Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse

<div><p>Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10⁷ TCID₅₀/mL 10 days after infection when using an MOI of 10⁻⁴. The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO₄) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO₄-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.</p></div

T helper cell responses in vaccinated mice.

<p>The levels of IL-4 and IFN-γ spot-forming cells in the spleens of mice were determined by ELISpot assay. Mice groups (n = 5) were immunized with PBS and various dosages of EV71 vaccines including AddaVAX-EV71, AlPO₄-EV71, or EV71 antigen as indicated. Splenocytes from PBS- or vaccine-treated mice were counted and stimulated <i>in vitro</i> with heat-inactivated whole viruses of EV71(E59-B4), 5746 (C2), or 3340 (C4) strains. The cells were stimulated with ConA (1 μg/mL) as a positive control for the IFN-γ ELISpot assay or were mock stimulated as a negative control (w/o) for both ELISpot assays. Bars represent the means ± SEM of spot counts in triplicate wells. The <i>p</i> value between vaccine groups relative to the PBS control and two vaccine groups were labeled.</p

<i>In situ</i> detection of EV71 distribution in the brain stems of Tg mice after challenge.

<p>The uninfected Tg mice either pre-treated with PBS or vaccines were used as the negative control, and the images of immunohistochemistry (IHC) staining with the Mab979 antibody are shown in A-D. The PBS- and vaccine-treated Tg mice infected with the EV71 C2 or C4 strains were sacrificed on day 6 post-infection, and the waxed sections of brain stem stained by IHC are representatively shown twice in E-R as labeled. All pictures were taken at 200X magnification. Viral particles in the sections are indicated with arrows.</p

Antibody response to the EV71 vaccine in rabbits, hSCARB2 transgenic (Tg) mice and BALB/c mice.

<p>Measurement of vaccine elicited neutralizing antibody titers against EV71 viruses. The used vaccine formulations to immunize animals and the tested subgenotypes of EV71 virus are as labeled. “Prime” or “boost” indicate the detected viral neutralizing titers (NT) in antisera collected from animals that had been immunized once or twice.</p

Neurological symptoms in Tg mice after lethal challenge with EV71 C2 or C4 strains.

<p>Tg mice receiving PBS (A-B), 2 μg of the EV71 vaccine with AddaVAX (C-D), or 2 μg (E-F) or 6 µg (G) of the EV71 vaccine with AlPO₄ were challenged with EV71 C2 or C4 viruses. The representative photographs showed the immunized Tg mice at 6 days post-challenge.</p

Vaccine treatments protect Tg mice against a lethal-dose challenge of subgenotypes of the EV71 virus.

<p>The survival rate of immunized Tg mice challenged with 3×10⁶ pfu of the C2 strain (A) or 1×10⁶ pfu of the C4 strain (B). Mouse body weight change after EV71 C2 (C) or C4 (D) virus challenges. Scores of central nervous system (CNS)-like hind limb paralysis caused by EV71 C2 (E) or C4 (F) viral challenges. The severity of CNS symptoms was scored from 0 to 5 using the following criteria for scoring CNS diseases: 5 = severe front and rear limb paralysis (LP) and no movement, 4 = moderate two rear LP and hesitant movement, 3 = one rear LP with bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, and 0 = normal movement. LP is defined as the rigidness of mouse legs that are resist movement. The PBS and vaccination groups are indicated as symbols. *: <i>p</i> < 0.05, **：<i>p</i> < 0.01, and ***: <i>p</i> < 0.001.</p

List of the sequence of primer pairs specific to target genes.

<p>F:Forward primer; R:Reverse primer</p><p>List of the sequence of primer pairs specific to target genes.</p

The purification and characterization of the EV71 virus.

<p>(A) Purification of EV71 by sucrose density gradient using zonal ultracentrifugation and fractionation analysis by sucrose percentage (Sucrose), contents of total protein (TP) and Vero cell-derived protein (HCP), and EV71-specific units (ELISA). (B) Definition of viral particles distributed in fractions using SDS-PAGE. (C) Electromicrographs of EV71 particles by transmission electron microscopy. (a) Fraction 10 presented a full-particle (F-particle) with infectious capability and a solid particle structure; (b) fraction 13 presented a non-infectious particle (E-particle) with defective particle structure; and (c) pooled viral particles for the vaccine bulk preparation. (D) Quality confirmation of EV71 pooled viral particles before and after formalin inactivation using SDS-PAGE and western blot analysis with a specific monoclonal antibody (Mab979). The molecular weight of VP0, 1, 2, and 3 are as indicated. The black triangle indicates the quantities of loading antigen from high to low.</p

Expression of pro-inflammatory cytokines/chemokines in the CNS compartments and muscle tissue of EV71 (C4)-infected mice.

<p>The vaccinated Tg mice were subcutaneously infected with 1×10⁶ pfu of the EV71 3340 (C4) strain, and the RNAs extracted from the brain stem, spinal cord, and muscle at 6 days post-infection were subjected to quantitative RT-PCR analysis. Mock (M): uninfected mice group; Virus (V): EV71 3340 (C4) infected mice group. A schematic representation of the target gene expression and the statistical average from 5 mice per group is shown. The used vaccine formulations are as labeled. Significant difference between each group was shown as *: <i>p</i> ﹤ 0.05, **: <i>p</i> ﹤ 0.01, and ***: <i>p</i> ﹤ 0.001.</p

The muscle pathological changes in Tg mice challenged with EV71 viruses.

<p>The hematoxylin and eosin (H&E) staining of muscle tissues collected from PBS- or vaccine-treated mice without EV71 challenge (A-D) as control group. H&E staining of muscle tissues collected from PBS- or vaccine-treated mice at 6 days post-infection with the EV71 C2 (E-H) or C4 (I-K) strains. The vaccine formulations and viruses are labeled. All pictures were taken at 200X magnification.</p