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. 2011;6(11):e28127.
doi: 10.1371/journal.pone.0028127. Epub 2011 Nov 21.

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus

Naomi J Logsdon  1 , Meghan K Eberhardt, Christopher E Allen, Peter A Barry, Mark R Walter
Affiliations

Design and analysis of rhesus cytomegalovirus IL-10 mutants as a model for novel vaccines against human cytomegalovirus

Naomi J Logsdon et al. PLoS One. 2011.

Abstract

Background: Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.

Methods and findings: Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.

Conclusion: This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV's ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Purification and quaternary structure of RhCMVIL-10.
GF Chromatographs of cIL-10 (grey) and RhCMVIL-10 (black) plotted with X-axis in mL and Y-axis in optical density (OD) at 280 nm. (Inset) SDS-PAGE gel of affinity purified RhCMVIL-10 (lane 1) and pooled fractions (black bar) of the major RhCMVIL-10 GF peak (lane 2).
Figure 2
Figure 2. Sequence and structure model of RhCMVIL-10 binding residues.
(A) Sequence alignment of cellular and viral human and rhesus IL-10s. The predicted site for N-linked glycosylation in RhCMVIL-10 is underlined. GenBank Accession numbers: RhCMVIL-10, AAF59907; cIL-10, AAA63207; RhIL-10, AAA99975; CMVIL-10, AAF63437. CIL-10 helices are denoted on the alignment (A–F [28]), which also highlights residues chosen for mutagenesis. (B) Structure model of the RhCMVIL-10/RhIL-101R1 interface based on the crystal structure of the HuIL10/HuIL-10R1 complex (pdbid 1Y6K, [23] ). Residues chosen for mutagenesis that disrupt IL-10R1 binding are shown in red, while the Arg-34Glu mutant (corresponding to Lys-34 in CMVIL-10) that exhibits essentially WT activity is colored green (see Fig. 4A).
Figure 3
Figure 3. Expression and IL-10R1 binding of RhCMVIL-10 point mutants.
Expression of RhCMVIL-10 point mutants in Drosophila cell media was characterized by western-blotting (WB). Cell supernatants containing the point mutants were incubated with HuIL-10R1 coupled beads. After washing, the beads were loaded onto a 12% SDS-PAGE gel and subsequently stained with coomassie blue.
Figure 4
Figure 4. Biological activity of RhCMVIL-10 mutants M1 and M2 on TF1/IL-10R1 cells.
(A) Cell supernatants containing RhCMVIL-10 single point mutants (described in the Figure legend) (Lys-34Glu, Gln-38Arg, and Asp-144His) were evaluated for their ability to stimulate proliferation of TF-1/IL-10R1 cells in relation to cIL-10 and WT RhCMVIL-10. (B) TF-1/IL-10R1 cell proliferation assay for RhCMVIL-10 M1 (Q38R, D144H) and M2 (E142Q, D144H) cell supernatants. Error bars represent estimated standard error from duplicate measurements.
Figure 5
Figure 5. Ability of RhCMVIL-10 M1 and M2 to suppress IL-12 levels in LPS-activated rhesus PBMC.
IL-12 levels produced by LPS-activated rhesus PBMCs were measured by ELISA in the presence, or absence, of purified RhCMVIL-10 M1, RhCMVIL-10 M2, or RhCMVIL-10 WT over a concentration range of 0.1–1,000 ng/ml. Also shown are results for cells incubated with media alone and with LPS alone. Assays were performed using PBMC from three rhesus macaques (Animal 1–3).
Figure 6
Figure 6. SPR analysis of RhCMVIL-10 wild-type, M1, and M2 binding to IL-10R1.
Panels A, B, and C, show experimental sensorgrams (colored) and bivalent model fits (black lines) for RhCMVIL-10WT, M1, and M2, respectively. Note the Y axis in panels B and C only extends to +/- 5RU compared to −5 to 25RU in panel A. The maximum RU observed for RhCMVIL-10 M1 and M2 is below that of RhCMVIL-10WT observed at 3.125 nM; the lowest concentration tested.
Figure 7
Figure 7. Stimulation of RhCMVIL-10 binding and neutralizing antibodies (Ab) following immunization of RhCMV-infected rhesus macaques.
(A) RhCMVIL-10-binding antibodies were analyzed throughout the immunization schedule (Table 2) by ELISA measured at absorbance at 450 nm (see Methods for details). (B) Neutralization of RhCMVIL-10 WT biological activity by plasma collected throughout the immunization schedule (Table 2). Percent (%) neutralization of RhCMVIL-10 denotes the ratio ([ IL-12 ] Plasma+RhCMVIL-10 / [ IL-12 ]Plasma )*100, such that 100% corresponds to complete inhibition of RhCMVIL-10 and 0% is no inhibition. Values greater than 100 reflect errors/variations in the measured levels of IL-12 in the two samples. The times of DNA (cyan) and protein (red) vaccination are shown on the Figure with arrows. Three animals (A1–A3) were immunized with M1 (solid lines), and three animals (A4–A6) were immunized with M2 (dashed lines). The times of blood draws are noted by the solid symbols for M1 immunized animals and open symbols for M2 immunized animals. Where immunization and blood draws were performed on the same day, blood was taken prior to immunization. The shape of the symbols denotes different animals, with M1 immunized animals A1–A3 solid diamond, square, and circle, respectively. M2 immunized animals A4–A6 are denoted by open square, diamond, and circle, respectively. The same designations are used for panels A and B.
Figure 8
Figure 8. RhCMVIL-10-NAbs generated against RhCMVIL-10 M1 and M2 do not cross react with cellular RhIL-10.
Plasma from animals (A1–A6) pre- (pre-Imm, week 0, Figure 7), and post-immunization (week 19 plasma sample, 1 week after the second protein boost, Figure 7), was tested for its ability to neutralize cellular RhIL-10 (grey bars) or RhCMVIL-10 (black bars) bioactivity, where bioactivity was measured as suppression of LPS-induced IL-12 production in rhesus PBMC. The data show the biological activity of RhIL-10 is not neutralized by plasma from animals immunized with RhCMVIL-10 M1 or M2. However, RhCMVIL-10 biological activity, except for A6, is almost completely neutralized by the same plasma sample. Percent (%) neutralization is calculated as described in Figure 7 and in the “Materials and methods” section.
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