A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics

Two recent papers tested the effectiveness of topical antibiotics in preventing Borrelia burgdorferi infection in mice following a tick bite.  Infection by the Lyme disease spirochete was successfully halted in the Knauer et al. study from Germany¹ but not in the Wormser et al. study conducted in New York.²  However a flaw in the Knauer study may have unfairly tipped the outcome in the antbiotic's favor.  (I'll save the Wormser study for another post.)

The paper by Knauer and colleagues¹ presented two trials, which differed in how the mice were inoculated with B. burgdorferi,  In the first trial the spirochetes were injected into the skin, and azithromycin was applied topically one hour, three days, and five days later at the injection site.  In the second trial infected ticks transmitted the spirochetes to the mice.   Azithromycin was applied topically to the feeding site immediately after the ticks stopped feeding.  In both trials azithromycin was dissolved in ethanol for application to the inoculation site.  Disseminated infection of the mice was assessed by culturing the heart, bladder, ear, and tarsus 56 days after inoculation.

The results from the first trial reveals the problem with the study (Table 1).  Among the ten mice in the placebo group (first row), which received only ethanol, only one (10%) had any culture positive organs 56 days later.  The spirochetes failed to establish a persistent infection in the other nine mice, suggesting that the investigators were working with a weakened strain of B. burgdorferi.  The ethanol could have had a slight killing effect (see the second trial) yet could not have accounted for the poor infection rate in the placebo group.

The table legend claims that the difference between the placebo and treatment groups was significant, but the statistics were done on the numbers in the column labeled "Infection Status."  According to the text of the paper, a positive "Infection Status" refers to those animals that managed to produce antibodies against B. burgdorferi antigens.  Infection status is therefore not the proper metric to assess the infectivity of B. burgdorferi.  When the statistics are performed with the appropriate numbers, located in the column under "Culture," the effect of azithromycin (1/10 culture positive in control group vs. 0/10 culture positive in any treatment group) is not significant (P = 0.9 for control vs. any treatment group).

Results from the second trial are shown in Table 2.  This trial included an extra control group ("No treatment") that received neither antibiotic nor ethanol.  Four of the seven mice in the untreated group (57%) ended up culture positive.  This is still a low infection rate compared to the rates observed in other studies, in which 90-100% of the control animals end up infected following tick inoculation of B. burgdorferi.  Two of the nine mice treated with ethanol alone (placebo) were culture positive, suggesting that ethanol alone helps prevent infection (57% culture positive in "no treatment" group vs. 22% in placebo group, P = 0.152), although the experiment would need to be repeated with larger groups of animals to make a statistically convincing case.

None of the animals treated with azithromycin were culture positive.  However the number of animals was again too low to conclude that antibiotic treatment was effective (2/9 culture positive in placebo group vs. 0/9, 0/8, and 0/5 in the treatment groups, P > 0.4 for comparison of each treatment group with placebo group).  The authors were able to claim statistical significance by combining the two control groups (no treatment and placebo) and the treatment groups.  However it is inappropriate to combine groups in this manner to attain statistical significance.

Even if the investigators had used a larger number of animals, the problem of their weakened challenge strain remains.  Application of topical antibiotics may turn out to be effective in preventing Lyme disease after a tick bite, but the study presented by Knauer and colleagues was not a fair test of this treatment approach.

References

1. Knauer, J., Krupka, I., Fueldner, C., Lehmann, J., & Straubinger, R. (2011). Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkr371

2. Wormser, G., Daniels, T., Bittker, S., Cooper, D., Wang, G., & Pavia, C. (2011). Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice Journal of Infectious Diseases DOI: 10.1093/infdis/jir382

BAM! A rare outer membrane protein of the stealth pathogen Treponema pallidum hidden in plain sight

Our immune system is capable of generating antibody against bacterial proteins located inside and outside of the bacterial cell.  However only those antibodies targeting surface proteins (or other surface components) have the potential to eliminate the bacteria.

Unlike typical Gram negative bacteria, the external surface of the Treponema pallidum outer membrane is bare except for a tiny number of proteins protruding from the membrane.  Most of the proteins targeted by the host antibody response are safely tucked away beneath the surface, inaccessible to the antibodies that recognize them.  T. pallidum even lacks LPS, which is a major target found on typical Gram negative bacteria.  The barren surface of T. pallidum is therefore one factor that may allow the "stealth pathogen" to persist in the host despite a strong antibody response.

None of the rare T. pallidum outer membrane proteins (Omps) have been identified with certainty, until now.  Scientists at the University of Connecticut have finally confirmed that TP0326 is one of these rare Omps.¹  Desrosiers and colleagues showed that the protein TP0326 was digested when proteinase K was added to live T. pallidum.  Proteins known to be located in the periplasm were left untouched by the protease, indicating that the fragile outer membrane remained intact while the spirochetes were being harvested for the experiment.  These results indicated that TP0326 was exposed on the surface of T. pallidum.

TP0326 (also called "Tp92") was first identified as a candidate Omp over a decade ago when rabbit antibodies against the protein were shown to promote opsonophagocytosis (engulfment) of T. pallidum.²   Antibodies must physically link bacteria to phagocytes for opsonophagocytosis to proceed.  Opsonophagocytosis therefore occurs only when antibodies against surface-exposed proteins are present.  The amino acid sequence of TP0326 also gave clues to its location.  TP0326 was identified as a homolog of the Omp85 family,² a set of proteins known to reside in the outer membrane of other Gram negative bacteria.  Omp85 was later renamed BamA when it was shown to be the core component of the outer membrane protein complex "BAM" that inserts newly expressed Omps into the outer membrane.³

A number of computer programs predicted that TP0326 spanned the outer membrane as a β-barrel structure, which I described in an earlier post.⁴  The gallery of E. coli transmembrane Omps displayed below shows that the loops on one side of the barrel are exposed on the surface of the bacterium.  The transmembrane portion of BamA is depicted as a box because its structure has yet to be determined experimentally, but it is also likely to have a β-barrel structure.

Figure 1 from Burgess 2008.  The outer membrane is colored gray.  The numbers indicate the number of β strands that cross the membrane.  The periplasm is located below the outer membrane.

The amino terminus of BamA consists of five repeating structures called POTRA, which are thought to guide the β strands of new transmembrane Omps into the outer membrane.³  Note that the POTRA domains protrude into the periplasmic space.  The amino terminus of TP0326 is thought to possess the POTRA domains as well.

Given the prediction that TP0326 structurally resembles BamA, it wasn't too surprising that TP0326 was exposed on the surface of T. pallidum.  What was surprising was how TP0326 was targeted by the immune system in syphilis patients.  Among the six patients examined by Desrosiers et al., three lacked any antibody whatsover against TP0326.  Although the other three syphilis patients managed to generate antibody against TP0326, the antibodies reacted weakly (1 patient) or not at all (2 patients) with the β-barrel domain, which contained the surface-exposed loops.  Instead, the antibodies targeting TP0326 reacted strongly with the subsurface POTRA domains in these three patients.  Assuming that the results with these six patients can be extrapolated to other syphilis patients, humans infected with T. pallidum are incapable of generating a strong antibody response against the surface-exposed loops of TP0326.

Since the exposed regions of TP0326 appear to be an Achilles heel of T. pallidum, TP0326 may have evolved to avoid generation of antibodies targeting its vulnerable segments.  Experimentally infected rabbits, which are not a natural host of T. pallidum, succeeded in generating antibody against the TP0326 β-barrel domain. Unfortunately the authors didn't present any data indicating whether the surface-exposed loops were recognized by the rabbit antibodies.  However, as I mentioned above, earlier work showed that infected rabbits produce opsonic antibody against TP0326.²  The same paper showed that TP0326 was somewhat effective as a subunit vaccine in the rabbit model of syphilis.²  Both of these observations suggest that rabbits are able to produce antibody that reacts with the surface-exposed regions of TP0326.  The rabbit model could be used to figure out conclusively whether the effectiveness of TP0326 as a vaccine relies upon generation of antibodies against the surface-exposed loops of the protein.  If so, an approach for developing a syphilis vaccine would be to coax the human immune system into generating antibodies that target the surface-exposed loops of TP0326.


Featured paper

1. Desrosiers DC, Anand A, Luthra A, Dunham-Ems SM, LeDoyt M, Cummings MAD, Eshghi A, Cameron CE, Cruz AR, Salazar JC, Caimano MJ, and Radolf JD (June 2011).  TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein.  Molecular Microbiology 80(6):1496-1515.  DOI: 10.1111/j.1365-2958.2011.07662.x

Related papers

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


3. Knowles TJ, Scott-Tucker A, Overduin M, and Henderson IR (March 2009).  Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.  Nature Reviews Microbiology 7(3):206-214.  DOI: 10.1038/nrmicro2069

4. Cox DL, Luthra A, Dunham-Ems S, Desrosiers DC, Salazar JC, Caimano MJ , and Radolf JD (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

Image source

Burgess NK, Dao TP, Stanley AM, and Fleming KG (September 26, 2008).  β-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro.  Journal of Biological Chemistry 283(39):26748-26758.  DOI: 10.1074/jbc.M80275420

Related posts

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

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.

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

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).

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

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

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:

• 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

Since GspG is not a surface component of other bacteria and nothing is known about where LA1939 is located on Leptospira, Loa22 and OspL36 are the best candidates to test as potential cross-protective vaccines.

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