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=OspA: The Lead to a Cure for Lyme Disease=
=='''Introduction'''==
=='''Introduction'''==


First recognized in 1957, [http://en.wikipedia.org/wiki/Lyme_disease Lyme disease] has been estimated to affected between 20 and 100 cases per 100,000 individuals in the United States (Rupprecht, 2008). This bacterial vector of [http://www.ucmp.berkeley.edu/bacteria/spirochetes.html spirochetes], called [http://en.wikipedia.org/wiki/Borrelia_burgdorferi Borrelia burgdorferi], is spread through the bite and feeding of ticks allowing sever skin lesions to erupt, chronic [http://en.wikipedia.org/wiki/Arthritis arthritis] to develop, and neurologic and cardiac abnormalities (Burgdorferi, 1982). From 10-12 weeks of infestation, other symptoms like [http://en.wikipedia.org/wiki/Erythema_chronicum_migrans erythema chronicum migrans] begin to appear as well(Burgdorferi, 1982). Studies were first conducted through New Zealand white rabbits (Burgdorferi, 1982) through the use of [http://en.wikipedia.org/wiki/Immunofluorescence indirect immunofluorescence].
=== Spirochetes and Lyme Disease ===


[[Image:Picture_of_borellia.jpg|thumb|300px|alt=text|''Borrelia Burgdorferi'']]
[[Image:220px-Erythema_migrans_-_erythematous_rash_in_Lyme_disease_-_PHIL_9875.jpg|thumb|250px|alt=text|''Erythema Migrans, also known as the "Bull's Eye Wound"'']]  




Outer surface protein A, or OspA is found to inhibit bacterial transmission <ref name=Ding >PMID: 11183781</ref>. Outer surface protein A, or OspA was found to initiate an immune response that contributed towards the development of Lyme disease vaccinations. It prevented the transmission of [http://en.wikipedia.org/wiki/Borrelia_burgdorferi Borrelia_burgdorferi], the causative bacterial agent of Lyme disease after the attachment of an infected tick <ref name=Ding >PMID: 11183781</ref>. Once entered the host through the skin or blood stream, Borrelia burgdorferi downregulated and suppressed OspA to minimize all of the host’s immunogenic characteristics <ref name=Ruprecht >PMID: 18097481</ref>. When OspA on the spirochete migrated to an inflammatory environment, it induced apoptosis on the bacteria through the activation of B-cells <ref name=Ruprecht >PMID: 18097481</ref>. Only OspA positive in borrelia bacteria were unable to establish an infection compared to OspA negative bacteria successfully hosted in studies of mice <ref name=Ruprecht >PMID: 18097481</ref>. OspA known as a potent stimulator of neutrophils was able to kill the pathogen and attract leukocytes allowing the release of proinflammatory cytokines in a host <ref name=Ruprecht >PMID: 18097481</ref> and avoid contracting Lyme disease as it affects the heart, joints, and central nervous system <ref name=Ruprecht >PMID: 18097481</ref>.
First recognized in 1957, [http://en.wikipedia.org/wiki/Lyme_disease Lyme disease] has been estimated to affect between 20 and 100 cases per 100,000 individuals in the United States <ref name=Ruprecht >PMID: 18097481</ref>. The peak infection rates of this disease occur approximately between the months of May and August in North America <ref name=Onrust>PMID: 10730551</ref>. The causative agent for this disease is a bacterial vector of [http://www.ucmp.berkeley.edu/bacteria/spirochetes.html spirochetes], called [http://en.wikipedia.org/wiki/Borrelia_burgdorferi Borrelia burgdorferi], which were found in the gut of the [http://en.wikipedia.org/wiki/Ixodes Ixodes] tick. The bacteria spread through the bite of the tick forming severe skin lesions. Other health complications include chronic [http://en.wikipedia.org/wiki/Arthritis arthritis], and neurologic and cardiac abnormalities <ref name=Burgdorferi>PMID: 7043737</ref>. From 10-12 weeks of infestation, other symptoms like [http://en.wikipedia.org/wiki/Erythema_chronicum_migrans erythema chronicum migrans] begin to appear as well <ref name=Burgdorferi>PMID: 7043737</ref>. Studies were first conducted through New Zealand white rabbits through the use of [http://en.wikipedia.org/wiki/Immunofluorescence indirect immunofluorescence].  
The reactive LA-2 antibody was found to serve as an important [http://en.wikipedia.org/wiki/Epitope epitope] of Osp-A binding <ref name=Ding >PMID: 11183781</ref> towards developing vaccinations. The free state of the 3D model exposed the C-terminal where there were 49 residues from the “three loops” involved that significantly affected by LA-2 binding, through findings from [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance nuclear magnetic resonance, or NMR] and crystallization <ref name=Ding >PMID: 11183781</ref>.


[[Image:Bulls_eye.jpg|thumb|200px|alt=text|''Erythema migrans, as known as Bulls Eye Wound'']]


=='''About OspA'''==


OspA is a lipoprotein found on the outer surface of the bacteria that is found to inhibit bacterial transmission <ref name=Ding >PMID: 11183781</ref>.  In order for Lyme Disease to start and progress in the human or animal body, the initial levels of OspA is underregulated, inactivated, hidden, or OspA negative <ref name=Ding >PMID: 11183781</ref>. The bacteria underregulates OspA for the reason that it does not want to trigger any innate immune response from the host <ref name=Ding >PMID: 11183781</ref>. Experiments that showed the success of the Borrelia bacteria progressing in the body, were done through mice. Only OspA- positive borrelia admitted to apoptosis in the host; therefore, when the outer surface protein was present the Borrelia bacteria cannot survive <ref name=Ding >PMID: 11183781</ref>. Unless the vaccine with OspA is administered, or the bacteria has moved to a different environment of the cerebrospinal fluid or an inflammatory environment, the bacteria will activate and start to regulate OspA <ref name=Ding >PMID: 11183781</ref> by activating B-cells. Later this induced astrogliosis.
[[Image:Tick-1.jpg|thumb|150px|alt=text|''Ixodes Tick'']]




The whole system of OspA on the antigen interacting with the antibodies was a cascade complement system. Once the complement found OspA on borrelia, it induced an innate response of [http://en.wikipedia.org/wiki/Phagocytosis phagocytosis]. Not only did OspA allowed opsonization, but also attracted [http://en.wikipedia.org/wiki/White_blood_cell leukocytes] (<ref name=Ding >PMID: 11183781</ref>.


These spirochetes however do not enter the host through salivary gland secretions into the blood stream. The host becomes infected because the tick's blood meal enters the gut where the spirochetes are located where some of the blood that returns to the bitten area is infected with borrelia. This results in the transmission into the host's blood stream <ref name=Onrust>PMID: 10730551</ref>). Once the bacteria is in the blood stream, the immune system may, or may not destroy this [http://en.wikipedia.org/wiki/Pathogen pathogen], depending on the presence of a [http://en.wikipedia.org/wiki/Vaccine vaccine]. The type of proteins on the surface of the borrelia detected by the host's immune system determines the fate of the survival of this disease. One of the main outer surface proteins, OspA, plays a major role in the ability of the immune system to destroy these [http://en.wikipedia.org/wiki/Antigen antigens] <ref name=Ding >PMID: 11183781</ref>.


=='''Structure'''==
[[Image:Picture_of_borellia.jpg|thumb|300px|alt=text|''Borrelia Burgdorferi'']]


<Structure load='1FJ1' size='400' align='left' caption='Structure of OspA (PDB entry[[1FJ1]])'scene="/>


The model presented by the Protein Data Bank is OspA containing two [http://en.wikipedia.org/wiki/Immunoglobulin_light_chain light chains] or Hybridoma Antibody LA2 (chains A and C), two [http://en.wikipedia.org/wiki/Heavy_chain heavy chains] or Hyrbridoma Antibody LA2 (chains B and D), and two outer surface protein A (chains E and F). In addition, the original PDB image suggests that the C-terminal domain was unchanged by the LA-2 binding, other than minor shifts in the conformations of all 3-loops to accommodate interactions with the Fab <ref name=Ding >PMID: 11183781</ref>. 
=='''About OspA'''==


The reactive LA-2 antibody was found to serve as an important epitope of Osp-A binding <ref name=Ding >PMID: 11183781</ref> towards developing vaccinations. The protein, OspA obtained from PDB was dissected to isolate and concentrate the F chain from the molecule of OspA from the crystallized structure to show the free state of the 3D model exposing the C-terminal to express the interaction with the Fab antigen combing site exposing the 3-loops where LA-2 makes direct contact <ref name=Ding >PMID: 11183781</ref>.  
The Borrelia bacteria has proteins on its surface that are different in immunogenic expression <ref name=Wagner>PMID: 22336289</ref>. One of these outer surface proteins, OspA, is a [http://en.wikipedia.org/wiki/Lipoprotein lipoprotein] that was found to inhibit bacterial transmission <ref name=Ding >PMID: 11183781</ref>. OspA, known as a potent stimulator of [http://en.wikipedia.org/wiki/Neutrophils nuetrophils], was able to kill the pathogen and attract [http://en.wikipedia.org/wiki/Leukocytes leukocytes]. The interaction of OspA on the antigen and the antibodies was a cascade [http://en.wikipedia.org/wiki/Complement_system complement system]. Once the complement found OspA on borrelia, it induced an innate response of [http://en.wikipedia.org/wiki/Phagocytosis phagocytosis] allowing the release of [http://en.wikipedia.org/wiki/Proinflammatory_cytokines proinflamatory cytokines] in a host <ref name=Ruprecht >PMID: 18097481</ref> and avoid contracting Lyme disease as it affects the heart, joints, and [http://en.wikipedia.org/wiki/Central_nervous_system central nervous sytem] <ref name=Ruprecht >PMID: 18097481</ref>.


The following is the fit mechanism where the conformations recognize LA-2 and shifts to optimize the complementary antigen combing site (Ding, 2000). There were 49 residues from the “three loops” involved that significantly affected by LA-2 binding, through findings from NMR and crystallization <ref name=Ding >PMID: 11183781</ref>.  Residues 207 and 227 were excluded from analysis because of the peak overlap <ref name=Ding >PMID: 11183781</ref>. The portion of the protein chain detected through 15N-HSQC NMR that were affected by the binding of LA2 <ref name=Ding >PMID: 11183781</ref> was highlighted.<scene name='OspA_L03_Group2/Residues_203-220_loop1/1'>Residues 203 to 220 in "loop 1"</scene>” were represented by pale green, <scene name='OspA_L03_Group2/Residues_224-233_loop2/1'>residues 224-233 in loop 2</scene> were colored purple and <scene name='OspA_L03_Group2/Residues_246-257_loop3/1'>residues 247-257 in "loop 3"</scene>” were colored medium slate blue. The cool coloring of the residues shows the location of LA-2’s direct contact on the “3 loops”. Primary colors were used to represent <scene name='OspA_L03_Group2/Ala_208/3'>Ala-208</scene> as red and <scene name='OspA_L03_Group2/Ala_215/3'>Ala-215</scene> as blue in spacefills for the primary or initial identification of the LA-2 epitope on the beta-strands. The coloration of resides are all at one end, C-terminal of the isolated OspA molecule showing the side where LA-2 binds. The rest of the model were beta-sheets that were left yellow, as the neutral color, and the only alpha-helix was colored pink, to show the general overall structure of 21 anti-parrallel beta-strands to 1 alpha-helix <ref name=Ding >PMID: 11183781</ref>.


==== Pathology of OspA ====
Once entered the host through the skin or blood stream, Borrelia burgdorferi downregulated and suppressed OspA to minimize all of the host’s [http://en.wikipedia.org/wiki/Immunogenic immunogenic] characteristics <ref name=Ruprecht >PMID: 18097481</ref>, while expression of OspC was active. Usually, the initial stages of Lyme disease diagnosed individuals with peaked elevations of OspC up until 7-11 weeks, then later declined <ref name=Wagner>PMID: 22336289</ref>). When OspA on the spirochete migrated to an inflammatory environment, it induced [http://en.wikipedia.org/wiki/Apoptosis apoptosis] on the bacteria through the activation of [http://en.wikipedia.org/wiki/B-cells B-cells] <ref name=Ruprecht >PMID: 18097481</ref>.




==''Structure''==


=='''Vaccination (La-2)'''==
<scene name='OspA_L03_Group2/Default/1'>Restore to default</scene>


OspA is the least variable protein located on the outer surface of the borrelia bacteria <ref name=Ding >PMID: 11183781</ref>.  This results in fewer mutations to occur allowing the same [http://en.wikipedia.org/wiki/Vaccine vaccine] to stay in use for a longer time.  Individuals who were bit by an infected tick and received the ospA vaccine were able to produce anti-opsA antibodies. These antibodies enter the tick through its blood meal into the gut where spirochetes are found and are destroyed by the antibody’s detection of opsA. Therefore, before the spirochete enters the host, it will be inactivated and Lyme Disease may be prevented (Marks, 2011).
<Structure load='1FJ1' size='400' align='left' caption='OspA (magenta, cyan) complex with antibody light chain (grey, pink) and heavy chain (gree, yellow) (PDB code [[1fj1]])' scene='OspA_L03_Group2/Default/1' />


The complement system of the host also has a beneficial effect on destroying the borrelia located in the tick’s gut. It works by opsonizing these pathogens and attracting leukocytes which are a main component of the defense system against pathogens <ref name=Ruprecht >PMID: 18097481</ref>. Overall, the large percentage of the antibodies raised during the OspA vaccination must bind to antigen surfaces that overlap with the LA-2 epitope <ref name=Ding >PMID: 11183781</ref> in order for the Borrelia bacteria to enter apoptosis.  
=== OspA ===
The model presented by the Protein Data Bank is OspA containing two [http://en.wikipedia.org/wiki/Immunoglobulin_light_chain light chains] or Hybridoma Antibody LA2 (chains A and C), two [http://en.wikipedia.org/wiki/Heavy_chain heavy chains] or Hyrbridoma Antibody LA2 (chains B and D), and two outer surface protein A (chains E and F). In addition, the original PDB image suggests that the C-terminal domain was unchanged by the LA-2 binding, other than minor shifts in the conformations of all 3-loops to accommodate interactions with the Fab <ref name=Ding >PMID: 11183781</ref>.


===''Side Effects''===   
<scene name='OspA_L03_Group2/Labeling_chains/1'>Chain Locations</scene>
Recent studies however have shown that neurological complications may appear in patients with this vaccination. This occurs because the down regulation of OspA from the vaccine would not be an abundant amount to activate the immune system for defense (Marks, 2011). This may then cause a wide range of adverse events such as headaches and acute neuroborreliosis(Marks, 2011).


=='''Comparing OspA , OspB , OspC'''==
=== LA-2 ===
The reactive <scene name='OspA_L03_Group2/La-2/2'>LA-2 antibody</scene> was found to serve as an important epitope of Osp-A binding <ref name=Ding >PMID: 11183781</ref> towards developing vaccinations. The protein, OspA obtained from PDB was dissected to isolate and concentrate the F chain from the molecule of OspA from the crystallized structure to show the free state of the 3D model exposing the  C-terminal to express the interaction with the Fab antigen combing site exposing the 3-loops and 49 residues involved where LA-2 makes direct contact <ref name=Ding >PMID: 11183781</ref>. Studies were shown through findings from nuclear magnetic resonance, or [http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance  NMR] and [http://en.wikipedia.org/wiki/Protein_crystallization crystallization] <ref name=Ding >PMID: 11183781</ref>.




==== Binding Sites ====
The following is the fit mechanism where the conformations recognize LA-2 and shifts to optimize the complementary antigen combing site <ref name=Ding >PMID: 11183781</ref>. There were 49 residues from the “three loops” involved that significantly affected by LA-2 binding, through findings from NMR and crystallization <ref name=Ding >PMID: 11183781</ref>.  Residues 207 and 227 were excluded from analysis because of the peak overlap <ref name=Ding >PMID: 11183781</ref>. The portion of the protein chain detected through 15N-HSQC NMR that were affected by the binding of LA2 <ref name=Ding >PMID: 11183781</ref> was highlighted.<scene name='OspA_L03_Group2/Residues_203-220_loop1/2'>Residues 203 to 220 in "loop 1"</scene>” were represented by purple, <scene name='OspA_L03_Group2/Residues_224-233_loop2/2'>residues 224-233 in loop 2</scene> were colored green and <scene name='OspA_L03_Group2/Residues_246-257_loop3/3'>residues 247-257 in "loop 3"</scene>” were colored aqua blue The cool coloring of the residues shows the location of LA-2’s direct contact on the “3 loops”. Primary colors were used to represent <scene name='OspA_L03_Group2/Ala_208/4'>Ala-208</scene> as orange and <scene name='OspA_L03_Group2/Ala_215/4'>Ala-215</scene> as yellow in spacefills for the primary or initial identification of the LA-2 epitope on the beta-strands. The coloration of resides are all at one end, C-terminal of the isolated OspA molecule showing the side where LA-2 binds. The rest of the model were beta-sheets that were left yellow, as the neutral color, and the only alpha-helix was colored pink, to show the general overall structure of 21 anti-parrallel beta-strands to 1 alpha-helix <ref name=Ding >PMID: 11183781</ref>.


=='''Vaccinations'''==


OspA is the least variable protein located on the outer surface of the borrelia bacteria <ref name=Ding >PMID: 11183781</ref>.  This results in fewer [http://en.wikipedia.org/wiki/Mutations mutations] to occur allowing the same [http://en.wikipedia.org/wiki/Vaccine vaccine] to stay in use for a longer time.  Individuals who were bit by an infected tick and received the ospA vaccine were able to produce anti-opsA antibodies. These antibodies enter the tick through its blood meal into the gut where spirochetes are found and are destroyed by the antibody’s detection of opsA. Therefore, before the spirochete enters the host, it will be inactivated and Lyme Disease may be prevented <ref name=Marks>PMID: 21673416</ref>.


The complement system of the host also has a beneficial effect on destroying the borrelia located in the tick’s gut. It works by opsonizing these pathogens and attracting leukocytes which are a main component of the defense system against pathogens <ref name=Ruprecht >PMID: 18097481</ref>. Overall, the large percentage of the antibodies raised during the OspA vaccination must bind to antigen surfaces that overlap with the LA-2 epitope <ref name=Ding >PMID: 11183781</ref> in order for the Borrelia bacteria to enter apoptosis.


===''Side Effects''===   
Recent studies however have shown that neurological complications may appear in patients with this vaccination. This occurs because the down regulation of OspA from the vaccine would not be an abundant amount to activate the immune system for defense <ref name=Marks>PMID: 21673416</ref>. This may then cause a wide range of adverse events such as headaches and acute neuroborreliosis <ref name=Marks>PMID: 21673416</ref>.


==Past Experiments on the Effects of OspA==
Experiments were done using mice to test the effects of OspA on Borrelia bacteria as Lyme diseases  progressed in the host. Only when OspA expression was positive the borrelia bacteria in the host admitted to apoptosis. When the outer surface protein was not present the Borrelia bacteria survived and the mice later suffered through skin lesions <ref name=Ding >PMID: 11183781</ref>. On the other hand, if the vaccine with OspA is administered, or the bacteria has moved to a different environment of the [http://en.wikipedia.org/wiki/Cerebrospinal_fluid cerebrospinal fluid] or an inflammatory environment, the bacteria will activate and start to regulate OspA <ref name=Ding >PMID: 11183781</ref> by activating B-cells. Later this induced [http://en.wikipedia.org/wiki/Astrogliosis astrogliosis].




Line 55: Line 65:
<ref name=Ding >PMID: 11183781</ref>
<ref name=Ding >PMID: 11183781</ref>
<ref name=Ruprecht >PMID: 18097481</ref>
<ref name=Ruprecht >PMID: 18097481</ref>
<ref name=Wagner>PMID: 22336289</ref>
<ref name=Onrust>PMID: 10730551</ref>
<ref name=Marks>PMID: 21673416</ref>
<ref name=Burgdorferi>PMID: 7043737</ref>

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Chris DiFiore, Yimei Miao, Kareema Roushdy, Michal Harel
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