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

Maternal antibodies protected neonatal mice from lethal CA16 virus challenge. Adult female BABL/c mice were immunized with VLPs or other control antigen as indicated. (A) A representative picture of wasting caused by CA16/JB141030026 at 3 dpi is shown (indicated by arrows). The mouse on the left-hand side was an age-matched control from the PBS-immunized group. (B) A representative picture of right-hind-limb paralysis caused by CA16/JB141030026 (indicated by arrows). The neonatal mice born to the immunized dams were i.p. challenged with CA16/JB141030026 (B1a) or CA16/JB141030230 (B1b) within 24 h after birth and monitored daily for clinical scores (C, E) and survival (D, F) for a period of 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The logrank test was used to compare the survival curves between each vaccine group and the PBS control 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


getmorefigures.php?uid=PMC5036384&req=5

f6-medscimonit-22-3370: Maternal antibodies protected neonatal mice from lethal CA16 virus challenge. Adult female BABL/c mice were immunized with VLPs or other control antigen as indicated. (A) A representative picture of wasting caused by CA16/JB141030026 at 3 dpi is shown (indicated by arrows). The mouse on the left-hand side was an age-matched control from the PBS-immunized group. (B) A representative picture of right-hind-limb paralysis caused by CA16/JB141030026 (indicated by arrows). The neonatal mice born to the immunized dams were i.p. challenged with CA16/JB141030026 (B1a) or CA16/JB141030230 (B1b) within 24 h after birth and monitored daily for clinical scores (C, E) and survival (D, F) for a period of 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The logrank test was used to compare the survival curves between each vaccine group and the PBS control group. Statistical significance was indicated as follows: n.s. p>0.05; * p<0.05; and ** p<0.01.

Mentions: The in vivo protective efficacy of the VLPs was evaluated by maternal immunization and passive protection assays. For the maternal challenge/protection experiment, the neonatal mice born to the immunized dams were i.p. challenged with a lethal dose of CA16 within 2–4 h after birth. As shown in Figure 6, mice from control groups began to get sick at 3 days post-infection (dpi) (Figure 6A), gradually developed limb paralysis at 5 dpi (Figure 6B), and eventually died at 7 dpi. Mice born to the VLPs-immunized and VLPs+FA-immunized dams were partially protected from the homologous CA16 challenge, with a final survival rate of 60% and 87.5% at 14 dpi, respectively (Figure 6C, 6D). When challenged with the heterologous CA16, the survival rate increased to 62.5% and 87.5% at 14 dpi for the VLPs and VLPs+FA groups (Figure 6E, 6F). Maternal VLPs antisera conferred higher protection against homologous and heterologous CA16 challenge to the neonatal mice than that of inactivated CA16 at the same sample treatment, with lower mean clinical scores. For the passive protection assay, groups of one-day-old BABL/c mice were i.p. inoculated with antisera from VLPs-immunized or empty pGAPZαA vector antigen-immunized mice, followed by i.p. challenge with a lethal dose of CA16 at 24 h post-inoculation. As shown in Figure 7, the anti-VLPs serum conferred full protection against homologous and heterologous CA16 challenge for the neonatal mice. In contrast, neonatal mice that received antiserum from pGAPZαA-immunized mice died by 7 dpi.


Virus-Like Particles Produced in Pichia Pastoris Induce Protective Immune Responses Against Coxsackievirus A16 in Mice
Maternal antibodies protected neonatal mice from lethal CA16 virus challenge. Adult female BABL/c mice were immunized with VLPs or other control antigen as indicated. (A) A representative picture of wasting caused by CA16/JB141030026 at 3 dpi is shown (indicated by arrows). The mouse on the left-hand side was an age-matched control from the PBS-immunized group. (B) A representative picture of right-hind-limb paralysis caused by CA16/JB141030026 (indicated by arrows). The neonatal mice born to the immunized dams were i.p. challenged with CA16/JB141030026 (B1a) or CA16/JB141030230 (B1b) within 24 h after birth and monitored daily for clinical scores (C, E) and survival (D, F) for a period of 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The logrank test was used to compare the survival curves between each vaccine group and the PBS control 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

f6-medscimonit-22-3370: Maternal antibodies protected neonatal mice from lethal CA16 virus challenge. Adult female BABL/c mice were immunized with VLPs or other control antigen as indicated. (A) A representative picture of wasting caused by CA16/JB141030026 at 3 dpi is shown (indicated by arrows). The mouse on the left-hand side was an age-matched control from the PBS-immunized group. (B) A representative picture of right-hind-limb paralysis caused by CA16/JB141030026 (indicated by arrows). The neonatal mice born to the immunized dams were i.p. challenged with CA16/JB141030026 (B1a) or CA16/JB141030230 (B1b) within 24 h after birth and monitored daily for clinical scores (C, E) and survival (D, F) for a period of 14 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The logrank test was used to compare the survival curves between each vaccine group and the PBS control group. Statistical significance was indicated as follows: n.s. p>0.05; * p<0.05; and ** p<0.01.
Mentions: The in vivo protective efficacy of the VLPs was evaluated by maternal immunization and passive protection assays. For the maternal challenge/protection experiment, the neonatal mice born to the immunized dams were i.p. challenged with a lethal dose of CA16 within 2–4 h after birth. As shown in Figure 6, mice from control groups began to get sick at 3 days post-infection (dpi) (Figure 6A), gradually developed limb paralysis at 5 dpi (Figure 6B), and eventually died at 7 dpi. Mice born to the VLPs-immunized and VLPs+FA-immunized dams were partially protected from the homologous CA16 challenge, with a final survival rate of 60% and 87.5% at 14 dpi, respectively (Figure 6C, 6D). When challenged with the heterologous CA16, the survival rate increased to 62.5% and 87.5% at 14 dpi for the VLPs and VLPs+FA groups (Figure 6E, 6F). Maternal VLPs antisera conferred higher protection against homologous and heterologous CA16 challenge to the neonatal mice than that of inactivated CA16 at the same sample treatment, with lower mean clinical scores. For the passive protection assay, groups of one-day-old BABL/c mice were i.p. inoculated with antisera from VLPs-immunized or empty pGAPZαA vector antigen-immunized mice, followed by i.p. challenge with a lethal dose of CA16 at 24 h post-inoculation. As shown in Figure 7, the anti-VLPs serum conferred full protection against homologous and heterologous CA16 challenge for the neonatal mice. In contrast, neonatal mice that received antiserum from pGAPZαA-immunized mice died by 7 dpi.

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