Epitope mapping in Lyme disease

Introduction

A new study from Columbia University, Stony Brook University and the CDC – that has seen the collaboration of Brian Fallon and W. Ian Lipkin, among others – has proposed a new serological test for Lyme disease and coinfections (Babesia microti, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Rickettsia ricketsii and some viruses that are not present in Europe) (Tokarz R et al. 2018). Current serologic analyses consist of a two-tiered algorithm:  a measure of serum activity against a main immunogenic protein of B. burgdorferi (whether it is VlsE or its peptide C6) is followed by a western blot, where the immune response to a set of several full-length proteins is performed. This method lacks sensitivity for early Lyme disease (Aguero-Rosenfeld ME et al 2005) and – according to studies on the animal model of Lyme disease – it might miss some cases of disseminated infection too (Embers ME, 2012), (Nicholas A, 2017), (Embers ME 2017). So, there is common agreement that a better test is urgently needed.

The quest for immunogenic peptides in Lyme patients: the set of proteins

Most B cells epitopes on non-denaturated proteins (i.e. proteins that conserve their tertiary structure) are believed to be conformational (Morris, 2007) but it is also true that in the average B cell epitope, a linear stretch of 5 amino acids is reported (Kringelum, et al., 2013). This means that it is conceivable to search for new immunogenic peptides in Lyme disease with the following method: each protein from Borrelia, known to be immunogenic in humans, is divided in peptides with a fixed length, then each of these peptides is exposed to sera from patients. Those peptides that strongly bind sera from patients are eligible as immunogenic peptides useful for diagnostic purposes. This is exactly what has been done in this study, and this elaborate analysis has been performed not only for Borrelia burgdorferi but also for the other tick-borne pathogens already mentioned. In particular, the length of the peptides has been fixed to be 12, and contiguous peptides have an overlapping of 11 amino acids. The set of immunogenic proteins chosen for each pathogen are reported in table 1.

A new array of diagnostic peptides in Lyme disease

The analysis was conducted using sera from 66 Lyme patients (27 early Lyme, 19 with positive IgG Western blot, 10 with acute neuroborreliosis). The end result is the set of peptides reported in table 2. There is also a set of peptides specific for neuroborreliosis (table 3).

Flagellin B: a new specific peptide for diagnostic purposes is born

It is widely recognized that reactivity of sera to Flagellin B (FlaB, p41) is common in healthy persons too (Chandra A et al. 2011). This is because this protein is highly conserved among bacteria, so there is cross-reactivity and FlaB is almost without utility for diagnostic purposes. That said, one of the results of this study is that peptide 211-223 fro FlaB is highly immunogenic in patients and is also not-cross-reactive with flagellar proteins from other bacteria (I have checked using this peptide as query sequence in a BLAST search among bacteria, with a word of 3 and with standard settings: no match has been found). So we have a new specific peptide for diagnostic purposes. In figure 1 this epitope is reported in yellow, on the 3D structure of Flagellin B.

The strange case of VlsE and C6

Vmp-like sequence expressed (VlsE) is a major immunogenic protein of Borrelia burgodorferi, widely used for the first-tier test of Lyme disease (Aguero-Rosenfeld ME et al 2005). This protein goes through a process of variation during mammalian infection that involves six variable regions (VR1-VR6) and it has been postulated that this gene recombination of the locus that codes for VlsE is responsible – at least in part – for the ability of B. burgodorferi of evading the immune system (Zhang JR et al. 1997). Nevertheless, six regions of this protein are invariable (IR1-6) and these ones have been studied as possible peptides to use for diagnosis. One of them, peptide IR6 (also called C6), has been used as a diagnostic tool (Liang FT et al. 1999). In the present study, the peptide that pops up from the analysis is the same C6 (peptide 274-290 in table 1), while in neuroborreliosis there are two more peptides on VlsE that appear to be useful markers. I have reported these three peptides on the 3D structure of VlsE, the one experimentally determined in (Eicken C et al. 2002), see figure 2. It is worth noting now that peptide 274-290 (C6) is buried inside the protein, so it is difficult to understand how it could be a B cell epitope since B cell epitope protrudes from the surface (Thornton, et al., 1986) and this is probably because B cell receptors need to bind their specific antigen, for B cell to be activated. This is the very first time that I realize that C6 is not surface exposed. So, how could the immune system generate an antibody to a hidden peptide? I have to think about that.

OspC and the buried epitope

Another weird case is that of OspC, where one of the two main epitopes found in patients with neuroborreliosis is buried in the cytoplasmic membrane of Borrelia burgdorferi (figure 2, right). This is an uncommon case, I guess, for the same reason mentioned above: BCRs need surface exposure to bind their specific epitope. The other peptide (132-144) appears to be a classic surface-exposed B cell epitope.