The document discusses the complexities and challenges in diagnosing Lyme disease using serological assays, highlighting advancements like peptide-based diagnostics but also emphasizing issues like cross-reactivity and sensitivity. It reviews the history and evolution of Lyme disease laboratory diagnostics, detailing specific antigens, serologic criteria, and the performance of various tests, including the C6 peptide assay. Future diagnostic approaches are suggested, including metabolomics and cytokine release assays, with the need for further studies to refine diagnostic methods.

1. FACULTY/PRESENTER DISCLOSURE
• Faculty:
• Relationships with commercial interests:
• The epitope mapping presented was done at Biopeptides
Corp. and the IP is owned by Biopeptides Corp.

2. DISCLOSURE OF COMMERCIAL SUPPORT
• Potential for conflict(s) of interest:
• Raymond Dattwyler and Biopeptides Corp have received NIH
grants
• BioRad has licensed technology developed by Biopeptides Corp
• Qiagen has licensed technology developed by Biopeptides Corp

3. LABORATORY DIAGNOSTICS OF LYME
DISEASE, PAST, PRESENT, FUTURE:
RAYMOND J. DATTWYLER, MD
PROFESSOR OF MICROBIOLOGY/IMMUNOLOGY AND MEDICINE
NEW YORK MEDICAL COLLEGE, VALHALLA, NY USA
PRESIDENT BIOPEPTIDES CORP

4. LYME DISEASE LABORATORY DIAGNOSIS
1980 1990 2000 2010

5. SEROLOGIC Assays
Antigen targets: whole cell Bb or Bb sonicates
• Problems:
• Whole bacteria contain many epitopes that are
common among other bacterial species – “non-specific
epitopes”-
• Cultured Borrelia can lose plasmids coding key antigens
-
• Some important antigens are not express in in vitro

6. CDCCRITERIA
TWO-TIERSEROLOGY
First Month of Infection:
First tier: IgG and/or IgM antibody to whole-cell sonicate by either EIA or IFA
Second-tier: IgG and IgM Western blotting if first tier positive or equivocal
2 of 3 IgM bands: 23-, 39-, and 41-kDa
5 of 10 IgG bands: 18-, 23-, 28-, 30-, 39-, 41-, 45-, 58-, 66-, and 93-kDa
After the first month :
First tier: IgG antibody to whole-cell sonicate by either EIA or IFA
Second-tier: IgG Western blotting if first tier positive or equivocal
5 of 10 IgG bands: 18-, 23-, 28-, 30-, 39-, 41-, 45-, 58-, 66-, and 93-kDa

7. WESTERN BLOT CRITERIA
1. The Criteria were developed using cultured Bb
2. At the time, these criteria were formulated, many of
these antigens were not defined.
3. In Vivo expressed antigen are poorly represented
7

8. SENSITIVITY OF STANDARD2-TIER
• Early acute disease: 30% to 40%
• Early convalescent disease: 70%
• Early neurological and carditis (Acute Disseminated): 85%
• Late disease: 98+%

9. 2D GEL WESTERN HUMAN LATE DISSEMINATED INFECTION
Nowalk, A. J., R. D. Gilmore, Jr., and J. A. Carroll. 2006. Serologic proteome
analysis of Borrelia burgdorferi membrane-associated proteins. Infect. Immun.
74:3864–3873.

10. ANTIGENS ASSOCIATED WITH WB
10
PROTEIN Kd Early Intermediate Late
p93 93 + +
Hsp90 90 +
P66* 66 +
OppA2 58 +
ErpB 58 +
BBK32 47 +
FtsZ 45 +
FlaB* 41 +
BmpA 39 +
BBO323* 30 +
OspF 26 + +
OspC 21 +
LA7 20 + +
ErpP 18 + +
DbpA 18 + +
RevA 18 +

11. ANTIGEN CROSSREACTIVITY
• Crossreactivity is a significant issue in any serodiagnostic assay,
particularly when using preparations derived from whole organisms or
highly conserved proteins.
• Borrelia flagellin generates positive responses in >40% of healthy
individuals with no history of Lyme disease (Liang, FT, et al, J. Clin.
Microbiol. December 1999 vol. 37 no. 12 3990-3996.)
• A 60kDa antigen has seropositivity in >16% of healthy controls
(Liang, FT, et al, J. Clin. Microbiol. December 1999 vol. 37 no. 12
3990-3996.)
• BBO323 Homolgus with periplasmic substrate binding proteins of
Gram Negatives.
• Whole cell sonicate ELISAs can detect antibody at rates greater
than 90% in tropical countries that don’t have Lyme disease
(Burkitt TR, et al, J Infect Dis. (1997) 175 (2): 466-469.)

12. PROTEINS CONTAIN CROSS REACTIVE REGIONS SIMILAR TO
OTHER BACTERIA
B.BURGDORFERI P41 FLAGELLIN IS A GOOD EXAMPLE

13. • P66 is a prominent antigen included in
most conventional Lyme seroassays
• We found a significant amount of
antibody binding to 6 different
peptides in serum from healthy and
disease controls
• In concordance, we find that the p66
band on most western blots is
positive regardless of serum source
P66 a lesson in
crossreactivity.

14. OTHER ANTIGENS ALSO HAVE CROSS REACTIVE EPITOPES
• Peptides were generated from OspC epitope sequences
and screened Lyme and healthy control sera

15. BENEFITS OF PEPTIDE EPITOPES
• Preservation of specificity by selection epitopes unique to
Borrelia spp., and eliminating ‘cross-reactive’ epitopes
present in other bacterial species
• Conceptually, this improves both the specificity and
sensitivity of seroassays

16. C6
• The first FDA approved peptide based serodiagnostic assay
• A conserved peptide from the invariable region 6 of the VlsE protein
• Has performed well as a single antigen assay; however, has not performed well
enough to supplant the two-tier paradigm
• C6 does not bind well to IgM, it predominantly binds IgG
• The IR6 region of VlsE is more variable than was initially though, and peptide based
diagnostics are exquisitely sensitive to variances in AA sequence
• The VlsE antigen is not expressed in the tick (<1% of bacteria), therefore it needs to be
upregulated prior to generating immunity, limiting utility in very early responses
• Despite the drawbacks observed, it clearly demonstrates the utility of peptide based
serodiagnostics

17. EPITOPE MAPPING of the OppA2 protein
Signorino G, Arnaboldi PM, Petzke MM, Dattwyler RJ. Identification of OppA2 linear epitopes as
serodiagnostic markers for Lyme disease. Clin Vaccine Immunol.2014 May;21(5):704-11.
• For OppA2, many sequences were identified, for other proteins only one or two
epitopes were identified per protein
Table 1. Peptide sequences containing immunodominant epitopes of OppA2.
Peptide name
Amino acid sequence
OppA (11-25) IFFLTFLCCNNKERK
OppA (191-225) YGQNWTNPENMVTSGPFKLKERIPNEKIVFEKNNK
OppA (276-290) SDYYSSAVNAIYFYS
OppA (276-300) SDYYSSAVNAIYFYSFNTHIKPLD
OppA (286-300) IYFYSFNTHIKPLD
OppA (286-310) IYFYSFNTHIKPLDNVKIRKALTLA
OppA (356-375) LAEAGYPNGNGFPILKLKYN
OppA (381-400) KKICEFIQNQWKKNLNIDVE
OppA (491-505) APIYIYGNSYLFRND

18. • Each peptide was run with a
panel of 114 early Lyme, 44
healthy controls, 30 RA, 28 RPR+
• Cutoffs for positivity were
determined as >3SD from mean
of healthy control (positive) and
>2SD from mean of healthy
control (equivocal)
OppA2 ELISA

19. LESSONS FROM SEROASSAYS DEVELOPMENT
• Identification of new Peptide targets continue to improve
specificity and sensitivity compared to current first tier EIAs
• However, antigen selection must be carefully performed
• No ‘blanket approach’ for epitope identification is effective
• Individual epitopes from different antigens have different
attributes.
• OspC1 binds IgM better than IgG, DbpA4-DbpB6 only works
well as a dipeptide, many p66 epitopes are crossreactive,
p41 has a useful epitope

20. • It takes time for antibody formation
• IgG levels remain elevated for years after infection, seroassays
cannot distinguish new infection from previous exposures
• Antibody levels do not correlate treatment success
Seroassays have issues that cannot be
overcome

21. NEW APPROACHES TO LABORATORY DIAGNOSIS
THE FUTURE?
• Metabolomics
• Transcriptome analysis
• Monitoring T cell activity

22. T CELL RESPONSE
• Distinct kinetics of T cell responses
• Following resolution of infection:
• T cell effector function wanes
• Activated T cell numbers contract
• Cytokine release can be used as a indirect measure
of T cell activation

23. CYTOKINE RELEASE ASSAY
• The QuantiFERON technology platform
• Whole blood
• Incubate O/N, Harvest plasma
• IFN-γ ELISA
Nil Lyme Mito

24. TARGET ANTIGENS
• Sequences for each antigen were
evaluated against protein databases
using the NCBI Basic Local Alignment
Search Tool (BLAST).
• Overlapping peptide libraries were
generated for regions with:
• <50% identity with other proteins
and
• >80% identity among Borrellia spp.
• These data were generated using a
mixture of 33 peptides from OspC,
DbpB, p66, and FlaB
Antigens evaluated for T cell
reactivity:
• OspC
• DbpB
• p66
• FlaB
• CRASP2
• Bbk32
• BmpA
• OppA2
• LA-7
• FlilB
• Bdf2
• RecA

25. PATIENT CHARACTERISTICS
Subjects Median
Age
(range)
Gender
(F/M)
Median
Duration of
Symptoms
(range)
Single vs.
Multiple
EM
Symptomsb
Erythema
migrans+a
(n=29)
58 (19-74) 9/20 5d (2-30) 23/6
headache (n=23)
stiff neck (n=15)
myalgia (n=15)
fever (n=13)
fatigue (n=13)
arthalgia (n=10)
lymphadenopathy (n=1)
none (n=2)
a- >5 cm round lesion, often with partial central clearing
b- symptoms resolved following 10d treatment with Doxycycline
• Blood was collected at initial visit (n=29), 2 months post treatment (n=27), and 6
months post treatment (n=5)

26. INTERFERON-GAMMA SECRETION
• IFNγ was detected in 3.1% (6 of 192) healthy and
Anaplasmosis controls
Acute Convalescent
(2 mo.)
Convalaescent
(6 mo.)
Positivea
20/29
(69.0%)
IFNγ
(IU/ml)
1.46 ±
2.26
4/27
(15.3%)
1/5
(20.0%)
0.27 ±
0.59
0.13 ±
0.19
a- >3 SD (0.33 IU/ml) from the mean of IFNγ produced in
blood of healthy volunteers (n=187) and Anaplasmosis (n=5).

27. COMPARISON WITH SEROLOGY
QuantiFERON
ELISA
C6
ELISA
Western Blot
IFNγ (IU) IgM or IgG IgM or IgG
Acute
(n=29)
69.0%
(20/29)
58.6%
(17/29)
17.2%
(5/29)
• When combined the C6 and QuantiFERON ELISAs positively identified
82.8% (24/29) of acute Lyme disease patients
• Adding peptides from additional antigens can increase the sensitivity
of the assay.

28. SUMMARY
• IFNγ secretion can be used to detect infection with Borrelia burgdorferi
• Requires multiple peptides from different proteins
• IFNγ secretion is reduced following successful antibiotic therapy
• No patients complained of ongoing symptoms following doxycycline
treatment
• Additional studies are warranted to:
• more critically define cut-off values,
• evaluate the inclusion of other antigens
• evaluate localized vs. disseminated Lyme disease
• evaluate late Lyme disease

29. ACKNOWLEDGEMENTS
• NIH NIAID for their funding
• Biopeptides, Corp./NYMC
• Paul M. Arnaboldi, PhD
• Mariya Sambir, MS
• NYMC
• Mary Petzke, PhD
• Giacomo Signore, PhD
• Gary Wormser, MD
• Gundersen-Lutheran, Wisc.
• Steven Callister, PhD
• Dean Jobe, MS
29