Advertisement

Molecular Biotechnology

, Volume 60, Issue 11, pp 773–782 | Cite as

DNA Vectors Generating Engineered Exosomes Potential CTL Vaccine Candidates Against AIDS, Hepatitis B, and Tumors

  • Flavia Ferrantelli
  • Francesco Manfredi
  • Chiara Chiozzini
  • Simona Anticoli
  • Eleonora Olivetta
  • Claudia Arenaccio
  • Maurizio Federico
Original Paper
  • 174 Downloads

Abstract

Eukaryotic cells constitutively produce nanovesicles of 50–150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nefmut) with the antigen of interest. The strong efficiency of Nefmut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen. When translated in animals, the injection of Nefmut-based DNA vectors generates engineered exosomes whose internalization in antigen-presenting cells induces cross-priming and antigen-specific CTL immunity. Here, we describe the molecular strategies we followed to produce DNA vectors aimed at generating immunogenic exosomes potentially useful to elicit a CTL immune response against antigens expressed by the etiologic agents of major chronic viral infections, i.e., HIV-1, HBV, and the novel tumor-associated antigen HOXB7. Unique methods intended to counteract intrinsic RNA instability and nuclear localization of the antigens have been developed. The success we met with the production of these engineered exosomes opens the way towards pre-clinic experimentations devoted to the optimization of new vaccine candidates against major infectious and tumor pathologies.

Keywords

Exosomes HIV-1 HBV HOXB7 Nef Constitutive transport elements 

Abbreviations

AchE

Acetylcholinesterase

cART

Combination anti-retroviral therapy

IE-CMV

Immediate-early cytomegalovirus

CTE

Constitutive transport elements

CTL

Cytotoxic T lymphocytes

FCS

Fetal calf serum

HBV

Hepatitis C virus

HIV

Human immunodeficiency virus

HOXB7

Homeobox-B7

mAb

Monoclonal antibody

MVBs

Multivesicular bodies

RRE

Rev-responsive elements

Notes

Acknowledgements

This work was supported by the grant of “Ricerca Finalizzata” project RF-2010-2308334 from the Ministry of Health, Italy. We thank Kazuaki Chayama, Department of Gastroenterology and Metabolism, Applied Life Science, Institute of Biomedical & Health Science, Hiroshima University, Hiroshima, Japan, for kindly providing the HBV Core expressing vector, Alessandra Carè and Maria Cristina Errico, ISS, for kindly providing HOXB7 vectors, and Pietro Arciero, ISS, for technical support.

References

  1. 1.
    Arenaccio, C., & Federico, M. (2017). The multifaceted functions of exosomes in health and disease: An overview. Advances in Experimental Medicine and Biology, 998, 3–19.CrossRefPubMedGoogle Scholar
  2. 2.
    Booth, A. M., Fang, Y., Fallon, J. K., Yang, J. M., Hildreth, J. E., & Gould, S. J. (2006). Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. Journal of Cell Biology, 172, 923–935.CrossRefPubMedGoogle Scholar
  3. 3.
    Romancino, D. P., Paterniti, G., Campos, Y., De Luca, A., Di Felice, V., d’Azzo, A., & Bongiovanni, A. (2013). Identification and characterization of the nano-sized vesicles released by muscle cells. FEBS Letters, 587, 1379–1384.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kalra, H., Drummen, G. P., & Mathivanan, S. (2016). Focus on extracellular vesicles: Introducing the next small big thing. International Journal of Molecular Sciences, 17, 170.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Ciardiello, C., Cavallini, L., Spinelli, C., Yang, J., Reis-Sobreiro, M., de Candia, P., Minciacchi, V. R., & Di Vizio, D. (2016). Focus on extracellular vesicles: New frontiers of cell-to-cell communication in cancer. International Journal of Molecular Sciences, 17, 175.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Percario, Z. A., Ali, M., Mangino, G., & Affabris, E. (2015). Nef, the shuttling molecular adaptor of HIV, influences the cytokine network. Cytokine & Growth Factor Reviews, 26, 159–173.CrossRefGoogle Scholar
  7. 7.
    Lenassi, M., Cagney, G., Liao, M., Vaupotic, T., Bartholomeeusen, K., Cheng, Y., Krogan, N. J., Plemenitas, A., & Peterlin, B. M. (2010). HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells. Traffic, 11, 110–122.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Muratori, C., Cavallin, L. E., Krätzel, K., Tinari, A., De Milito, A., Fais, S., D’Aloja, P., Federico, M., Vullo, V., Fomina, A., Mesri, E. A., Superti, F., & Baur, A. S. (2009). Massive secretion by T cells is caused by HIV Nef in infected cells and by Nef transfer to bystander cells. Cell Host & Microbe, 6, 218–230.CrossRefGoogle Scholar
  9. 9.
    Lattanzi, L., & Federico, M. (2012). A strategy of antigen incorporation into exosomes: Comparing cross-presentation levels of antigens delivered by engineered exosomes and by lentiviral virus-like particles. Vaccine, 30, 7229–7237.CrossRefPubMedGoogle Scholar
  10. 10.
    D’Aloja, P., Santarcangelo, A. C., Arold, S., Baur, A., & Federico, M. (2001). Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HIVnef allele. Journal of General Virology, 82, 2735–2745.CrossRefPubMedGoogle Scholar
  11. 11.
    Di Bonito, P., Ridolfi, B., Columba-Cabezas, S., Giovannelli, A., Chiozzini, C., Manfredi, F., Anticoli, S., Arenaccio, C., & Federico, M. (2015). HPV-E7 delivered by engineered exosomes elicits a protective CD8+ T cell-mediated immune response. Viruses, 7, 1079–1099.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Di Bonito, P., Chiozzini, C., Arenaccio, C., Anticoli, S., Manfredi, F., Olivetta, E., Ferrantelli, F., Falcone, E., Ruggieri, A., & Federico, M. (2017). Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation. International Journal of Nanomedicine, 12, 4579–4591.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Anticoli, S., Aricò, E., Arenaccio, C., Manfredi, F., Chiozzini, C., Olivetta, E., Ferrantelli, F., Lattanzi, L., D’Urso, M. T., Proietti, E., & Federico, M. (2018) Engineered exosomes emerging from muscle cells break immune tolerance to HER2 in transgenic mice and induce antigen-specific CTLs upon challenge by human dendritic cells. Journal of Molecular Medicine, 96, 211–221.CrossRefPubMedGoogle Scholar
  14. 14.
    Siliciano, R. F., & Greene, W. C. (2011). HIV latency. Cold Spring Harbor Perspectives in Medicines, 1, a007096.Google Scholar
  15. 15.
    Deeks, S. G. (2012). HIV: Shock and kill. Nature, 487, 439–440.CrossRefPubMedGoogle Scholar
  16. 16.
    Jones, R. B., & Walker, B. D. (2016). HIV-specific CD8+ T cells and HIV eradication. Journal of Clinical Investigation, 126, 455–463.CrossRefPubMedGoogle Scholar
  17. 17.
    Bell, N. M., & Lever, A. M. (2013). HIV Gag polyprotein: Processing and early viral particle assembly. Trends in Microbiology, 21, 136–144.CrossRefPubMedGoogle Scholar
  18. 18.
    Zhang, J., Tamilarasu, N., Hwang, S., Garber, M. E., Huq, I., Jones, K. A., & Rana, T. M. (2000). HIV-1 TAR RNA enhances the interaction between Tat and cyclin T1. Journal of Biological Chemistry, 275, 34314–34319.CrossRefPubMedGoogle Scholar
  19. 19.
    European Association for the Study of the Liver, European Association for the Study of the Liver, Lampertico, P., Agarwal, K., Berg, T., Buti, M., Janssen, H. L. A., Papatheodoridis, G., Zoulim, F., Tacke, F. (2017) EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. Journal of Hepatology, 67, 370–398.CrossRefGoogle Scholar
  20. 20.
    McMahon, B. (2010). Natural history of chronic hepatitis B. Journal of Clinics in Liver Disease, 14, 381–396.CrossRefPubMedGoogle Scholar
  21. 21.
    Boonstra, A., Woltman, A. M., & Janssen, H. L. (2008). Immunology of hepatitis B and hepatitis C virus infections. Best Practice & Research Clinical Gastroenterology, 22, 1049–1061.CrossRefGoogle Scholar
  22. 22.
    Boni, C., Fisicaro, P., Valdatta, C., Amadei, B., Di Vincenzo, P., Giuberti, T., Laccabue, D., Zerbini, A., Cavalli, A., Missale, G., Bertoletti, A., & Ferrari, C. (2007). Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. Journal of Virology, 81, 4215–4225.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Seeger, C., & Mason, W. S. (2015). Molecular biology of hepatitis B virus infection. Virology, 479–480, 672–686.CrossRefPubMedGoogle Scholar
  24. 24.
    Duboule, D. (2007). The rise and fall of Hox gene clusters. Development, 134, 2549–2560.CrossRefPubMedGoogle Scholar
  25. 25.
    Errico, M. C., Jin, K., Sukumar, S., & Carè, A. (2016). The widening sphere of influence of HOXB7 in solid tumors. Cancer Research, 76, 2857–2862.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mautino, M. R., Keiser, N., & Morgan, R. A. (2000). Improved titers of HIV-based lentiviral vectors using the SRV-1 constitutive transport element. Gene Therapy, 7, 1421–1424.CrossRefPubMedGoogle Scholar
  27. 27.
    Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., & Martin, M. A. (1986). Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. Journal of Virology, 59, 284–291.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Balboni, P. G., Bozzini, R., Zucchini, S., Marconi, P. C., Grossi, M. P., Caputo, A., Manservigi, R., & Barbanti-Brodano, G. (1993). Inhibition of human immunodeficiency virus reactivation from latency by a tat transdominant negative mutant. Journal of Medical Virology, 41, 289–295.CrossRefPubMedGoogle Scholar
  29. 29.
    Théry, C., Amigorena, S., Raposo, G., & Clayton, A. (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Current Protocols in Cell Biology, 30, 3–22.CrossRefGoogle Scholar
  30. 30.
    Rieu, S., Géminard, C., Rabesandratana, H., Sainte-Marie, J., & Vidal, M. (2000). Exosomes released during reticulocyte maturation bind to fibronectin via integrin alpha4beta1. European Journal of Biochemistry, 267, 583–590.CrossRefPubMedGoogle Scholar
  31. 31.
    Wodrich, H., Schambach, A., & Krausslich, H. G. (2000). Multiple copies of the Mason-Pfizer monkey virus constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic Acids Research, 28, 901–910.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Bray, M., Prasad, S., Dubay, J. W., Hunter, E., Jeang, K. T., Rekosh, D., & Hammarskjold, M. L. (1994) A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proceedings of the National Academy of Science USA, 91, 1256–1260.CrossRefGoogle Scholar
  33. 33.
    Tabernero, C., Zolotukhin, A. S., Valentin, A., Pavlakis, G. N., & Felber, B. K. (1996). The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. Journal of Virology, 70, 5998–6011.PubMedPubMedCentralGoogle Scholar
  34. 34.
    Gasmi, M., Glynn, J., Jin, M. J., Jolly, D. J., Yee, J. K., & Chen, S. T. (1999). Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors. Journal of Virology, 73, 1828–1834.PubMedPubMedCentralGoogle Scholar
  35. 35.
    Kosaka, N., Iguchi, H., Yoshioka, Y., Hagiwara, K., Takeshita, F., & Ochiya, T. (2012). Competitive interactions of cancer cells and normal cells via secretory microRNAs. Journal of Biological Chemistry, 287, 1397–1405.CrossRefPubMedGoogle Scholar
  36. 36.
    Trajkovic, K., Hsu, C., Chiantia, S., Rajendran, L., Wenzel, D., Wieland, F., Schwille, P., Brügger, B., & Simons, M. (2008). Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science, 319, 1244–1247.CrossRefPubMedGoogle Scholar
  37. 37.
    Yuyama, K., Sun, H., Mitsutake, S., & Igarashi, Y. (2012). Sphingolipid-modulated exosome secretion promotes clearance of amyloid-beta by microglia. Journal of Biological Chemistry, 287, 10977–10989.CrossRefPubMedGoogle Scholar
  38. 38.
    Wei, Y., Neuveut, C., Tiollais, P., & Buendia, M. A. (2010). Molecular biology of the hepatitis B virus and role of the X gene. Pathologie Biologie, 58, 267–272.CrossRefPubMedGoogle Scholar
  39. 39.
    Schreiber, R. D., Old, L. J., & Smyth, M. J. (2011). Cancer immunoediting: Integrating immunity’s roles in cancer suppression and promotion. Science, 331, 1565–1570.CrossRefGoogle Scholar
  40. 40.
    Hyman, E., Kauraniemi, P., Hautaniemi, S., Wolf, M., Mousses, S., Rozenblum, E., Ringnér, M., Sauter, G., Monni, O., Elkahloun, A., Kallioniemi, O. P., & Kallioniemi, A. (2002). Impact of DNA amplification on gene expression patterns in breast cancer. Cancer Research, 62, 6240–6245.PubMedGoogle Scholar
  41. 41.
    Anticoli, S., Falcone, E., Ruggieri, A., & Federico, M. (2016). Engineered exosomes boost the HCV NS3-specific CD8+ T lymphocyte immunity in humans. Trials in Vaccinology, 5, 105–110.CrossRefGoogle Scholar
  42. 42.
    Elliott, J. H., Wightman, F., Solomon, A., Ghneim, K., Ahlers, J., Cameron, M. J., Smith, M. Z., Spelman, T., McMahon, J., Velayudham, P., Brown, G., Roney, J., Watson, J., Prince, M. H., Hoy, J. F., Chomont, N., Fromentin, R., Procopio, F. A., Zeidan, J., Palmer, S., Odevall, L., Johnstone, R. W., Martin, B. P., Sinclair, E., Deeks, S. G., Hazuda, D. J., Cameron, P. U., Sékaly, R. P., & Lewin, S. R. (2014). Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy. PLoS Pathogens, 10, e1004473.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Rasmussen, T. A., Tolstrup, M., Brinkmann, C. R., Olesen, R., Erikstrup, C., Solomon, A., Winckelmann, A., Palmer, S., Dinarello, C., Buzon, M., Lichterfeld, M., Lewin, S. R., Østergaard, L., & Søgaard, O. S. (2015). Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: A phase 1/2, single group, clinical trial. The Lancet HIV, 1, e13–e21.CrossRefGoogle Scholar
  44. 44.
    Sogaard, O. S., Graversen, M. E., Leth, S., Olesen, R., Brinkmann, C. R., Nissen, S. K., Kjaer, A. S., Schleimann, M. H., Denton, P. W., Hey-Cunningham, W. J., Koelsch, K. K., Pantaleo, G., Krogsgaard, K., Sommerfelt, M., Fromentin, R., Chomont, N., Rasmussen, T. A., Østergaard, L., & Tolstrup, M. (2015). The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathogens, 11, e1005142.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Ho, Y. C., Shan, L., Hosmane, N. N., Wang, J., Laskey, S. B., Rosenbloom, D. I., Lai, J., Blankson, J. N., Siliciano, J. D., & Siliciano, R. F. (2013). Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell, 155, 540–551.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Kiepiela, P., Ngumbela, K., Thobakgale, C., Ramduth, D., Honeyborne, I., Moodley, E., Reddy, S., de Pierres, C., Mncube, Z., Mkhwanazi, N., Bishop, K., van der Stok, M., Nair, K., Khan, N., Crawford, H., Payne, R., Leslie, A., Prado, J., Prendergast, A., Frater, J., McCarthy, N., Brander, C., Learn, G. H., Nickle, D., Rousseau, C., Coovadia, H., Mullins, J. I., Heckerman, D., Walker, B. D., & Goulder, P. (2007). CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nature Medicine, 13, 46–53.CrossRefPubMedGoogle Scholar
  47. 47.
    Sanchez, G., Xu, X. Y., Chermann, J. C., & Hirsch, I. (1997). Accumulation of defective viral genomes in peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected individuals. Journal of Virology, 71, 2233–2240.PubMedPubMedCentralGoogle Scholar
  48. 48.
    Fourati, S., Lambert-Niclot, S., Soulie, C., Malet, I., Valantin, M. A., Descours, B., Ait-Arkoub, Z., Mory, B., Carcelain, G., Katlama, C., Calvez, V., & Marcelin, A. G. (2012). HIV-1 genome is often defective in PBMCs and rectal tissues after long-term HAART as a result of APOBEC3 editing and correlates with the size of reservoirs. Journal of Antimicrobial Chemotherapy, 67, 2323–2326.CrossRefPubMedGoogle Scholar
  49. 49.
    Eriksson, S., Graf, E. H., Dahl, V., Strain, M. C., Yukl, S. A., Lysenko, E. S., Bosch, R. J., Lai, J., Chioma, S., Emad, F., Abdel-Mohsen, M., Hoh, R., Hecht, F., Hunt, P., Somsouk, M., Wong, J., Johnston, R., Siliciano, R. F., Richman, D. D., O’Doherty, U., Palmer, S., Deeks, S. G., & Siliciano, J. D. (2013) Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PloS Pathogens, 9, e1003174.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Chen, M., Sällberg, M., Hughes, J., Jones, J., Guidotti, L. G., Chisari, F. V., Billaud, J. N., & Milich, D. R. (2005). Immune tolerance split between hepatitis B virus precore and core proteins. Journal of Virology, 79, 3016–3027.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Li, H. J., Zhai, N. C., Song, H. X., Yang, Y., Cui, A., Li, T. Y., & Tu, Z. K. (2015). The role of immune cells in chronic HBV infection. Journal of Clinical and Translational Hepatology, 3, 277–283.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Care, A., Silvani, A., Meccia, E., Mattia, G., Stoppacciaro, A., Parmiani, G., Peschle, C., & Colombo, M. P. (1996). HOXB7 constitutively activates basic fibroblast growth factor in melanomas. Molecular and Cellular Biology, 16, 4842–4851.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Naora, H., Yang, Y. Q., Montz, F. J., Seidman, J. D., Kurman, R. J., & Roden, R. B. (2001) A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells. Proceedings of the National Academy of Science USA, 98, 4060–4065.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Flavia Ferrantelli
    • 1
  • Francesco Manfredi
    • 1
  • Chiara Chiozzini
    • 1
  • Simona Anticoli
    • 1
  • Eleonora Olivetta
    • 1
  • Claudia Arenaccio
    • 1
  • Maurizio Federico
    • 1
  1. 1.Istituto Superiore di Sanità (ISS)National Center for Global HealthRomeItaly

Personalised recommendations