24 May 2012 

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CDC Greed (won't answer the FOIA)

ELISA = arbitrary cutoff.

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Overview

TUSKEGEE - By Jerry Leonard

1998, CIA Oilmen & Israelis plan to overthrow Saddam for the oil.

Bush/Gore  Oil/War-(Oct,2000)  

Bush's own explainer (Oct 2000) re: Iraq Oil

The Lyme Western Blot Bands (underconstruction)

Don't miss "Strain Tricks" Here

'Might be considered "Controversial", in terms of the vaccine trials. >>  Not being able to actually read the blots.

What's an antigen and what's a virulence determinant?  An antigen is something that stimulates the immune reponse, but is not necesarily a virulence determinant.  A virulence determinant is something generally that enhances invasiveness.  Neither of these terms is always used in these contexts.  When we are talking about burgdorferi, the lipoproteins seem to be both.  They may be part of a transporter complex, which makes sense, since these are parasites.  The Osps are generally ligands for various types of tissue.

For Example OspA probably binds connective tissue/perhaps chitin/collagen, and plasminogen, and OspC seems to prefer integrins (likely, on red blood cells, hence is a BIG virulence determinant, since the RBCs then carry the bug all through the body). 

The porins seem to be part of the transporter complex.  One would expect that since borrelia do not synthesize lipids (or much else for that matter, which is why they are parasites), they draw lipid components from host cell membrane, since trypsin is blocked by OspA.  

So, the various types of antigens/virulence determinants, are obviously flagellin, anything that looks like a heat shock protein/transporter/porin, anything that is part of the transporter complex, such as the ligands for the hosts (mammals, in our case), and then these things that are not lipoprotein >> the Variable Major Proteins, or the Variable Surface Proteins or whatever is today's flavor of jargon that means, not only are antigens coded from the Osps plasmids, variable, but also are the non-Osp, non-lipid membrane components.  And those, even more so, which is the reason, researchers looked for an invariable region of the variable surface membrane code, and they claim is the "C6 Peptide", or IR-6 for Invariable Region 6.   From this phenomenon, scientists became aware of the mechanism of Relapsing Fever.  Antigen Switch.

So, when borrelia are sonicated in a lab, and blotted as antigen, what fragments will we be looking at on a Western Blot, a method in which the substrate binds proteins?

The lipoproteins, the porins, the heat shock proteins, the flagellar polypeptide, something that anchors the flagella, and primarily things that are non-lipid and will be extracted into a non-lipid solvent.

The identity and function of all of them are not known.

But certainly lipoproteins of borrelia have high specificity.

The Porins, about the same specificity.

            In the mid-90s.  That means if you have one of these bands, and symptoms, you can be 90+ % certain, they are from Borrelia burgdorferi.

The Heat Shock Proteins, more specificity than they are given credit for:  Syntex, antibodies and T cells specific for the Bb HSP60t

(Steere, 1986)

The presense of antibodies to non-lipid, phospholipid (Lupus) and glycolipid, seem to be associated with more severe neurological illness.  (But nobody looks for those.)

What Blots look like :  What Blots look like: Compare the blots here, Arthritis and Acrodermatitis (on the right), vs. Neuroborreliosis (two sets of Western Blot lanes, on the left)   IGNORE THE REST OF THE ARTICLE  (Bologna Detector score 90%)  http://alpha1.mpk.med.uni-muenchen.de/bak/nrz-borrelia/miq-lyme/Frame-MiQ-microbiological53.html

The antibody concentration in the hyperresponsive ACA and arthritis presentation *is* the CDC's criteria for "Lyme Disease".   Most people do not have the genetic predisposition for reactive arthritis (Steere's HLA-DR4 or HLA-DR2), and so very few people actually have what the CDC calls "Lyme diosease", but plenty have all kinds of borelioses.

Abundant Silliness :  Essential to understanding your Western Blot test results, is that although the CDC currently requires a person to have 5 of 10 bands for a positive result, the vaccine antigens, OspA, were only one band.  Many physicians do not understand that any one specific band on a Western Blot is diagnostic.  For example, one of the first specific antibodies to appear after infection is OspB, which is 34 kilodaltons.

Although OspA and OspB are encoded on the same plasmid, and expressed early in infection, and are "primary" antigens, these were left out of the CDC's recommendations for diagnosis.

OspA and OspB are specific and primary, and OspsA were made into vaccines, but according to the CDC. if you have only one of these specific antibodies, you don't have "Lyme disease".  Enough to protect, but not enough to diagnose.  (This is difficult to comprehend, so this concept must be repeated in several different ways.)

Apparent molecular weights: (antigen is spotted, and antibody binding is read as concentration of absorbed light.  Technically, these are approximate molecular weights.  They are compared to each other in migration rates of the various sized (weight) antigens.)  GEL ELECTROPHORESIS

---------------------------------

BANDS------  Remember that these Western Blots are sloppy chromatography..  It's clear that better separation is possible.

7.4  hemolysin

7.5   lipoprotein  Philipp

10  (below)

11  (below)

12  (below)

13  (Barbour patent)

14  (lipoprotein)

18  (unknown, Weinstein and Steere identified it)

19  (Osp E)

22  (PC)

23  (OspC)

26  (OspF)

27  (below, Magnarelli)

28  (porin/toxin?  James Miller  )/ OspD 

29  (? OspF)

30  (Barbour- "virulence-associated antigen)

31  (OspA)

33  - 36 could be a/the   Vmp /VlsE and borrelia   ("The C6 Peptide") 

34 (OspB)

37  (Flagellin component - Barbara Johnon)

39 

41  Flagellin

45  (porin/toxin?)  (James Miller)

47

50  (Larry Zemel, seen in children)

55  (Fikrig)

57

58 (a protein, not a heat shock, function unidentified)

60  (heat shock protein)

62  (heat shock protein)

66  (porin/heat shock protein (different, but the same weight)

68 (heat shock protein)

72 (heat shock protein)

74

80 kDa

83/93/100 (coded on the chromosome, protoplasmi cylinder antigen, Dattwyler)

http://iai.asm.org/cgi/content/full/66/9/4115?view=full&pmid=9712756#B51

Lyme Disease-Causing Borrelia Species Encode Multiple Lipoproteins Homologous to Peptide-Binding Proteins of ABC-Type Transporters

Among the various cell envelope proteins of B. burgdorferi that have been described are the outer surface proteins OspA (29 kDa) (11), OspB (32 kDa) (11), OspC (23 kDa) (30), OspD (28 kDa) (57), OspE (19 kDa) (48), and OspF (26 kDa) (48), the 41-kDa flagellin protein (83), and other proteins with sizes of 18 kDa (17), 22 kDa (84), 27 kDa (65), 28 kDa (73), 35 kDa (33), 36 kDa (86), 39 kDa (72), 55 kDa (25), 66 kDa (13), 80 kDa (63), and 93 kDa (51). OspA has been shown to bind to human plasminogen (29). The flagellin protein is the major component of the periplasmic flagella (83). Although functional roles for the other cell envelope proteins are currently unknown, the 36-kDa surface-exposed lipoprotein VlsE undergoes extensive antigenic variation that may contribute to the ability of B. burgdorferi to evade the host immune response (86). In addition, several putative envelope proteins of B. burgdorferi appear to be expressed only in the infected mammalian host (17, 77, 82).

11.  Bergstrom, S., V. G. Bundoc, and A. G. Barbour. 1989. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol. Microbiol. 3:479-486[Medline].

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=2761388&dopt=Abstract

30.  Fuchs, R., S. Jauris, F. Lottspeich, V. Preac-Mursic, B. Wilske, 1and E. Soutschek. 1992. Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22 kDa protein (pC) in Escherichia coli. Mol. Microbiol. 6:503-509[Medline].

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=1560779&dopt=Abstract

57.  Norris, S. J., C. J. Carter, J. K. Howell, and A. G. Barbour. 1992. Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein. Infect. Immun. 60:4662-4672[Abstract].

http://iai.asm.org/cgi/content/abstract/60/11/4662?ijkey=LCNQLJnmLR6ZU

48.  Lam, T. T., T.-P. K. Nguyen, R. R. Montgomery, F. S. Kantor, E. Fikrig, and R. A. Flavell. 1994. Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease.  Infect. Immun. 62:290-298[Abstract]. link

83.  Wallich, R., S. E. Moter, M. M. Simon, K. Ebnet, A. Heiberger, and M. D. Kramer. 1990. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect. Immun. 58:1711-1719[Medline]. limk

18.  Chang, C., W.-J. Kuang, and E. Chen. 1986. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli.  Gene 44:121-125[Medline]. link

84.  Wallich, R., M. M. Simon, H. Hofmann, S. E. Moter, U. E. Schaible, and M. D. Kramer. 1993. Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi.  Infect. Immun.  61:4158-4166[Abstract]. link

65.  Reindl, M., B. Redl, and G. Stoffler. 1993. Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli. Mol. Microbiol. 8:1115-1124[Medline]. link

73.  Skare, J., C. Champion, T. Mirzabekov, E. Shang, D. Blanco, H. Erdjument-Bromage, P. Tempst, B. Kagan, J. Miller, and M. Lovett. 1996. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi. J. Bacteriol. 178:4909-4918[Abstract]. link

33.  Gilmore, R., K. Kappel, and B. Johnson. 1997. Molecular characterization of a 35-kilodalton protein of Borrelia burgdorferi, an antigen of diagnostic importance in early Lyme disease. J. Clin. Microbiol. 35:86-91[Abstract]. link

86.  Zhang, J., J. Hardham, A. Barbour, and S. Norris. 1997. Antigenic variation in Lyme disease Borreliae by promiscuous recombination of VMP-like sequence cassettes. Cell 89:275-285[Medline].link

72.  Simpson, W., W. Cieplak, M. Schrumpf, A. Barbour, and T. Schwan. 1994. Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen. FEMS Microbiol. Lett. 119:381-387[Medline]. link

25.  Feng, S., S. Das, T. Lam, R. Flavell, and E. Fikrig. 1995. A 55-kilodalton antigen encoded by a gene on a Borrelia burgdorferi 49-kilobase plasmid is recognized by antibodies in sera from patients with Lyme disease. Infect. Immun. 63:3459-3466[Abstract]. link

13.  Bunikis, J., L. Noppa, Y. Ostberg, A. Barbour, and S. Bergstrom. 1996. Surface exposure and species specificity of an immunoreactive domain of a 66-kilodalton outer membrane protein (P66) of the Borrelia spp. that cause Lyme disease. Infect. Immun. 64:5111-5116[Abstract]. link

[80/83]

63.  Perng, G., R. LeFebvre, and R. Johnson. 1991. Further characterization of a potent immunogen and the chromosomal gene encoding it in the Lyme disease agent, Borrelia burgdorferi. Infect. Immun. 59:2070-2074[Medline].

51.  Luft, B., S. Mudri, W. Jiang, R. Dattwyler, P. Gorevic, T. Fischer, P. Munoz, J. Dunn, and W. Schubach. 1992. The 93-kilodalton protein of Borrelia burgdorferi: an immunodominant protoplasmic cylinder antigen. Infect. Immun. 60:4309-4321[Abstract] link

"The derived amino acid sequence predicted it to be predominantly alpha helical."

Patent: Inventors: Dunn; John J. (Bellport, NY); Luft; Benjamin J. (Port Jefferson, NY)

Assignee: Research Foundation State University of New York (Stony Brook, NY); Brookhaven  Science Associates (Upton, NY)  link

A human mAb secreting EBV-transformed B cell line, D7, has been developed that is specific for a 93-kDa B. burgdorferi protein and has been used to characterize this potentially important Ag. D7 produces an IgG3 antibody that detects the 93-kDa Ag as well as smaller fragments at 46 kDa and lower molecular mass. The antibody detects similar epitopes on all B. burgdorferi isolates tested and on a Borrelia hermsii protein with molecular mass greater than 100 kDa but binds poorly to Treponema species. In contrast, polyclonal sera from Lyme disease patients show little binding to the homologous Ag in B. hermsii. Structurally, the 93-kDa protein is associated with the flagellum and may be firmly anchored in the protoplasmic cylinder.

In addition to these:

55kD:   Feng S, Barthold SW, Telford SR 3rd, Fikrig E., P55, an immunogenic but nonprotective 55-kilodalton Borrelia burgdorferi protein in murine Lyme disease., Infect Immun. 1996 Jan;64(1):363-5. PMID: 8557366

Feng S, Das S, Lam T, Flavell RA, Fikrig E., A 55-kilodalton antigen encoded by a gene on a Borrelia burgdorferi 49-kilobase plasmid is recognized by antibodies in sera from patients with Lyme disease. Infect Immun. 1995 Sep;63(9):3459-66. PMID: 7642278

3-5kD Glycolipid

Oschmann P, Wellensiek HJ, Zhong W, Dorndorf W, Pflughaupt KW.  Relationship between the Borrelia burgdorferi specific immune response and different stages and syndromes in neuroborreliosis.  Infection. 1997 Sep-Oct;25(5):292-7. PMID: 9334864

"Antibodies against certain proteins and the glycolipid of B. burgdorferi seem to have a prognostic value as to the development of more severe disease or transition to stage III."

Eiffert H, Lotter H, Jarecki-Khan K, Thomssen R., Identification of an immunoreactive non-proteinaleous component in Borrelia burgdorferi., Med Microbiol Immunol (Berl). 1991;180(5):229-37. PMID: 1722277

14kD:

Honarvar N, Schaible UE, Galanos C, Wallich R, Simon MM., A 14,000 MW lipoprotein and a glycolipid-like structure of Borrelia burgdorferi induce proliferation and immunoglobulin production in mouse B cells at high frequencies., Immunology. 1994 Jul;82(3):389-96., PMID: 7959873

Virulence associated proteins in Borrelia burgdorferi (BB)

Abstract

The invention relates to a DNA segment encoding a Borrelia burgdorferi antigenic polypeptide. The invention also relates to a purified 30 kDa polypeptide isolated from a virulent strain of B. burgdorferi and to epitopic segments of the polypeptide with immunogenic potential. The 30 kDa protein provides a route for the development of immunodiagnostics for Lyme disease and related disorders. The 30 kDa protein and related amino acid and DNA sequences may also be used for the immunization, for the detection of B. burgdorferi in human or animal tissues or body fluids, and also for the generation of specific antibodies for use in diagnosis, epidemiology, and prevention of Lyme disease.

Methods and compositions including a 13kD B. burgdorferi protein

Abstract

All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osp). Mutants of B. burgdorferi lacking Osp proteins were selected with polyclonal or monoclonal antibodies at a frequency of 10.sup.-6 to 10.sup.-5. One mutant that lacked OspA, B, C and D was further characterized in the present study. It was distinguished from the OspA.sup.+ B.sup.+ cells by its (i) auto-aggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum- and complement-sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in the skin the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two different classes, A and B, of monoclonal antibodies (mAb) with growth inhibiting properties for mutant cells were produced. Class A mAbs bound to 13 kDa surface proteins of B. burgdorferi sensu stricto and of B. afzelii. The minimum inhibitory concentration of the Fab fragment of one mAb of this class was 0.2 .mu.g/ml. Class B mAbs did not bind by Western Blot to B. burgdorferi cells but reacted with cells in an unfixed cell immunofluorescence assay and growth inhibition assay. These studies revealed hitherto unknown functional aspects of Osp proteins, notably serum-resistance, and indicated that in the absence of Osp proteins other antigens are expressed or become accessible at the cell's surface.

K. M. Dickson