Preclinical Development of a Novel Epitope-based DNA Vaccine Candidate against SARS-CoV-2 and Evaluation of Immunogenicity in BALB/c Mice.

The protective efficacies of current licensed vaccines against COVID-19 have significantly reduced as a result of SARS-CoV-2 variants of concern (VOCs) which carried multiple mutations in the Spike (S) protein. Considering that these vaccines were developed based on the S protein of the original SARS-CoV-2 Wuhan strain, we designed a recombinant plasmid DNA vaccine based on highly conserved and immunogenic B and T cell epitopes against SARS-CoV-2 Wuhan strain and the Omicron VOC. Literature mining and bioinformatics were used to identify 6 immunogenic peptides from conserved regions of the SARS-CoV-2 S and membrane (M) proteins. Nucleotide sequences encoding these peptides representing highly conserved B and T cell epitopes were cloned into a pVAX1 vector to form the pVAX1/S2-6EHGFP recombinant DNA plasmid vaccine. The DNA vaccine was intranasally or intramuscularly administered to BALB/c mice and evaluations of humoral and cellular immune responses were performed. The intramuscular administration of pVAX1/S2-6EHGFP was associated with a significantly higher percentage of CD8+ T cells expressing IFN-γ when compared with the empty vector and PBS controls. Intramuscular or intranasal administrations of pVAX1/S2-6EHGFP resulted in robust IgG antibody responses. Sera from mice intramuscularly immunized with pVAX1/S2-6EHGFP were found to elicit neutralizing antibodies capable of SARS-CoV-2 Omicron variant with the ACE2 cell surface receptor. This study demonstrated that the DNA vaccine construct encoding highly conserved immunogenic B and T cell epitopes was capable of eliciting potent humoral and cellular immune responses in mice.

The Wound Healing Potential of Lignosus rhinocerus and Other Ethno-myco Wound Healing Agents.

Wound care has become increasingly important over the years. Various synthetic products for wound care treatment have been reported to cause toxic side effects and therefore natural products are in significant demand as they have minimal side effects. The presence of bioactive compounds in medicinal mushrooms contributes to various biological activities which assist in the early inflammatory phase, keratinocyte proliferation, and its migration enhancement which are pertinent to wound rehabilitation. Lignosus rhinocerus (tiger milk mushroom) can reduce the inflammation phase in wound healing by fighting off bacterial infection and modulating pro-inflammatory cytokines expression in the early stage to avoid prolonged inflammation and tissue damage. The antibacterial, immunomodulating, and anti-inflammatory activities exhibited by most macrofungi play a key role in enhancing wound healing. Several antibacterial and antifungal compounds sourced from traditional botanicals/products may prevent further complications and reoccurrence of injury to a wounded site. Scientific studies are actively underway to ascertain the potential use of macrofungi as a wound healing agent.

The uncoating of EV71 in mature late endosomes requires CD-M6PR.

Enterovirus 71 (EV71) is one of the causative agents of hand-foot-and-mouth disease, which in some circumstances could lead to severe neurological diseases. Despite of its importance for human health, little is known about the early stages of EV71 infection. EV71 starts uncoating with its receptor, human scavenger receptor B2 (hSCARB2), at low pH. We show that EV71 was not targeted to lysosomes in human rhabdomyosarcoma cells overexpressing hSCARB2 and that the autophagic pathway is not essential for EV71 productive uncoating. Instead, EV71 was efficiently uncoated 30 min after infection in late endosomes (LEs) containing hSCARB2, mannose-6-phosphate receptor (M6PR), RAB9, bis(monoacylglycero)phosphate and lysosomal associated membrane protein 2 (LAMP2). Furthering the notion that mature LEs are crucial for EV71 uncoating, cation-dependent (CD)-M6PR knockdown impairs EV71 infection. Since hSCARB2 interacts with cation-independent (CI)-M6PR through M6P-binding sites and CD-M6PR also harbor a M6P-binding site, CD-M6PR is likely to play important roles in EV71 uncoating in LEs.

Neuronal infection is a major pathogenetic mechanism and cause of fatalities in human acute Nipah virus encephalitis.

OBJECTIVES: Acute Nipah (NiV) encephalitis is characterised by a dual pathogenetic mechanism of neuroglial infection and ischaemia-microinfarction associated with vasculitis-induced thrombotic occlusion. We investigated the contributions of these two mechanisms in fatal cases. MATERIALS AND METHODS: We analysed brain tissues (cerebrum, brainstem and cerebellum) from 15 autopsies using light microscopy, immunohistochemistry (IHC), in situ hybridisation and quantitative methods. RESULTS: Three types of discrete plaque-like parenchymal lesions were identified: Type 1 with neuroglial IHC positivity for viral antigens and minimal or no necrosis; Type 2 with neuroglial immunopositivity and necrosis; and Type 3 with necrosis but no viral antigens. Most viral antigen/RNA-positive cells were neurons. Cerebral glial immunopositivity was rare, suggesting that microinfarction played a more important role in white matter injury. Type 1 lesions were also detected in the brainstem and cerebellum, but the differences between cerebral cortex and these two regions were not statistically significant. In the cerebral cortex, Type 1 lesions overwhelmingly predominated, and only 14% Type 1 vs 69% Type 2 lesions were associated with thrombosis. This suggests that neuronal infection as a mechanism of pathogenesis was more important than microinfarction, both in general and in Type 1 lesions in particular. Between the 'early' group (<8-day fever) and the 'late' group (≥8-day fever), there was a decrease of Type 1 and Type 2 lesions with a concomitant increase of Type 3 lesions, suggesting the latter possibly represented late-stage microinfarction and/or neuronal infection. CONCLUSION: Neuronal infection appears to play a more important role than vasculopathy-induced microinfarction in acute NiV encephalitis.

Immunomodulatory Effect and an Intervention of TNF Signalling Leading to Apoptotic and Cell Cycle Arrest on ORL-204 Oral Cancer Cells by Tiger Milk Mushroom, Lignosus rhinocerus.

Research background: Tiger milk mushroom (Lignosus rhinocerus) is a medicinal mushroom that is geographically distributed in the region of South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. Consumption of its sclerotium has been reported to treat various ailments. However, its anticancer potential towards oral cancer cell lines is yet to be determined considering the traditional method of its consumption by biting/chewing of the sclerotium. Experimental approach: Mushroom sclerotial powder of cultivar TM02® was extracted and fractionated in a chromatographic column prior to cytotoxicity testing against a panel of human oral cancer cell lines. The capability of the identified bioactive fraction in regulating several molecules associated with its tumour necrosis factor (TNF) pathway was investigated. Results and conclusions: 2,5-Diphenyl-2H-tetrazolium bromide (MTT) proliferation assay indicated that cell lines ORL-48 (derived from gingiva), ORL-188 (derived from the tongue) and ORL-204 (derived from buccal mucosa) were inhibited by cold water extract of L. rhinocerus sclerotia and its high-molecular-mass fraction (HMM) in varying degrees with ORL-204 being most affected. Hence, the treatment of ORL-204 with HMM mushroom extract was further investigated. HMM mushroom extract induced apoptosis and G0/G1 phase cell cycle arrest through caspase-3/7 cleavage. Activities of MIP2 and COX-2 were downregulated by 0.2- and 4.6-fold respectively in the HMM mushroom extract-treated ORL-204 cells. Novelty and scientific contribution: Using ORL-204, we showed that HMM mushroom extract may act via the TNF pathway at various network sites as a potential dietary compound for cancer prevention and natural adjunct therapeutic to conventional cancer treatment.

Molecular mechanism of L-SP40 peptide and in vivo efficacy against EV-A71 in neonatal mice.

AIMS: Enterovirus A71 (EV-A71) is an etiological agent of hand foot and mouth disease (HFMD) and has the potential to cause severe neurological infections in children. L-SP40 peptide was previously known to inhibit EV-A71 by prophylactic action. This study aimed to identify the mechanism of inhibition in Rhabdomyosarcoma (RD) cells and in vivo therapeutic potential of L-SP40 peptide in a murine model. MAIN METHODS: A pull-down assay was performed to identify the binding partner of the L-SP40 peptide. Co-immunoprecipitation and co-localization assays with the L-SP40 peptide were employed to confirm the receptor partner in RD cells. The outcomes were validated using receptor knockdown and antibody blocking assays. The L-SP40 peptide was further evaluated for the protection of neonatal mice against lethal challenge by mouse-adapted EV-A71. KEY FINDINGS: The L-SP40 peptide was found to interact and co-localize with nucleolin, the key attachment receptor of Enteroviruses A species, as demonstrated in the pull-down, co-immunoprecipitation and co-localization assays. Knockdown of nucleolin from RD cells led to a significant reduction of 3.5 logs of viral titer of EV-A71. The L-SP40 peptide demonstrated 80% protection of neonatal mice against lethal challenge by the mouse-adapted virus with a drastic reduction in the viral loads in the blood (~4.5 logs), skeletal muscles (1.5 logs) and brain stem (1.5 logs). SIGNIFICANCE: L-SP40 peptide prevented severe hind limb paralysis and death in suckling mice and could serve as a potential broad-spectrum antiviral candidate to be further evaluated for safety and potency in future clinical trials against EV-A71.

Laser-heated needle for biopsy tract ablation: In vivo study of rabbit liver biopsy.

PURPOSE: To investigate the efficacy of a newly-developed laser-heated core biopsy needle in the thermal ablation of biopsy tract to reduce hemorrhage after biopsy using in vivo rabbit's liver model. MATERIALS AND METHODS: Five male New Zealand White rabbits weighed between 1.5 and 4.0 kg were anesthetized and their livers were exposed. 18 liver biopsies were performed under control group (without tract ablation, n = 9) and study group (with tract ablation, n = 9) settings. The needle insertion depth (~3 cm) and rate of retraction (~3 mm/s) were fixed in all the experiments. For tract ablation, three different needle temperatures (100, 120 and 150 °C) were compared. The blood loss at each biopsy site was measured by weighing the gauze pads before and after blood absorption. The rabbits were euthanized immediately and the liver specimens were stained with hematoxylin-eosin (H&E) for further histopathological examination (HPE). RESULTS: The average blood loss in the study group was reduced significantly (p < 0.05) compared to the control group. The highest percentage of bleeding reduction was observed at the needle temperature of 150 °C (93.8%), followed by 120 °C (85.8%) and 100 °C (84.2%). The HPE results show that the laser-heated core biopsy needle was able to cause lateral coagulative necrosis up to 14 mm diameter along the ablation tract. CONCLUSION: The laser-heated core biopsy needle reduced hemorrhage up to 93.8% and induced homogenous coagulative necrosis along the ablation tract in the rabbits' livers. This could potentially reduce the risk of tumor seeding in clinical settings.

A novel orally infected hamster model for Coxsackievirus A16 hand-foot-and-mouth disease and encephalomyelitis.

Coxsackievirus A16 (CV-A16) is one of the major causes of mild and self-limiting hand-foot-and-mouth disease (HFMD) in young children, which may occasionally leads to serious neurological complications. In this study, we had developed a novel, consistent, orally infected CV-A16 HFMD hamster model with encephalomyelitis. Four groups of 7-day-old hamsters in a kinetic study were orally infected with mouse-adapted CV-A16 strains and sacrificed at 1-4 days post infection (dpi), respectively. Tissues were studied by light microscopy, immunohistochemistry to detect viral antigens, in situ hybridization to detect viral RNA, and by viral titration. In a separate transmission experiment, orally infected index hamsters were housed together with contact hamsters to investigate oral and fecal viral shedding by virus culture and reverse transcription polymerase chain reaction (RT-PCR). At severe infection/death endpoints, index and contact hamster infection were also histopathologically analyzed. In the kinetic study, infected hamsters developed signs of infection at 4 dpi. Viral antigens/RNA were localized to brainstem (medulla/pons; reticular formation and motor trigeminal nucleus) and spinal cord anterior horn neurons, oral squamous epithelia and epidermis from 3 to 4 dpi. Salivary and lacrimal glands, myocardium, brown adipose tissue, intestinal smooth muscle, and skeletal muscle infection was also demonstrated. Viremia at 1 dpi and increasing viral titers in various tissues were observed from 2 dpi. In the transmission study, all contact hamsters developed disease 3-5 days later than index hamsters, but demonstrated similar histopathological findings at endpoint. Viral culture and RT-PCR positive oral washes and feces confirmed viral shedding. Our hamster model, orally infected by the natural route for human infection, confirmed CV-A16 neurotropism and demonstrated squamous epitheliotropism reminiscent of HFMD, attributes not found in other animal models. It should be useful to investigate neuropathogenesis, model person-to-person transmission, and for testing antiviral drugs and vaccines.

Japanese Encephalitis Virus Infects the Thalamus Early Followed by Sensory-Associated Cortex and Other Parts of the Central and Peripheral Nervous Systems.

Japanese encephalitis (JE) is a known CNS viral infection that often involves the thalamus early. To investigate the possible role of sensory peripheral nervous system (PNS) in early neuroinvasion, we developed a left hindlimb footpad-inoculation mouse model to recapitulate human infection by a mosquito bite. A 1-5 days postinfection (dpi) study, demonstrated focal viral antigens/RNA in contralateral thalamic neurons at 3 dpi in 50% of the animals. From 4 to 5 dpi, gradual increase in viral antigens/RNA was observed in bilateral thalami, somatosensory, and piriform cortices, and then the entire CNS. Infection of neuronal bodies and adjacent nerves in dorsal root ganglia (DRGs), trigeminal ganglia, and autonomic ganglia (intestine, etc.) was also observed from 5 dpi. Infection of explant organotypic whole brain slice cultures demonstrated no viral predilection for the thalamus, while DRG and intestinal ganglia organotypic cultures confirmed sensory and autonomic ganglia susceptibility to infection, respectively. Early thalamus and sensory-associated cortex involvement suggest an important role for sensory pathways in neuroinvasion. Our results suggest that JE virus neuronotropism is much more extensive than previously known, and that the sensory PNS and autonomic system are susceptible to infection.

Development of live attenuated Enterovirus 71 vaccine strains that confer protection against lethal challenge in mice.

Besides causing mild hand, foot and mouth infections, Enterovirus A71 (EV-A71) is associated with neurological complications and fatality. With concerns about rising EV-A71 virulence, there is an urgency for more effective vaccines. The live attenuated vaccine (LAV) is a more valuable vaccine as it can elicit both humoral and cellular immune responses. A miRNA-based vaccine strain (pIY) carrying let-7a and miR-124a target genes in the EV-A71 genome which has a partial deletion in the 5'NTR (∆11 bp) and G64R mutation (3Dp°l) was designed. The viral RNA copy number and viral titers of the pIY strain were significantly lower in SHSY-5Y cells that expressed both let-7a and miR-124a. Inhibition of the cognate miRNAs expressed in RD and SHSY-5Y cells demonstrated de-repression of viral mRNA translation. A previously constructed multiply mutated strain, MMS and the pIY vaccine strain were assessed in their ability to protect 4-week old mice from hind limb paralysis. The MMS showed higher amounts of IFN-γ ex vivo than the pIY vaccine strain. There was absence of EV-A71 antigen in the skeletal muscles and spinal cord micrographs of mice vaccinated with the MMS and pIY strains. The MMS and pIY strains are promising LAV candidates developed against severe EV-A71 infections.

A monoclonal antibody to ameliorate central nervous system infection and improve survival in a murine model of human Enterovirus-A71 encephalomyelitis.

BACKGROUND: Enterovirus A71 (EV-A71) encephalomyelitis is an often fatal disease for which there is no specific treatment available. Passive immunization with a specific monoclonal antibody to EV-A71 was used on a murine model of EV-A71 encephalomyelitis to evaluate its therapeutic effectiveness before and after established central nervous system (CNS) infection. METHODS: Mice were intraperitoneally-infected with a mouse-adapted EV-A71 strain and treated with a dose of monoclonal antibody (MAb) daily for 3 days on day 1, 2 and 3 post-infection or for 3 days on 3, 4 and 5 post-infection. Treatment effectiveness was evaluated by signs of infection and survival rate. Histopathology and qPCR analyses were performed on mice sacrificed a day after completing treatment. RESULTS: In mock-treated mice, CNS infection was established from day 3 post-infection. All mice treated before established CNS infection, survived and recovered completely without CNS infection. All mice treated after established CNS infection survived with mild paralysis, and viral load and antigens/RNA at day 6 post-infection were significantly reduced. CONCLUSIONS: Passive immunization with our MAb could prevent CNS infection in mice if given early before the establishment of CNS infection. It could also ameliorate established CNS infection if optimal and repeated doses were given.

Immunity and clinical efficacy of an inactivated enterovirus 71 vaccine in healthy Chinese children: a report of further observations.

BACKGROUND: To investigate the long-term effects on immunity of an inactivated enterovirus 71 (EV71) vaccine and its protective efficacy. METHODS: A sub-cohort of 1,100 volunteers from Guangxi Province in China was eligible for enrolment and randomly administered either the EV71 vaccine or a placebo on days 0 and 28 in a phase III clinical trial and then observed for the following 2 years with approval by an independent ethics committee of Guangxi Zhuang Autonomous Region, China. Serum samples from the 350 participants who provided a full series of blood samples (at all the sampling points) within the 2-year period were collected. Vaccine-induced immune effects, including the neutralizing antibody titres and cross-protection against different genotypes of EV71, were examined. This study also evaluated the protective efficacy of this vaccine based upon clinical diagnosis. RESULTS: This sub-cohort showed a >60% drop-out rate over 2 years. The seroconversion rates among the 161 immunized subjects remained >95% at the end of study. The geometric mean titres of neutralizing antibodies (anti-genotype C4) 360 days after vaccination in 350 subjects were 81.0 (subjects aged 6-11 months), 98.4 (12-23 months), 95.0 (24-35 months), and 81.8 (36-71 months). These titres subsequently increased to 423.1, 659.0, 545.0, and 321.9, respectively, at 540 days post-immunization (d.p.i.), and similar levels were maintained at 720 d.p.i. Higher IFN-γ/IL-4-specific responses to the C4 genotype of EV71 and cross-neutralization reactivity against major EV71 genotype strains were observed in the vaccine group compared to those in the placebo group. Five EV71-infected subjects were observed in the placebo-treated control group and none in the vaccine-immunized group in per-protocol analysis. CONCLUSION: These results are consistent with the induction of dynamic immune responses and protective efficacy of the vaccine against most circulating EV71 strains. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, NCT01569581, Trial registration date: March 2012.

Enterovirus 71 can directly infect the brainstem via cranial nerves and infection can be ameliorated by passive immunization.

Enterovirus 71 (EV71)-associated hand, foot, and mouth disease may be complicated by encephalomyelitis. We investigated EV71 brainstem infection and whether this infection could be ameliorated by passive immunization in a mouse model. Enterovirus 71 was injected into unilateral jaw/facial muscles of 2-week-old mice, and hyperimmune sera were given before or after infection. Harvested tissues were studied by light microscopy, immunohistochemistry, in situ hybridization, and viral titration. In unimmunized mice, viral antigen and RNA were detected within 24 hours after infection only in ipsilateral cranial nerves, motor trigeminal nucleus, reticular formation, and facial nucleus; viral titers were significantly higher in the brainstem than in the spinal cord samples. Mice given preinfection hyperimmune serum showed a marked reduction of ipsilateral viral antigen/RNA and viral titers in the brainstem in a dose-dependent manner. With optimum hyperimmune serum given after infection, brainstem infection was significantly reduced in a time-dependent manner. A delay in disease onset and a reduction of disease severity and mortality were also observed. Thus, EV71 can directly infect the brainstem, including the medulla, via cranial nerves, most likely by retrograde axonal transport. This may explain the sudden cardiorespiratory collapse in human patients with fatal encephalomyelitis. Moreover, our results suggest that passive immunization may still benefit EV71-infected patients who have neurologic complications.