A novel mucosal bivalent vaccine of EV-A71/EV-D68 adjuvanted with polysaccharides from Ganoderma lucidum protects mice against EV-A71 and EV-D68 lethal challenge

Abstract

Background: Human enteroviruses A71 (EV-A71) and D68 (EV-D68) are the suspected causative agents of hand-foot-and-mouth disease, aseptic meningitis, encephalitis, acute flaccid myelitis, and acute flaccid paralysis in children. Until now, no cure nor mucosal vaccine existed for EV-A71 and EV-D68. Novel mucosal bivalent vaccines are highly important for preventing EV-A71 and EV-D68 infections.

Methods: In this study, formalin-inactivated EV-A71 and EV-D68 were used as antigens, while PS-G, a polysaccharide from Ganoderma lucidum, was used as an adjuvant. Natural polysaccharides have the characteristics of intrinsic immunomodulation, biocompatibility, low toxicity, and safety. Mice were immunized intranasally with PBS, EV-A71, EV-D68, or EV-A71 + EV-D68, with or without PS-G as an adjuvant.

Results: The EV-A71 + EV-D68 bivalent vaccine generated considerable EV-A71- and EV-D68-specific IgG and IgA titres in the sera, nasal washes, saliva, bronchoalveolar lavage fluid, and feces. These antibodies neutralized EV-D68 and EV-A71 infectivity. They also cross-neutralized infections by different EV-D68 and EV-A71 sub-genotypes. Furthermore, compared with the PBS group, EV-A71 + EV-D68 + PS-G-vaccinated mice exhibited an increased number of EV-D68- and EV-A71-specific IgA- and IgG-producing cells. In addition, T-cell proliferative responses, and IFN-γ and IL-17 secretion in the spleen were substantially induced when PS-G was used as an adjuvant with EV-A71 + EV-D68. Finally, in vivo challenge experiments demonstrated that the immune sera induced by EV-A71 + EV-D68 + PS-G conferred protection in neonate mice against lethal EV-A71 and EV-D68 challenges as indicated by the increased survival rate and decreased clinical score and viral RNA tissue expression. Taken together, all EV-A71/EV-D68 + PS-G-immunized mice developed potent specific humoral, mucosal, and cellular immune responses to EV-D68 and EV-A71 and were protected against them.

Conclusions: These findings demonstrated that PS-G can be used as a potential adjuvant for EV-A71 and EV-D68 bivalent mucosal vaccines. Our results provide useful information for the further preclinical and clinical development of a mucosal bivalent enterovirus vaccine against both EV-A71 and EV-D68 infections.

Keywords: Acute flaccid myelitis; Acute flaccid paralysis; Adjuvant; Enterovirus A71; Enterovirus D68; Ganoderma lucidum polysaccharide; Intranasal; Mucosal vaccine.

Fig. 1

Long-term memory immunity induced by inactivated EV-A71 or EV-D68 mucosal vaccines in mice. Mice were intranasally immunized with PBS, formalin-inactivated EV-A71 (2.5 μg/mouse), or EV-D68 (2.5 μg/mouse) thrice at 3-week intervals. a Immunization schedules for the EV-A71 mucosal vaccine. Serum and faecal samples were collected from immunized mice at weeks 8, 11, 14, 22, and 24. The levels of EV-A71-specific IgG and IgA in the serum (b, c) and feces (d, e) were measured using ELISA. f Neutralization titre against EV-A71 C2 infection in the serum of immunized mice. g Immunization schedules for the EV-D68 mucosal vaccine. Serum, feces, and saliva samples were collected from immunized mice at weeks 8, 11, 14, 25, and 27. The levels of EV-D68-specific IgG and IgA in the serum (h, i), feces (j, k), and saliva (l, m) were measured using ELISA. n Neutralization titre against EV-D68 1788 infection in the serum of immunized mice. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

Effect of the EV-A71 + EV-D68 bivalent vaccine on EV-D68- and EV-A71-specific IgG and IgA and neutralization titres against infection by different enteroviruses in immunized mice. Mice were intranasally immunized with PBS, formalin-inactivated EV-A71 (2.5 μg/mouse), formalin-inactivated EV-D68 (2.5 μg/mouse), and combined formalin-inactivated EV-A71 (2.5 μg/mouse) and EV-D68 (2.5 μg/mouse) thrice at 3-week intervals. a Three-dose immunization schedules. b The levels of EV-D68-specific IgA and IgG and c EV-A71-specific IgA and IgG in the saliva, nasal wash, BALF, and feces of mice after the third immunization were measured using ELISA. d The levels of EV-D68-specific IgG and IgA and e EV-A71-specific IgG and IgA in the sera of mice after the third intranasal immunization were measured using ELISA. Sera were serially diluted (2³–2¹²), mixed with f EV-D68 and g EV-A71 virus, and used to infect RD cells. After 4 d, the the highest dilution that resulted in the virus producing no cytopathic effect was considered to be the neutralization titre. All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Effect of the EV-A71 + EV-D68 bivalent vaccine on the number of antibody-secreting cells (ASCs), T-cell proliferation, and cytokine release in splenocytes. Mice were intranasally immunized with PBS, formalin-inactivated EV-A71 (2.5 μg/mouse) alone, formalin-inactivated EV-D68 (2.5 μg/mouse) alone, and combined formalin-inactivated EV-A71 (2.5 μg/mouse) with EV-D68 (2.5 μg/mouse) thrice at 3-week intervals. Spleen was isolated 2 weeks after the third immunization, and then the numbers of a EV-D68-specific IgG and IgA ASCs and b EV-A71-specific IgG and IgA ASCs were measured using ELISPOT. c, d Splenocytes from immunized mice were harvested and cultured in medium containing 10 μg/mL heat-inactivated EV-D68 or EV-A71. After culture for 5 d, proliferation was measured as [³H]-thymidine incorporation in splenocytes. Culture supernatants of splenocytes were analysed for levels of IFN-γ, IL-17, and IL-4 after 2 d in response to heat-inactivated EV-D68 or EV-A71. Thymidine uptake was determined by harvesting cells and using a scintillation counter to measure the level of incorporation (as counts per minute; cpm). All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Protective efficacy of the inactivated EV-A71 + EV-D68 vaccine in the EV-D68 or EV-A71 infection mouse model. The passive sera transfer and virus challenge schedules. Briefly, 2-d-old ICR mice were i.p. injected with 20 μL anti-PBS, anti-EV-A71, anti-EV-D68, or anti-EV-A71 + EV-D68 sera. After 6 h, the suckling mice were intracerebrally (i.c.) administered with RD medium, a EV-D68 (CDC_NO 2016-05298), or e EV-A71 (MP4). Mock mice were administered medium only. Mice were monitored daily for b, f clinical score and c, g survival rate for 14 d following infection. Clinical scores were graded as described in “Methods”. d, h Brain, spinal cord, and muscle were collected after infection, and viral loads within the indicated tissues were determined using real-time quantitative PCR (qRT-PCR). The levels of expression were normalized to those of GAPDH. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Effect of the EV-A71 + EV-D68 bivalent vaccine with or without PS-G or CpG as adjuvant on the levels of EV-D68-specific and EV-A71-specific IgA and IgG in the saliva, nasal wash, BALF, and feces of mice. Mice were intranasally immunized with PBS, formalin-inactivated EV-A71 (2.5 μg/mouse) and EV-D68 (2.5 μg/mouse), and formalin-inactivated EV-A71 (2.5 μg/mouse) and EV-D68 (2.5 μg/mouse) combined with PS-G (10 μg/mouse) or CpG (20 μg/mouse) as adjuvant thrice at 3-week intervals. The levels of a EV-D68-specific IgA and IgG, and b EV-A71-specific IgA and IgG in the saliva, nasal wash, BALF, and feces of mice after the third immunization were measured using ELISA. All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Effect of the EV-A71 + EV-D68 bivalent vaccine with or without PS-G or CpG as adjuvant on the levels of EV-D68- and EV-A71-specific IgG and IgA, and neutralization titre against infection by different enteroviruses in the sera of mice. The levels of a EV-D68-specific IgG and IgA and b EV-A71-specific IgG and IgA in the sera of mice after the third intranasal immunization were measured using ELISA. Sera were serially diluted (2³–2¹²), mixed with c EV-D68 and d EV-A71 virus, and used to infect RD cells. After 4 d, the highest dilution that resulted in the virus producing no cytopathic effect was considered to be the neutralization titre. All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Effect of the EV-A71 + EV-D68 bivalent vaccine with or without PS-G as adjuvant on the number of antibody-secreting cells (ASCs), T-cell proliferation, and cytokine release in splenocytes. Mice were intranasally immunized with PBS, formalin-inactivated EV-A71 (2.5 μg/mouse) and EV-D68 (2.5 μg/mouse), and formalin-inactivated EV-A71 (2.5 μg/mouse) and EV-D68 (2.5 μg/mouse) combined with PS-G (10 μg/mouse) as adjuvant thrice at 3-week intervals. Spleen was isolated at 2 weeks after the third immunization, and then the numbers of a EV-D68-specific IgG and IgA ASCs and b EV-A71-specific IgG and IgA ASCs were measured using ELISPOT. c, d Splenocytes from immunized mice were harvested and cultured in medium containing 10 μg/mL heat-inactivated EV-D68 or EV-A71. After culture for 5 d, proliferation was measured as [³H]-thymidine incorporation in splenocytes. Culture supernatants of splenocytes were analysed for the levels of IFN-γ, IL-17, and IL-4 after 2 d in response to heat-inactivated EV-D68 or EV-A71. e, f Information on IFN-γ and IL-17 production by CD4⁺ and CD8⁺ T-cells in the spleen were obtained from all mice. Splenocytes were collected from mice immunized with PBS, formalin-inactivated EV-A71 + EV-D68, or PS-G-adjuvanted EV-A71 + EV-D68 and cultured in RPMI medium containing 10 μg/mL of heat-inactivated EV-A71 or EV-D68. Cells were cultured for 5 d, and the levels of IFN-γ and IL-17 produced by CD4⁺ and CD8⁺ T-cells were measured by flow cytometry. All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Protective efficacy of the inactivated EV-A71 + EV-D68 vaccine with or without PS-G as adjuvant in the EV-D68 or EV-A71 infection mouse model. The passive sera transfer and virus challenge schedules are shown. Briefly, 2-d-old ICR mice were i.p. injected with 20 μL anti-PBS, anti-EV-A71 + EV-D68, or anti-EV-A71 + EV-D68 + PS-G sera. After 6 h, the suckling mice were intracerebrally (i.c.) administered with RD medium, a EV-D68 (CDC_NO 2016-05298), or f EV-A71 (MP4). Mock mice were given medium only. b, g Representative images showing symptoms of healthy mice in the EV-A71 + EV-D68 or EV-A71 + EV-D68 + PS-G immunized group and mice with limb paralysis in the PBS-immunized group on day 9 post-infection with EV-D68 or EV-A71. Mice were monitored daily for c, h clinical score and d, i survival rates for 14 d following infection. Clinical scores were graded as described in “Methods”. e, j Brain, spinal cord, and muscle were collected after infection, and viral loads within the indicated tissues were determined using real-time quantitative PCR (qRT-PCR). The levels of expression were normalized to those of GAPDH. All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001