Towards sterilizing immunity against Leptospira with a DNA vaccine

In my previous post, I described the failure of researchers to come up with a conventional protein-based subunit vaccine that confers sterilizing immunity against leptospirosis.  What I mean by "conventional" subunit vaccine is a mixture of purified recombinant Leptospira protein with an adjuvant (either aluminum hydroxide or Freund's).  Although an antibody response was detected against most proteins tested, immunization failed to prevent kidney colonization in every case, including those animals that survived challenge with lethal strains of Leptosipra.  It's becoming clear that the leptospirosis vaccine field must move beyond simple formulations of protein plus adjuvant if sterilizing immunity is desired.

Several labs have explored more modern approaches to delivering leptospirosis vaccines.  Odir Dellagostin's group down in Brazil tested the efficacy of DNA vaccines in protecting hamsters against leptospirosis, as described in this paper in Clinical and Vaccine Immunology.  Forster and colleagues targeted the Leptospira interrogans LigA and LigB proteins, which are surface proteins that disrupt (or exploit) multiple host functions.

The investigators cloned various fragments of the lengthy ligA and ligB genes downstream of the human cytomegalovirus promoter of the commercial expression plasmid pTARGET.  They mixed the plasmid DNA with aluminum hydroxide adjuvant and injected the material into the muscle of the hind leg of hamsters.  The animals were given a booster with the same material 21 days later.  An IgG immune response was detected against four of the the five Lig protein fragments being tested (see figure below).

Figure 2 from Forster et al., 2013.  Sera were drawn before immunization and after the first and second immunizations with pTARGET-based lig plasmid DNA.  Purified recombinant protein encoded by each plasmid was used as antigen in ELISAs.  Left, middle, and right bar for each DNA: before immunization, after first immunization, and after second immunization, respectively.

21 days after the boost, the animals were challenged with a lethal strain of L. interrogans.  The survival curves are shown below.  Note that animals immunized with the vector alone (small filled circles) were all dead by day 11.

Figure 3 from Forster et al., 2013.

Among the lig gene fragments, the one expressing the "LigBrep" fragment was effective, protecting five of the eight animals in the group (62.5%) from death.  LigBrep comprises amino acid residues 1 through 628 of LigB, whose total length is 1891 residues.  Although the survival rate is nothing to get excited over, what distinguishes the LigBrep DNA vaccine from the conventional subunit vaccines tested in earlier studies is that the kidneys from 4 of the 5 survivors were culture negative, indicating that sterilizing immunity was achieved in 80% of the animals that survived infection.

Another notable outcome of the study was that protection was achieved even though the challenge strain and the vaccine's lig gene originated from different Leptospira serovars.  One of the problems with killed whole-cell vaccines is that they only protect against Leptospira serovars present in the vaccine formulation because they target LPS, whose structure varies among different serovars.  Leptospira proteins tend to be similar in amino acid sequence across different species and are therefore more attractive as vaccines.  (The "killed-whole leptospires" control plotted in the graph above was generated from the challenge strain).

So how does DNA vaccination induce sterilizing immunity against Leptospira?  As always, more studies are needed to explore this issue, but I will go ahead and speculate. DNA vaccines that are administered by standard injection stimulate a Th1-biased immune response.  Studies with cattle have suggested that vaccines must stimulate Th1 immunity to minimize kidney colonization by Leptospira (see this study, for example).  Moreover, an earlier study by Dellagostin's group demonstrated sterilizing immunity against L. interrogans in some animals immunized with a Mycobacterium bovis BCG strain that was engineered to express LipL32, the major outer membrane protein of L. interrogans.  BCG also stimulates Th1 immunity.

Why would a Th1 response be necessary for sterilizing immunity against Leptospira?  Th1 cytokines help steer B cells into producing an IgG isotype that is strongly recognized by Fc receptor on phagocytes.  Consequently, bacteria bound by these IgG molecules are engulfed by opsonophagocytosis.  During Leptospira infections, opsonophagocytosis clears spirochetes from the circulation during the antibody response, raising the possibility that opsonophagocytosis also leads to sterilizing immunity by vaccines that induce production of the "right" IgG.

Th1 cells are also necessary for cellular immunity, which enhances the killing functions of macrophages so that they can rid themselves of intracellular pathogens.  Leptospira is considered to be an extracellular pathogen.  Nevertheless, there may be a transient intracellular phase that is critical during infection.  Although intracellular Leptospira has not been observed in vivo, L. interrogans is known to survive and replicate in cultured macrophages.

References

Forster KM, Hartwig DD, Seixas FK, Bacelo KL, Amaral M, Hartleben CP, & Dellagostin OA (2013). A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis. Clinical and Vaccine Immunology : CVI, 20 (5), 725-731 PMID: 23486420

Zuerner RL, Alt DP, Palmer MV, Thacker TC, & Olsen SC (2011). A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clinical and Vaccine Immunology : CVI, 18 (4), 684-91 PMID: 21288995

Seixas FK, da Silva EF, Hartwig DD, Cerqueira GM, Amaral M, Fagundes MQ, Dossa RG, & Dellagostin OA (2007). Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine, 26 (1), 88-95 PMID: 18063449

Toma C, Okura N, Takayama C, & Suzuki T (2011). Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages. Cellular microbiology, 13 (11), 1783-1192 PMID: 21819516


Related posts

• Is sterilizing immunity against Leptospira possible with protein subunit vaccines?
• A new attentuated leptospirosis vaccine protects hamsters from lethal infection by more than one serovar of Leptospira

Is sterilizing immunity against Leptospira possible with protein subunit vaccines?

With complete bacterial genome sequences now available, "reverse vaccinology" can be conducted to identify proteins that can function as subunit vaccines.  The "gene first" approach of reverse vaccinology relies upon computer analysis of the genome sequence to identify encoded proteins with  features common to known surface-exposed and secreted bacterial proteins.  The selected genes can then be cloned and expressed as recombinant proteins.  The proteins, which may number in the hundreds, are then purified for vaccine testing in the animal model appropriate for the bacterial pathogen.  Reverse vaccinology has been employed successfully to find protective protein antigens against Neisseria meningitidis serogroup B, Streptococcus pneumoniae, group B Streptococcus, Bacillus anthracis, Porphyromonas gingivalis, and other bacterial pathogens (reviewed in this paper).

This approach sounds straightforward but in practice may not always lead to identification of effective subunit vaccines.  An important study from Ben Adler's group down in Monash University illustrates the challenges of finding a subunit vaccine that prevents chronic Leptospira infections.  They focused on serovar Hardjo, which causes chronic infections in cattle.  They selected 263 Hardjo genes that were predicted to encode surface-exposed, secreted, or lipid-modified proteins.  Among these they successfully cloned and expressed 223 genes as 238 protein antigens in E. coli.  Some genes were expressed as two or more fragments because of their large size.  210 of the 238 (88%) aggregated into inclusion bodies during expression and had to be kept dissolved in urea during their purification.  (Strangely, the urea was not removed by dialysis prior to immunization.)  The 238 purified proteins were mixed with an aluminum hydroxide adjuvant and injected into hamsters.  169 of the 238 proteins (71%) generated an antibody response, yet none succeeded in preventing colonization of the kidneys following challenge with a Hardjo strain.

It's been hard enough to find leptospiral proteins that protect hamsters from lethal disease when tested as vaccines (see this article for a review), yet Murray and colleagues sought proteins that protected against Leptospira colonization, a more difficult endeavor that has never been achieved with subunit vaccines.  The Hardjo strain they used easily colonizes the kidneys yet fails to produce any signs of disease in hamsters.  Although several studies have demonstrated that certain versions of the LigA and LigB proteins, when administered as vaccines, protect hamsters and mice from being killed by lethal strains of Leptospira, survivors are left with infected kidneys.  A vaccine that protects against disease or death but not infection may be adequate for humans, who eventually clear the spirochetes from their kidneys even following a natural infection (assuming the disease doesn't kill them).  However, vaccinated cattle infected with Hardjo may not be able to clear the spirochetes and will continue to shed infectious Leptospira into the environment, placing the entire herd and the workers handling them at risk of infection.  Hardjo infections generally don't cause signs of disease in cattle, but they can cause fetal death and drop in milk production in cows.

The choice of adjuvant and destruction of protective conformational epitopes by urea are possible reasons for failure to find a protective antigen.  On the other hand, perhaps a different method for delivery of protein antigens into animals should been considered.  Stay tuned.

Featured paper

Murray GL, Lo M, Bulach DM, Srikram A, Seemann T, Quinsey NS, Sermswan RW, Allen A, & Adler B (2013). Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation. Vaccine, 31 (3), 495-499 PMID: 23176980

Helpful reviews

Dellagostin, O.A., Grassmann, A.A., Hartwig, D.D., Felix, S.R., da Silva, E.F., & McBride, A.J.A. (November 2011).  Recombinant vaccines against leptospirosis.  Human Vaccines 7(11):1215-1224. DOI: 10.4161/hv.7.11.17944

Serruto, D., Serino, L., Masignani, V., & Pizza, M. (May 26, 2009).  Genome-based approaches to develop vaccines against bacterial pathogens.  Vaccine 27(25-26):3245-3250.  DOI: 10.1016/j.vaccine.2009.01.072

Triggering OspC production in Borrelia burgdorferi during tick feeding: Is temperature the real signal?

The Ixodes tick, the vector of the Lyme disease spirochete, goes months without a meal.  During this time, the Borrelia burgdorferi spirochetes living in its midgut live quiet lives, sipping on the tick's antifreeze to sustain themselves. When the tick finally takes a blood meal from a warm-blooded victim, B. burgdorferi responds by producing a number of new proteins, some of which are needed for transmission to and infection of the mammalian host.  Among these proteins is the outer surface lipoprotein OspC, whose function involves capture of tick (see this post) and mammalian host proteins.  How does B. burgdorferi know when to start making these critical proteins?  The favored model has been that the the warmth of the blood entering the tick triggers B. burgdorferi to make these proteins.  It's been known for almost two decades that B. burgdorferi growing in culture medium produces miniscule amounts of OspC at low temperatures (23º-24ºC) and larger amounts at higher temperatures (32º-37ºC), as shown in the figure below from the classic 1995 report by Tom Schwan and colleagues.

Figure 4 from Schwan et al., 1995.  B. burgdorferi incubated at 24ºC (lanes 2 and 6), transferred from 24ºC to 37ºC (lanes 3 and 7), incubated at 37ºC (lanes 4 and 8), or transferred from 37ºC to 24ºC (lanes 5 and 9).  Panel A, SDS-PAGE gel stained for total proteins with Coomassie  brilliant blue.  Arrow marks location of OspC.  Panel B, Western blot with flagellin antibody (Fla) and OspC antibody.

As reasonable as this model sounds, findings from a recent paper from Brian Stevenson's group (Jutras et al., 2012) challenge the model.  Although not emphasized in earlier papers, the authors noted that B. burgdorferi multiplies much more quickly at higher temperatures.  In their hands, B. burgdorferi proliferated with a doubling time of 32 hours at 23ºC and 12 hours at 34ºC.  As expected, their Western blots showed that more OspC was produced by the spirochetes growing at the higher temperature.  Members of the Erp family of surface proteins, whose levels also rise during tick feeding, were produced at higher levels at the higher temperature as well, as shown in earlier studies.  The investigators devised an experiment to test whether B. burgdorferi could tie OspC and Erp expression to its growth rate instead of temperature.

The standard culture medium for Borrelia is BSK-II with 6% rabbit serum, a complex nutrient-rich concoction.  They made two new formulations of the culture medium to slow the growth rate:  (1) quarter strength BSK-II with the rabbit serum concentration remaining at 6%; (2) full-strength BSK-II with the rabbit serum concentration reduced to 1.2%.  Medium #1 slowed the doubling time at 34ºC to 40 hours, and medium #2 reduced it to 32 hours.  Western blots of the spirochetes harvested from both cultures revealed low levels of the OspC and Erp proteins.  When these spirochetes were inoculated into the standard culture medium (BSKII/6% rabbit serum) and incubated at 34ºC, high levels of the proteins were again detected.  Therefore, B. burgdorferi is capable of adjusting OspC and Erp expression by monitoring its growth rate, even if the surrounding temperature does not change.

The final experiment from the study demonstrates that not even growth rate is the direct signal.  The authors froze B. burgdorferi at -80ºC for at least a month and then inoculated the bacteria into standard culture medium for incubation at 23ºC.  As a control, bacteria being maintained at 34ºC were also transferred to standard culture for incubation at 23ºC.  Both cultures grew with the same doubling time.  Nevertheless, the spirochetes that were revived from the frozen state produced more OspC and Erp proteins that those that were initially maintained at 34ºC.

So what's the real cue?  Going back to the natural life cycle of B. burgdorferi, the spirochetes living in the unfed tick's midgut do not really grow or divide.  The metabolism of B. burgdorferi is slowed by the nutrient-poor conditions in the tick's midgut.  When the tick finally takes a blood meal, the surge of nutrients entering the tick signals B. burgdorferi to rev up its metabolism, triggering production of OspC.  This model would explain why the frozen spirochetes, whose metabolism was undoubtedly slowed, were able to produce large amounts of OspC and Erp proteins when inoculated into standard culture medium at 23ºC, the temperature usually associated with diminished production of the proteins.  The challenge will be to figure out how B. burgdorferi is sensing its metabolic state at the molecular level.

References

Jutras, B.L., Chenail, A.M., & Stevenson, B. (2012). Changes in bacterial growth rate govern expression of the Borrelia burgdorferi OspC and Erp infection-associated surface proteins. Journal of Bacteriology, 195 (4), 757-764 DOI: 10.1128/JB.01956-12

Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., & Rosa, P.A. (1995). Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proceedings of the National Academy of Sciences, 92 (7), 2909-2913 DOI: 10.1073/pnas.92.7.2909

Related posts

• Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut
• The Lyme disease spirochete feasts on tick antifreeze
• A fresh approach towards a Lyme disease vaccine: targeting the tick

Is the major outer membrane lipoprotein LipL32 really exposed on the surface of Leptospira?

Here's a study that may come as a surprise to those in the leptospirosis field.  The outer membrane lipoprotein LipL32 is believed to be the dominant protein on the cell surface of pathogenic species of Leptospira.  However, according to a new PLoS One article written by Pinne and Haake at UCLA, LipL32 may not be present on the surface at all.  This is an important issue to get right because the function proposed for LipL32, attachment to the extracellular matrix during infection, assumes that the lipoprotein is exposed on the surface of the spirochete.  More importantly, a number of research groups have already committed a lot of time and resources towards generating LipL32-based vaccines, which in current formulations confer (at best) weak protection against leptospirosis in rodent models (see this review for a critical analysis of the vaccine studies).

Pinne and Haake assessed surface exposure of LipL32 by two methods.  The first involved adding proteinase K to suspensions of Leptospira to digest proteins exposed on the surface of the spirochete.  When they did this, they saw that the known surface-exposed proteins OmpL37 and OmpL47 were degraded.  On the other hand, LipL32 didn't break down at all unless the spirochetes were first lysed by boiling them in a detergent (see Western blot below).

Figure 1B from Pinne and Haake (2013).  Increasing concentrations of proteinase K (up to 150 ug/ml) were added to suspensions (first five lanes) or lysates (last five lanes) of L. interrogans.  Following incubation, LipL32 was examined in a Western blot.  Source.

They next added LipL32 antibodies to Leptospira to see if they bound to the surface of the spirochete.  They did not, providing additional evidence that LipL32 was not exposed on the cell surface.  The authors tested LipL32 antibodies from different sources in an attempt to rule out the  possibility that failure of antibody binding was due to the surface-exposed portion of LipL32 not being antigenic.  LipL32 antiserum raised in rabbits, monoclonal LipL32 antibodies raised in mice, and LipL32 antibodies purified from the sera of leptospirosis patients all failed to bind the surface of Leptospira unless the outer membrane was chemically (with methanol or EDTA) or physically disrupted.  Note that antibodies raised against OmpL54, a known suface-exposed protein, reacted strongly with intact Leptospira (last pair of images below).

Figure 3A from Pinne and Haake (2013).  Bound antibody was detected with a secondary fluorescent antibody (green).  The spirochetes were also stained with DAPI, a penetrating dye that stains DNA (blue).  Left column, intact Leptospira, right column, methanol-treated Leptospira. Source.

In light of these results, the authors took another look at the 2005 study by Cullen and coauthors, who claimed LipL32 was surface exposed.  In contrast to Pinne and Haake, Cullen and colleagues detected binding of LipL32-specific antibodies to intact Leptospira in three different assays.  However, Pinne and Haake point out that antibody binding in their assays was extremely weak when  the abundance of LipL32 is considered.  The most striking example was the immunoelectron microscopy image of Leptospira treated with gold-labeled LipL32 antibody (see next image).  Yes, the surface ended up labeled, with a mean of 10.8 gold particles per spirochete cell.  However, we now know that there are 38,000 copies of LipL32 in each bacterial cell, making LipL32 the most abundant protein of L. interrogans (see this blog post about the Leptospira protein census).  If LipL32 were really surface exposed, the surface of the spirochete should have been packed with gold particles.

Figure 5 from Cullen et al. (2005).

Cullen and colleagues also mixed suspensions of Leptospira with a biotin probe that reacts with primary amines (mostly on lysine side chains).  The probe should have reacted solely with surface-exposed proteins since it's unable to penetrate the outer lipid bilayer and is assumed to be too large to diffuse through outer membrane porins.  Biotinylated proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry (see next figure).  Although LipL32 was one of the few proteins labeled with biotin, it's hard to make a firm conclusion about its surface exposure because proteins known to be located underneath the outer membrane, FlaB1 (a flagellar protein) and GroEL (a cytoplasmic heat shock protein), were also labeled with biotin. It's possible that LipL32 was labeled despite being located underneath the surface because the membrane was damaged while the spirochetes were being harvested for the experiment.

Modified from Figure 2 of Cullen et al., 2005.  Biotinylated proteins were separated by two-dimnesional electrophoresis.  Spots were removed and analyzed by mass spectrometry to identify proteins.  Multiple spots for each protein in the result of members of the population of each protein reacting with different numbers of biotin molecules.  "LipL32.16" was generated by proteolysis of LipL32.

Based on the intense labeling of LipL32 with biotin, Cullen and coauthors declared LipL32 the most abundant protein on the cell surface.  They speculated that LipL32 is poorly accessible to large molecules such as antibodies and proteases (which in their study failed to digest any protein when added to intact Leptospira) because the LPS side chains act as a "rainforest canopy" that can be penetrated only by smaller molecules such as biotin. This is a reasonable supposition because LipL32 is up to 60Å in length, whereas the distance between the outer membrane and the surface of the LPS layer is 92Å, according to a cryoelectron microscopy study of L. interrogans.  On the other hand, Pinne and Haake concluded that LipL32 is entirely or almost entirely subsurface since their assays failed to detect even a hint of the lipoprotein on the surface of Leptospira.  They maintain that the reactivity of surface probes with LipL32 observed by Cullen and colleagues was an artifact generated by the presence of damaged spirochetes in their assays and the massive copy number of LipL32. 

The results from Pinne and Haake's study do not rule out the "rainforest canopy" model since they did not test smaller surface probes that could penetrate into the LPS side chain layer.  Additional studies are needed to pin down the location of LipL32 relative to the surface of Leptospira.

References

Pinne, M., & Haake, D.A. (2013). LipL32 is a subsurface lipoprotein of Leptospira interrogans: Presentation of new data and reevaluation of previous studies. PLoS ONE, 8 (1) DOI: 10.1371/journal.pone.0051025

Cullen, P.A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A.I., Haake, D.A., & Adler, B. (2005). Surfaceome of Leptospira spp. Infection and Immunity, 73 (8), 4853-4863 DOI: 10.1128/IAI.73.8.4853-4863.2005

Related posts

• Protein census of Leptospira interrogans
• The major outer membrane protein of Leptospira interrogans: Not essential for infection?

An autoantigen targeted during Lyme arthritis

Infection by the spirochete Borrelia burgdorferi, if left untreated, can lead to a form of Lyme disease called Lyme arthritis.  About 10% of Lyme arthritis patients end up with a chronic form that doesn't go away with antibiotic treatment.  Allen Steere's group has long suspected that the antibiotic-refractory form of Lyme arthritis involves an autoimmune process.  This notion seems reasonable since those with antibiotic-refractory Lyme arthritis tend to have certain forms of the HLA-DR gene that are also common among those afflicted with the autoimmune disease rheumatoid arthritis.

Several groups have been searching for autoantigens (self antigens) that could drive the joint inflammation seen in Lyme arthritis patients.  Several candidate protein autoantigens were identified based on their short sequence similarities (molecular mimicry) to a T-cell or antibody epitopes in the B. burgdorferi OspA protein, which is targeted by the immune system in many Lyme arthritis patients, especially those with the antibiotic-refractory form.  However, further studies demonstrated that none of these autoantigens were likely to stimulate a sufficiently robust T-cell or antibody response that could account for the prolonged joint swelling experienced by patients with antibiotic-refractory Lyme disease (see this excellent review article for the complete story).  Therefore, an additional approach is needed to identify additional autoantigen candidates, an approach that does not assume that molecular mimicry underlies antibiotic-refractory Lyme arthritis.

An unbiased approach for finding autoantigens is to gather all of the different self-peptides being displayed by the HLA-DR molecules in the synovial tissue of the swollen joint and then figure out which of these peptides are capable of stimulating T cells.  At one time this approach wasn't possible since the individual peptides presented by HLA-DR molecules are found in such tiny amounts in human tissues, but the sensitivity of today's liquid chromatography/tandem mass spectrometry systems have improved to the point where many of the peptides can now be sorted and sequenced.

Steere's new study, which appeared in January's print issue of Arthritis and Rheumatism, was conducted in collaboration with Catherine Costello's group in Boston University.  The study was a follow-up to an earlier one published two years ago.  The authors extracted the inflamed synovial tissue from the swollen knee of a 12 year old boy suffering from antibiotic-refractory Lyme arthritis (see picture below).  He had gone through three months of antibiotic therapy a year prior to the procedure.  The tissue was culture and PCR negative.  When the patient's HLA-DR genes were examined, he turned out to have a copy of the DRB1*0101 allele, one of the HLA-DRB gene variants that places individuals at a higher risk for antibiotic-refractory Lyme arthritis.

Figure 1A from Drouin et al., 2013

The boy's synovial tissue was ground up, and HLA-DR-specific antibodies were used to capture the HLA-DR molecules with their bound peptides.  The peptides were then analyzed by liquid chromatography/tandem mass spectrometry.  The authors identified 120 different self-peptides from this analysis.  When each peptide was chemically synthesized and mixed with the boy's blood mononuclear cells, one peptide turned out to stimulate proliferation of his T cells.  This peptide came from a human protein called endothelial cell growth factor, or ECGF.

What's the function of ECGF?  The protein stimulates angiogenesis, the sprouting of new blood vessels from pre-existing ones.  Angiogenesis is a general feature of inflammatory arthritis, including Lyme arthritis and rheumatoid arthritis.

The authors went on to examine the T- and B-cell responses to ECGF in other Lyme arthritis patients.  The T-cell response was determined by measuring the amount of interferon-γ secreted by the patients' blood mononuclear cells upon exposure to ECGF in vitro.  In antibiotic-refractory patients, the T-cell response was observed in 38% (14/37) subjects against 30% (8/27) among Lyme arthritis patients who responded to antibiotics. The difference between the two groups was not statistically significant.  The B-cell (antibody) response was examined by ELISA in a larger group of patients.  17% (19/109) of antibiotic-refractory patients and 8% (6/77) of antibiotic-responsive patients had an IgG antibody response against ECGF that was higher than among healthy controls, yet the difference between the antibiotic-refractory and -responsive groups again was not statistically significant (P = 0.09).  So a link between an autoimmune response to ECGF and antibiotic-refractory arthritis was not clear-cut.  However, in support of a link, the authors mentioned that almost all of the Lyme arthritis patients with a T-cell response to ECGF (20/21, 98%) had one of the HLA-DR alleles known to be a risk factor for antibiotic-refractory arthritis.

The authors also looked at the levels of ECGF in the swollen joints of Lyme arthritis patients.  Those with antibiotic-refractory Lyme arthritis had much higher levels of ECGF in their joint fluid (mean 448 ng/ml, 37 subjects) than those whose arthritis responded to antibiotic treatment (mean 154 ng/ml, 19 subjects, P < 0.0001)

Further evidence for a link between an immune response to ECGF and chronic Lyme arthritis came from a group of untreated Lyme disease patients who were followed in the late 1970s, before the cause of Lyme disease was known.  Sera from sequential bleeds were still available from many of these patients.  If an autoimmune process involving ECGF was responsible for the disease, then the immune response to the autoantigen should have appeared before the disease symptoms.  This turned out to be the case.  Six of the seven Lyme arthritis patients who had antibodies against ECGF developed the antibody response before their joints swelled up.  The duration of the arthritis attack was longer in Lyme arthritis patients with an immune response to ECGF, lasting a median of 67 weeks in the seven patients with an ECGF antibody response and only 17 weeks in the 20 Lyme arthritis lacking the response (P = 0.004).

Steere's paper proposes that the immune response to ECGF leads to a persisting, autoimmune form of arthritis in those who have a high level of ECGF in their joint fluid.  In those patients, T cells that recognize ECGF would be activated more easily because of the high levels of ECGF available for phagocytes to engulf, process, and display to the T cells.  These events would lead to a chronic form of arthritis that would persist even when the spirochetes were cleared from the joints by the immune system or antibiotics.  These patients also have a lot of ECGF in their synovial tissue.  Antibody against ECGF could bind to the tissue and trigger attack by complement, contributing to the tissue damage.

Molecular mimicry doesn't appear to be involved in triggering an immune response to ECGF.  The authors were unable to identify any B. burgdorferi proteins that could cross-react with ECGF.

The immune response to ECGF can't be the whole story since most patients with antibiotic-refractory Lyme arthritis don't generate a T-cell or antibody response to the protein.  An autoimmune process in these other patients may involve other self-antigens waiting to be discovered.  Other host and spirochete factors also influence the course of Lyme arthritis (see this post, which gives the spirochete's point of view).


References

Drouin, E.E., Seward, R.J., Strle, K., McHugh, G., Katchar, K., Londoño, D., Yao, C., Costello, C.E., & Steere, A.C. (2013). A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease. Arthritis & Rheumatism, 65 (1), 186-196 DOI: 10.1002/art.37732

Seward, R.J., Drouin, E.E., Steere, A.C., & Costello, C.E. (2010). Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis. Molecular & Cellular Proteomics, 10 (3) DOI: 10.1074/mcp.M110.002477

A helpful review

Steere, A.C., Drouin, E.E., & Glickstein, L.J. (2011). Relationship between immunity to Borrelia burgdorferi Outer-surface protein A (OspA) and Lyme arthritis. Clinical Infectious Diseases, 52 (Supplement 3) DOI: 10.1093/cid/ciq117

Related post

• The genetics of both host and pathogen matter in antibiotic-refractory Lyme arthritis