Limits...
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

Expression of VLPs in the SMD1168 yeast strain. The supernatant of recombinant SMD1168 yeast was harvested after 96 h of culture and detected by SDS-PAGE (A) and Western blot (B). The PAGE gel was stained by Coomassie blue R-250. The primary antibody used in the Western blot was rabbit anti-CA16 antiserum diluted at 1: 1500. Lane M: 10–170 KD protein marker; Lane 1: SMD1168 culture supernatant; Lane 2: pGAPZαA/SMD1168 culture supernatant; Lane 3: pGAPZαA-3CD/SMD1168 culture supernatant; Lane 4: pGAPZαA-P1/SMD1168 culture supernatant; Lane 5: pGAPZαA-VLPs/SMD1168 culture supernatant; Lane 6: pGAPZαA-VLPs/SMD1168 concentrated supernatant.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036384&req=5

f1-medscimonit-22-3370: Expression of VLPs in the SMD1168 yeast strain. The supernatant of recombinant SMD1168 yeast was harvested after 96 h of culture and detected by SDS-PAGE (A) and Western blot (B). The PAGE gel was stained by Coomassie blue R-250. The primary antibody used in the Western blot was rabbit anti-CA16 antiserum diluted at 1: 1500. Lane M: 10–170 KD protein marker; Lane 1: SMD1168 culture supernatant; Lane 2: pGAPZαA/SMD1168 culture supernatant; Lane 3: pGAPZαA-3CD/SMD1168 culture supernatant; Lane 4: pGAPZαA-P1/SMD1168 culture supernatant; Lane 5: pGAPZαA-VLPs/SMD1168 culture supernatant; Lane 6: pGAPZαA-VLPs/SMD1168 concentrated supernatant.

Mentions: The P1 and 3CD genes were cloned from the local endemic genotype CA16 B1a strain (JB141030268), which was also the predominant genotype currently circulating in mainland China. The full length of P1 was 2586 bp, which encoded 862 amino acids. The full length of 3CD was 1935 bp, which encoded 645 amino acids. The P1 and 3CD genes were inserted into the yeast expression vector pGAPZαA, and the resultant recombinant plasmids pGAPZαA-3CD and pGAPZαA-P1 were electroporated successively into the SMD1168 yeast strain to make the double transformants pGAPZαA-VLPs/SMD1168. CA16 VLPs were secreted in culture supernatant of positive transformants and purified by ultracentrifugation for SDS-PAGE and Western blot analysis. As shown in Figure 1, the yeast strain SMD1168 and empty vector pGAPZαA transformant did not express P1 (lane 1 and lane 2), whereas the pGAPZαA-P1/SMD1168 expressed an obvious protein band, approximately 97 kDa (lane 4). Lane 5 (VLPs harvested at 96 h) and lane 6 (concentrated VLPs) show three protein bands ranging from 25 to 40 KDa, and further analysis of Western blot with rabbit anti-CA16 antiserum confirmed that the purified VLPs contained three major subunit proteins, with molecular weight corresponding to VP0 (39 KDa), VP1 (36 KDa,) and VP3 (28 KDa), respectively. The TEM observation further unraveled that VLPs presented as 27~30 nm spherical particles consistent with the native CA16 virions (Figure 2). All these results indicated that co-expression of P1 and 3CD in the transformed P. pastoris successfully led to the cleavage of P1 polyprotein into VP0, VP1, and VP3 by functional 3CD protease and subsequent self-assembly into the icosahedral VLPs. The yield of VLPs was approximately 2.45 mg/L, and the purity was greater than 83%.


Virus-Like Particles Produced in Pichia Pastoris Induce Protective Immune Responses Against Coxsackievirus A16 in Mice
Expression of VLPs in the SMD1168 yeast strain. The supernatant of recombinant SMD1168 yeast was harvested after 96 h of culture and detected by SDS-PAGE (A) and Western blot (B). The PAGE gel was stained by Coomassie blue R-250. The primary antibody used in the Western blot was rabbit anti-CA16 antiserum diluted at 1: 1500. Lane M: 10–170 KD protein marker; Lane 1: SMD1168 culture supernatant; Lane 2: pGAPZαA/SMD1168 culture supernatant; Lane 3: pGAPZαA-3CD/SMD1168 culture supernatant; Lane 4: pGAPZαA-P1/SMD1168 culture supernatant; Lane 5: pGAPZαA-VLPs/SMD1168 culture supernatant; Lane 6: pGAPZαA-VLPs/SMD1168 concentrated supernatant.
© Copyright Policy
Related In: Results  -  Collection

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

f1-medscimonit-22-3370: Expression of VLPs in the SMD1168 yeast strain. The supernatant of recombinant SMD1168 yeast was harvested after 96 h of culture and detected by SDS-PAGE (A) and Western blot (B). The PAGE gel was stained by Coomassie blue R-250. The primary antibody used in the Western blot was rabbit anti-CA16 antiserum diluted at 1: 1500. Lane M: 10–170 KD protein marker; Lane 1: SMD1168 culture supernatant; Lane 2: pGAPZαA/SMD1168 culture supernatant; Lane 3: pGAPZαA-3CD/SMD1168 culture supernatant; Lane 4: pGAPZαA-P1/SMD1168 culture supernatant; Lane 5: pGAPZαA-VLPs/SMD1168 culture supernatant; Lane 6: pGAPZαA-VLPs/SMD1168 concentrated supernatant.
Mentions: The P1 and 3CD genes were cloned from the local endemic genotype CA16 B1a strain (JB141030268), which was also the predominant genotype currently circulating in mainland China. The full length of P1 was 2586 bp, which encoded 862 amino acids. The full length of 3CD was 1935 bp, which encoded 645 amino acids. The P1 and 3CD genes were inserted into the yeast expression vector pGAPZαA, and the resultant recombinant plasmids pGAPZαA-3CD and pGAPZαA-P1 were electroporated successively into the SMD1168 yeast strain to make the double transformants pGAPZαA-VLPs/SMD1168. CA16 VLPs were secreted in culture supernatant of positive transformants and purified by ultracentrifugation for SDS-PAGE and Western blot analysis. As shown in Figure 1, the yeast strain SMD1168 and empty vector pGAPZαA transformant did not express P1 (lane 1 and lane 2), whereas the pGAPZαA-P1/SMD1168 expressed an obvious protein band, approximately 97 kDa (lane 4). Lane 5 (VLPs harvested at 96 h) and lane 6 (concentrated VLPs) show three protein bands ranging from 25 to 40 KDa, and further analysis of Western blot with rabbit anti-CA16 antiserum confirmed that the purified VLPs contained three major subunit proteins, with molecular weight corresponding to VP0 (39 KDa), VP1 (36 KDa,) and VP3 (28 KDa), respectively. The TEM observation further unraveled that VLPs presented as 27~30 nm spherical particles consistent with the native CA16 virions (Figure 2). All these results indicated that co-expression of P1 and 3CD in the transformed P. pastoris successfully led to the cleavage of P1 polyprotein into VP0, VP1, and VP3 by functional 3CD protease and subsequent self-assembly into the icosahedral VLPs. The yield of VLPs was approximately 2.45 mg/L, and the purity was greater than 83%.

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