LymeNet Newsletter Volume 3 Issue 09

Volume: 3
Issue: 09
Date: 14-Jun-95

Table of Contents:

I. LYMENET: Vancouver LD Conference Notes - Part 1 II. About The LymeNet Newsletter

Newsletter:

*********************************************************************** * The National Lyme Disease Network * * LymeNet Newsletter * ***********************************************************************

IDX# Volume 3 - Number 09 - 6/14/95 IDX# INDEX IDX# IDX# I. LYMENET: Vancouver LD Conference Notes - Part 1 IDX# II. About The LymeNet Newsletter IDX#

I. LYMENET: Vancouver LD Conference Notes - Part 1 ----------------------------------------------------- Sender: Lloyd E. Miller, DVM <[email protected]>

Notes from the VIII Annual Lyme Disease Foundation Scientific Conference on Lyme Borreliosis and other Spirochetal and Tick-Borne Diseases - Held April 28-29 1995 -- at Vancouver, British Columbia, Canada.

Part 1

Prepared by Lloyd E. Miller, DVM

Every effort has been made to be accurate. Please report any additions or corrections. Always confirm drug doses and names of medications with another source before using.

EPIZOOLOGY, EPIDEMIOLOGY

1. Update on the epizootiology of Borrelia burgdorferi in southeastern U.S. - J. Oliver, Ph.D.

Documented the finding Bb infected ticks (Ixodes scapularis and Ixodes dentatus), on cotton rats and wood rats in of southeastern states (Missouri, So. Carolina, Georgia, and Florida). In some areas Infectivity rates of rodents can be as high as 75%. Showed that the populations of Bb and Ix. Scapularis have more genetic variability in the south than in the north. Made the case that this implies the populations with the most variability are the oldest populations and that instead of Bb and Ixodes scapularis spreading from north to south the opposite may have occurred. He pointed out that in LD a lot of assumptions are made - forgetting that exceptions do occur.

2. Ecology of Lyme disease in Northeastern United States - J.F. Anderson, Ph.D.

Ixodes scapularis has been recorded feeding on 120 species of animals (birds, mammals, lizards). Infectivity rates in ticks in northeast varies by locality from 12 to 100% as compared to 1-3% in the southeast and west. Part of reason may be that in the northeast ticks feed primarily on mammals where in the southeast and west ticks also feed on lizards which can break the Infectivity cycle.

3. Isolation of B. burgdorferi in British Columbia during 1992-1994 - S. N. Banerjee, Ph.D.

Showed evidence of the endemic status of British Columbia through the isolation of Bb from Ixodes angustus and Ixodes pacificus ticks.

4. Lyme disease in British Columbia - P. Daly, M.D

Lyme disease is a reportable disease in British Columbia since June 1994. As in the USA the Canadian surveillance definition of Lyme disease is quite restricted.

5. Lyme Disease cases acquired in British Columbia 1992-1994 - R. Gill

Reported the acquisition of Lyme disease within the boundaries of British Columbia in 11 patients (out of 43 known cases), further demonstrating the endemic status of British Columbia. Only two of the 11 had an EM rash.

6. Surveillance for Tick-borne Relapsing Fever in Texas Caves - J. Rawlings MPH

Evidence was presented that Tick-borne Relapsing Fever can be acquired in caves. The disease is also transmitted by tick bite (Ornithodorus turicata) and is caused by Borrelia turicatae.

VACCINE PROGRESS

7. Induction of Lyme arthritis and role of Borrelia burgdorferi specific T-lymphocytes - R. Schell, Ph.D.

This presentation was a continuation of research reported at this same conference last year in which it was reported that hamsters challenged with homologous stains of Bb developed severe arthritis post vaccination and prior to the development of immunity. It was also reported that when the protective titer waned arthritis would also develop on challenge. This report shows that Bb specific T-lymphocytes were responsible for the development of the arthritis. The importance of this is related to the development of safe and effective vaccination and in the development of medications that may prevent or treat the arthritis caused by Bb.

8. Efficacy of recombinant OspA formulations in Rhesus monkey - M. Philip, Ph.D

* Monkeys were vaccinated with 3 doses intramuscularly at four week intervals and then challenged by bite of nymphal Ixodes scapularis 5 weeks post vaccination. * One of 130 ticks that fed on vaccinates was found to contain Bb following feeding. From control monkeys 48 of 50 were still infected. * Control animals developed significant response to Bb antigens on western blot at 4 and 40 weeks post challenge. Bb was found in the skin by PCR during the 4 weeks post challenge. Inflammation and positive PCR was observed in urinary tract (2/4), heart (3/4), joints (1/4) and nerves (4/4), lung (4/4). * None of the vaccinates reacted to any Bb antigens on western blot except the OspA of the vaccine. Bb was not found in the skin by PCR during the 4 weeks post challenge. Inflammation and positive PCR were not observed in the urinary tract or joints. However, *very significantly*, inflammation and positive PCR was observed in nerves (2/6), heart (2/6) and lung (5/6). * The abstract states that: "The difference in the tick infection rate
between ticks that fed on vaccinated animals, the lack of seroconversion in the vaccinated animals and the absence of spirochetal DNA in the skin of the vaccinated animals in the weeks following the challenge, indicate that the vaccinated monkeys were protected against tick challenge," * Note the affinity of Bb for the nerves, heart and lung. * Were the monkeys really protected against *infection*? Note not all monkeys were examined so it will be possible in the future to determine whether the Bb found in the vaccinates were transiently infecting or whether the vaccine prevented all but a small number of Bb to infect (a number low enough to not cause immune response). This will be very important to determine for unless the vaccine can prevent infection it may not prevent chronic infection and possible long term disease consequences. The authors are continuing their study and hopefully will answer this question.

[ Comment - Were the monkeys really protected against *infection*? Note not all vaccinated monkeys have been examined so hopefully it will be possible in the future to determine whether the Bb found in the vaccinates were transiently infecting or whether the vaccine prevented all but a small number of Bb to infect (a number low enough to not stimulate immune response). This will be very important to determine for unless the vaccine can prevent infection it may not prevent chronic infection and possible long term disease consequences. The authors are continuing their study and hopefully will answer this question. The reason(s) for this finding must be addressed before vaccination programs for humans are developed. ]

9. Update on recombinant Lyme disease vaccine development - A. Barbour, M.D.

Several of the Osp proteins are candidates for incorporation in a vaccine OspA,B and C are known to produce immune protection in animals. OspD is found in some Bb that do not express OspA or B. OspE and F are described but it is not known if they can produce protective immunity. It is now reported that Bb expresses OspA when in ticks but OspA is rarely found when Bb infects humans and animals (mouse, hamster, monkey and dog). Some Bb isolates process the OspA gene but don't express OspA. Once Bb is transmitted to mammals OspA production declines. Therefore, if an OspA based vaccine is going to work Anti-OspA antibody must be present at the site of tick bite at the time the bite occurs. *This means the vaccine will not help anyone who already has been infected*. Safety studies on the OspA vaccines have shown them to be safe. There are no reactions other than what is seen with any other vaccine. Studies have shown that the vaccine produces an immune response resulting in the production of anti-OspA antibodies.

[ Comment - I was disappointed to learn that endpoint of the vaccination trials currently in progress as I understand them, is observing whether or not EM develops in vaccinated individuals. Many of the attendees I spoke to thought this was an inadequate test of efficacy. We know that EM is observed in only approximately 50% of those infected. There is no study, as far as I could ascertain, that attempts to determine whether prevention of infection occurs following vaccination. (If I'm wrong I hope someone out there will reassure us). It should be noted, based on the monkey study reported herein, that total prevention of infection by vaccination must be determined for it may do no good to vaccinate to prevent the early development of Lyme disease symptoms only to have later symptoms develop. As noted also herein, treatment of the early symptoms is far more successful than the treatment of later stage symptoms. Vaccination in this case will give a false sense of security and may result in even more cases of
chronic debilitating disease! Given the ability of Bb to persist, total prevention of infection MUST be the goal of vaccination. ]

PATHOGENESIS OF DISSEMINATION

10. Antigenic variation of Borrelia burgdorferi sensu lato: implications for pathogenesis, diagnosis and prophylaxis - B. Wilske, MD

This work was done in Europe using Ixodes ricinus ticks but it was stated that the information held true for the American ticks too. Apparently Bb OspA is regularly expressed in ticks but once Bb is transmitted to humans there is strong expression of OspC and low expression of OspA. They identified 7 types of OspA and 13 Types of OspC. They were able to show that in European patients disease expression was significantly associated with OspA type expressed by Bb isolates from the patient. For instance Type 2 was associated predominately with skin disease. It is therefore predicted that antigenic variation does have influence on the expression of disease. Significance of these findings regarding diagnostic testing; if the test is only looking for antibodies to OspA and not OspC then a false negative test might result. Antibodies against OspC will not protect against OspA and vise versa thus a vaccine based only one may not provide the required level of protection.

11. Virulent Borrelia burgdorferi specifically attach to, activate, and kill TIB-215 Human B-Lymphocytes - D. Dorward, Ph.D.

Experiments were described showing that when Bb were incubated with B-lymphocytes 50% of the cells were lysed. The amount of lysing was dose dependent and required live spirochetes. A video was shown demonstrating the entry of Bb into lymphocytes and the rupture of a lymphocyte. This type of interaction may play a roll in colonization and persistence of Bb in mammals.

12. The Borrelia turicatea-mouse model of Lyme disease - A. Barbour, M.D.

Using Borrelia turicatea which infects the peripheral and CNS of mice it was determined that ONLY certain serotypes exhibit neurotropism. Serotype A infects the brain; serotype B doesn't. Serotype B causes a severe polyarthritis; Serotype A causes only a mild arthritis. The only difference identified between Serotypes A and B is an outer membrane protein that is homologous to OspC proteins of Bb. This finding suggests that disease manifestations of borrelial (including LD) infections are at least in part determined by features of the infecting organisms. In this model ceftriaxone eliminated Borrelia turicatea 100% from the brain; vancomycin did not.

INNOVATIVE BIOLOGY

13. Further evidence for a spirochetal etiology of Alzheimer's disease - J. Miklossy, M.D.

Information was presented reporting on the finding of spirochetes by dark field microscopy in the blood, CSF and brain tissue of Alzheimer's disease cases (14 of 14 cases). Spirochetes were cultured from 3 of 4 Alzheimer's cases. No spirochetes have been found in control patients. Using a monoclonal antibody against Bb a positive immunoreaction was seen in senile plaques and in neurons was observer in the brain of a patient with concurrent Alzheimer's disease and Lyme disease. Individual spirochetes were observed. The observations seem to reinforce the author's hypothesis that Alzheimer's disease may correspond to the tertiary stage of neurospirochetosis. Exactly which spirochetes are involved is yet to be determined.

[ Comment - This information needless to say has met with some skepticism in both the Alzheimer's community and the Lyme community. The information as presented is compelling. We should keep an open mind until more research is completed. Alzheimer's disease may have multiple causes - could spirochetes be involved? I include the following three references for anyone interested: * Miklossy J. Alzheimer's disease - A spirochetosis? Neuroreport 4:841-848, 1993 * Miklossy J. Kasas S. Janzer RC. Ardizzoni F. Van der Loss H. Further ultrastructural evidence that spirochetes may play a role in the etiology of Alzheimer's disease. Neuroreport 5:1201-1204. 1994 * Miklossy J. Gern L. Darekar P. Janzer RC. Van der Loss H. Senile Plaques, Neurofibrillary Tangles, and Neuropil Threads Contain DNA? Journal of spirochetal and Tick-Borne Diseases 2:9-13.1995 ]

14. Borrelia burgdorferi surface DNA represents a possible target for a new category of antimicrobial agents - C. Garon, Ph.D.

Report of work being done to identify agents that might be used to interrupt DNA in Bb and thus find alternatives to antibiotics to eradicate Bb from the system. With the exception of trioxale (not a clinically useful drug) crosslinking, Bb seemed relatively insensitive to known eukaryotic and prokaryotic DNA replication inhibitors. Cis-platinum didn't appear to work.

NEW THERAPEUTIC APPROACHES

15. Cellular infection of human fibroblasts, Langerhans cells and leukocytes by isolation of Borrelia with emphasis to antibiotic and antibody treatment - D. Hulinska, Ph.D.

This presentation demonstrated that Bb enters fibroblasts. Langerhans cells and leukocytes and in these locations can be protected from the effect of antibiotics and antibodies.

16. Evidence of the intracellular borreliae localization - S. Donta, M.D.

That Bb persists after initial infection is well established. Bb can rarely be found following initial infection and dissemination. Despite treatment for months and even years Bb can persist and be detected by PCR and/or culture in blood, spinal fluid and urine. Viruses in the latent state can not be found by DNA testing. Could the same be true for Bb? Mechanism of persistence is yet to be definitively worked out. Bb has a strong tropism to neural tissue; brain, spinal cord and peripheral nerves. Neural tissue invasion alone can produce all the symptoms of Lyme disease according to Dr. Donta. Many examples (not all are enumerated here) of recurrent disease or persistent infection were enumerated including viruses (herpes, hepatitis B and C), parasites (malaria, leishmania, toxoplama), fungi (cryptococcus, pneumocystus) and bacteria (chlamydia, legionella, M. tuberculosis, T. pallidum, salmonella, staphylococcus). It is known that some of these persist inside cells. For others the location of persistence is unknown (T.
pallidum). Tissue culture models of intracellular infection by Bb have been described and in these models ceftriaxone was* ineffective* in eliminating Bb from the cultures. Dr. Donta theorized that recurrent disease and persistence of Bb can be explained by the intracellular localization of Bb. He stated he knows of no other plausible explanation for the persistence of any organism other than in an intracellular location. There are many good examples of intracellularly localizing persistent organisms but no examples of extracellular persistence are known. Organisms can persist in endosomes and endolysosomes within cells. In this location they can remain protected from the action of most antibiotics and antibodies. He suggests that treatment of chronic Lyme disease needs antibiotics that enter the cells. Macrolide antibiotics enter cells but have been less effective than expected. He suggests it's because macrolides do not work well in an acid environment, such as that in endolysomal-like vesicles, that by using the lysosomotrophic agent
hydroxychlorlquine to increase the Ph of the endolysome along with the administration of macrolide antibiotics the efficacy of the macrolide antibiotics (clarithromycin, azithromycin, erythromycin) would be increased. Doxycycline and tetracycline also enter the cell so using hydroxychloquine along with them may also improve treatment outcomes. He also stated that amantadine 100mg three times daily has been helpful in some Lyme patients.

[ Comment - Neurotropism of Bb according to Dr. Donta can account for all the symptoms of LD. When you think about this it makes sense. Every organ and system in the body is dependent on the nervous system. Certainly the obvious neurologic symptoms are real. But consider the pain syndromes that do not appear to be especially responsive to the usual analgesics - this is typical of neurogenic pain. There are also reported effects of Bb on the endocrine system (stated by Dr. B. and others) - which may be explained by Bb's effects on the neuroendocrine system. It stands to reason that if the nerve supply to any part or organ is affected by Bb symptoms referable to this organ or system may be expressed. ]

DIAGNOSTIC ADVANCEMENTS

17. Use of recombinant chimeric proteins for the diagnosis of Lyme Disease - B. Luft, M.D.

Work to develop a Lyme test which is accurate and economical using recombinant antigens was described. The information presented showed it to be feasible to develop such a test based on a recombinant multivalent antigen. Peptides from OspA, OspB, OspC, p41 and p93 are being studied.

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