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. 2014 Apr;141(4):531-9.
doi: 10.1111/imm.12210.

Virus-encoded ectopic CD74 enhances poxvirus vaccine efficacy

Crystal C Walline  1 , Sarah N Deffit, Nan Wang, Lynette M Guindon, Victoria L Crotzer, Jianyun Liu, Kristin Hollister, Laurence C Eisenlohr, Randy R Brutkiewicz, Mark H Kaplan, Janice S Blum
Affiliations

Virus-encoded ectopic CD74 enhances poxvirus vaccine efficacy

Crystal C Walline et al. Immunology. 2014 Apr.

Abstract

Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast, virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4⁺ T-cell responses to viral and tumour antigens.

Keywords: CD74; MHC; class II; invariant chain; virus.

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Figures

Figure 1
Figure 1
Ectopic expression of CD74 provided partial protection for MHC class II (MHCII)-mediated but not CD1d-mediated antigen presentation during infection with vaccinia virus (VV). (a) M1DR4 and M1DR4CD74 cells were cultured for 6 hr with glutamic acid decarboxylase (GAD) peptide in the presence of VV [multiplicity of infection (MOI) 5 or 10] or mock-treated (Cont.). Cells were fixed (0·5% paraformaldehyde) and peptide presentation to GAD-specific CD4+ T cells was monitored for each antigen-presenting cell (APC). T-cell responses were monitored using a bioassay to detect interleukin-2 production. T-cell responses to Cont. (uninfected) cells were normalized to 1 to permit relative comparisons with VV-infected APC in panels (a) and (e). (b) Expression levels of CD74, viral H3L and actin were measured by immunoblot analysis of APC lysates. (c) HeLa cells expressing CD1d (HCD1d) and those with ectopic CD74 (HCD1dCD74) were infected with VV (MOI 10) or mock treated (Cont.) overnight. Cells were then cultured with CD1d-restricted natural killer T (NKT) cells and granulocyte–macrophage colony-stimulating factor (GM-CSF) secretion was measured. (d) Similar levels of VV infection were detected in HCD1d cells with or without ectopic CD74 by flow cytometric staining using an antibody specific for the viral E3L antigen or an isotype-matched control antibody. Cell staining after mock treatment without virus (Cont.) confirms the virus-specificity of the E3L antibody. (e) T2DR4 and T2DR4DM cells were incubated with GAD peptide and VV (MOI 10) or mock-treated (Cont.) for 6 hr. T-cell interleukin-2 production in response to these APC was monitored. (f) Virus H3L antigen and cellular actin expression were measured by immunoblotting of T2 cell lysates. Studies are representative of two or three independent experiments, mean ± SD. (a) One-way analysis of variance, Dunnett's multiple comparisons tests compared virus-infected cells (MOI 5 and 10) with the Cont. cells. ++ P < 0·01, Two-way analysis of variance, Sidak's multiple comparisons tests comparing M1DR4CD74 cells with M1DR4 at virus (MOI 5). (c, e) Unpaired Student's t-test compared analysis of virus-treated cells with Cont. cells.
Figure 2
Figure 2
Virus-encoded murine CD74 enhanced intracellular antigen presentation. (a) PriessGAD cells were cultured with vaccinia virus VV, mCD74-VV [multiplicity of infection (MOI) 10] or without virus (Cont.). Cells were harvested at 6, 14 and 24 hr and fixed before co-culture with glutamic acid decarboxylase (GAD)-specific T cells. T-cell activation was monitored by a bioassay to detect interleukin-2 production relative to untreated cells (Cont.). Aliquots of cells infected with mCD74-VV (MOI 10) were also harvested and lysed for immunoblot analysis to detect virus encoded mCD74, actin and viral antigens (VV) (inset). (b) PriessGAD cells were treated as in (a) and cell lysates were harvested 24 hr post infection and immunoblotted to detect human CD74 (hCD74) and actin. Densitometry was used to quantify cellular hCD74 protein levels relative to actin as a loading control. (a) Studies are representative of three independent experiments, mean ± SD. Two-way analysis of variance, Bonferroni's multiple comparisons test to compare VV-treated cells with mCD74-VV treated cells. (b) Study depicts the average of three independent experiments, ± SD. One-way analysis of variance, Dunnett's multiple comparisons test.
Figure 3
Figure 3
Reactivation of virus specific CD4+ T cells was enhanced upon antigen-presenting cell (APC) exposure to vaccinia virus (VV) encoding murine CD74 (mCD74-VV). (a) Splenic APC were cultured in vitro with VV [multiplicity of infection (MOI) 0·1], mCD74-VV (MOI 0·1), or without virus (Cont.) for 14 hr, followed by incubation with VV-primed CD4+ T cells. T-cell proliferation in response to virus-treated APC was monitored. T-cell responses to VV-infected APC were set equal to 1 for comparison to uninfected (Cont.) or mCD74-VV-infected splenic APC. (b) Flow cytometric analysis of splenic APC revealed a similar pattern and level of virus-infected B cells, dendritic cells (DC), and macrophages after in vitro infection with VV or mCD74-VV (MOI 1) at 24 hr (VV was not detectable at an MOI of 0·1). Virus-infected cells were detected using a polyclonal anti-VV antibody. Studies are representative of three independent experiments, mean ± SD. One-way analysis of variance, Dunnett's multiple comparisons test.
Figure 4
Figure 4
Protection against sub-lethal vaccinia virus (VV) challenge was enhanced by previous immunization with VV expressing mCD74. (a) C57BL/6 mice were injected 14 days in advance with PBS (Cont.), a recombinant control VV (rVV) or mCD74-VV [1000 plaque-forming units intraperitoneally (PFU, i.p.)]. At 14 days post-immunization (dpi), mice were challenged with VV (106 PFU, intranasally) and animal weight was monitored as a sign of animal health. (b) VV titres were determined using homogenized lung at 24 dpi. VV titres were compared per mg of lung tissue. The dotted line shows the threshold of detection for the viral plaque assay. (c) Mice were bled after i.p. immunization (0, 7, and 14 dpi) or 4 and 8 days after VV challenge (18 and 22 dpi), and plasma virus-specific antibody was monitored by ELISA. There were five to eight mice per group. Studies are representative of three independent experiments, mean ± SD. (a, b) Two-way analysis of variance, Bonferroni's multiple comparisons test rVV compared with mCD74-VV. (c) Unpaired Student's t-test.
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