and measured by ELISA as described in the Material and Methods section. 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 comparing between each vaccine group and the PBS control group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036384&req=5

f3-medscimonit-22-3370: Titer profiles of total IgG and neutralizing antibody. (A) Titers of total anti-CA16 IgG. (B) Neutralization titers. Eight groups of adult female BABL/c mice received priming and boosting injection at two-week intervals with PBS, FA, pGAPZaA, P1, VLPs, VLPs+FA, CA16, and CA16+FA, respectively. The serum samples were collected at the indicated times and measured by ELISA as described in the Material and Methods section. 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 comparing between each vaccine group and the PBS control group.
Mentions: Humoral immune response elicited by VLPs immunization was evaluated by levels of anti-CA16 IgG and neutralizing antibodies. Groups of female BALB/c mice were i.p. immunized twice at two-week intervals with PBS, Freund’s adjuvant (FA), empty pGAPZαA vector antigen (pGAPZαA), P1 antigen (P1), purified VLPs (VLPs) or purified VLPs with Freund’s adjuvant (VLPs+FA), purified heat-inactivated CA16 virus (CA16), and purified CA16 virus with FA (CA16+FA), respectively. The serum samples were collected and pooled at different time points after priming and boosting. Levels of specific IgG against CA16 in sera were measured by ELISA as described in the Material and Methods section. Figure 3 shows that the FA, pGAPZαA, and P1 groups induced negligible anti-CA16 IgG titers, flatting against the background level induced by negative control PBS. Conversely, the VLPs or VLPs+FA triggered significantly higher anti-CA16 IgG antibodies than those provoked by negative controls since week 1. The titers gradually increased and peaked at 2020 or 7132 at week 2 after booster immunization (p<0.05), respectively. Despite the slight drop, the anti-CA16 IgG antibodies elicited by the VLPs or VLPs+FA were sustained at high levels at week 4 after booster immunization (the last time point tested). Compared with the VLPs or VLPs+FA, the inactivated CA16 or CA16+FA induced statistically similar IgG antibodies with the same changing trends (p>0.05). Furthermore, the profiles of specific IgG subclasses were measured in the sera harvested from VLPs-immunized and inactivated CA16-immunized mice at week 2 after booster immunization. The results showed that IgG1 and IgG2b were dominantly induced by the VLPs or inactivated CA16, followed by IgG2a and IgG3; the ratio of IgG1 to IgG2a was 1.54 in VLPs and 1.16 in inactivated CA16 (Table 1).

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.