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. 2014 Feb;22(2):420-429.
doi: 10.1038/mt.2013.249. Epub 2013 Oct 25.

Maraba virus as a potent oncolytic vaccine vector

Jonathan G Pol  1 , Liang Zhang  1 , Byram W Bridle  2 , Kyle B Stephenson  3 , Julien Rességuier  1 , Stephen Hanson  1 , Lan Chen  1 , Natasha Kazdhan  1 , Jonathan L Bramson  1 , David F Stojdl  4 , Yonghong Wan  1 , Brian D Lichty  5
Affiliations

Maraba virus as a potent oncolytic vaccine vector

Jonathan G Pol et al. Mol Ther. 2014 Feb.

Abstract

The rhabdovirus Maraba has recently been characterized as a potent oncolytic virus. In the present study, we engineered an attenuated Maraba strain, defined as MG1, to express a melanoma-associated tumor antigen. Its ability to mount an antitumor immunity was evaluated in tumor-free and melanoma tumor-bearing mice. Alone, the MG1 vaccine appeared insufficient to prime detectable adaptive immunity against the tumor antigen. However, when used as a boosting vector in a heterologous prime-boost regimen, MG1 vaccine rapidly generated strong antigen-specific T-cell immune responses. Once applied for treating syngeneic murine melanoma tumors, our oncolytic prime-boost vaccination protocol involving Maraba MG1 dramatically extended median survival and allowed complete remission in more than 20% of the animals treated. This work describes Maraba virus MG1 as a potent vaccine vector for cancer immunotherapy displaying both oncolytic activity and a remarkable ability to boost adaptive antitumor immunity.

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Figures

Figure 1
Figure 1
Maraba MG1 infects, replicates in and kill B16-F10 melanoma cells in vitro. A monolayer culture of murine B16-F10 melanoma cells was incubating with Maraba MG1-GFP at a multiplicity of infection of 0.01. (a) Maraba virus infects B16-F10 cells and spread across the culture (GFP signal) leading to complete cell population clearance within 36 hours (visible field). (b) Replication of Maraba MG1-GFP illustrated by viral titers (pfu/ml) in B16-F10 culture supernatant. (c) Population of viable B16-F10 cells, monitored by MTT assay, in absence or presence of Maraba MG1-GFP. (b,c) Curves from a representative experiment realized in triplicate where each timepoint is illustrated as mean ± SD. GFP, green fluorescent protein; pfu, plaque-forming units.
Figure 2
Figure 2
Maraba MG1 selectively replicates at the B16-F10 melanoma metastases site in vivo. (a) C57Bl/6 mice received 2.5 × 105 syngeneic B16-F10 cells by intravenous injection in order to establish syngeneic lung melanoma metastases. Fourteen days after tumor challenge, MG1-GFP was administered by intravenous injection at a dose of 109 pfu. To address Maraba tumor selectivity, virus replication in tumor-free mice was compared to replication in lung metastases-bearing animals. Viral titers were quantified at various timepoints after infection in the lungs (d), where metastases are located in tumor-bearing animals, as well as in secondary lymphoid organs: (b) spleen and (c) inguinal lymph nodes. Titers were quantified in three samples per tissue and illustrated as mean ± SD. **P < 0.01, ***P < 0.001. GFP, green fluorescent protein; NS, nonsignificant; pfu, plaque-forming units.
Figure 3
Figure 3
Maraba MG1-hDCT alone is insufficient to improve the therapeutic outcome and to induce antitumor immunity. (a) C57Bl/6 mice received 2.5 × 105 syngeneic B16-F10 cells by intravenous injection (i.v.) in order to establish syngeneic lung melanoma metastases. Five days after tumor challenge, Maraba MG1 expressing either GFP or the melanoma antigen hDCT was administered i.v. at a dose of 109 pfu; untreated mice received phosphate-buffered saline (PBS). Despite viral replication at the tumor site, MG1-hDCT failed to increase mouse survival as illustrated by Kaplan–Meier curves (n = 5 per group). (b,c) C57Bl/6 mice received or not 2.5 × 105 syngeneic B16-F10 cells by i.v. injection in order to establish syngeneic lung melanoma metastases. Five days later, tumor-free (TF) and tumor-bearing (TB) mice were administered i.v. with 109 pfu MG1-hDCT or PBS. Nine days after Maraba injection, immune responses against the tumor antigen DCT were measured in the blood. (b) Percentage of CD8+ T cells secreting IFN-γ after ex vivo exposure to the MHC-I restricted DCT immunodominant epitope SVYDFFVWL (SVY). (c) Percentage of CD4+ T cells secreting IFN-γ after ex vivo exposure to the MHC-II restricted DCT immunodominant epitope KFFHRTCKCTGNFA (KFF). Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values (n = 5 mice per group). P value considered nonsignificant (NS) when >0.05. DCT, dopachrome tautomerase; GFP, green fluorescent protein; IFN-γ, interferon-gamma; pfu, plaque-forming units.
Figure 4
Figure 4
Maraba MG1 is a potent vector for boosting DCT-specific response. (a) C57Bl/6 mice received 2 × 108 pfu of Ad-hDCT by intramuscular injection (DCT prime) before receiving, or not, 109 pfu of VSV-hDCT or Maraba MG1-hDCT by intravenous injection (DCT boost). Negative controls received 2 × 108 pfu of Ad-empty. DCT-specific T-cell responses were measured 10 days after Ad (peak of the prime response) and 5 days after VSV/MG1 (peak of the boost response). Immune responses represented as the percentage of CD8+ T cells secreting IFN-γ after ex vivo exposure to SVY peptide corresponding to the MHC-I restricted immunodominant epitope of DCT (n = 5 mice for Ad-empty and Ad-hDCT + VSV-hDCT groups, n = 10 mice for Ad-hDCT and Ad-hDCT + MG1-hDCT groups). (b) Representative dot plots of DCT-specific CD8+ T-cell responses measured in Ad-hDCT and Ad-hDCT + MG1-hDCT immunized mice. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values. *P < 0.05 and ***P < 0.001. Ad, adenoviral vector; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.
Figure 5
Figure 5
Systemic administration of Maraba MG1 vaccine is required for generating optimal tumor-specific boost responses. C57Bl/6 mice received 2 × 108 pfu Ad-hDCT by intramuscular injection (DCT prime). Twelve days later, 109 pfu of Maraba MG1-hDCT were administered through various routes (DCT boost): intravenous (i.v.), intranasal (i.n.), or intramuscular (i.m.). DCT-specific T-cell responses were measured 5 days after Maraba injection in the blood (a,b) and in the spleen (c,d). Immune response measured at the same timepoint in Ad-hDCT prime only animals is indicated (“No boost”). (a,c) Percentage of CD8+ T cells secreting IFN-γ after ex vivo exposure to the MHC-I restricted DCT immunodominant epitope SVY. (b,d) Percentage of CD4+ T cells secreting IFN-γ after ex vivo exposure to the MHC-II restricted DCT immunodominant epitope KFF. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values (n = 10 mice for the “i.v.” group, n = 5 for the “i.n.” and “i.m.” groups). P value considered nonsignificant (NS) when >0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. Ad, adenoviral vector; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.
Figure 6
Figure 6
Maraba MG1 vaccine accelerates secondary CD8+ T-cell responses. C57Bl/6 mice received 2 × 108 pfu Ad-hDCT by intramuscular injection (DCT prime). At 12, 9 or 4 days after Ad-hDCT prime, MG1-hDCT was administered by intravenous injection at a dose of 109 pfu. DCT-specific T-cell responses were measured 5 days after Maraba injection in the blood. Immune response measured at the same timepoint in Ad-hDCT prime only animals is indicated (“No boost”). (a) Percentage of CD8+ T cells secreting IFN-γ after ex vivo exposure to the MHC-I restricted DCT immunodominant epitope SVY. (b) Percentage of CD4+ T cells secreting IFN-γ after ex vivo exposure to the MHC-II restricted DCT immunodominant epitope KFF. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values (n = 10 mice per group). P value considered nonsignificant (NS) when >0.05, *P < 0.05 and ***P < 0.001. Ad, adenoviral vector; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.
Figure 7
Figure 7
Maraba MG1-hDCT administered in a heterologous prime-boost setting allowed to generate potent antitumor immunity and to extend survival of melanoma lung tumor-bearing animals. (a) C57Bl/6 mice were challenged intravenously with 2.5 × 105 B16-F10 cells in order to establish syngeneic lung melanoma metastases. Five days later, mice received 2 × 108 pfu Ad-hDCT by intramuscular injection. Control mice received empty Ad vector. Nine days after Ad injection, animals were administered intravenously with 109 pfu MG1-hDCT or its GFP control. Immune responses against the tumor antigen DCT were measured 5 days after Maraba injection in the blood and mouse survival was monitored daily. Percentage of CD8+ or CD4+ T cells reacting to SVY (b) or KFF (c) peptide exposure, respectively. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values. Pooled data from several experiments: n = 9 for Ad-empty group, n = 23 for Ad-hDCT group, n = 29 for Ad-hDCT + MG1-hDCT group, n = 11 for Ad-hDCT + MG1-GFP group. (d) Kaplan–Meier curves illustrating survival of treated melanoma lung-tumor bearing mice. Pooled data from several experiments: n = 16 for Ad-empty group, n = 23 for Ad-hDCT group, n = 30 for Ad-hDCT + MG1-hDCT group, n = 11 for Ad-hDCT + MG1-GFP group. (e) T-cell populations were selectively depleted to evaluate their respective therapeutic contribution. Kaplan–Meier curves illustrating survival of treated melanoma lung-tumor bearing mice (n = 5 per group). (-CD8) indicates CD8+ T-cells depletion while (-CD4) indicates CD4+ T-cells depletion. (f) Lungs extracted at day 19 from untreated and Ad-hDCT + MG1-hDCT treated mice. (g) Autoimmunity (vitiligo) induced in long-term survivors following Ad-hDCT + MG1-hDCT treatment. Pictures taken 280 days (mice on the left) or 220 days (mice on the right) after tumor challenge. P value considered nonsignificant (NS) when >0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. Ad, adenoviral vector; GFP, green fluorescent protein; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.
Figure 8
Figure 8
Maraba MG1-hDCT efficiently boosted DCT-specific response in melanoma brain tumor-bearing mice and significantly extended their survival. C57Bl/6 mice were challenged with 103 B16-F10 cells by intracranial injection in order to establish syngeneic brain melanoma tumor. Five days later, mice received 2 × 108 pfu Ad-hDCT by intramuscular injection while control mice received an empty Ad vector. Nine days after Ad injection, animals were administered intravenously with 109 pfu MG1-hDCT. Mouse survival was monitored daily. (a) Percentage of CD8+ T cells secreting IFN-γ in response to DCT-specific SVY peptide exposure measured 5 days after Maraba injection in the blood. Box plots representing 25–75 percentile including median and whiskers illustrating the range between minimal and maximal values. Pooled data from surviving mice at day 19 after tumor challenge from several experiments: n = 2 for Ad-empty group (only 2 control mice out of 17 still alive at the time of immune analysis), n = 8 for Ad-hDCT group, n = 15 for Ad-hDCT + MG1-hDCT group. (b) Kaplan–Meier curves illustrating survival of treated melanoma brain-tumor bearing mice. Pooled data from several experiments: n = 17 for Ad-empty group, n = 10 for Ad-hDCT group, n = 15 for Ad-hDCT + MG1-hDCT group. **P < 0.01, and ***P < 0.001. Ad, adenoviral vector; DCT, dopachrome tautomerase; IFN-γ, interferon-gamma; pfu, plaque-forming units.
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