Summary of immunogenicity of V5040 CHIKV vaccine in BALB/c and C57BL/6 mice.

Summary of immunogenicity of V5040 CHIKV vaccine in BALB/c and C57BL/6 mice.</p

Primers used for preparation of CHIKV 181/25 clone expressing nLuc (CHIKV 181/25-nLuc) and for sequencing PCR fragment from cDNA of V5040 virus.

The nLuc gene was inserted within the nsP3 gene by Gibson cloning method at 5217 bp site as described in Materials and Methods.</p

Preparation of V5040 CHIKV in Vero cells.

(a) Plaque morphology of attenuated V5040 and 181/25 vaccine viruses. (b). Comparison of the growth kinetics of V5040 in Vero cell infected with V5040 (102 PFU, blue diamonds; 103 PFU, blue circles) or transfected with pMG5040 (100 ng, grey triangles). As control, CHIKV 181/25 vaccine virus was used (103 PFU, red squares).</p

Neutralization antibodies from C57BL/6 mice vaccinated with V5040 or CHIKV 181/25.

Antisera collected from terminal bleeding of mice vaccinated either with V5040 or CHIKV 181/25 were tested for PRNT50 assays against their corresponding homologous virus. Blue circles and red squares indicate antisera of CHIKV 181/25 and V5040, and each symbol represents serum from an individual mouse (n = 8/group/timepoint). The bar and whiskers indicate the means and ± their standard deviation. Ns: not significant. **, P < 0.0021, Two-Way ANOVA with Sidak’s multiple comparisons test (Prism).</p

Genetic maps depicting the pMG5040 plasmid encoding the full-length rearranged RNA.

(a) Indicated are approximate locations of cytomegalovirus (CMV) promoter (solid arrow), 26S promoter (open arrows) and attenuating mutations (asterisks). The wild type CHIKV genome is shown in red. CHIKV pMG5040 plasmid encoded rearranged full-length infectious CHIKV RNA under transcriptional control of the CMV promoter. The plasmid pMG5040 contained both attenuating mutations (asterisks) from the IND vaccine 181/25. (b) 1% Agarose/TAE gel showing pMG5040 plasmid as compared to control VEEV vaccine. M, 1kB Plus DNA ladder (Thermo); 1, VEE control pMG4020: 2, CHIKV pMG5040.</p

Representative bioluminescence images from each group are shown (upper and lower panels).

C57/BL6 were vaccinated either with PBS (sham), CHIKV 181/25, or V5040 via the s.c. route at the both flanks with 2x105 pfu). PBS and vaccine groups (CHIKV 181/25 and V5040) are indicated on the top of the Figure. Three weeks later animals were infected with 5x105 pfu of CHIKV181/25-nLuc at the right footpad via the s.c. route. Two days later animals were subjected with bioluminescence imaging with 9 μmole/kg of FFz (i.p. injection) as described in Materials and Methods. Four mice on the left were sham-vaccinated (PBS group) and showed strong bioluminescence signal at the footpad (arrows) after infection with CHIKV181/25-nLuc virus indicating replication of the challenge virus. In contrast, four mice vaccinated with CHIKV 181/25 vaccine (center panels) or V5040 vaccine (four mice on the right panels) did not show bioluminescence signal suggesting protective effect of both CHIKV 181/25 and V5040 vaccines against infection with CHIKV181/25-nLuc virus.</p

Immunogenicity of V5040 virus in mice.

(a) western blot and (b) IFA with antisera from BALB/c mice vaccinated with V5040 virus. (a) For western blot, mouse serum was probed at 1:100 dilution followed by AP-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:1000 dilution). Lane 1, SeeBlue Plus2 standard, Lane 2, control Vero cell lysate; Lane 3, blank; Lane 4, lysate of Vero cells infected with CHIKV 181/25 virus. Predicted band of E2 is indicated. (b) For IFA, Vero cells seeded in chamber slides were infected with V5040 at MOI 0.01. At 24 h post-infection, Infected monolayer cells were fixed with acetone and probed with mouse serum diluted 1:25, followed by FITC-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:25 dilution). Infected Vero monolayers developed foci of V5040-infected cells shown in green (indicated with arrows). Nuclei are stained in red using VectaShield mounting medium containing propidium iodide.</p

Anti-CHIKV neutralization activity of antisera immunized with CHIKV 181/25 and V5040 vaccine.

Diluted serum was mixed with the same volume of diluted CHIKV-nLuc virus (600 pfu) for 1h, then used to infect Vero 76 cells in a 96-well cell culture plate for two days. The nLuc activity from the virus replication was measured by using Nano-Glo Luciferase Assay System (Promega) following the manufacturer’s protocol using a plate reader (HT4 Biotek). Each data points represents a group of 8 mice (4 male and 4 female), with p = 0.0152 between 181/25 and V5040 groups. The statistical analysis was performed with GraphPad Prism using two-way ANOVA.</p

Transfusion of blood during reperfusion impacted HCV kinetics.

<p>Increasing volumes of blood transfused during the reperfusion period increased k (<b>A</b>: r = 0.53, p = 0.05) and shortened half-life (<b>B</b>: r = 0.59, p = 0.03) (n = 14).</p

Allografts rapidly remove HCV from patient circulation.

<p><b>(A)</b> and <b>(C)</b>: Each line represents an individual patient. The dotted (patient 1) and dashed (patient 5) lines represent the patients with the slowest rates of viral clearance. <b>(B)</b> and <b>(D)</b>: Line represents the average of all patients with error bars representing standard error. <b>(A)</b> and <b>(B)</b>: Absolute values for HCV are presented. <b>(C)</b> and <b>(D)</b>: HCV values relative to starting amount are presented. During reperfusion, the rate of viral clearance varied more than 20-fold with a median rate constant of 0.0357 1/minute (range 0.0089–0.2169 1/minute) and median half-life of 19.4 minutes (range 3.2–77.8 minutes).</p