OspA Downregulation and Host Adaptation
Significance of Latency
Can We Predict a Future Vaccine Failure?
References for "Limitation ..."
Note: "OspA" is "outer surface protein A"
From Journal of Spirochetal and Tick-borne Diseases
J Spiro Tick Diseases 5(4):69-74, 1998.
Limitations of the OspA Vaccine for Humans: A Review
Denise M. Foley, PhD, Department of Biomolecular Science, Chapman University Division of Natural Sciences, Orange, CA; David R. Blanco, PhD, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles; Michael A. Lovett, MD, PhD, Department of Microbiology and Immunology; and the Division of Infectious Diseases, UCLA School of Medicine, Los Angeles; James N. Miller, PhD, Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles
Address correspondence to: Denise M. Foley, PhD, Divison of Natural Science, Chapman University, 333 North Glassell, Orange, CA 92866.
Due to the lengthy review process, this report was written and revised prior to the recent FDA approval of the vaccine for Lyme disease.
J Spiro Tick Diseases 5(4):69-74, 1998.
Presently, the OspA vaccine is the most developed and defined candidate for generating protection against Lyme disease. As has been reported in the literature, significant protection has been achieved for several animal models including mice,[1-9] dogs, rabbits, and monkeys. An important issue for any vaccine is that it not only be effective, but also safe. In this regard, vaccination studies in monkeys and humans[14-16] have indeed shown that the OspA vaccine is safe with only minor reactions being reported in a small percentage of persons.
More importantly, both the Pasteur Merieux Connaught and SmithKline Beecham Laboratories have now reported on phase III human vaccine trials where it was demonstrated that OspA provides significant protection against Lyme disease.[17,18]
While these studies indicate that OspA is a very promising vaccine against Lyme disease, there are limitations based upon experimental studies in animals that may have important implications for humans. The purpose of this paper is to discuss the recombinant OspA vaccine and, in particular, raise issues regarding its limitations for humans by reviewing data obtained from published studies.
Many investigators have described their rationale for these limitations that tend to support the necessity for utilizing other protective immunogens in concert with OspA in a "cocktail" vaccine. These concerns will be included in the context of this review.
... The ability of some OspA serotypes to avoid killing with antibodies raised against other serotypes has been shown.[24,31] In the study by Lovrich et al, the authors concluded that although cross protection occurred against some strains expressing different antigenic types of OspA, vaccination with a single OspA type did not provide complete protection against challenge with all strains. Even more surprising was the finding that the presence of ANTI-OspA ANTIBODIES ELICITED FROM SOME ISOLATES DID NOT RESULT IN PROTECTION against challenge with the homologous strain.
The current OspA vaccine utilizes a Borrelia burgdorferi sensu stricto OspA molecule which, to date, has been found in the majority of the isolates from North America.[19,26] However, one type of North American OspA variant, typified by strain 250, has been shown to infect mice vaccinated with N40 OspA, a molecule similar to the current OspA vaccinogen. This variant type, isolated from upstate New York, has also been isolated from Illinois, (presented by Maria Picken, 11th Annual Scientific Conference on Lyme Borreliosis, New York, April 25-27, 1998) ...
While the greatest variation of the OspA molecule occurs in European isolates, the increasing evidence of OspA variability in North America, together with the observation that cross protection is not always achieved with OspA vaccination, implies that even a vaccine that includes several serotypes of OspA molecules will not result in complete protection of the vaccinated North America population.
OspA Downregulation in the Vertebrate and Host Adaptation
Another key issue to be considered when using OspA as the sole vaccinogen is the widely accepted fact that OspA is not expressed during vertebrate infection. The studies by Schwan et al and de Silva et al demonstrate that OspA is present on B burgdorferi before tick feeding but is lost after initiation of the bloodmeal.
Furthermore, in the study by Schwan et al, it was demonstrated that OspC, a more heterogeneous molecule than OspA, is unregulated after tick feeding. They suggest this downregulation of OspA and corresponding upregulation of OspC is crucial for the ability of the organism to infect the vertebrate host.
Further evidence that OspA is not expressed in the vertebrate host can be gathered from studies in which animals inoculated with low numbers of B burgdorferi, whether it be from needle injection or tick transmission, do not develop antibodies to OspA in spite of developing an antibody response to other B burgdorferi antigens.[45,47-51] It has also been observed that many Lyme borreliosis patients either do not produce antibodies that react with OspA or produce relatively low OspA antibody titers.[52-57]
In a study by Schutzer et al, 12 of 16 early Lyme disease patients with neurological involvement were found to have CerebroSpinal Fluid (CSF) and serum IgM directed against OspC and 5 of these 12 also had IgM to OspA that was restricted to the CSF. These data suggest that in certain neurological Lyme disease patients, OspA may be selectively expressed in the central nervous system (CNS) and not in the peripheral blood or skin.
When considering the abundance of the OspA protein in ex vivo cultured organisms and the evidence for its downregulation during tick feeding, the absence of a universal serum antibody response to OspA in humans would seem to support the theory that the majority of spirochetes will not initially express this protein in the infected human. If some organisms do revert to expression of OspA once they reach the CNS or other specific site, this may explain why some patients develop a response to this protein.
IT IS ASSUMED THAT VACCINATED PERSONS WILL DESTROY THESE ORGANISMS IF THE APPROPRIATE OspA SEROTYPE IS BEING EXPRESSED. HOWEVER, THE POPULATION OF ORGANISMS WITHIN THE OspA-VACCINATED HOST, WHICH CONTINUE TO KEEP OspA DOWNREGULATED, WOULD REMAIN UNAFFECTED.
The studies of Barthold et al provide additional evidence that OspA is not expressed by B burgdorferi while in the host. These data are particularly convincing because they demonstrate that OspA vaccinated mice exposed to organisms taken directly from a vertebrate host via a skin transplant from an infected syngeneic mouse are susceptible to infection.
These studies were extended by de Silva et al who demonstrated that mice passively administered immune mouse serum were still susceptible to infection by B burgdorferi following homologous challenge with infected mouse skin or by tick bite. All mice became infected despite being administered immune sera over the course of 14 days. These investigators concluded that the organisms may be resistant to immune serum antibodies through a mechanism of "host adaptation" that results in immune evasion.
The ability of these organisms to evade host immune defenses is clear from the course of natural infection where the establishment of chronic infection and late debilitating manifestations is a common feature. This is also illustrated by the fact that IN SPITE OF HIGH LEVELS OF BORRELIACIDAL ANTIBODIES PRESENT In humans during stages of Lyme disease,[61-63] THESE PATIENTS REMAIN INFECTED ...
It has been shown that infected ticks feeding upon an OspA-vaccinated host results in the destruction of the majority of spirochetes in the tick. However, it has also been shown that some spirochetes survive within some ticks after a bloodmeal containing OspA antibodies.[6,9,12,46] Presumably, these organisms were not expressing OspA or expressed an OspA variant resistant to the killing antibodies present in the bloodmeal.
A relevant question is, what is the disposition of the few spirochetes which do survive in the engorged ticks after feeding upon an OspA-vaccinated host? One would predict that these organisms, for perhaps a significant period of time, do not express OspA in response to the downregulating effects of the bloodmeal.
Therefore, do these organisms represent a real or merely a theoretical danger to the individual vaccinated with only OspA? If these organisms gain entry to the vaccinated human host, their ability to quickly adapt and resist the anti-OspA immune response creates a potentially dangerous scenario. Transmission in presumably very low numbers might establish an UNDETECTED ASYMPTOMATIC INFECTION WHICH LATER EXACERBATES AS DEBILITATING CHRONIC MANIFESTATIONS OF LATE STAGE LYME DISEASE.
The Significance of Latency
The issue of the potential development of a latent infection in a previously vaccinated individual or animal has not been vigorously investigated. This is particularly pertinent in view of the recognized capacity for spirochetal pathogens, including B burgdorferi, to cause latent infection. Others have recognized this gap in the literature as evidenced by this statement:
"...it is surprising that so little attention has been paid to the question of asymptomatic infection (as manifested by seroconversion) in experimental test systems of vaccine candidates, given the presumption that latency may occur in human B burgdorferi infections." GP Wormser. Infection 1996;24:203 ...
The question remains as to whether some vaccinated individuals, after exposure to B burgdorferi, harbor a low level latent infection. THE POSSIBILITY THAT A LOW LEVEL INFECTION MAY NOT STIMULATE A MEASURABLE ANTIBODY RESPONSE BUT MAY EXACERBATE INTO CLINICAL lYME DISEASE AT A LATER TIME, IS SUGGESTED BY SEVERAL OspA ANIMAL VACCINE STUDIES.
IN OspA-VACCINATED RHESUS MONKEYS, THE DETECTION OF B burgdorferi DNA AND ANTIGENS IN TISSUES WAS FOUND AT A TIME WHEN OVERT SYMPTOMS OF THE DISEASE AND WESTERN BLOT REACTIVITY WERE ABSENT. These findings suggest the presence of organisms in these tissues although an attempt to "activate" this potential latent infection by administering immunosuppressive drugs was not successful.
Although many have argued that the detection of DNA does not indicate the presence of living organisms, a study by Malawista et al65 has shown a very high correlation between the detection of DNA and positive cultures ...
Another question to be considered among OspA-vaccinated persons is whether partial immunity, either from a waning resistance or from an incomplete vaccination regimen, results in an altered disease state upon exposure or a "masking" phenomenon in which infection in the absence of characteristic clinical manifestations such as erythema migrans (EM) occurs. In support of this hypothesis, we found that 4 of 11 OspA-vaccinated rabbits became infected upon challenge with B burgdorferi strain B31.
WE BELIEVE THAT THESE OBSERVATIONS SUPPORT THE THEORY THAT WHEN STATES OF PARTIAL IMMUNITY EXIST, ALTERED FORMS OF DISEASE MAY ARISE IN SOME VACCINATED AND EXPOSED INDIVIDUALS WHICH MAY MAKE DIAGNOSIS MORE DIFFICULT.
Because the clinical manifestations may be inconsistent with typical disease, a differential diagnosis that includes Lyme disease, might not be considered given that some physicians may conclude that vaccination reduces or eliminates the chances of acquiring the disease. Furthermore, as suggested by these studies, the infection may be subclinical but could emerge at a later time as more difficult to treat late manifestations.
Can We Predict a Future Vaccine Failure?
Information pointing to the prediction of when spirochetes are likely to evade the immune response of the vaccinated host may currently be available. In two separate studies, a correlation was demonstrated between protective antibody and a specific epitope on OspA, defined by the monoclonal antibody LA-2.[68,69].
It was shown in the study by Golde et al that the LA-2 antibody titer is a reliable indicator of immune status following immunization with OspA; vaccinated mice and dogs with a low LA-2 antibody response were susceptible to infection upon challenge ...
Future Directions Toward the Development of a More Efficacious Vaccine
For almost as long as recombinant OspA has been tested as a vaccine candidate, many investigators have recognized the need for an improved vaccine. Recognizing the proven and potential limitations of an OspA vaccine, several investigators have suggested the addition of other components to the vaccine. Such a "cocktail" may include one or more additional recombinant proteins including various OspA serotypes as well as other B burgdorferi molecules ...
In conclusion, although the OspA vaccine is the most promising candidate thus far, there clearly remains a need for a Lyme disease vaccine that stimulates high levels of long lasting protection against all strains of Borrelia responsible for Lyme disease.
References for: "Limitations of the OspA Vaccine for Humans"
Journal of Spirochetal and Tick-Borne Diseases 5(4):69-74, 1998.
1. Fikrig E, Barthold SW, Kantor F, Flavell R. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science 1990;250:553-556.
2. Simon M, Schaible U, Kramer M, et al. Recombinant outer surface protein A from Borrelia burgdorferi induces antibodies protective against spirochetal infection in mice. J Infect Dis 1991;164:123-132.
3. Fikrig E, Barthold S, Kantor F, Flavell R. Protection of mice from Lyme borreliosis by oral vaccination with Escherichia cold expressing OspA. J Infect Dis 1991;164:1224-1227.
10. Chang YF, Appel MJG, Jacobson RH, et al. Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi. Infect Immun 1995;63:3543-3549.
11. Foley DM, Wang YP, Wu XY, et al. Acquired resistance to Borrelia burgdorferi infection in the rabbit: comparison between outer surface protein A vaccine- and infection-derived immunity. J Clin Invest 1997;99:2030-2035.
12. Philipp MT, Lobet Y, Bohm RP, et al. The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey. Vaccine 1997;15:1872-1887.
13. Philipp MT, Lobet Y, Bohm RP, et al. Safety and immunogenicity of recombinant outer surface protein A (OspA) vaccine formulations in the rhesus monkey. Journal of Spirochetal Tick-borne Diseases 1996;3:1-12.
14. Keller D, Koster FT, Marks DH, et al. Safety and immunogenicity of a recombinant outer surface protein A Lyme vaccine. JAMA 1994;271:1764-1768.
15. Schoen RT, Meurice F, Brunet CM, et al. Safety and immunogenicity of an outer surface protein A vaccine in subjects with previous Lyme disease. J Infect Dis 1995;172:1324-1329.
6. Van Hoecke C, Comberbach M, De Grave D, et al. Evaluation of the safety, reactogenicity and immunogenicity of three recombinant outer surface protein (OspA) Lyme vaccines in healthy adults. Vaccine 1996;14:1620-1626.
17. Steere AC, Sikand VK, Meurice F, et al. Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. N Engl J Med 1998;339:209-215.
18. Sigal LH, Zahradnik JM, Lavin P, et al. A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease. N Engl J Med 1998;339:216-222.
19. Barbour AG, Heiland RA, Howe TR. Heterogeneity of major proteins in Lyme disease Borrelia: a molecular analysis of North American and European isolates. J Infect Dis 1985;152:478-484.
20. Anderson JF, Magnarelli LA, McAninch JB. New Borrelia burgdorferi antigenic variant isolated from Ixodes dammini from upstate New York. J Clin Microbiol 1988;26:2209-2212.
21. Fikrig E, Barthold SW, Persing DH, et al. Borrelia burgdorferi strain 25015: characterization of OspA vaccination against infection. J Immunol 1992;148:2556-2560.
2. Wilske B, Preac-Mursic V, Gobel UB, et al. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol 1993;31:340-350.
23. Zingg BC, Anderson JF, Johnson RC, LeFebvre RB. Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulsed-field gel electrophoresis. J Clin Microbiol 1993;31:3109-3114.
24. Lovrich SD, Callister SM, Lim LCL, Schell RF. Seroprotective groups among isolates of Borrelia burgdorferi. Infect Immun 1993;61:4367-4374.
25. Zingg BC, Brown RN, Lane RS, LeFebvre RB. Genetic diversity among Borrelia burgdorferi isolates from wood rats and kangaroo rats in California. J Clin Microbiol 1993;31:3109-3114.
26. Marconi RT, Konkel ME, Garon CF. Variability of osp genes and gene products among species of Lyme disease spirochetes. Infect Immun 1993;61:2611-2617.
27. Lovrich SD, Callister SM, Lim LCL, et al. Seroprotective groups of Lyme borreliosis spirochetes from North America and Europe. J Infect Dis 1994;170:115-121.
8. Lovrich SD, Callister SM, DuChateau BK, et al. Abilities of OspA from different seroprotective groups of Borrelia burgdorferi to protect hamsters from infection. Infect Immun 1995;2113-2119.
29. Mathiesen DA, Oliver JH Jr, Kolbert CP, et al. Genetic heterogeneity of Borrelia burgdorferi in the United States. J Infect Dis 1997;197:98-107.
30. Anderson JF, Magnarelli LA, LeFebvre RB, et al. Antigenically variable Borrelia burgdorferi isolated from cottontail rabbits and Ixodes dentatus in rural and urban areas. J Clin Microbiol 1989;27:13-20.
31. Hanson MS, Cassatt DR, Guo BP, et al. Active and passive immunity against Borrelia burgdorferi Decorin binding protein (DbpA) protects against infection. Infect Immun 1998;66:2143-2153.
32. Picken RN, Cheng Y, Han D, et al. Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois. J Clin Microbiol 1995;33:2304-2315.
33. Rosa PA, Schwan T, Hogan D. Recombination between genes encoding major outer surface proteins A and B of Borrelia burgdorferi. Mol Microbiol 1992;6:3031-3040.
34. Sadziene A, Rosa P, Hogan D, Barbour A. Antibody resistant mutants of Borrelia burgdorferi: in vitro selection and characterization. J Exp Med 1992;176:799-809.
5. Fikrig E, Liu B, Fu LL, et al. An OspA frame shift, identified from DNA in Lyme arthritis synovial fluid, results in an outer surface protein A that does not bind protective antibodies. J Immunol 1995;155:5700-5704.
36. Fikrig E, Tao H, Barthold SW, Flavell RA. Selection of variant Borrelia burgdorferi isolates from mice immunized with outer surface protein A or B. Infect Immun 1995;63:1658-1662.
37. Sole M, Bantar C, Indest K, et al. Borrelia burgdorferi escape mutants that survive in the presence of antiserum to the OspA vaccine are killed when complement is also present. Infect Immun 1998;66:2540-2546.
38. Caporale DA, Kocher TD. Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi. Mol Biol Evol 1994;11:51-64.
39. Schwan TG, Schrumpf ME, Karstens RH, et al. Distribution and molecular analysis of Lyme disease spirochetes, Borrelia burgdorferi, isolated from ticks throughout California. J Clin Microbiol 1993;31:3096-3108.
40. Barbour AG, Maupin GO, Teltow GJ, et al. Identification of an uncultivatible Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness. J Infect Dis 1996;173:403-409.
41. Oliver JH Jr, Kollers TM Jr, Chandler FW Jr, et al. First isolation and cultivation of Borrelia burgdorferi sensu lato from Missouri. J Clin Invest 1998;36:1-5.
42. Oliver JH Jr, Chandler FW Jr, James AM, et al. Unusual strain of Borrelia burgdorferi isolated from Ixodes dentatus in central Georgia. J Parisitol 1996;82:36-940.
43. Oliver JH Jr, Chandler FW Jr, James AM, et al. Natural occurrence and characterization of the Lyme disease spirochete, Borrelia burgdorferi, in cotton rats (Sigrnodon hispidus) from Georgia and Florida. J Parisitol 1995;81:30-36.
44. Oliver JH Jr, Chandler FW Jr, Luttrell MP, et al. Isolation and transmission of the Lyme disease spirochete from the southeastern United States. Proc Natl Acad Sci USA 1993;90:7371-7375.
5. Schwan TG, Piesman J, Golde WT, et al. Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci USA 1995;92:2909-2913.
46. deSilva AM, Telford SR, Bruntet LR, et al. Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine. J Exp Med 1996;183:271-275.
47. Greene RT, Walker RL, Nicholson WL, et al. Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs. J Clin Microbiol 1988;26:648-653.
48. Schaible UK, Gern L, Wallich R, et al. Distinct patterns of protective antibodies are generated against Borrelia burgdorferi in mice experimentally inoculated with high and low doses of antigen. Immunol Lett 1993;36:219-226.
49. Gern L, Schaible UK, Simon MM. Mode of inoculation of the Lyme disease agent Borrelia burgdorferi influences infection and immune responses in inbred strains of mice. J Infect Dis 1993;167:971-975.
50. Roehrig JT, Piesman J, Hunt AR, et al. The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms. J Immunol 1992;149:3648-3653.
1. Aydintug MK, Gu Y, Philipp M. Borrelia burgdorferi antigens that are targeted by antibodydependent, complement-mediated killing in the rhesus monkey. Infect Immun 1994;62:4929-4937.
52. Zoller L, Burkard S, Schafer H. Validity of Western immunoblot band patterns in the serodiagnosis of Lyme borreliosis. J Clin Microbiol 1991;29:174-182.
53. Ma B, Christen B, Leung D, Vigo-Pelfry C. Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. J Clin Microbiol 1992;30:370-376.
54. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400.
55. Kalish RJ, Leong JM, Steere AC. Association of treatment resistant Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi. Infect Immun 1993;61:2774-2779.
56. Aguero-Rosenfeld MA, Nowakowski J, McKenna DF. Serodiagnosis in early Lyme disease. J Clin Microbiol 1993;31:3090-3095.
57. Schutzer SE, Coyle PK, Dunn JJ, et al. Early and specific antibody response to OspA in Lyme disease. J Clin Invest 1994;94:454-457.
8. Schutzer SE, Coyle PK, Krupp LB, et al. Simultaneous expression of Borrelia burgdorferi OspA and OspC and IgM response in cerebrospinal fluid in early neurologic Lyme disease. J Clin Invest 1997;100:763-767.
59. Barthold SW, Fikrig E, Bockenstedt LK, Persing DH. Circumvention of outer surface protein A immunity by host-adapted Borrelia burgdorferi. Infect Immun 1995;63:2255-2261.
60. de Silva AM, Fikrig E, Hodzic E, et al. Immune evasion by tickborne and host-adapted Borrelia burgdorferi. J Infect Dis 1998;177:395-400.
61. Padilla ML, Callister SM, Schell RF, et al. Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine. J Infect Dis 1996;174:739-746.
62. Fikrig E, Bockenstedt LK, Barthold SW, et al. Sera from patients with chronic Lyme disease protects mice from Lyme borreliosis. J Infect Dis 1994;169:568-574.
63. Sadziene A, Thompson PA, Barbour AG. In vitro inhibition of Borrelia burgdorferi growth by antibodies. J Infect Dis 1993;167:165-172. 4.Steere AC. Medical progress: Lyme disease. N Engl J Med 1989;321:586-596.
65. Malawista SE, Barthold SW, Persing DH. Fate of Borrelia burgdorferi DNA in tissues of infected mice after antibiotic treatment. J Infect Dis 1994;170:1312-1316.
66. Mcleod CP, Magnuson HJ. Effect of cortisone on latent syphilis in rabbits and mice. J Immunol 1956;76:373-376.
67. Mcleod CP, Magnuson HJ. Development of Treponemal immobilizing antibodies in mice following injection of killed Treponema pallidum (Nichols strain). J Ven Dis Infor 1951;32:274-279.
68. Golde WT, Piesman J. Dolan MC, et al. Reactivity with a specific epitope of outer surface protein A predicts protection from infection with the Lyme disease, Borrelia burgdorferi. Infect Immun 1997;65:882-889.
69. Johnson BJB, Svia SL, Happ CM, et al. Incomplete protection of hamsters with unlipidated OspA from Borrelia burgdorferi infection associated with low levels of antibody to an epitope defined by Mab LA-2. Vaccine 1995;13:1086-1094.
70. Sunlian F, Hodzic E, Stevenson B, Barthold SW. Humoral immunity to Borrelia burgdorferi N40 decorin binding proteins during infection of laboratory mice. Infect Immun 1998;66:2827-2835.
71. Hagman KE, Lahdenne P, Popova TG, et al. Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis. Infect Immun 1998;66:2674-2683.