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...