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. 2018 Jul 17;92(15):e00569-18.
doi: 10.1128/JVI.00569-18. Print 2018 Aug 1.

Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors

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Free PMC article

Efficient Delivery of Human Cytomegalovirus T Cell Antigens by Attenuated Sendai Virus Vectors

Richard Kiener et al. J Virol. .
Free PMC article

Abstract

Human cytomegalovirus (HCMV) represents a major cause of clinical complications during pregnancy as well as immunosuppression, and the licensing of a protective HCMV vaccine remains an unmet global need. Here, we designed and validated novel Sendai virus (SeV) vectors delivering the T cell immunogens IE-1 and pp65. To enhance vector safety, we used a replication-deficient strain (rdSeV) that infects target cells in a nonproductive manner while retaining viral gene expression. In this study, we explored the impact that transduction with rdSeV has on human dendritic cells (DCs) by comparing it to the parental, replication-competent Sendai virus strain (rcSeV) as well as the poxvirus strain modified vaccinia Ankara (MVA). We found that wild-type SeV is capable of replicating to high titers in DCs while rdSeV infects cells abortively. Due to the higher degree of attenuation, IE-1 and pp65 protein levels mediated by rdSeV after infection of DCs were markedly reduced compared to those of the parental Sendai virus recombinants, but antigen-specific restimulation of T cell clones was not negatively affected by this. Importantly, rdSeV showed reduced cytotoxic effects compared to rcSeV and MVA and was capable of mediating DC maturation as well as secretion of alpha interferon and interleukin-6. Finally, in a challenge model with a murine cytomegalovirus (MCMV) strain carrying an HCMV pp65 peptide, we found that viral replication was restricted if mice were previously vaccinated with rdSeV-pp65. Taken together, these data demonstrate that rdSeV has great potential as a vector system for the delivery of HCMV immunogens.IMPORTANCE HCMV is a highly prevalent betaherpesvirus that establishes lifelong latency after primary infection. Congenital HCMV infection is the most common viral complication in newborns, causing a number of late sequelae ranging from impaired hearing to mental retardation. At the same time, managing HCMV reactivation during immunosuppression remains a major hurdle in posttransplant care. Since options for the treatment of HCMV infection are still limited, the development of a vaccine to confine HCMV-related morbidities is urgently needed. We generated new vaccine candidates in which the main targets of T cell immunity during natural HCMV infection, IE-1 and pp65, are delivered by a replication-deficient, Sendai virus-based vector system. In addition to classical prophylactic vaccine concepts, these vectors could also be used for therapeutic applications, thereby expanding preexisting immunity in high-risk groups such as transplant recipients or for immunotherapy of glioblastomas expressing HCMV antigens.

Keywords: HCMV; MCMV; Sendai virus; cytomegalovirus; dendritic cells; vaccine.

Figures

FIG 1
FIG 1
Sendai virus is capable of replicating in moDCs. (A) Schematic representation of viral genomes highlighting transgene insertion sites (not to scale; N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; HN, hemagglutinin-neuraminidase; L, large protein). The modified SeV P gene is highlighted as Pmut. For MVA, letters refer to genome fragment sizes after HindIII digestion (66). (B) Western blot analysis of transgene expression 48 h postinfection (hpi) of Vero cells at an MOI of 1 with replication-competent (rcSeV) or replication-deficient (rdSeV) Sendai virus strains expressing the indicated genes, IE-1, pp65, or GFP. (C) Titration of cell culture supernatants at different time points after infection of human monocyte-derived dendritic cells (moDCs) with rcSeV-GFP or rdSeV-GFP at an MOI of 1. rdSeV was also used to infect the Vero cell line V3-10 (trans-complementing a full-length version of the viral P gene) at the same MOI. Three h postinfection, cells were washed once with medium and an aliquot was collected to determine baseline virus levels (residual virions that did not enter target cells and were not removed by washing). Viral titers are given as cell infectious units (CIU) per ml (bd, below detection limit).
FIG 2
FIG 2
SeV efficiently infects moDCs with rcSeV, eliciting higher transgene expression than rdSeV. MoDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs, and the intracellular presence of the transgenes GFP, IE-1, and pp65 was quantified via flow cytometry after 24 (A and B) and 48 h (C and D) (nd, not determined). (A and C) Results are presented as the percentage of cells positive for a given antigen, with connected lines indicating values that were obtained using cells from an individual donor. (B and D) Median fluorescence intensity (MFI) values were normalized to the signals obtained from uninfected cells, with bars representing the means and standard deviations of values from all donors.
FIG 3
FIG 3
Infection with both Sendai vectors leads to efficient restimulation of T cells by infected moDCs. (A) Direct presentation assay. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. At 24 hpi, antigen-specific T cell clones were added at an effector/target ratio of 1:1. After 6 h of cocultivation in the presence of brefeldin A (BFA), cells were stained for CD8 and intracellular IFN-γ and analyzed via flow cytometry. (B) Removal of extracellular virions before cross presentation: HeLa cells were transduced at an MOI of 10 with rdSeV-pp65. At 24 hpi, the supernatant from the overnight culture (lane 0) as well as 4 subsequent washing steps with cell culture medium (lanes 1 to 4) was collected and added to moDCs. Twenty-four h later, a pp65-specific, HLA-matched T cell clone was added to moDCs at an effector/target ratio of 1:1. After 6 h in the presence of BFA, CD8/IFN-γ staining and flow cytometry analysis were performed. (C) Cross-presentation assay. HeLa cells were transduced at the indicated MOIs with rdSeV-pp65. At 24 hpi, cells were washed 4 times and added to moDCs from 3 individual donors at a 1:1 ratio. After 24 h of cocultivation, an antigen-specific T cell clone was added for a HeLa/DC/T cell ratio of 1:1:1. T cell restimulation was measured after 6 h as described for panel A. Bars represent the means and standard deviations of values from all donors (A and C) or 3 independent experiments (B) (nd, not determined).
FIG 4
FIG 4
Attenuated rdSeV is less cytotoxic than rcSeV. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. After 24 or 48 h, samples were costained with annexin V/7-AAD, and cells positive for one or both markers were quantified by flow cytometry. Bars represent the means and standard deviations (SD) of values from all donors (nd, not determined).
FIG 5
FIG 5
SeV induces maturation of dendritic cells. moDCs from 3 different HCMV seronegative blood donors were infected at the indicated MOIs with IE-1- or pp65-expressing vectors. After 24 or 48 h, samples were stained for CD80, CD83, CD86, or HLA-DR and analyzed via flow cytometry. Obtained median fluorescence intensity (MFI) values were normalized to those of uninfected cells. Log2 values which represent the means (± standard deviations) from all donors are displayed in a heatmap indicating upregulation (blue) or downregulation (red) of a given marker (nd, not determined).
FIG 6
FIG 6
SeV infection induces secretion of IL-6 and IFN-α. moDCs from 3 different HCMV seronegative blood donors were infected with either GFP-, IE-1-, or pp65-expressing vectors. At 48 h after infection, the presence of 13 different cytokines in the conditioned cell culture medium was assessed by a bead-based multiplex immunoassay. Uninfected cells that were untreated or stimulated with LPS (1 μg/ml) served as controls. Selected cytokine concentrations are depicted with bars representing the means and standard errors of the means (SEM) of 3 measurements from the same donor (performed in one experiment). Levels were not reproducibly above the limit of detection for IL-1β, IFN-γ, IL-12p70, IL-17A, IL-23, and IL-33, and no significant differences in secretion were detected for MCP-1 and IL-8 (data not shown).
FIG 7
FIG 7
SeV is capable of infecting T cells, NK cells, and monocytes. (A) Gating strategy for discriminating different leukocyte populations. PBMCs from 3 different HCMV seronegative blood donors were infected at an MOI of 1 or 10 with rcSeV-GFP. (B) At 24 hpi, the amount of GFP-positive cells in the indicated populations was determined by flow cytometry. Bars represent the means and standard deviations (SD) of values from all donors.
FIG 8
FIG 8
Vaccination with rdSeV-pp65 limits viral replication after MCMV-NLV challenge in mice. HLA-A2 transgenic mice were separated into two groups of 5 animals each and immunized intranasally at weeks 0, 3, and 6 with rdSeV-pp65 (6 × 106 CIU per dose) or an equivalent volume of PBS as a negative control. Two weeks after the last immunization, mice were challenged intravenously with 5 × 105 PFU of MCMV-NLV. (A) Viral infectivity was quantified in the indicated organ homogenates 4 days after challenge infection by plaque assay. (B) Viral genome load was quantified in the same samples by qPCR. P values were calculated after log transformation by using unpaired, two-sided t test with Welch's correction.

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