Difference between revisions of "Team:Freiburg/Project/Diseases"

The Rubella Virus is the only member of the genus of Rubivirus. It contains single-stranded positive-sense RNA that encodes three structural and two non-structural proteins. Two of the structural proteins are the envelope proteins E1 and E2 which form heterodimers that are arranged in groups of three all over the viruses surface. In our project we express the protein E1 that was shown to provoke an immunogenic response and to have „hemagglutinating and virus-neutralizing activities“ ¹⁾.
The virus is transmitted via airborne infection with humans as the only known hosts. It causes the rubella disease, also known as German measles or Rubeola. After infection the virus persists in the body for the whole life providing lifelong immunity and can therefore be referred to as a typical children’s disease. The incubation time amounts to 14 to 21 days and goes along with exanthemas, fever, headache, rheumatic pains and swollen lymph nodes. The patient is contagious from one week before the first exanthema until one week after the last one. The infection can also be asymptomatic for some patients as opposed to possible complications in rare incidents. Arthritis, bronchitis, encephalitis and inclusion of heart damages are conceivable in such cases. As the virus can also cross the placenta, pregnant women getting infected in the beginning of their pregnancy might lose their child due to a spontaneous abort. The children can also still be born but might then suffer from deformities of the heart, cataracts or labyrinthine deafness.
Therapies are only symptomatic but there is a vaccination available that could prevent the disease.
http://de.wikipedia.org/wiki/Rötelnvirus
http://flexikon.doccheck.com/de/Röteln

A lot of people might have already had little blisters in the area of the lip that disappeared after some days. In most cases those might have been provoked by the Herpes Simplex Virus Type 1 (HSV1).
HSV1 belongs to the Human Herpesviruses and is therefore part of the family of Herpesviridae. It consists of double-stranded DNA and an envelope containing at least 10 viral proteins surrounds the capsid. Most of these proteins are glycoproteins, one of which is the glycoprotein G that we express (Bergström et al.: Variability of the Glycoprotein G Gene in Clinical Isolates of Herpes Simplex Virus Type 1 ²⁾. It is the glycoprotein differing the most among the different species so it allows their discrimination ³⁾. Glycoprotein G is important for the attachment to the cell and the entry into it.
There are two different types of Herpes Simplex Virus with about 99% of identity in the gene regions. Both types are transmitted via the contact of mucous membranes. Type 1 – the one we are working with – can be transmitted via kissing, type 2 is mainly transmitted via sexual contact. Below we are only referring to the Herpes Simplex Virus Type 1. It remains in the body in a latent state and several outbreaks during life are possible. As about 85-90% of the world’s population is seropositive the primary infection often takes place prior to the age of five due to a transmission from the parents to their child. The infection of newborns is dangerous to life.
After infection the virus proliferates and is shed via the mucous membrane. It also infiltrates neurons in which it persists life-long. In very severe cases the virus can cause encephalitis as well all meningitis. The reasons for sudden outbreaks of the virus are not clearly understood. A suppression of the immune system or stress are only some possible explanations.
A therapy with anti-viral drugs is possible, for cutaneous infections in facial or rather labial areas the application of corticoids is however usually sufficient.
http://www.gesundheitsamt-bw.de/ML/DE/Aids-STI-Beratung/Aids-STI/Sexuell_uebertragbare_Krankheiten/Seiten/Herpessimplex.aspx
http://de.wikipedia.org/wiki/Herpes-simplex-Viren
http://flexikon.doccheck.com/de/Herpes-simplex-Virus
http://www.rki.de/DE/Content/InfAZ/H/Herpes/Herpes_Forschung.html

If the ambulance is called to take care of an injured person often the first thing they do is to perform a preventing vaccination against Clostridium tetani. This is a little anaerobic gram-positive rod-shaped bacillus that forms spores. These can even survive in inhospitable areas and are present in soil. They can enter the human body easily via small wounds which is why injured people are at high risk of getting infected with C. tetani but cannot transmit the infection to other individuals.
In regions where people are not vaccinated and good health care is not provided, tetanus, which is caused by the bacterium, is a very common cause of death following injuries. The bacterium produces two toxins, named tetanospasmin and tetanolysin. The former was found to cause tetanus by reaching the bone marrow via the nerves. There it is responsible for provoking hypersensitivity, increased reflexes and spasms. The latter causes damage to the heart muscle and blood components.
After an infection with C. tetani the first symptoms are headache, muscle and dorsal pain and a feeling of being tired. Additionally, the patient may feel some tautness in the area of the injury and reveal sensitivity to light and noise. If the patient is not taken care of, the infection is manifested by local stiffening of muscles, mainly those in the area of the jaw and neck. In the following progression the patient will suffer from high fever and muscle spasms. Those will at first be located in the face but spread over the whole body what causes the typical extended position. The immense tension in the muscles due to the effect of the toxins can cause lesions as well as dislocations of the joints or broken bones. As a consequence, many patients suffer from shortened muscles, ankylosis (stiffness of the joints) or spine deformity. In case of the lack of appropriate health care, death due to suffocation or cardiovascular failure is common and occurs partially despite previous vaccination.
To avoid long-term effects due to an infection with C. tetani or even death, the wound is excised and surgically taken care of. Additionally, the patient will be treated with antibiotics and will be given antibodies targeting the toxin.
For our DiaCHIP we expressed a part of the tetanospasmin protein that is generally referred to as tetanustoxin. It consists of a heavy and a light chain that are linked by disulfide bonds ⁴⁾. We worked with the carboxyl-terminal domain of the heavy chain that is able to bind to the target membrane and allows the internalization of the actual toxic region of the light chain ⁵⁾. As we did not express any of the toxic fragments there was no need for special safety measures. The toxin fragment interfering with the neuronal system was not expressed and the part we used is not able to reproduce itself.

An infection going along with red and itching skin that nearly every person in the world suffers from…most might know it as chickenpox and might not even remember the first infection as this often takes place during childhood. The infection is caused by a virus that belongs to the family of Herpesviridae, the same family as the Herpes Simplex Virus, and contains double-stranded DNA. It is transmitted via droplet infection or by having contact with blisters or mucous membranes. Following the first contact with the virus and with an incubation time of about two weeks the patient suffers from fever and exanthemas that normally heal without leaving scars. This first infection is called Varicella Zoster or, as mentioned above, chicken pox. All over the world it is thought that >95% of adults have antibodies against the Varicella Zoster Virus in their blood.
As the virus resides in some ganglions of the body, mostly elderly or immune deficient people might again be afflicted with the disease that is then called Herpes Zoster. It manifests itself in exanthemas restricted to the area of the respective ganglion that is affected. Additionally, the patients are very sensitive to skin contact, have a fever and feel pain. In some cases the reactivation of the virus can lead to neuritis.
Normally the infection with the virus, the primary infection as well as the reactivation of the virus, are not life threatening and in most cases end without consequences for the patient. The primary infection is only a threat for newborns and immune deficient patients where a hemorrhagic development can be lethal. If adults are for the first time exposed to the virus it can also cause much more severe damage. The development of nervous defects is as well possible as pneumonia.
For our DiaCHIP we expressed the glycoprotein E of the virus. It forms heterodimers with the glycoprotein I and was found to play an important role in cell-cell attachment as well as facilitating the entry of the virus and the assembly of the virion ⁶⁾. The sequence of the glycoprotein was obtained after the description of ⁷⁾ and given to IDT for synthesis.

As specific antigen for S. typhimurium we used the enzyme dihydroxyacid dehydratase that is, among others, important in catalyzing the biosynthesis of isoleucine and valine ⁸⁾. ⁹⁾

The sequence was obtained from the group of Prof. Dr. Michael Hust (TU Braunschweig) who identified immunogenic proteins of S. typhimurium using phage display libraries ¹⁰⁾. The antigen sequence was delivered in an expression vector and additionally included a C-terminal His-tag so that we could subsequently use it for overexpression and purification. The same group kindly provided us with an expression vector carrying the sequence for a specific single chain antibody against this antigen, also containing a His-tag for purification. Both parts were overexpressed and purified via Ni-NTA. Figure 1 shows the analysis of the overexpression by SDS page. ……. The specific binding of the single chain antibody against the antigen was verfied by Western Blot analysis (figure 2).

Fig. 2: Western blot analysis of the S. typhimurium antigen with the self-purified specific single chain antibody verified its expression. The antigen has a size of about ~63 kDa.

Fig. 1: SDS PAGE analysis of overexpression and purification of the S. typhimurium antigen (1501) and the corresponding single chain antibody (1301).

There are many epitopes known from the Human Immunodeficiency Virus 1, comprising envelope proteins as well as glycoproteins amongst others. For our purposes we found a paper introducing a multi-epitopic recombinant protein that is constructed with six different epitopes ¹¹⁾. The single epitopes are two of the HIV-1-trans-activating (tat) encoding region, one epitope of the reverse transcriptase, one of the p24 protein, one of the envelope protein gp41 and one of gp120.
The HI Virus itself is highly immunogenic and underlies strict safety regulations. The great advantage of the multi-epitopic protein is that it does not have any immunogenic properties and is only built with small parts of functional proteins but still combines some of the most important epitopes present on the virus.

The group of Wu et al. ¹²⁾ investigated antigens of T. pallidum that could be used for serodiagnosis of syphilis at different stages as many antigens used until then only allowed the conclusion for later stages of syhpilis. They encountered the bacterioferritin protein TpF1, which was expressed in E. coli with a His-tag for purification and subsequently used for serodiagnosis. The protein is known to bind ferric iron and plays an important role in different biological processes involving iron ions, such as stress responses and oxidoreductase activity for example ¹³⁾.