Therapeutic Antibodies Against Prion Disease Biology Essay

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To produce Therapeutic Antibodies against prion disease i.e. variant CREUTZFELDT-JAKOB DISEASE is a challenge. It is a rare disorder but fatal. The disease, vCJD, principally occurs in the UK where some 170 deaths have been recorded. The main cause of disease in uk population is their exposure via blood transfusion and blood products to the infective agent.

All cases of vCJD have been in PRNP-129 methionine homozygote (MM) and heterozygote individuals. Mutations in PRNP results in different pathological changes of prion protein metabolism (The Lancet, 1997).

Variant Creutzfeldt-Jakob disease (vCJD) is a human transmissible spongiform encephalopathy whose main cause of infection is agent of bovine spongiform encephalopathy (BSE). It got transferred into the food chain in the UNITED KINGDOM in 1980 and 1990. In 2004 the UK Blood Service refused to take blood from the transfusion recipients which were acting as blood donors (University of Liverpool, 2009).

Median age at death is about 28 years. Duration of illness is about 13-14 months. it has prominent psychiatric symptoms with painful dyesthesiasis and with delayed neurological signs. The periodic sharp waves in electroencephalogram is often absent. Marked accumulation of protease resistence prion protein is observed.

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vCJD occur with the ingestion of infected beef and beef products and people in the UK also get this infection when they receive blood from the donors who later got affected with vCJD. So far there is no report of vCJD getting transmitted due to personal contact with the infected person (University of Liverpool, 2009).

It is a neurological disorder which affects the brain adversely. It has long incubation period sometimes even decades and after the onset of symptoms death usually occurs within 24 months. Production of antibodies for this disease will not only benefit the company commercially but also will act as a blessing for many patients and their families who are in distress. It will benefit the company because of high cost i.e big profit.

The peptide sequence found is

PrP FAB18 light chain Distance, Å

Ser143 N Tyr31 OH 3.4

Asp144 N Asp49 OD1 3.2

Asp144 OD2 His33 NE2 3.3

Arg151 NH2 Tyr95 OH 3.1

Arg151 NH1 Arg91 O 3.2

Arg151 NH2 Trp90 O 2.9

PrP FAB18 heavy chain Distance, Å

Tyr145 OH Asp31 OD2 3.4

Arg148 NH2 Asp35 OD1 2.7

Arg148 NH1 Asp35 OD2 3.2

Glu152 OE1 Tyr OH 2.7

Glu152 OE2 Asn50 OD1 3.3

Lys204 NZ Tyr101 OH 3.6

Antibodies are the main therapeutics here which can be used against this disease. Many Pharmaceutical companies are emerging these days to produce Antibodies for such Rare Diseases. Treatment will be of high cost with high profit. We as biotechnologist would like to commercialize these antibodies and got it patent (Antonyuk, Trevitt, Strange, Jackson & et al., 2009)

1.1 INTRODUCTION TO PRION DISEASE:

Prion is a misfolded protein. The word Prion was termed by Stanley B. Prusiner in 1982 which was derived from word protein and infection. Prions are an exceptional group of pathogens that cause fatal neurodegenerative diseases in animals and humans. Human Prion diseases consist of - CJD: Creutzfeldt - Jakob disease, GSS: Gerstmann-Straussler-Scheinker syndrome, FFI: Fatal familial Insomnia, Kuru, and Alpers Syndrome. Animal Prion diseases include - Scrapie: in sheep, BSE (Bovine Spongiform Encephalopathy): or mad cow disease in cows, chronic wasting disease (CWD) in deer and elk (Wells, G et al., 1987). There are four different types of CJD and they are divided according to their cause of transmission and symptoms: Sporadic CJD, Variant CJD, Iatrogenic CJD, and Familial CJD. Sporadic CJD was found in every country in the world. The cause of sporadic is not known but it has been suggested that it might be the result of spontaneous conversion of normal protein into the abnormal form. It is not caused due to the risk from diet, previous surgery, and blood transfusion, occupational or animal exposure. (Anthony, et al 2003).Variant CJD the first wave of case of Variant Creutzfeldt-Jacob Disease was caused in human by eating the beef products obtained from the cattle infected with BSE Prion disease. vCJD is also known as the human BSE. Iatrogenic CJD is transmitted during the course of medical or surgical treatments. For example, human pituitary hormone therapy, human Dura mater grafts corneal grafts or neurological instruments. Familial CJD is also known as genetic CJD because it can be transmitted from individuals to their offsprings. It is an autosomal dominant type of inheritance. Familial CJD is due to mutations in the Prion gene which produce the abnormal form of protein.

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Patients with sporadic CJD show the loss of memory, brain cell lose can cause the blindness, disability to walk, stand and speak. People with vCJD disease demonstrate behavioral symptoms which includes depression, personality changes. In addition to this, individuals may suffer from neurological symptoms including forgetfulness, involuntary movements (jerky movements).GCJD and ICJD sufferers exhibit the same symptoms like SPCJD. PrPc Prion proteins contain 253 amino acids in humans. It comprises the molecular weight of 35-36 kDa. Mutation in the Prion gene cause the formation of abnormal form of protein called PrPSc. PRNP gene is found on the short (p) arm of chromosome 20. PrPSc can convert the normal Prion protein PrPc, into more PrPsc. The abnormal protein accumulates in brain forming clumps that may damage brain and microscopic sponge-like holes are produced due to the loss of the cells in the brain. Therefore, Prion diseases are also known as spongiform encephalopathy (Adriano, 2004.) PrPc is the normal form of cellular protein and contain alpha helical structure. This normal protein is sensitive to the protease and soluble in the detergent while PrPsc shows resistant to proteases and not soluble in detergent. It comprises beta sheet structure. The Prion is infectious rather than contagious. Prion disease is untreatable but many compounds are used to treat it such as quinacrine, branched polyamines, phthalocyanines, porphyrin derivatives beta breaker peptides, anti - PrP antibodies, Congo red, compound 60. These compounds not only block the replication but also remove the existing Prion. Although many of the compounds are able to clear the Prion in Scrapie infected neuroblastoma cells but they are not effective in animals or humans (Baron, 2006).

1.2 HISTORY:

Creutzfeldt-Jacob Disease was reported by Hans Gerhard Creutzfeldt and Alfons Maria Jakob. vCJD mainly occurs in younger aged people mainly at the age of 28.

Scrapie was first observed in 1730s. It was first found in English sheep in Europe.

Kuru was observed in the fore tribe people of New Guinea. In 1960, Scientists transmitted the Kuru and CJD to chimpanzees with artificially to describe the transmissible nature of CJD (Velev, 1997).In 1980, about 60 people died from CJD via contaminated surgical instruments. 85 people died due to receival of Prion-infected growth hormone injections. In 1982, Dr. Stanley Prusiner proposed the term "Prion" (Proteinaceous infectious particle). He won Nobel Prize in medicine in 1997.

In 1985, Scientists discovered the PrP gene and discovered that people who are unaffected produce normal form of the PrP protein. In 1986, Mad cow disease was first recognized in United Kingdom. After that 36,680 cattle were infected in 1992 in Great Britain and then it showed decline in the numbers. Thousands of cattle were killed to stop the transmission of infection (Will, R. S. 2004). In 1996, vCJD was identified due to the consumption of Prion infected beef. Sporadic Creutzfeldt-Jakob disease is very rarely occurring disease. It influences about one person in a million people per year. The cause of Variant CJD is associated with BSE in the cattle. CJD usually manifests in later life. Beginning of symptoms appears about at the age of 60 and within 1 year 90% of them died. vCJD causes the effect on younger individuals and they suffer from longer duration of illness. In may 2006, 161 cases of vCJD was reported in UK, 4 cases in Ireland, 17 cases in France, 2 cases in USA and one each cases in other countries ( Stanley B. Prusiner, M. R. 1998).

1.3 PRION STRUCTURE:

Isoforms

(Structure can be seen in Figure 1 and 2.)

PrP can be found in the whole body in all human beings and animals. The only difference is that the PrP which exist in infectious material has different shape which has its own characteristics i.e. it is resistant to proteases, the enzyme which helps in the breakdown of proteins. PrPC is the normal protein and, PrPSc refers to the infectious protein.

''C'' is a short form for 'cellular' or 'common' PrP ''Sc'' is for 'Scrapie', a Prion disease which occurs in sheep. PrPC has structure with less folds where as PrPSc has dispersed structure having many folds. They are called Isoforms because they have change in their shape. There is no change in amino acids, its structure. The only change is in shape. PrPC is normal or cellular. It has about 3% beta sheets. It is monomer and soluble. It is protease sensitive. On the other hand, PrPSC is disease associated with 43% of beta sheets. Beta sheets allows it to aggregate and thus further making it to become insoluble and resistant to protease thus damage in the brain (John Collinge, M. A., 1994 ).

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PrPC (Structure can be seen in the file attached). PrPC occurs on the membranes of cells with 209 amino acids (humans) with one disulfide bond. Its molecular weight is 35-36 kDa and structurally it is alpha-helical. PrPC binds copper (II) ions with high affinity to give stability to the cell. Proteinase K can breakdown PrPC to liberate it from the cell surface by the enzyme phosphoinositide phospholipase C (PI-PLC), which helps in the cleavage of the glycophosphatidylinositol (GPI) glycolipid. PrP helps mainly in cell-cell adhesion and intracellular signaling. Basically it helps in cell-cell communication in the Brain (Mastrianni JA). 1998.)

PrPSC

The abnormal or contagious form of PrP is P rPSC. It converts normal PrPC proteins into the abnormal one by changing its shape. Alpha helix structure is changed into the ß-sheet structure. It aggregates and form amyloid fibers; it builds up to form plaques. Each fiber has free ends on which protein molecules can attach which helps in the growth of fiber. PrP molecules with same amino acid sequence to the only PrPSC fit into the fiber and helps in its growth (Gambetti, Petersen, et al., 1999).

Figure 1: Isoforms of PrPC and PrPSc with conformational changes. One with blue bands is the PrPSc i.e. the infectious protein.

Figure 2: Cellular Prion protein and PrPC structure.

1.4 REPLICATION PROCESS IN PRION PROTEIN:

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Model of Prion replication: it can be seen in the figure attached.

Normal cellular Prion protein PrPc is non-pathogenic, found attached to surface of neuron. PrPc is soluble, cell surface glycoprotein, functions of which not yet understood. Misfolding of cellular PrPc protein result in different conformations. The mechanism involved in structural conversion of normal cellular PrPc protein into PrPSc is not well established. The non pathogenic PrpC protein is single polypeptide comprised more of spiral conformation known as Alpha-helical sheet, which is converted in pathogenic PrPSc protein. PrPc can be easily degraded by proteolytic digestion. PrPSc is extended structure of PrPc conformational strands, which consists of 95% beta sheets aggregate and less alpha helical sheets. It is conformational variant of PrPc which is highly resistant to heat and protease activity. Also this conformational isomer is insoluble in non-ionic detergents, favors aggregation and formation of fibrils (John Collinge, M. A. 1994).

In the process of infection, a chain reaction takes place. PrPSc binds to PrPc, converting the structural conformation of PrPc in more stable PrPSc. The process is carried over until there is no single PrPc is left over. These PrPSc are absorbed by vesicles, when it detaches from cell membrane after forming complete beta sheet structures. This leads to accumulation of large number of pathogenic PrPSc in lysosome-related organelles of neuron system, causing severe irreparable damage to brain activity. The aggregation of PrPSc proteins helps in understanding the pathogenesis of Prion disease. (Baron, B. C. 2006). No nucleic acid is involved in it. So whole Information appears to be stored in the Prion structure. Prion diseases have long incubation periods as decades in humans. The incubation period in lab conditions is remarkably reproducible. (John Collinge, M. A. 1994). PrPc is the normal cellular protein which is a transmembrane glycoprotein, alpha helices ruled the secondary structure and encoded by a gene represetatative PRNP. PrPSc is abnormal disease causing protein and having similar amino acid sequence as PrPC as they have similar primary structure but beta confirmation dominates the secondary structure. As soon as PrPSc get contact with PrPc it modifies into PrPc, and forms aggregates (John Collinge, M. A. 1994). Prion species is encoded by chromosomal PrP gene, in tertiary structure of PrPSc, Prion seems to encipher strain-specific properties. (Telling et al., 1998). As per transgenetic studies PrPSc work as template on which PrPc refolds into nascent PrPSc molecule. At least 20 mutations of the PrP gene can cause inherited Prion diseases in human and in which genetic linkage is found to be in five mutations (Dlouhy et al., 1992).

Figure 3- Replication cycle of Prion.

Experiments on Knock-out Mice-

Clinical Trials have been carried on Mice to understand the infectivity of Prion diseases and its Trans infection. Transmission of Spongiform encephalopathy such as Bovine Serum Albumin (BSE) or Creutzfeldt-Jakob disease (CJD) caused by designated Prion, which is known as transmissible agents. Researchers had predicted that animal devoid of normal cellular Prion protein should able to resist Prion disease and should fail to propagate disease symptoms. This prediction was proven and paved clear understanding for 'Protein Only' hypothesis. Reverse genetics had been carried on PrP Knock-out Mice to understand Prion disease and its functions (Weissmann C and Flechsig E.2003).

By Homologous recombination, several lines of mice devoid of normal cellular Prion protein were produced. "Radical Strategy" was followed, which involves deletion of reading frame and flanking region in the site of third exon. Knock-out PrP-/- of this kind showed normal life style and was resistant to pathogenic PrpSC. But the knock-out mice Prnp-/- showed symptoms of severe Ataxia and loss of Purkinje cell (Weissmann C and Flechsig E. 2003).

1.5 CLINICAL DIAGNOSIS OF PRION DISEASE

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The clinical diagnosis of Prion disease is established in the late stage because the incubation period of the disease can take decades with no conventional immune response (Adriano, 2004). It consists of different combinations of neurologic signs and symptoms such as: dementia, psychiatric symptoms, ataxia, myoclonus, chorea etc. In Neuropathology findings spongiform deterioration and astrogliosis, multiple amyloid plaques take place. Though biochemical test are in progress, but clinically neurologic examination is the most effective tool in diagnostic. Variant CJD can be diagnosed by tonsil biopsy, however, anyone experiencing signs of progressive dementia must be considered and rapid alternating movements and point-to-point movements are typically evident early in the disease (Anthony et al., 2003). Other diagnosis includes brain imaging, EEG, and/or cerebrospinal fluid assessment, but not even one diagnostic is sufficient. Mostly these studies are carrying out to assess for other potential diseases of CNS. But these tests are able to diagnose of non-genetic Prion disease. An EEG is supportive to assess the patient for typical changes in brain wave activity, but no one diagnostic are sufficient. Brain imaging is also done in this disease. Magnetic resonance imaging (MRI) can be used. DWI: Diffusion-weighted MRI (DWI) gives more response than other devices. It is more accurate as well (Azra, 2000). PET or SPECT scanning is useful at early stage of disease. It shows major and selective fall in activity in the thalamus.

1.6 VARIANT CREUTZFELDT JAKOB DISEASE

After epidemic of Mad Cow disease by BSE this variant CJD appeared in humans (155 by 2005) after some years transmitted by livestock flocks in Great Britain. This disease transmitted by eating contaminated beef. It is associated with the accumulation in the brain of an abnormal partially protease-resistant Isoforms of a host-encoded glycoprotein known as Prion protein. The disease-related Isoforms, PrPSc, is derived from its normal cellular precursor, PrPc, by a post-translational process that involves a conformational change. (Baron, B. C. 2006). PrPc is rich in α-helical structure, whereas PrPSc seems to be composed mainly of a β-sheet structure. PrPSc is postulated to act as a conformational template that promotes the conversion of PrPc to further PrPSc. Prion-strain diversity seems to be encoded by differences in PrP conformation and pattern of glycosylation. A molecular approach to strain typing has enabled the identification of four main types of CJD-sporadic and iatrogenic CJD are of PrPSc types 1-3, and all variant CJD cases are associated with a distinctive type 4 PrPSc. (Adriano, M. H. 2004). Factors that are important in determining the probability of BSE transmission to an individual include dose, route of exposure, genetic susceptibility, and the height of the species barrier between cattle and human beings.

1.7 MOLECULAR GENETIC TESTING:

The Prion protein (PrP) also termed as CD230 and PRNP gene name. Both forms of PrP, pathological and normal have identical sequences of amino acid. The differences are in biochemical properties and way the tertiary structure is folded. PO4156 (Accession number) is specific for PrP human protein has 253 amino acid.

>sp|P04156|

MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQP

HGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGA

VVGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCV

NITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSSMVLFSSPPV

ILLISFLIFLIVG

Using the EMBOSS software (pepstatats/pepwindow/pepinfo) programs for basic protein sequence analysis divides the Prion protein into different categories: polar residues, non polar residues, aromatic residues etc. The EMBOSS is useful to predict epitope mapping based on polar residues also known as hydorophilicity .The table below shows the most polar region in PrPc amino acid sequence:

Table 1:

1

KQHTVTTTTK

2

TQYERESQAY

3

PMDEYSNQNN

4

SDYEDRYYREN

5

MHRYPNQVYY

6

GGTHSQWNKP

7

SKPKTNMKHM

1.8 ANTIBODY THERAPUETICS:

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Therapeutic approaches to Prion disease:

Prion infection is caused by the conversion of PrPc into PrPSc. Experiments showed that this conversion can be inhibited by using the monoclonal antibodies which bind with their affinity to the PrPc. The reduction of the infection depends on the interaction between antibodies and PrPC. Research revealed that ICSM is the effective monoclonal antibodies. X-ray crystallography showed the interaction between Fab fragment antibody and Prion protein (Antonyuka et al., 2009).

Table2: Interaction between Fab ICSM Fab18 and PrPc

PrP FAB18

light chain

Distance (Å)

PrP FAB18

heavy chain

Distance (Å)

Ser143 N

Tyr31 OH

3.4

Tyr145 OH

Asp31 OD2

3.4

Asp144 N

Asp49 OD1

3.2

Arg148 NH2

Asp35 OD1

2.7

Asp144 OD2

His33 NE2

3.3

Arg148 NH1

cAsp35 OD2

3.2

Arg151 NH2

Tyr95 OH

3.1

Glu152 OE1

Tyr52 OH

2.7

Arg151 NH1

Arg91 O

3.2

Glu152 OE2

Asn50 OD

3.3

Arg151 NH2

Trp90 O

2.9

Lys204 NZ

Tyr101 OH

3.6

An amino acid residue which resides at 129 position of the Prion protein plays an important role in the determining the person's risk for the Prion disease. There are two types of Prion protein depending on the amino acid residue at 129 position either valine (v) or methionine (m). Around 10% of the UK population

have MM genotype, 10% have VV genotype and rest of the population have MV genotype. Individuals who have MM or VV genotype are more susceptible than the MV genotype. This genetic variation also determines the susceptibility of an individual for the infection.

When the PrPSC infected mouse neuroblastoma cells (ScN2a) treated with recombinant antibodies, they showed the inhibition of the Prion infection. Prion replication can be eliminated and pre-existiting PrPSC can be cleared the when the cells were treated with the potent antibody Fab D18. This activity of the Fab D18 is because of its better binding affinity for the PrpC.

CONCLUSION AND FUTURE DIRECTIONS

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It can be concluded that there is a prevalence of vCJD infection in the UK and a development of a sensitive and specific screening test is vital for detection of the disease during the incubation period. There should be better improvement in the decontamination of surgical instruments which are in direct contact with the central nervous system and monitor the progress in treatment and prophylaxis.