Dual role of TLR8 during the engulfment of Lyme disease spirochetes by human monocytes

For the first time scientists have shown that Toll-like receptor 8 (TLR8), a microbial RNA sensor located inside phagocytes, detects what is primarily an extracellular pathogen, the Lyme disease spirochete Borrelia burgdorferi.¹  As one may expect, the phagocytes secreted a mixture of inflammatory cytokines in response to the spirochete.  But they also expressed at least one of the type I interferons (IFNs), which until recent years were thought to be produced only in response to viral and intracellular bacterial infections.²

The phagocytes used for the study were human monocytes, which are the more easily available bloodstream form of the macrophages found in our tissues.  Macrophages are designed to capture and engulf microbial pathogens invading our bodies.  While sopping up the invaders, the macrophages send out warning signals in the form of cytokines and other inflammatory molecules to alert nearby cells and to get the immune system to send more immune cells to help defend the tissue under attack.

Most of the macrophage's microbial sensors belong to a family of related membrane proteins called Toll-like receptors (TLRs).  TLR1, TLR2, TLR4, and TLR6 span the plasma membrane, whereas TLR3, TLR7, TLR8, and TLR9 are located in membrane structures inside the cell.  Each TLR recognizes a specific component of microbes.  For example, TLR4 latches onto LPS; TLR2 forms a complex with TLR1 or TLR6 to bind the lipidated amino terminus of lipoproteins; TLR7 and TLR8 recognize single-stranded RNA; and TLR9 recognizes DNA.  Engagement of a TLR by a microbial component triggers a signaling cascade within the cell leading to the transcription of genes encoding inflammatory cytokines, which are then secreted.  Stimulation of TLR3, TLR4, TLR7, TLR8, and TLR9 can also activate transcription of genes encoding type I IFNs.  The exact response of the macrophage depends on which TLRs are engaged by the pathogen.³

In the PNAS paper by Cervantes and colleagues, the authors searched for the sensors triggered by B. burgdorferi.¹ It had been known for a decade that one of the sensors of B. burgdorferi is TLR2, which recognizes the many lipoproteins that populate the surface of the spirochete.  However, TLR2 can't be the only B. burgdorferi sensor in macrophages because a more recent study showed that mouse macrophages missing its Tlr2 gene continued to produce inflammatory cytokines, albeit at lower levels, while engulfing B. burgdorferi.⁴ This same study also showed that B. burgdorferi stimulated human monocytes to transcribe genes encoding type I IFNs and a number of genes known to be induced by type I IFNs.

The investigators first examined the effects of blocking phagocytosis of B. burgdorferi by treating the monocytes with cytochalasin D, a chemical that blocks phagocytosis.  They found that cytochalasin D prevented transcription of the gene encoding the type I interferon IFN-β and reduced the amount of the inflammatory cytokine TNFα secreted from the monocytes.  The little bit of TNFα that continued to be produced was probably a consequence of TLR2 being stimulated by B. burgdorferi lipoproteins on the surface of the monocytes.

Since the spirochetes had to be engulfed to observe the monocyte's complete response, an intracellular sensor must participate in sensing B. burgdorferi.  The investigators therefore focused their attention on the intracellular nucleic acid sensors TLR7, TLR8, and TLR9.  Earlier studies by many other labs have shown that engagement of these intracellular TLRs activated production of type I IFNs, so it made sense to examine these TLRs.

To figure out which TLR functioned as the intracellular sensor of B. burgdorferi, the investigators used synthetic fragments of DNA that specifically blocked each TLR without interfering with phagocytosis.  When they applied the inhibitors individually to the monocytes, they found that only the TLR8 inhibitor blocked induction of the IFN-β gene by B. burgdorferi (see right half of panel A below).  A synthetic DNA fragment that does not inhibit any TLR failed to block induction of IFN-β transcription.  Therefore, TLR8 was implicated as being the intracellular sensor that detects B. burgdorferi.  As a control, LPS, which is sensed by TLR4 but not TLR8, continued to induce synthesis of the IFN-β transcript in the presence of the TLR8 inhibitor (left half of panel A).

B. burgdorferi was added at a 10:1 ratio (bacteria:monocytes).  Following the 4-hour incubation period, IFN-β transcript levels were measured by quantitative real-time RT-PCR.  ODN, a control synthetic oligodeoxynucleotide; N.S., not significant; *, P < 0.05; **, P < 0.01 (Mann-Whitney U test)

When the investigators looked at the inflammatory cytokines being produced by the TLR8-inhibited monocytes, they discovered that TLR8 had another role in the monocyte's response to B. burgdorferi.  Blocking TLR8 reduced (but did not eliminate) the secretion of the inflammatory cytokines TNFα, IL-6, IL-1β, and IL-10 (see panel B below).  These results indicated that TLR2 and TLR8 both had to send signals the nucleus to maximize the amount of cytokines produced during engulfment of the spirochete.  The authors also found by indirect immunofluorescence microscopy that TLR2 and TLR8 were together in the phagosomal membrane surrounding the engulfed B. burgdorferi, which they saw were being destroyed.

The amount of cytokines secreted by the human monocytes was measured following the 4-hour incubation period.  "Un," no spirochetes added; "Bb," B. burgdorferi added at a 10:1 ratio (bacteria:monocytes)

So here's what the authors believe is happening during the encounter of human monocytes with B. burgdorferi.  The monocyte first contacts the spirochete at the surface of the plasma membrane.  The lipoproteins on the surface of the spirochete activate the TLR2 sensor on the surface of the monocyte, but the low local concentration of TLR2 in the plasma membrane hampers its full signaling potential.  As the monocyte engulfs the spirochete by phagocytosis, TLR8 and TLR2 are recruited to the phagosomal membrane surrounding the spirochete, which at this point is being destroyed by antimicrobial substances being dumped into the phagosome.  Destruction of the spirochete releases its RNA to stimulate TLR8, while the crowding of TLR2 in the phagosomal membrane enhances the signaling stimulated by lipoproteins.  TLR8 and TLR2 work together to send a signal to the nucleus to activate transcription of numerous genes, including those encoding inflammatory cytokines.  Signaling from TLR8 by a separate pathway also stimulates transcription of type I interferons.

So is TLR8 relevant to Lyme disease?  The authors make the reasonable assertion that TLR8 activation benefits those infected with B. burgdorferi in part by turning on the type I IFN response.

Given that type I IFNs can shape a variety of downstream inflammatory responses through positive and/or negative regulation of hundreds of additional genes involved in secondary host defenses, TLR8 activation is likely to play a critical role in clearance of the spirochete and more importantly, disease control. [emphasis mine]

Unfortunately, the research literature tells us that type I IFNs have a dark side.  Although the beneficial role of type I IFNs in fighting off viral infections is well established, whether they help or hurt us during bacterial infections is not always obvious.  Type I IFNs are clearly essential in combating some bacterial infections such as lethal bloodstream infections caused by group B streptococci, Streptococcus pneumoniae, and E. coli.²  As for Lyme disease, type I IFNs may help kill spirochetes, but they also promote joint inflammation in infected mice.⁵  To determine whether TLR8 contributes to Lyme arthritis, scientists will need to perform B. burgdorferi infection studies with Tlr8-knockout mice.


Featured paper

1. Cervantes, J.L., Dunham-Ems, S.M., La Vake, C.J., Petzke, M.M., Sahay, B., Sellati, T.J., Radolf, J.D., & Salazar, J.C. (2011). Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-β Proceedings of the National Academy of Sciences, 108 (9), 3683-3688 DOI: 10.1073/pnas.1013776108


Key references

2. Mancuso G, Midiri A, Biondo C, Beninati C, Zummo S, Galbo R, Tomasello F, Gambuzza M, Macrì G, Ruggeri A, Leanderson T, & Teti G (2007). Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria. Journal of Immunology, 178 (5), 3126-3133 PMID: 17312160

3. Kawai, T., & Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors Nature Immunology, 11 (5), 373-384 DOI: 10.1038/ni.1863

4.Salazar, J.C., Duhnam-Ems, S., La Vake, C., Cruz, A.R., Moore, M.W., Caimano, M.J., Velez-Climent, L., Shupe, J., Krueger, W., & Radolf, J.D. (2009). Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-β PLoS Pathogens, 5 (5) DOI: 10.1371/journal.ppat.1000444

5. Miller JC, Ma Y, Bian J, Sheehan KC, Zachary JF, Weis JH, Schreiber RD, & Weis JJ (2008). A critical role for type I IFN in arthritis development following Borrelia burgdorferi infection of mice. Journal of Immunology, 181 (12), 8492-8503 PMID: 19050267


Related posts

• Antigen presentation in the bloodstream:  How invariant NKT cells are activated by Lyme disease spirochetes

Serologic testing for syphilis: missing the point

You may have seen several news sources touting the recent CDC finding that nearly one in five positive reactions with a newer syphilis test are wrong.  These headlines may grab the reader's attention, but the press took the finding out of context and failed to deliver the real message that the CDC was trying to convey.  Worse, the press reports may needlessly confuse and worry those who are being treated for syphilis.

Serological tests for syphilis are grouped into two categories.  Nontreponemal tests such as the VDRL and RPR are based on antibody generated against the lipid cardiolipin.  Presumably cardiolipin is released from damaged tissue in syphilis patients and gets incorporated into the membrane of Treponema pallidum.  The reason that these tests are "nontreponemal" is that antibodies to cardiolipin accompany many other conditions.  On the other hand, treponemal tests use T. pallidum proteins or even the entire spirochete as antigen to detect antibodies against the spirochete.  Although the classic treponemal tests such as the FTA-ABS (fluorescent treponemal antibody-absorption) and TP-PA (Treponema pallidum particle agglutination) are still used, the newer automated EIA (enzyme immunoassay) and CIA (immunochemiluminescence) treponemal tests enable clinical laboratories to rapidly screen a large number of sera.

The traditional approach to syphilis testing is to first screen the patient's serum with a nontreponemal test.  Since nontreponemal tests can give false positive reactions, reactive sera are retested with one of the treponemal tests.  However, the low cost of executing the automated treponemal tests have led some high-volume clinical laboratories to reverse the order of the assays:  they screen with the EIA/CIA treponemal test and confirm positive results with a nontreponemal test.  The CDC report in the Morbidity and Mortality Weekly Report deals with this so-called "reverse sequence" testing.

So where did the "nearly one in five" figure come from?  From 2006 to 2010, five large clinical laboratories screened 140,176 sera specimens with the reverse sequence procedure.  Of the 4,834 reactive with the EIA/CIA treponemal test, 2,743 gave negative results with the nontreponemal RPR test.  When the samples that gave discrepant results were tested further with one of the classic treponemal tests, 866 of the 2,743 samples were negative.  Overall, among the 4,834 samples that were reactive with the newer treponemal test, 866 or 18% were nonreactive with two subsequent tests.  These 866 were assumed to be false positives.

The news media pounced on the 18% figure and declared that hundreds may have been given antibiotics to treat a disease that they didn't have.  But they ignored the fact that doctors don't diagnose syphilis on the basis of a single lab test.  It is standard practice to perform a second test when the first comes back positive and to do even a third one if warranted.  Doctors also take into account the physical exam and the sexual and medical history of the patient before making the decision to treat with antibiotics.

Here's how the CDC responded to the assertion that those among the 18% may have been falsely diagnosed and treated unnecessarily with antibiotics:

There are two problems with this assertion. First, the current report does not document whether or not treatment was provided. Second, in those cases where treatment was provided, it may have been justified based on sexual risk and findings on clinical evaluation. It is also important to note that syphilis is not diagnosed on the basis of a single blood test. Many labs routinely will do additional testing when the first test is positive, without notifying the patient. Doctors diagnose syphilis after considering at least two syphilis tests, the patient's history, the physical exam, and a review of past syphilis test results. The MMMR analysis, while important, does not allow us to conclude that the newer tests led to inaccurate syphilis diagnosis or inappropriate treatment.

So what was the message that the CDC was trying to communicate to readers of the MMMR report?  Their intention was to provide guidance in the management of cases for which the reverse sequence screening is performed instead of the traditional sequence, which is still recommended by the CDC.  Specifically, when conflicting results occur (positive with the treponemal test, negative with the nontreponemal test), a third test should be done with the TP-PA.  (The CDC does not recommend the FTA-ABS because it is less specific and probably less sensitive.)  A positive reaction with the TP-PA indicates past or present syphilis; a negative reaction indicates that syphilis is unlikely.  As always, the clinical observations and medical history of the patient should also be considered in making an informed treatment decision.


Reference

Centers for Disease Control and Prevention (February 11, 2011).  Discordant results from reverse sequence syphilis screening -- five laboratories, United States, 2006-2010.  MMMR. Morbidity and Mortality Weekly Report 60(5):133-137.  link

Another reason not to own a pet rat

A pair of European doctors wrote about the trials of a 37-year-old man who came down with Weil's disease, a severe form of leptospirosis.  The case report appeared in the recent issue of The Lancet Infectious Diseases.

The individual showed up in the emergency room complaining of a sudden fever, muscle pain, and a severe headache.  Notable were the signs of jaundice and conjunctival suffusion (reddening of the eye), which suggested leptospirosis.

Figure from Jansen and Schneider, 2011

Shortly after he was admitted, the patient had to be moved to the intensive care unit because his kidneys were failing.  Blood tests for Leptospira antibodies came back negative, most likely because it was too early in the infection for antibodies to appear.

An infectious diseases specialist noted that the patient suffered from the classic signs of Weil's disease:  kidney failure, jaundice, and an enlarged spleen.  Accordingly, the patient was started on intravenous penicillin, the favored treatment for severe leptospirosis.  Further blood tests conducted during the second week of treatment finally revealed antibodies to Leptospira.  The patient completely healed within several weeks.  The only question that remained is how the patient got infected with Leptospira.

At some point the patient admitted that he owned four pet rats.  Since rats can silently carry Leptospira in their kidneys, his pets were sacrificed to check their kidneys for the presence of the spirochete.  All four kidneys turned out to be culture positive for Leptospira interrogans, serovar Icterohaemorrhagiae or Copenhageni (the typing test couldn't distinguish the two serovars).  The patient's antibodies reacted against the same serovars, suggesting that the source of the infection was tainted urine from his pet rats.

Although cases of leptospirosis acquired from pet rats appear to be rare, anyone who wishes to own a rat should be aware that rats can be carriers of a potentially deadly spirochete.  This is yet another reason why rats should be obtained from a responsible breeder.

For another case, check out this post from the Worms & Germs Blog (make sure you read the comments for the complete story).

Reference

Jansen, A. and Schneider, T. (February 2011).  Weil's disease in a rat owner.  The Lancet Infectious Diseases 11(2):152.  DOI: 10.1016/S1473-3099(10)70106-7

The quest for outer membrane proteins of the stealth pathogen Treponema pallidum: the cliffhanger episode

Syphilis patients are able to generate antibodies against the spirochete Treponema pallidum.  However, if you were to mix sera from these patients with T. pallidum in a test tube, very few of the antibodies in the sera would bind to the spirochetes.  The reason is that the strange outer membrane architecture of T. pallidum makes the spirochete invisible to the antibodies.  Most of the proteins and lipid molecules targeted by the antibodies lie beneath the outer membrane.

The outer membrane of T. pallidum differs considerably from that of a typical Gram-negative bacterium.  The most glaring difference is that T. pallidum lacks lipopolysaccharide (LPS), a favorite target of the immune response.  The outer membrane is also bare of other potential surface antigens except for a very small number of transmembrane outer membrane proteins (Omps) and (possibly) surface lipoproteins.  The poor surface antigencity may help the so-called "stealth pathogen" persist in the body despite a robust immune response to the infection.

The few Omps displayed on the surface of T. pallidum must be doing something really important if the spirochete is willing to risk exposing them to attack by antibodies.  For this reason, scientists have been seeking the identity of these Omps to figure out what they do.  These rare Omps could also be fashioned into a long-desired syphilis vaccine.

Unfortunately, the scarcity of Omps and the delicate nature of the outer membrane of T. pallidum have stymied efforts to identify Omps.  The routine centrifugation and washing steps used to prepare other bacteria for analysis easily damage the outer membrane of T. pallidum, causing the loss of Omps and exposing the abundant periplasmic and inner membrane proteins.  Consequently, probes used to identify exposed proteins may react with the periplasmic and inner membrane proteins, which are normally shielded by the outer membrane.  Without the proper controls, this would lead one to conclude wrongly that a non-Omp that reacts with the probe (such as antibodies raised against the protein of interest) is surface exposed.  In addition, when the outer membrane is purified with the intention to identifying Omps, it is hard to distinguish the tiny amounts of Omps from proteins from other bacterial compartments contaminanting the outer membrane preparation.

Despite these technical challenges, several Omp candidates have been proposed, but those proteins are either mired in controversy (TprK, for example) or await experimental confirmation of their surface exposure.  To date, no protein that has been demonstrated unambiguously to be displayed on the exterior of T. pallidum.

The past decade has seen the development of several computer programs that can be used to predict whether a given gene encodes a transmembrane Omp.  The algorithms differ, but all of these programs attempt to identify amino acid sequences that fold into a β-barrel, which forms the core of transmembrane Omps whose 3D structures are known.  The β-barrel forms when an anti-parallel β-sheet rolls into the shape of a barrel.  Most of the β-barrel is embedded in the outer membrane so that the loops connecting the β-strands stick out from the two surfaces of the membrane.  The loops displayed on the external face of the outer membrane would be accessible to antibodies.  The size and composition of the loops vary among different Omps, but all β-strands tend to have alternating hydrophobic amino acids with their nonpolar side chains acid protruding out from the barrel into the hydrophobic interior of the lipid bilayer.  Each β-strand consists of 9-11 amino acid residues and is tilted up to 45° out of the transmembrane axis.  Different β-barrels have as few as 8 and as many as 22 transmembrane β-strands.

The ribbon representation of OmpA, an 8-stranded transmembrane Omps from E. coli, is shown below as one example.

From Figure 1b of Smith et al., 2007.  The N- and C-terminal β-strands are colored brown and blue, respectively.  The side chains of the "aromatic girdle" are shown.

Below, OmpA is unfurled to show the topology of the protein:

From Figure 1a of Smith et al., 2007.  β-strand amino acid residues are depicted as diamonds, and loop residues are depicted as circles.  Alternating hydrophobic amino acid residues within the  β-strand are colored red (aromatic residues of the girdle) and yellow.

Assuming that the rare transmembrane Omps of T. pallidum share the β-barrel structure, the obvious computational approach to finding these Omps would be to run all of the proteins encoded by the T. pallidum genome through one of these programs.  One problem with these programs is that they will pick up a few proteins that are not truly transmembrane Omps.  To minimize this problem, Justin Radolf's group, as reported in the December 2010 issue of Infection and Immunity, ran the 1038 protein-coding sequences of T. pallidum through seven different Omp-predicting programs.  They found that two proteins were predicted by all seven programs to have the β-barrel structure; another four candidates were identified by six programs.

One of the proteins at the top of the list, identified by all seven programs, was TP0326, a BamA homolog encoded by the genomes of many Gram-negative bacteria.  Experiments with other bacteria have shown that BamA is a member of an outer membrane protein complex that assembles other transmembrane Omps into the outer membrane, so it would make sense for T. pallidum to possess such a protein.  BamA itself is thought to be a transmembrane Omp. 

This wasn't the first time that a syphilis researcher has encountered TP0326.  In a study published 11 years ago, before the function of BamA was known, Caroline Cameron and colleagues demonstrated that antibodies raised against TP0326 (also called "Tp92" in their paper) stimulated macrophages to engulf T. pallidum in a process called opsonophagocytosis.  In addition, TP0326 was somewhat effective as a vaccine in the rabbit model of syphilis:  rabbits that had been immunized with TP0326 experienced milder skin lesions than unimmunized rabbits following inoculation of T. pallidum into the skin.  These observations indirectly supported the localization of TP0326 to the outer membrane since opsonophagocytosis and effective vaccination require a target that is accessible on the surface of the spirochete.  However, this earlier work lacked a more direct test such as the indirect immunofluorescence assay to confirm that TP0326 was exposed on the surface.

The problem with the standard two-step indirect immunofluorescence assay is that it is not sensitive enough to detect the rare Omps of T. pallidum.  Therefore, as described in the Infection and Immunity paper, Radolf's group tinkered with the assay and managed to amplify the output signal by adding a third step to the procedure.  To minimize damage to the outer membrane during the centrifugation and washing steps, the spirochetes were encased in gel microdroplets, which protected the delicate outer membrane while allowing antibodies to permeate to probe the T. pallidum surface.

With the modified immunofluorescence assay, the investigators were able to detect surface proteins with syphilitic antibodies for the first time, although only in a small minority of the spirochetes in the field of view lit up with the red color (see figure below).  Presumably, the other spirochetes failed to react with the antibodies because they didn't quite have enough Omp antigens being expressed on their surface (although a more interesting explanation would be that the nonreactive spirochetes had down-regulated their surface Omps).  Regardless of the true explanation, these results indicated that at least some of the antibodies generated by syphilis patients were directed against surface components of T. pallidum.  When the investigators treated the spirochetes with the detergent Triton X100 to intentionally damage the outer membrane, all of the spirochetes glowed, indicating that most of the antibodies targeted proteins beneath the surface of T. pallidum.  As a negative control, they demonstrated that sera from healthy patients failed to react with intact spirochetes.

To keep track of how many spirochetes were damaged by the procedure, the investigators added antibody raised against the periplasmic flagella along with the patient antibodies.  The flagellar antibodies would bind to the spirochetes only if the integrity of the outer membrane was compromised by handling the spirochetes.  The assay was designed so that bound flagellar antibodies would glow green.

From Figure 4 of Cox et al., 2010.   (A) All spirochetes, whether or not they fluoresced, could be seen with darkfield optics (DF).  Spirochetes that bound to antibodies from syphilis patients (HSS) glowed red.   Spirochetes with a disrupted outer membrane reacted with the flagellar antibody (anti-FlaA) and glowed green.  (B) 5.8% of the spirochetes observed were undamaged and reacted with patient antibodies (glowed red but not green).  Another 5.1% were damaged (glowed red and green).  89.0% of the spirochetes failed react with the patient antibodies.  100% of the spirochetes fluoresced when treated with the detergent Triton X100 before adding the antibodies.

With an improved immunofluorescence assay, the investigators were poised to test the proteins at the top of the list for surface exposure.  As I was nearing the end of the paper, I was expecting the authors to describe their test of the BamA homolog TP0326 for surface exposure.  Surprisingly, they ended the paper without testing any of the proteins near the top of the list.

I can only assume that the authors are planning to submit a separate manuscript in the future describing the successful detection of TP0326 or another protein near the top of the list.  But the problem with ending the paper without demonstrating surface localization of even a single protein is that one can question whether even the 3-step immunofluorescence assay is sensitive enough to detect an Omp exposed on the T. pallidum surface.  They did test two proteins lower down on the list that other labs believe are surface exposed (TprK and a fibronectin-binding lipoprotein)  but neither protein was detected on the outer membrane surface by the modified immunofluorescence assay.  So we are left with an assay that certainly has more sensitivity, but is it sensitive enough?

To be continued...(?)

Featured paper

Cox, D.L., Luthra, A., Dunham-Ems, S., Desrosiers, D.C., Salazar, J.C., Caimano, M.J.., and Radolf, J.D. (December 2010).  Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum.  Infection and Immunity 78(12):5178-5194.  DOI: 10.1128/IAI.00834-10

Other references

Radolf, J.D. (June 1995).  Treponema pallidum and the quest for outer membrane proteins.  Molecular Microbiology 16(6):1067-1073.

Cameron, C.E., Lukehart, S.A., Castro, C., Molini, B., Godornes, C., and Van Voorhis, W.C. (April 2000).  Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92.  Journal of Infectious Diseases 181(4):1401-1413.  DOI: 10.1086/315399

Cox, D.L., Akins, D.R., Porcella, S.F., Norgard, M.V., and Radolf, J.D. (1995).  Treponema pallidum in gel microdroplets:  a novel strategy for investigation of treponemal molecular architecture.  Molecular Microbiology 15(6):1151-1164.

Image source

Smith, S.G.J, Mahon, V., Lambert, M.A., and Fagan, R.P. (August 2007).  A molecular Swiss army knife:  OmpA structure, function and expression.  FEMS Microbiology Letters 273(1):1-11.  DOI: 10.1111/j.1574-6968.2007.00778.x

Designing a Lyme disease vaccine to attack the tick vector

Conventional vaccines target the surface components or secreted toxins of pathogens.  Erol Fikrig's group at Yale University has been exploring an unconventional approach towards developing a vaccine for Lyme disease, which is caused by a tick-borne pathogen.  Their recent work, published in the November issue of PLoS Pathogens, demonstrated partial success in protecting laboratory mice by immunization with a protein found in the saliva of the Ixodes tick vector.

Ixodes ticks spend several days feeding on blood while attached to the victim's skin.  B. burgdorferi is carried into the victim's skin in the Ixodes tick's saliva starting 3-4 days (on average) after attachment.  Tick saliva contains a blend of biological substances that aid the tick in drinking blood from its victim.  These substances include cement proteins to keep the tick's feeding apparatus tightly bound to the skin, anti-coagulants to keep the blood flowing into the tick, and anti-inflammatory factors that ward off the local inflammatory response.  The activity of these substances also promote transmission of B. burgdorferi from the feeding tick to the victim.  Hence a vaccine that targets a saliva component may protect humans from Lyme disease.

A Lyme vaccine that targets the tick has a few advantages over one that targets the spirochete.  First, a tick-based Lyme disease vaccine is unlikely to interfere with laboratory diagnosis, which currently relies on detection of antibodies against the Lyme Borrelia spirochete.  Second, an effective vaccine that targets the tick may also prevent transmission of other pathogens carried by the Ixodes tick by interfering with tick feeding or with the tick's countermeasures against the host inflammatory response at the feeding site.

In their recent work, the investigators focused their efforts on a salivary protein called tick histamine release factor (tHRF).  Because tHRF levels in the salivary glands of feeding Ixodes ticks were higher when B. burgdorferi was present in the tick, they guessed that tHRF was doing something to help transmit B. burgdorferi from the tick to the victim.  The authors turned out to be correct.  Transmission of B. burgdorferi was impaired when they knocked down the tick's production of tHRF by RNAi.

The investigators went on to test the vaccine potential of tHRF in their mouse model.  They passively immunized mice with antiserum raised against tHRF or actively immunized the rodents with recombinant tHRF.  Actively immunized mice were also given booster injections with tHRF (the paper did not say how many).  Control mice were not immunized.  They then challenged the mice with ticks infected with B. burgdorferi.  One or three weeks later, tissues were removed from the mice, and the bacterial load of B. burgdorferi in skin, heart, and joints was measured by quantitative PCR.

Their data showed that immunization with tHRF was somewhat effective.  Depending on the experiment, B. burgdorferi DNA could not be detected in any of the three tissues in 20-33% of immunized mice, whereas the spirochete's DNA was detected in at least one tissue in all control mice.  Even in immunized mice with detectable B. burgdorferi DNA, the levels were often lower than the average level found in the control mice.  It would have been nice to know how much inflammation was present in the tissues of the immunized mice.  Unfortunately, the histopathology of the tissues was not presented in the paper.

Figure 4, panels E-G from Dai et al., 2010.  Bacterial burden in skin (day 7 after challenge) and joint and heart (day 21) was determined by quantitative PCR with flaB primers.  Horizonal lines represent the mean value ± SEM.  * p < 0.05 and ** p < 0.01.  Results were pooled from 3 independent experiments.

tHRF is not the first Lyme vaccine candidate to target a protein found in tick saliva.  An earlier report from Fikrig's group demonstrated that active and passive immunization with Salp15, another tick salivary protein, was also somewhat effective in protecting mice from colonization with B. burgdorferi.  tHRF was superior to Salp15 in impairing feeding by ticks. Ticks feeding on Salp15-immunized mice were able to complete their blood meal.  In contrast, most of the ticks had a hard time feeding on mice immunized with tHRF and could not complete their blood meal, as assessed by tick weights following detachment from the mice.

Although immunization with tHRF and Salp15 prevented colonization in only some mice, Fikrig's work shows for the first time that it may be possible to design a Lyme disease vaccine that targets the tick vector.  Ultimately, the most effective vaccine may be a mixture that targets multiple components in both the tick and the spirochete.

References

Dai, J., Narasimhan, S., Zhang, L., Liu, L., Wang, P., & Fikrig, E. (2010). Tick histamine release factor is critical for Ixodes scapularis engorgement and transmission of the Lyme disease agent PLoS Pathogens, 6 (11) DOI: 10.1371/journal.ppat.1001205

Dai, J., Wang, P., Adusumilli, S., Booth, C.J., Narasimhan, S., Anguita, J., & Fikrig, E. (2009). Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent. Cell Host & Microbe, 6 (5), 482-492 DOI: 10.1016/j.chom.2009.10.006

Related post

• A fresh approach towards a Lyme disease vaccine: targeting the tick