Calcium Chloride Treatment Enhances Antigen Production in Foot-and-Mouth Disease Vaccines for Serotypes SAT1 and SAT3.

Foot-and-mouth disease (FMD) is a highly contagious viral infection causing acute and severe vesicular lesions in cattle and pigs, which has prompted global vaccination policies. This study presents a technique for enhancing antigen yield in SAT1 BOT and SAT3 ZIM by treatment with calcium chloride (CaCl2). We tested changes in cell viability in BHK-21 suspension cells treated with varying concentrations of CaCl2. The optimal CaCl2 concentration was determined based on antigen yield. The timing of CaCl2 supplementation relative to FMD virus inoculation was tested. Finally, the optimal medium for antigen production was identified. We observed a concentration-dependent decrease in BHK-21 cell viability at >7.5 mM CaCl2. A CaCl2 concentration of 3 mM yielded the most antigens. CaCl2 supplementation relative to FMD virus infection was optimal 2 h before or with viral inoculation. CD-BHK 21 medium supplemented with CaCl2 was the most productive medium. Specifically, SAT1 BOT and SAT3 ZIM showed improved antigen production in CD-BHK 21 medium with 3 mM CaCl2, while Provero-1 and Cellvento BHK-200 media showed no significant enhancement. Overall, CaCl2 supplementation enhanced FMD antigen productivity. This study provides a useful framework for enhancing antigen production efficiently in the FMD vaccine industry.

Isoprinosine as a foot-and-mouth disease vaccine adjuvant elicits robust host defense against viral infection through immunomodulation.

Background: Commercial foot-and-mouth disease (FMD) vaccines have limitations, such as local side effects, periodic vaccinations, and weak host defenses. To overcome these limitations, we developed a novel FMD vaccine by combining an inactivated FMD viral antigen with the small molecule isoprinosine, which served as an adjuvant (immunomodulator). Method: We evaluated the innate and adaptive immune responses elicited by the novel FMD vaccine involved both in vitro and in vivo using mice and pigs. Results: We demonstrated isoprinosine-mediated early, mid-term, and long-term immunity through in vitro and in vivo studies and complete host defense against FMD virus (FMDV) infection through challenge experiments in mice and pigs. We also elucidated that isoprinosine induces innate and adaptive (cellular and humoral) immunity via promoting the expression of immunoregulatory gene such as pattern recognition receptors [PRRs; retinoic acid-inducible gene (RIG)-I and toll like receptor (TLR)9], transcription factors [T-box transcription factor (TBX)21, eomesodermin (EOMES), and nuclear factor kappa B (NF-kB)], cytokines [interleukin (IL)-12p40, IL-23p19, IL-23R, and IL-17A)], and immune cell core receptors [cluster of differentiation (CD)80, CD86, CD28, CD19, CD21, and CD81] in pigs. Conclusion: These findings present an attractive strategy for constructing novel FMD vaccines and other difficult-to-control livestock virus vaccine formulations based on isoprinosine induced immunomodulatory functions.

Development and efficacy evaluation of a novel water-in-oil-in-water adjuvant for an inactivated foot-and-mouth disease vaccine.

To develop a novel water-in-oil-in-water (W/O/W) adjuvant and evaluate the effect on foot-and-mouth disease (FMD) inactivated vaccine, in this study, we prepared the novel nano-emulsion adjuvant based on QS-21 (BEA) which is composed of the mixture of mineral oil Marcol52, surfactant Tween80, oleate polyoxyethylene ether ester, polyoxyethylene palmitic acid ester and span80, cosurfactant polyethylene glycol and QS-21. The two-step emulsification method formed the W/O/W nano-emulsion with two films and three-phase structures. The effective particle diameter of the BEA was about 184 nm, and it has good thermal stability. Then, BEA was emulsified as an adjuvant to prepare for the inactivated FMDV vaccine, and BALB/c mice and pigs were immunized to evaluate its safety and immunization effect. The results showed that the inactivated BEA-FMDV vaccine significantly increased BALB/c mice and pigs' antibodies and cytokine IFN-γ in serum. Meanwhile, the pig-neutralizing antibodies were higher than control group. Safety tests found no symptoms of FMD or significant toxic reactions. After 28 days of immunization, the protection rate can reach 93.3%. The BEA vaccine had good stability at 4 °C, no stratification after 180 days, and the content of 146S in the vaccine did not decrease. In conclusion, the BEA prepared in this study is suitable for FMDV inactivated vaccine and is an effective adjuvant.

On Prevention and Treatment of Infectious Diseases in Summer Based on TCM Theory of Heat Epidemic / 浙江中医药大学学报

[Objective]To provide insights into the clinical diagnosis and treatment of infectious diseases in summer through probing into the theory of heat epidemic and its characteristics formulated by famous doctors in the past dynasties.[Methods]The etiology,pathogenesis and treatment of heat epidemic were analyzed by summarizing the discussions of various doctors from different angles and at different levels,and the differentiation and treatment rules of several common summer infectious diseases were explored on the basis of the application of heat epidemic theory.[Results]The etiology and pathogenesis of heat epidemic can be classified as excessive main Qi,evil abundance and Yin deficiency,transpiration of summer heat and diffuse ptomaine.Its treatment can be roughly divided into interior-clearing and exterior-harmonizing,blood-cooling and orifice-unblocking.On the basis,the article explored the etiology,pathogenesis and treatment of summer infectious diseases,such as hand-foot-and-mouth disease,dengue and Corona Virus Disease 2019(COVID-19),which have certain similarities with heat epidemic.[Conclusion]The theory of heat epidemic,closely related to the syndrome and treatment rules of three types of summer infectious diseases,can be the tentative basis for clinical diagnosis and treatment of summer infectious diseases with traditional Chinese medicine.

Bestatin, A Pluripotent Immunomodulatory Small Molecule, Drives Robust and Long-Lasting Immune Responses as an Adjuvant in Viral Vaccines.

An inactivated whole-virus vaccine is currently used to prevent foot-and-mouth disease (FMD). Although this vaccine is effective, it offers short-term immunity that requires regular booster immunizations and has several side effects, including local reactions at the vaccination site. To address these limitations, herein, we evaluated the efficacy of bestatin as a novel small molecule adjuvant for inactivated FMD vaccines. Our findings showed that the FMD vaccine formulated with bestatin enhanced early, intermediate-, and particularly long-term immunity in experimental animals (mice) and target animals (pigs). Furthermore, cytokines (interferon (IFN)α, IFNß, IFNγ, and interleukin (IL)-29), retinoic acid-inducible gene (RIG)-I, and T-cell and B-cell core receptors (cluster of differentiation (CD)28, CD19, CD21, and CD81) markedly increased in the group that received the FMD vaccine adjuvanted with bestatin in pigs compared with the control. These results indicate the significant potential of bestatin to improve the efficacy of inactivated FMD vaccines in terms of immunomodulatory function for the simultaneous induction of potent cellular and humoral immune response and a long-lasting memory response.

The role of zinc sulfate in enhancing cellular and humoral immune responses to foot-and-mouth disease vaccine.

Foot-and-mouth disease (FMD) is a rapidly propagating infectious disease of cloven-hoofed animals, especially cattle and pigs, affecting the productivity and profitability of the livestock industry. Presently, FMD is controlled and prevented using vaccines; however, conventional FMD vaccines have several disadvantages, including short vaccine efficacy, low antibody titers, and safety issues in pigs, indicating the need for further studies. Here, we evaluated the efficacy of a novel bivalent vaccine containing zinc sulfate as an immunostimulant and FMD type O and A antigens (O PA2 and A YC, respectively) against FMD virus in mice and pigs. Zinc sulfate induced cellular immunity in murine peritoneal exudate cells (PECs) and porcine peripheral blood mononuclear cells (PBMCs) by increasing IFNγ secretion. Additionally, FMD vaccine containing O PA2 and A YC antigens and zinc sulfate induced early, mid-, and long-term immune responses in mice and pigs, and enhanced cellular and humoral immunity by regulating the expression of pathogen recognition receptors (PRRs), transcription factors, co-stimulatory molecules, and cytokines in porcine PBMCs from vaccinated pigs. Overall, these results indicated that the novel immunostimulant zinc sulfate induced potent cellular and humoral immune responses by stimulating antigen-presenting cells (APCs) and T and B cells, and enhanced long-term immunity by promoting the expression of co-stimulatory molecules. These outcomes suggest that zinc sulfate could be used as a novel vaccine immunostimulant for difficult-to-control viral diseases, such as African swine fever (ASF) or COVID-19.

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication.

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

Hematological parameters, antioxidant status, and gene expression of γ-INF and IL-1ß in vaccinated lambs fed different type of lipids.

This study was aimed to evaluate the effects of vegetable oils as calcium salt on immune responses and the expression of immune-related genes in vaccinated lambs. Twenty-four lambs (35 kg body weight, 6 months old) were assigned to four treatments with six replicates in a completely randomized design for 40 days. Four concentrates were formulated in which the calcium salts of palm oil, canola oil, corn oil, and flaxseed oil were used. On day 30 of the experiment, lambs were vaccinated by a dose of foot-and-mouth disease virus. The blood samples were collected from jugular vein 10 days after vaccination. The level of malondialdehyde and the activity of liver enzymes were the highest in lambs receiving corn oil and the lowest in lambs receiving flaxseed oil. The highest lymphocytes and the lowest neutrophil percentages were observed in lambs receiving flaxseed oil. There was a significant difference among treatments for the relative genes expression. Flaxseed oil significantly upregulated interferon-γ and corn oil upregulated interleukin-1ß. The highest titer against foot-and-mouth disease virus was related to lambs receiving flaxseed oil, and the lowest titer was related to lambs that received corn oil. Flaxseed oil had more beneficial effects on immune response than other oils.

Dose-responses of zinc as zinc-methionine supplements on antioxidant status, hematological parameters, immune response and the expression of IL-4 and IL-6 genes of ewes in the hot season.

This study was implemented to evaluate the effects of different zinc doses as Zinc-Met supplement (Zinpro®) on the antioxidant status, blood immune cells, antibody titers, and the expression of IL-4 and IL-6 genes of ewes in the hot season. In a completely randomized design, 24 ewes were assigned to treatments as follow: 0, 15, 30 and 45 mg/kg zinc as Zinc-Met supplementation for 40 days in region with 40 °C and vaccinated against food-and-mouth disease as an immune challenge at day 30, and then blood samples were collected on day 40. Ewes were fed a basal diet containing 29.9 mg zinc/kg. The highest activity of the antioxidant enzyme and the lowest lipid peroxidation values were found in ewes receiving 30 and 45 mg/kg zinc following a linear trend. The highest lymphocytes count and antibody titers were found in ewes received 30 mg zinc/kg. There were no significant differences among treatments for the relative expression of genes. In overall, zinc supplementation non-significantly up-regulate interleukin-4 and down-regulate interleukin-6. It was concluded that zinc supplementation as Zinc-Met could enhance the antioxidant status and immune response of ewes under heat stress; supplementation of diet with 30 mg zinc/kg (300 mg/kg Zinpro®) appeared to be the most effective dose.

Local and systemic immune responses induced by intranasal immunization with biomineralized foot-and-mouth disease virus-like particles.

Introduction: Foot-and-mouth disease virus (FMDV) infects the host by invading mucosal epithelial cells of the respiratory or digestive tract. Therefore, establishing a specific antiviral mucosal immune barrier can effectively block viral invasion. Methods: We evaluated local mucosal and systemic immune responses elicited by intranasal immunization of mice with foot-and-mouth disease (FMD) calcium phosphate mineralized virus-like particles (CaP-VLPs) and tested whether three commercial mucosal adjuvants enhanced the immunogenicity of the antigen. The biosafety of the vaccine was verified through gross observation and pathological analysis of the lungs. Results: CaP-VLPs effectively induced secretion of IgA (sIgA) from multiple sites in mouse mucosa and produced anti-FMD-specific IgG in the serum. Splenic lymphocytes specifically proliferated and secreted IFN-γ following antigen stimulation, indicating the vaccine can induce a certain level of cellular immune response. Finally, the pathological examination confirmed that CaP-VLPs did not cause substantial damage to the lungs of animals after immunization via mucosal administration. Notably, the vaccine mixed with S adjuvant increased the content of sIgA and serum IgG, and the high level of IgG in serum was maintained at least 7 weeks. Discussion: Overall, this study reveals that FMD CaP-VLPs can induce good local mucosal immune and systemic immune response through intranasal immunization, and the immune response was specifically enhanced by S adjuvant. These data support that CaP-VLPs-S as a candidate mucosal vaccine for the prevention of FMD vaccine infection.

[Overview of Meta-analysis of Lianhua Qingwen preparations in treatment of viral diseases].

This study aims to obtain higher-level evidence by overviewing the Meta-analysis of Lianhua Qingwen preparations in the treatment of viral diseases including influenza, coronavirus disease 2019(COVID-19), and hand, foot and mouth disease(HFMD). CNKI, Wanfang, VIP, China Clinical Trial Registry(ChiCTR), PubMed, EMbase, Web of Science, and Cochrane Library were searched for the Meta-analysis about the treatment of viral diseases with Lianhua Qingwen preparations from the database establishment to April 1, 2022. After literature screening and data extraction, AMSTAR2 and the grading of recommendations assessment, development and evaluations(GRADE) system were used to assess the methodological quality and evidence quality, respectively, and then the efficacy and safety outcomes of Lianhua Qingwen preparations in the treatment of viral diseases were summarized. Thirteen Meta-analysis were finally included, three of which were rated as low grade by AMSTAR2 and ten as very low grade. A total of 75 outcome indicators were obtained, involving influenza, COVID-19, and HFMD. According to the GRADE scoring results, the 75 outcome indicators included 5(6.7%) high-level indicators, 18(24.0%) mediate-level indicators, 25(33.3%) low-level evidence indicators, and 27(36.0%) very low-level indicators.(1)In the treatment of influenza, Lianhua Qingwen preparations exhibited better clinical efficacy than other Chinese patent medicines and Ribavirin and had similar clinical efficacy compared with Oseltamivir. Lianhua Qingwen preparations were superior to other Chinese patent medicines, Oseltamivir, and Ribavirin in alleviating clinical symptoms. They showed no significant differences from Oseltamivir or conventional anti-influenza treatment in terms of the time to and rate of negative result of viral nucleic acid test.(2)In the treatment of COVID-19, Lianhua Qingwen preparation alone or combined with conventional treatment was superior to conventional treatment in terms of total effective rate, main symptom subsidence rate and time, fever clearance rate, duration of fever, time to fever clearance, cough subsidence rate, time to cough subsidence, fatigue subsidence rate, time to fatigue subsidence, myalgia subsidence rate, expectoration subsidence rate, chest tightness subsidence rate, etc. Lianhua Qingwen preparations no difference from conventional treatment in terms of subsiding sore throat, nausea, diarrhea, loss of appetite, headache, and dyspnea. In terms of chest CT improvement rate, rate of progression to severe case, cure time, and hospitalization time, Lianhua Qingwen alone or in combination with conventional treatment was superior to conventional treatment.(3)In the treatment of HFMD, Lianhua Qingwen Granules was superior to conventional treatment in terms of total effective rate, average fever clearance time, time to herpes subsidence, and time to negative result of viral nucleic acid test.(4)In terms of safety, Lianhua Qingwen preparations led to low incidence of adverse reactions, all of which were mild and disappeared after drug withdrawal. The available evidence suggests that in the treatment of influenza, COVID-19, and HFMD, Lianhua Qingwen preparations can relieve the clinical symptoms, shorten the hospitalization time, and improve the chest CT. They have therapeutic effect and good safety in the treatment of viral diseases. However, due to the low quality of available studies, more high-quality clinical trials are needed to support the above conclusions.

Effect of the extract made from Rhizoma Atractylodis Macrocephalae (RAM) on the immune responses of mice to a commercial foot-and-mouth disease vaccine.

OBJECTIVES: Foot-and-mouth disease (FMD) is an economically important animal disease because of the speed of its transmission. Routine vaccination may not be effective; RAM can be considered as a potential facilitator for this. Present study was designed to evaluate the effects of feeding different treatment of the RAM in different days on the immune responses in mice immunised with FMDV type O vaccine. MATERIAL AND METHODS: In experiment 1, 50 ICR mice were randomly divided into five groups with 10 animals in each group, and the basic diet containing 1% Crush of RAM for 1-week ad libitum feeding period, 1% Crush of RAM for 6-week ad libitum feeding period, 1% Decoction of RAM for a 1-week ad libitum feeding period, 1% Decoction of RAM for a 6-week ad libitum feeding period, respectively. Blood samples were collected 2 weeks after boosting for measurement of FMDV-specific IgG level and the IgG subclasses, lymphocyte proliferation as well as production IL-5 and IFN-γ. In experiment 2, four groups mice were fed basic diet and basic diet containing 5% Decoction of RAM for 2-, 4- and 6-day ad libitum feeding periods, respectively. Then we collected blood samples for detecting IgG and IgG subclasses, splenocytes for lymphocyte proliferation as well as production IL-5 and IFN-γ, and tissue samples of small intestine for sIgA. RESULTS: The results indicated that 1% Decoction of RAM for a 1-week ad libitum feeding period group and 5% Decoction of RAM for 2-, 4- and 6-day ad libitum feeding period group enhance the FMDV-specific immune responses significantly. CONCLUSIONS: Taken together, the results demonstrate that doses and feeding time of RAM are important to affect the immune responses.

Oil-in-ionic liquid nanoemulsion-based adjuvant simultaneously enhances the stability and immune responses of inactivated foot-and-mouth disease virus.

Maintaining structural integrity and enhancing stability of inactivated foot-and-mouth disease virus (iFMDV) antigen in adjuvants is crucial to ensure the vaccine potency. Unfortunately, formulation with most reported adjuvants leads to the accelerated dissociation of iFMDV into inactive pentamers. Here, an ionic liquid, i.e., choline and niacin ([Cho][Nic]), which was found to stabilize iFMDV against the acid- and thermo- induced dissociation in buffer solution, was applied to construct a novel oil-in-ionic liquid (o/IL) nanoemulsion adjuvant composed of [Cho][Nic], squalene, and Tween 80. The o/IL nanoemulsion formulated with iFMDV has a monodisperse diameter of 135.8 ± 40.4 nm. The thermostability and long-term stability of iFMDV were remarkably enhanced in o/IL nanoemulsion compared with that in the o/w emulsion without [Cho][Nic] and in the commercial Montanide ISA 206 adjuvant. The o/IL nanoemulsion exerted its adjuvant effects by improving the humoral immune responses. Immunization of o/IL nanoemulsion adjuvanted iFMDV induced specific IgG titers similar to that adjuvanted by Montanide ISA 206 and about 4-fold higher than the un-adjuvanted iFMDV, also promoted the activation of B lymphocytes and the secretion of interleukin-4 in the mice model. This [Cho][Nic]-based o/IL nanoemulsion can serve as a promising adjuvant platform for the foot-and-mouth disease vaccine.

[Effect of Pien Tze Huang against EV71].

This study aims to investigate the inhibitory effect of Pien Tze Huang(PZH) on enterovirus 71(EV71). To be speci-fic, chemiluminescence method was adopted to evaluate the toxicity of PZH to African green monkey kidney(Vero) cells and human rhabdomyosarcoma(RD) cells, and cytopathic effect(CPE) method to assess the inhibition on EV71-GFP reporter virus and EV71 C4 wild-type virus. The results showed that PZH had low cytotoxicity to Vero cells and RD cells, with the half-maximal cytotoxic concentration(CC_(50)) of about 0.691 3-0.879 2 mg·mL~(-1) for the two. In addition, PZH can effectively inhibit the replication of EV71 within the non-cytotoxic concentration range, and dose-dependently alleviate the cytopathic changes caused by virus infection, with the half-maximal effective concentration(EC_(50)) of 0.009 2-0.106 3 mg·mL~(-1). On the basis of the above results, the green fluorescent protein(GFP), indirect immunofluorescence assay(IFA), and median tissue culture infective dose(TCID_(50)) were employed to assess and verify the anti-EV71-GFP and anti-EV71 C4 activity of PZH. The results demonstrated that PZH can dose-dependently lower the expression of GFP by EV71-GFP and structural protein VP-1 by EV71 C4 and decrease the production of progeny infectious viruses. The EC_(50) of PZH for EV71-GFP and EV71 C4 was about 0.006 0-0.006 2 mg·mL~(-1) and 0.006 6-0.025 6 mg·mL~(-1), respectively. This study suggested that PZH may exert antiviral activity by acting on EV71 and interfering with the expression of VP-1. At the moment, there is still a lack of specific anti-EV71 drugs. This study proposed a new idea for the symptomatic treatment of EV71 infections such as hand-foot-mouth disease and verified an effective drug for the treatment of EV71 infections.

Relationship between the expression of peripheral blood stimulating molecules CD80, CD86 and the therapeutic effects of children with severe hand, foot, and mouth dise / 中国热带医学

@#Abstract: Objective　To observe the expression of peripheral blood stimulating molecules CD80 and CD86 in children with severe hand, foot, and mouth disease (HFMD), and to analyze the relationship between them and the therapeutic effects of children. Methods　The clinical data of 252 children with severe HFMD treated in Wuhan Hospital of Traditional Chinese Medicine from March 2017 to March 2021 were collected retrospectively. All children were treated with standardized treatment and the therapeutic effects was evaluated. The baseline data and laboratory test results of children were recorded, and the positive rates of CD80 and CD86 cells in peripheral blood were detected by flow cytometry. Logistic regression was used to analyze the relationship between the above indexes and the therapeutic effects of children. The receiver operating curve (ROC) was drawn to evaluate the value of the above indicators in predicting the therapeutic effects of children. Results　After standardized treatment, 48 children were ineffective, and 204 children were effective; the levels of serum CD80 [(2.28±0.84)% vs (2.12±0.33 )%] and CD86 [(3.35±0.96)% vs (2.23±0.41)%] in children were significantly lower than those at admission (t=2.851, 16.991; P<0.05). The levels of blood lactic acid, serum C-reactive protein (CRP), matrix metalloproteinase-9 (MMP-9), CD80 and CD86 at admission in the ineffective group were significantly higher than those of the effective group (P<0.05). Logistic regression analysis showed that the overexpression of serum CRP (OR=10.929), MMP-9 (OR=1.926), CD80 (OR=3.943) and CD86 (OR=1.947) at admission might be the risk factors of ineffective (all P<0.05). The results of the goodness of fit test for the model showed that, the goodness of fit was high (χ2=6.245, P=0.620); the model collinearity results showed that the variance inflation factors (VIF) values of each variable were <2, and there was no collinearity among the main indicators; the results of the individual independence test for the model showed that Durbin-Watson statistics (D-W)=0.279 and there was poor mutual independence among main indicators. ROC curve analysis showed that the area under the curve(AUC) of serum CD80 at admission in predicting the therapeutic effects of children was 0.762, the cut-off value was 2.390%, and the specificity, sensitivity and Youden index were 0.598, 0792 and 0.390 respectively; the AUC predicted by CD86 was 0.739, the cut-off value was 3.280%, and the specificity, sensitivity and Youden index were 0.510, 0.896 and 0.406 respectively; the AUC by combined prediction was 0.823, and the specificity, sensitivity and Youden index were 0.696, 0.833 and 0.529 respectively. Conclusions　Peripheral blood stimulating molecules CD80 and CD86 are involved in the progression of HFMD. Their overexpression may suggest a high risk of treatment ineffectiveness in children with severe HFMD. Early dynamic monitoring of the expression of serum CD80 and CD86 has a certain predictive value for the therapeutic effect of children.

Effect of Pien Tze Huang against EV71 / 中国中药杂志

This study aims to investigate the inhibitory effect of Pien Tze Huang(PZH) on enterovirus 71(EV71). To be speci-fic, chemiluminescence method was adopted to evaluate the toxicity of PZH to African green monkey kidney(Vero) cells and human rhabdomyosarcoma(RD) cells, and cytopathic effect(CPE) method to assess the inhibition on EV71-GFP reporter virus and EV71 C4 wild-type virus. The results showed that PZH had low cytotoxicity to Vero cells and RD cells, with the half-maximal cytotoxic concentration(CC_(50)) of about 0.691 3-0.879 2 mg·mL~(-1) for the two. In addition, PZH can effectively inhibit the replication of EV71 within the non-cytotoxic concentration range, and dose-dependently alleviate the cytopathic changes caused by virus infection, with the half-maximal effective concentration(EC_(50)) of 0.009 2-0.106 3 mg·mL~(-1). On the basis of the above results, the green fluorescent protein(GFP), indirect immunofluorescence assay(IFA), and median tissue culture infective dose(TCID_(50)) were employed to assess and verify the anti-EV71-GFP and anti-EV71 C4 activity of PZH. The results demonstrated that PZH can dose-dependently lower the expression of GFP by EV71-GFP and structural protein VP-1 by EV71 C4 and decrease the production of progeny infectious viruses. The EC_(50) of PZH for EV71-GFP and EV71 C4 was about 0.006 0-0.006 2 mg·mL~(-1) and 0.006 6-0.025 6 mg·mL~(-1), respectively. This study suggested that PZH may exert antiviral activity by acting on EV71 and interfering with the expression of VP-1. At the moment, there is still a lack of specific anti-EV71 drugs. This study proposed a new idea for the symptomatic treatment of EV71 infections such as hand-foot-mouth disease and verified an effective drug for the treatment of EV71 infections.

In Vivo Sustained Release of Peptide Vaccine Mediated by Dendritic Mesoporous Silica Nanocarriers.

Mesoporous silica nanoparticles have drawn increasing attention as promising candidates in vaccine delivery. Previous studies evaluating silica-based vaccine delivery systems concentrated largely on macromolecular antigens, such as inactivated whole viruses. In this study, we synthesized dendritic mesoporous silica nanoparticles (DMSNs), and we evaluated their effectiveness as delivery platforms for peptide-based subunit vaccines. We encapsulated and tested in vivo an earlier reported foot-and-mouth disease virus (FMDV) peptide vaccine (B2T). The B2T@DMSNs formulation contained the peptide vaccine and the DMSNs without further need of other compounds neither adjuvants nor emulsions. We measured in vitro a sustained release up to 930 h. B2T@DMSNs-57 and B2T@DMSNs-156 released 23.7% (135 µg) and 22.8% (132 µg) of the total B2T. The formation of a corona of serum proteins around the DMSNs increased the B2T release up to 61% (348 µg/mg) and 80% (464 µg/mg) for B2T@DMSNs-57 and B2T@DMSNs-156. In vitro results point out to a longer sustained release, assisted by the formation of a protein corona around DMSNs, compared to the reference formulation (i.e., B2T emulsified in Montanide). We further confirmed in vivo immunogenicity of B2T@DMSNs in a particle size-dependent manner. Since B2T@DMSNs elicited specific immune responses in mice with high IgG production like the reference B2T@Montanide™, self-adjuvant properties of the DMSNs could be ascribed. Our results display DMSNs as efficacious nanocarriers for peptide-based vaccine administration.

Enhancing immune responses to inactivated foot-and-mouth virus vaccine by a polysaccharide adjuvant of aqueous extracts from Artemisia rupestris L.

BACKGROUND: New-generation adjuvants for foot-and-mouth disease virus (FMDV) vaccines can improve the efficacy of existing vaccines. Chinese medicinal herb polysaccharide possesses better promoting effects. OBJECTIVES: In this study, the aqueous extract from Artemisia rupestris L. (AEAR), an immunoregulatory crude polysaccharide, was utilized as the adjuvant of inactivated FMDV vaccine to explore their immune regulation roles. METHODS: The mice in each group were subcutaneously injected with different vaccine formulations containing inactivated FMDV antigen adjuvanted with three doses (low, medium, and high) of AEAR or AEAR with ISA-206 adjuvant for 2 times respectively in 1 and 14 days. The variations of antibody level, lymphocyte count, and cytokine secretion in 14 to 42 days after first vaccination were monitored. Then cytotoxic T lymphocyte (CTL) response and antibody duration were measured after the second vaccination. RESULTS: AEAR significantly induced FMDV-specific antibody titers and lymphocyte activation. AEAR at a medium dose stimulated Th1/Th2-type response through interleukin-4 and interferon-γ secreted by CD4⁺ T cells. Effective T lymphocyte counts were significantly elevated by AEAR. Importantly, the efficient CTL response was remarkably provoked by AEAR. Furthermore, AEAR at a low dose and ISA-206 adjuvant also synergistically promoted immune responses more significantly in immunized mice than those injected with only ISA-206 adjuvant and the stable antibody duration without body weight loss was 6 months. CONCLUSIONS: These findings suggested that AEAR had potential utility as a polysaccharide adjuvant for FMDV vaccines.

Regulation of RDN on Th1/ILC1 cell imbalance in HFMD patients caused by EV71 infection.

Enterovirus 71 (EV71) infection is more likely to cause hand, foot and mouth disease (HFMD) in children, which can lead to neurogenic complications and higher mortality. As a commonly used clinical medicine, Reduning injection (RDN) helps to shorten the symptoms of patients with HFMD and facilitate the early recovery of children. However, the regulatory mechanism of RDN on the HFMD immune system disorder caused by EV71 remains to be discussed. This study collected detailed treatment data of 56 children with HFMD who entered the affiliated Children's Hospital of Nanjing Medical University during 2019. Retrospective analysis of clinical data showed that the symptoms of the RDN treatment group were improved compared with the untreated group. To explore its mechanism, the relevant detection indicators were detected by flow cytometry, enzyme-linked immunosorbent assay and real-time quantitative PCR. It was found that the number and function of innate immune (ILCs) and adaptive immunity (Th1, Th2 and secreted cytokines) were reduced, suggesting that RDN plays a role by regulating cellular immunity. The in vitro differentiation inhibition test further confirmed that RDN affected Th1 differentiation by inhibiting the expression of transcription factors on the basis of Th1 cell differentiation in vitro.

Ginsenoside Rb1 is an immune-stimulatory agent with antiviral activity against enterovirus 71.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the theory of traditional Chinese medicine, the main pathogenesis of severe hand, foot and mouth disease (HFMD) is that the heat and wet poisons are deeply trapped in the viscera, which causes the deficiency of Qi and Yin in the patient's body. Ginsenoside Rb1 (Rb1) is the most abundant triterpenoid saponin in Panax quinquefolius L., which has the function of Qi-invigorating and Yin-nourishing. Enterovirus 71 (EV71) is one of the causative pathogens of HFMD, especially the form associated with some lethal complications. Therefore, the therapeutic effect of Rb1 on this disease caused by EV71 infection is worth exploring. AIM OF THE STUDY: We explored the effective antiviral activities of Rb1 against EV71 in vitro and in vivo and investigated its preliminary antiviral mechanisms. MATERIAL AND METHODS: EV71-infected two-day-old suckling mice model was employed to detect the antiviral effects of Rb1 in vivo. To detect the antiviral effects of Rb1 in vitro, cytopathic effect (CPE) reduction assay was performed in EV71-infected Rhabdomyosarcoma (RD) cells. Interferon (IFN)-ß interference experiment was employed to detect the antiviral mechanism of Rb1. RESULTS: In this paper, we first found that Rb1 exhibited strong antiviral activities in EV71-infected suckling mice when compared to those of ribavirin. Administration of Rb1 reduced the CPE of EV71-infected RD cells in a dose-dependent manner. Moreover, EV71-induced viral protein-1 (VP-1) expression was significantly reduced by Rb1 administration in vitro and in vivo. Furthermore, Rb1 treatment could induce high cellular and humoral immune responses in vivo. Meanwhile, Rb1 contributed to the enhanced Type I IFN responses and IFN-ß knockdown reversed the antiviral activity of Rb1 in vitro. CONCLUSION: In summary, our findings suggest that Rb1 is an immune-stimulatory agent and provide an insight into therapeutic potentials of Rb1 for the treatment of EV71 infection.