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 minutes 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

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

[[abstract]]Background: Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10?5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion:These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification

The immunomodulatory activity of meningococcal lipoprotein Ag473 depends on the conformation made up of the lipid and protein moieties.

We have previously demonstrated that the meningococcal antigen Ag473 in the presence of Freund's adjuvant can elicit protective immune responses in mouse challenge model. In this study, we evaluated the structural requirement for the immunological activity and the possible signaling pathway of recombinant Ag473 antigen produced in E. coli. We found that lipidated Ag473 (L-Ag473) possesses an intrinsic adjuvant activity that could be attributed to its ability to activate dendritic cells and promote their maturation. In addition, we found that L-Ag473 can activate human monocytes and promote maturation of human monocyte-derived dendritic cells. These results provide an indirect support that L-Ag473 may also be immunogenic in human. Interestingly, the observed activity is dependent on the overall conformation of L-Ag473 because heating and proteinase K treatment can diminish and abolish the activity. Furthermore, our data suggest a species-differential TLR recognition of L-Ag473. Overall, these data suggest a new paradigm for the ligand-TLR interaction in addition to demonstrating the self-adjuvanting activity of the vaccine candidate L-Ag473

The Immunomodulatory Activity of Meningococcal Lipoprotein Ag473 Depends on the Conformation Made up of the Lipid and Protein Moieties

We have previously demonstrated that the meningococcal antigen Ag473 in the presence of Freund’s adjuvant can elicit protective immune responses in mouse challenge model. In this study, we evaluated the structural requirement for the immunological activity and the possible signaling pathway of recombinant Ag473 antigen produced in E. coli. We found that lipidated Ag473 (L-Ag473) possesses an intrinsic adjuvant activity that could be attributed to its ability to activate dendritic cells and promote their maturation. In addition, we found that L-Ag473 can activate human monocytes and promote maturation of human monocyte-derived dendritic cells. These results provide an indirect support that L-Ag473 may also be immunogenic in human. Interestingly, the observed activity is dependent on the overall conformation of LAg473 because heating and proteinase K treatment can diminish and abolish the activity. Furthermore, our data suggest a species-differential TLR recognition of L-Ag473. Overall, these data suggest a new paradigm for the ligand-TLR interaction in addition to demonstrating the self-adjuvanting activity of the vaccine candidate L-Ag473

Adjuvant effects of TcdA rRBD and its truncated fragments.

<p>To demonstrate the adjuvant effects of TcdA rRBD, enhancement of the anti-OVA IgG response was evaluated by co-administration of TcdA rRBD and OVA. (A) BALB/c mice were immunized with 2 × 2 μg of OVA formulated with or without 10 μg of TcdA rRBD and alum as a positive control. (B) BALB/c mice were immunized with 2 μg of OVA formulated with 10 μg of TcdA rRBD, F1, F2, or F3. The anti-OVA IgG titer was determined by OVA-specific ELISA. The symbols ** and *** indicate <i>p</i><0.01 and <i>p</i><0.005, respectively.</p

Cytokine secretion from BMDCs treated with TcdA rRBD or its truncated fragments.

<p>After BMDCs were treated with TcdA rRBD or its truncated fragments at day 6 for 18 h, the culture supernatants were collected and analyzed for cytokine profiles using specific cytokine ELISAs: (A) IL-6, (B) IL12p40, and (C) TNF- α. The symbols * and ** indicate <i>p</i><0.05 and <i>p</i><0.01, respectively.</p

Several functional assays were used to evaluate the biological properties of TcdA rRBD and its fragments.

<p>(A) A hemagglutinin (HA) assay was performed starting with either 15 pmol of TcdA or 250 pmol of TcdA rRBD, F1, F2 and F3 mixed with 2% rabbit erythrocytes in a 1-to-1 ratio (v/v). TcdA rRBD cell-binding ability was characterized by flow cytometry (B) and western blot (C) using an anti-TcdA specific monoclonal antibody. Different amounts of TcdA rRBD were used to treat cells to evaluate binding ability in a dose-dependent manner. (D) Vero and Caco-2 cell-binding abilities of TcdA rRBD and its fragments (1 μM) were characterized by FACS analysis as described in the Materials and Methods. The MFI from TcdA rRBD fragment binding assays were measured to evaluate statistical significance, as shown in the bar charts within the FACS figures. The symbols *, ** and *** indicate <i>p</i><0.05, <i>p</i><0.01 and <i>p</i><0.005, respectively.</p

The consensus sequence of the C-terminal repeats and putative receptor-binding domain from <i>C</i>. <i>difficile</i> toxin A (TcdA rRBD).

<p>(A) The amino acid sequence (911 residues) of TcdA rRBD was identified using online software (<a href="http://www.ebi.ac.uk/Tools/pfa/radar/" target="_blank">http://www.ebi.ac.uk/Tools/pfa/radar/</a>). Sequence alignment with a reference TcdA (strain VPI10463) was performed, and the sequence differences are highlighted. (B) The localization and sequence of each short repeat are represented in the left and right columns. (C) The RBD fragments are F1 (residues 1–411), F2 (residues 296–701) and F3 (residues 524–911); the 7 lectin-like receptor-binding (LR) sites are putatively located at residues 93–140 (LR1), 228–275 (LR2), 362–409 (LR3), 475–522 (LR4), 610–657 (LR5), 723–790 (LR6) and 815–881 (LR7).</p

Up-regulation of surface biomarkers of BMDCs by either TcdA rRBD or its truncated fragments.

<p>BMDCs from C57BL/6 were collected and treated with GM-CSF at days 0 and 3. TcdA rRBD was added at day 6 for 18 h, and then, DCs were collected to analyze their surface markers, including CD40 (A), CD80 (B), CD86 (C), and MHC II (D), by flow cytometry. All groups were divided as polymyxin B-treated (PMB) (black-net bar) or non-treated (gray-net bar) to exclude LPS contamination. All surface marker signaling was normalized by calculating the ratio of mean fluorescence intensity (MFI) between the medium control and treatments. The symbols *, ** and ns indicate <i>p</i><0.05, <i>p</i><0.01 and no significant difference, respectively.</p

The immunogenicity of TcdA rRBD and its truncated fragments in mice.

<p>BALB/c mouse anti-RBD antibody responses elicited by 3 × 10 μg of either TcdA rRBD or its fragments. Anti-RBD IgG titers at 6 weeks were determined by RBD-specific ELISA.</p