Cancer Immunology, Immunotherapy

, Volume 61, Issue 11, pp 1941–1951 | Cite as

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects

  • Takuya Osada
  • Peter Berglund
  • Michael A. Morse
  • Bolyn Hubby
  • Whitney Lewis
  • Donna Niedzwiecki
  • Xiao Yi Yang
  • Amy Hobeika
  • Bruce Burnett
  • Gayathri R. Devi
  • Timothy M. Clay
  • Jonathan Smith
  • H. Kim LyerlyEmail author
Original article


We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.


Interleukin-12 Cancer vaccine CEA Alphavirus 


Conflict of interest

Peter Berglund, Bolyn Hubby, Whitney Lewis, and Jonathan Smith were previously employed by Alphavax,Inc. Takuya Osada, Michael A. Morse, Donna Niedzwiecki, Xiao Yi Yang, Amy Hobeika, Bruce Burnett, Gayathri R. Devi, Timothy M. Clay, and H. Kim Lyerly have no conflict of interest.

Supplementary material

262_2012_1248_MOESM1_ESM.pptx (1.3 mb)
Supplementary material 1 (PPTX 1377 kb)


  1. 1.
    Yang S, Kittlesen D, Slingluff CL, Vervaert CE, Seigler HF, Darrow TL (2000) Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3. J. Immunol 164:4204–4211PubMedGoogle Scholar
  2. 2.
    zum Büschenfelde CM, Metzger J, Hermann C, Nicklisch N, Peschel C, Bernhard H (2001) The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells. J Immunol 167:1712–1719Google Scholar
  3. 3.
    Alexopoulou L, Holt AC, Medzhitov R, Flavell RA (2001) Recognition of double-stranded RNA and activation of NF-kappa B by Toll-like receptor 3. Nature 413:732–738PubMedCrossRefGoogle Scholar
  4. 4.
    Diebold SS, Montoya M, Unger H, Alexopoulou L, Roy P, Haswell LE et al (2003) Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 424:324–328PubMedCrossRefGoogle Scholar
  5. 5.
    Pushko P, Parker M, Ludwig GV, Davis NL, Johnston RE, Smith JF (1997) Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 239:389–401PubMedCrossRefGoogle Scholar
  6. 6.
    Caley IJ, Betts MR, Irlbeck DM, Davis NL, Swanstrom R, Frelinger JA et al (1997) Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector. J Virol 71:3031–3038PubMedGoogle Scholar
  7. 7.
    MacDonald GH, Johnston RE (2000) Role of dendritic cell targeting in Venezuelan equine encephalitis virus pathogenesis. J Virol 74:914–922PubMedCrossRefGoogle Scholar
  8. 8.
    Moran TP, Collier M, McKinnon KP, Davis NL, Johnston RE, Serody JS (2005) A novel viral system for generating antigen-specific T cells. J Immunol 175:3431–3438PubMedGoogle Scholar
  9. 9.
    Gardner JP, Frolov I, Perri S, Ji Y, MacKichan ML, zur Megede J et al (2000) Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein. J Virol 74:11849–11857PubMedCrossRefGoogle Scholar
  10. 10.
    Mazzoni A, Segal DM (2004) Controlling the Toll road to dendritic cell polarization. J Leukoc Biol 75:721–730PubMedCrossRefGoogle Scholar
  11. 11.
    Morse MA, Hobeika A, Osada T, Berglund P, Negri S, Niedzwiecki D et al (2010) A novel recombinant alphaviral vector breaks tolerance to self-antigen in the setting of elevated regulatory T cells. J Clin Invest 120:3234–3241PubMedCrossRefGoogle Scholar
  12. 12.
    Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K et al (1993) Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci USA 90:3539–3543PubMedCrossRefGoogle Scholar
  13. 13.
    Trinchieri G (1998) Proinflammatory and immunoregulatory functions of interleukin-12. Int Rev Immunol 16:365–396PubMedCrossRefGoogle Scholar
  14. 14.
    Robbins PF, Kantor JA, Salgaller M, Hand PH, Fernsten PD, Schlom J (1991) Transduction and expression of the human carcinoembryonic antigen gene in a murine colon carcinoma cell line. Cancer Res 51:3657–3662PubMedGoogle Scholar
  15. 15.
    Talarico T, Maughan M, Pancorbo B, Ruiz J, Graham A (2006) Development and manufacture of alphavaccines. Bioprocessing Fall:8–14Google Scholar
  16. 16.
    Hubby B, Talarico T, Maughan M, Reap EA, Berglund P, Kamrud KI et al (2007) Development and preclinical evaluation of an alphavirus replicon vaccine for influenza. Vaccine 25:8180–8189PubMedCrossRefGoogle Scholar
  17. 17.
    Mosca PJ, Hobeika AC, Clay TM, Nair SK, Thomas EK, Morse MA et al (2000) A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood 96:3499–3504PubMedGoogle Scholar
  18. 18.
    Trinchieri G (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3:133–146PubMedCrossRefGoogle Scholar
  19. 19.
    Robertson MJ, Soiffer RJ, Wolf SF, Manley TJ, Donahue C, Young D et al (1992) Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF. J Exp Med 175:779–788PubMedCrossRefGoogle Scholar
  20. 20.
    Grufman P, Kärre K (2000) Innate and adaptive immunity to tumors: IL-12 is required for optimal responses. Eur J Immunol 30:1088–1093PubMedCrossRefGoogle Scholar
  21. 21.
    Gherardi MM, Ramírez JC, Esteban M (2001) Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens. Histol Histopathol 16:655–667PubMedGoogle Scholar
  22. 22.
    Little RF, Pluda JM, Wyvill KM, Rodriguez-Chavez IR, Tosato G, Catanzaro AT et al (2006) Activity of subcutaneous interleukin-12 in AIDS-related Kaposi sarcoma. Blood 107:4650–4657PubMedCrossRefGoogle Scholar
  23. 23.
    Lenzi R, Rosenblum M, Verschraegen C, Kudelka AP, Kavanagh JJ, Hicks ME et al (2002) Phase I study of intraperitoneal recombinant human interleukin 12 in patients with Müllerian carcinoma, gastrointestinal primary malignancies, and mesothelioma. Clin Cancer Res 8:3686–3695PubMedGoogle Scholar
  24. 24.
    Younes A, Pro B, Robertson MJ, Flinn IW, Romaguera JE, Hagemeister F et al (2004) Phase II clinical trial of interleukin-12 in patients with relapsed and refractory non-Hodgkin’s lymphoma and Hodgkin’s disease. Clin Cancer Res 10:5432–5438PubMedCrossRefGoogle Scholar
  25. 25.
    Robertson MJ, Pelloso D, Abonour R, Hromas RA, Nelson RP Jr, Wood L et al (2002) Interleukin 12 immunotherapy after autologous stem cell transplantation for hematological malignancies. Clin Cancer Res 8:3383–3393PubMedGoogle Scholar
  26. 26.
    Peeva E, Fishman AD, Goddard G, Wadler S, Barland P (2000) Rheumatoid arthritis exacerbation caused by exogenous interleukin-12. Arthritis Rheum 43:461–463PubMedCrossRefGoogle Scholar
  27. 27.
    Wadler S, Levy D, Frederickson HL, Falkson CI, Wang Y, Weller E et al (2004) A phase II trial of interleukin-12 in patients with advanced cervical cancer: clinical and immunologic correlates. Eastern Cooperative Oncology Group study E1E96. Gynecol Oncol 92:957–964PubMedCrossRefGoogle Scholar
  28. 28.
    Ansell SM, Witzig TE, Kurtin PJ, Sloan JA, Jelinek DF, Howell KG et al (2002) Phase 1 study of interleukin-12 in combination with rituximab in patients with B-cell non-Hodgkin lymphoma. Blood 99:67–74PubMedCrossRefGoogle Scholar
  29. 29.
    Parihar R, Nadella P, Lewis A, Jensen R, De Hoff C, Dierksheide JE et al (2004) A phase I study of interleukin 12 with trastuzumab in patients with human epidermal growth factor receptor-2-overexpressing malignancies: analysis of sustained interferon gamma production in a subset of patients. Clin Cancer Res 10:5027–5037PubMedCrossRefGoogle Scholar
  30. 30.
    Eisenbeis CF, Lesinski GB, Anghelina M, Parihar R, Valentino D, Liu J et al (2005) Phase I study of the sequential combination of interleukin-12 and interferon alfa-2b in advanced cancer: evidence for modulation of interferon signaling pathways by interleukin-12. J Clin Oncol 23:8835–8844PubMedCrossRefGoogle Scholar
  31. 31.
    Alatrash G, Hutson TE, Molto L, Richmond A, Nemec C, Mekhail T et al (2004) Clinical and immunologic effects of subcutaneously administered interleukin-12 and interferon alfa-2b: phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma. J Clin Oncol 22:2891–2900PubMedCrossRefGoogle Scholar
  32. 32.
    Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL et al (1999) Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine. J Immunol 163:1690–1695PubMedGoogle Scholar
  33. 33.
    Hamid O, Solomon JC, Scotland R, Garcia M, Sian S, Ye W et al (2007) Alum with interleukin-12 augments immunity to a melanoma peptide vaccine: correlation with time to relapse in patients with resected high-risk disease. Clin Cancer Res 13:215–222PubMedCrossRefGoogle Scholar
  34. 34.
    Peterson AC, Harlin H, Gajewski TF (2003) Immunization with Melan-A peptide-pulsed peripheral blood mononuclear cells plus recombinant human interleukin-12 induces clinical activity and T-cell responses in advanced melanoma. J Clin Oncol 21:2342–2348PubMedCrossRefGoogle Scholar
  35. 35.
    Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB et al (1997) Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 90:2541–2548PubMedGoogle Scholar
  36. 36.
    Rakhit A, Yeon MM, Ferrante J, Fettner S, Nadeau R, Motzer R et al (1999) Down-regulation of the pharmacokinetic-pharmacodynamic response to interleukin-12 during long-term administration to patients with renal cell carcinoma and evaluation of the mechanism of this “adaptive response” in mice. Clin Pharmacol Ther 65:615–629PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Takuya Osada
    • 1
  • Peter Berglund
    • 5
  • Michael A. Morse
    • 2
    • 4
  • Bolyn Hubby
    • 5
  • Whitney Lewis
    • 6
  • Donna Niedzwiecki
    • 4
  • Xiao Yi Yang
    • 1
  • Amy Hobeika
    • 1
  • Bruce Burnett
    • 4
  • Gayathri R. Devi
    • 1
    • 4
  • Timothy M. Clay
    • 1
    • 3
    • 4
  • Jonathan Smith
    • 5
  • H. Kim Lyerly
    • 1
    • 3
    • 4
    Email author
  1. 1.Department of SurgeryDuke University Medical CenterDurhamUSA
  2. 2.Department of MedicineDuke University Medical CenterDurhamUSA
  3. 3.Department of ImmunologyDuke University Medical CenterDurhamUSA
  4. 4.Duke Comprehensive Cancer CenterDurhamUSA
  5. 5.Liquidia Technologies, RTPDurhamUSA
  6. 6.Precision BiosciencesDurhamUSA

Personalised recommendations