A Small Antigenic Determinant of the Chikungunya Virus E2 Protein Is Sufficient to Induce Neutralizing Antibodies which Are Partially Protective in Mice

<div><p>Background</p><p>The mosquito-borne Chikungunya virus (CHIKV) causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses.</p><p>Methodology/Principal Findings</p><p>E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L) and surface-exposed parts of the E2 domain A (sA) alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+) was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+) induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA), MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice.</p><p>Conclusions/Significance</p><p>The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.</p></div

CHIKV neutralizing activity of mouse sera.

<p>A: Schematic representation of the immunization schedule. Immunizations are indicated by an arrow and blood sampling by a red line. B: Neutralization assay with mouse sera. Serum of immunized mice was serially diluted, incubated with CHIKV Env or VSV-G pseudotyped vector particles and analyzed for its neutralizing activity by detection of relative luciferase activities. The AUC of the luciferase activity obtained with preimmune serum was divided by the values obtained with serum after the last immunization. The data represent the mean values of three mice and * and ** indicate statistical significance. Statistical analyses were done using the GraphPad Prism 5.04 software and the p-values were determined by the paired two-tailed t-test.</p

Summary of the recombinant genes and the proteins purified from <i>E</i>.<i>coli</i>.

<p>A: Schematic representation of the combinations of E2 fragments. Red- main early neutralizing epitope; yellow- surface exposed domains of A; red/white—one peptide of L; orange- domain B with ß-ribbon connector. B: Overview of the recombinant proteins purified by Ni²⁺ affinity chromatography from <i>E</i>.<i>coli</i>. Proteins were separated on a 15% SDS-PAGE and stained with Coomassie. The prominent faster migrating bands of protein LB⁺ were identified by mass spectrometry as degradation products of LB⁺ (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003684#pntd.0003684.s003" target="_blank">S3 Fig</a>). Lanes 1–5 from the left were loaded with the indicated amounts of BSA and used to estimate the protein amount of the recombinant proteins.</p

Challenge infection of immunized mice.

<p>Mice (n = 5 per CHIKV antigen immunized group, n = 4 for the control groups) were immunized with the indicated antigens and infected two weeks after the final immunization with 1x10⁶ IU CHIKV. Two and four days after infection, the CHIKV titer was determined as RNA copies/μl serum by RT-PCR. At the end of the experiments, spleen, lung and brain were isolated and the CHIKV titer was determined as RNA copies/mg organ by RT-PCR. Lane 6 shows data from mice that were infected seven weeks before and also subjected to a challenge infection (n = 5). * indicates statistical significance. Statistical analyses were done using the GraphPad Prism 5.04 software and the p-values were determined by the unpaired two-tailed t-test was performed. * P ≤ 0.05; ** P ≤ 0.01. Indicated on the x-axis are the recombinant proteins or vaccinia viruses used for immunization.</p

Characterization of MVA-CHIKV-sAB⁺.

<p>A: Expression of the recombinant sAB⁺ protein. HEK 293T cells were infected with MVA wt or MVA-CHIKV-sAB⁺ at a MOI of 5 and the cells were harvested at the indicated time points. The cell lysates were analyzed by Western blot with an antibody directed against the myc-tag. Equal loading was controlled by detection of ß-actin (lower panel). B: sAB⁺ is secreted from cells. HEK 293T cells were infected at a MOI of 0.1 and harvested after 72 hours. Cell lysates and supernatants were analyzed by Western blot with an antibody directed against the myc-tag. Transiently transfected/MVA infected cells served as a positive control.</p

Strategisches Krisenmanagement, Ökonomische Krisenresistenz durch risikobasiertes Kapitalkostenmanagement (Teil B)

<p><b>Binding of recombinant CHIKV E2 protein domains A, B, and C, and E2ex to 293T, CHO-K1, and pgsA-745 cells in the presence of soluble glycosaminoglycans (GAGs).</b> 10 μg of the indicated recombinant proteins were incubated with the indicated soluble GAGs (500 μg/ml) for 30 minutes at 4°C. 293T (top), CHO-K1 (middle), and pgsA-745 (bottom) cells were then incubated with this mixture. Binding was measured by flow cytometry using an anti-His-tag and an anti-mouse FITC conjugated antibody. The results are shown as relative values to the control (incubation of cells with the respective recombinant protein alone). A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate inhibition of binding. Data represent the average of three independent experiments. * and ** indicate significant differences to the GAG-free controls. n.s. means not significant. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001) and **** (P ≤ 0.0001).</p

Binding of Fc-fusion proteins containing variants of the CHIKV E2 domain A to CHO-K1 and pgsA-745 cells.

<p>A: Fc-E2 domain A-fusion proteins and Fc protein were expressed from HEK293T cells, affinity purified by protein-A chromatography and separated by SDS-PAGE. The Western blot was detected with an HRP-labeled anti-human IgG antibody. The calculated molecular weights are: Fc-CHIKV-E2-A, E79K and E166K 53 kDa; Fc-CHIKV-E2-A-ß 43,4 kDa. B: Separation of the Fc-fusion proteins under native conditions. The Western blot was detected with an HRP-labeled anti-human IgG antibody. C: CHO-K1 (black) and pgsA-745 (grey) cells were incubated with the indicated recombinant Fc-fusion proteins. Binding was measured by flow cytometry using an anti-human IgG FITC conjugated antibody. The results are shown as fold induction compared to Fc binding. A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate binding. Data represent an average experiment of three independent experiments performed.</p

Identification of Functional Determinants in the Chikungunya Virus E2 Protein - Fig 6

<p><b>Binding of recombinant CHIKV E2 protein domains A, B, and C, and E2ex to 293T, CHO-K1, and pgsA-745 cells in the presence of soluble glycosaminoglycans (GAGs).</b> 10 μg of the indicated recombinant proteins were incubated with the indicated soluble GAGs (500 μg/ml) for 30 minutes at 4°C. 293T (top), CHO-K1 (middle), and pgsA-745 (bottom) cells were then incubated with this mixture. Binding was measured by flow cytometry using an anti-His-tag and an anti-mouse FITC conjugated antibody. The results are shown as relative values to the control (incubation of cells with the respective recombinant protein alone). A value of one represents the mean FITC value of the control and is labeled by the dashed line. Values above this line indicate inhibition of binding. Data represent the average of three independent experiments. * and ** indicate significant differences to the GAG-free controls. n.s. means not significant. * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001) and **** (P ≤ 0.0001).</p

Infection and cell entry by CHIKV-mCherry-490 in the presence of soluble GAGs.

<p>A: 293T cells were seeded in 24-well plates and 500 μg/ml of the indicated GAG was added. Afterwards, cells were infected with CHIKV-mCherry-490 using an MOI of 1. The viral replication was determined 6 hrs post-infection by flow cytometry detecting mCherry. B: 500 μg/ml of the indicated GAG and CHIKV-mCherry-490 (MOI 1) were incubated together at 4°C for 30 minutes. After addition to 293T cells, another incubation of 30 minutes at 4°C followed. Then, the unbound virus together with the respective GAG were washed away and fresh medium was added to the cells. The viral replication was determined 6 hrs post-infection by flow cytometry detecting mCherry. Data represent the average of three independent experiments. *** and **** indicate significant differences in infection rates to untreated control cells. ** (P ≤ 0.01) and *** (P ≤ 0.001).</p

Transduction or infection of cells with CHIKV Env pseudotyped vector particles.

<p>A: 293T, CHO-K1, and pgsA-745 cells were seeded in 24-well plates and transduced with <i>gfp</i>-encoding CHIKV Env-pseudotyped lentiviral vector particles. The cells were analyzed by flow cytometry for GFP expression 48 hrs post transduction. The proportion of GFP-positive cells is given relative to the proportion of positive cells following transduction with VSV-G-pseudotyped vectors (control). Data represent the average of three independent experiments. *** (P ≤ 0.001) indicates significant differences in transduction rates compared to CHO-K1 cells. B: CHO-K1 and pgsA-745 cells were seeded in 24-well plates and infected with CHIKV-mCherry at an MOI of 1. The viral replication was determined 6 and 24 hrs post-infection, respectively, by flow cytometry detecting mCherry. Data represent the average of three independent experiments. ** and *** indicate significant differences in infection rates to CHO-K1 cells after 6 and 24 hrs, respectively. * (P ≤ 0.05) and ** (P ≤ 0.01).</p