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. 2013 Oct 8;110(41):16550-5.
doi: 10.1073/pnas.1310215110. Epub 2013 Sep 19.

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ

Tihana Trsan  1 , Andreas Busche, Maja Abram, Felix M Wensveen, Niels A Lemmermann, Maja Arapovic, Marina Babic, Adriana Tomic, Mijo Golemac, Melanie M Brinkmann, Wiebke Jäger, Annette Oxenius, Bojan Polic, Astrid Krmpotic, Martin Messerle, Stipan Jonjic
Affiliations

Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ

Tihana Trsan et al. Proc Natl Acad Sci U S A. .

Abstract

Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell-based vaccines.

Keywords: CD8 T cell vaccine; RAE-1 gamma; vaccine vector.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression of RAE-1γ by an MCMV vector improves the specific CD8 T-cell response to a vectored antigen. (A) RAE-1γMCMVList and RAE-1γMCMV-SIINFEKL were constructed on the RAE-1γMCMV backbone by replacing the Dd-restricted antigenic m164 peptide 167AGPPRYSRI175 with either the Kd-restricted listeriolysin O (LLO)-derived peptide 91GYKDGNEYI99 or the H-2Kb–restricted ovalbumin-derived peptide SIINFEKL. (B) BALB/c mice were infected i.v. with 2 × 105 pfu per mouse of the indicated viruses, and viral titers were determined by plaque assay. DL, detection limit. (C) Splenocytes of MCMVList- or RAE-1γMCMVList–infected BALB/c mice (105 pfu per mouse, via f.p.) were stained for LLO tetramer-specific CD8 T cells. (D) C57BL/6 mice were f.p. infected with 105 pfu per mouse of MCMV-SIINFEKL or RAE-1γMCMV-SIINFEKL. At indicated time points, splenocytes were isolated, stimulated with SIINFEKL-peptide, and stained for the intracellular IFN-γ production. For panels B–D, individual animals (circles or triangles) and median values are shown.
Fig. 2.
Fig. 2.
RAE-1γMCMV expressing antigenic peptides protects mice against L. monocytogenes. (A) Mice were immunized via f.p. with 105 pfu per mouse of WT MCMV or MCMV vector with or without RAE-1γ or were left nonimmunized. At 3 wk p.i. or later, mice were challenged with L. monocytogenes. On day 4 postchallenge, mice were sacrified and analyzed for an immune response and protective capacity to L. monocytogenes. (B) BALB/c mice were challenged 4 wk postimmunization with 2 × 104 cfu per mouse of L. monocytogenes. Bacterial load was determined in spleen and liver. Individual animals (circles) and median values are shown. † indicates the death of a mouse. (C) Three weeks postimmunization, BALB/c mice were challenged with 104 cfu per mouse of L. monocytogenes. Paraffin-embedded spleen sections were stained for CD3ε expression. * indicates central artery. PALS, periarteriolar lymphoid sheath. (Magnification: 20×.) (D) Splenocytes were isolated and either stained with LLO-tetramers or stimulated with LLO-peptides and intracellularly stained for cytokine production (mean ± SEM, n = 4–5). (E) Three weeks postvaccination, C57BL/6 mice were challenged with 5 × 104 cfu per mouse of OVA-Listeria. Individual animals and median values are shown.
Fig. 3.
Fig. 3.
Long-term protection against L. monocytogenes challenge in RAE-1γMCMVList–immunized mice. BALB/c mice were f.p. immunized with 105 pfu per mouse of indicated viruses. (A) Three weeks postimmunization, mice were challenged with 104 cfu per mouse of L. monocytogenes. One day before the challenge, mice were depleted of CD8 T cells or left undepleted. Four days later, the bacterial loads were determined (mean ± SEM, n = 4–5). (B) Challenge with 2 × 104 cfu per mouse of L. monocytogenes was performed 60 d postimmunization. Survival and body weight loss (mean ± SEM) on day 3 (n = 9–12) is shown. (C) Bacterial load of survived mice on day 4 postchallenge in spleen is shown (individual animals and median values, n = 9; † indicates the death of a mouse). (D) Challenge with 4 × 104 cfu per mouse of L. monocytogenes was performed 60 d postimmunization, and the survival rate was followed (n = 9). (E) Mice were injected with an equal ratio of unstimulated and LLO-peptide–stimulated CSFE-stained splenocytes 2 and 11 mo postimmunization. Percentage of listeriolysin peptide-specific killing is shown (mean ± SEM, n = 3–4).
Fig. 4.
Fig. 4.
NKG2D-independent attenuation and immune function of RAE-1γ. (A) BALB/c mice were infected i.v. with 2 × 105 pfu per mouse of the indicated viruses. One day before infection and on days 2 and 5 p.i., mice were treated with αNKG2D antibody. Virus titer was determined on day 8 p.i. (B) Mice were infected with 105 pfu per mouse via f.p. and treated with αNKG2D as in A. LLO-specific CD8 T-cell response was determined on day 8 p.i. (n = 5). (C) C57BL/6 mice were infected with 2 × 105 pfu per mouse i.v. and treated with αNKG2D. The frequency of SIINFEKL-specific CD8 T cells was determined on day 8 p.i. (n = 3–4). (D) C57BL/6 and NKG2D−/− mice were infected i.v. with 2 × 105 pfu per mouse. On day 7 p.i., virus titer is shown. (E) The frequency of SIINFEKL-specific CD8 T cells was determined on day 7 p.i. (n = 3–4). (F) Virus titer in organs was determined on day 7 p.i. For A, D, and F, individual animals and median values are shown. For B, C, and E, mean ± SEM is shown.
Fig. 5.
Fig. 5.
RAE-1γMCMV expressing SIINFEKL-peptide protects mice against OVA-Listeria in NKG2D−/− mice. C57BL/6 and NKG2D−/− mice were immunized with 105 pfu per mouse via f.p. of indicated viruses or left nonimmunized. Three weeks postimmunization, mice were challenged with 5 × 104 cfu per mouse of OVA-Listeria. The bacterial load was determined. Individual animals (circles) and median values are shown.
Fig. 6.
Fig. 6.
RAE-1γ expression promotes epitope-specific CD8 T cells priming. BALB/c mice were i.v. infected with 2 × 105 pfu, and the following parameters were analyzed: (A) the frequency of DC subsets in spleen, (B) the frequency of proliferating CD8 T cells, and (C) IFN-α level in the sera. For A–C, mean ± SEM is shown; n = 3–5. (D) C57BL/6 and 3d mice were i.p. infected with 2 × 105 pfu of indicated viruses and the specific CD8 T-cell response in spleen was determined on day 7 p.i. (E) C57BL/6 and Batf3−/− mice were infected i.v. with 2 × 105 pfu of indicated viruses and SIINFEKL-specific CD8 T cells in spleen were determined on day 8 p.i. Mean ± SEM is shown. (F) Naive recipients (CD45.1) were transferred with 104 NKG2DΔ/ΔOT1 cells (CD45.2) and infected with 104 pfu per mouse of indicated viruses 24 h later. On days 7 and 14 p.i., donor CD8 T cells (CD45.1) were analyzed for the frequency of MPECs (KLRG1−CD127+ CD8 T cells) in blood (n = 3). Representative FACS plots of donor MPECs expansion are shown; n = 3–4.
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