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. 2018 May 7:9:991.
doi: 10.3389/fimmu.2018.00991. eCollection 2018.

Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

Lea Hiršl  1 , Ilija Brizić  1 , Tina Jenuš  1 , Vanda Juranić Lisnić  1   2 , Johanna Julia Reichel  1 , Slaven Jurković  3   4 , Astrid Krmpotić  2 , Stipan Jonjić  1   2
Affiliations

Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

Lea Hiršl et al. Front Immunol. .

Abstract

The development of a vaccine against human cytomegalovirus (CMV) has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV) expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1) (MULT-1MCMV) inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.

Keywords: CD8+ T cells; NK cells; NKG2D; cytomegalovirus; murine CMV; murine UL16 binding protein-like transcript 1; vaccine.

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Figures

Figure 1
Figure 1
Recombinant viruses used in this study. (A) Recombinant murine CMV (MCMV) was made by insertion of genes for NKG2D ligands, RAE-1γ, and murine UL16 binding protein-like transcript-1 (MULT-1) in place of m152 and m145, respectively. OVA-derived Kb restricted CD8+ T cell epitope SIINFEKL was swapped with Dd restricted viral CD8+ T cell epitope of m164 167AGPPRYSRI175. (B) Multi-step growth kinetics assay on mouse embryonic fibroblasts (MEF) comparing wild-type (WT) MCMV, RAE-1γMCMV, and MULT-1MCMV is shown. (C,D) MEFs were infected with 1.5 PFU/cell of indicated viruses and expression of NKG2D ligands was evaluated 24 h after infection by staining either with (C) mouse NKG2D-Fc fusion protein (black line) or (D) αRAE-1γ (upper row, black line), αMULT-1 (lower row, black line), and appropriate isotype controls (gray). (E) SVEC4-10 cells were infected with 3 PFU/cell of WT MCMV, Δm145MCMV, and MULT-1MCMV for 16 h. Surface expression of MULT-1 was detected with αMULT-1 (black line) or isotype control (gray).
Figure 2
Figure 2
Recombinant murine CMV (MCMV) expressing MULT-1 is controlled by NK cells in NKG2D-dependent manner. (A) BALB/c mice were infected intravenously (i.v.) with 2 × 105 PFU of wild-type (WT) MCMV, RAE-1γMCMV, and MULT-1MCMV. On day 4 and 14 after infection viral titer was determined in organs by plaque assay. (B) Newborn C57BL/6 mice were infected with 200 PFU of WT MCMV, RAE-1γMCMV, and MULT-1MCMV intraperitoneally (i.p.) 24 h after birth. Viral titer was determined in organs by plaque assay on day 8 and 14 after infection. (C) BALB/c mice received 20 µl of αAGM1 or 250 µg of NKG2D blocking antibody i.p. 2 h before infection and additional dose of NKG2D blocking antibody on day 2 after i.v. infection with 2 × 105 PFU of WT MCMV, RAE-1γMCMV, and MULT-1MCMV. Viral titer was determined in organs by plaque assay on day 4 after infection. (D) C57BL/6 and NKG2D−/− mice were i.v. infected with 5 × 105 PFU of WT MCMV, RAE-1γMCMV, and MULT-1MCMV. On day 4 after infection viral titer was determined in organs by plaque assay. Each circle represents an individual animal and lines represent medians. Data were analyzed using Mann–Whitney U test. Asterisks denote significant values: *P < 0.05; **P < 0.01. Abbreviation: DL, detection limit.
Figure 3
Figure 3
Attenuated MULT-1MCMV induces strong antigen-specific CD8+ T cell response and efficiently stimulates CD8+ T cells in vitro. C57BL/6 mice were infected footpad with 2 × 105 PFU of wild-type (WT) MCMV, RAE-1γMCMV, and MULT-1MCMV. (A) At different times after infection CD8+ T cells from spleen were analyzed after peptide stimulation for cytokine production (n = 5). (B) C57BL/6 bone marrow-derived dendritic cells (BMDCs) were infected with 2 PFU/cell of WT MCMV, RAE-1γMCMV, or MULT-1MCMV. After 24 h, infected BMDCs were co-incubated with splenocytes from Maxi or OT-1 mice for 6 h and IFN-γ production was measured by flow cytometry. Representative data from two to five independent experiments are shown. Data are presented as mean ± SEM and were analyzed using Student’s t-test. Asterisks denote significant values: *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4
MULT-1MCMV induces protective anti-viral CD8+ T cell response. (A) C57BL/6 CD45.1+ mice were infected footpad (f.p.) with 2 × 105 PFU of wild-type (WT) MCMV, RAE-1γMCMV, and MULT-1MCMV. After 6 weeks 106 CD8+ T cells sorted from spleens were intravenously transferred into NK depleted, irradiated C57BL/6 recipients challenged f.p. with 105 PFU of WT MCMV. Eight days after infection organs of recipient animals were collected and viral titer was determined by plaque assay (n = 4–8). (B) C57BL/6 mice were infected f.p. with 2 × 105 PFU of WT MCMV, RAE-1γMCMV, and MULT-1MCMV and after 6 weeks 105 CD8+ T cells sorted from spleen were transferred into C57BL/6 newborn mice challenged i.p. with 500 PFU of WT MCMV. On day 14 after challenge infection viral titer was determined in organs by plaque assay (n = 4–6). Representative data of two independent experiments are shown. Data are presented as medians with interquartile range and analyzed using Mann–Whitney U test. Asterisks denote significant values: *P < 0.05; **P < 0.01. Abbreviation: DL, detection limit.
Figure 5
Figure 5
Vaccination of female mice with MULT-1MCMV induces MCMV-specific antibodies which protect their offspring from MCMV challenge. (A) BALB/c females were immunized intravenously with 2 × 105 PFU of wild-type (WT) MCMV, RAE-1γMCMV, and MULT-1MCMV 2 weeks prior mating. Six hours after birth, their offspring were either bled to collect sera or intraperitoneally challenged with 500 PFU of WT MCMV. (B) Titers of MCMV-specific antibodies in sera of immunized mothers (in average 1.5 month after infection) and their offspring (6 h after birth) are shown. (C) WT MCMV challenged newborn mice were sacrificed 9 days after infection and viral titer was determined in their organs by plaque assay. Each circle represents an individual animal. Results are presented as pooled data from two independent experiments. Data are presented as mean ± SEM (B) or medians (C) and were analyzed using two-way ANOVA (B) and Mann–Whitney U test (C). Asterisks denote significant values: *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviation: DL, detection limit.
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