Advances & challenges in leptospiral vaccine development

Live vaccine

These vaccines are known to elicit a strong cellular and humoral immune response and confer lifelong immunity with a few doses. These vaccines are produced by attenuating a pathogenic organism but retaining its ability to replicate and induce protective immune response without being able to cause disease. Live leptospiral vaccines have not gained prominence due to a lack of detailed knowledge about the pathogenicity, diversity in serovar distribution and the need for genetic tools that allow for manipulation of pathogenic Leptospira species1920. Experimental live attenuated Pomona vaccine has been shown to be effective in protecting pigs against leptospiral challenge and also prevented establishment of renal reservoirs of the organism2122. Serovar Pomona attenuated by laboratory passage was also used as a live vaccine in the cattle. The leptospires were attenuated by passage through egg and the vaccine was used in an aborting cattle herd. The vaccine was shown to elicit protective immune response and reduced the abortion rate23. The application of these vaccines in leptospirosis was limited due to their instability, undefined genetic basis for attenuation and lack of strict quality control measures necessary for their production24. Efforts by Srikram et al24 in developing a transposon mutant with LPS-associated mutations of Leptospira interrogans serovar Manilae conferred a significant cross-protective immunity in hamsters against homologous as well as heterologous challenges. Although the live-attenuated vaccines were able to induce higher antibody titres compared to bacterins, their acceptance and use among veterinary biologic manufacturers were hampered by the difficulty of maintaining their viability during storage as well as the potential for reversion to virulent state in the host animal25. Thus, most commercially available Leptospira vaccines are bacterins26.

Bacterin vaccines

Killed whole-cell bacterins against leptospirosis have been in use for over five decades. This follows successful immunization of guinea pigs with phenol inactivated leptospires9. However, side effects such as reactivity due to components of the growth media, local and systemic reactions and restriction of protection to closely related serovars have hindered its application18. Furthermore, local variability of endemic pathogenic leptospiral strains from region to region as well as virulence and lack of cross-reactivity have greatly hampered its application in the prevention of leptospirosis. Others factors include lack of consistency of production or repeatability, high production costs, lack of immunological memory and short duration of immunity requiring several booster doses18.

Efforts to grow leptospires in protein-free medium have yielded mixed results with some reporting no significant difference in the side effects produced between leptospires grown in bovine serum albumin-supplemented medium and protein-free medium9. Due to lack of cross-reactivity among different serovars, a polyvalent vaccine containing multiple serovars has been used largely in dogs, swine and cattle and yielded a satisfactory response27. Despite reports indicating lack of heterologous protection, they are able to confer protection against serovars included in the vaccine as well as antigenically related serovars9. The serovar-specific LPS component of Leptospira is responsible for inducing antibody response, although the response lacks immunological memory except for Leptospira interrogans serovar Hardjo infection in cattle which is characterized by T-helper I and interferon gamma (INFγ) T-cell response928. The duration of immunity is also said to range from six months to three years depending on the choice of adjuvants1718. Recent reports in the United States indicate up to 30 per cent of vaccinated dogs do not respond to vaccinations and immunizations are also associated with adverse effects10. This is attributed to change in the epidemiology of the disease and ingress of new serovars in endemic areas. Not only is there failure to provide protective immunity by certain commercial bacterin vaccines, but also claims of vaccines (Nobivac L4) attributed death or transmission of infection among vaccinated dogs29. These controversies further highlight the importance of leptospirosis as important zoonosis and emphasized the need for safe and effective vaccine for both humans and animals. This has led to development of new strategies such as the use of sub-unit vaccines (Duramune, by Fort Dodge, USA) composed only of the immunogenic component of the Leptospira organism instead of the entire organism30.

As observed in animals, use of bacterins in humans also has limited protection against serovars contained in the vaccine. The bacterins for human use are composed of monovalent or polyvalent cellular suspension, for example, French SPIROLET (IMAXIO) from Pasteur Merieux against Leptospira icterohaemorrhagiae serovar. Although this vaccine is specific for icterohaemorrhagiae infection, it is capable of protecting against antigenically related serovars31. In addition to the SPIROLET vaccine, there is a bivalent vaccine from Shanghai Institute of Biological Products and a trivalent vaccine (Vax-Spiral) from Cuba, both of which have successfully undergone phase four clinical trial as reported by Martinez et al31. The vaccine was registered in 1998 and hasshown improved safety with low reactivity3132. However, these vaccines are only recommended for workers with high risk of contracting leptospirosis infection. Other benefits of bacterins include anti-shedding effect which helps reduce transmission, stability during storage, unlikely to cause disease due to residual disease-causing characteristics, induction of strong antigen-specific proliferative response by peripheral blood mononuclear cells and production of cross-protective immunity against closely related serovars173334.

Leptospiral lipopolysaccharide antigen vaccine

Leptospiral lipopolysaccharide (L-LPS) constitutes the major component of the outer membrane which greatly influences the virulence of pathogenic Leptospira19. It is a complex structure with approximately 100 genes all encoded in a single double-stranded DNA molecule within its biosynthetic loci35. However, there is a lack of adequate knowledge on its structure and the role of individual proteins involved in its synthesis1219. During infectious process, L-LPS plays a significant role in the stimulation and activation of innate immune response by eliciting a differential recognition via toll-like receptors (TLRs 2 and 4)3637. L-LPS is a protective immunogen as demonstrated by protections rendered by monoclonal antibodies against lethal infection in hamsters38; however, there are conflicting views on the ability of LPS-based Leptospira vaccine to protect against heterologous challenge1739. Hence, there is a need to carry out further studies to determine whether LPS vaccines are serovar-specific or heterologous. The disadvantage of the strong biological activity of LPS is its contribution to vaccine reactivity40. As mentioned earlier, there is lack of knowledge on the complex structure of L-LPS and the genes involved in its biosynthesis; thus, modifications of the LPS structure that would have allowed triggering of immune response needed in a vaccine and the lowering of its toxicity would make LPS vaccines more potent and efficient.

Recombinant subunit protein vaccine

Live-attenuated vaccine mimics natural infection, and as a result, antibody production is achieved for a prolonged period of time. However, the use of live-attenuated vaccines against leptospirosis poses a risk due to its potential ability to revert to virulence, cause infection, continuous shedding in the urine and accidental infection resulting from handling of live strains.

An ideal vaccine for human and animal use should be effective, avirulent and produce long-lasting neutralizing immunity47. Subunit vaccines such as recombinant protein vaccines have been reported to be promising vaccine candidates because these are avirulent, less bio-hazardous, non-infectious, non-viable and well-defined4748.

Recombinant proteins vaccines

Selection of an optimal antigen is paramount for designing an effective vaccine. These antigenic proteins, depending on the response desired, should contain appropriate epitopes to B-cell receptors and can be recognized by the T-cell receptor in a complex with major histocompatibility complex molecule31.

Several recombinant protein vaccines such as the outer membrane protein (OMPs), lipoproteins and virulence factor vaccines have been developed using various biotechnological methods532.

The first successful recombinant leptospirosis vaccine was reported in 19995. Immunization with recombinant OmpL1, a transmembrane OMP that functions as a porin in leptospiral outer membrane and lipoprotein LipL41 conferred significant protection against intraperitoneal challenge with virulent Leptospira kirschneri49. The leptospiral outer membrane contains both transmembrane proteins exposed on the leptospiral surface and may play a potential role in the pathogenesis of the disease. The location of OmpL1 on the leptospiral surface has also been demonstrated by immune-electron microscopy and is presumed to have surface-exposed epitopes5. Evaluation of the immunoprotective ability of OmpL1 and the outer membrane lipoprotein LipL41 in the Golden-Syrian hamster revealed a synergistic effect49. The two membrane-associated proteins OmpL1 and LipL41 were expressed in E. coli using specialized expression plasmids to enhance their expression and reduce toxicity to the cells. Active immunization of hamsters with the expressed recombinant proteins provided significant protection against challenge with Leptospira kirschneri serovar Grippotyphosa49. The survival rate was 75 per cent compared with 25 per cent in the control group, although immunization with OmpL1 alone did not produce significant level of protection at 28 days after challenge. Analysis of time course of survival of hamsters after immunization with OmpL41 alone indicated significant protection at nine days after challenge5. Surface-exposed proteins are potential candidates for inducing immune responses following infection and are also said to act as adhesins that mediate the initial process of pathogen attachment to host cells. Furthermore, well-conserved outer membrane proteins and leptospiral lipoprotein LipL41 are promising vaccine candidates with potentials to induce cross-protective immunity49.

LipL32 [haemolysis-associated protein 1 (Hap1)], is the most abundant outer membrane protein and it is highly expressed both during infection and by in vitro culture50. The nucleotide sequence coding for LipL32 is conserved among pathogenic Leptospira species and absent in non-pathogenic leptospires51. Branger et al11 have reported that adenovirus-mediated Hap1 vaccinations induce significant immune response against heterologous challenge with virulent Leptospira interrogans serovar Canicola in gerbils, whereas a similar OmpL1 construct failed to protect the animals52. The 31- to 34-kDa protein fraction of L. interrogans serovar Autumnalis was obtained by cloning of the gene in pET-29b expression vector using PCR fragments digested and ligated into the NotI and XhoI site of the vector. The adenovirus construct was generated by ligation into the pET-29b vector carrying the desired gene. The 6-12 wk old Mongolian gerbils were immunized with 26-31 kDa for Hap1 and 31-34 kDa for OmpL1 intramuscularly via the quadriceps. The result of this experiment showed that the cross-protective effect exhibited by pathogenic Leptospira was shared by Hap1 protein mediated by an adenovirus vector11. In another study, immunization of hamsters with recombinant BCG expressing LipL32 were found to render protection against lethal challenge with L. interrogans serovar Copenhageni32. For the purpose of this experiment, four E. coli-mycobacteria shuttle vectors were constructed by cloning of the gene into the expression vectors and introduced into Mycobacterium bovis BCG Pasteur strain by electroporation. Hamsters immunized with recombinant BCG expressing LipL32 were protected against lethal challenge with L. interrogans serovar Copenhageni. Histopathological examination did not indicate any clinical sign indicative of leptospirosis during the 28-day period after challenge. Importantly, recombinant rBCG-LipL32 was able to induce sterilizing immunity in animals that survived the lethal challenge as indicated by the absence of clinical and histopathological signs of the disease as well as negative result from lung and kidney culture32. These results show that immunogenicity increases with the expression of multiple genes and is enhanced by the use of suitable adjuvant.

Recombinant LipL32 purified from an E. coli expression system was assessed for protective immunity in a group of five hamsters challenged with virulent L. interrogans serovar Canicola strain Kito. However, no significant survival was observed as compared with the unvaccinated control group53. Subsequent histological analysis revealed reduced amount of L. interrogans in the kidneys of vaccinated hamsters53. Furthermore, due to conflicting reports on the inability of LipL32 subunit vaccine to stimulate protective immune response, the protein was co-administered with the B subunit of E. coli heat-labile enterotoxin in a hamster model of leptospirosis to enhance its immune stimulating capability. The highly conserved lipL32 coding sequence was amplified by PCR after obtaining the DNA material from L. interrogans serovar Copenhageni strain Fiocruz L1-130 and then cloned into the corresponding enzyme sites on pAE/ltb vector to form pAE/ltb-lipL32. The construct was used to transform competent BL21 E. coli cells and the expressed protein was purified using affinity chromatography. The 4-5 wk old hamsters were injected in the quadriceps muscle with the vaccines (one group received LipL32 coupled with heat labile enterotoxin while another group received LipL32 co-administered with heat labile enterotoxin) and control groups received the recombinant heat-labile enterotoxin alone. After challenge with 5×50 per cent lethal dose of L. interrogans, hamsters vaccinated with LipL32 coupled with heat-labile E. coli enterotoxin had significantly higher survival rates than animals in the control group54. Based on this finding, it was claimed that this was the first reported protective immune response afforded by a subunit vaccine using LipL32 and could serve as an important contribution to the development of improved leptospirosis vaccine54. This may be a valid argument considering a number of subunit proteins have been evaluated as potential vaccine candidates55.

DNA vaccines

DNA vaccines are a novel approach for inducing an immune response. These are the simplest embodiment of vaccines that consist of the antigen itself, and provide genes encoding the antigen56. In this approach, purified plasmid DNA containing the coding sequences of an immunogenic gene and the essential regulatory elements to transcribe and translate them is introduced into the tissue intramuscularly. This is preceded by tissue expression and induction of potent, long-lasting heterologous immune response. The efficiency of leptospiral DNA vaccines has been demonstrated in animal models4857. This approach is seen as a positive development in the efforts to prevent leptospirosis for which the conventional vaccines have failed. In addition, DNA vaccines also allow for inclusion of multiple genes so as to improve the coverage and ability to protect against infection due to a wide range of serovars.

DNA vaccines provide prolonged antigen expression and amplification of the immune response, while simultaneously offering several advantages over other vaccine preparations such as ease of construction, low cost of mass production, high-level temperature stability and the ability to elicit both humoral and cell-mediated immune response5859.

A DNA construct encoding L. interrogans Hap1 was designed to enhance direct gene transfer of the protein into gerbils followed by challenge with virulent strain of serovar Canicola. The genomic DNA from L. interrogans Autumnalis strain 32 was amplified by PCR and cloned in PCRII.1 vector. The amplified fragment was later digested and sub-cloned into pUC 19 expression vector using the corresponding restriction enzyme site. The DNA construct was used to immunize hamster, and after the second immunization, the hamsters were challenged with virulent L. interrogans Canicola. The result revealed significant protection against the Canicola challenge4, and the survival rate of the gerbils vaccinated with the vaccine was significantly higher than the control4. These findings indicated the potentials of DNA vaccine and it was able to protect gerbils against the pathogenic challenge similar to the protection conferred by Hap1 expressed by adenovirus vector.

In a related study60, OmpL1 plasmid DNA vaccine rescued some vaccinated animals from lethal challenge with heterologous L. interrogans serovar Pomona at one week after vaccination. The vaccine also delayed death time and reduced morbidity and the number of Leptospira in the tissue of vaccinated animals. In this study, the OmpL1 plasmid DNA vaccine was generated by cloning PCR amplified fragment of the gene into expression vector pcDNA3.1 and propagated by transforming competent Top10 E. coli cells. Outer membrane proteins although occur in small amounts, play a significant role as porins or receptors for soluble molecules60. However, further efforts are required to optimize the dose and formulation to maximize its efficacy.

Immunization with LigA DNA vaccine provided significant protection against virulent L. interrogans serovar Pomona challenge in hamsters42. This protection was achieved by both humoral and cell-mediated immunity as revealed by increase in antibody titres after administration of a booster, significant proliferation of lymphocytes from vaccinated animals and the enhancement of both Th1 and Th2 cytokines42. The vaccine was constructed in two truncated forms with the conserved and variable portion of the LigA gene. The conserved and variable regions of the gene were cloned in a eukaryotic expression vector and used to immunize hamsters. The high-level stimulation of cell-mediated immunity was indicated by substantial lymphocytes proliferation among the LigA-vaccinated animals. However, there was greater response in the conserved LigA group compared to the variable region of LigA42.

Although the ability of plasmid DNA vaccine to induce strong and specific immune response has been established, there are still concerns regarding its safety specifically the potential to induce deleterious autoimmune disease and development of tolerance in response to the persistent exposure to a foreign antigen58. However, questions on how to maximize immune responses by optimization of the route of application and delivery methods of the plasmid are being researched. Some bacterial and viral vectors been shown to be a good source of plasmid DNA due to their ability to transfer plasmids across phylogenetic borders to mammalian host cells61.

In the last few years, immense progress has been made in the field of DNA vaccine and it has been proven to be a successful approach using animal models. Despite these achievements using animal models, the same cannot be said about human clinical trials due to the low immunogenicity observed. Currently, efforts are under way to enhance the immunogenicity of plasmid DNA to allow for its application in humans.