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
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[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.
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[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.
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[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.
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[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.
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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