Efficacy of Coxsackievirus A5 Vaccine Candidates in an Actively Immunized Mouse Model.

Coxsackievirus A5 (CV-A5) has recently emerged as a main hand, foot, and mouth disease (HFMD) pathogen. Following a large-scale vaccination campaign against enterovirus 71 (EV-71) in China, the number of HFMD-associated cases with EV-71 was reduced, especially severe and fatal cases. However, the total number of HFMD cases remains high, as HFMD is also caused by other enterovirus serotypes. A multivalent HFMD vaccine containing 4 or 6 antigens of enterovirus serotypes is urgently needed. A formaldehyde-inactivated CV-A5 vaccine derived from Vero cells was used to inoculate newborn Kunming mice on days 3 and 10. The mice were challenged on day 14 with a mouse-adapted CV-A5 strain at a dose that was lethal for 14-day-old suckling mice. Within 14 days postchallenge, groups of mice immunized with three formulations, empty particles (EPs), full particles (FPs), and a mixture of the EP and FP vaccine candidates, all survived, while 100% of the mock-immunized mice died. Neutralizing antibodies (NtAbs) were detected in the sera of immunized mice, and the NtAb levels were correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak or not observed in the immunized mice compared with those in alum-inoculated control mice. Another interesting finding was the identification of CV-A5 dense particles (DPs), facilitating morphogenesis study. These results demonstrated that the Vero cell-adapted CV-A5 strain is a promising vaccine candidate and could be used as a multivalent HFMD vaccine component in the future.IMPORTANCE The vaccine candidate strain CV-A5 was produced with a high infectivity titer and a high viral particle yield. Three particle forms, empty particles (EPs), full particles (FPs), and dense particles (DPs), were obtained and characterized after purification. The immunogenicities of EP, FP, and the EP and FP mixture were evaluated in mice. Mouse-adapted CV-A5 was generated as a challenge strain to infect 14-day-old mice. An active immunization challenge mouse model was established to evaluate the efficacy of the inactivated vaccine candidate. This animal model mimics vaccination, similar to immune responses of the vaccinated. The animal model also tests protective efficacy in response to the vaccine against the disease. This work is important for the preparation of multivalent vaccines against HFMD caused by different emerging strains.

Polyethylene glycol 400 significantly enhances the stimulation of 2-phenoxyethanol on Vibrio qinghaiensis sp.-Q67 bioluminescence.

Previous studies demonstrated long-term stimulation of some commercial personal care products (PCPs) on freshwater luminescent bacteria Vibrio qinghaiensis sp.-Q67 (Q67). However, whether a certain component can affect mixture's hormetic effect is still unknown. In this paper, two of ingredients in PCPs, 2-phenoxyethanol (PhE) and polyethylene glycol 400 (PEG400), were selected as object compounds to explore the relationship between concentration-response (CR) of mixtures and that of a single component. It was found that PEG400 has monotonic CR (MCR) on Q67 both at the short-term (0.25 h) and long-term (12 h) exposures while PhE has MCR at 0.25 h and hormetic CR (HCR) at 12 h. Here, the concentration-response curves (CRCs) of PEG400 at 0.25 and 12 h are overlapped each other and the CRCs of PEG400 are on the right of PhE. If the pEC50 is taken as a toxic index, the toxicities of PEG400 at two times are basically the same, and those of PhE are the same, too, but PhE is twice as toxic as PEG400. For the mixtures of PEG400 and PhE, all rays except R1 have MCRs at 0.25 h while all rays have HCRs at 12 h where the higher the mixture ratio of PhE is, the more negative the maximum stimulation effect is. More importantly, the Emin values of all rays are more negative (1.79-3.17-fold) than that of PhE worked alone, which implies that the introduction of PEG400 significantly enhances stimulative effect of PhE. At 0.25 h, all binary mixture rays but R1 produce a low-concentration additive action and high-concentration synergism. At 12 h, all rays display additive action, antagonism, additive action, and synergism in turn when the concentration changes from low to high. The overall findings suggested toxicological interactions should be considered in the risk assessment of PCPs and their potential impacts on ecological balances.

Inhibition of Nav1.7 channels by methyl eugenol as a mechanism underlying its antinociceptive and anesthetic actions.

AIM: Methyl eugenol is a major active component extracted from the Chinese herb Asari Radix et Rhizoma, which has been used to treat toothache and other pain. Previous in vivo studies have shown that methyl eugenol has anesthetic and antinociceptive effects. The aim of this study was to determine the possible mechanism underlying its effect on nervous system disorders. METHODS: The direct interaction of methyl eugenol with Na(+) channels was explored and characterized using electrophysiological recordings from Nav1.7-transfected CHO cells. RESULTS: In whole-cell patch clamp mode, methyl eugenol tonically inhibited peripheral nerve Nav1.7 currents in a concentration- and voltage-dependent manner, with an IC50 of 295 µmol/L at a -100 mV holding potential. Functionally, methyl eugenol preferentially bound to Nav1.7 channels in the inactivated and/or open state, with weaker binding to channels in the resting state. Thus, in the presence of methyl eugenol, Nav1.7 channels exhibited reduced availability for activation in a steady-state inactivation protocol, strong use-dependent inhibition, enhanced binding kinetics, and slow recovery from inactivation compared to untreated channels. An estimation of the affinity of methyl eugenol for the resting and inactivated states of the channel also demonstrated that methyl eugenol preferentially binds to inactivated channels, with a 6.4 times greater affinity compared to channels in the resting state. The failure of inactivated channels to completely recover to control levels at higher concentrations of methyl eugenol implies that the drug may drive more drug-bound, fast-inactivated channels into drug-bound, slow-inactivated channels. CONCLUSION: Methyl eugenol is a potential candidate as an effective local anesthetic and analgesic. The antinociceptive and anesthetic effects of methyl eugenol result from the inhibitory action of methyl eugenol on peripheral Na(+) channels.

Humoral and cellular immunogenicity and efficacy of a coxsackievirus A10 vaccine in mice.

Coxsackievirus A10 (CV-A10) has become one of the major pathogens of hand, foot and mouth disease (HFMD), and studies on the vaccine and animal model of CV-A10 are still far from complete. Our study used a mouse-adapted CV-A10 strain, which was lethal for 14-day-old mice, to develop an infected mouse model. Then this model was employed to establish an actively immunized-challenged mouse model to evaluate the efficacy of a formaldehyde-inactivated CV-A10 vaccine, which was prepared from a Vero cell-adapted strain. CV-A10 vaccine at a dose of 0.5 or 2.0 µg was inoculated intraperitoneally in neonatal Kunming mice on the third and ninth day. Then the mice were challenged on day 14. The survival rate of mice immunized with 0.5 or 2.0 µg vaccine were 90% and 100%, respectively, while all Alum-inoculated mice died. Compared to those in the two vaccinated groups, the Alum-inoculated mice showed severe pathological damage, strong viral protein expression and high viral loads. The antisera from vaccinated mice showed high level of neutralizing antibodies against CV-A10. Meanwhile, three potential T cell epitopes located at the carboxyl-terminal regions of the VP1 and VP3 were identified and exhibited CV-A10 serotype-specific. The humoral and cellular immunogenicity analysis showed that immunization with two doses of the vaccine elicited CV-A10 specific neutralizing antibody and T cell response in BALB/c mice. Collectively, these findings indicated that this actively immunized-challenged mouse model will be invaluable in future studies on CV-A10 pathogenesis and evaluation of vaccine candidates.

Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice.

Coxsackievirus A5 (CV-A5) is a re-emerging enterovirus that causes hand, foot, and mouth disease in children under five years of age. CV-A5-M14-611 is a mouse-adapted strain that can infect orally and lead to the death of 14-day-old mice. Here, recombinants based on CV-A5-M14-611 were constructed carrying three reporter genes in different lengths. Smaller fluorescent marker proteins, light, oxygen, voltage sensing (iLOV), and nano luciferase (Nluc) were proven to be able to express efficiently in vitro. However, the recombinant with the largest insertion of the red fluorescence protein gene (DsRed) was not rescued. The construction strategy of reporter viruses was to insert the foreign genes between the C-terminus of VP1 and the N-terminus of 2A genes and to add a 2A protease cleavage domain at both ends of the insertions. The iLOV-tagged or Nluc-tagged recombinants, CV-A5-iLOV or CV-A5-Nluc, exhibited a high capacity for viral replication, genetic stability in cells and pathogenicity in mice. They were used to establish a rapid, inexpensive and convenient neutralizing antibody assay and greatly facilitated virus neutralizing antibody titration. Living imaging was performed on mice with CV-A5-Nluc, which exhibited specific bioluminescence in virus-disseminated organs, while fluorescence induced by CV-A5-iLOV was weakly detected. The reporter-gene-tagged CV-A5 can be used to study the infection and mechanisms of CV-A5 pathogenicity in a mouse model. They can also be used to establish rapid and sensitive assays for detecting neutralizing antibodies.

Identification of specific and shared epitopes at the extreme N-terminal VP1 of Coxsackievirus A4, A2 and A5 by monoclonal antibodies.

Hand, foot and mouth disease (HFMD) is caused by a variety of serotypes in species A of the Enterovirus genus, including recently re-emerged Coxsackievirus A2 (CV-A2), CV-A4 and CV-A5. For development of diagnostic reagents, for surveillance, and the development of multivalent vaccines against HFMD, the antigenicity of HFMD-associated enteroviruses warrants investigation. The purified virions of CV-A4 were inoculated into Balb/c mice and hybridomas were obtained secreting monoclonal antibodies (mAbs) directed against CV-A4 and cross-reacting with other closely related species A enteroviruses. The mAbs were characterized by ELISA, Western blotting and in vitro neutralizing assays. The majority of mAbs was non-neutralizing, with only 2% of the mAbs neutralizing CV-A4 specifically. Most of mAbs bound to linear VP1 epitopes of CV-A4. Interestingly, four types of mAbs were obtained which bound specifically to CV-A4 or were broadly to CV-A4/-A2, CV-A4/-A5 and CV-A4/-A2/-A5, respectively. Mapping with overlapping or single-amino-acid mutant peptides revealed that the four types of mAbs all bound to the first 15 amino acids at the N-terminus of the VP1. This region of picornaviruses is functionally important as it is involved in uncoating and releasing of viral RNA into the cytosol. The binding footprints of four type mAbs are composed of conserved and variable residues and are different from each other. The newly discovered broadly cross-reactive mAbs reflect the high homology of CV-A4/ CV-A2/CV-A5. The results also demonstrate that it is possible and beneficial to develop the diagnostic reagents to detect rapidly the main pathogens of enteroviruses associated with HFMD cause by CV-A4/CV-A2/CV-A5.

Daphnia magna uptake and excretion of luminescence-labelled polystyrene nanoparticle as visualized by high sensitivity real-time optical imaging.

The environmental and ecological consequences of nanoplastics (NPs) draw increasing research interests and social concerns. However, the in situ and real-time detection of NPs from living organisms and transferring media remains as a major technical obstacle for scientific investigation. Herein we report a novel time-gated imaging (TGI) strategy capable of real-time visualizing the intake of NPs by an individual living organism, which is based on the polystyrene NPs labelled with lanthanide up-conversion luminescence. The limit of detection (LOD) of the TGI apparatus was 600 pg (SNR = 3) in a field of view of 2.4 × 3.8 mm. Taking Daphnia magna as the aquatic model, we investigated the dynamics of uptake and accumulation of NPs (500 µg/L) for 24 h, and the subsequent excretion process (in clean medium) for 48 h, and quantitively analyzed the distribution and the overall mass of NPs deposited in D. magna. The uptake of NPs via filter-feeding occurred in a few minutes, whereas a longer accumulation was found, in a timescale of several hours. And similar behaviors (bi-phase elimination) were also seen in the excretion, indicating the migration of NPs into the circulatory system. The average mass of NPs accumulated in an individual D. magna was ∼12 ng after 24 h exposure, indicating that D. magna as a filter feeder tends to retain NPs. The observed NPs accumulation in D. magna exemplifies the potential risk of aquatic ecosystem on exposure to NP contamination.

Identification of a Conserved, Linear Epitope on VP3 of Enterovirus A Species Recognized by a Broad-Spectrum Monoclonal Antibody.

Outbreaks of hand, foot and mouth disease (HFMD) have occurred frequently in the Asian-Pacific region over the last two decades, caused mainly by the serotypes in Enterovirus A species. High-quality monoclonal antibodies (mAbs) are needed to improve the accuracy and efficiency of the diagnosis of enteroviruses associated HFMD. In this study, a mAb 1A11 was generated using full particles of CV-A5 as an immunogen. In indirect immunofluorescence and Western blotting assays, 1A11 bound to the viral proteins of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 of the Enterovirus A and targeted VP3. It has no cross-reactivity to strains of Enterovirus B and C. By mapping with over-lapped and truncated peptides, a minimal and linear epitope 23PILPGF28 was identified, located at the N-terminus of the VP3. A BLAST sequence search of the epitope in the NCBI genus Enterovirus (taxid: 12059) protein database indicates that the epitope sequence is highly conserved among the Enterovirus A species, but not among the other enterovirus species, first reported by us. By mutagenesis analysis, critical residues for 1A11 binding were identified for most serotypes of Enterovirus A. It may be useful for the development of a cost-effective and pan-Enterovirus A antigen detection for surveillance, early diagnosis and differentiation of infections caused by the Enterovirus A species.

Efficacy of Coxsackievirus A2 vaccine candidates correlating to humoral immunity in mice challenged with a mouse-adapted strain.

BACKGROUND: In recent years, Coxsackievirus A2 (CV-A2) has become one of the main serotypes of enterovirus species A associated with hand, foot and mouth disease (HFMD) in China. It has also caused HFMD epidemics in many countries all over the world. Currently, there are no effective, preventive vaccines against it. METHODS: A CV-A2 strain was isolated in RD cells and then adapted to grow in Vero cells. This is in compliance with guidelines for cell substrates allowed for human vaccines by the Chinese regulatory authority. Groups of newborn Kunming mice were inoculated on day 3 and day 9 using two formulations of candidate vaccines, empty particles and full particles. They were then challenged on day 14 at a lethal dose with a mouse-adapted strain. RESULTS: The mice in the control group all died within 14 days post-challenge whereas most of the mice in the candidate vaccine groups survived. It was found that the titers of neutralizing antibodies was dose-dependent in sera of immunized mice. The results also showed that the vaccine candidates stimulated a strong humoral immune response and protected the mice from disease and death. The virus loads in tissues or organs were significantly reduced and pathological changes were either weak or not observed in the immunized groups compared with those in Al(OH)3 control group. Preliminary mapping of the nucleotide and amino acid residues potentially related to cell tropism of the vaccine strain and virulence of the challenge strain was performed. CONCLUSION: The results showed that the RD cell-isolated and Vero cell-adapted CV-A2 strain is a promising vaccine candidate. This active immunization-challenge mouse model mimics the vaccination and then exposure to wildtype viruses, compared with passive immunization-challenge model, and is invaluable for efficacy evaluation in studies on multivalent vaccines containing CV-A2 against HFMD.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.