Preclinical Validation

The center for animal experimentation is a preclinical development hub within the Fraunhofer IZI network. Our qualified members of staff, who are particularly experienced in the hygienic management of animals, have access to modern facilities which fulfil the highest standards in animal husbandry. At the same time, different pharmacological and pharmacokinetic issues are processed and discussed by the unit, both in vitro and in vivo. The potentialities of work involving organisms range here from biological safety level 1 through to safety level 3 in accordance with the genetic engineering law / ordinance on biological agents.

The unit develops and investigates drug and vaccine candidates in cell culture experiments and various trials involving animal experiments, optionally under GLP standards. This research is focused in part on the development and efficacy testing of innovative vaccines for humans and animals.

Currently, human Herpes Simplex Virus (HSV) infection affects about 82 percent of Germany’s population. The pathogen is categorized into two types, which differ in their predilection for the site of infection. HSV type 1 (HSV-1) is associated with a wide range of clinical manifestations including cold sores. In contrast, HSV type 2 (HSV-2) is linked to genital herpes. Both types are able to develop severe disease progression leading to fatal Herpes Simplex Encephalitis (inflammation of the brain). Until now nucleoside analogues, such as Acyclovir and Valacyclovir, are still the treatment of choice for HSV infections. However, due to the existence of nucleoside-resistant viral strains alternative therapies are needed.

Recently, this alternative has been represented by helicase-primase inhibitors (HPIs), which use a novel mechanism of action to inhibit viral replication. In a drug development trial we analyzed the antiviral efficacy of new drug candidates for the treatment of HSV infections in a mouse model. The mice were infected with HSV and treated daily for five days post infection with the compounds of the novel drug class, Valacyclovir or placebo.

Despite the lower dose, we observed a better outcome in clinical parameters in comparison to Valacyclovir control. We could not observe toxic side effects during the monitoring period of 3 weeks post infection. The subsequent analysis showed that treated animals harbor a significantly lower viral load compared with placebo animals.

In this project we showed that treatment with the new development candidates can significantly reduce or prevent clinical symptoms. HPI‘s are at least one order of magnitude more potent and efficacious compared to Valacyclovir. Thus, candidates of the new drug class are promising inhibitors of HSV infections in vivo and should be translated into clinical trials.

The use of anesthetics can lead to unwanted and sometimes life-threatening side effects. One of the most commonly used anesthetics is propofol. During the use of propofol for longterm anesthesia and during the anesthesia of children, propofol can cause a rare but fatal side effect, the propofol infusion syndrome (PRIS). PRIS is a symptom complex that can lead to severe disorders of the cardiovascular system, kidney failure, a drastic reduction in blood pH (lactic acidosis) as well as the resolution of striated muscles (rhabdomyolysis). In most cases these disorders lead to fatal multi-organ failure. In cooperation with a large industrial partner, a model system in the rabbit was established to investigate PRIS. Based on a publication from 2007 (Ypsilantis et al., 2007), a pilot study was carried out at the Fraunhofer IZI to adapt the described model to the questions. After intubation and successful initiation of propofol anesthesia, it was possible to keep the animals stable under anesthesia for a period of up to 48 hours. Meanwhile, the oxygen and carbon dioxide levels as well as the acid-base balance of the animals were closely monitored. In addition, reflex tests were carried out to ensure a safe depth of anesthesia and the heart functions as well as the temperature were monitored at regular intervals. The successful development of PRIS resulted in an irreversible lethal multiple organ failure. After each experiment, all organs of the animals were removed, fixated and stained for histological examinations. Furthermore, mass spectroscopic analyses of the bile fluid and detailed examinations of the blood work were performed. The pathological findings of most animals were normal. However, a new biomarker could already be identified in this pilot study, which may be of possible use for monitoring anesthetized patients. This biomarker will be validated in human blood samples over the next few months. 

DNA vaccines are gaining popularity due to their inexpensive production and good stability even at room temperature. While it was possible to overcome the initially unsatisfyingly low antigen-specific antibody response in larger animals and humans by use of electroporation, which greatly increases the cellular uptake of the DNA, this method is comparatively laborious and painful for the vaccinee. Novel delivery methods are thus necessary. Being the DNA delivery specialists that viruses are, pseudoviruses (PsVs), which package the vaccine-plasmid inside their capsid, can mediate the delivery of the vaccine and ensure the efficient shuttling of the DNA vaccine into the cells. Different animal papilloma viruses were detected and analyzed for their ability to form PsV particles, package DNA in form of a reporter plasmid and transduce cells in vitro. While most of the tested non-human papilloma viruses bearly showed a transfer of DNA in vitro, two candidates – papillomaviruses that normally infect the puma (PcPV1) and the macaque (MfPV11) – transduced especially effectively. PcPV1 and MfPV11 PsVs were therefore studied further in in-vivo experiments. Both candidates mediated the transduction of a luciferase reporter plasmid after intramuscular application in mice, leading to the expression of firefly luciferase. This expression lasted several weeks after injecting PcPV1 PsVs. Further, in a vaccination including intramuscular and intranasal application, it was tested whether the papilloma PsV mediated the delivery of a DNA vaccine against the respiratory syncytial virus (RSV) in mice. Finally, the mice were infected with infectious RSV and the viral load was quantified. The application of PcPV1 and MfPV11 PsVs carrying a plasmid coding for RSV-F led to a significantly reduced viral load in the lungs of the vaccinated mice upon challenge. Human papilloma PsVs have successfully been used for gene delivery in the past, but have limitations due to vector immunity, which would occur in all individuals that have previously been exposed to these viruses. The project results show that non-human papilloma viruses have the potential of being promising gene delivery vectors and present a vaccine platform for intramuscular or mucosal application.

Project Manager
Dr. Thomas Grunwald

Human respiratory syncytial virus (RSV) usually only leads to mild complaints such as cold, cough or hoarseness. However, this virus is the greatest infectiological issue affecting premature babies and babies younger than six months old. The virus exhibits a severe disease progression among these infants which often has to be treated in hospital. At present, there is neither a therapy nor a vaccine which effectively protects against the RSV infection. A test among children in the 1960s involving a chemically inactive vaccination had a contrary effect: The RSV infection was seen to strengthen when contracted naturally.

Genetic vaccines are currently being tested to be developed for a series of indications. An innovative vaccination method was investigated with the aid of genetic vaccines as part of a major study. This involved a circular DNA molecule being administered as a vaccine, followed by a pain-free vaccination sprayed into the throat. This combination of vaccinations demonstrated surprisingly complete protection against infection with the virus. These promising, preclinical successes are now to be further tested in humans and expanded to other vaccine candidates.

• Helmholtz Centre for Environmental Research – UFZ
• Ichor Medical Systems
• InstrAction GmbH
• Pilot Pflanzenöltechnologie Magdeburg e.V.
• University of Leipzig, Faculty of Veterinary Medicine, Institute for Virology

• Bayer L, Fertey J, Ulbert S, Grunwald T. Immunization with an adjuvanted low-energy electron irradiation inactivated respiratory syncytial virus vaccine shows immunoprotective activity in mice. Vaccine 36 (2018), 12, S. 1561-1569. dx.doi.org/10.1016/j.vaccine.2018.02.014
• Bayer L, Gümpel J, Hause G, Müller M, Grunwald T. Non-human papillomaviruses for gene delivery in vitro and in vivo. PLoS one 13 (2018), Art. e0198996, 14 S. dx.doi.org/10.1371/journal.pone.0198996
• Fingas F, Rückner A, Heenemann K, Volke D, Sieg M, Bielefeldt P, Grunwald T, Vahlenkamp TW, Hassert R, Hoffmann R. Highly sensitive ELISA for the serological detection of murine rotavirus EDIM based on its major immunogen VP6. Journal of Virological Methods 262 (2018), S. 72-78. dx.doi.org/10.1016/j.jviromet.2018.07.016
• Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Benga L, Grunwald T, Müller U, Schrödl W, Baums CG. Comparative analysis of humoral immune responses and pathologies of BALB/c and C57BL/6 wildtype mice experimentally infected with a highly virulent Rodenti­bacter pneumotropicus (Pasteurella pneumotropica) strain. BMC microbiology 18 (2018), Article 45, 11 S. dx.doi.org/10.1186/s12866-018-1186-8
• Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Eisenberg T, Schrödl W, Grunwald T, Müller U, Baums CG. Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis. Microbes and infection 20 (2018), 2, S. 101-110. dx.doi.org/10.1016/j.micinf.2017.10.001
• Ambrosius B, Faissner S, Guse K, von Lehe M, Grunwald T, Gold R, Grewe B, Chan A. Teriflunomide and mono-methylfumarate target HIV-induced neuro-inflammation and neurotoxicity. Journal of neuroinflammation 14 (2017), Art. 51, 10 S. dx.doi.org/10.1186/s12974-017-0829-2
• Fornefett J, Krause J, Klose K, Fingas F, Hassert R, Eisenberg T, Schrödl W, Grunwald T, Müller U, Baums CG. Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis. Microbes and infection. 20 (2018), 2, S. 101-110. dx.doi.org/10.1016/j.micinf.2017.10.001
• Haid S, Grethe C, Bankwitz D, Grunwald T, Pietschmann T. Identification of a human respiratory syncytial virus cell entry inhibitor by using a novel lentiviral pseudotype system. Journal of virology. 90 (2016), Nr. 6, S. 3065-3073. dx.doi.org/10.1128/JVI.03074-15
• Grunwald T, Ulbert S. Improvement of DNA vaccination by adjuvants and sophisticated delivery devices: vaccine-platforms for the battle against infectious diseases. Clin Exp Vaccine Res. 2015 Jan;4(1):1-10. DOI http://dx.doi.org/10.7774/cevr.2015.4.1.1
• Faissner S, Ambrosius B, Schanzmann K, Grewe B, Potthoff A, Münch J, Sure U, Gramberg T, Wittmann S, Brockmeyer N, Uberla K, Gold R, Grunwald T, Chan A. Cytoplasmic HIV-RNA in monocytes determines microglial activation and neuronal cell death in HIV-associated neurodegeneration. Exp Neurol. 2014 Nov;261:685-97. DOI http://dx.doi.org/10.1016/j.expneurol.2014.08.011
• Grunwald T, Tenbusch M, Schulte R, Raue K, Wolf H, Hannaman D, de Swart RL, Uberla K, Stahl-Hennig C. Novel vaccine regimen elicits strong airway immune responses and control of respiratory syncytial virus in nonhuman primates. J Virol. 2014 Apr;88(8):3997-4007. DOI http://dx.doi.org/10.1128/JVI.02736-13
• Sharma A, Wendland R, Sung B, Wu W, Grunwald T, Worgall S. Maternal immunization with chimpanzee adenovirus expressing RSV fusion protein protects against neonatal RSV pulmonary infection. Vaccine. 2014 Sep 29;32(43):5761-8. DOI http://dx.doi.org/10.1016/j.vaccine.2014.08.049
• Lai D, Odimegwu DC, Esimone C, Grunwald T, Proksch P. Phenolic compounds with in vitro activity against respiratory syncytial virus from the Nigerian lichen Ramalina farinacea. Planta Med. 2013 Oct;79(15):1440-6. DOI http://dx.doi.org/10.1055/s-0033-1350711
• Pachernegg S, Joshi I, Muth-Köhne E, Pahl S, Münster Y, Terhag J, Karus M, Werner M, Ma-Högemeier ZL, Körber C, Grunwald T, Faissner A, Wiese S, Hollmann M. Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs. Front Cell Neurosci. 2013 Dec 3;7:241. DOI http://dx.doi.org/10.3389/fncel.2013.00241
• Stab V, Nitsche S, Niezold T, Storcksdieck Genannt Bonsmann M, Wiechers A, Tippler B, Hannaman D, Ehrhardt C, Uberla K, Grunwald T, Tenbusch M. Protective efficacy and immunogenicity of a combinatory DNA vaccine against Influenza A Virus and the Respiratory Syncytial Virus. PLoS One. 2013 Aug 14;8(8):e72217. DOI http://dx.doi.org/10.1371/journal.pone.0072217
• Décard BF, von Ahsen N, Grunwald T, Streit F, Stroet A, Niggemeier P, Schottstedt V, Riggert J, Gold R, Chan A. Low vitamin D and elevated immunoreactivity against Epstein-Barr virus before first clinical manifestation of multiple sclerosis. J Neurol Neurosurg Psychiatry. 2012 Dec;83(12):1170-3. DOI http://dx.doi.org/10.1136/jnnp-2012-303068
• Grewe B, Hoffmann B, Ohs I, Blissenbach M, Brandt S, Tippler B, Grunwald T, Uberla K. Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag. J Virol. 2012 Mar;86(6):2990-3002. DOI http://dx.doi.org/10.1128/JVI.06874-11
• Schneeweiss A, Chabierski S, Salomo M, Delaroque N, Al-Robaiy S, Grunwald T, Bürki K, Liebert UG, Ulbert S. A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII. Vaccine. 2011 Aug 26;29(37):6352-7. DOI http://dx.doi.org/10.1016/j.vaccine.2011.04.116
• Tenbusch M, Grunwald T, Niezold T, Storcksdieck Genannt Bonsmann M, Hannaman D, Norley S, Uberla K. Codon-optimization of the hemagglutinin gene from the novel swine origin H1N1 influenza virus has differential effects on CD4(+) T-cell responses and immune effector mechanisms following DNA electroporation in mice. Vaccine. 2010 Apr 26;28(19):3273-7. DOI http://dx.doi.org/10.1016/j.vaccine.2010.02.090
• Esimone CO, Grunwald T, Nworu CS, Kuate S, Proksch P, Uberla K. Broad spectrum antiviral fractions from the lichen Ramalina farinacea (L.) Ach. Chemotherapy. 2009;55(2):119-26. DOI http://dx.doi.org/10.1159/000194974
• Kohlmann R, Schwannecke S, Tippler B, Ternette N, Temchura VV, Tenbusch M, Uberla K, Grunwald T. Protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized F protein of respiratory syncytial virus. J Virol. 2009 Dec;83(23):12601-10. DOI http://dx.doi.org/10.1128/JVI.01036-09
• Potthoff A, Schwannecke S, Nabi G, Hoffmann D, Grunwald T, Wildner O, Brockmeyer NH, Uberla K, Tenbusch M. Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines. Vaccine. 2009 May 11;27(21):2768-74. DOI http://dx.doi.org/10.1016/j.vaccine.2009.03.001
• Röhrs S, Kutzner N, Vlad A, Grunwald T, Ziegler S, Müller O. Chronological expression of Wnt target genes Ccnd1, Myc, Cdkn1a, Tfrc, Plf1 and Ramp3. Cell Biol Int. 2009 Apr;33(4):501-8. DOI http://dx.doi.org/10.1016/j.cellbi.2009.01.016
• Stang A, Petrasch-Parwez E, Brandt S, Dermietzel R, Meyer HE, Stühler K, Liffers ST, Uberla K, Grunwald T. Unintended spread of a biosafety level 2 recombinant retrovirus. Retrovirology. 2009 Sep 22;6:86. DOI http://dx.doi.org/10.1186/1742-4690-6-86
• Eckardt-Michel J, Lorek M, Baxmann D, Grunwald T, Keil GM, Zimmer G. The fusion protein of respiratory syncytial virus triggers p53-dependent apoptosis. J Virol. 2008 Apr;82(7):3236-49. DOI http://dx.doi.org/10.1128/JVI.01887-07
• Esimone CO, Eck G, Duong TN, Uberla K, Proksch P, Grunwald T. Potential anti-respiratory syncytial virus lead compounds from Aglaia species. Pharmazie. 2008 Oct;63(10):768-73.
• Brandt S, Blissenbach M, Grewe B, Konietzny R, Grunwald T, Uberla K. Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation. PLoS Pathog. 2007 Apr;3(4):e54.
• Neuhoff S, Moers J, Rieks M, Grunwald T, Jensen A, Dermietzel R, Meier C. Proliferation, differentiation, and cytokine secretion of human umbilical cord blood-derived mononuclear cells in vitro. Exp Hematol. 2007 Jul;35(7):1119-31.
• Ternette N, Stefanou D, Kuate S, Uberla K, Grunwald T. Expression of RNA virus proteins by RNA polymerase II dependent expression plasmids is hindered at multiple steps. Virol J. 2007 Jun 5;4:51.
• Ternette N, Tippler B, Uberla K, Grunwald T. Immunogenicity and efficacy of codon optimized DNA vaccines encoding the F-protein of respiratory syncytial virus. Vaccine. 2007 Oct 10;25(41):7271-9.
• Brandt S, Grunwald T, Lucke S, Stang A, Uberla K. Functional replacement of the R region of simian immunodeficiency virus-based vectors by heterologous elements. J Gen Virol. 2006 Aug;87(Pt 8):2297-307.
• Jogler C, Hoffmann D, Theegarten D, Grunwald T, Uberla K, Wildner O. Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species. J Virol. 2006 Apr;80(7):3549-58.
• Quack I, Rump LC, Gerke P, Walther I, Vinke T, Vonend O, Grunwald T, Sellin L. beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity. Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):14110-5.
• Esimone CO, Grunwald T, Wildner O, Nchinda G, Tippler B, Proksch P, Uberla K. In vitro pharmacodynamic evaluation of antiviral medicinal plants using a vector-based assay technique. J Appl Microbiol. 2005;99(6):1346-55.
• Lucke S, Grunwald T, Uberla K. Reduced mobilization of Rev-responsive element-deficient lentiviral vectors. J Virol. 2005 Jul;79(14):9359-62.
• Grunwald T, Pedersen FS, Wagner R, Uberla K. Reducing mobilization of simian immunodeficiency virus based vectors by primer complementation. J Gene Med. 2004 Feb;6(2):147-54.