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Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

ABSTRACT: The efficacy of DNA vaccines may be improved by small interfering (si)RNA adjuvants targeting pro-apoptotic genes. The aim of the present study was to investigate the capacity of siRNAs targeting B-cell lymphoma 2 homologous antagonist killer (BAK) and B-cell lymphoma 2-associated X protein (BAX) to improve the efficacy of a cytomegalovirus (CMV) vaccine. BALB/c mice were divided into four groups (n=18 in each): unimmunized and immunized with pcDNA 3.1-pp65 expressing CMV 65 kDa matrix phosphoprotein and BAK + BAX siRNAs, pcDNA 3.1-pp65 and control siRNA, or control pcDNA 3.1 and BAK + BAX siRNAs. Immunizations were performed twice with an interval of 3 weeks. CMV-specific mouse splenocyte interferon (IFN)-? secretion was assessed by ELISPOT; furthermore, an in vivo cytotoxic T lymphocyte assay was performed 2 weeks after the last immunization. After lethal CMV challenge of the mice, body weight, virus titers in the spleens and salivary glands as well as survival were recorded. The amount of splenocytes secreting IFN-? in response to CMV pp65 peptides and specific lysis of peptide-pulsed target cells were significantly higher in mice administered pcDNA3.1-pp65 and BAK + BAX siRNAs than those in mice administered pcDNA3.1-pp65 and control siRNA (P<0.05 for each). After the virus challenge, the virus titers in the spleens and salivary glands of mice given pcDNA3.1-pp65 and BAK + BAX siRNAs were significantly lower than those in mice immunized with pcDNA3.1-pp65 and control siRNA (P<0.05 for each). Furthermore, mice immunized with pcDNA 3.1-pp65 and control siRNA or BAK + BAX siRNAs survived for longer, and at 21 days after lethal CMV challenge, 66 and 100% of these mice survived, respectively. These mice also experienced less weight loss compared with mice immunized with pcDNA3.1-pp65 and control siRNA (P<0.05). In conclusion, intradermal administration of siRNAs targeting BAK and BAX improved the efficacy of CMV pp65 DNA vaccine.

SUBMITTER: Liu J

PROVIDER: S-EPMC5450512 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

Liu Jixiao J Feng Keke K Zhao Lu L Luo Haining H Zhu Yingjun Y

Experimental and therapeutic medicine 20170426 6

The efficacy of DNA vaccines may be improved by small interfering (si)RNA adjuvants targeting pro-apoptotic genes. The aim of the present study was to investigate the capacity of siRNAs targeting B-cell lymphoma 2 homologous antagonist killer (BAK) and B-cell lymphoma 2-associated X protein (BAX) to improve the efficacy of a cytomegalovirus (CMV) vaccine. BALB/c mice were divided into four groups (n=18 in each): unimmunized and immunized with pcDNA 3.1-pp65 expressing CMV 65 kDa matrix phosphopr ...[more]

PMID: 28587400

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Project description:Abstract: We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-{kappa}B is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response. Polyadenylated RNA from 107 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.). A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Keywords: disease_state_design, pathogenicity_design

Project description:Purpose: Patients with glioblastoma have less than 15-month median survival despite surgical resection, high-dose radiation, and chemotherapy with temozolomide. We previously demonstrated that targeting cytomegalovirus pp65 using dendritic cells (DC) can extend survival and, in a separate study, that dose-intensified temozolomide (DI-TMZ) and adjuvant granulocyte macrophage colony-stimulating factor (GM-CSF) potentiate tumor-specific immune responses in patients with glioblastoma. Here, we evaluated pp65-specific cellular responses following DI-TMZ with pp65-DCs and determined the effects on long-term progression-free survival (PFS) and overall survival (OS).Experimental Design: Following standard-of-care, 11 patients with newly diagnosed glioblastoma received DI-TMZ (100 mg/m2/d × 21 days per cycle) with at least three vaccines of pp65 lysosome-associated membrane glycoprotein mRNA-pulsed DCs admixed with GM-CSF on day 23 ± 1 of each cycle. Thereafter, monthly DI-TMZ cycles and pp65-DCs were continued if patients had not progressed.Results: Following DI-TMZ cycle 1 and three doses of pp65-DCs, pp65 cellular responses significantly increased. After DI-TMZ, both the proportion and proliferation of regulatory T cells (Tregs) increased and remained elevated with serial DI-TMZ cycles. Median PFS and OS were 25.3 months [95% confidence interval (CI), 11.0-∞] and 41.1 months (95% CI, 21.6-∞), exceeding survival using recursive partitioning analysis and matched historical controls. Four patients remained progression-free at 59 to 64 months from diagnosis. No known prognostic factors [age, Karnofsky performance status (KPS), IDH-1/2 mutation, and MGMT promoter methylation] predicted more favorable outcomes for the patients in this cohort.Conclusions: Despite increased Treg proportions following DI-TMZ, patients receiving pp65-DCs showed long-term PFS and OS, confirming prior studies targeting cytomegalovirus in glioblastoma. Clin Cancer Res; 23(8); 1898-909. ©2017 AACR.

Dataset Information

Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

Publications

Improvement of cytomegalovirus pp65 DNA vaccine efficacy by co-administration of siRNAs targeting BAK and BAX.

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