Horizon 2020 – The Framework Programme for Research and Innovation Projects

Personalized in Vtro tests to detect immune responses to infectious agents – Acronym PERVIMMUNE

Horizon 2020 – The Framework Programme for Research and Innovation

Participant organisation name
Gennaro De Libero: co-ordinator, immunology of infectious diseasesUniversity Hospital BaselSwitzerland
Roberto Nisini: Scientific partner; detection of immune response in patients with infectious diseasesIstituto Superiore di Sanità, RomeItaly
Peter Seeberger: Scientific partner; synthesis of oligosaccharides and lipidsMax Planck Institute of Colloids and Interfaces, BerlinGermany
Giovanni Delogu: Clinical partner; diagnosis of tuberculosis and other infectionsUniversitá Cattolica del Sacro Cuore, RomeItaly
Riccardo Manganelli: Scientific partner; recombinant bacterial proteinsUniversity of PadovaItaly
Amit Singhal: Scientific partner; development of POC devicesSingapore Immunology Network, A-STAR, SingaporeSingapore
Mircea I. Popa: Clinical partner; tuberculosis diagnosisIPNEU “Carol Davila” and ”Marius Nasta” National Institute for Pneumophtysiology, BucarestRomania
Beate Kampmann: Clinical partner; pediatric tuberculosis diagnosisImperial College LondonUK
Utkan Demirci: Scientific partner; expertise in development of POC devicesStanford UniversityUSA
Immunotools. New generation ELISASMEGermany
Koek Biotech. Microfluidic devicesSMETurkey
Mamadou DafféCNRS, ToulouseFrance


An important unmet medical need in current clinical practice is the use of appropriate diagnostic tools to implement early identification of diseases, proper stratification of patients and the possibility to take fast therapeutic decisions. Innovative diagnostic tools should utilize reliable technologies, be based on robust biomarkers and also possibly rely on novel physio-pathological concepts capable of improving final clinical decisions.

A diagnostic device has also to provide highly sensitive and disease-specific information, and if it works with novel technologies, these must be highly reproducible, low in cost, applicable in every clinical setting and should be effective at point-of-care (POC).

PERVIMMUNE proposes the development of a device allowing novel in vitro tests, with the aim of using them as diagnostic tools for infectious diseases.

In preliminary work we have generated a prototype chip that uses nanobeads and that is capable of performing enzyme-linked immunoassays (ELISA) to detect anti-tuberculosis responses in clinical samples in a short time (15 minutes). Other important features of our prototype are its sensitivity (already in the same range as classical plate ELISA assays and better than the currently available commercial test for TB), specificity (no crossreactivity has been found with different protein and lipid antigens) and robustness (a high degree of reproducibility has been found in repeated evaluations of the same sera against the same antigens). In addition, the prototype works without the need for complex motors or optics, and all reagents are in place within the microchip, with no need for additional pipetting.

This chip can be implemented to perform T cell activation assays and antigen detection assays in addition to ELISA assays. These new applications will rely on the utilisation of the novel technologies described below.

The aims of PERVIMMUNE are to develop an affordable device using multidisciplinary scientific and technological knowledge:

  1. to detect the antibody response to microbial protein, lipid and oligosaccharide antigens;
  2. to detect T cell responses specific for microbial antigens relevant to disease diagnosis;
  3. to detect microbial products in biofluids;
  4. to validate the clinical applications of the chip;
  5. to promote the clinical use of the chip and implement sustainability of the health-care system.

The disease that will be used as clinical proof of principle is Tuberculosis (TB) because of the following important reasons. TB, caused by Mycobacterium tuberculosis (Mtb) infection, remains one of the world’s most important infectious diseases, with an estimated 9 million cases and 1.4 million deaths per year. It is a major threat to the global health, particularly due to the emergence of multiple drug-resistant (MDR) and extensively drug-resistant (XDR) TB strains. Therefore, novel and more stringent diagnostic tests and therapeutic regimens are absolutely needed. Upon infection about 5% of individuals develop an active disease, whereas most infected individuals control the bacterial growth and become latently infected. Patients with latent disease may undergo reactivation during their life and represent an immense reservoir of infecting mycobacterial bacilli.

Diagnosis of TB infection is essential not only for treatment of the infected individual but also for controlling its spread within the human population. Treatment and management of TB remains difficult due to the low specificity of clinical diagnosis and poor performance of diagnostic methods available. Currently, the routine diagnosis of active TB is based mainly on sputum microscopy (provides answer in 2-3 days, labour intensive and has modest sensitivity of 34-80%) and culture of MTB bacilli (require 2-3 weeks and has sensitivity of >95%). Recently PCR-based tests have been developed, however their costs and requirements for appropriate laboratory settings, make their use very cumbersome. An immunological-based test would be of great value if capable of restricting the number of people who required screening with more costly tests. It might also discriminate patients with active vs. latent disease, thus facilitating the therapeutic decisions to be taken. The same test might also identify individuals who may develop active disease before clinical symptoms appear. This would be useful in clinical settings where there is a predisposition to TB reactivation following the use of immunobiologics (anti-TNF-α monoclonal antibodies) or immunodeficiencies (patients with organ transplantations or HIV-infection).


  1. Jasmer, R.M., Nahid, P. & Hopewell, P.C. Clinical practice. Latent tuberculosis infection. N Engl J Med 347, 1860-6 (2002).
  2. WHO. Global tuberculosis control 2011. http://www.who.int/tb/publications/global_report/en/accessed on January 26th, 2012 (2011). (2012).
  3. Van Rie, A., Page-Shipp, L., Scott, L., Sanne, I. & Stevens, W. Xpert((R)) MTB/RIF for point-of-care diagnosis of TB in high-HIV burden, resource-limited countries: hype or hope? Expert Rev Mol Diagn 10, 937-46 (2010).
  4. Niemz, A., Ferguson, T.M. & Boyle, D.S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 29, 240-50 (2011).
  5. Wang, S., Xu, F. & Demirci, U. Advances in developing HIV-1 viral load assays for resource-limited settings. Biotechnol Adv 28, 770-81 (2010).
  6. Pai, N.P. & Pai, M. Point-of-care diagnostics for HIV and tuberculosis: landscape, pipeline, and unmet needs. Discov Med 13, 35-45 (2012).
  7. Wang, S., Inci, F., De Libero, G., Singhal, A. & Demirci, U. Point-of-care assays for tuberculosis: role of nanotechnology/microfluidics. Biotechnol Adv 31, 438-49 (2013).
  8. Mani, V., Wang, S., Inci, F., De Libero, G., Singhal, A. & Demirci, U. Emerging technologies for monitoring drug-resistant tuberculosis at the point-of-care. Adv Drug Deliv Rev (2014).
  9. Barry, C.E., 3rd, Boshoff, H.I., Dartois, V., Dick, T., Ehrt, S., Flynn, J., Schnappinger, D., Wilkinson, R.J. & Young, D. The spectrum of latent tuberculosis: rethinking the biology and intervention strategies. Nat Rev Microbiol 7, 845-55 (2009).
  10. Hobby, G.L., Holman, A.P., Iseman, M.D. & Jones, J.M. Enumeration of tubercle bacilli in sputum of patients with pulmonary tuberculosis. Antimicrob Agents Chemother 4, 94-104 (1973).
  11. Tostmann, A., Kik, S.V., Kalisvaart, N.A., Sebek, M.M., Verver, S., Boeree, M.J. & van Soolingen, D. Tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the Netherlands. Clin Infect Dis 47, 1135-42 (2008).
  12. Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S. & Small, P.M. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520-3 (1999).
  13. Siddiqi, K., Lambert, M.L. & Walley, J. Clinical diagnosis of smear-negative pulmonary tuberculosis in low-income countries: the current evidence. Lancet Infect Dis 3, 288-96 (2003).
  14. WHO. Commercial serodiagnostic tests for diagnosis of tuberculosis. http://whqlibdoc.who.int/publications/2011/9789241502054_eng.pdf Accessed on January 26th, 2012. (2011).
  15. Dowdy, D.W., Steingart, K.R. & Pai, M. Serological testing versus other strategies for diagnosis of active tuberculosis in India: a cost-effectiveness analysis. PLoS Med 8, e1001074 (2011).
  16. Wu, X., Yang, Y., Zhang, J., Li, B., Liang, Y., Zhang, C., Dong, M., Cheng, H. & He, J. Humoral immune responses against the Mycobacterium tuberculosis 38-kilodalton, MTB48, and CFP-10/ESAT-6 antigens in tuberculosis. Clin Vaccine Immunol 17, 372-5 (2010).
  17. Delogu, G., Sanguinetti, M., Posteraro, B., Rocca, S., Zanetti, S. & Fadda, G. The hbhA gene of Mycobacterium tuberculosis is specifically upregulated in the lungs but not in the spleens of aerogenically infected mice. Infect Immun 74, 3006-11 (2006).
  18. Menozzi, F.D., Rouse, J.H., Alavi, M., Laude-Sharp, M., Muller, J., Bischoff, R., Brennan, M.J. & Locht, C. Identification of a heparin-binding hemagglutinin present in mycobacteria. J Exp Med 184, 993-1001 (1996).
  19. Pethe, K., Alonso, S., Biet, F., Delogu, G., Brennan, M.J., Locht, C. & Menozzi, F.D. The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination. Nature 412, 190-4 (2001).
  20. Masungi, C., Temmerman, S., Van Vooren, J.P., Drowart, A., Pethe, K., Menozzi, F.D., Locht, C. & Mascart, F. Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis. J Infect Dis 185, 513-20 (2002).
  21. Delogu, G., Chiacchio, T., Vanini, V., Butera, O., Cuzzi, G., Bua, A., Molicotti, P., Zanetti, S., Lauria, F.N., Grisetti, S., Magnavita, N., Fadda, G., Girardi, E. & Goletti, D. Methylated HBHA produced in M. smegmatis discriminates between active and non-active tuberculosis disease among RD1-responders. PLoS One 6, e18315 (2011).
  22. Zanetti, S., Bua, A., Delogu, G., Pusceddu, C., Mura, M., Saba, F., Pirina, P., Garzelli, C., Vertuccio, C., Sechi, L.A. & Fadda, G. Patients with pulmonary tuberculosis develop a strong humoral response against methylated heparin-binding hemagglutinin. Clin Diagn Lab Immunol 12, 1135-8 (2005).
  23. Delogu, G., Bua, A., Pusceddu, C., Parra, M., Fadda, G., Brennan, M.J. & Zanetti, S. Expression and purification of recombinant methylated HBHA in Mycobacterium smegmatis. FEMS Microbiol Lett 239, 33-9 (2004).
  24. Molicotti, P., Bua, A., Cubeddu, M., Cannas, S., Delogu, G. & Zanetti, S. Tuberculosis patients are characterized by a low-IFN-gamma/high-TNF-alpha response to methylated HBHA produced in M. smegmatis. Diagn Microbiol Infect Dis 71, 449-52 (2011).
  25. Shin, A.R., Lee, K.S., Lee, J.S., Kim, S.Y., Song, C.H., Jung, S.B., Yang, C.S., Jo, E.K., Park, J.K., Paik, T.H. & Kim, H.J. Mycobacterium tuberculosis HBHA protein reacts strongly with the serum immunoglobulin M of tuberculosis patients. Clin Vaccine Immunol 13, 869-75 (2006).
  26. Singh, K.K., Zhang, X., Patibandla, A.S., Chien, P., Jr. & Laal, S. Antigens of Mycobacterium tuberculosis expressed during preclinical tuberculosis: serological immunodominance of proteins with repetitive amino acid sequences. Infect Immun 69, 4185-91 (2001).
  27. Kunnath-Velayudhan, S., Davidow, A.L., Wang, H.Y., Molina, D.M., Huynh, V.T., Salamon, H., Pine, R., Michel, G., Perkins, M.D., Xiaowu, L., Felgner, P.L., Flynn, J.L., Catanzaro, A. & Gennaro, M.L. Proteome-scale antibody responses and outcome of Mycobacterium tuberculosis infection in nonhuman primates and in tuberculosis patients. J Infect Dis 206, 697-705 (2012).
  28. Kunnath-Velayudhan, S., Salamon, H., Wang, H.Y., Davidow, A.L., Molina, D.M., Huynh, V.T., Cirillo, D.M., Michel, G., Talbot, E.A., Perkins, M.D., Felgner, P.L., Liang, X. & Gennaro, M.L. Dynamic antibody responses to the Mycobacterium tuberculosis proteome. Proc Natl Acad Sci U S A 107, 14703-8 (2010).
  29. Mustafa, A.S. In silico analysis and experimental validation of Mycobacterium tuberculosis -specific proteins and peptides of Mycobacterium tuberculosis for immunological diagnosis and vaccine development. Med Princ Pract 22 Suppl 1, 43-51 (2013).
  30. Abraham, P.R., Latha, G.S., Valluri, V.L. & Mukhopadhyay, S. Mycobacterium tuberculosis PPE protein Rv0256c induces strong B cell response in tuberculosis patients. Infect Genet Evol 22, 244-9 (2014).
  31. Zhang, M.M., Zhao, J.W., Sun, Z.Q., Liu, J., Guo, X.K., Liu, W.D. & Zhang, S.L. Identification of RD5-encoded Mycobacterium tuberculosis proteins as B-cell antigens used for serodiagnosis of tuberculosis. Clin Dev Immunol 2012, 738043 (2012).
  32. Nguyen, T.K., Wieland, W., Santema, W., Hoeboer, J., van Eden, W., Rutten, V., Koets, A. & Van Rhijn, I. Immune response of cattle immunized with a conjugate of the glycolipid glucose monomycolate and protein. Vet Immunol Immunopathol 142, 265-70 (2011).
  33. de la Salle, H., Mariotti, S., Angenieux, C., Gilleron, M., Garcia-Alles, L.F., Malm, D., Berg, T., Paoletti, S., Maitre, B., Mourey, L., Salamero, J., Cazenave, J.P., Hanau, D., Mori, L., Puzo, G. & De Libero, G. Assistance of microbial glycolipid antigen processing by CD1e. Science 310, 1321-4 (2005).
  34. Yu, X., Prados-Rosales, R., Jenny-Avital, E.R., Sosa, K., Casadevall, A. & Achkar, J.M. Comparative evaluation of profiles of antibodies to mycobacterial capsular polysaccharides in tuberculosis patients and controls stratified by HIV status. Clin Vaccine Immunol 19, 198-208 (2012).
  35. Gilleron, M., Stenger, S., Mazorra, Z., Wittke, F., Mariotti, S., Bohmer, G., Prandi, J., Mori, L., Puzo, G. & De Libero, G. Diacylated sulfoglycolipids are novel mycobacterial antigens stimulating CD1-restricted T cells during infection with Mycobacterium tuberculosis. J Exp Med 199, 649-59 (2004).
  36. Biselli, R., Mariotti, S., Sargentini, V., Sauzullo, I., Lastilla, M., Mengoni, F., Vanini, V., Girardi, E., Goletti, D., R, D.A. & Nisini, R. Detection of interleukin-2 in addition to interferon-gamma discriminates active tuberculosis patients, latently infected individuals, and controls. Clin Microbiol Infect 16, 1282-4 (2010).
  37. El-Masry, S., El-Kady, I., Zaghloul, M.H. & Al-Badrawey, M.K. Rapid and simple detection of a mycobacterium circulating antigen in serum of pulmonary tuberculosis patients by using a monoclonal antibody and Fast-Dot-ELISA. Clin Biochem 41, 145-51 (2008).
  38. Wood, R., Racow, K., Bekker, L.G., Middelkoop, K., Vogt, M., Kreiswirth, B.N. & Lawn, S.D. Lipoarabinomannan in urine during tuberculosis treatment: association with host and pathogen factors and mycobacteriuria. BMC Infect Dis 12, 47 (2012).
  39. Rotherham, L.S., Maserumule, C., Dheda, K., Theron, J. & Khati, M. Selection and application of ssDNA aptamers to detect active TB from sputum samples. PLoS One 7, e46862 (2012).
  40. Zhu, C., Liu, J., Ling, Y., Yang, H., Liu, Z., Zheng, R., Qin, L. & Hu, Z. Evaluation of the clinical value of ELISA based on MPT64 antibody aptamer for serological diagnosis of pulmonary tuberculosis. BMC Infect Dis 12, 96 (2012).
  41. Douglas, T.A., Tamburro, D., Fredolini, C., Espina, B.H., Lepene, B.S., Ilag, L., Espina, V., Petricoin, E.F., 3rd, Liotta, L.A. & Luchini, A. The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease. Biomaterials 32, 1157-66 (2011).
  42. Shafagati, N., Narayanan, A., Baer, A., Fite, K., Pinkham, C., Bailey, C., Kashanchi, F., Lepene, B. & Kehn-Hall, K. The use of NanoTrap particles as a sample enrichment method to enhance the detection of Rift Valley Fever Virus. PLoS Negl Trop Dis 7, e2296 (2013).
  43. Luchini, A., Fredolini, C., Espina, B.H., Meani, F., Reeder, A., Rucker, S., Petricoin, E.F., 3rd & Liotta, L.A. Nanoparticle technology: addressing the fundamental roadblocks to protein biomarker discovery. Curr Mol Med 10, 133-41 (2010).
  44. Fredolini, C., Meani, F., Reeder, K.A., Rucker, S., Patanarut, A., Botterell, P.J., Bishop, B., Longo, C., Espina, V., Petricoin, E.F., 3rd, Liotta, L.A. & Luchini, A. Concentration and Preservation of Very Low Abundance Biomarkers in Urine, such as Human Growth Hormone (hGH), by Cibacron Blue F3G-A Loaded Hydrogel Particles. Nano Res 1, 502-518 (2008).
  45. Righetti, P.G., Boschetti, E., Lomas, L. & Citterio, A. Protein Equalizer Technology : the quest for a “democratic proteome”. Proteomics 6, 3980-92 (2006).
  46. Peter, J.G., Theron, G., van Zyl-Smit, R., Haripersad, A., Mottay, L., Kraus, S., Binder, A., Meldau, R., Hardy, A. & Dheda, K. Diagnostic accuracy of a urine lipoarabinomannan strip-test for TB detection in HIV-infected hospitalised patients. Eur Respir J 40, 1211-20 (2012).