There are many types of enteroviruses (EV), which infect humans via the gastrointestinal and respiratory tracts. While most infections they cause are mild, a small number can lead to severe neurological symptoms such as meningitis, encephalitis, acute flaccid myelitis, and acute flaccid paralysis, or even result in death.

 

For example, enterovirus A71 (EV-A71, formerly known as enterovirus 71) and D68 (EV-D68) are the two major strains that cause severe disease, and are also the main targets of prevention for scientists when developing enterovirus vaccines.

 

In 2020, Professor Bor-Luen Chiang of the Institute of Immunology, National Taiwan University College of Medicine, and Associate Researcher Yu-Li Lin of the Department of Medical Research, National Taiwan University Hospital, demonstrated that the PS-G (polysaccharide purified from Ganoderma lucidum) significantly enhances the immune protection of the intranasal monovalent EV-A71 vaccine.

 

By the end of 2023, this team published a paper in the Journal of Biomedical Science further proving that the PS-G also enhances the immune response induced by the intranasal bivalent EV-A71 and EV-D68 vaccine, significantly reducing the mortality threat posed by these two enteroviruses in young mice.

 

 

Instill the bivalent enterovirus vaccine and PS-G into the nasal cavity to stimulate mucosal immunity.

 

The bivalent vaccine in this study uses inactivated (killed) enterovirus types A71 and D68 as antigens to induce the immune system to produce antibodies. An adjuvant added to the vaccine to stimulate the immune response and enhance vaccine efficacy is PS-G (polysaccharide purified from Ganoderma lucidum), extracted from the fruiting bodies of Ganoderma lucidum through a specific process.

 

PS-G is a β-D-glucan containing 5% protein, with a molecular weight of approximately 150,000. It has a basic structure consisting of a β-(1→3)-linked main chain and β-(1→6)-linked branches. Previous studies have confirmed that PS-G can accelerate dendritic cell activation (accurately recognizing and processing pathogens) and maturation (correctly initiating subsequent immune responses), thereby enhancing the immune system's ability to combat infections and tumors through T-cell activation.

 

The administration method involves slowly instilling 12 μL of vaccine drops into the nostrils (6 μL per nostril), containing 2.5 μg each of inactivated enterovirus A71 and D68 (bivalent enterovirus vaccine group). For those with the Ganoderma lucidum polysaccharide as an adjuvant, an additional 10 μg of PS-G is included (PS-G + bivalent enterovirus vaccine group). The vaccine is administered once every three weeks, for a total of three doses, with the immune response induced by the vaccine being evaluated two weeks after the final dose.

 

The experiment also includes a control group where 20 μg of a human adjuvant, CpG, replaces PS-G (control adjuvant CpG + bivalent enterovirus vaccine group) to compare the effects of PS-G. Additionally, a placebo group (mimicking the vaccination procedure but instilling physiological saline into the mice's nasal cavity) is used to simulate the absence of vaccine protection.

 

PS-G can enhance the immune response induced by the bivalent enterovirus vaccine.

 

The results indicate that, under the same experimental conditions, compared to the bivalent enterovirus vaccine without adjuvant or with CpG adjuvant, the mice vaccinated with "PS-G + bivalent enterovirus vaccine" showed the following:

 

● Mucosa - Higher levels of specific antibodies: The levels of specific IgA and IgG antibodies in the saliva (oral mucosa), nasal lavage fluid and bronchoalveolar lavage fluid (respiratory mucosa), and feces (intestinal mucosa) were significantly increased. This indicates a stronger mucosal immune barrier to prevent viral invasion (Figure 1).

 

● Serum - Stronger virus-neutralizing ability of antibodies: The levels of specific IgA and IgG antibodies in the serum were significantly increased, with stronger virus-neutralizing abilities (binding to the virus to prevent it from infecting cells). There was also broad neutralization against multiple genetic subtypes of enterovirus A71 and D68, suggesting a higher likelihood of immediate antibody-mediated interception of the virus upon entry into the body (Figure 2).

 

● Spleen - Faster and more robust immune cell response: The number of cells (B cells) in the spleen responsible for producing specific IgA and IgG antibodies was higher. Additionally, spleen cells showed greater proliferation of specific T cells (specialized to combat enterovirus A71 and D68) and higher secretion of antiviral cytokines IFN-γ (interferon gamma) and IL-17 (interleukin 17) upon stimulation with enterovirus A71 or D68. This indicates a more efficient immune system response in mounting a supportive attack against successfully invading viruses (Figure 3).

 

Figure 1: Virus-specific antibodies in various mucosal sites of mice two weeks after vaccination with the bivalent vaccine (Note: In this and subsequent figures, *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between the groups, with smaller p-values representing greater differences between the groups).

 

Figure 2: The quantity of virus-specific antibodies in the serum of mice two weeks after vaccination with the bivalent vaccine, and the neutralizing ability of serum antibodies against different genetic subtypes of enterovirus.

 

Figure 3: Two weeks after mice were vaccinated with the bivalent vaccine, (A, B) the number of antibody-producing cells in the spleen, and (C, D) the proliferation of T cells and cytokine secretion by spleen cells induced by inactivated virus.

 

PS-G can enhance the protective efficacy of the bivalent enterovirus vaccine against life.

 

To confirm that the immunity induced by the vaccine can indeed combat the virus, researchers conducted another animal experiment. They injected the serum from mice that had been vaccinated with either the "bivalent enterovirus vaccine" or the "PS-G + bivalent enterovirus vaccine" intraperitoneally into 2-day-old young mice. Six hours later, they injected a lethal dose of enterovirus D68 or A71 into the young mice' brains and observed them for 14 days.

 

The results revealed that both types of serum could help the young mice combat enterovirus D68 and A71. However, the young mice that received serum from mice vaccinated with "PS-G + bivalent enterovirus vaccine" exhibited a higher level of protection: they not only maintained health comparable to uninfected young mice or rapidly recovered from wasting illness (rather than deteriorating into weakness or paralysis) but also achieved a 100% survival rate (Figure 4). Further analysis showed significantly lower viral loads in their brain, spinal cord, and muscle tissues (Figure 5).

 

Figure 4: Protective efficacy of "serum from mice vaccinated with the bivalent enterovirus vaccine" against infection in newborn young mice: reduction in severe disease rate and increase in survival rate.

Figure 5: Protective efficacy of "serum from mice vaccinated with the bivalent enterovirus vaccine" against infection in newborn young mice: reduction in viral load.

 

The enterovirus vaccine, supplemented with Ganoderma polysaccharides, ensures a comprehensive immune response.

 

Ideally, vaccines should simultaneously trigger mucosal and systemic immune responses. The former can strengthen the mucosal barriers of the respiratory tract, oral cavity, and intestinal tract to block viral invasion while the latter can increase the levels of antibodies and white blood cells in the blood circulation to neutralize viruses that have invaded the body.

 

However, the vast majority of current vaccines are administered via intramuscular or subcutaneous injection, which can only induce a systemic immune response. The mucosal immune response, induced through stimulation of mucosal surfaces (such as the nasal mucosa), is often not robust enough, making the choice of adjuvants a critical opportunity to overcome this challenge.

 

Existing enterovirus vaccines are administered intramuscularly and are only available for enterovirus A71. There are currently no vaccines for enterovirus D68, which is likely to become a significant concern in the future. Therefore, the nasal drop bivalent enterovirus vaccine with PS-G as an adjuvant, which emerged as the winner in this study, can significantly enhance both mucosal and systemic immunity. This dual approach can prevent lethal attacks by enteroviruses. The painless administration process is also more convenient and user-friendly, clearly outlining an ideal blueprint for the next generation of enterovirus vaccines. Additionally, it further validates the safety and efficacy of Ganoderma polysaccharides as an adjuvant for enterovirus vaccines.

 

References: Yu-Li Lin, et al. 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. J Biomed Sci. 2023;30(1):96.

 

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★ The original manuscript of this article was authored in Chinese by Wu Tingyao and subsequently translated into English by Alfred Liu. In the event of any inconsistencies between the English translation and the original Chinese text, the latter shall take precedence. For any queries, pls reach out to the original author, Ms. Wu Tingyao.