CVI Accepts, published online ahead of print on 20 June 2012
Clin. Vaccine Immunol. doi:10.1128/CVI.00228-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
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Dynamic Longitudinal Antibody Responses during Borrelia burgdorferi Infection and
Antibiotic Treatment of Rhesus Macaques
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Monica E. Embers*, Nicole R. Hasenkampf, Mary B. Jacobs, and Mario T. Philipp
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Division of Bacteriology & Parasitology, Tulane National Primate Research Center, Tulane
University Health Sciences Center, Covington, LA
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Running title: Longitudinal Antibody Responses to B. burgdorferi
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*Corresponding author: Monica E. Embers Division of Bacteriology and Parasitology, Tulane
National Primate Research Center (TNPRC), Tulane University Health Sciences Center, 18703
Three Rivers Road, Covington, LA 70433. Phone: (985) 871-6607, E-mail:
[email protected];
ABSTRACT
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Infection with B. burgdorferi elicits robust, yet disparate antibody responses in infected
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individuals. A longitudinal assessment of antibody responses to multiple diagnostic antigens
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following experimental infection and treatment has not previously been reported. Our goal was
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to identify a combination of antigens that could indicate infection at all phases of disease and
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response to antibiotic treatment. Because the rhesus macaque recapitulates the hallmark signs
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and disease course of human Lyme disease, we examined the specific antibody responses to
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multiple antigens of B. burgdorferi following infection of macaques. Five macaques infected
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with strain B31 and 12 macaques infected with strain JD1 were included in the analysis.
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Approximately half of these animals were treated with antibiotics at 4-6 months post-inoculation.
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Antibody responses to several B. burgdorferi recombinant antigens, including OspC, DbpA,
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BBK32, OspA and OppA-2 were measured at multiple points throughout infection. We have
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previously shown a decline in the response to the C6 peptide following antibiotic treatment.
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Responses to OspA and OspC, however, were variable over time among individuals, irrespective
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of antibiotic treatment. Not every individual responded to BBK32, but anti-DbpA IgG levels
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were uniformly high and remained elevated for all animals. All responded to OppA-2, with a
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decline post-treatment that was slow and incomplete. This is the first demonstration of B.
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burgdorferi OppA-2 antigenicity in nonhuman primates. The combination of DbpA, OspC,
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OspA, and OppA-2 with the C6 diagnostic peptide has potential to detect infection throughout all
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disease phases.
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INTRODUCTION
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The etiologic agent of Lyme disease, Borrelia burgdorferi, is transmitted to humans
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predominantly by ticks of the genus Ixodes, resulting in a disease of protean manifestations due
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to the organisms’ systemic spread and promiscuous organ tropism. Those infected may exhibit a
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skin rash, arthritis, carditis, extreme fatigue, myalgia, and neurological dysfunction. In 2009,
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over 30,000 confirmed cases were reported to the Centers for Disease Control and Prevention.
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Hence, Lyme disease is the most commonly reported vector-borne disease in the United States
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(http://www.cdc.gov/lyme/stats/chartstables/casesbyyear.html). Treatment with antibiotics is
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generally effective, especially when administered soon after onset (5). As such, early and
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reliable diagnosis is critical for effective cure of Lyme disease patients.
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Laboratory diagnosis of Lyme disease is typically made by the detection of patient serum
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antibodies specific for B. burgdorferi antigens. Detection of antibodies instead of antigens is
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necessitated by the absence of detectable spirochetes in the bloodstream once the organism has
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disseminated. Detection of the bacteria or bacterial antigens from blood or skin biopsy is both
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invasive and of relatively low sensitivity (2). Antigen can sometimes be detected in urine and
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cerebrospinal fluid, but these tests are neither reliable nor recommended (46). Two of the most
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commonly used tests for diagnosis in North America are: (1) the two-tier test (that includes an
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ELISA and western blot using antigen derived from whole cell lysates); and (2) the C6 test,
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where antibodies to a specific peptide within a conserved region of the B. burgdorferi antigen
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VlsE, are detected (6, 31, 33). The C6 test has also been used experimentally for evaluation of
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treatment efficacy (37, 38).
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While the two-tier and C6 tests are suitable for a majority of patients, neither is
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completely specific or sensitive enough to diagnose all patients. The C6 test, for instance is more
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sensitive for human patients with early or late disseminated disease than for patients in the
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localized phase (6, 17). While the C6 peptide is highly-conserved, other ELISA and western blot
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tests utilize whole cell lysates or recombinant proteins from one species/strain/isolate, despite the
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enormous potential for antigenic variability that can preclude recognition by antibody. The
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western blot will also specifically detect antibodies that recognize the antigen in a full- or
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partially-denatured state, so those antibodies that target conformational epitopes are missed.
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Furthermore, the potential for patient serum cross-reactivity with antigens shared with other
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bacteria has led to stringent diagnostic criteria that can confound western blot interpretation and
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thus hinder accurate diagnosis (7).
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Along with C6 or VlsE, several other B. burgdorferi proteins that are known to elicit
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antibody responses in natural infections and have been incorporated into immunoblot-based
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diagnostics include outer surface protein C (OspC) (45), the fibronectin-binding protein BBK32
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(30), decorin-binding protein A (DbpA) (19), flagellar protein, FlaB, and outer surface protein A
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(OspA). The temporal induction and magnitude of the B cell response to each of these,
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characterized primarily in mice, is different. OspC, for example, is highly immunogenic,
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contains antigenic regions that vary among isolates, and is expressed early in infection, then
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repressed at the advent of humoral immune responses (29). Antibodies (IgM and IgG) to OspC
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often appear early in infection (25, 35).
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infection and continues post-dissemination, so the antibody response can remain, even in late
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disease (19). FlaB is constitutive and immunogenic, but holds the potential to be detected by
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cross-reactive antibodies from other bacterial species, especially when denatured as in a western
DbpA is expressed within the first few days of
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blot (18). OspA is expressed when B. burgdorferi is in the tick, but its expression is repressed as
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the spirochetes traverse to the host during tick feeding (41). However, studies indicate that
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expression of OspA and subsequent antibody responses may return during long-term infections
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in Lyme arthritis patients (4, 27). As a model for human Lyme disease, B. burgdorferi-infected
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nonhuman primates (NHP) are known to elicit bold and specific antibody responses to multiple
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antigens (34). Importantly, the responses may differ from those produced by mice (34). We also
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report here that nonhuman primates develop antibodies to the peptide transporter protein OppA-
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2. This response appears to decline with antibiotic treatment, but more slowly and incompletely
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than the anti-C6 response (16).
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This report presents the first assessment of temporal changes in levels of specific
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antibody to multiple antigens following experimental infection with B. burgdorferi. By
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examining the responses in nonhuman primates before and after antibiotic treatment, we
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empirically determined antigens that would be appropriate for inclusion into a multiplex test to
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detect infection at multiple phases and as an indicator of treatment outcome.
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MATERIALS AND METHODS
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Animals, infection, and treatment. Practices in the housing and care of animals
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conformed to the regulations and standards of the PHS Policy on Humane Care and Use of
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Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. The Tulane
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National Primate Research Center is fully accredited by the Association for the Assessment and
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Accreditation of Laboratory Animal Care-International. The Institutional Animal Care and Use
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Committee (IACUC) of the Tulane National Primate Research Center approved all animal-
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related protocols, including the infection, treatment, and sample collection from nonhuman
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primates. All animal procedures were overseen by veterinarians and their staff. For blood
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collection, monkeys were anesthetized with ketamine (10 mg/kg) by intramuscular injection.
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The infection and treatment of these animals was described previously (16). The experimental
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design reflects the assessment of antimicrobial efficacy against early disseminated infection
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(B31-infected macaques) or late disseminated infection (JD1-infected macaques). The repository
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of serum samples collected over time from these animals afforded the opportunity to assess
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serological responses to multiple antigens before, during, and after antibiotic treatment.
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Strain B31-infected animals: Five male rhesus macaques (Chinese origin), 3-4 years of age, were
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given 3 inoculations in the ventral midline of in vitro-cultured late log-phase B. burgdorferi
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strain B31, isolate 5A19 (40): two subcutaneous 1.0 mL injections and one intradermal 0.1 mL
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injection, each containing 1 x 108 organisms diluted in sterile Hanks Balanced Salt Solution
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(HBSS), for a total inoculum of 3 x 108. At four months post-inoculation (PI), three of the five
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animals received antibiotic treatment consisting of one 50 mg tablet of doxycycline (Bio-Serv)
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twice/day for 28 consecutive days. This dose corresponded to >12 mg/kg/day to ensure that an
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effective blood level was achieved. Blood was collected at the following time points: 0, 2, 6, 10,
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14, 16, 18, 22, 26, 28, 34, 40 and 47 weeks PI.
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Strain JD1-infected animals: Briefly, twenty-four rhesus macaques (Chinese origin) were given
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an inoculation of 3.2 x 108 spirochetes of the JD1 strain (39) each via needle and syringe. Four
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additional animals were sham-inoculated as uninfected controls. At 27 weeks PI, 12 of the
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infected animals and two of the controls were treated with ceftriaxone, followed by doxycycline.
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Animals received intravenous ceftriaxone, 25 mg/kg, once per day for 30 days followed by 60
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days of oral doxycycline, 2 mg/kg, twice per day. Twelve infected and 2 control animals were
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sham-treated. Blood was collected prior to inoculation and every week for more than a year,
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until the time of euthanasia (54-60 weeks PI). The serum antibody responses of half of the
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animals (randomly-selected) in each test group were included in our assessment.
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Recombinant protein expression and purification. Each B. burgdorferi antigen was
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produced as a glutathione S-transferase (GST) fusion protein by inserting the gene of interest
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into the pGex 4T-1 vector (GE Healthcare). Genes were cloned by primer insertion of BamHI
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and XhoI restriction enzyme sites for digestion and ligation into the expression vector. The full
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open reading frames of DbpA and BBK32 were amplified from B. burgdorferi strain B31
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template DNA using the following primers: 5’-
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CATCGGATCCATGATTAAATGTAATAATAAAAC-3’(DbpA forward), 5’-
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CATCCTCGAGTTAGTTATTTTTGC-3’(DbpA reverse), 5’-
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CATCGGATCCATGAAAAAAGTTAAAAGC-3’ (BBK32 forward), 5’-
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CATCCTCGAGAATATAATTTAATAAGGTC-3’(BBK32 reverse). OspA genes from B31 and
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JD1 were cloned by insertion of BamHI and XhoI restriction enzyme sites, so as to omit the
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leader sequence, beginning with the 20th amino acid of the coding sequence, using the conserved
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forward primer: 5’-CATCATGGATCCAATGTTAGCAGCCCTGACGAG-3’ and reverse
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primers: 5’-GACTAGCTCGAGTTATTTTAAAGCGTTTTTAATTTCATCAAG-3’ (B31) and
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5’-GACTAGCTCGAGTTATTGTAAAGCGTTTTTAATTTCATCAAG-3’(JD1); ospC was
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amplified beginning at the 20th amino acid, downstream of the leader sequence, using primers:
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5’-CATAGGATCCAATTCAGGGAAAGATGGGAAT-3’ (forward) and 5’-
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CATACTCGAGTTAAGGTTTTTTTGGACTTTCTG-3’(reverse); the entire oppA2 gene was
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amplified with primers and cloned into the pGex 4T-1 vector: 5’-
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GATAGGATCCATGAAATTACAAAGGTC (forward) and 5’-
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GAGCCTCGAGTTATTTATTTTTTAATTTTAGCTG-3’. Plasmids with productive insertions
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were used to transform Top10 cells and screened. To improve protein expression, BL21
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competent cells were transformed with each plasmid. Protein expression was induced with
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Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hours or overnight. GST-fusion proteins
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were extracted from E. coli and purified with the Pierce BPER GST fusion purification kit
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(ThermoScientific). Protein expression and purity were confirmed by Coomassie Blue gel
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staining and immunoblot using a mouse monoclonal anti-GST antibody clone GST-2 (Sigma
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Life Science).
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ELISAs, antibodies, and measurement of end point dilution titer (EPDT). Specific
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IgG and IgM titers were measured and compared. Specificity was measured with pre-immune
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serum and immune serum tested on GST only purified from bacterial lystate, of which none
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reacted.
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Standard IgG and IgM ELISAs. The ELISAs were performed essentially as described previously
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(22). Briefly, plates were coated with 0.5-1.0 µg/well of recombinant protein. After blocking
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with nonfat milk, serum was added at a dilution of 1:200. Anti-GST antibody (Sigma) was used
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as a positive control and the peroxidase-labeled goat anti-Monkey IgG or goat anti-monkey IgM
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(KPL, Gaithersburg, MD) secondary antibodies were used at 1:1000 dilution. Absolute OD
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450nm values for each sample were determined by subtracting the average value given by pre-
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immune serum from that animal. The number of infected animals that were positive by each test
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(above the mean value + 3x the SD for pre-immune serum) versus those that were negative was
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compared by the Fisher’s exact test for significance.
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Determination of the EPDT. For determining antibody titers, serum samples were diluted 2-fold
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until reactivity was absent. Specifically, end point titers were calculated as the dilution at which
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the sample reached <0.05 when the average pre-immune serum values + 2 x the SD were
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subtracted. The reported values are the reciprocal of the EPDT. To compare changes in
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antibody titer with treatment, the percent change was calculated as: {peak titer}- {end point
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titer}/{peak titer}.
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RESULTS
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Variation among longitudinal IgG responses to multiple antigens. Serum samples from
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macaques were tested for IgG antibodies against four different proteins (DbpA, BBK32, OspA,
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OspC) that have been used in Lyme disease diagnosis by standard ELISA or immunoblot. The
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data presented here are from five macaques followed for 11 months of infection; two were
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untreated and three were treated with antibiotics. We have supplemented these results with other
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serum specimens from strain JD1-infected and treated macaques. A summary of the antigen
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reactivity amongst these groups can be found in Table 1.
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DbpA and BBK32. In mice, DbpA is known to be a T-cell independent antigen to which a high
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proportion of the antibody response is directed (10, 43). It has been tested as a vaccine
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immunogen (12, 21, 22) and as a diagnostic antigen in immunoblots (46) and ELISA (36). The
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infected monkeys all produced high-titer, sustained responses to DbpA (Figure 1). The anti-
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DbpA titers remained elevated in the five animals that were infected with strain B31 spirochetes
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(figure 1A, B), while reactivity was more variable in animals infected with spirochetes of the
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JD1 strain (Figure 1C, D). The origin of the recombinant detection antigen (B31) may affect
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reactivity, but all animals showed a detectable response to DbpA. BBK32 is another
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extracellular matrix (fibronectin) binding protein that has been tested both as a vaccine (12) and
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diagnostic antigen (36). Only three of the five B31-infected macaques produced strong
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responses to BBK32, though the titer dropped substantially for one of the treated monkeys
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(Figure 2). Due to this low responsiveness, we did not test monkeys that were inoculated with
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JD1 spirochetes for responses to BBK32.
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OspA and OspC. For interpretation of the antibody response to OspA, it should be mentioned
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that these animals were infected by needle-inoculation of in vitro-cultured spirochetes, which
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express OspA. Because expression of OspA is downregulated in the feeding tick and remains as
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such in the mammalian host immediately after transmission, the initial responses are not
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representative of a natural infection. Thus, all animals responded to OspA, as predicted (Figure
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3). However, fluctuations in antibody titers ensued with time in both treated and untreated
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animals. Following an initial decline between weeks 2 and 10 for each B31-infected animal
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(Figure 3A,B), the titers either declined and rose again or remained elevated, as shown for the
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JD1-infected animals between week 27 and necropsy (figure 3C,D). OspC is known to be an
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immunodominant antigen that is expressed upon transition to the mammalian host environment
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(20, 41). As such, responses to OspC arose very early (within 2 weeks for most animals) after
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infection and steadily declined over time among individuals, irrespective of antibiotic treatment
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(Figure 4A,B). Importantly, all animals generated an early response to OspC, and antibodies
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from JD1-infected animals recognized strain B31 OspC.
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OppA-2 as a potential diagnostic antigen. The oppA-2 gene belongs to a B. burgdorferi
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oligopeptide permease operon that is induced during changes in environmental conditions (44).
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A similar protein also has been used as a vaccine antigen with other bacterial species (42, 47).
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All infected animals generated anti-OppA-2 antibody responses (Figure 5). A decline was
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evident for all three treated strain B31-infected animals shortly after treatment (Fig. 5A,B). This
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decline was not marked by a return to background levels, but instead leveled off and may have
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even increased, with induction of IgM late in 2 of 3 treated animals. (see Figure 7). An
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additional 12 animals that were infected with B. burgdorferi strain JD1 all had high-titer anti-
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OppA-2 antibodies as well (Figure 5 C-F). Here, a decline was seen initially for most treated
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animals, whereas the response generally remained elevated in the untreated animals.
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Comparison of EPDTs to different antigens at the peak response and study end point. We
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also determined the reciprocal end point dilution titers of serum antibodies to each antigen from
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animals at the peak response (16-22 weeks) and at necropsy (47 weeeks). Figure 6 shows a
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comparison between the EPDTs of antibodies to the five recombinant antigens and the C6
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peptide. The levels of IgG produced in response to DbpA were the highest and were lowest for
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C6. The order of relative responses is: DbpA > OspA/OspC/OppA2 >C6. The percent decline
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(Table 2) in C6 titer for treated animals was 93.8%for animal GB56 and 98.4% for animals
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GA59 and FK38. The only other antigen against which antibody titers declined with treatment in
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all animals but not in both untreated animals was OppA-2. Here, a decline in titers occurred for
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treated animals GA59 (87.5%) and FK38 (96.9%) but not GB56 (50%). This will need to be
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tested on more animals to determine if these changes are statistically significant.
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Early and late stage IgM responses.
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Longitudinal measurement of the IgM response to each full-length antigen was also included in
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the assessment (Figure 7). The anti- OspA IgM responses followed a predictable pattern (Fig.
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7A). However, fluctuations in IgM to the other antigens tested, including OspC (Fig. 7B), DbpA
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(Fig.7C), and OppA2 (Fig. 7D) were evident beyond primary infection. For example, treated
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animal GB56 generated increased anti-OspC IgM levels at weeks 22 and 40. Fluctuations in
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both the anti-DbpA and anti-OppA2 IgM response were evident for each of the few animals
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tested, regardless of antibiotic treatment status.
DISCUSSION
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The importance of rational design for diagnostic tests is predicated by diversity in the
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immune responses produced by infected individuals. The use of multiplex testing with
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cytometric bead-based technology has expanded vigorously in recent years and has become a
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platform for serological testing. In the case of Lyme disease, the C6 peptide has demonstrated
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superior reliability as a standalone antigen for diagnostic testing. However, the inclusion of
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multiple antigens into a serologic test may increase sensitivity. This must be accomplished
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without compromising test specificity.
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To gain insight into antigens that may offer the potential for broad (encompassing all
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phases of disease) sensitivity, we examined longitudinal responses: (1) in the most appropriate
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animal model for human Lyme disease—the rhesus macaque; and (2) both pre- and post-
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antibiotic treatment to include assessment of responses that may be linked to disease resolution.
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In doing so, we discerned a combination of antigens that would be appropriate for inclusion in a
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multi-antigen serologic test.
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In mice, DbpA is known to be a T-cell independent antigen to which a high proportion of
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the antibody response is directed (10, 43). It has been tested as a vaccine immunogen (12, 21,
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22) and as a diagnostic antigen (36, 46). All NHP infected with either B. burgdorferi strain in
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this study also produced staunch antibody responses to DbpA. Likely this is a T-independent
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antigen for NHP as well. BBK32 is another extracellular matrix binding protein that has been
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tested both as a vaccine (12) and as a diagnostic antigen (36). Though the titer dropped
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substantially for one treated B31-infected monkey, only three of the five macaques produced
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veritable antibody responses to this protein. Due to the strong and sustained responses to DbpA
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in all animals tested to date, it appears a valuable diagnostic antigen. BBK32 has a lower
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potential for sensitivity and may not add value to a multiplex test.
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Despite the artificial induction of primary anti-OspA responses by inoculation of in vitro-
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cultured spirochetes, we found the fluctuations in response over time compelling. We detected
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ospA transcript from persistently-infected macaques (16) so we reasoned that their anti-OspA
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antibody responses may not decline predictably over time. In addition, OspA antibodies have
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been associated with arthritis during disseminated infection (3), and more recently shown to be
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elevated in patients with post-treatment Lyme disease syndrome (13). For its potential to detect
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infection at the late-disseminated phase, inclusion of OspA may benefit a multiplex test. OspC
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is a well-characterized, highly immunogenic B. burgdorferi lipoprotein. Heterogeneity in ospC
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among different isolates and strains has been well-documented. However, this does not appear to
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result in type-specific responses to OspC (24), so it remains a good candidate diagnostic antigen.
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Antibody responses to OspC may be expected to arise early and steadily decline, as this antigen’s
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expression is known to be shut off after the advent of the humoral response (29). This is indeed
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what we observed.
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OppA-2 transcript was detected in heart tissue of mice post-antibiotic treatment (9). Over
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a decade ago, two-dimensional electrophoresis and immunoblotting revealed antibody responses
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of infected individuals that targeted an ABC transporter protein of Borrelia garinii (26). Human
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patients are known to produce antibodies to the oligopeptide permease A-type protein from B.
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burgdorferi (8, 15, 32). The OppA-2 protein was also demonstrated to be antigenic in rabbits
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(14) and in mice (8, 32) This gene is on the chromosome, whereas the gene from which C6 is
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derived is located on a plasmid that can be lost during infection (40) and possibly also post-
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treatment (11). OppA-2 transcript was shown to be produced by B. burgdorferi in mice post-
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antibiotic treatment, as an indicator of their metabolic viability. Given the broad reactivity of
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macaques to OppA-2 and the decline in response, albeit moderate, post-treatment, this protein
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could prove to have utility as a diagnostic antigen both before and after treatment. While we did
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not see a response to OppA-2 using pre-immune serum, the modest homology (28) that this
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protein may have with similar bacterial proteins necessitate its careful screening with multiple
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samples for specificity.
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Continuous IgM responses beyond primary introduction of antigen are peculiar, but have
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been observed in B. burgdorferi infection previously (1, 23). Mostly using immunoblot, the
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continual detection of B. burgdorferi antigen-specific IgM is well documented; however,
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fluctuations in specific antibody levels over time have not generally been presented. The
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induction of IgM-producing cells, particularly by T cell independent antigens (43) may provide a
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mechanism for such fluctuations and persistent IgM titers.
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The results of this study indicate that the antigens OspA, OspC, DbpA and OppA-2 may
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offer distinct benefits when combined with the C6 peptide into a multi-antigen diagnostic test.
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However, there are limitations to this study, including the inoculum dose, route and the few
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animals tested. While the inoculum dose used here may not accurately reflect the dose that
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naturally-infected humans would receive, the dynamic responses to particular antigens may be
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comparable in a general sense. Use of an outbred animal model and two different B. burgdorferi
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strains yielded patterns of antibody response that were remarkably similar per antigen. We
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cannot assert that these responses reflect those which may be observed in humans, but future
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studies are aimed at testing the responses to these specific antigens from a broad panel of Lyme
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disease patients.
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For a multi-antigen quantitative diagnostic test, specifically, the inclusion of OspC could
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allow for improved sensitivity in the localized phase, and that of OspA has the potential to alert a
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physician about the possibility of development of late arthritis. Detection of OppA-2 increases in
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antibody levels post-treatment may be a sign of persistent spirochetes or recrudescence, as all
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three treated animals in the strain B31-inoculated experimental group had indications of
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persistent B. burgdorferi (16) . The early (~4 weeks p.i.) and strong response to DbpA by a
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majority of animals indicates that DbpA may also lend diagnostic value to a multiplex test.
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Observation of dynamic longitudinal responses to various antigens over time may provide
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insight for optimal diagnostic antigen combination in developing diagnostic tests for other
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infections with pathogens that possess multiple candidate diagnostic antigens.
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Acknowledgements
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This work was supported by a TNPRC Pilot Study Grant (MEE), NIAID grant R01-AI042352
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(MTP), and NCRR grant RR00164. We thank Lisa Bowers for technical assistance.
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Figure Legends
509
Figure 1. Anti-DbpA IgG responses in macaques infected with B. burgdorferi strain B31 (A, B)
510
and JD1 (C, D). Antibiotic-treated animal responses are depicted with a gray line and untreated
511
animals with a black line. Treatment was administered from weeks 16-20 for B31-infected
512
monkeys (A, B) and between weeks 27-39 for JD1-infected monkeys. Shown is the average of
513
duplicate or triplicate OD450 readings with the average value for each individual animal’s pre-
514
immune serum response subtracted. The EPDTs, shown in (B) were determined for B31-infected
515
macaques at the height of the response (week 22) and the end point of the study (week 47).
516
517
Figure 2. Anti-BBK32 responses in strain B31-infected macaques. The relative longitudinal
518
responses are shown in (A) and the reciprocal EPDTs are shown in (B).
519
520
Figure 3. Anti-OspA responses in strain B31 (A, B) and strain JD1 (C,D)-infected macaques.
521
The relative longitudinal responses are shown in (A, C and D) and the reciprocal EPDTs for B31
522
–infected monkeys are shown in (B). Animal BR99 was an uninfected control.
523
524
Figure 4. Anti-OspC responses in macaques infected with B. burgdorferi strain B31 (A, B) and
525
JD1 (C, D). The relative longitudinal responses are shown in (A, C and D) and the reciprocal
526
EPDTs for B31 –infected monkeys are shown in (B). Animal BR99 was an uninfected control.
527
528
Figure 5. Anti-OppA-2 responses amongst macaques infected with B. burgdorferi strain B-31
529
(A,B) and strain JD1 (C-F). For B31-infected monkeys, the relative longitudinal responses are
530
shown in (A) and the reciprocal EPDTs are shown in (B). For JD1-infected monkeys, the
531
longitudinal responses over the full study period are shown in panels C and D, whereas the time
532
interval immediately before, during, and after treatment is shown in panels E and F. Treated
533
animals are shown in C and E, whereas untreated animals are shown in D and F. The results in E
534
and F are from separate ELISA plates, compared to those shown in C and D.
535
536
Figure 6. Reciprocal EPDTs for each antigen at the peak response (A) and at the study end point
537
(B) for each of the strain B31-infected macaques. Here, the relative abundance of specific
538
antibodies for each antigen, with and without treatment, can be compared.
539
540
Figure 7. Longitudinal IgM responses by macaques infected with B. burgdorferi strain B31
541
against four different antigens. Shown are the relative responses to OspA (A), OspC (B), DbpA
542
(C) and OppA-2 (D).
543
544
545
546
547
548
Table 1. Summary of monkey groups, antigens tested and results.
# positive/total tested:
Strain B31
2
C6 {reference
(16)}
2/2
DbpA
BBK32
OspA
OspC
2/2
2/2
2/2
2/2
OppA2
2/2
3/3 (early)
3/3
1/3
3/3
3/3
3/3
5/5
nd
6/6
6/6
6/6
0/12
5/5
nd
5/5
5/5
6/6
0/4
nd
nd
0/1
0/1
0/1
15/15
3/5
16/16
16/16
17/17
untreated
3 treated
3/3 very low
titer (late)
Strain JD1
untreated
12/12 (early)
5/12 (late)
treated
uninfected
549
Total positive*/infected
animals tested
nd=not determined
550
551
Table 2. Percent change in antibody titer with antibiotic treatment
animal
designation
GA59
(treated)
FK38
(treated)
GB56
(treated)
GC84
(untreated)
FT47
(untreated)
552
553
C6
{reference
(16)}
DbpA
BBK32
OspA
OspC
OppA-2
98.4%
no
decline
75%
75%
93.8%
87.5%
87.5%
87.5%
96.9%
75%
50%
50%
75%
nd
no
decline
50%
50%
50%
50%
no
decline
98.4%
93.8%
no decline
no decline
75%
no
decline
no
decline
no
decline
no
decline
no
decline
B
0.8
0.7
OD 450 nm
0.6
GA59
0.5
FK38
0.4
GC84
0.3
FT47
0.2
GA59
FK38
1.0E+05
0
FT47
GB56
1.0E+04
wk2 wk10 wk16 wk22 wk34 wk40 wk47
C
week22
D
1.4
1
AL70
0.8
BH50
0.6
AG02
AH77
0.4
treatment
AM38
0.2
0
wk2
wk4
wk15
wk27
wk37
wk49
OD 450 nm
12
1.2
OD 450 nm
GC84
GB56
treatment
0.1
1.0E+06
reciprocal end point
dilution titer
A
week 47
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
BD71
BH31
BT02
BG48
BH18
wk2
wk4
wk15 wk27 wk37 wk49
B
0.35
OD 4
450 nm
0.3
reciprrocal end point
diilution titer
A
1.4E+03
1.2E+03
1.0E+03
GA59
8.0E+02
FK38
6.0E+02
GC84
4.0E+02
FT47
0.05
2.0E+02
GB56
0
0.0E+00
0.25
GA59
0.2
FK38
treatment
GC84
0.15
FT47
0.1
GB56
wk2 wk10 wk16 wk22 wk34 wk40 wk47
week22
week 47
B
09
0.9
0.8
OD 45
50 nm
0.7
0.6
GA59
0.5
FK38
0.4
GC84
0.3
treatment
FT47
GB56
0.2
0.1
1 00E+05
1.00E+05
reciprocal end po
oint dilution titer
A
wk2
D
1.2
FT47
1.00E+03
GB56
22 wk
47 wk
1.2
1
1
AL70
0.8
BH50
0.6
AG02
0.4
AH77
0.2
AI97
treatment
AM38
OD 450 nm
m
OD 450 nm
m
FK38
2 wk
wk10 wk16 wk22 wk34 wk40 wk47
1.4
0
GA59
1.00E+02
0
C
1.00E+04
0.8
BD71
BH31
0.6
BT02
0.4
BG48
0.2
BH18
0
BR99-uninf
BR99
uninf
0.5
B
0.45
OD 450 nm
0.4
0.35
GA59
0.3
FK38
0.25
GC84
0.2
FT47
0 15
0.15
GB56
0.1
treatment
0.05
0
Reciprocal e
end point dilution tite
er
A
9.0E+04
8.0E+04
7.0E+04
6.0E+04
GA59
5.0E+04
FK38
4.0E+04
GC84
3.0E+04
FT47
2.0E+04
GB56
1.0E+04
0.0E+00
wk2 wk10 wk16 wk22 wk34 wk40 wk47
C
week16
14
1.4
D
1.2
12
1.2
1
treatment
BH50
0.8
AG02
0.6
AH77
0.4
AI97
0.2
AM38
0
BG48
OD 450 nm
AL70
1
OD 450 nm
week 47
0.8
BH18
BD71
0.6
BH31
0.4
BT02
BR99-uninf.
0.2
0
wk2
wk4
wk15
wk27
wk37
wk49
wk2
wk4
wk15
wk27
wk37
wk49
B
0.5
0.45
OD 450 nm
0.4
0.35
GA59
0.3
FK38
0.25
treatment
0.2
GC84
FT47
0.15
GB56
0.1
0.05
0
recipro
ocal end point dilutio
on titer
A
>5 0E+04
>5.0E+04
GA59
FK38
1.0E+04
GC84
FT47
GB56
1.0E+03
wk2
wk10 wk16 wk22 wk34 wk40 wk47
week 16
week 47
Figure 5, cont.
C
D
E
F
treatment
A
B
A
0.8
B
0.7
1.2
0.6
GA59
0.5
FK38
0.4
GC84
0.3
FT47
02
0.2
OD 450 nm
OD 450 nm
1.4
1
GA59
0.8
FK38
0.6
GC84
GB56
0
0
wk2 wk10 wk16 wk22 wk34 wk40 wk47
wk2 wk10 wk16 wk22 wk34 wk40 wk47
0.6
D
0.7
0.6
0.5
FK38
0.3
GC84
FT47
0.2
GB56
0.1
0.5
OD 4
450 nm
GA59
0.4
OD
D 450
GB56
0.2
0.1
C
FT47
0.4
GA59
0.4
FK38
0.3
FT47
0.2
GB56
0.1
0
0
wk2 wk10 wk16 wk22 wk34 wk40 wk47
wk2 wk10 wk16 wk22 wk34 wk40 wk47