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:
  • 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 interrogansPLoS One 4(6):e6071. DOI: 10.1371/journal.pone.0006071