Monday, February 21, 2011

7 Patent Watch: VMP-Like Sequences of Pathogenic Borrelia

To the dude that keeps writing in his blog that chronic Lyme patients are hypochondriacs, and to A.C. Steere, who in his March 2010 Powerpoint presentation insisted that there is no such thing as Chronic Lyme Disease and it is a misnomer, I have one question to ask:

If Lyme disease is easy to diagnose and treat and not chronic - as you and the IDSA have stated - why do people in the field put this stuff in a patent application posted December 2010?

Application number: 12/853,019
Publication number: US 2010/0317026 A1
Filing date: Aug 9, 2010

Check it out on Google Patents. You can download it as a PDF file on the upper right corner of your window.

Select snippets for your viewing enjoyment:

First, we'll start with the abstract so you know what they plan to do with these VMP-Like Sequences of DNA, anyway. Oh, vaccines? Why yes. But also possibility of therapeutic applications and in immunoblots as reagents.


If you download and read the entire thing, though, you either need to a) have some focus or b) a little of insanity or c) possibly both to get through it. If you do bother to download the PDF in its entirety, I recommend that you start looking at the early pages and skip over a few pages about a third through, then read again, then skip the DNA sequencing pages at the end - unless you are a molecular biologist or geneticist... then it will be more fun for you to read all of it.



[0006] These organisms are closely related and cause similar manifestations with multiple stages: an expanding rash at the site of the tick bite (erythema migrans), fever, lymphadenopathy, fatigue, and malaise; effects of disseminated infection, including carditis, meningoradiculitis, and polyarthritis; and chronic manifestations including arthritis and neurologic disorders. Lyme disease is often difficult to diagnose because of shared manifestations with other disorders, and it can also be refractory to treatment during late stages of the disease.

re·frac·to·ry
 (r-frkt-r)

adj.
1. Resistant to treatment, as a disease.
2. Unresponsive to stimuli, as a muscle or nerve fiber.

(Did anyone make a checklist out of reading the above symptoms and nod "yes" to them? I did...)

[0007] B. burgdorferi, the etiologic agent of Lyme disease, is able to persist for years in patients or animals despite the presence of an active immune response (Steere, 1989; Schutzer, 1992).


[0009] Lyme disease may be disabling (particularly in its chronic form), and thus there is a need for effective therapeutic and prophylactic treatment. (Noooo...  You think?)

[0010] However, animal studies indicate that OspA vaccination may not be effective against all strains of Lyme disease Borreliae. OspA is also not useful for immunodiagnosis, due to weak antibody responses to OspA in Lyme disease patients. (Wait... but... Lymerix... I thought you guys said you pulled it due to lack of sales? Oh shhhh... that's not what I heard...)

[0020] An important aspect of the invention is the recognition that Borrelia VMP-like sequences recombine at the vls site, with the result that antigenic variation is virtually limitless. Multiclonal populations therefore can exist in an infected patient so that immunological defenses are severely tested if not totally overwhelmed. Thus there is now the opportunity to develop more effective combinations of immunogens for protection against Borrelia infections or as preventative inoculations such as in the form of cocktails of multiple antigenic variants based on a base series of combinatorial VMP-like antigens.


[0127] The present work discloses the identification and characterization of an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, vlsE. A 28-kilobase plasmid of B. burgdorferi B31 (pBB28La) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 non-expressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. The resulting combinatorial variation will potentially produce millions of unique antigenic variants and thereby contribute to immune system evasion, long-term survival, and pathogensis in the mammalian host.

(Note: C3H/HeN mice are reported to develop severe arthritis when infected with B. burgdorferi.)




These observations suggest that the vls locus may provide the Lyme disease Borreliae with the capability of antigenic variation analogous to the vmp system of B. hermsii (Barbour, 1993). The above similarities also indicate that the vlsE gene, silent vls cassettes, and large vmp genes of relapsing fever organisms, all evolved from a common ancestral gene. Their relatively high G+C compositions (e.g. 45% for vlsE and 37% for vmp17) when compared with Borrelia G+C content (~28%) are also consistent with this evolutionary relationship, and further suggest the possibility of lateral transfer from other organisms(Okay, these are more "may" and "indicate" and "suggest" statements, but given the weight of the evidence so far... something to consider.)

[0130] Lastly, each phase of B. hermsii infection is caused predominantly by organisms expressing a single vmp allele (Meier et al. 1985; Plasterk et al. 1985), whereas a high degree of vlsE allele variation occurs among organisms isolated even from a small ear biopsy specimen during B. burgdorferi infection.
[0137] Variation of B. burgdorferi surface proteins such as VlsE may also effect the organism's virulence and its ability to adapt to different micro-environments during infection of the mammalian host. Recent studies of a Borrelia turicatae mouse infection model that resembles Lyme disease showed that one serotype expressing VmpB exhibited more severe arthritic manifestations, whereas another expressing VmpA had more severe central nervous system involvement (Cadavid et al, 1994). The numbers of Borreliae present in the joints and blood of serotype B-infected mice were much higher than those of mice infected with serotype A, consistent with a relationship between Vmp serotype and disease severity (Pennington et al, 1997). (And? Where was the Borreliae present in mice in serotype A? Hm?)
[0138] The importance of the vls-containing plasmid, pBB28La, during infection is supported by the following evidence: (i) all high-infectivity clones and strains tested thus far contain the vls-containing plasmid pBB28LA and loss of this plasmid correlates with a decrease in infectivity; (ii) pBB28La was maintained in all animal isolates tested thus far, and (iii) the vls sequences are preserved among three Lyme genospecies despite their genetic heterogeneity (Casjens et al, 1995).

[0139] VlsE (or, potentially, other genes encoded by pBB28La) appears to have another important but undefined function which is unrelated to antigenic variation. Low-infectivity clones lacking the vls-encoding plasmid pBB28La do not propagate in severe combined immunodeficiency (SCID) mice, indicating that the required factor(s) provides an important function unrelated to evasion of the adaptive immune system.

Also, in vivo selection against Bb clones lacking pBB28La appears to occur early in infection (within the first week), before the adaptive immune response would be expected to exert significant selection pressure. Therefore, it is likely that vlsE plays an important role in some aspect of infection (e.g. colonization, dissemination, adherence, extravasation, evasion of innate immune mechanisms, or nutrient acquisition), and that antigenic variation merely permits surface expression of this protein without leading to elimination of bacteria by the host's immune response.

[1041]  A genetic locus (called vmp-like sequence or vls) has been identified and characterized in B. burgdorferi that surprisingly resembles the vmp system of B. hermsii. [...] Examination of ear and blood isolates from C3H/HeN mice infected 4 weeks previously with B31 clone 5A3 demonstrated the occurrence of promiscuous recombination at the vlsE site, such that each of B. burgdorferi clones examined was unique and appeared to have undergone multiple recombination events with portions of the silent vls cassettes. The resultant vlsE variants exhibited a decreased reactivity to antiserum directed against the parental Vls1 cassette region. This elaborate genetic system permits combinatorial antigenic variation of vlsE in the mammalian host, thereby contributing to evasion of the immune response and long-term survival in the mammalian host.
Etc...
[0145] This mechanism of genetic switching appears to be different from any other antigenic variation mechanism described in bacteria or protozoa and has important implications in Lyme disease. By combining different regions of the silent vls cassettes, it is possible for many different vlsE serotypes to coexist the same patient. It may be impossible for the host to mount a protective response against any one of these clonal populations, because of the small number of each type. Even mounting a response against one serotype would not protect against rapidly evolving, new serotypes. The fact that B. burgdorferi has evolved such an elaborate mechanism for varying the sequence of VlsE indicates the importance of the protein in pathogenesis and/or immune evasion.
[0294] Since the C3H/HeN mice were infected with a large number (105)  of the organisms, it was possible that the antibody response against vlsE had resulted from the intial inocolum. To test this possibility, sera from the white-footed mice (Peromyscus leucopus) infected with B. burgdorferi B31 via tick bite and from human Lyme disease patients were used to react with the similar immunoblots. The representative results depicted showed that tick-infested Peromyscus leucopus mice also had strong reactivity to the VlsE protein of B. burgdorferi B31-5A3 and GST-Vsl fusion protein but not with GST alone. These results were further confirmed with sera from Lyme disease patients. [...] These results indicate that VlsE is expressed and is highly immunogenic in the mammalian host, but that genetic variation may generate unique VlsE variants which are no longer fully recognized by the immune response against the parental vlsE. They also indicate that antibodies generated against VlsE may be useful in immunodiagnosis of Lyme disease. (Got new tests, anybody? I hope this is a good thing!)
[0295] (Contains test data that just confirms more of what was said further upstream, but thought I'd add it here...)

Seriously, this is fascinating stuff, and I really hope that the knowledge about vlsE can be put to good use. My immediate thoughts, of course, are to ask how this can be used to create new treatments for Lyme disease and improve testing - as well as if a safe and effective vaccine can be developed. The vaccine issue - as always - is touchy, and is no different in this case... especially when they are proposing multiple shots will be needed over time. Also, there is more detailed information in the remainder of the patent describing ways of using bacteriophage therapy or attaching DNA to recombinant adenoviruses for  gene therapy treatment.

But the take home point I'm making here by sharing portions of this patent (and it is a multipage document, with lots of pages of data and genetic sequencing that most people will not want to plow through) is that Lyme diseases's Borrelia burgdorferi is unique, and closely related to relapsing fever, and has genetic behavior which is similar to - yet different from - relapsing fever.

Borrelia burgdorferi is highly complex in its presentation, multiple sources have stated that it can be refractory to treatment, and it has a chronic manifestation. It's all right here.
"This mechanism of genetic switching appears to be different from any other antigenic variation mechanism described in bacteria or protozoa and has important implications in Lyme disease. By combining different regions of the silent vls cassettes, it is possible for many different vlsE serotypes to coexist the same patient. It may be impossible for the host to mount a protective response against any one of these clonal populations, because of the small number of each typeEven mounting a response against one serotype would not protect against rapidly evolving, new serotypes."
We can't ignore this. The scientific truth isn't going to go away, whether it is posted in this patent or in the papers to which it refers.

ADDENDUM

There are more entries posted here related to this one. If you were interested in this post, check out these  - especially the one on the vlsE test kit package insert:
http://campother.blogspot.com/2011/02/more-on-that-vmp-like-sequence-aka-vlse.html
http://campother.blogspot.com/2011/02/package-insert-excerpt-athena-multi.html
http://campother.blogspot.com/2011/02/history-of-antigenic-variation-in.html

7 comments:

  1. great sleuthing! Couldn't help but notice the citation of a Steere study to support persistence...who does AS think he's fooling? Apparently, legions! how quickly they "forget" when the incentives are great enough.
    -NB

    ReplyDelete
  2. Thanks, NB. I was up late researching the effects of certain ketamine doses on mice immune systems when I came across this patent... long story short, I couldn't resist reading it and had to post it. I hope you've reread it now that I've included more excerpts from the patent, and if you can, get a chance to download and read it yourself.

    If a lot of it is incomprehensible, it's because it has to be worded to cover potential future applications for the patent (one can continue to add more sections to tighten up loopholes presented by the original patent) and because the authors are Bb researchers. But that doesn't mean that a) we can't glean something from it, even if we are not experts in this field, and b) can't ask our friends who are working in molecular and micro to take a look at it.

    ReplyDelete
  3. Thank you for such an excellent post I hope you don't mind me sharing this on Eurloyme (Chat line) and on my blog http://lookingatlyme.blogspot.com/2011/02/idsa-guideline-author-on-failure-of.html

    ReplyDelete
  4. Joanne, you're welcome - feel free to share - just credit Camp Other and link back when you pass it along. Thanks!

    I've read portions of your journal before and found it interesting. What you posted here in October was significant: http://lookingatlyme.blogspot.com/2010/10/tipping-point-in-science-surrounding.html.

    I really think it is exposing the science around Borrelia that is going to push things entirely over the tipping point - those who are funding the research and paying for vaccines and drug development need to be accountable (regardless of their position on the Lyme controversy) when the science is out there and recognized.

    May I link back to your journal on my blog roll?

    ReplyDelete
  5. The citations you give for the patent entitled "VMP-like sequences of pathogenic Borrelia" are incorrect. The application was submitted by Steven Norris et al. and filed on 8/16/2002. The application number is: 10/222,566. The Publication Number is: US 2004/0044192 A1. It should be noted that some of the scientists in this group are actively involved is research on a relapsing form of infection caused by Borrelia hermsii, which is quite different from B. burgdorferi, the agent of Lyme disease. In fairness to Alan Steere, the quote attributed to him re: persistent infection was derived from an old paper published in 1989. Since we now have much more information on Lyme disease now than we did then, his views and the views of others have changed. Remember: Lyme disease was first reported in the U.S. in 1985.

    ReplyDelete
  6. Anonymous:

    Please refer to this later post:
    http://campother.blogspot.com/2011/02/history-of-antigenic-variation-in.html

    It addresses the statements you make - I discuss the earlier patent application from 2002 there as well as the connection to Borrelia hermsii.

    If old citations no longer support a patent and its introductory background - and the state of the science has moved on - then maybe applicants should remove them and use only the most recent citations which support their patent and its claims.

    I would be interested in hearing Steere's story about why his views have changed and what the turning point was for him. There has been much speculation in the Lyme disease patient community about Steere and IDSA Lyme disease guideline members' focusing more on Chronic Lyme disease/PLDS as an autoimmune condition - but in their eyes, not enough explanation as to why he/they think this is so.

    ReplyDelete
  7. One more thing, Anonymous...

    "Remember: Lyme disease was first reported in the U.S. in 1985."

    Which reference do you use for this date, offhand? I thought Steere was reviewing cases in Polly Murray's backyard in 1975, and the causative agent for Lyme disease was discovered by Willy Burgdorfer in 1982 and showed up in Science magazine shortly thereafter. 1985 as the year in which Lyme disease was first reported is a later date than I have seen from any reference source cited.

    ReplyDelete

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