Today Viral Genetics posted a letter to shareholders with the following information which relates to their experimental chronic Lyme disease candidate, VGV-L.
The letter stated:
"We are still finalizing scheduling a meeting with the FDA and our team to discuss our Lyme disease pre-IND submission from earlier this spring. Scheduling has been difficult with the number of people involved and the looming summer season. Following this meeting we expect to have a clear road map to follow towards clinical trials"
I am looking forward to hearing more about this meeting. It seems there have been a number of delays on the pre-IND road for VGV-L so far, though, and I can't predict when one is going to see progress on getting this treatment to trial stage.
Now might be the time to contact Viral Genetics and begin asking them questions about what sort of ideas they are coming up with in terms of clinical trial design. As a chronic Lyme disease patient reading along, your input may be informative and useful for researchers.
Stay tuned for more updates on VGV-L as I get them.
The National Institute of Allergy and Infectious Disease (NIAID) is conducting some Lyme disease related research which I think readers should know about. There are a number of projects to be found on the Project Reporter web site which may be fascinating, but I took the time to select and highlight a few projects which would be of greater interest to patients suffering with Lyme disease and/or its coinfections.
Project: AN INTRACELLULAR NICHE FOR BORRELIA BURGDORFERI Institution: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR PI: Skare, Jonathan
Description (by applicant):
Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is the leading arthropodborne infection in the United States and causes significant morbidity in endemic areas. If untreated B. burgdorferi can persistently infect individuals even though the host mounts a potent adaptive immune response such that antibodies obtained from infected patients or experimentally infected animals effectively kills in vitro cultivated B. burgdorferi. In addition, a robust cell-mediated proinflammatory response is observed that induces IL-6, IL-12 and IFN- and inhibits IL-10. Furthermore, the spirochete can resist complement killing demonstrating that this important component of the innate immune response is not sufficient to eliminate B. burgdorferi infection.
The observation that B. burgdorferi persists in such a hostile environment indicates that the spirochete is adept at evading the host immune response via mechanisms that have not been completely elucidated. One possibility is that B. burgdorferi invades host cells and survives at low levels. Recently we have determined that B. burgdorferi invade both immortalized and, more importantly, primary cells (both fibroblasts and endothelial cells) and persist as viable cells in o-culture. In addition we have preliminary data suggesting that the ability to invade host cells involves both integrin binding and Src kinase activity.
In this application we propose to further characterize the internalization of B. burgdorferi and track the fate of B. burgdorferi within thes infected cells to determine how they affect the localized host response following infection. To accomplish this we will use both in vitro correlates of invasion and intracellular survival as well as in vivo imaging of experimentally infected mice as readouts for our studies.
Specifically, we propose to:
(1) Characterize the invasion of Borrelia burgdorferi into primary fibroblasts. The working hypothesis here is that B. burgdorferi exploits invasion as an additional mechanism to avoid host clearance. Our preliminary studies demonstrate that B. burgdorferi invasion is not dependent on host fibronectin, but does involve B1 integrins other than a5B1. In this Aim we will identify the subunit that pairs with B1 to promote invasion and will also evaluate how B. burgdorferi traffics within these cells; and
(2) Determine if invasion is required for B. burgdorferi persistence in vivo. Our working hypothesis is that invasion contributes to persistence by providing an immunoprotected niche for B. burgdorferi. Since Src kinases are required for borrelial internalization in vitro, we will determine whether Src kinase inhibitors alter the infectivity potential of B. burgdorferi in vivo. In addition to standard cultivation and molecuar approaches, novel in vivo imaging will be employed to assess how the inhibitor affects colonization.
The overall goal of these studies is to determine the extent in which an intracellular locale contributes to borrelial persistence.
PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod-borne infectious agent in the United States, and, as such, represents an important Public Health issue. The studies described in this application are designed to address how B. burgdorferi is able to persist effectively in infected mammals despite effective innate immune killing mechanisms and a potent adaptive immune response directed against this pathogen. The hypothesis being tested herein is that B. burgdorferi is capable of low-level intracellular survival in non-immune cells as an additional strategy to prevent borrelial host clearance.
Comment: This really begins fulfilling my wishlist, and I look forward to the imaging study videos that I hope will be made and posted online. If there is some sort of confirmation of intracellular Bb in vivo this may explain why some patients need additional antibiotics and why existing treatments may be inadequate as a matter of timing.
This next project is bound to generate discussion, as it involves the potential role of toxins in Borrelia burgdorferi. In this case, the researcher is looking for gene clusters in Borrelia burgdorferi which may create cytolysins similar to the toxins which are found in Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum.
Project: A COMMON DENOMINATOR OF PATHOGENESIS; A RARE OPPORTUNITY FOR NOVEL THERAPEUTIC DE(VELOPMENT) Institution: UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN PI: Mitchell, Douglas
Description (by applicant):
Abstract: The 20th century witnessed several major advances in medicine. Perhaps most important were the discovery of antibiotics for bacterial infections and effective vaccines for several major viruses. Unfortunately, the creation of effective vaccines for bacteria has lagged behind analogous anti-viral strategies. Compounded with the rise in antibiotic resistance and a lack of interest from the pharmaceutical industry in pursuing novel antibiotics, we risk losing the fight against bacterial pathogens.
Described herein is an unconventional strategy to exploit bacterial toxins as both novel targets for antibacterial agents and antigens for vaccine development. To intelligently address the increasing threat posed by bacterial pathogens, more effort is needed to uncover the molecular underpinnings of virulence. Our group specializes in the use of bioinformatics, in vitro reconstitution, and genetic manipulation to identify and characterize gene clusters that are responsible for the biosynthesis of virulence-promoting cytolysins. The best-known toxin in this family is the highly modified peptide, streptolysin S (SLS, produced by Streptococcus pyogenes).
SLS production is required for the infective process, but not essential life processes. Our work has uncovered SLS-like toxins are synthesized by at least three other notorious human pathogens, including Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum. We aim to study the potential role of the SLS-like toxin in an additional organism, Borrelia burgdorferi (Bb), which causes Lyme disease.
Although widely known, the Bb molecular mechanism of pathogenesis is inadequately defined. If the SLS-like toxin was indeed employed during Bb infections, this would represent the first demonstration of toxin utilization in this family of organisms and would prompt a major revision of borrelioses.
Because bacteria typically employ disparate pathogenic mechanisms, the conserved, SLS-like pathway provides a rare opportunity to develop more broadly applicable, yet targeted countermeasures. From our perspective, new antimicrobial strategies should directly target the pathogenic mechanism, rather than DNA replication, protein synthesis, or the cell wall. This approach holds enormous potential, as these drugs will theoretically be resistant to resistance.
This project will identify inhibitors of SLS toxin biosynthesis for the specific purpose of developing novel antibacterials. Moreover, SLS is non-immunogenic, rendering it an unfeasible candidate for vaccine development.
We have succeeded in generating attenuated variants with the anticipation that these can be used for raising toxin-neutralizing antibodies. The notion of immunizing against a bacterial toxin represents a potentially general strategy for future vaccine development.
With this proposal, we aim to not only fundamentally shift the accepted view of Bb pathogenesis, but also to challenge the paradigm that antibiotics must kill bacteria and non-immunogenic toxins are intractable vaccine candidates. These seemingly unrelated goals are actually quite intertwined. Our approach rests on the philosophy that a more complete understanding of toxin biosynthetic pathways and chemical structure can be rationally exploited to design novel therapeutics.
Public Health Relevance: Bacterial pathogens employ numerous mechanisms to evade the human immune system. We have discovered a novel strategy within the organism that causes Lyme Disease, who's pathogenesis remains largely enigmatic. A greater understanding of these processes will lay the foundation for developing the next generation of antimicrobial drugs.
Wait... I thought Radolf & co. said Borrelia burgdorferi does not produce a toxin? I know Donta patented some genes in Bb he saw as being analogous to a toxin.
Is there now evidence of newly researched genes which create a toxin in Bb? Or is this an old hypothesis which is being revisited?
Project: ASSESSMENT OF PATIENTS WITH BORRELIA INFECTION Institution: NIAID PI: Marques, Adriana
Description (by applicant):
Lyme disease is a multisystem illness caused by infection with the spirochete Borrelia burgdorferi and it is the leading vector-borne disease in the United States. Our current work addresses the following areas in Lyme disease: development of new tests and biomarkers for infection, investigation of persistence of infection with B. burgdorferi in humans, search for the cause of Southern Tick-associated Rash Illness (STARI), and investigation of the role of immune response in Lyme disease and PLDS.
One of the main problems in Lyme diagnosis has been the lack of highly specific and sensitive assays for B. burgdorferi and the lack of a test that could be used to assess response to therapy. Such assays should greatly facilitate the accurate diagnosis of Lyme disease and assessment of response to therapy in individual patients. Currently, no such test is available.
We have developed a new test using the luciferase immunoprecipitation systems (LIPSs) for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. A synthetic protein consisting of a repeated antigenic peptide sequence, named VOVO, had the best diagnostic performance, similar to the C6 test (a diagnostic test using a peptide ELISA that we have helped develop and is highly sensitive and specific). The VOVO LIPS test displays a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution; and offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.
Recent studies have shown that B. burgdorferi may persist in animals after antibiotic therapy and can be detected by using the natural tick vector (Ixodes scapularis) to acquire the organism through feeding. Whether this occurs in humans is unknown.
We have implemented a new clinical protocol to investigate the utility of this approach for identifying persistence of B. burgdorferi in treated human Lyme disease.
STARI is a rash similar to the rash of Lyme disease that occurs in persons residing in southeastern and south-central states and is associated with the bite of the lone star tick, Amblyomma americanum. The cause of the rash is unknown, as it is the natural course of the disease.
We have a clinical protocol to investigate the cause of STARI, and we are applying new genomic tools that identify bacteria based on species-specific sequences in the 16S rRNA ribosomal genes to the skin biopsies from patients with STARI.
Inflammatory innate immune responses are critical in the control of early disseminated infection, while adaptive immune responses are vitally important, particularly the humoral immune response, in controlling spirochete levels in tissues and resolution of Lyme arthritis in animal models. We are examining the antibody response to immunogenically dominant antigens of B. burgdorferi in PLDS patients and controls.
Further investigation of the anti-borrelia immune response may help in elucidating the pathogenic mechanism of PLDS and yield important information for future approaches to diagnosis and treatment. We have a clinical protocol in which we use DNA microarrays to characterize gene expression patterns in skin biopsies from individuals with EM, with the aim of capturing the human host response to pathogen exposure.
We are also investigating the differences in immunological response between predominantly lymphocytic meningitis and predominantly neutrophilic meningitis. Results from these studies will serve as a window into the fundamental biology of the infection.
The existence of the VOVO LIPS test is nothing new - reports on the development of this test have been around since 2010. Also, there is already information about a chronic Lyme disease xenodiagnosis study out there.
It seems like this project has a large scope - or consists of more than one project under the same umbrella. So far, no project end date has been posted for this entry.
What would be of most interest to me would be finding differences in immunological response between patients with acute Lyme disease and those with assumed PLDS - something Alaedini has already been studying.
(Side note: I thought that it was already determined that Borrelia lonestari, a relapsing fever spirochete, was the cause of STARI or Masters disease - did I miss something?)
I have a few strange but true facts about spirochetes to share which you may not know. A few are ones I have shared here before - but most are not something about which I've already written. What you read here today may surprise you...
Many people call Borrelia burgdorferi spirochetes Gram negative bacteria. However, Borrelia burgdorferi are not Gram-negative bacteria even if a Gram negative stain works on them:
"Borrelia were thought to be Gram negative because of their double membrane structure, but genetic analysis places them - along with other spirochetes - into a separate eubacterial phylum. Ultrastructural molecular and biochemical studies have emphasized the wide taxonomic gap between spirochetes and Gram-negative bacteria."
- From "The Genus Borrelia" by Melissa Caimano. Prokaryotes (2006) 7:235-293.
Unlike Leptospira and Brachyspira, spirochetes in the Borrelia and Treponema genera appear to have acquired Phenylalanyl-tRNA synthetase (PheRS) genes from Archaea through horizontal gene transfer. [1] Borrelia and Treponema have Archaea genes.
Somewhere along the line, an ancient Spirochaeta relative picked up genes from Archaea's order, Thermococcales. Borrelia and Treponema have close affinities with Thermococcus and Pyrococcus(not depicted on tree).[1]
The fascinating thing about this genetic relationship is that these genes come from organisms which are extremely thermophilic organisms. They are extremophiles - which means they can live in extreme environments. Thermophilic extremophiles thrive in hot environments such as volcanic vents and hot springs. That genes from extremophiles would end up in mesophilic organisms which thrive in lower temperatures - such as in mammalian and acarian hosts - seems surprising. The highest temperature Borrelia garinii can still grow in is around 41-42 C. That's not anywhere near the high temperatures in which one finds Archaean Thermococcales (often over 60 C, sometimes as high as 100 C).
This all does seem really weird. But the reason why it isn't too far fetched to see genes from extremely thermophilic organisms in moderately warm Borrelia and Treponema is more easily understood once you know more about the wide diversity found within the genus Spirochaeta in general. A number of Spirochaeta species live in extreme environments and not just in humans, animals, or ticks. For example:
S. halophila lives in a high salinity pond on the Sinai shore.[2]
S. thermophila lives in marine hot springs in New Zealand and Russia.[3]
S. americana lives in alkaline, hypersaline Mono Lake in California.[4]
When looking at a phylogenetic tree, Spirochaeta is at the base of the tree and Borrelia and Treponema branch off later. Based on this, the best assessment one can make about the gene transfer from Archaea to Spirochaeta is that the most recent common ancestor of Spirochaeta, Borrelia, and Treponema had to have been very similar to thermophilic Spirochaeta.
My running joke on this is to imagine a pile of thermophilic Archaea and thermophilic Spirochaeta hanging out around a hot spring together, laughing, joking, and flirting. Before you know it, horizontal gene transfer occurs, and a new form of spirochete is born. (This would make for a good Far Side comic, I just know it.)
As if having Borrelia acquire Archaea genes wasn't interesting enough, it's been thought that ProS prolyl-tRNA synthetase (BB402) was acquired from a eurkaryote.
Treponema spirochetes have a symbiotic relationship with termites. These spirochetes help termites in breaking down cellulose in wood in the termites' guts. So it isn't just ticks which have a symbiotic relationship with spirochetes - termites have one, too.[1, 5]
Both Borrelia hermsii and Borrelia burgdorferi metabolize chitobiose and N acetyl-glucosamine, a nutrient of these spirochetes and the major constituent of chitin for the exoskeletons of ticks.[6]
Borrelia have most of the genes required for the enzymes which make up the mevalonate pathway - a metabolic pathway used by the bacteria for synthesis of isoprenoid precursors. Isoprenoids are very important compounds which are found in over 30,000 products from the three domains of life (Eukaryotes, Prokaryotes, and Archaea). One interesting proposal about how Borrelia has the genes required for these enzymes for this pathway is that they come from the genetic cenancestor - an ancestor which predates the split into the three domains.[7]
Spirochetes continue to hold surprises and mysteries for us all... both good and bad. Another interesting installment of strange spirochete facts could be posted here - probably not too far in the future.
References:
1) Cheryl P Andam and J Peter Gogarten. Biased gene transfer and its implications for the concept of lineage. Biology Direct 2011, 6:47 doi:10.1186/1745-6150-6-47
2) Greenberg EP, Canale-Parola E: Spirochaeta halophila sp. n., a facultative anaerobe from a high-salinity pond. Arch Microbiol 1976, 110:185-19
3) Aksenova H, Rainey F, Janssen P, Zavarzin G, Morgan H: Spirochaeta thermophila sp. nov., an obligately anaerobic, polysaccharolytic, extremely thermophilic bacterium. Int J Syst Bacteriol 1992, 42:175-177
4) Hoover RB, Pikuta EV, Bej AK, Marsic D, Whitman WB, Tang J, Krader P: Spirochaeta americana sp. nov., a new haloalkaliphilic, obligately anaerobic spirochaete isolated from soda Mono Lake in California. Int J Syst Evol Microbiol 2003, 53:815-821.
5) Droge S, Frohlich J, Radek R, Konig H: Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus. Appl Environ Microbiol 2006, 72:392-397.
6) Tilly, K., Elias, A.F., Errett, J., Fischer, E., Iyer, R., Schwartz, I., et al. Genetics and regulation of chitobiose utilization in Borrelia burgdorferi. J Bacteriol 183: 5544–5553.
7) Jonathan Lombard and David Moreira. Origins and Early Evolution of the Mevalonate Pathway of Isoprenoid Biosynthesis in the Three Domains of Life. Mol Biol Evol 2011, 28 (1): 87-99. doi: 10.1093/molbev/msq177 http://mbe.oxfordjournals.org/content/28/1/87.full
I came across this video on Youtube which I haven't seen mentioned elsewhere. It is a presentation by Dr. Jorge Benach on tickborne diseases, mostly focused on cases in New York State and much of it on Lyme disease - but there is also discussion on tickborne diseases in a more general sense as well.
I watched the video and made a note on topics of discussion during various points of time during the presentation which may be of interest to others.
Note that it is a little over an hour long, but you can skip the first three minutes as they are only an introduction. The last fifteen minutes are dedicated to a question and answer session with the audience - including one person who walked out because she was not satisfied with Dr. Benach's response.
[Time: 1:06:41]
11:39 Benach discusses Lone Star tick as primary tick on Long Island and that the number of cases of Lyme disease are going down in Eastern Long Island - possibly due to this tick's expansion.
16:33 Lifestyle of Ixodes tick described.
23:17 Early Babesia microti case on Long Island identified in 1970's - opens discussion on Babesiosis. Risk categories: over 50, elderly, asplenic, immunosuppressed, and/or alcoholism history.
29:00 Beginning of Lyme disease discussion... history of discovery, use of dark field microscopy for detection; electromicroscopy.
36:12 60% of patients have EM rash that is noticed. 40% do not.
37:20 Secondary Disseminated symptoms - refractory to treatment - Benach does not understand what happens with chronic Lyme disease patients. Audience member brings up infection-related damage, Benach agrees with him that this is a problem - then goes back to discussing acute Lyme disease.
39:40 A rash that enlargens is clearly an EM rash. This is key to early diagnosis with a rash.
40:20 Multiple EM rash is sign of disseminated Lyme disease and requires IV or parenteral antibiotics.
40:57 Discusses spirochetes affecting the CNS and how it is similar to syphilis, and that a dementia-like form of Lyme disease is controversial. Audience member mentions person who was completely messed up by neurological Lyme disease; had CSF that was positive for Lyme disease and improved with IV treatment.
43:00 Benach thinks neurologic involvement in Lyme disease is underreported.
43:10 Explanation of Bells palsy in a child, says it is very common but not malignant.
43:57 Mentions Lyme arthritis in the classic sense. Discusses symptoms as relapsing and remitting.
44:38 Benach is under impression that most people's cases of Lyme disease are caught early and treated early due to presence of EM rash.
44:50 Epidemiology of Lyme disease in New York State and counties in NY. Benach thinks doctors in some counties are treating Lyme disease and are not reporting their cases to the state any more - they are "Lyme tired". For other counties, there is active surveillance, and the numbers are going up as more cases are new to their area.
47:00 Quip that LD now threatens politicians in Albany.
47:48 Is Lonestar tick driving other ticks away? Maybe… someone needs to study it.
48:13 Audience member asks about birds. Catbirds and robins have ticks, but don't carry a lot because they like the rims near eyes (bare skin). Birds are dead ends for the spirochetes because of their high temperature, according to Benach…
49:30 Start of Q & A session
51:38 Do people have natural immunity to Lyme disease? Benach does not think so - there is universal susceptibility to LD.
53:00 Jury still out on whether or not people have genetic susceptibility to Lyme disease. Hard to know if you are bitten multiple times if you have new instance of disease or preexisting disease because Lyme disease can last for 30 (possibly more) years in the human body.
54:40 No known existence of antibiotic resistant Lyme disease. Does he rule it out completely? No. But he states Borrelia are genetically challenged and have so few genes they need them to do housekeeping; they have a very small genome. He says there is no presence of those genes and he is 90% sure there is no antibiotic resistance.
57:09 Vaccine discussion - brief.
58:00 Pesticide soaked cotton balls used to fight ticks locally. (Damminix)
1:00 Opinion on prolonged chronic Lyme IV treatment: If my child or I myself had a very strong titer for Lyme disease, I would use antibiotics for as long as it did good. If I did not have a very strong titer, then I would be reluctant to use antibiotics due to side effects.
Recurring arthritis and neurological manifestations come with strong serology according to Benach.
Benach leaves the audience with a confusing opinion: On one hand, he states he would not take antibiotics long term. On the other, he states that if he continued to be sick in the presence of strong serology then he would take antibiotics.
1:05 IgM doesn't drop over time in Lyme disease. We cannot culture Lyme disease easily, doesn't grow well in vitro - it is very slow growing. Only mycobacteria divides more slowly. You need 5 weeks to culture Borrelia. Benach's implication is no one would wait for those results - test is too difficult; takes too long.
Migrating birds can easily carry Borrelia spirochetes because their average daytime temperature is around 42.5C and goes down to 33C at night - the birds temporarily have hypothermia. They do this to save energy during long trips.
While some strains of Borrelia are sensitive to the birds' higher temperature range, some birds are actually conducive of supporting Borrelia spirochetal infections. Catharus fuscescens is one example.
B. garinii, at 41C has the highest growth temperature on record. However, just because Borrelia stop growing doesn't indicate it is not present. Under varying temperature conditions, some Borrelia may be able to survive.
Another comment is that Dr. Benach mentions that Borrelia burgdorferi does not show signs of antibiotic resistance or genes for antibiotic resistance mechanism.
I posted some of my notes on VGV-L on LNE, related to the previous entry on VGV-L which I posted on Monday. While there is a lot I posted about VGV-L based on interviews, press releases, and patents - and while I outlined how the immune system normally should work - I did not give a concise explanation of how the immune system doesn't work relative to polyclonal b cell activation.
I think another entry on this topic is in order. But in the meantime, I have decided to share the notes I posted on LNE about VGV-L here before I write a more concise post on the immune system and polyclonal b cell activation...
I did as much poking around about Viral Genetics' new chronic Lyme disease candidate drug, VGV-L, as I could and it wasn't easy. One CV, several patents, and a few Dr. Karen Newell Roger interviews later, and the best I could get out of it is that it is not just a synthetic thymus peptide that they patented but a method of replacing a peptide, CLIP, on the surface of B cells with this synthetic thymus peptide (which somehow maps to the MHC genetic type (HLA) of the patient) so that it activates Treg cells. The activation of these Treg cells is supposed to lead to reduction of non-specific B cells (polyclonal B cell activation).
In the patent, there is also mention of using bacterial antigens and antibiotics as adjunct treatments which are optional. The impression I'm left with is the bacterial antigens are used to prime new B cells and if there is any existing infection, antibiotics are used.
So the entire method of treating patients may be: a) VGV-L alone, b) VGV-L and antigen exposure, or c) VGV-L, antigen exposure, and antibiotics are used. (antivirals and antiparasitics may also be used, depending on the patient's diagnosis)
[...] I don't get the impression after reading everything I've read thus far that chronic Lyme disease is a pure B-cell disorder. But maybe it's a variant on that? Maybe cell-mediated immunity is somehow affected by B. burgdorferi?
I've made a major edit on my blog entry. I still think I need a revised version of the post to be made in the future, but I am so exhausted at the moment that I think it won't be for a few days at least. Maybe call it the revised executive summary version, because the current post is rambling even relative to rambling for me.
Why do I mention this edit here? Because it is very important to note, relative to how I infer the way VGV-L has been hypothesized to work:
The edit was about Tunev and Barthold's research on lymphadenopathy in Borrelia burgdorferi infection in mice. In their research, they noticed an outsized B-cell response to the presence of spirochetes. However, what they found differed from what has been found in polyclonal B-cell activation in other infections - where it's clear runaway non-specific polyclonal B-cell activation leads to autoimmune disorders. In Tunev and Barthold's research, the outsized immune response had B-cells which were specific for Borrelia burgdorferi yet were of low quality. This is notably different from typical polyclonal B-cell activation.
"From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality. By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed. The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3. Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally. Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies. If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host."
I don't know how this difference in response would work with VGV-L. It isn't clear to me, and I have to read through more research to understand it - at least hypothetically. I also want to know if anyone else has used similar technology to treat pure B-cell disorders and if so, what the pros and cons were. If this is a completely novel invention, then it's harder to evaluate and all one can do is look at the animal trials if one has access to them and examine the underlying hypothesis for its application.
The paper that this technology primarily appears to be based on is this one:
M. K. Newell, R. P. Tobin, J. H. Cabrera, M. B. Sorensen, A. Huckstep, E. M. VillalobosMenuey, M. Burnett, E. McCrea, C. P. Harvey, A. Buddiga, A. Bar-Or, M. S. Freedman, J. Nalbantoglu, N. Arbour, S. S. Zamvil, and J. P. Antel. 2010. TLR-Mediated B Cell Activation Results in Ectopic CLIP Expression that Promotes B Cell-Dependent Inflammation. Journal of Leukocyte Biology.
Online e-Pub. July 14, 2010.
I'll just post the abstract here, and you can read the entire text at the link above:
Abstract
Infectious pathogens produce compounds called Toll ligands that activate TLRs on lymphocytes. Acute activation triggered by certain TLRs appears to "jump start" the innate immune response, characterized by the release of inflammatory cytokines and cellular expansion.
In some individuals, there is a failure to control acute inflammation, resulting in postinfectious, chronic inflammation. Susceptibility to chronic inflammation is strongly associated with an individual's MHC genes. Recent clinical trials for several autoimmune diseases characterized by chronic inflammation suggest that B lymphocyte depletion therapies dampen chronic immune activation. However, currently, there is no known mechanism that accounts for the correlation among TLR activation, MHC genetics, and a pathological role for B-lymphocytes.
Our hypothesis is that TLR-activated B cells (B cells that have been polyclonally activated in the absence of antigen-specific signals) are not controlled properly by T cell-dependent B cell death, thereby causing B cell-dependent chronic inflammation.
Here, we show that treatment with Toll ligands results in polyclonal B cell activation accompanied by ectopic expression of CLIP. Furthermore, by adoptively transferring purified CLIP+ B cells in syngeneic animals, we find that CLIP+ B cells induce production of TNF-α by host T cells. Finally, we demonstrate that CLIP-targeted peptide competition results in the death of polyclonally activated CLIP+ B cells.
I think I know at this point what my missing pieces are now:
- describe the role of Treg cells (regulatory T cells) and how they relate to killing off B cells and generating more targeted immune responses.
- describe the hypothesis that Foxp3+ Treg generation in the thymus is somehow dysfunctional in those with persisting symptoms (I think this has some relationship to the core hypothesis behind VGV-L's use).
- And I have to more clearly state that even if the Foxp3+ Treg generation is what it is happening, there may be some risk involved in proceeding with this kind of treatment. Wikipedia managed to explain some of it, and while Wikipedia is not the most reliable resource on everything, it is relatively easy to follow and this particular entry (so far) jives with what I've seen in other sources:
Plus, of course, there are the papers I link to on the blog.
The more I delve into this, the more questions I have... Tunev and Barthold's paper, in particular, lead me to wonder if the hypothesis underlying VGV-L's design developed with the scenario described in their research being taken into account. Between T & B's research plus other publications, I get the impression that in at least the animal model of immune response to Bb, that there is a mixed state response to it - It is both immune suppressing and immune stimulating. Some research has even indicated a certain amount of tolerizing is involved. (http://en.wikipedia.org/wiki/Peripheral_tolerance - anergy plus inflammation; see also http://users.ox.ac.uk/~path0116/tig/tolg2.html for a more detailed explanation)
Adding a bacterial antigen/adjuvant (and possibly antimicrobial medicine) to the treatment may be a way of working around this combination, in order to produce new B cells which are stimulated and respond specifically to Bb and not be nonspecific - and in order to eliminate any remaining infection if there is one present.
This is complicated - and while more and more is being learned about immunology every day, there are still an enormous number of unknowns...
On Friday I posted about the use of Filgrastim and Ceftriaxone for treating persisting symptoms in a Lyme disease case study as well as the use of Rituximab for treating CFS/ME. I also touched upon Viral Genetics' VGV-L or targeted peptide therapy for treating chronic Lyme disease, and wanted to write an entry about this treatment on its own.
What I can tell you is to some degree limited by the fact that VGV-L's exact design and mechanism is proprietary in nature, so I can only report based on what the researchers and Viral Genetics choose to disclose. But hopefully, what I post here and future publications by Dr. Karen Newell Rogers will shed some light on the matter.
Dr. Karen Newell Rogers from Texas A & M is in the middle of contributing to the following three papers which seem to have a relationship between VGV-L and chronic Lyme disease:
S. Harris, E. W. Newell, R. P. Tobin, C. P. Harvey, N. Kurzman, E. M. Hechinger, P.
Cipriani, and M. K. Newell. 2010. Comparative Analysis of Peptide Binding, MHC alleles,
and B cell activation in patients meeting CDC criterion for Chronic Lyme Disease.
(manuscript in preparation).
E. Connick, R. Schlichtemeier, J. Folkvord, R. Tobin, C. P. Harvey, and M. K. Newell.
2010. TLR activation of human peripheral blood B cells can be reversed by peptide
treatment. 2010. Manuscript in preparation.
Cabrera, J. and M. K. Newell. 2010. Polyclonal TLR-induced B cell activation is
controlled by Peptide-dependent B cell death (manuscript in preparation).
All three in preparation, but I think they are tightly related to the same research and stem from this previous publication:
M. K. Newell, R. P. Tobin, J. H. Cabrera, M. B. Sorensen, A. Huckstep, E. M. VillalobosMenuey, M. Burnett, E. McCrea, C. P. Harvey, A. Buddiga, A. Bar-Or, M. S. Freedman, J. Nalbantoglu, N. Arbour, S. S. Zamvil, and J. P. Antel. 2010. TLR-Mediated B Cell Activation Results in Ectopic CLIP Expression that Promotes B Cell-Dependent Inflammation.Journal of Leukocyte Biology.
Online e-Pub. July 14, 2010.
In this patent, the portion attributed to Lyme disease states:
"[0116] It is believed according to the invention that Borrelia burgdorferi also produces a Toll ligand for TLR2. Replacement of the CLIP on the surface of the B cell by treatment with a thymus derived peptide with high affinity for the MHC fingerprint of a particular individual, would result in activation of the important Tregs that can in turn cause reduction in antigen-non-specific B cells. Thus treatment with thymus derived peptides could reactivate specific Tregs and dampen the pathological inflammation that is required for the chronic inflammatory condition characteristic of Lyme Disease. With the appropriate MHC analysis of the subject, a specific thymus derived peptide can be synthesized to treat that subject. Thus individuals with all different types of MHC fingerprints could effectively be treated for Lyme disease."
However, I just found out that there are additional patents on this technology of which I was previously unaware. These patents contain a great deal of detail about what these targeted peptides can do and their effect on polyclonal B cells:
In addition to the above published paper on CLIP expression, Viral Genetics published the following excerpt in its research newsletter which explains what VGV-L does for HIV in easy-to-understand terms - substitute "Lyme disease" for "HIV" here:
"The conventional approach to HIV vaccines, for example, is to develop therapeutic vaccines to stimulate immune system response. The problem with the conventional approach is that the infected cells are camouflaged and not visible to the body’s immune system. The body’s powerful T-cells are unable to seek out and destroy the infected camouflaged cells because they cannot recognize that the cell is infected.
To understand the issue, think of the Klingon space ship on Star Trek that has its cloaking device activated. The U.S.S. Enterprise has no way of knowing where the enemy is in space. The only hope it has in winning the battle is for the Klingon vessel to be de-cloaked and, once revealed, use their ammunition to destroy it. What’s worse in the case of HIV is that while the infected cell is cloaked, it is also effectively setting off an alarm that triggers the immune system to create inflammation. Why is this important? It turns out that this inflammation is critical for allowing the HIV virus to spread to even more cells.
Many other viruses and bacteria also trigger inflammation but, unlike HIV, the inflammation does not necessarily allow or facilitate the spread of the virus or bacteria itself. * However, in these cases, the inflammation itself is harmful because it creates a hostile and inflamed environment that provides the necessary components for a potential autoimmune reaction that can cause the immune system to attack and damage one’s own body. Viral believes that diseases such as Lyme Disease, Multiple Sclerosis and others involve this inflammatory mechanism.
To use the Star Trek metaphor, what Dr. Newell Rogers has developed with TPT is a de-cloaking device for the body’s immune system to use in its pursuit of invaders. Through the development and use of computational biology programs and databases, Dr. Newell Rogers and her team havecreated a way to remove the camouflage that is cloaking the infected cells, flagging them with custom peptides that allow the body’s immune system to seek out and destroy them.
The key discovery of the TPT platform is that a self-peptide (in other words, one that is naturally produced and a healthy part of one’s normally functioning immune system) called ―CLIP2 that was until now thought only to exist primarily inside certain immune system cells, is sometimes displayed on the outside of cells, thus leading to harmful inflammation. Dr. Newell Rogers discovered that the products of some pathogen invaders such as viruses and bacteria, when picked up on the surface of certain immune system cells, sometimes incorrectly cause those cells to display CLIP externally (i.e. ―ectopically).
Normally, when an invader strikes, this process may promote needed inflammation early in infection, but it is quickly controlled when a more specific, immune response takes over, allowing a highly-targeted immune response to be marshaled against the pathogen. However, when CLIP is improperly displayed, displayed for too long or displayed chronically, the immune system is marshaled to promote a broad and unspecified inflammation without the specific targeting, leaving open the possibility that this inflammation actually turns against one’s own cells. Replacing CLIP is the focus of Viral’s Targeted Peptides because it turns off the harmful alarm."
One thing which I have thought of (and heard a few patients mention in passing) is that this candidate drug is only for treating inflammation and would only address an autoimmune angle relating to chronic Lyme disease.
However, this is not the case:
If you read the full patents, VGV-L's technology works not only to reduce inflammation, it also works to rebalance the immune system so that it is focused on fighting infection in a targeted manner. And in terms of treatment with VGV-L, patients may not just receive VGV-L alone - but also receive a bacterial antigen and antibacterial (possibly also antiparasitic and/or antiviral) therapy concurrently to treat their condition.
Here is the excerpt from the patent concerning the treatment of infections using this technology - including Lyme disease:
[0169] Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, mycobacteria, rickettsia, mycoplasma, neisseria, Borrelia and legionella.
[0170] Although Applicant is not bound by a specific mechanism of action it is believed that the CLIP inhibitors of the invention displace CLIP from MHC class I and cause down regulation of Treg activity and/or activation of effector T cells such as γδT cells.Downregulation of regulatory function of Treg activity prevents suppression of the immune response and enables the subject to mount an effective or enhanced immune response against the bacteria.At the same time the Treg cell may shift to an effector function, producing an antigen specific immune response. Thus, replacement of CLIP with a peptide of the invention results in the promotion of an antigen specific CD8+ response against the bacteria, particularly when the peptide is administered in conjunction with a tumor specific antigen. Activation of effector T cells also enhances the immune response against the bacteria, leading to a more effective treatment.
[0171] One component of the invention involves promoting an enhanced immune response against the bacteria by administering the compounds of the invention. The compounds may be administered in conjunction with an antigen to further promote a bacterial specific immune response. A "bacterial antigen" as used herein is a compound, such as a peptide or carbohydrate, associated with a bacteria surface and which is capable of provoking an immune response when expressed on the surface of an antigen presenting cell in the context of an MHC molecule. Preferably, the antigen is expressed at the cell surface of the bacteria.
[0172] The compounds of the invention may be used in combination with anti-bacterial agents. Examples of such agents to treat bacterial infections include, but are not limited to, folate antagonists (e.g., mafenide, silver sulfadiazine, succinylsulfathiazole, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, pyrimethoamine, trimethoprim, co-trimoxazole), inhibitors of cell wall synthesis (e.g., penicillins, cephalosporins, carbapenems, monobactams, vacomycin, bacitracin, clavulanic acid, sulbactam, tazobactam), protein synthesis inhibitors (e.g., tetracyclines, aminoglycosides, macrolides, chloramphenicol, clindamycin), fluoroquinolones (e.g., ciproloxacin, enoxacin, lomefloxacin, norfloxacin, ofloxacin), nalidixic acid, methenamine, nitrofurantoin, aminosalicylic acid, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampin, clofazimine, and dapsone.
I don't know entirely what the researchers intend to use as a bacterial antigen... An Osp? They are suggesting a peptide or carbohydrate, though, and not a highly immunogenic lipoprotein from the cell's outer membrane - even though that's what I think they would have to use if they were to use an antigen. Reading ahead, though, there is the potential that any of a number of Borrelia burgdorferi antigenic products may be used.
Both items #0171 and #0172 have wording which implies that they are optional treatments, as they use the word, "may be administered" or "may be used" rather than "will be administered" or "will be used", respectively. I would assume that whether or not these individual treatments are applied depends entirely on the individual patient and their needs and clinical diagnosis.
So, it seems that whether there is current infection or not, VGV-L may be one way to effectively treat chronic Lyme disease and lower inflammation due to runaway immune dysregulation. And if infection is currently present, then it looks like VGV-L will trigger a more targeted immune response towards bacteria rather than the overload that polyclonally expanded B cells can be.
One of the more fascinating sections of the patent is towards the end. The researchers give a number of examples of how their technology was applied and what the results were. Example 13 of this patent appears relevant to demonstrating how Borrelia burgdorferi activators affect tissue and about eliminating excessive B cells which cause inflammation in tissues. They did an in vitro post-mortem study of these actions in mice:
Example 13 - TLR Activators Promote CLIP-MHC HLA Association and CLIP Inhibitor Peptides Reduce an TLR Activator Promoted CLIP-MHC HLA Association
[0480] Methods
[0481] Preparation of Cells: Mice were Sacrificed by Cervical Dislocation. Spleens and lymph nodes were removed; the tissues were minced through cell strainers to create single cell suspensions; red cells were lysed using buffered ammonium chloride followed by addition of phosphate buffered saline and centrifugation to wash out the ammonium chloride; and the cells were counted using trypan blue exclusion to determine live versus dead cell discrimination and to determine the number of cells per tissue.
[0482] Treatments: The spleen or lymph node cells were treated in vitro with various stimuli (TLR activators: CpG ODN (Alexis), LPS (Sigma), Polyl:C (BD Pharmagen), Pam3Cys (Genway); IL-4 (BD Pharmagen), anti-CD40 monoclonal antibody (BD Pharmagen), both IL-4 and anti-CD40 antibody and OspA and Osp C (Genway) and the cells were cultured for the indicated time periods. The cells were grown in RPMI 1640 medium supplemented with standard supplements, including 10% fetal calf serum, gentamycin, penicillin, streptomycin, sodium pyruvate, HEPES buffer, 1-glutamine, and 2-ME as well as (where indicated) the stimuli listed above. The cells were incubated at 37° C. in an atmosphere containing 5% CO2 and approximately 92% humidity. The cells were incubated for 3, 24, and 48 hours. At each time point, the cells from that experimental time were harvested and stained for flow cytometric analysis of cell surface expression of CLIP (MHC Class II invariant peptide/IAb, Santa Cruz) by using the commercially available anti-mouse CLIP/IAb peptide, anti-mouse B220, anti-mouse CD4, anti-mouse CD8, and anti-mouse FoxP3 (all commercially available from Becton Dickinson/Pharmingen). Harvested cells were stained using standard staining procedure that called for a 1:100 dilution of Fitc-anti-mouse CLIP/IAb or isotype control. Following staining on ice for 25 minutes, cells were washed with PBS/FCS and resuspended in 100 microliters and added to staining tubes containing 400 microliters of PBS. Samples were acquired and analyzed on a Coulter Excel Flow Cytometer. The data were analyzed using FloJo software.
[0483] Results
[0484] B cell death, including total B cell death and % CLIP positive B cell death in cells treated with a TLR activator (CpG ODN) alone or in combination with MKN3 in the presence or absence of CLIP was assessed. The results are shown in FIG. 12. FIG. 12 is a line graph having a double Y axis, on one side depicting % total B cell death (diamonds, representing CpG ODN alone and squares representing CpG ODN+MKN3) and on the other side depicting % CLIP+ B cell death (triangles, representing CpG ODN and CLIP alone and Xs representing CpG ODN+MKN3 and CLIP). The data reveal that CpG ODN cause an initial increase in B cell death which after 72 hours appears to level off. The CpG ODN+MKN3 data demonstrate that MKN3 is capable of preventing the increase in B cell death.
[0485] Changes in CLIP positive B cells in spleen versus lymph nodes were also assessed. FIG. 13 is a line graph having a double Y axis, on one side depicting % CLIP+ B cell numbers in spleen (light gray square with solid lines representing CpG ODN alone and dark gray square with solid lines representing CpG ODN+MKN3) and on the other side depicting % CLIP+ B cell numbers in lymph nodes (diamonds with dashed lines representing CpG ODN alone and light gray square with dashed lines representing CpG ODN+MKN3). In both spleen and lymph nodes the addition of the peptide to the cells with CpG ODN resulted in less CLIP positive B cells.
[0486] CLIP positive B6.129 cultured B cells (H-2b haplotype) and H2M-/- (from C3H HeJ mice) cultured B cells were also examined in the presence or absence of treatment with a number of different TLR activators. The data is shown in FIGS. 14A and 14B. As shown in the Figures, several TLR activators were able to induce levels of CLIP+ B cells.
Just so it's clear, this isn't the treatment a patient would receive - Dr. Newell Rogers and her colleagues won't be breaking your neck and removing your tissues if you sign up for a clinical trial, okay?
This is an example of an experiment they did to show that VGV-L technology is effective in reducing the number of ineffective B cells which cause inflammation. The end result measured this change, and also measured the end of the sordid relationship between TLR-promoted CLIP MHC-HLA association in the immune system.
[Edited Apr. 3, 2012: Removed mention of CLIP positive cells - these cells need to be removed not added. ]
Now time for a brief lesson in immunology, based on what normally happens in immune response:
Terminology: MHC = major histocompatibility complex; key components of T cell immunity. Think of them as immune response genes. HLA = human leukocyte antigen (think of earlier discussions on this blog about HLA-DR4 and HLA-DR11, and different alleles which respond to infection differently)
So the story goes, B cells express MHC class II. Once antigen has been bound on the antigen receptor on the B cell, the antigen and its receptor are sucked into an endosomal compartment inside the B cell. Then the endsomal compartment fuses with another compartment, the lysosome.
Antigens are broken down into smaller pieces inside the lysosome and then loaded onto the MHC class II component, then the MHC is transported to the B cell surface where the B cell displays the antigen to a CD4+ T cell. This T cell is also known as a helper cell, of which there are two types - Th1 and Th2.
Susceptibility or resistance to many diseases appears to be determined by the genes encoding Major Histocompatibilty Complex (MHC) molecules. Often referred to as immune response genes (or IR genes), these molecules are the key players in restricting T cell activation.
T cells, both CD8 and CD4 positive T cells, recognize antigens only when the antigen is presented to the T cell in association with MHC class I (expressed on all nucleated cells) or MHC class II molecules (expressed on cells that present antigens to CD4+ T cells), respectively.
To sum up:
B cells express MHC class II.
Different people produce different levels of allele variation in MHC locus.
Because of this genetic difference, some people are more or less vulnerable to certain diseases.
The B cell's expression of MHC class II is noticed by CD4+ T cells.
These CD4+ T cells are known as helper cells - of which there are two types, Th1 and Th2.
CD4+ T cells are a major player in our immune systems for fighting infection.
These helper cells do not kill - they activate and direct other immune cells. They are essential in B cell antibody class switching, in the activation and growth of cytotoxic T cells, and in maximizing bactericidal activity of phagocytes such as macrophages.
Look at these handy diagrams as an overview to what happens with B cells, MHC molecules, and T-cells...
Or, if your learning style is better geared towards watching videos, watch this one (warning: might be preceded by an ad, which you can mostly skip):
A simple overview of the immune system [Time: 5:56]
If you've gotten this far, right about now you might be asking yourself, "So what's the big deal? Why is it an issue that there are excess B cells, and how getting rid of them going to make a difference in fighting off Lyme disease if I have a chronic infection?"
Good questions.
Obviously, you can see so far that one reason to not have certain B cells around is that they trigger autoimmune responses. No one wants that. But there are other reasons to avoid an overzealous non-specific B cell response.
Let me unwind the answer, step by step.
Google "polyclonally expanded B cells Borrelia" and tell me what you find. Or, read on and I'll tell you what I found:
Remember that study on Borrelia burgdoferi that Tunev and Barthold did, where it was noted that there was an outsized yet seemingly inadequate immune response to Borrelia burgdorferi found in lymph nodes? One with ill-formed B cells? This one: http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002066 ?
Well, the phenomenon that's happening there is somewhat different from what is happening in polyclonal B cell activation in general. In polyclonal B cell activation, there can be lots of B cells which are produced which are non-specific. In this case, in Tunev and Barthold's research, a notable percentage of the B cells were Borrelia burgdorferi antigen specific - yet the cells were of low quality and inadequate to the task.
That's different than what is generally understood to be the typical polyclonal B cell activation, which is what I think is the hypothesis behind this treatment, VGV-L. In both cases, what one observes is an overwhelming number of B cells being produced.
[Edit Apr. 3. 2012: Updated correction to describe Tunev and Barthold difference in outcome of B cell activation.]
Polyclonal b cell activation has been thought to be a useful immune defense mechanism early in acute infection. What has not been investigated as much is how it might be a damaging process in chronic infection and immune dysregulation.
It's important to settle this argument because dysregulated and mis-targeted B cell antibody responses could result in autoimmunity, whereas impaired antibody responses during an actual infection could result in an immune deficiency.
Either way, over time it has become clearer that the production of these B cells relates to the appearance of an IgM response, and the relevance of the presence of an IgM response - particularly a prolonged IgM response - has also become a subject of heated discussion. See: "IgM in microbial infections: Taken for granted?"
"We have used the murine model for Lyme disease described by Barthold et al. (S. W. Barthold, D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody, J. Infect. Dis. 162:133-138, 1990) to determine whether the B. burgdorferi B-cell mitogen is expressed during active infection.
To correlate arthritic changes with immune events, we have studied two strains of mice injected with B.
burgdorferi; one of them, C3H/HeJ, developed severe disease, and the other, BALB/c, developed only mild
disease.C3H/HeJ mice displayed a persistent 10-fold increase in circulating immunoglobulin G (IgG) levels, a
2-fold increase in IgM levels, and a 15-fold increase in peripheral lymph node B-cell numbers, providing
evidence of mitogenic activity. Infected BALB/c mice also had evidence for mitogen activity, since the IgG level
in serum increased three- to fourfold.
The bulk of the increase in circulating IgG levels was not directed against B. burgdorferi antigens, supporting the occurrence of polyclonal B-cell activation. Analysis of IgG isotpes pointed out a contrast between C3H/HeJ and BALB/c mice in that levels of all isotypes were elevated somewhat
in both strains of infected mice but IgG2a levels were much more dramatically increased in the C3H/HeJ mice
(28-fold) than in the BALB/c mice (4-fold). In this study, interleukin-6 levels were found to be persistently
elevated in the serum of infected C3H/IHeJ mice. Interestingly, interieukin-6 levels in serum were much lower
in the infected BALB/c mice. These findings indicate that the B. burgdorferi mitogen is active in infected
animals and may contribute to the inflammatory and immune response to infection."
Right from the start, you get the idea that this paper is going to tell you that the presence of these polyclonally activated B cells have a relationship to IgM and IgG levels.
This is relevant, very relevant - because it can reflect how antibodies to Borrelia burgdorferi are present and how they are picked up in serological testing like ELISA and Western Blots.
Meaningful excerpts from this paper include:
"Immunological abnormalities, including hyperactive B cells, elevated IgM levels in serum, lymphadenopathy, impaired natural killer function, and delayed development of humoral immunity, have been documented in patients with Lyme disease (11, 16, 29, 30, 32). This has suggested a possible involvement of the specific or innate host responses
in the pathogenesis associated with stage 2 and 3 disease(32)."
"Because of the persistent nature of infection and the ability of the organism to gain access to the joint and other tissues (5, 15, 29), a B-cell mitogen present during infection could play a role in the pathology of Lyme disease. To support this possibility, it was important to determine whether the mitogen functioned in vivo. This paper provides evidence that a B. burgdorferi mitogen is active in vivo in infected animals.
Three lines of
evidence support the conclusion that B-cell activation in vivo is polyclonal or oligoclonal in addition to being antigen specific.
First, the level of IgG in serum in infected mice was elevated about 10- to 15-fold, with the value ranging from 10
to 15 mg/ml (Fig. 1C). In comparison, the amount of IgG specific for B. burgdorferi antigens was approximated at 0.6
mg/ml (Fig. 2).
Second, the number of B lymphocytes in peripheral lymph nodes of infected animals was increased
10- to 15-fold, with a 5-fold rise in the ratio of B to T cells (Fig. 4). The number of B cells also increased about twofold relative to the number of T cells in spleens from C3I/HeJinfected mice.
Third, the IgG titer in the serum of infected animals to an unrelated antigen, ovalbumin, was increased 10- to 15-fold, which resembles the increase in the total IgG level (Fig. 3).
These findings suggest that levels of autoreactive antibodies might be also expanded in infected animals, although anti-collagen antibodies were not identified. Because CD5+ B cells have been shown to produce autoreactive antibodies and are selectively increased in patients with rheumatoid arthritis (7), we determined whether they were expanded in B. burgdorferi-infected animals.No selective increase in the number of B cells of this lineage were found in C3H/HeJ animals at any stage of infection. Further studies are required to determine whether autoreactive antibodies are generated during infection."
So their initial experiment to see if there was an overwhelming B cell response provides us with evidence that yes, there is, and also - while there is a high IgG response, only a small percentage of IgG produced is B. burgdorferi specific. There was at the time no indication that autoreactive antibodies were involved.
(This process can be a precursor to autoimmunity developing - but that's later on.)
A later paper, from 1997, "Why is chronic Lyme Borreliosis chronic?"(full text), also brings up a host of issues related to TLRs, MHC class II, and the relationship between B and T cells in lymph nodes.
Doesn't it seem a little prescient?
"The question remains whether downregulation or even loss of MHC class II molecules on LCs might influence a patient's disease susceptibility. It is MHC class II molecules that bind antigenic peptide fragments, present them to CD4+ Th cells, and induce cytokine secretion and IgG secretion by B cells [55]. In vitro investingations have shown that MHC II class molecules are downregulated on antigen-presenting cells after coculture with Th cell clones in the presence of antigenic peptides of tetanus toxoid or staphylococcal superantigen, which elicit a strong HLA-DR-restricted T cell response.
Several hypotheses were suggested as the cause of this down-regulation.
(1) Downregulation occurs when antigenic peptides catabolized in macrophages are recognized by CD+4 helper T cells, in order to control the size of a T cell clone and provide a homeostatic mechanism [55]. (2) Downregulation occurs for completion of T-B cell collaboration after antigen presentation, limiting excessive T cell help to the triggered B cells, or (3) it occurs for focusing the T cell repines to one or a few immunodominant peptides.
(4) LCs of patients with AIDS express decreased amounts of MHC class II molecules. Polyclonal B-cell activation, as seen in these patients and in patients with ACA, could cause the appearance of autoantibodies or immunocomplexes that interact with LCs and block their surface-staining characteristics [45]. (5) IL-10, originally identified as a product of Th2 cells, has a significant inhibitory influence on the antigen-presenting functions of macrophages and LCs by downregulation of MHC class II molecules. In fact, LCs pretreated with IL-10 were converted from specifically sensitizing to specifically tolerogenic antigen-presenting cells in vitro and in vivo [56]. In other studies treatment of LC cultures with IL-10 inhibited to upregulation of HLA-DR [57].
(6) Downregulation is initiated for establishment of self-tolerance. This downregulation can protect the antigen-presenting cell by inhibiting the presentation of self-antigens [58]. On the other hand, the downregulation of MHC class II antigens on LCs could result in inadequate presentation of antigens in lymph nodes, which in turn may reduce activation and proliferation of both B and T cells and the secretion of relevant cytokines. This may be what happens in CLB."
Dr. Karen Newell Rogers et al recent patent has this to say about TLRs (Toll Like Receptors) and B cells:
"Many bacteria and viruses produce substances, collectively called Toll ligands, that elicit an immediate response from an individual's immune system. These Toll ligands appear to promote inflammation by activating a wide variety of immune cells to bring them rapidly into battle against the invading pathogen.
In most cases, these events correlate with a healthy and productive immune response to the pathogen. However, in some cases the Toll ligand binds to a Toll-like Receptor (TLR) on lymphocytes and non-specifically activates immune cells called B and T lymphocytes that would normally to respond to infectious pathogens with an exquisitely specific response. When Toll ligands activate B cells in a non-specific way, the non-specific activation is a pro-inflammatory event that may result in uncontrolled, or even auto-reactive, production of antibodies. When a B cell is activated non-specifically, we have discovered that the B cell expresses an important, small self-peptide called MHC class II invariant peptide, CLIP. In most individuals, a control cell, known as a T regulatory cell (Treg for short), has been shown, to kill the activated B cell.
During a viral or bacterial infection, non-antigen specific B cells in close proximity to an inflammatory or inciting lesion could manage to become activated in a bystander fashion. In those cases, CLIP would remain in the groove and get transported to the cell surface of the B cell. Its presence on the cell surface can be undesirable because if CLIP gets removed from the groove by a self antigen, the B cell would be in a position to present self antigens to self-reactive T cells, a process that could lead to autoreactivity and autoimmune disease.
For some B cells this may result in death to the B cell by a nearby killer cell, perhaps a natural killer (NK) cell, unless the antigen receptor on the B cell has engaged antigen. Antigen recognition would thereby provide a survival signal for the B cell. However, if a killer cell doesn't remove the potentially autoreactive B cell and it encounters a CD4+ T cell that can recognize that antigen (most likely one that was not in the thymus) the B cell might receive additional help from a T cell specific for the antigen that now occupies the groove (antigen binding location in the MHC molecule). Alternatively, a nearby cell whose job it is to detect damaged self cells, may become activated by the self antigen-presenting B cell. Such a damage detecting cell is, for example, an effector T cell (Teff) such as a gamma delta T cell, also referred to as a γδT cell (γδ refers to the chains of its receptor). The γδT cell can then seek out other sites of inflammation (for example in the brain in MS, in the heart for autoimmune myocarditis, in the pancreas in the case of Type I Diabetes). Alternatively, the γδT cell might attempt to kill the CD4+ T cell that may respond to self antigens."
So based on all this, I think one has to consider that the complex interactions within the immune system related to B. burgdorferi infection have to be paid close attention to - and not just any persisting spirochetes themselves.
I am really interested in seeing what VGV-L - along with supportive and antibacterial treatment together - can do for chronic Lyme disease. It appears it not only prevents autoimmune responses to infection, but redirects the immune response so it can better target infection.
I do wonder, though, how VGV-L would handle a situation where many B cells are being created and a good percentage of them are antigen specific but are of low quality - such as those mentioned in Tunev and Barthold's research.
* It may be that Lyme disease is more like HIV in that inflammation may allow or facilitate the spread of spirochetes as it encourages vlsE recombination. See: http://www.jimmunol.org/content/167/6/3383.long for one example.
On the heels of Embers et al having published their statement on PLoSONE, a number of patients are already questioning its content.
Some are claiming that Embers et al statement about how their findings should not be used to oppose current IDSA treatment guidelines for Lyme disease is something they were asked to write - rather than something the authors included on their own.
I don't know. For this claim - whether it's true or not - I have no evidence. However, one thing I do know is that there are solid scientific reasons which back the need for more research on spirochetes which survive after prolonged antibiotic treatment.
The question, of course, which weighs heavily on every patient's mind has been this one:
Why aren't persisting spirochetes enough evidence of infection?
It's become a political hot button question, and it's a scientific question. But most people think that as long as the spirochetes Embers found are alive and metabolically active, that is enough evidence to state that yes, Lyme disease is a chronic infection - let's stop all this nonsense right now and change the treatment guidelines!
Given my own experience and how longer than standard treatment helped me improve, I totally get this. I've been there, done that - and I think that a standard course of antibiotic treatment does not work for everyone. Particularly if there is a delay in proper diagnosis and treatment. Particularly if a coinfection is present. Particularly if there is some abnormality in one's immune system.
But if you are a scientist and you are researching this phenomenon of persistence - whether you as a scientist suspect these spirochetes can cause persisting infection or not; whether the above claim by other patients is true or not - you will be called upon by other scientists to support your findings.
It isn't just going to be the IDSA or the ALDF or other organizations which deny the possibility of persistent infection as a cause of chronic Lyme disease which are going to want to know the outcome of your study.
It's going to be the American Society for Microbiology (ASM) that wants to know the outcome. It's going to be researchers in Europe like the Brorsons who study the "cyst" form of Borrelia burgdorferi and want confirmation of their own findings about persistence.
It's also going to be universities and health departments and many different organizations which may not have any particular position on whether or not Lyme disease can be chronic who will want to know the outcome of your study.
They're all going to want to know the outcome of a study such as Embers et al, so these researchers must be certain about what they found and its significance, and conduct additional research related to their findings in order to confirm them.
They must find evidence that no one can argue against - even the most skeptical - if they are to support their own hypotheses. And it may be that at this stage they genuinely do not know what to make of these persistent spirochetes and not only their ability to cause disease - but how they cause it.
I can easily imagine that Embers et al is being very cautious about the interpretation of their results and wanting further studies as easily as it is for other people to imagine that Embers at al were somehow instructed to downplay the significance of their spirochetes surviving antibiotic treatment.
Why do I say this? I say this because I have learned a few things about these stealthy bacteria and think there is good reason for Embers et al to be cautious about the interpretation and approaching their results either way.
Borrelia burgdorferi spirochetes, plasmids, and infectivity
After doing some research on this issue, the issue of whether or not these spirochetes were infectious and pathogenic or not is a more complex issue than it at first appears.
First, here's a refresher of some basic microbiological definitions. (Bear with me, I'll try to get through this part quickly.)
Infection = the replication of organisms in the tissue of a host; when defined in terms of infection, disease is overt clinical manifestation. In an inapparent or subclinical infection, an immune response can occur without overt clinical disease.
Colonization = A carrier (colonized individual) is a person in whom organisms are present and may be multiplying, but who shows no clinical response to their presence.
Pathogenicity = The pathogenicity of an agent is its ability to cause disease; pathogenicity is further characterized by describing the organism's virulence and invasiveness.
Virulence = refers to the severity of infection, which can be expressed by describing the morbidity (incidence of disease) and mortality (death rate) of the infection.
Invasiveness = invasiveness of an organism refers to its ability to invade tissue.
Now that we're past these definitions, I'll cut to the chase and say there are two important things to know upfront:
1) During invitro passage or certain stressors, Borrelia burgdorferican lose some of their plasmids. How soon this happens varies depending on the strain and particular isolates of Borrelia.
2) When Borrelia burgdorferi loses specific plasmids with specific genes on them, it can lose infectivity and pathogenicity. It should be noted that specific genes for specific purposes can show up on different plasmids on different strains. (For example: Bb strain N40's VlsE locus is different from the one found on commonly studied B31, and it shows up on a different plasmid than on B31.)
The essential bit of information here is that the loss of a particular gene or set of genes can affect spirochetes' ability to cause infection - and even if these genes are lost, spirochetes may still survive for a while. They can become attentuated or less infectious.
Numerous studies on Borrelia burgdorferi's plasmids have shown that lp28-1 is a linear plasmid which makes Borrelia burgdorferi infectious. VlsE genes found on lp28-1 are thought to be essential for mammalian infection with Borrelia burgdorferi.
When the lp28-1 and yet a different plasmid, lp25, are missing from spirochetes, they are unable to infect mice. The lack of lp25 completely abolishes infectivity since this plasmid encodes a gene (bbe22) which is essential for Borrelia burgdoferi's survival in mice.
Spirochetes which lose lp28-1 plasmids will still live for a while - but the immune system tends to mop them up in a few weeks without antibiotic usage.
Specific research on mutant spirochetes with a lack of the lp28-1 plasmid has shown the following:
"While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1− mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1− mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1− mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis."
To translate the above:
Normal Bb spirochetes infected C3H/HeN (mice which are specifically bred for the ability to demonstrate joint swelling and arthritic symptoms similar to those found in the average person who gets Lyme disease) mice and these spirochetes could not be cleared by the immune system despite the fact that these mice had a strong humoral response.
However, mutant Bb spirochetes which did not contain linear plasmid 28-1 were completely cleared by these C3H/HeN mice.
What's fascinating about this study is even though the mutant Bb spirochetes lacked lp28-1, these spirochetes could still disseminate. Only in severely compromised immune deficient mice (scid mice) could the spirochetes both disseminate and persist - acquired immunity must be functional in animals infected with such mutants in order to clear the spirochetes.
So, here is one example of how you can have spirochetes which are alive and metabolically active and can even disseminate - yet they are no longer causing disease. In this instance, they were cleared by the immunocompetent mice without the use of any antibiotics within a mere 18 days. (I wish I were that lucky!)
More recently, other plasmids have been found to contribute to infectivity in mammalian hosts - such as lp36. lp36 is viewed as being another major contributor to persistent infection in mice, and spirochetes become attenuated when lp36 is removed.
Linear plasmid 28-1 and lp25 have a much longer history of their role in infectivity and pathogenicity, and they are two of the most studied linear plasmids thus far - lp28-1 the most because of its VlsE genes in strain B31.
So, keep this in mind when you think of the Embers et al study, and realize why this part of their paper on Rhesus macaques caught my attention:
"A few spirochetes grew in cultures of organ tissues collected post-mortem from each animal after > 9 weeks, but we were unable to subculture any spirochetes from either treated or untreated animals due to their slow growth. We therefore pelleted these cultures to confirm their identity and test their viability by DNA/RNA analysis. Transcription was detected in culture pellets and the tissues of treated animals, indicating that the bacteria were metabolically active (Figure 6C, D). Figure 6D shows ospA transcription detected directly in tissues harvested from treated and untreated animals.We also hypothesized that persistent spirochetes may lose linear plasmid 28-1 (lp28-1), which encodes the VlsE antigen bound by the anti-C6 antibody. Transcription of a lp28-1 gene (bbf26) was verified in organ tissue from both untreated animals and one treated animal (Figure 6D).
In the case of Embers et al study on Rhesus macaques, one antibiotic treated animal was found to have evidence of transcription of a lp28-1 gene (bbf26 - protein; purpose unknown) from a sample taken from heart tissue (Fig. 6D) and that transcription should only be able to occur if the lp28-1 plasmid is intact and functional. lp28-1 is a linear plasmid which is very specific to infection both in vitro and in vivo, whether a tick or needle inoculation is used.
In Embers study, in addition to transcription of a gene from lp28-1, OspA transcription from lp54 was found in three treated animals. OspA transcription was detected in two tissue samples taken from the bladder and one tissue sample taken from the spleen. Additional OspA transcription was found in different organs in two out of three of the same animals using organ tissue culture pellets.
Overall, this sounds interesting and points to the possibility of chronic infection after antibiotic treatment.
But if I have seemed cautiously optimistic about this study, it's because of a few factors*:
1) Only one treated animal had evidence of a infection where lp28-1 transcription was taking place - had more treated animals shown evidence of transcription on this plasmid, I would have been more excited. How long could spirochetes maintain these plasmids while being treated? What about lp25?
2) It is unknown to me if the genetic background and/or immune system of the treated Rhesus macaques somehow played a role in their inability to clear the spirochetes which remain after antibiotic treatment. (Refer to this post on HLA-DR types, read what's before and after the "=" signs, and you'll see what I mean.)
3) it is unknown to me how different the results would be if the Rhesus macaques had been infected using ticks instead of needle inoculations. It seems to make sense to me to do this study again using ticks because that mimics what happens in nature.
On the other hand, I find it very interesting that three animals showed evidence of transcription of OspA. Given how much inflammation people experience during Lyme disease - plus evidence of later stage antibody reactivity to OspA - it at least gives me pause to think about how often OspA has been a culprit for my own symptoms, directly or indirectly.
The only kind of spirochete
you don't mind getting close to.
So it's a mixed bag how I look at the results of the Embers Rhesus macaque study. I think it's a positive step in the right direction establishing what happens with spirochetes in their host after antibiotic treatment. And yet the unanswered questions for me seem related to the same unanswered questions the researchers themselves wrote in their paper.
Is There Anything Positive To Glean From Dr. Baker?
Of Dr. Baker's two major stated issues with the Embers study, the only one now left is whether or not the spirochetes which were transmitted by ticks to new hosts (xenodiagnosis) were in fact infectious. His other concern was over the use of ceftiofur in the study rather than ceftriaxone - however, the authors of the study have since posted a correction to PLoSONE stating that ceftriaxone - not ceftiofur - was used throughout the entire study.
If there are any remaining minor issues he has with the study, he has yet to share them on the Lyme Policy Wonk blog. Mostly, he seemed to reiterate his concern about these two issues and focused on the single mention of ceftiofur in the paper repeatedly.
About the most positive response I heard from Dr. Baker on that blog thus far was about his view of how Lyme disease research should be conducted:
"...I favor a multi-disciplinary approach that moves the field in a different direction, rather than solutions based on the assumed yet to be proved existence of a persistent infection that can only be cured by antibiotics. I don’t really discount such a view; rather, I feel we are neglecting other possibilities that may provide the answers we all are looking for. A case in point, would be the recent work of good friend, Armin Alaedini — who I helped support when I was at the NIH– using specimens collected by Mark Klempner as part of his clinical trial. These valuable specimens are being maintained by Mark in a specimen repository for use in just such cutting-edge research. They are available free of charge on request."
Like Pamela Weintraub, I agree that a multi-disciplinary approach to research on Lyme disease is important. And while Dr. Baker also supports a multi-disciplinary approach to research on Lyme disease and he states he doesn't discount the view of persistent infection in the above paragraph - his direct responses to patients suffering with CLD/PTLDS state that most patients are suffering from some other non-Lyme disease related condition - something I find particularly unhelpful to my situation. That and a lack of sufficient research on other treatment approaches has been an issue for ages.
In my opinion, Dr. Baker's response to the Embers Rhesus macaque study was more negative than it warranted. I wouldn't have viewed it negatively at all - I see it as a stepping stone in getting a better understanding about Lyme disease. And just because it leaves unanswered questions does not mean it was inherently flawed - which was what Dr. Baker seemed to suggest.
To quote someone else on that blog:
"My question to Dr. Baker is why don’t you and your colleagues offer some expert advice, according to your best opinions and hunches if science really has proven inadequate for your epistemic standards of validity, without having to officially disclose any sensitive data that might get you in trouble with your career, that could actually HELP these affected people lessen their pain and disability? Just disparaging some controversial or technically flawed research as being invalid does not seem helpful enough to me."
Yes. This.
Regardless of anyone's opinion - Dr. Baker, or LLMDs, or my friends and family - researchers will be expected to provide evidence to the world that these remaining spirochetes are pathogenic. They will need to provide evidence that that they can cause infection and reproduce - even if they are already proven to be alive.
Researchers who are trying to work without bias will want to cover all the bases and check their postulates twice to be 100% certain that Borrelia burgdorferi either causes a chronic infection or it does not after standard antibiotic treatment.
This may be so - but I'm impatient about it.
References:
The Absence of Linear Plasmid 25 or 28-1 of Borrelia burgdorferi Dramatically Alters the Kinetics of Experimental Infection via Distinct Mechanisms. Maria Labandeira-Rey, J. Seshu, and Jonathan T. Skare. Infect Immun. 2003 August; 71(8): 4608–4613. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC166013/
Correlation between plasmid content and infectivity in Borrelia burgdorferi. Purser JE, Norris SJ. Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13865-70. http://www.ncbi.nlm.nih.gov/pubmed/11106398
High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi. Norris, SJ, Howell, JK, Garza, SA, Ferdows, MS, and Barbour, AG. Infect. Immun. 63:2206-2212.
Plasmid Stability during In Vitro Propagation of Borrelia burgdorferi Assessed at a Clonal Level. Dorothee Grimm, Abdallah F. Elias, Kit Tilly and Patricia A. Rosa. Infect. Immun. June 2003 vol. 71 no. 6 3138-3145
http://iai.asm.org/content/71/6/3138.full
Experimental assessment of the roles of linear plasmids lp25 and lp28-1 of Borrelia burgdorferi throughout the infectious cycle. Grimm D, Eggers CH, Caimano MJ, Tilly K, Stewart PE, Elias AF, Radolf JD, Rosa PA. Infect Immun. 2004 Oct;72(10):5938-46. http://www.ncbi.nlm.nih.gov/pubmed/15385497
The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mollie W Jewett, Kevin Lawrence, Aaron C Bestor, Kit Tilly, Dorothee Grimm, Pamela Shaw, Mark VanRaden, Frank Gherardini, and Patricia A Rosa. Mol Microbiol. 2007 June 1; 64(5): 1358–1374. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1974800/?tool=pubmed
Basic Epidemiology. Beaglehole R, Bonita R, Kjellstrom T. World Health Organization, Geneva, Switzerland, 1993
* Factors which concern others but I did not originally think of are included in comments below.
[Edited March 9, 2012 - Removed item above about brain tissue after reviewing Embers paper again - multiple brain samples were taken; one treated animal was positive for B. burgdorferi RNA in both heart and brain.]
