Protective Efficacy of VP1-Specific Neutralizing Antibody Associated with a Reduction of Viral Load and Pro-Inflammatory Cytokines in Human SCARB2-Transgenic Mice

<div><p>Hand-foot-mouth diseases (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) in children have now become a severe public health issue in the Asian-Pacific region. Recently we have successfully developed transgenic mice expressing human scavenger receptor class B member 2 (hSCARB2, a receptor of EV71 and CVA16) as an animal model for evaluating the pathogenesis of enterovirus infections. In this study, hSCARB2-transgenic mice were used to investigate the efficacy conferred by a previously described EV71 neutralizing antibody, N3. A single injection of N3 effectively inhibited the HFMD-like skin scurfs in mice pre-infected with clinical isolate of EV71 E59 (B4 genotype) or prevented severe limb paralysis and death in mice pre-inoculated with 5746 (C2 genotype). This protection was correlated with remarkable reduction of viral loads in the brain, spinal cord and limb muscles. Accumulated viral loads and the associated pro-inflammatory cytokines were all reduced. The protective efficacy of N3 was not observed in animals challenged with CVA16. This could be due to dissimilarity sequences of the neutralizing epitope found in CVA16. These results indicate N3 could be useful in treating severe EV71 infections and the hSCARB2-transgenic mouse could be used to evaluate the protective efficacy of potential anti-enterovirus agent candidates.</p></div

Survival of N3 treated hSCARB2-transgenic mice preinfected with 5746.

<p>Seven-day old mice preinfected with 1×10⁵ pfu of 5746 were given 200 µg N3 at time points of 3 h (▾), 24 h (▴), 48 h (▪), and 48 h twice (•) post infection. Mice injected without N3 (O), with the same amounts of isotype antibody (♦), and mice treated with a low dose of N3 (70 µg) (□) 3 h post infection were included. Mice were monitored daily and survival rates were recorded. Each group consisted of 7 to 10 mice and the results were representative of 2 independent experiments. The Logrank test was used for statistical analysis.</p

Administration of N3 reduced pro-inflammatory chemokines expression in CNS and muscle tissues of 5746-preinfected hSCARB2-transgenic mice.

<p>RNAs were extracted from the brain, spinal cord, and muscle of 5746-infected 7-day old hSCARB2-transgenic mice treated with N3 or isotype antibody, and antibody-untreated mice as described in Fig. 6 and were subjected to quantitative RT-PCR specific to (<b>A</b>) CXCL10, (<b>B</b>) CCL3, and (<b>C</b>) IL-6. The number of PCR cycles (Ct) required for fluorescent detection of target genes was calculated and presented as the relative expression after normalization with the internal control of β-actin expression from the same tissue. Each normalized 2^Ct value was the ratio to the value from the mean of 2^Ct obtained from the antibody-untreated tissues. A schematic representation of the target gene expression and the statistical average from 10 mice per group is shown. Unpaired student <i>t</i> test with Welch correction was used for statistical analysis. (*<i>p</i>< = 0.05, **<i>p</i>< = 0.01).</p

N3 conferred the reduction of hair loss and scurf on E59-preinfected hSCARB2-transgenic mice.

<p>(<b>A</b>) and (<b>B</b>) 1-day old hSCARB2-transgenic mice (n = 9–10/group) were preinfected with a HFMD symptom-causing dose of E59 at 1×10⁷ pfu subcutaneously. After 3 h of infection, 200 µg of N3 or isotype antibody was injected i.p. Mice without any antibody treatment were included. The Experiments were repeated twice, and 100× magnification of pictures (<b>A</b>) shown the representative data were taken. The graph represents the scoring of severe hair loss and skin scurf (<b>B</b>) from each group of mice. The scores were monitored on a daily basis. One-way ANOVA and the Kruskal-Wallis test were used for statistical analysis (*<i>p</i>< = 0.05, **<i>p</i>< = 0.01).</p

N3 reduced the pathogenesis of EV71 in the limb muscles and in the intestinal mucosa and villi.

<p>(<b>A</b>) Brainstem and (<b>B</b>) limb muscle tissues were isolated from the 1×10⁵ pfu of 5746-infected 7-day old hSCARB2-Tg mice which treated i.p. with 200 µg of N3 or isotype antibodies (3 h post infection), and from antibody-untreated mice staining with anti-EV71 antibody on Day 4 post infection. The normal tissues (MOCK) with anti-EV71 staining were also prepared. (<b>C</b>) H/E staining of intestine (>8 days post infection) from the groups of mice described in (<b>A</b>) was shown. Observations were made at a magnification of x200 for (<b>A</b>) and x100 for (<b>B</b>) and (<b>C</b>).</p

N3-mediated neutralization against B4 genotype of E59 and C2 genotype of 5746 viruses in vitro.

<p>(<b>A</b>) Both E59 and 5746 were pre-incubated (m.o.i. = 0.5) with various amounts of N3 for 1 h at 37°C before adding them to 3T3-SCARB2 cells. RNA was extracted 2 h after infection, and subjected to RT-PCR to detect the expression of viral genome P1. The amounts of N3 in different lanes were Lane 1∶50 µg, Lane 2∶10 µg, Lane 3∶2 µg, Lane 4∶0.4 µg, Lane 5: uninfected 3T3-SCARB2 cells as the negative control, and Lane 6: E59 viral cDNA as the positive control were included. The same loading scheme was applied to (<b>B</b>) where Lane 6 was replaced by 5746 virus for the control. Expression of cytosolic GADPH as the internal control of RT-PCR was detected. (<b>C</b>) and (<b>D</b>) The bar graph represents the densitometric quantification of the band intensities of viral genome P1 and GADPH from (<b>A</b>) and (<b>B</b>), respectively. The error bar of each group generated from three independent experiments was included.</p

N3 downregulated TNF-α and IL-1β in CNS and muscle tissues of 5746-infected hSCARB2-transgenic mice.

<p>RNA was extracted from the brain, spinal cord, and muscles of 5746-infected 7-day old hSCARB2-transgenic mice treated with N3 or isotype antibody, and antibody-untreated mice as described in Fig. 4 and were subjected to quantitative RT-PCR specific to (<b>A</b>) TNF-α and (<b>B</b>) IL-1β. The relative expression of target gene normalized with the internal control of β-actin expression from the same tissue was calculated as described in Fig. 7. Unpaired student <i>t</i> test with Welch correction was used for statistical analysis. (*<i>p</i>< = 0.05, **<i>p</i>< = 0.01).</p

N3 inhibited viral loads of the tissues from hSCARB2-transgenic mice preinfected with EV71 5746.

<p>7-day old hSCARB2-transgenic mice were challenged with 1×10⁵ pfu of 5746 and followed by receiving 200 µg N3 or isotype antibodies at 3 h post infection. Infected mice received no antibody were included. On day 6 post infection, mice were sacrificed and RNAs were extracted from the brainstem, spinal cord, muscle, skin, and intestine for quantitative RT-PCR using primers specific to VP1 region of P1 transcripts. Virus copy numbers were calculated using a standard curve shown by purified EV71. A schematic representation of the virus copy number and the statistical average from 11 mice per group is shown. The unpaired student <i>t</i> test with Welch correction was used for statistical analysis.</p

Heat Shock protein 90: Role in Enterovirus 71 Entry and Assembly and Potential Target for Therapy

<div><p>Although several factors participating in enterovirus 71 (EV71) entry and replication had been reported, the precise mechanisms associated with these events are far from clear. In the present study, we showed that heat shock protein 90 (HSP90) is a key element associated with EV71 entry and replication in a human rhabdomyosarcoma of RD cells. Inhibition of HSP90 by pretreating host cells with HSP90β siRNA or blocking HSP90 with a HSP90-specific antibody or geldanamycin (GA), a specific inhibitor of HSP90, as well as recombinant HSP90β resulted in inhibiting viral entry and subsequent viral replication. Co-immunprecipitation of EV71 with recombinant HSP90β and colocalization of EV71-HSP90 in the cells demonstrated that HSP90 was physically associated with EV71 particles. HSP90 seems to mediate EV71 replication by preventing proteosomal degradation of the newly synthesized capsid proteins, but does not facilitate viral gene expression at transcriptional level. This was evident by post-treatment of host cells with GA, which did not affect the expression of viral transcripts but accelerated the degradation of viral capsid proteins and interfered with the formation of assembled virions. <i>In vivo</i> studies were carried out using human SCARB2-transgenic mice to evaluate the protection conferred by HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), an analog of geldanamycin, that elicited similar activity but with less toxicity. The results showed that the administration of 17-AAG twice conferred the resistance to hSCARB2 mice challenged with C2, C4, and B4 genotypes of EV71. Our data supports HSP90 plays an important role in EV71 infection. Targeting of HSP90 with clinically available drugs might provide a feasible therapeutic approach to treat EV71 infection.</p> </div

EV71 infection is blocked by dynamin-2 specific siRNA.

<p>3T3-SCARB2 cells were treated with 100 and 200 pmoles of dynamin-2 siRNA or 200 pmole of control siRNA (MOCK) prior to the infection of EV71 as following the treatment protocol as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030507#pone-0030507-g004" target="_blank">Figure 4</a> described above. After 24 or 48 hours of transfection, the detection of dynamin-2 by western blot using anti-dynamin antibody was conducted, or following the transfectants were infected with MOI = 0.04 of EV71 and incubated for 24 or 48 hours before lysate preparation. Expression of dynamin-2 in the lysates was shown in the top panel. Relative dynamin-2 protein levels in specific siRNA-treated cells compared to the levels in 48 hours of cells transfected with 200 pmoles control siRNA (Mock) as 1.0 was shown. The lower panel showed the expression of EV71 capsid protein which was detected by western blotting of MAB979 antibody. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.</p