2000 Conference

Lyme Test Information

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We would like to thank MEDSCAPE for allowing us to use the following abstracts.

Presented March 25th 1:25 PM at the 13th International Scientific Conference on Lyme Disease and other Tick-Borne Disorders, Hartford Connecticut.

 

Immunologic Aspects of VlsE, a B. burgdorferi Antigenic Variation Protein
Steve J. Norris, PhD, University of Texas -- Houston Medical School

Lyme disease borrelia can persistently infect Lyme disease patients and infected animals for months to years despite a vigorous host immune response. Our laboratory recently discovered the vls (VMP-like sequence) antigenic variation system in B. burgdorferi B31 that may in part explain the ability of this organism to evade an active immune response. The vls locus consists of the expression site for a surface lipoprotein, VlsE, and 15 'silent' cassettes that represent variations of the central cassette region of the vlsE gene. Segments of the silent cassettes begin to recombine into the vlsE gene within 4 days following infection, and this ongoing process could produce as many as 1030 different sequence variations. Lyme disease patients consistently produce a strong antibody response against VlsE, which may actually be useful in the diagnosis of Lyme disease. Although immunization with a single form of VlsE fully protects mice against infection with B. burgdorferi expressing the identical protein, it is only partially protective against variants expressing slightly different versions of VlsE; this finding demonstrates the role of VlsE sequence variation in immune evasion. DNA sequences homologous to vlsE have been identified in every infectious Lyme disease isolate examined to date, and may represent a common immune evasion mechanism in Lyme disease borrelia. Further characterization of the vls system may lead to improved immunodiagnosis and additional immunoprotective vaccines against Lyme disease.

 

Presented March 25th 1:50 PM at the 13th International Scientific Conference on Lyme Disease and other Tick-Borne Disorders, Hartford Connecticut.

 

Invariable Regions of VlsE, the Variable Surface Antigen of Borrelia burgdorferi: Their Application in Diagnosis and Immunoprophylaxis of Lyme Disease
Mario Philipp, PhD, Tulane University Medical Center; Tulane Regional Primate Research Center, Department of Parasitology

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable antigens such as the variable major protein (Vmp) of Borrelia hermsii and the variant surface glycoprotein (VSG) of African trypanosomes include an immunodominant variable domain and one or more invariable domains which are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain.
VlsE (variable major protein-like sequence), the variable surface antigen of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR11-6) which together amount to one half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Unlike the invariable regions of Vmp and VSG, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. The utilization of IR6 as a universal diagnostic reagent for Lyme disease and the possible application of other IRs as vaccines will be discussed.

 

 

 

The following are a reviews of the presentations.

 

Presented March 25th 1:25 PM at the 13th International Scientific Conference on Lyme Disease and other Tick-Borne Disorders, Hartford Connecticut.

 

Using Cassettes to Evade the Immune System
Steven J. Norris, PhD, and Julie Rawlings, MPH

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable antigens--such as the variable major protein (Vmp) of Borrelia hermsii and the variant surface glycoprotein (VSG) of African trypanosomes--include an immunodominant variable domain and 1 or more invariable (constant) domains that are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain.

Dr. Steven Norris, Professor and Vice Chair for Research, Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, discussed immunologic aspects of VlsE, a Borrelia burgdorferi antigenic variation protein, during the first day of the 13th Lyme Disease Conference. He said that Borrelia sp can persistently infect humans and other animals for months to years despite a vigorous host immune response. His laboratory recently discovered the vls (VMP-like sequence) antigenic variation system in B burgdorferi B31, which may in part explain the ability of this organism to evade an active immune response.

 

The vls System: Creating Variation

Dr. Norris explained that the vls locus consists of the expression site for a surface lipoprotein, VlsE, and 15 "silent" cassettes that represent variations of the central cassette region of the vlsE gene (Figure 1). Segments of the silent cassettes begin to recombine into the vlsE gene within 4 days following infection, and between 9 and 13 recombination events have been found to occur in mice over a 28-day period of infection. This ongoing process could produce as many as 1030 different sequence variations.

In B burgdorferi B31, vlsE recombination does not occur at a detectable rate in vitro, and the recombination is unidirectional (ie, from silent cassette to the vlsE expression site). Therefore, this variation appears to involve a specialized recombination mechanism that is induced during infection. Because these changes occur rapidly during the infection of each mammalian host, they represent true antigenic variation rather than heterogeneity, which is sequence variation that is observed between strains and requires hundreds of generations to occur.

 

Immune Response to VlsE: Only Partial Protection

Dr. Norris has found that Lyme disease patients consistently produce a strong antibody response against VlsE, which may be useful in the diagnosis of this disease. He cited studies by Liang and colleagues, which indicate that at least part of this shared reactivity is induced by invariant regions within the vlsE cassette region.

 

Immunization with a single form of VlsE fully protects mice against infection with B burgdorferi expressing the identical protein, but it is only partially protective against variants expressing slightly different versions of VlsE. This finding demonstrates the role of VlsE sequence variation in immune evasion. Further characterization of the vls system may lead to improved immunodiagnosis and additional immunoprotective vaccines against Lyme disease.

 

Consistent Presence of the vls System in Lyme Disease Isolates

DNA sequences homologous to vlsE have been identified in every infectious Lyme disease isolate examined to date, including B burgdorferi sensu stricto, B garinii, and B afzellii strains. Analysis of a vls locus of B garinii, Ip90, indicates that it contains at least 11 vls silent cassettes, although some of these are truncated.

Dr. Norris said that in a recent study by Iyer and associates, each of the 22 B burgdorferi isolates from Lyme disease patients in the Hudson Valley area of New York were found to contain a vls sequence-containing plasmid, based on Southern blot hybridization. Ten of these strains were found to have vlsE sequences very similar to those of the B31 strain, whereas the other 12 had divergent sequences. Eight of the 10 strains with B31-like vlsE sequences had a genotype found in disseminated (blood-borne) infections. Moreover, absence of the plasmid containing the vls locus (lp28-1) correlates with a reduction in infectivity, providing further evidence that VlsE is involved in pathogenesis through an as-yet undetermined mechanism.

 

Conclusions: Effective Immune Evasion

Dr. Norris concluded by saying the vls system appears to represent a common immune evasion mechanism in Lyme disease Borrelia. There is evidence that protective antibodies are produced against the variable regions of VlsE and that antibodies against the conserved regions are not protective (perhaps because they are not readily assessable on the surface). However, it may be possible to design an effective "combinatorial" Lyme disease vaccine by including many different variants of VlsE.

 

 

Presented March 25th 1:50 PM at the 13th International Scientific Conference on Lyme Disease and other Tick-Borne Disorders, Hartford Connecticut.

 

Invariant VlsE Regions

Dr. Mario Philipp, Department of Parasitology, Tulane Regional Primate Research Center, Tulane University Medical Center, New Orleans, Louisiana

Dr. Mario Philipp, has performed more detailed studies of the invariant regions of VlsE. Fang-Teng Liang and other members of his laboratory have shown that a particular invariant 'loop' of VlsE is responsible for much of the strong antibody response observed against this protein.
Interspersed within the VlsE variable cassette region (Figure 1), there are 6 invariable regions (IR 1-6) which together amount to one half of this portion's primary structure. These IRs are conserved among strains and genospecies of the B burgdorferi sensu lato complex. Unlike the invariable regions of Vmp and VSG, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B burgdorferi.

 

A Universal Diagnostic Reagent

Dr. Philipp showed that IR6 could be utilized as a universal diagnostic reagent for Lyme disease and the possible application of other IRs as vaccines. His laboratory has developed a simple enzyme immunoassay (EIA) that uses a peptide (C6) corresponding to IR6 as the antigen. Sera from most patients with erythema migrans and nearly all patients with disseminated Lyme disease are highly reactive by this assay. The C6 immunoassay is reactive with sera from both North American and European Lyme disease patients, because the C6 sequence is shared among Lyme disease isolates from both continents. By contrast, sera from healthy individuals or patients with autoimmune or a variety of other infectious diseases were uniformly nonreactive. The C6 immunoassay also had a high degree of precision, ie, was highly reproducible from test to test. Therefore, this assay shows great promise in resolving the many diagnostic dilemmas associated with Lyme disease serodiagnosis.

Suggested Reading

Iyer R, Hardham JM, Wormser GP, et al. Conservation and heterogeneity of vlsE among human and tick isolates of Borrelia burgdorferi. Infect Immun. 2000;68:1714-1718.
Lawrenz MB, Hardham JM, Owens RT, et al. Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi. J Clin Microbiol. 1999;37:3997-4004.
Liang FT, Alvarez AL, Gu Y, et al. An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi. J Immunol. 1999;163:5566-5573.
Liang FT, Philipp MT. Analysis of antibody response to invariable regions of VlsE, the variable surface antigen of Borrelia burgdorferi. Infect Immunol. 1999;67:6702-6706.
Liang FT, Steere AC, Marques AR, et al. Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi VlsE. J Clin Microbiol. 1999;37:3990-3996.
Philipp MT, Lobet Y, Bohm RP, et al. Safety and immunogenicity of recombinant outer surface protein A (OspA) vaccine formulations in the rhesus monkey. J Spirochetal and Tick-Borne Diseases. 1996;3:67-79.
Zhang JR, Norris SJ. Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion. Infect Immun. 1998;66:3698-3704.

 

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