Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jul 16;94(15):e00398-20.
doi: 10.1128/JVI.00398-20. Print 2020 Jul 16.

Orf Virus-Based Therapeutic Vaccine for Treatment of Papillomavirus-Induced Tumors

M Schneider  1 , M Müller  2 , A Yigitliler  1 , J Xi  1 , C Simon  1 , T Feger  2 , H-J Rziha  2 , F Stubenrauch  1 , H-G Rammensee  2   3 , T Iftner  4 , R Amann  5
Affiliations

Orf Virus-Based Therapeutic Vaccine for Treatment of Papillomavirus-Induced Tumors

M Schneider et al. J Virol. .

Abstract

Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.

Keywords: CRPV; ORFV; Orf virus; cottontail rabbit papillomavirus; papillomavirus; therapeutic vector vaccine.

PubMed Disclaimer

Figures

FIG 1
FIG 1
In vitro characterization of D1701-V-E2, D1701-V-LE6, and D1701-V-E7. (A) Genomic map of ORFV D1701-V with inserted CRPV genes. ITR, inverted terminal repeat. (B) Vero cells were infected (MOI = 0.5) with D1701-V-E2, D1701-V-LE6, and D1701-V-E7 for 20 h. Cells were stained with polyclonal chicken antibodies to detect either the CRPV E2, LE6, or E7 protein by immunofluorescence microscopy (red). Noninfected and mock-infected (D1701-V-GFP) cells (green) were used as controls. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (C) Flow cytometry-based analysis of ORFV-CRPV-infected Vero cells. Cells were infected (MOI = 3.0) with D1701-V-E2, D1701-V-LE6, and D1701-V-E7 for 20 h. Black, noninfected cells; red, cells infected with D1701-V-E2, D1701-V-LE6, or D1701-V-E7; green, cells infected with D1701-V-GFP mock virus; dark red, cells infected with D1701-V-E2, D1701-V-LE6, or D1701-V-E7 and stained with 2nd antibody only. (D) Immunofluorescence microscopy of ORFV-infected rabbit PBMCs. PBMCs were infected with D1701-V-mCherry for 24 h (MOI = 3.0). ORFV-infected cells were detected by mCherry expression (red). Bright-field imaging revealed that mCherry is expressed only in larger (monocytes) and not in surrounding smaller cells like T, B, or NK cells. (E) Rabbit monocytes were isolated from PBMCs by adherence and subsequently infected with D1701-V-E2, D1701-V-LE6, and D1701-V-E7 for 24 h (MOI = 3.0). Staining with antibodies specific for E2, LE6, and E7 confirmed transgene expression in rabbit monocytes. DNA was stained with DAPI (blue).
FIG 2
FIG 2
Therapeutic immunization with ORFV-CRPV recombinants reduces tumor growth. (A) Schematic representation of the experimental design of the first rabbit experiment. Nine rabbits were transfected at a total of 22 spots with CRPV-DNA using the gene gun infection method as illustrated. As indicated, rabbits were immunized 35, 43, and 55 days after transfection. The experiment was terminated on day 118. (B) Boxplots depicting the tumor volume distributions of the control (orange) and the vaccine group (green) at the indicated times postinfection. Black dots indicate the tumors of the nonresponder rabbit 9 (euthanized on day 104). On day 118, statistical analysis was performed without rabbit 9 (vaccine group). Each box spans quartiles; whiskers represent minimum and maximum. Arrows mark the days of immunization. Each dot equates to the volume of a single tumor. An unpaired one-tailed t test was used to determine statistical significance (ns, P > 0.05; *, P < 0.1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 3
FIG 3
Therapeutic immunization with the ORFV-CRPV recombinants can lead to a complete tumor regression. (A) Schematic representation of the experimental design of the second rabbit experiment. Fourteen rabbits were transfected at a total of 12 spots with CRPV-DNA using the gene gun infection method as illustrated. Arrows indicate the days of immunization. The DTH skin test was performed between days 158 and 160. The experiment was terminated on day 187. (B) Boxplots depicting the tumor volume distributions of the control (orange) and the vaccine group (green) for the indicated time points. Arrows mark the days of immunization. Each box spans quartiles; whiskers represent minimum and maximum. Each dot equates to the volume of a single tumor. The disappeared tumors of the full responder rabbit 22 are highlighted by pink crosses. An unpaired one-tailed t test was used to determine statistical significance (*, P < 0.1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The red P value summary indicates that papillomas of the vaccine cohort are significant bigger; the black P value summary indicates that papillomas of the vaccine cohort are significant smaller.
FIG 4
FIG 4
Immunization with the combined ORFV-CRPV vaccine induces a specific DTH response. (A) Photodocumentary results of the DTH skin test are shown for the indicated rabbits. DTH reaction was photographed 24 h after the intradermal antigen injection. Shown is one representative rabbit from the control cohort (rabbit 15) and the seven rabbits of vaccine cohort (rabbits 17 to 23). The day before, each animal was injected with 100 μg of purified antigen (MBP-E1, MBP-E2, MBP-LE6, or MBP-E7) dissolved in 100 μl of PBS. PBS served as a negative control. The number in the picture represents the intensity of the skin reddening (0, no reddening; 1, reddening; 2, strong reddening; 3, very strong reddening) (B) Shown are the mean scores for reddening of the control (orange) and the vaccine group (green) in the delayed-type-hypersensitivity reaction. The numbers within the bars indicate the mean for the corresponding cohort and antigen (as depicted on the x axis). An unpaired two-tailed t test was used to determine statistical significance (ns, P > 0.05; *, P < 0.1).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chan WM, McFadden G. 2014. Oncolytic poxviruses. Annu Rev Virol 1:119–141. doi:10.1146/annurev-virology-031413-085442. - DOI - PMC - PubMed
    1. Zhang YQ, Tsai YC, Monie A, Wu TC, Hung CF. 2010. Enhancing the therapeutic effect against ovarian cancer through a combination of viral oncolysis and antigen-specific immunotherapy. Mol Ther 18:692–699. doi:10.1038/mt.2009.318. - DOI - PMC - PubMed
    1. Mastrangelo MJ, Lattime EC. 2002. Virotherapy clinical trials for regional disease: in situ immune modulation using recombinant poxvirus vectors. Cancer Gene Ther 9:1013–1021. doi:10.1038/sj.cgt.7700538. - DOI - PubMed
    1. Gomez CE, Najera JL, Krupa M, Perdiguero B, Esteban M. 2011. MVA and NYVAC as vaccines against emergent infectious diseases and cancer. Curr Gene Ther 11:189–217. doi:10.2174/156652311795684731. - DOI - PubMed
    1. Rziha HJ, Rohde J, Amann R. 2016. Generation and selection of Orf virus (ORFV) recombinants. Methods Mol Biol 1349:177–200. doi:10.1007/978-1-4939-3008-1_12. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources