Journal of Postgraduate Medicine Medknow Publications and Staff Society of Seth GS Medical College and KEM Hospital, Mumbai, India ISSN: 0022-3859 EISSN: 0972-2823 Vol. 51, Num. 3, 2005, pp. 210-214

Journal of Postgraduate Medicine, Vol. 51, No. 3, July-September, 2005, pp. 210-214

Symposium

Leptospirosis vaccines: Past, present, and future

Koizumi N, Watanabe H

Department of Bacteriology, National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640
Correspondence Address:1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, haruwata@nih.go.jp

Code Number: jp05076

Abstract

Keywords: Leptospira, Leptospirosis, Vaccine, Killed whole cell vaccine, Serovar, LPS, Protein antigen, Genome, Cellular immunity

Leptospirosis is an important zoonotic disease that is distributed worldwide. [1],[2],[3] In mammals, leptospirosis is transmitted either by direct contact with infected animals or by exposure to water or soil contaminated by the urine of infected animals. The efficacy of vaccine for preventing leptospirosis was shown soon after Leptospira was proven to be a causative agent of Weil′s disease in Japan. The heat-killed whole cell vaccine made from leptospiral cultures dramatically protected coal miners from Weil′s disease in the Kyushu area where Weil′s disease was endemic.[4] The development of killed whole cell vaccine against leptospirosis in the middle of the last century has been reviewed,[5] and subsequently results of vaccine trials were published.[1], [6],[7],[8]

Killed whole cell vaccine

Killed whole cell leptospiral vaccines for humans are available in some countries, including Japan. The Japanese leptospiral vaccine consists of formalin-killed leptospires. The concentrations and serovars are 250 million/ml each of Australis, Autumnalis, and Hebdomadis and 500 million/ml of Copenhageni. The leptospires are grown in media containing rabbit serum and/or bovine serum albumin, inactivated with formalin, and washed by centrifugation. The standard procedures for production and verification of the vaccine were established in 1952.[9] The vaccine is administered initially using two subcutaneous injections of 1.0 ml given at a 7-day interval. The booster injection is 1.0 ml of vaccine injected subcutaneously and given within 5 years after the second initial dose. The antibody titres (MAT titres) after vaccination were significantly lower than those developed after natural infection and seroconversion occurred with low frequency (about 20-60%).[10],[11],[12],[13] However, protection was reported to be high in such populations and efficacy rates of whole cell vaccines were about 60-100%.[6],[7],[8],[14] The effectiveness of the killed vaccine is serovar-specific. The studies done in the Izena-jima Island in Okinawa prefecture showed that people who were inoculated with a serovar Pyrogenes monovalent vaccine were protected from the infection with serovar Pyrogenes, but not from infection with serovars Autumnalis and Hebdomadis. However, polyvalent vaccine, consisting of serovars Autumnalis, Hebdomadis and Pyrogenes, prevented infection with the three leptospiral serovars strains for at least for 7 years after immunization.[14] The serovar-specific protection of killed leptospiral vaccine has also been documented in other studies.[6],[15] There are more than 230 serovars among the pathogenic leptospires. The local variability in serovars of endemic leptospiral strains complicates the development of a vaccine that can be used worldwide.[1],[16] The study done in the Izenajma Island also demonstrated that the killed vaccine induced long-term immunity, although the prevalence of leptospirosis among the unvaccinated population after the introduction of the vaccine dramatically decreased from those rates previously documented. However, one study has reported that the duration of immunity induced by killed vaccine ranges between 6 months to a 1 year at the longest and a second study reported a duration of at least 3 years.[5],[6] Persistence of immunity in dogs is also controversial.[17],[18] The side effects of the whole cell vaccines were reported, which included both systemic and local reactions at a various frequency. To reduce side effects ascribable to serum in cultures, a vaccine that consisted of leptospires grown in the protein-free medium was developed.[11],[19],[20] A Japanese research group, however, reported that there was no difference in the frequency of side-effects between the vaccines derived from either cultures in Korthof′s or protein-free medium.[21] In addition to side-effects, the whole cell vaccine may induce autoimmune diseases, such as uveitis.[22]

There is a report on the outer envelope vaccine for leptospirosis which was studied in China.[23] The results of the study showed the good protection with less side effects and higher agglutinating titre than those in a whole cell vaccine. Vaccination using the outer envelope vaccine also reduced the number of the patients with vaccine-unrelated serogroup strains.

Lipopolysaccharides

Protein antigens

The immunogenic proteins, especially the outer membrane surface proteins, of pathogenic Leptospira, may be effective as vaccinogens. The identification of proteins, which are conserved among pathogenic leptospires and can generate cross-protection against various serovars, has become a major focus of leptospirosis research. [32],[33],[34],[35],[36],[37],[38],[39],[40],[41],[42],[43],[44],[45],[46],[47],[48],[49],[50],[51],[52],[53],[54] Subunit vaccines may also have fewer side-effects than killed whole cell vaccine. Sera from patients with leptospirosis have antibodies against several protein antigens.[39] Protein extracts prepared from a pathogenic Leptospira can induce protective immunity against challenge with a heterologous serovar strain in an experimental animal model.[55] These data point to the potential use of leptospiral protein(s) as candidates for a new vaccine that could induce good protection against diverse serovars.

Some leptospiral protein antigens have been shown to elicit protective immunity in animal models. These are described as follows:

1. OmpL1 and LipL41
OmpL1 is a transmembrane protein,[33],[56] and LipL41 is an outer membrane lipoprotein.[34] Both proteins are surface-exposed. OmpL1 and LipL41 act synergistically to induce immunoprotection in the hamster model of leptospirosis, although neither of the individual proteins induces protective immunity.[37] Although the mechanism of this synergistic protection remains to be solved, a steric hindrance of OmpL1 by LipL41 has been proposed.[52] The results from this same study also indicated that immunization using the membrane-associated OmpL1/LipL41 (also referred to as lipidated LipL41) was critical for inducing immunoprotection. The authors suggested that conformational integrity of OmpL1/LipL41 epitopes for antibody binding might require the association with membrane and/or lipid attached to LipL41 for immunogenicity. A similar condition for immunogenicity exists with Borrelia burgdorferi lipoprotein, OspA, in which immunogenicity and immunoprotective capacity were enhanced by lipidation of the protein.[57], [58] Southern and western blot analyses revealed that ompL1 gene product and LipL41 protein were present in various pathogenic leptospiral serovars, but not in non-pathogenic leptospires.[33],[34] Patients with leptospirosis have antibodies against these proteins in their sera.[38],[59] Whether or not these proteins will confer cross-protection against a heterologous challenge remains to be determined.

2. LipL32/Hap-1
LipL32 is one of the most abundant proteins in Leptospira .[38] LipL32 is an outer membrane lipoprotein that is conserved, both genetically and immunologically, in the various pathogenic leptospires. LipL32 antigen induces antibodies in patients with leptospirosis. A recombinant LipL32 antigen has good sensitivity and specificity when used in an ELISA for human leptospirosis IgG.[59] LipL32 is also called haemolysis associated protein-1 (Hap-1) because E. coli harbouring the plasmid encoding this gene showed some haemolytic activity on sheep, but not on human erythrocytes.[60] LipL32 stimulates the expression of both MCP-1 and iNOS mRNAs and augments the nuclear binding of NF-kB and AP-1 transcription factors in cultured mouse proximal tubule cells.[61] Vaccination using an adenovirus vector encoding the lipL32/hap-1 gene induced cross-protection in the gerbil model of leptospirosis.[40] The lipL32/hap-1 gene derived from L. interrogans serovar Autumnalis conferred protective immunity against a challenge with a heterologous strain of L. interrogans serovar Canicola. In addition, OmpL1, either alone or in combination with LipL32/Hap-1, had no protective activity. This result is in contrast to the synergistic activity observed with OmpL1 and LipL41.

3. Leptospiral immunoglobulin-like proteins
Leptospiral immunoglobulin-like (Lig) proteins are surface-exposed outer membrane proteins punctuated by tandem repeats of about 90 amino acids of bacterial immunoglobulin (Ig) like domains.[45],[49],[51],[62] LigA consists only of Ig-like domains, whereas LigB has an additional unique domain at the C terminus. The bacterial immunoglobulin-like domains are found in various adhesion proteins of pathogenic bacteria; for example, the intimin of E. coli ,[63], [64] and the invasin of Y. pseudotuberculosis ,[65] and may function as adhesion molecules. Both LigA and LigB contain an N-terminal lipobox and LigA was lipidated when expressed in E. coli .[51] Expression of Lig protein and lig mRNA was lost after attenuation by culture of L. kirshneri and L. interrogans , suggesting that Lig proteins are associated with virulence. Lig proteins were not detected in some virulent strains during in vitro culture, although specific mRNAs were transcribed.[45],[62] We also failed to detect expression of LigA protein in fresh human isolates of serogroup Autumnalis and Hebdomadis.[66] Recent studies show that expression of Lig proteins, surface exposure of LigB and extracellular release of LigA are all enhanced by physiological osmolarity.[67] Sera from either rats immunized with extracts of in vitro cultured low-passage strain or sera from vaccinated dogs failed to react with Lig proteins in Western blot analysis, while sera from captured rat reservoirs or dogs infected with Leptospira gave a positive response.[49], [62] Therefore expression of Lig proteins is upregulated during infection of a mammalian host. The lig genes are present among various pathogenic, but not non-pathogenic, leptospires.[49],[51],[62] Sera from human patients infected with different leptospiral serogroup strains reacted with Lig proteins.[51] Furthermore, in a mouse model of leptospirosis, the Lig proteins elicited protective immunity against challenges, not only with the homologous serovar Manilae infection,[51] but also the heterologous serovar Icterohaemorrhagiae.[66] These data suggest that Lig proteins could induce protective immunity against the challenge with strains of various leptospiral serovars.

Exploration of the full-length leptospiral genome to detect genes encoding candidate vaccine proteins

Cell-mediated immunity

Concluding remarks

Both the mechanisms of pathogenesis of Leptospira and nature of protective immunity against leptospiral infection are poorly understood. The availability of full-length genome sequences of two Leptospira strains will be helpful for future studies. The development of the genetic analysis tools for pathogenic leptospires, which are currently available only for non-pathogenic leptospires, [81],[82],[83] will accelerate both the identification of virulence factors, as well as our understanding of the pathogenesis of leptospires. As new information evolves, insights will be gained into ways to improve existing, and develop effective new, vaccines for leptospirosis.

References

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