0 A History of Antigenic Variation in Borrelia hermsii, burgdorferi

I decided to investigate more of Norris et al's patent history, and found that the first patent Norris filed was back in 1991, entitled Virulence Associated Proteins In Borrelia Burgdorferi (BB).

I also learned that the patent I had posted earlier this week - VMP-LIKE SEQUENCES OF PATHOGENIC BORRELIA - was first applied for as early as 2002, and decided to do a head-to-head comparison of the 2010 and 2002 patent. Out of all the points I had highlighted and posted earlier this week, they were about 99% the same.

Essentially, what has been known about vlsE's role in antigenic variation in Borrelia burgdorferi has been known for a number of years before I posted about it this week.

From VMP-LIKE SEQUENCES OF PATHOGENIC BORRELIA
Patent number: 6719983
Filing date: Aug 16, 2002
Issue date: Apr 13, 2004
Application number: 10/222,566

This application is a Divisional Application of U.S. application Ser. No. 09/125,619 filed on Jan. 27, 1999, now 5 U.S. Pat. No. 6,437,116 which is a continuation of PCT Application PCT/US97/02952 filed Feb. 20, 1997, which is a continuation-in-part and claims priority to Provisional Application Ser. No. 60/012,028 filed on Feb. 21, 1996.

That's a lot of patent applications there.

What was known in 2010 was known back in 2002, and some time before then.

The 1991 patent is different in one major respect from its successors: There is no mention of antigenic variation as either a possible mechanism for evading the immune system or as a known fact.

From this one may guess that there were huge strides made in understanding antigenic variation in Borrelia burgdorferi that happened within that decade.

According to research, Borrelia hermsii was known to have antigenic variation many years prior to the discovery of Borrelia burgdorferi's antigenic variation.

The first record of Borrelia hermsii's antigenic variation was in the Journal of Experimental Medicine, Volume 156, Issue 5, 1982, Pages 1297-1311:

Antigenic variation of Borrelia hermsii

Stoenner, H.G., Dodd, T., Larsen, C.
Rocky Mountain Lab., Natl. Inst. Allergy Infect. Dis., NIH,
Hamilton, MT 59840, United States

Abstract 

At least 24 different serotypes were detected in populations of Borrelia hermsii that originated from a single organism. These serotypes were identified by staining with specific fluoresceinated antisera prepared against cloned populations of living organisms of each type.

In the order of decreasing frequency, the 10 types more often encountered were 7, which was clearly dominant, and 2, 17, 24, 13, 2, 1, 21, 11, and 12. Each of the 24 types were shown to change to 7 or more other serotypes. Spirochetemia in mice was persistent, and relapses occurred when the concentration of organisms was sufficient for detection by visual means.

After mice were inoculated with a single organism, peak spirochetemia usually occurred on day 4, after which clearance of organisms occurred, and an apparently pure population was replaced by a mixed population consisting of as many as seven variants.

These types persisted for 2-3 d before being replaced by other types. Conversions occurred constantly and were independent of relapses.

The rate of conversion in mice treated with cyclophosphamide to delay antibody production was comparable to that of controls.

Spontaneous conversion was clearly demonstrated in tubes of fortified Kelly's medium inoculated with a single organism of type 7 or 21. 11 different variants appeared in eight cultures of type 21 by the time growth had reached 4 x 10⁶-10⁷ organisms/ml. The rate of spontaneous change was estimated to be
~ 10^-4-10^-3 per cell per generation.

That was in 1982.

And who should be working with Stoenner back at this time, but Barbour?

Indeed, in the same year, Barbour published another paper, with S. L. Tessier, and H. G. Stoenner - Variable major proteins of Borrelia hermsii. J. Exp. Med. 156:1312-1324.

So they were right there at the beginning, when antigenic variation was discovered in Borrelia hermsii.

So why did it take so long to find out Borrelia burgdorferi has antigenic variation?

Because the bacteria known to cause Lyme disease wasn't identified until that same year - 1982.

It actually didn't take all that long.

This particular spirochete had yet to really be studied, and studied with an increasing level of technology that bacteriologists did not possess in earlier generations.

According to research, Borrelia burgdorferi's antigenic variation wasn't known until the late 1990's.

This article was posted in Current Biology,Volume 7, Issue 9, 1 September 1997, Pages R538-R540:

Bacterial pathogenesis: A variation on variation in Lyme disease

Michael Koomey
Department of Microbiology and Immunology,
University of Michigan Medical School,
Ann Arbor, Michigan 48109, USA

Abstract

The discovery of antigenic variation in Borrelia burgdorferi, the bacterium that causes Lyme disease, provides a potential explanation for the chronic nature of infection as well as new insights into the genetic structure of highly recombinogenic loci responsible for combinatorial genetic diversification.

Text

Microbial pathogens evolve many strategies aimed at evading the immunoprotective surveillance systems that operate in mammals. Perhaps the most direct countermeasure to the humoral wing of the immune system — the part involving recognition by specific antibodies — is antigenic variation, the process in which the primary structure and antigenicity of key surface proteins are altered without perturbing their function(s). These changes are achieved almost without exception by the reassortment and recombination of repeated genes or gene segments [1]. Such combinatorial mechanisms of genetic diversification have been found to underlie high frequency changes in the surface components of a wide variety of pathogens — trypanosomes [2], malaria parasites [3], relapsing-fever-causing Borrelia species [4] and the gonorrheal agent Neisseria gonorrhoeae [5]. And now the Lyme disease pathogen, Borrelia burgdorferi can be added to this list; the recent discovery of antigenic variation in this species may explain the chronic nature of Lyme disease.

In each of the pathogens that have been found to exhibit antigenic variation, the phenomenon has been discovered and characterized by a sequence of observations starting with biochemical and immunochemical documentation of inter-strain, and subsequently intra-strain, variation of a predominant surface component. This initial observation was followed by cloning of the structural gene encoding the variable surface component, and the identification of multiple copies of related but divergent gene copies or elements. It then became a relatively straightforward matter (in retrospect) to document the DNA rearrangements and recombination of related genes that are responsible for the antigenic variation.

Relapsing fever, caused by Borreliae hermsii and related species, is arguably the best understood disease involving antigenic variation. The initial infection occurs during the bite from an infected tick, or a louse during epidemics, and following this the infected animal undergoes waves of spirochetemia and accompanying fever. The relapses are associated with the clonal emergence in the bloodstream of a variant expressing a novel variable major protein [6]. After up to ten such waves of parasitemia, occurring every four to seven days, the individual or animal can succumb or recover completely.

Lyme disease is a tick-borne infection caused by the spirochete B. burgdorferi, which is related to the relapsing fever borreliae [7]. In stark contrast to relapsing fever, which has been relatively quiescent for almost 50 years, Lyme disease is currently the most common arthropodborne disease in both North America and Europe. Although rarely fatal, the manifestations of Lyme disease can be physically and emotionally debilitating [7]. Early human infection is usually characterized by a spreading skin rash (erythema migrans) and flu-like illness, which is self-limiting. In the following weeks to months, most untreated infected individuals proceed into a chronic late disease characterized by systemic involvement of the joints, brain, nerves, eyes and heart [8].

From these presentations alone, it seemed likely that antigenic variation would play a role in the immune evasiveness displayed by B. burgdorferi. Subtle differences in highly expressed surface lipoproteins were found between B. burgdorferi strains, but no compelling evidence for major antigenic changes [9] or gross genetic rearrangements [10] was initially forthcoming, despite indirect evidence suggesting that antigenic shifts can occur during mouse infection [11] and [12]. One major problem has been that there are as yet no experimental animal models that mimic the course of Lyme disease and in which spirochetemias can be detected. To complicate matters further, basic methodologies for gene transfer and mutant isolation in borrelia spirochetes have been slow in developing.

In the absence of many of the reagents that play such an important part in current studies of bacterial infectious disease, it is not surprising that the identification of antigenic variation in Lyme disease borreliae came about through a rather circuitous series of findings and experiments. This story begins in much the same way as many of the early studies of microbial pathogenesis, with the observation that passage of the organism in the laboratory leads to the recovery of variants or mutants of reduced virulence. In the case of Lyme disease borreliae, it was well documented that they lose their ability to infect laboratory animals following ten or more blind in vitro passages [13] and [14]. Attempts to define the genetic basis for this attenuation phenomenon were complicated by the weaknesses of the system noted above. Moreover, B. burgdorferi contains a linear chromosome and a complex set of multiple circular and linear plasmid DNA species. The extrachromosomal plasmid profiles exhibit instability during laboratory cultivation, but it was difficult to correlate the presence of particular plasmids or known surface components with infectivity [15].

As the low-infectivity B. burgdorferi strains failed to give rise to virulent revertants in animals, Zhang et al. [16] surmised that the loss of one or more of the extrachromosomal elements might account for the irreversible genetic alteration. By using subtractive hybridization, they went on to identify sequences that are present only in high-infectivity strains. Characterization of one of the resulting clones revealed the presence of a single open reading frame, encoding a putative protein with greater than 25% amino acid sequence identity to a variable membrane lipoprotein (Vmp) of B. hermsii, the relapsing fever agent.

Using this clone as a DNA probe, Zhang et al. [16] were able to establish that the Vmp-like sequence (vls) resides on a 28 kilobase (kb) linear plasmid that was absent from the vast majority of low-infectivity strains enriched during in vitro passage. Further sequence characterization of the linear plasmid revealed the presence of an extensive vls locus consisting of a single telomeric gene expression site, vlsE, and 15 tandemly arrayed, non-expressed vls cassettes whose derived peptide sequences correspond to the central 200 residues of the predicted vlsE product. In light of the finding that the central segments encoded by the vls cassettes were closely related but not identical to one another, the genetic foundation for VlsE antigenic variability was established.

From findings in other systems that exhibit antigenic variation, it was expected that genetic recombination between the variant-encoding vls cassettes and the expressed locus would turn out to be the mechanism that generates VlsE antigenic variability. To examine this possibility, Zhang et al. [16] compared the vlsE DNA sequences from clones recovered from mice following four weeks of infection. By comparison with the vlsE of the inoculum clone, numerous nucleotide substitutions, insertions and deletions were found in the gene from each reisolate, which could only be accounted for by repeated rounds of templated recombination with individual vls cassettes. As a climax to the work, they demonstrated that the products of these vlsE alleles display altered levels of immunoreactivity with sera raised against the parental VlsE, sera from a white-footed mouse (the natural host for B. burgdorferi) infected by a tick bite, and sera from a human Lyme disease patient.

Given the similarity in sequence of the B. burgdorferi Vls and B. hermsii Vmp proteins, it is surprising that their mechanisms of antigen variation are so different. In relapsing fever, non-expressed B. hermsii vmp genes — of which there are more than 25 per strain — are carried in linear, storage plasmids whereas the single active gene — the expression locus — is telomerically located on a distinct linear plasmid [17]. Antigenic variation in B. hermsii occurs by partial or complete replacement of the vmp gene at the expression locus by any one of the silent vmp gene copies. [4], [18] and [19]. Many facets of this process are similar to the mechanism of antigenic variation of surface glycoproteins in African trypanosomes [2].

Antigenic variation of the Lyme disease agent B. burgdorferi, in contrast, involves the use of partial gene segments that are tightly clustered immediately upstream of the vls expression locus, an arrangement that is more similar in its overall design to what is seen in the avian immunoglobulin [20] and N. gonorrhoeae pilin [21] combinatorial diversification systems. In each of these three systems, despite the fact that a relatively small number of non-expressed alleles or pseudogenes are used as templates, an extremely high level of diversity is achieved by multiple rounds of recombination with different donor alleles or different length tracts of a single allele.

The key to each of these systems appears to be the ability to undergo efficient intragenic recombination within short homologous segments of nucleotides, which encode conserved domains within the proteins. A second common feature of these systems is the extremely high frequency with which variants arise in vivo compared with that occurring in vitro. For example, in the chicken, the complete pre-immune repertoire of immunoglobulin light chain genes is generated within the bursa of Fabricius in a matter of days by multiple segmental recombination events between a single rearranged variable (V) gene and 25 V pseudogenes [20]. Pilus expression by N. gonorrhoeae is quite stable in the laboratory, whereas an extensive mixture of antigenic variants are found at the earliest time points following infection [5] and [22]. For B. burgdorferi, Zhang et al. [16] have coined the term ‘promiscuous’ recombination to describe the extensive vls rearrangements that they suggest are induced when in the mammalian host. It will be very exciting to see what role the conserved gene organization of these systems plays in high frequency geneconversion-like events, and what signals are responsible for the enhanced recombination rates seen in vivo.

The potential impact of the discovery of Vls antigenic variation on the study of Lyme disease pathogenesis is enormous. As is so often the case with scientific break-throughs, many important and readily obvious questions can now be addressed. Does Vls antigenic variation occur in infected humans? What is the function of the Vls protein? Is the loss of infectivity of in vitro passaged clones accounted for by their lack of vls expression, or is it associated with another plasmid-encoded product?

With regard to immunization against Lyme disease, it will be of interest to determine if antibodies directed at conserved Vls domains are protective or modify the course of Vls variation. By chance, the new findings have come at a time when favorable results of phase III human vaccine trials with antigenically stable borrelia antigens are being reported. But as we have learned from other vaccines as well as from antibiotics and the development of resistance, current formulations can almost always be improved and the discovery of Vls may have important implications for a second generation of Lyme disease vaccines. The new findings also coincide with the impending completion of the B. burgdorferi genome sequence and the first report of gene transfer in borrelia [23], so it seems assured that research into this important pathogen will proceed at an accelerated pace.

References

[1] P Borst, Molecular genetics of antigenic variation, Immunol Today 12 (1991), pp. 29–33 91299105.

[2] P Borst, JH Gommers Ampt, MJ Ligtenberg, G Rudenko, R Kieft, MC Taylor, PA Blundell and F van Leeuwen, Control of antigenic variation in African trypanosomes, Cold Spring Harb Symp Quant Biol 58 (1993), p. 105 95044072.

[3] LH Van der Ploeg, K Gottesdiener and MG Lee, Antigenic variation in African trypanosomes, Trends Genet 8 (1992), pp. 452–457 93150551.

[4] AG Barbour, N Burman, CJ Carter, T Kitten and S Bergstrom, Variable antigen genes of the relapsing fever agent Borrelia hermsii are activated by promoter addition, Mol Microbiol 5 (1991), pp. 489–493 91251780.

[5] J Swanson, K Robbins, O Barrera, D Corwin, J Boslego, J Ciak, M Blake and JM Koomey, Gonococcal pilin variants in experimental gonorrhea, J Exp Med 165 (1987), pp. 1344–1357 87197065.

[6] HG Stoenner, T Dodd and C Larsen, Antigenic variation in Borrelia hermsii, J Exp Med 156 (1982), pp. 1297–1311 83032379.

[7] AG Barbour and D Fish, The biological and social phenomenon of Lyme disease, Science 260 (1993), pp. 1610–1616 93276286.

[8] AC Steere, Lyme disease, N Engl J Med 321 (1989), pp. 586–596 89344179.

[9] SW Barthold, Antigenic stability of Borrelia burgdorferi during chronic infections of immunocompetent mice, Infect Immun 61 (1993), pp. 4955–4961 94041611.

[10] B Stevenson, LK Bockenstedt and SW Barthold, Expression and gene sequence of outer surface protein C of Borrelia burgdorferi reisolated from chronically infected mice, Infect Immun 62 (1994), pp. 3568–3571 94314484.

[11] W Burgdorfer and TG Schwan, Lyme borreliosis: a relapsing fever-like disease?, Scand J Infect Dis Suppl 77 (1991), pp. 17–22 92054270.

[12] TG Schwan, RH Karstens, ME Schrumpf and WJ Simpson, Changes in antigenic reactivity of Borrelia burgdorferi, the Lyme disease spirochete, during persistent infection in mice, Can J Microbiol 37 (1991), pp. 450–454 92005004.

[13] SJ Norris, JK Howell, SA Garza, MS Ferdows and AG Barbour, High- and low-infectivity phenotypes of clonal populations of in vitro- cultured Borrelia burgdorferi, Infect Immun 63 (1995), pp. 2206–2212 95286265.

[14] TG Schwan, W Burgdorfer and CF Garon, Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation, Infect Immun 56 (1988), pp. 1831–1836 88284899.

[15] Y Xu, C Kodner, L Coleman and RC Johnson, Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31, Infect Immun 64 (1996), pp. 3870–3876 96355903.

[16] J-R Zhang, JM Hardham, AG Barbour and SJ Norris, Antigenic variation in lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes, Cell 89 (1997), pp. 275–285 97262068.

[17] AG Barbour, Linear DNA of Borrelia species and antigenic variation, Trends Microbiol 1 (1993), pp. 236–239 94184809.

[18] BI Restrepo, CJ Carter and AG Barbour, Activation of a vmp pseudogene in Borrelia hermsii: an alternate mechanism of antigenic variation during relapsing fever, Mol Microbiol 13 (1994), pp. 287–299 95075314.

[19] BI Restrepo and AG Barbour, Antigen diversity in the bacterium B. hermsii through ‘somatic’ mutations in rearranged vmp genes, Cell 78 (1994), pp. 867–876 94373822.

[20] C-A Reynaud, V Anquez, H Grimal and J-C Weill, A hyperconversion mechanism generates the chicken light chain gene preimmune repertoire, Cell 48 (1987), pp. 379–388 87102887.

[21] R Haas and TF Meyer, The repertoire of silent pilus genes in Neisseria gonorrhoeae: evidence for gene conversion, Cell 44 (1986), pp. 107–115 86079567.

[22] HS Seifert, CJ Wright, AE Jerse, MS Cohen and JG Cannon, Multiple gonococcal pilin antigenic variants are produced during experimental infections, J Clin Invest 93 (1994), pp. 2744–2749 94259840.

[23] P Rosa, DS Samuels, D Hogan, B Stevenson, S Casjens and K Tilly, Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi, J Bacteriol 178 (1996), pp. 5946–5953 96427327.

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The patent Norris filed in 1991 came 9 years after antigenic variation in Borrelia hermsii had been discovered - and there is no mention of Borrelia burgdorferi's antigenic variation until 1997.

What more has been learned about Borrelia burgdorferi in the past two decades and how is it relevant to current and future studies?

How is knowledge of the process of antigenic variation useful in finding a method of effectively treating affected patients?

More on this in a future post on Camp Other...

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0 More on that Vmp-like sequence - aka vlsE in Borrelia

So I know many of you have read my entry on the patent, VMP-like sequences of pathogenic Borrelia species and strains, with Steven J. Norris and Alan Barbour as primary patent holders.

For some of you, the news of this patent may come as a total surprise. For others, it may not.

For those of you who were listening in on or attended the October 2010 Institute of Medicine (IOM) workshop - A Workshop on the Critical Needs and Gaps in Understanding Prevention, Amelioration, and Resolution of Lyme and Other Tick-borne Diseases: the Short-Term and Long-Term Outcomes -well...you might have remembered this presentation:

DAY 2  Institute of Medicine October 13, 2010
11:30 am Antigenic Variation as a Mechanism for Persistent Borrelia Infection
Steven Norris, Ph.D. Greer Professor Vice Chair for Research Pathology & Laboratory Medicine University of Texas, Houston

For any Lyme patients and others watching the IOM webcast or otherwise listening in, Dr. Norris presented on that day the following (I got these notes off a mailing list - roughly transcribed and spelling corrections/minor changes made - I also listened to the original web cast to check on the accuracy of these notes) information:

Lyme Bb

• Motile invasive organism with unusual properties
• invasive, persistent infection
• disseminates fairly early on
• in mice, found in skin, joints, bladder, heart, spleen, and other organs 2 years after innoculation.
• virtually any tissue can contain them in lifetime of mice
• Persistence in humans is not well understood.
• Produce no known toxins or enzymes that are toxogenic - don’t have toxins that cause tissue damage. (compare to Botulin toxin and Gas gangrene - extreme toxin invasion of tissues)
• Different pathogenic strategies for different species (gives examples of different organisms at different extremes of toxogenicity)

Lyme and Syphilis

• both have local, disseminated, and long term stages
• can draw from diseases that are similar in examining Lyme Disease

Bb infection pathogenesis (outline from book chapter he put together - Pathology of Lyme Disease Borrelia - from Borrelia: Molecular Biology, Host Interaction and Pathogenesis By Justin Radolf, D. Scott Samuels)

To cause persistent infection, it must have multiple ways of evading immune response.
These are possible mechanisms:
1) protective niches: sequestration of organisms in dense tissue (Barthold talks about this later in conference)
2) hiding or downregulating of antigens - known as masking (regulation of genes in Bb) - occurs early in infection

• OspA is downregulated in ticks but not expressed in high levels in mammalian infection,
• OspC and other proteins are upregulated in early stages of infection
• There changes in gene expression are important in lifecycle of organism

3) inhibition of immune response (inhibition of complement cascade by proteins called CRASPS interfere with immune system, mentioned by Dr. Oliver earlier)
4) Today’s talk is on antigenic variation: a change in surface structure (usually) that occurs at rate higher what would be expected from mutation (what we’ll discuss today)

Vls

VMP-like sequence, resembles that of Relapsing Fever, but there are important differences.
We’re only going to talk about gene or locus today so I picked VLS in one plasmid of the organism.

In one plasmid of the organsim, expression site called VlsE and silent cassettes upstream from that, silent ones are 92% identical with central cassette site, called the expression cassette or region - if you align these areas next to another they show areas of identity and then areas of variability named variable regions 1,2,3,4,5, 6 (points to slide).

When this locus was present, we thought each silent cassette could exchange into central/expression site, and therefore cause 15 different variants - but instead we found there was a segmented exchange/recombination that occurs (we don’t know how this occurs) - which means gene conversion event occurs where the silent cassette donates genes to expression cassette

Can’t detect this segmented exchange in standard liquid culture or in ticks, but can we can detect this in infected mice.

There are so many different variations produced, so many that you can’t find the same VlsE combination or sequence in the same tissue twice 28 days post-infection.

• 10³² amino acid sequence combinations possible.
• Most sequence differences are very short - 1 or 2 amino acid differences (refer to Luft).
• Protein is anchored to outer membrane of organism like umbrella.
• Organism can change amino acids of the outer surface. These are colored regions with highest degree of sequence variation. Evolution favors this outer region to change to evade immune system; continual changing of this surface (amino acid sequence) leads to immune evasion.

VlsE is now used for a Lyme immunodiagnosis.
In particular, the IR6 or C6 region of organism has high antibody response. And other regions of the protein is reactive as well.

Dilemma: immune invasion but at the same time high antibody response to this protein.

How important is this system in terms of pathogenesis?

The real landmark study (Bankhead and Chaconas) was where they deleted out the LP281 plasmid that carries the VLS locus. (Removed right end and just preserve the left end of plasmid that carries this locus - see slide). They found when those organisms infected mice that they were now defective in infecting mice. They were LP281 deficient phenotype and they are quickly eliminated by immuno-competent mice. However, SCID mice (immuno-deficient) can’t clear the organism. Therefore this locus is very important in evading the adaptive immune response (B and T cells).

Little is known about VLS recombination.
VLS recombination involves gene conversion - replacement of VLS recipient sequence with donor silent cassette sequence.
Does not require RecA.
But gene conversion reduced in genes lack Holliday junction resolvase encoded by proteins RuvA and RuvB.
[...]
VLSe recombination occurs in mammalian host during infection, so far was not found in standard in vitro cultures of Bb.

One advanced study by (Dr. Diane Edmondson (sp?)):

• Tissue on gel foam (collagen) and incubated in medium
• Inoculated tissue implants with Bb (called explants)
• Then after one day explants were moved to fresh dish and excess removed
• And tissues monitored in artificial infection scenario - the ex-plants did quite well
• Up to 16 days post culture heart looks normal, spleen less so - has loss of lymphocytes (devolution of the tissue)
• Multiplication of Bb in tissue and it differed from tissue to tissue and with medium used
• Still trying to work out best conditions to examine this situation and maintain that replication.

Had to develop method to measure VlsE gene recombination - it is rare, only occurs in
1/10⁶ cells show VlsE recombinancy

in vitro used PCR technique where parental and recombinancy showed
Monitored change in PCR to identify sequences that underwent recombination
In 3 of 4 explant samples we found recombination - this is first detection of it in vitro (slide)
We’re trying to figure out exactly what the recombination events are - it’s not very easy

I’ll show you our final results:
In 3 of these instances, Cassette 7, 2, and 4 show recombination events - very long ones,
but in some incidences, shorter recombination events
This shows a model for the antigenic variation system.

Did a meta-analysis of Luft’s data of 13 different strains sequences
He looked at them evenings and weekends and analyzed the VLS sequences
The VLS sequences differ more than any LD Borrelia homologues
Within vlsE the identity is as low as 54% identity with a 69% similarity
OspC has overall the lowest sequence identity between diff OspC’s from different organisms/strains - 69% identity 79% similarity
VlsE system under high degree of evolutionary selection and pressure.

First identified this region in B31 strain (burgdorferi)
Emphasize that the silent cassettes are in a single continguous open frame - it is huge protein interrupted in only 3 places by stop codon and 2 frame shifts
We thought maybe that was important
There are direct repeats at the end of each junction between each of those silent cassettes

Analysis examples

64B - has 22 silent cassettes, opposed to only 15 in our initial characterised strain (B31)
3 frame shifts present
Initial annotation here (very hard to annotate the entire genome, let alone 13 genomes - this is what came out of their - Luft & co's - notes)
Took 2 days just to analyze this one locus
Borrelia 29805 - 17 silent cassettes and annotated initially as single long frame
strain 404 (B. garinii) - total 18 open [..] frames
There are no direct repeats in any of these that we can identify
most of the frame shifts are between silent cassettes and do not interrupt cassettes
a lot of theories of how the system worked were blown out of the water just by this meta analysis
I want to emphasize differences are quite marked in locii between these different strains
We are looking for correlate at differences in ribosomal types or OspC types
Also want to know how differences in VLS may relate to infectivity and virulence of different strains
Want to replace B VLS with VLS of other strains to see how it affects its pattern of pathogenesis
(how it operates or if it’s infectious)
Number of sequence differences obvious between B31 and 404 are there
[...]
VlsE antigenic variation important route of immune invasion
VlsE gene conversion not well understood but involves RuvA & RuvB resolvase
VlsE has higher sequence diversion than any other sequence including OspC

Questions

What are the cis- and transacting factors that regulate and carry out VLS recombination?
Can tissue explant models be used to study immune evasion better than other tissues such as in mice? [...]
Do differences in VLS recombination correlate to various outcomes (arthritis, chronic effects)?
How can protein that induces strong antibody response be involved in immune evasion?
What roles do other proteins or mechanisms play in infection by Lyme Disease infection? (i.e. sequestration, masking, inhibition of immune response)

See abstract http://www.ncbi.nlm.nih.gov/pubmed/12603744 for some of Norris’ earlier work (2003).

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You can still watch the recorded webcast of the IOM workshop and watch Steven Norris' webcast presentation there (scroll down to each section and click on the section saying "Play Flash Video" - apologies to viewers who cannot view Flash on their computer or device.)

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More people have been doing research on this sequence for some time - it is critical for beginning to grasp Borrelia's antigenic variation.

For example:

From CAP's web site:

"Mario T. Philipp, PhD, and colleagues first identified and characterized C6 for use in the serodiagnosis of Lyme disease. “We were hoping to try VlsE as a vaccine candidate,” says Dr. Philipp, pro­fessor of microbiology and immu­nology and chair of the Division of Bacteriology and Parasitology, Tulane National Primate Research Center. However, other researchers discovered that V1sE was a protein that changed its antigenic properties as infection progressed, making it possibly unsuitable as a vaccine. Attention then turned to invariant regions of VlsE, segments whose antigenic properties did not change over time. “What struck us was that invariant region six reacted with serum specimens taken from nonhuman primates early in infection,” Dr. Philipp says. “We thought we might have a candidate for early diagnosis.” When they tested C6 with a battery of human specimens, sensitivity ranged from 74 to 100 percent, depending on stage of infection. More important, specificity was close to 100 percent, with only two false-positives out of 176 samples (Liang FT, et al. J Clin Microbiol. 1999;37:3990–3996)."

Evidence That the Variable Regions of the Central Domain of VlsE Are Antigenic during Infection with Lyme Disease Spirochetes - 2002 -
John V. McDowell, Shian-Ying Sung,Linden T. Hu, and Richard T. Marconi

Immune responses to borrelial VlsE IR6 peptide variants - 2007 - Heidi Sillanpääa, Pekka Lahdenneb, Heikki Sarvasa, c, Maja Arnežd, Allen Steeree, Miikka Peltomaae and Ilkka Seppälä

Evaluation of the Recombinant VlsE-Based Liaison Chemiluminescence Immunoassay for Detection of Borrelia burgdorferi and Diagnosis of Lyme Disease - 2008 -
Thomas B. Ledue, Marilyn F. Collins, John Young, and Martin E. Schriefer

Oh, and by the way? There is already a test out there using vlsE for Lyme disease immunoblots.

Check out: http://www.zeusscientific.com/products/technology-systems/athena-multi-lyte/

Borrelia VlsE-1 IgG/pepC10 IgM Plus Test System

The ZEUS Scientific, Inc. AtheNA Multi-Lyte® Borrelia VlsE1/pepC10 IgM Plus Test System is a multiplexed sandwich assay for the qualitative detection of IgG class antibody to recombinant VlsE1 and the IgM class of antibody to synthetic pepC10 in human serum. The AtheNA Multi-Lyte® Borrelia VlsE1/pepC10 IgM Plus Test System is intended for use in testing human serum samples which have been found equivocal or positive by alternate serological procedures to provide supportive evidence of infection by Borrelia burgdorferi. This kit is for in vitro diagnostic use only. Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients, cord blood, neonatal specimens, or infants.

Product code: A90151

You can bet this isn't the last you'll be hearing about vlsE - from me, or elsewhere...

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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 type. Even 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

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