Monday, July 4, 2011
Does Borrelia burgdorferi cause an inadequate antibody response by altering B cell activation in the lymph node?
One of the characteristic features of Lyme disease is lymphadenopathy or swollen lymph nodes. It's not too surprising when a lymph node draining a site of infection swells. However when investigators looked at the lymph node draining the inoculation site of Borrelia burgdorferi in mice, they found that the spirochete had somehow altered the course of activation of B cells producing the antibodies that targeted the spirochete.
Before discussing the findings in the paper, a review of how the antibody response evolves in the lymph node is in order. An antibody response to microbial proteins is sparked when antigen from microbes breaching the skin layer flow into the draining lymph node or are carried to the lymph node by dendritic cells. Lymph nodes are where naive B cells, upon recognition of antigen, differentiate into plasma cells, which secrete large amounts of antibody that target the invading microbe. The antibodies which bind most tightly to protein antigens are made with T cell help in germinal centers, which emerge from the rare B cells in the lymph node that produce antibody capable of recognizing the antigen. (I say "rare" here because each B cell in the lymph node produces antibodies with different antigenic specificities to ensure that any microbe that the host may possibly encounter will be recognized by antibodies displayed by at least a few B cells.) Upon binding the antigen and reception of critical signals from T cells, the B cells migrate to areas in the lymph node containing fixed networks of follicular dendritic cells (FDCs), a type of immune cell with long branched processes that extend out from the body of the cell. (FDCs differ from the dendritic cells that bring antigen to the lymph node.) The B cells then start to proliferate wildly, doubling every 6 to 8 hours (faster than B. burgdorferi!). As the B cell numbers surge, they form germinal centers, which can be identified easily by standard histological stains (see image below). The lymph node may even swell, depending on how much the B cells proliferate.
As the B cells multiply in the germinal center, a process called somatic hypermutation, which is promoted by signals received from T cells, causes a large number of mistakes to be made within the segment of DNA encoding the antigen binding portion of the antibody. Consequently, the antibodies displayed by some germinal center B cells are no longer able to bind to the microbial antigen whereas those made by other B cells will bind better. The FDC processes, whose surfaces are loaded with antigen, continuously probe the antibodies expressed by the newly arising B cells. Since the new B cells are programmed to die unless they express antibody able to bind the antigen displayed by the FDCs, only B cells displaying antibody that bind most tightly to the antigen will survive. This process by which B cells expressing the antibodies with the highest affinity for antigen are selected is called affinity maturation. Somatic hypermutation and affinity maturation can only occur in germinal centers. The B cells also undergo class switching, in which the class of antibody expressed by the B cells switches from IgM and IgD, which are expressed by naive B cells, to IgA, IgE, or one of the IgG subclasses. The exact switch that occurs is governed by the cytokines that the B cells are exposed to. Which cytokines are present depends on the nature of the infection. Eventually the B cells expressing high-affinity antibodies of the appropriate class differentiate into antibody-secreting plasma cells, which are released from the lymph node to circulate throughout the body and fight the infection.
So what happens in the lymph nodes during a B. burgdorferi infection? When the investigators inoculated B. burgdorferi into or underneath the skin of mice, the lymph node draining the site swelled considerably, enlarging by more than a factor of 10 by the tenth day of infection.
What the investigators saw when they looked at lymph node sections under the microscope was very different from the textbook description of T-cell dependent B cell activation that I gave above. First of all, live B. burgdorferi was found in the lymph node draining the site of infection in mice. This is unusual since phagocytes would normally greet and destroy any microbe that managed to find its way into the lymph node.
Second, massive proliferation of B cells accounted for lymph node swelling, but the expansion of B cell numbers wasn't occurring in well-defined germinal centers. The authors also noted that T cells were not increasing in number. These observations suggested that T cells, which are required for germinal centers to form, were not fully participating in B-cell activation. The lack of germinal centers suggested that somatic hypermutation and affinity maturation were not occurring.
From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality. By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed. The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3. Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally. Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies. If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host.
Featured paper
Tunev SS, Hastey CJ, Hodzic E, Feng S, Barthold SW, and Baumgarth N (May 2011). Lymphadenopathy during Lyme borreliosis is caused by spirochete migration-induced specific B cell activation. PLoS Pathogens 7(5):e1002066. DOI: 10.1371/journal.ppat.1002066
Before discussing the findings in the paper, a review of how the antibody response evolves in the lymph node is in order. An antibody response to microbial proteins is sparked when antigen from microbes breaching the skin layer flow into the draining lymph node or are carried to the lymph node by dendritic cells. Lymph nodes are where naive B cells, upon recognition of antigen, differentiate into plasma cells, which secrete large amounts of antibody that target the invading microbe. The antibodies which bind most tightly to protein antigens are made with T cell help in germinal centers, which emerge from the rare B cells in the lymph node that produce antibody capable of recognizing the antigen. (I say "rare" here because each B cell in the lymph node produces antibodies with different antigenic specificities to ensure that any microbe that the host may possibly encounter will be recognized by antibodies displayed by at least a few B cells.) Upon binding the antigen and reception of critical signals from T cells, the B cells migrate to areas in the lymph node containing fixed networks of follicular dendritic cells (FDCs), a type of immune cell with long branched processes that extend out from the body of the cell. (FDCs differ from the dendritic cells that bring antigen to the lymph node.) The B cells then start to proliferate wildly, doubling every 6 to 8 hours (faster than B. burgdorferi!). As the B cell numbers surge, they form germinal centers, which can be identified easily by standard histological stains (see image below). The lymph node may even swell, depending on how much the B cells proliferate.
 |
| Lymph node: (1) capsule; (2) subcapsular sinus; (3) germinal centers; (4) lymphoide nodule; (5) trabeculae. Source |
As the B cells multiply in the germinal center, a process called somatic hypermutation, which is promoted by signals received from T cells, causes a large number of mistakes to be made within the segment of DNA encoding the antigen binding portion of the antibody. Consequently, the antibodies displayed by some germinal center B cells are no longer able to bind to the microbial antigen whereas those made by other B cells will bind better. The FDC processes, whose surfaces are loaded with antigen, continuously probe the antibodies expressed by the newly arising B cells. Since the new B cells are programmed to die unless they express antibody able to bind the antigen displayed by the FDCs, only B cells displaying antibody that bind most tightly to the antigen will survive. This process by which B cells expressing the antibodies with the highest affinity for antigen are selected is called affinity maturation. Somatic hypermutation and affinity maturation can only occur in germinal centers. The B cells also undergo class switching, in which the class of antibody expressed by the B cells switches from IgM and IgD, which are expressed by naive B cells, to IgA, IgE, or one of the IgG subclasses. The exact switch that occurs is governed by the cytokines that the B cells are exposed to. Which cytokines are present depends on the nature of the infection. Eventually the B cells expressing high-affinity antibodies of the appropriate class differentiate into antibody-secreting plasma cells, which are released from the lymph node to circulate throughout the body and fight the infection.
So what happens in the lymph nodes during a B. burgdorferi infection? When the investigators inoculated B. burgdorferi into or underneath the skin of mice, the lymph node draining the site swelled considerably, enlarging by more than a factor of 10 by the tenth day of infection.
 |
| From Fig. 2 of Tunev et al., 2011. Arrow points to lymph node draining the inoculation site. Source |
What the investigators saw when they looked at lymph node sections under the microscope was very different from the textbook description of T-cell dependent B cell activation that I gave above. First of all, live B. burgdorferi was found in the lymph node draining the site of infection in mice. This is unusual since phagocytes would normally greet and destroy any microbe that managed to find its way into the lymph node.
 |
| From Fig. 3 of Tunev et al., 2011. Day 8 of infection. The arrows point to intact extracellular B. burgdorferi in the subcapsular sinus of the lymph node, which was culture positive beginning on day 1 of infection. Source |
Second, massive proliferation of B cells accounted for lymph node swelling, but the expansion of B cell numbers wasn't occurring in well-defined germinal centers. The authors also noted that T cells were not increasing in number. These observations suggested that T cells, which are required for germinal centers to form, were not fully participating in B-cell activation. The lack of germinal centers suggested that somatic hypermutation and affinity maturation were not occurring.
From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality. By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed. The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3. Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally. Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies. If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host.
Featured paper
Tunev SS, Hastey CJ, Hodzic E, Feng S, Barthold SW, and Baumgarth N (May 2011). Lymphadenopathy during Lyme borreliosis is caused by spirochete migration-induced specific B cell activation. PLoS Pathogens 7(5):e1002066. DOI: 10.1371/journal.ppat.1002066
Saturday, June 4, 2011
Membrane fusion between Borrelia spirochetes, a new type of bacterial interaction
It's not unusual for bacteria to collide while swimming around in culture medium. When this happens, the bacteria simply bounce off each other and swim off in different directions. However scientists have discovered that the encounter between Borrelia spirochetes, the agents of Lyme disease and relapsing fever, can progress to something more intimate.1 When they looked at Borrelia cultures under the microscope, the spirochetes that bumped remained stuck to each other side-by-side as they swam. These encounters were usually brief, lasting for less than 10 seconds, although sometimes they lasted for more than a minute before separating. If you watch the video below, you'll see two Lyme disease B. afzelii cells near the bottom left corner coming together side-by-side and then separating several seconds later. The investigators saw similar interactions in cultures of other Lyme disease spirochetes (B. burgdorferi and B. garinii) and a relapsing fever spirochete (B. hermsii).
When I first saw the video, I thought that the spirochetes were simply getting tangled up and that it took several seconds for them to get untangled. However when the investigators examined the cultures by cryoelectron tomography (a type of electron microscopy), they saw that the Borrelia cells weren't merely tangled or stuck to each other. Their outer membranes were actually fused, sometimes so extensively that both cytoplasmic cylinders ended up in a single outer membrane sheath. Panel A below shows a cross-section of fused B. garinii cells. Panel B shows a 3-dimensional rendering of the the fused spirochetes from panel A. The two cytoplasmic cylinders (bright and dark magenta) are surrounded by a single outer membrane sheath. The flagellar filaments from both cells form a single bundle and are shown in yellow.
Artifacts are minimized in specimens observed by cryoelectron tomography because the specimen does not have to be fixed with harsh chemicals. Instead, the live specimen is placed on an electron microscope grid and plunge-frozen to preserve the structure of the biological sample. Still, as the authors point out, fusion of Borrelia cells could be an artifact of preparing the spirochetes for cryoelectron tomography. The outer membrane of Borrelia cells could have fused while collecting the spirochetes by centrifugation or when blotting excess liquid from the electron microscopy grid before freezing the specimen.
Assuming that this was not a preparation artifact, what could be the role of outer membrane fusion in the biology of Borrelia? The authors present two possibilities. First, Borrelia cells can share their outer membrane contents by fusing their outer membranes together. Sharing may be advantageous to Lyme Borrelia during transmission from the tick to the victim's skin, when the spirochetes are turning on genes encoding protective proteins such as OspC. One can imagine Borrelia cells sharing its protective surface proteins with others that have yet to express them so that a larger number of spirochetes can fend off host defenses and establish an infection. Another intriguing possibility is that DNA is exchanged between the two spirochetes. Out of the 110 pairs of fused Borrelia cells observed by cryoelectron tomography, the investigators found one pair whose inner membranes were also fused, providing a conduit (at least theoretically) for transfer of DNA. Cells in culture may not remain fused long enough to transfer DNA, but Lyme disease Borrelia lie dormant in the tick midgut for months, giving Borrelia cells lying next to each other plenty of time to exchange DNA, assuming that membrane fusion and DNA transfer can even occur in this setting.
The outer membrane of spirochetes is unique among diderm (double-membrane) bacteria because of its loose association with the underlying peptidoglycan layer. For this reason the outer membrane of all spirochetes, not just those of Borrelia, may be especially prone to fusing. This raises the possibility that the outer membranes of other spirochetes such as Leptospira and Treponema could also fuse.
Note: This work has also been described in the blog Small Things Considered.
Reference
1. Kudryashev M., Cyrklaff M., Alex B., Lemgruber L, Baumeister W, Wallich R, and Frischknecht F (May 2011). Evidence of direct cell-cell fusion in Borrelia by cryogenic electron tomography. Cellular Microbiology 13(5):731-741. DOI: 10.1111/j.1462-5822.2011.01571.x
Movie S2 from Kudryashev et al., 2011
When I first saw the video, I thought that the spirochetes were simply getting tangled up and that it took several seconds for them to get untangled. However when the investigators examined the cultures by cryoelectron tomography (a type of electron microscopy), they saw that the Borrelia cells weren't merely tangled or stuck to each other. Their outer membranes were actually fused, sometimes so extensively that both cytoplasmic cylinders ended up in a single outer membrane sheath. Panel A below shows a cross-section of fused B. garinii cells. Panel B shows a 3-dimensional rendering of the the fused spirochetes from panel A. The two cytoplasmic cylinders (bright and dark magenta) are surrounded by a single outer membrane sheath. The flagellar filaments from both cells form a single bundle and are shown in yellow.
 |
| Figure 4A and 4B from Kudryashev et al., 2011 |
Assuming that this was not a preparation artifact, what could be the role of outer membrane fusion in the biology of Borrelia? The authors present two possibilities. First, Borrelia cells can share their outer membrane contents by fusing their outer membranes together. Sharing may be advantageous to Lyme Borrelia during transmission from the tick to the victim's skin, when the spirochetes are turning on genes encoding protective proteins such as OspC. One can imagine Borrelia cells sharing its protective surface proteins with others that have yet to express them so that a larger number of spirochetes can fend off host defenses and establish an infection. Another intriguing possibility is that DNA is exchanged between the two spirochetes. Out of the 110 pairs of fused Borrelia cells observed by cryoelectron tomography, the investigators found one pair whose inner membranes were also fused, providing a conduit (at least theoretically) for transfer of DNA. Cells in culture may not remain fused long enough to transfer DNA, but Lyme disease Borrelia lie dormant in the tick midgut for months, giving Borrelia cells lying next to each other plenty of time to exchange DNA, assuming that membrane fusion and DNA transfer can even occur in this setting.
The outer membrane of spirochetes is unique among diderm (double-membrane) bacteria because of its loose association with the underlying peptidoglycan layer. For this reason the outer membrane of all spirochetes, not just those of Borrelia, may be especially prone to fusing. This raises the possibility that the outer membranes of other spirochetes such as Leptospira and Treponema could also fuse.
Note: This work has also been described in the blog Small Things Considered.
Reference
1. Kudryashev M., Cyrklaff M., Alex B., Lemgruber L, Baumeister W, Wallich R, and Frischknecht F (May 2011). Evidence of direct cell-cell fusion in Borrelia by cryogenic electron tomography. Cellular Microbiology 13(5):731-741. DOI: 10.1111/j.1462-5822.2011.01571.x
Sunday, May 1, 2011
A new attenuated leptospirosis vaccine protects hamsters from lethal infection by more than one serovar of Leptospira
Scientists have demonstrated that a new attenuated leptospirosis vaccine protects laboratory hamsters from being killed by Leptospira, even when the challenge and vaccine strains belong to different serovars (immune types).1,2 This is the first leptospirosis vaccine to confer complete cross-protection against lethal infection by a serovar different from the one used for immunization.
The leptospirosis vaccines that are out on the market are still formulated with killed Leptospira or sometimes their outer membrane. These traditional vaccines are administered primarily to dogs, cattle, and pigs. Human leptospirosis vaccines are not available in most countries, even in areas where leptospirosis is endemic.
New types of leptospirosis vaccines are needed since the traditional killed vaccines are flawed. One problem is that immunity is serovar specific. For this reason a vaccine must contain all the serovars that the target population may encounter. Even when the vaccine manufacturers figure out which serovars are circulating, a new serovar may emerge, rendering the vaccine ineffective as the new serovar spreads through the susceptible population. The vaccine must then be reformulated at substantial cost.
This is exactly what happened to the leptospirosis vaccines that are given to dogs.3 The early canine vaccines, first available in the 1970s, contained the serovars Canicola and Icterohemorrhagiae. These vaccines worked fine until the late 1980s or so, when new serovars started to appear in infected dogs, even in those that had been vaccinated. Since then vaccine makers have added the serovars Grippotyphosa and Pomona to their vaccines. Nevertheless with over 200 pathogenic serovars of Leptospira lurking out there, we don't know when or which additional serovars will emerge in the future.
It would be nice to have a single leptospirosis vaccine formulation that would protect against all serovars. The protective effect of traditional vaccines is due to antibodies generated against lipopolysaccharide (LPS), whose structure differs among the serovars of Leptospira. Since immunization elicits antibodies that recognize the LPS of only the serovars included in the vaccine, vaccinated individuals remain susceptible to infection by other serovars.
To get around this problem, scientists have been testing individual Leptospira surface proteins as potential vaccines in rodent models of leptospirosis. Leptospira surface proteins tend to be antigenically conserved among the different serovars: antibodies generated against a protein from one serovar often reacts against the same protein expressed by other serovars. According to many studies the LipL32 and Lig surface lipoproteins, when delivered as recombinant proteins, naked DNA, or by microbial vectors (adenovirus and Mycobacterium bovis), apparently protected hamsters or guinea pigs from lethal infection by Leptospira. Unfortunately one of the leaders in the leptospirosis field, Ben Adler (also an author of the two featured papers), has questioned the interpretation of these studies.4 He points out that the challenge strains used in several studies were not sufficiently lethal, making it easier to observe a protective effect of the vaccine. Moreover some studies claimed statistically significant protective effects of the protein vaccine when in fact there was none upon Adler's reanalysis of the data. The only protein to convincingly exert a protective effect in an appropriate animal model was LigA5 although the ability of the LigA subunit vaccine to cross-protect against different serovars of Leptospira has yet to be tested. However there is one major problem with using LigA as a vaccine--not all Leptospira strains have the ligA gene.6
In the two studies described here the investigators took a step back from looking at individual proteins and developed an attenuated strain to use for immunization. The properties of the attenuated strain, designated M1352, are described in the paper authored by Murray and colleagues.2 The M1352 strain was not developed by the classic approach of continuously growing and passaging the bacteria in culture until they lost their ability to cause disease. Instead the strain was one of a large collection of mutants generated by random transposon mutagenesis of L. interrogans serovar Manilae. The M1352 strain had the transposon inserted in a gene located in a large cluster of genes encoding enzymes that assemble LPS. The mutation had subtle effects on the reactivity of M1352 with various antibodies raised against leptospiral LPS, suggesting that the LPS structure itself was somehow changed in M1352 when compared with the wild-type Manilae strain.
Since LPS is a crucial surface component that interacts with the host, it was not too surprising that M1352 was not able to cause lethal infections like its wild-type Manilae parent. When they infected hamsters with the M1352 strain, the spirochetes were unable to kill the hamsters or even establish an infection in the kidneys. Despite the efficient clearance of M1352, the Leptospira lingered long enough in the hamsters to provoke an antibody response. Western blots of L. interrogans lysates revealed strong reactivity of antibodies from the M1352-infected hamsters to a number of proteins. Because the M1352 strain generated an antibody response without establishing an infection, the authors decided to test the weakened strain as a vaccine in the hamster model in a follow-up study.1
In the second study, Srikram and colleagues1 demonstrated that immunization of hamsters with a single dose of live M1352 was more effective than a dose of heat-killed wild-type strain in protecting hamsters from being killed by the wild-type Manilae strain. The M1352 vaccine also did a better job in preventing colonization of the kidneys by the spirochete and in minimizing lung hemorrhage than the heat-killed vaccine.
When they challenged the vaccinated hamsters with a different serovar, a Pomona strain, all the hamsters immunized with live M1352 survived whereas 60% of animals immunized with heat-killed wild-type Manilae perished. However the M1352 vaccine didn't work perfectly. Although all hamsters immunized with live M1352 survived the challenge with the Pomona strain, the kidneys from 90% of the animals were culture positive, and 90% had hemorrhaged lungs. Nevertheless this is the first time that complete protection from death was observed following challenge of vaccinated animals by a serovar unrelated to the vaccine strain. They also showed that the M1352 vaccine had to be administered alive. Heat-killed or chemically-killed M1352 vaccine failed to protect hamsters from lethal infection.
The investigators next tried to figure out which component of the M1352 strain was the protective cross-reactive antigen targeted by the hamster's immune system. They wondered whether the live M1352 and heat-killed wild-type Manilae vaccines generated antibody responses to different proteins. When they probed separate two-dimensional blots of L. interrogans membrane preparations of serovar Pomona with antibodies from hamsters immunized with M1352 and heat-killed wild-type Manilae, a number of protein spots lit up. Most proteins, including LipL32, reacted with both sets of antibodies. These proteins are unlikely to account for the cross-protection conferred by the M1352 vaccine since the presence of these antibodies in the hamsters immunized with heat-killed Manilae failed to protect the animals from being killed by the Pomona strain. On the other hand, four Pomona proteins were recognized only by hamsters receiving the attenuated vaccine:
Featured papers
1. Srikram A, Zhang K, Bartpho T, Lo M, Hoke DE, Sermswan RW, Adler B, and Murray GL (March 15, 2011). Cross-protective immunity against leptospriosis elicited by a live, attenuated lipopolysaccharide mutant. Journal of Infectious Diseases 203(6):870-879. DOI: 10.1093/infdis/jiq12
2. Murray GL, Amporn S, Henry R, Hartskeerl RA, Sermswan RW, and Adler B (November 2010). Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence. Molecular Microbiology 78(3): 701-709. DOI: 10.1111/j.1365-2958.2010.07360.x
Helpful references
3. Guerra MA (February 15, 2009). Leptospirosis. Journal of the American Veterinary Medical Association 234(4):472-478. DOI: 10.2460/javma.234.4.472
4. Adler B and de la Pena Moctezuma (January 27, 2010). Leptospira and leptospirosis. Veterinary Microbiology 140(3-4):287-296. DOI: 10.1016/j.vetmic.2009.03.012
5. Silva, ÉF, Medeiros MA, McBride AJA, Matsunaga J, Esteves GS, Ramos JGR, Santos CS, Croda J, Homma A, Dellagostin OA, Haake DA, Reis MG, and Ko AI (August 14, 2007). The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine 25(33):6277-6286. DOI: 10.1016/j.vaccine.2007.05.053
6. McBride AJA, Cerqueira GM, Suchard MA, Moreira MA, Zuerner RL, Reis MG, Haake DA, Ko AI, and Dellagostin OA (March 2009). Infection, Genetics and Evolution 9(2):196-205. DOI: 10.1016/j.meegid.2008.10.012
7. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre M, Ave P, Girons IS, Ko AI, and Picardeau M (July 2007). The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathogens3(7):e97. DOI: 10.1371/journal.ppat.0030097
8.Pinne M and Haake DA (June 2009). A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans. PLoS One 4(6):e6071. DOI: 10.1371/journal.pone.0006071
The leptospirosis vaccines that are out on the market are still formulated with killed Leptospira or sometimes their outer membrane. These traditional vaccines are administered primarily to dogs, cattle, and pigs. Human leptospirosis vaccines are not available in most countries, even in areas where leptospirosis is endemic.
New types of leptospirosis vaccines are needed since the traditional killed vaccines are flawed. One problem is that immunity is serovar specific. For this reason a vaccine must contain all the serovars that the target population may encounter. Even when the vaccine manufacturers figure out which serovars are circulating, a new serovar may emerge, rendering the vaccine ineffective as the new serovar spreads through the susceptible population. The vaccine must then be reformulated at substantial cost.
This is exactly what happened to the leptospirosis vaccines that are given to dogs.3 The early canine vaccines, first available in the 1970s, contained the serovars Canicola and Icterohemorrhagiae. These vaccines worked fine until the late 1980s or so, when new serovars started to appear in infected dogs, even in those that had been vaccinated. Since then vaccine makers have added the serovars Grippotyphosa and Pomona to their vaccines. Nevertheless with over 200 pathogenic serovars of Leptospira lurking out there, we don't know when or which additional serovars will emerge in the future.
It would be nice to have a single leptospirosis vaccine formulation that would protect against all serovars. The protective effect of traditional vaccines is due to antibodies generated against lipopolysaccharide (LPS), whose structure differs among the serovars of Leptospira. Since immunization elicits antibodies that recognize the LPS of only the serovars included in the vaccine, vaccinated individuals remain susceptible to infection by other serovars.
To get around this problem, scientists have been testing individual Leptospira surface proteins as potential vaccines in rodent models of leptospirosis. Leptospira surface proteins tend to be antigenically conserved among the different serovars: antibodies generated against a protein from one serovar often reacts against the same protein expressed by other serovars. According to many studies the LipL32 and Lig surface lipoproteins, when delivered as recombinant proteins, naked DNA, or by microbial vectors (adenovirus and Mycobacterium bovis), apparently protected hamsters or guinea pigs from lethal infection by Leptospira. Unfortunately one of the leaders in the leptospirosis field, Ben Adler (also an author of the two featured papers), has questioned the interpretation of these studies.4 He points out that the challenge strains used in several studies were not sufficiently lethal, making it easier to observe a protective effect of the vaccine. Moreover some studies claimed statistically significant protective effects of the protein vaccine when in fact there was none upon Adler's reanalysis of the data. The only protein to convincingly exert a protective effect in an appropriate animal model was LigA5 although the ability of the LigA subunit vaccine to cross-protect against different serovars of Leptospira has yet to be tested. However there is one major problem with using LigA as a vaccine--not all Leptospira strains have the ligA gene.6
In the two studies described here the investigators took a step back from looking at individual proteins and developed an attenuated strain to use for immunization. The properties of the attenuated strain, designated M1352, are described in the paper authored by Murray and colleagues.2 The M1352 strain was not developed by the classic approach of continuously growing and passaging the bacteria in culture until they lost their ability to cause disease. Instead the strain was one of a large collection of mutants generated by random transposon mutagenesis of L. interrogans serovar Manilae. The M1352 strain had the transposon inserted in a gene located in a large cluster of genes encoding enzymes that assemble LPS. The mutation had subtle effects on the reactivity of M1352 with various antibodies raised against leptospiral LPS, suggesting that the LPS structure itself was somehow changed in M1352 when compared with the wild-type Manilae strain.
Since LPS is a crucial surface component that interacts with the host, it was not too surprising that M1352 was not able to cause lethal infections like its wild-type Manilae parent. When they infected hamsters with the M1352 strain, the spirochetes were unable to kill the hamsters or even establish an infection in the kidneys. Despite the efficient clearance of M1352, the Leptospira lingered long enough in the hamsters to provoke an antibody response. Western blots of L. interrogans lysates revealed strong reactivity of antibodies from the M1352-infected hamsters to a number of proteins. Because the M1352 strain generated an antibody response without establishing an infection, the authors decided to test the weakened strain as a vaccine in the hamster model in a follow-up study.1
In the second study, Srikram and colleagues1 demonstrated that immunization of hamsters with a single dose of live M1352 was more effective than a dose of heat-killed wild-type strain in protecting hamsters from being killed by the wild-type Manilae strain. The M1352 vaccine also did a better job in preventing colonization of the kidneys by the spirochete and in minimizing lung hemorrhage than the heat-killed vaccine.
When they challenged the vaccinated hamsters with a different serovar, a Pomona strain, all the hamsters immunized with live M1352 survived whereas 60% of animals immunized with heat-killed wild-type Manilae perished. However the M1352 vaccine didn't work perfectly. Although all hamsters immunized with live M1352 survived the challenge with the Pomona strain, the kidneys from 90% of the animals were culture positive, and 90% had hemorrhaged lungs. Nevertheless this is the first time that complete protection from death was observed following challenge of vaccinated animals by a serovar unrelated to the vaccine strain. They also showed that the M1352 vaccine had to be administered alive. Heat-killed or chemically-killed M1352 vaccine failed to protect hamsters from lethal infection.
The investigators next tried to figure out which component of the M1352 strain was the protective cross-reactive antigen targeted by the hamster's immune system. They wondered whether the live M1352 and heat-killed wild-type Manilae vaccines generated antibody responses to different proteins. When they probed separate two-dimensional blots of L. interrogans membrane preparations of serovar Pomona with antibodies from hamsters immunized with M1352 and heat-killed wild-type Manilae, a number of protein spots lit up. Most proteins, including LipL32, reacted with both sets of antibodies. These proteins are unlikely to account for the cross-protection conferred by the M1352 vaccine since the presence of these antibodies in the hamsters immunized with heat-killed Manilae failed to protect the animals from being killed by the Pomona strain. On the other hand, four Pomona proteins were recognized only by hamsters receiving the attenuated vaccine:
- Loa22, the only surface protein known to be essential for L. interrogans to cause lethal infections7
- a homolog of GspG, a component of the type II secretion system
- LA1939, a possible lipoprotein of unknown function
- OmpL36, a surface-exposed outer membrane protein of unknown function8
Featured papers
1. Srikram A, Zhang K, Bartpho T, Lo M, Hoke DE, Sermswan RW, Adler B, and Murray GL (March 15, 2011). Cross-protective immunity against leptospriosis elicited by a live, attenuated lipopolysaccharide mutant. Journal of Infectious Diseases 203(6):870-879. DOI: 10.1093/infdis/jiq12
2. Murray GL, Amporn S, Henry R, Hartskeerl RA, Sermswan RW, and Adler B (November 2010). Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence. Molecular Microbiology 78(3): 701-709. DOI: 10.1111/j.1365-2958.2010.07360.x
Helpful references
3. Guerra MA (February 15, 2009). Leptospirosis. Journal of the American Veterinary Medical Association 234(4):472-478. DOI: 10.2460/javma.234.4.472
4. Adler B and de la Pena Moctezuma (January 27, 2010). Leptospira and leptospirosis. Veterinary Microbiology 140(3-4):287-296. DOI: 10.1016/j.vetmic.2009.03.012
5. Silva, ÉF, Medeiros MA, McBride AJA, Matsunaga J, Esteves GS, Ramos JGR, Santos CS, Croda J, Homma A, Dellagostin OA, Haake DA, Reis MG, and Ko AI (August 14, 2007). The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine 25(33):6277-6286. DOI: 10.1016/j.vaccine.2007.05.053
6. McBride AJA, Cerqueira GM, Suchard MA, Moreira MA, Zuerner RL, Reis MG, Haake DA, Ko AI, and Dellagostin OA (March 2009). Infection, Genetics and Evolution 9(2):196-205. DOI: 10.1016/j.meegid.2008.10.012
7. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre M, Ave P, Girons IS, Ko AI, and Picardeau M (July 2007). The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathogens3(7):e97. DOI: 10.1371/journal.ppat.0030097
8.Pinne M and Haake DA (June 2009). A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans. PLoS One 4(6):e6071. DOI: 10.1371/journal.pone.0006071
Tuesday, April 12, 2011
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).
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.
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.
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
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
Thursday, February 24, 2011
Serologic testing for syphilis: missing the point
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
