MHC class I genotypes of the chimpanzees.

A<p>Vac3 is likely to be homozygous A*0901.</p

Number of NS3-peptides that induce IFNγ production by CD4 T-cells or IL-2/IFNγ dual cytokine production by CD8 T-cells.

<p>PRE, pre challenge. POST, post challenge.</p

Cytolytic killing of CFSE-loaded target cells.

<p>(A) Specific lysis of NS3_1258–1272-pulsed Patr-A*03∶01 target cells (red peak) in comparison with unpulsed Patr-A*03∶01 target cells (yellow peak) by NS3-peptide-pool expanded PBMC from Vac1 isolated 10 weeks after HCV 1bJ4 challenge (blue peak). (B) No specific lysis of NS3_1258–1272-pulsed Patr-B*02∶01 target cells. (C) and (D) Control experiments showing no killing of unpulsed Patr-A*03∶01 or Patr-B*02∶01 target cells. (E,F,G) Percentage of lysis for all MHC class-I-peptide-combinations tested in Vac1, Vac2, Vac3 respectively.</p

Dose-inhibition curves for selected NS3-peptides to Patr-A*03∶01.

<p>Binding affinity of selected NS3-peptides to Patr-A*03∶01, established by peptide binding competition assay. Patr-A*0301 cells were incubated with increasing concentrations of NS3 p59 (top left), LGFGAYMSK (LGF, top right), AATLGFGAY (AAT, bottom left), or KGGLRPRAG (control, bottom right) in the presence of biotin-labeled indicator peptide. Binding of indicator peptide was quantified by incubation with europium labeled streptavidin. Indicated is reduction in percentage of europium positive cells relative to cells incubated with indicator-peptide only. The indicated IC₅₀ values (µM) are derived from the regression curves of three independent experiments in the case of p59, LGF and AAT, and of two individual experiments in the case of KGG. The IC₅₀ values were estimated using non-linear least-squares regression with the “R” platform for statistical computing.</p

Evaluation of peptide-specific IL-2, IFNγ and TNFα expression in CD4 and CD8 T-cells.

<p>A representative example of the analysis performed in Vac1 (left panel) and Vac2 (right panel), two weeks after the final vaccine boost, is shown. PBMC were first expanded for a 12 day period using a pool of all NS3 peptides, restimulated with individual peptides (medium alone, p59 or p64) and analyzed for induction of IFNγ, IL-2 and TNFα cytokine expression by expanded CD4 and expanded CD8 cells, using the gating strategy described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095103#pone.0095103.s001" target="_blank">Figure S1</a>. The numbers in the quadrants, represent the percentage of positive cells calculated from the parent-population.</p

Heat map of NS3-peptide-specific T-cell responses in four immunized chimpanzees at different study time points.

<p>(A) Percentage of expanded CD4 T-cells producing IFNγ upon restimulation with individual peptides, subdivided into three categories; i.e. low response (light green; 0.001%–0.1% specific IFNγ production), intermediate (green; 0.1–1% specific response), high (dark green;>1% specific response). (B) Percentage of expanded CD8 T-cells showing IL-2/IFNγ double cytokine production upon restimulation with individual peptide, subdivided into three categories; i.e. low response (light blue; 0.01%–0.1% specific IFNγproduction), intermediate (blue; 0.1–1% specific response), high (dark blue; > 1% specific response). White areas indicate where no responses were detected. The red boxes highlight areas within NS3 that are broadly recognized. Per animal 6 time points of analysis are shown lined up beneath each other; -12) Two weeks following the 1st MVA boost, cells stimulated with NS3_vaccine, -4) Four weeks following 2^nd MVA boost, cells stimulated with NS3_vaccine, 0) Day of HCV infection, stimulation with NS3_vaccine, 0) Day of HCV infection, stimulation with NS3_challenge, and 4 and 36 weeks after challenge, stimulation with NS3_challenge. The individual peptides tested are indicated at the top of the map by the numbers 1 to 156. The red boxes represent broadly recognized regions within NS3. Total frequency (all peptide responses combined) of (C) IFNγ production by expanded CD4 T-cells and (D) IFNγ/IL2 dual cytokine production by expanded CD8 T-cells per animal. (E) Frequency of p59 specific triple positive IL2/TNFα/IFNγ producing CD8 T-cells. The numbers on the X-axis represent the study week, relative to time of challenge. * Despite several attempts, frozen cells from Vac1 from 4 weeks following HCV infection did not respond to either NS3_vaccine or NS3_challenge peptide stimulation and no expansion could be achieved. @ Due to a high IFNγ background, peptide specific CD8 responses could not be detected</p

Antigen sensitivity of p59 specific cytokine induction.

<p>NS3-expanded T-cells from Vac1, Vac2 and Vac3, from 4 weeks prior to HCV challenge, were restimulated with a concentration range of p59 and evaluated for induction of IL-2/IFNγ expressing CD8 T-cells (left graph) and IFNγ expressing CD4 T-cells (right graph).</p

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets

<div><p>The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.</p></div

Characteristics of chimpanzee population.

<p>Characteristics of the chimpanzee population studied, the HCV-genotype, the time since HCV exposure, HCV-RNA load, serum IP-10levels, and the liver enzymes levels ALT, AST and γGT. The average values and the range are shown.</p

Relation between IP-10 levels and aminotransferases in serum.

<p>Relation between IP-10 and liver enzymes γGT, ALT and AST from HCV-infected individual chimpanzees (A) and HCV-infected patients (B) A significant correlation between the two parameters is defined as r²>0.85 and p<0.05 where “r²” is a measure for correlation and “p” is a measure for the quality if this correlation.</p