Preclinical Validation

Efficacy of novel helicase-primase based therapy for human Herpes Simplex Virus type (HSV)

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.

Establishment of a rabbit model for the propofol infusion syndrome

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. 

Non-human papilloma pseudoviruses for DNA delivery in vitro and in vivo

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

Development of a vaccine against Respiratory Syncytial Virus

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.