Human leptospirosis: In search for a better vaccine

Leptospirosis is a neglected disease caused by bacteria of the genus Leptospira and is more prevalent in tropical and subtropical countries. This pathogen infects humans and other animals, responsible for the most widespread zoonosis in the world, estimated to be responsible for 60 000 deaths and 1 million cases per year. To date, commercial vaccines against human leptospirosis are available only in some countries such as Japan, China, Cuba and France. These vaccines prepared with inactivated Leptospira (bacterins) induce a short‐term and serovar‐specific immune response, with strong adverse side effects. To circumvent these limitations, several research groups are investigating new experimental vaccines in order to ensure that they are safe, efficient, and protect against several pathogenic Leptospira serovars, inducing sterilizing immunity. Most of these protocols use attenuated cultures, preparations after LPS removal, recombinant proteins or DNA from pathogenic Leptospira spp. The aim of this review was to highlight several promising vaccine candidates, considering their immunogenicity, presence in different pathogenic Leptospira serovars, their role in virulence or immune evasion and other factors.


| LEPTOSPIROSIS
Leptospirosis is an infectious disease caused by pathogenic bacteria of the genus Leptospira with an estimated 1 million new cases close to 60 000 deaths yearly worldwide making the most widespread zoonosis. 1Greatest prevalence is generally observed in tropical regions due to warm and humid climates, which favours Leptospira survival. 2he dissemination of this microorganism in the environment and water occurs through the urine of infected animals since this pathogen colonizes their renal tubules 2,3 (Figure 1).The transmission of this disease occurs mainly through mucosa or damaged skin when in contact with infected urine or contaminated water and soil. 4The most affected population is usually made up of people exposed to unsanitary conditions, especially in areas prone to flooding.These high-risk populations are generally lowincome, making leptospirosis a socio-economic disease. 5 addition, professionals such as garbage collectors, sewage workers, farmers, military personnel and veterinarians are vulnerable due to higher chances of exposure with infected animals. 2 Pathogenic leptospires can infect domestic and wild animals, and rodents are the main carriers, especially in urban areas. 6,7Humans are considered accidental hosts since they are unable to transmit the disease, and become infected after Leptospira penetration through mucous membranes or damaged skin.Once pathogenic Leptospira reaches the bloodstream, it can cause local or systemic injury.
Different clinical manifestations can be observed during the early and late phases of this disease 2 (Figure 1) and severity varies according to serovar, inoculum size, patient age and health status. 8During the early phase (3-7 days), leptospires disseminate rapidly and replicate in the bloodstream, later migrating to the liver, lung, spleen, kidney and other organs. 2,3,9Most infected individuals (~90%) are asymptomatic or develop only mild symptoms such as fever, headache, myalgia, nausea, vomiting, malaise and conjunctival hyperaemia. 4These symptoms are common to other diseases such as influenza and dengue, which leads to misdiagnosis and consequent underestimation of leptospirosis cases worldwide.However, the conjunctival suffusion is considered a characteristic clinical hallmark of leptospirosis early phase. 10An estimated 10%-15% of infected individuals present severe clinical characteristics, often after the first week of infection (late phase).However, these severe manifestations are sometimes observed even in the early phase.The classical manifestation of the severe form of leptospirosis is known as Weil's Syndrome.These patients may develop jaundice, renal failure, myocarditis, pancreatitis, anaemia, splenomegaly, chronic interstitial nephritis and pulmonary haemorrhage 2,3 (Figure 1).In most cases, interstitial nephritis is the main pathological alteration observed in these patients. 11,12In experimental murine models, this pathology is due to Leptospira outer membrane proteins (OMPs), which induce the expression of pro-inflammatory cytokines by renal cells, resulting in local inflammation. 13n addition, severe pulmonary haemorrhage syndrome (SPHS) is one of the most concerning complications of leptospirosis, with the main symptoms being cough, dyspnoea, haemoptysis and dense pulmonary haemorrhage.Due to these complications, approximately 50% of patients with SPHS die. 2,14The aetiopathogenesis of SPHS is not yet completely understood and requires further investigation.The presence of the Complement fragment C3c and immunoglobulins such as IgM, IgG and IgA in the alveolar septum of guinea-pig infected with L. interrogans serovar Copenhageni was explored first by Nally et al and later confirmed in the lung of SPHS patients. 15,16Leptospira LA_2144 protein hydrolyses platelet aggregating factor (PAF), which impairs blood clot formation.Gerbils infected with L. interrogans serovar Icterohaemorrhagiae serovar Lai had increased levels of LA_2144, which was associated with high mortality. 17The role of Leptospira sphingomyelinases was investigated since they are able to cause damage to A549 human alveolar basal epithelial F I G U R E 1 Leptospirosis transmission and infection cycle.Rodents are the main reservoirs of Leptospira, and when infected, they can release these bacteria through their urine, contaminating the environment.In addition, other animals, like dogs, horses and cattle are reservoirs, contributing to the spread of leptospires.However, different from rodents, these animals present more severe clinical manifestations, such as abortion.Once soil and water are contaminated by Leptospira, humans can become infected through direct contact with these animals.After penetration, the bacteria reach the bloodstream and spreads to the organs causing pathological damage.Leptospirosis disease progression can be divided into the acute phase, which is characterized by milder, flu-like symptoms, and the chronic phase, which has more severe clinical manifestations, such as pulmonary diffuse haemorrhage and renal failure.This figure was created using Canva (https://canva.com).cells. 18As pointed out before by Sato and Coburn, 19 several leptospiral proteins might affect intercellular junctions and damage endothelial cells, which could possibly lead to vascular hyperpermeability and tissue lesions in severe leptospirosis patients.

| LEPTOSPIRA
Leptospira are Gram-negative spirochetes bacteria with hooked ends that contain two periplasmic flagella at each pole.These flagella are responsible for rotation and translational movements, which allow the bacteria to move rapidly in viscous environments such as blood, interstitial fluid and connective tissues, contributing to their survival in the host and infection. 2,3The outer membrane is composed of LPS, proteins, lipoproteins and lipids, while the inner membrane has a double layer containing lipoproteins and transport proteins and is closely associated with the peptidoglycan layer 2,3 (Figure 2).
][22][23] LPS plays a key role in virulence and varies considerably between different serovars, with its biological and pathological properties being dependent on the lipid A, phosphate and methyl groups, the acylation pattern of the acyl fat chains and the length of the fatty acids. 2,24Alterations in Leptospira LPS lipid A and O antigen affect its recognition by pathogen recognition receptors and consequently the innate immune response.One possible explanation for that is phosphate methylation in LPS resulting in Leptospira recognition by Toll-like receptor (TLR)2 instead of TLR4 by human macrophages. 24,25(see below in Item 3).The O-antigen combined with membrane Leptospira lipoproteins prevents recognition by CD14, essential for TLR4-mediated endocytosis and activation of TIR domain-containing adapter-inducing interferon-β (TRIF)-pathway.TRIF signalling is important for microorganism elimination since it induces the production of IFN-β and nitric oxide, which contributes to the bacteria killing.Furthermore, higher amounts of O antigen in the LPS composition is associated with the severity of disease observed in chronic kidney infection. 26][36] Usually, lipoproteins are not transmembrane proteins, but they are strongly associated with the outer membrane, which is evidenced by their presence even after treatment with reagents that remove peripheral proteins from membranes. 37The lipoprotein moieties are projected from the outer membrane and are exposed on the surface, thus allowing interaction with components external to the bacteria and recognition by the host immune system early during infection (Figure 2).

LEPTOSPIRA INFECTION
The innate immune system is the first line of defence against infection, and it is important for recognizing and killing several microorganisms. 38The complement system is formed by more than 40 proteins and can limit bacterial infection after activation of the Alternative, Lectin or classical pathways during the acquired immunity.Once activated, this system will generate important biological functions, such as: (1) release of pro-inflammatory mediators, like C3a and C5a, which attract inflammatory cells and promote degranulation of mastocytes and basophils locally or in the blood, releasing important inflammatory mediators; (2) increase of reactive oxygen species released by phagocytic cells; (3) opsonization by C3b and iC3b, which facilitates internalization of microorganisms by phagocytic cells; (4) lysis of microorganisms after formation of the membrane attack complex; and, (5) B lymphocyte activation followed by increased antibody production against C3d-bound antigens, reviewed in 39-42.Pathogenic and non-pathogenic Leptospira species are able to initiate the activation of all three complement pathways. 43However, pathogenic species have several mechanisms (see below in Item 3.1) to escape from complement and survive in the host.In addition, the soluble complement proteins C3 43 and Ficolin-3 44 bind to leptospires and may act as soluble pattern recognition receptors.
TLRs are localized in the outer plasma membrane or in the endosome of several host cells, including macrophages, monocytes, neutrophils and dendritic cells, being crucial for pathogen recognition by the innate immune system.6][47] However, Leptospira LPS is different and less endotoxic than LPS from other Gram-negative bacteria (ex: Escherichia coli), which may explain the unexpected recognition of Leptospira by TLR2 instead of TLR4 by human macrophages. 24,25,48,49In rodents, especially mice, which are resistant to Leptospira infection, LPS is recognized by both TLR2 and TLR4. 49Leptospira can also be recognized by their flagellin subunits through TLR5. 48,50][53] However, Holzapfel et al 54 showed that the recognition of the Leptospira does not depend on TLR5 since TLR5 knock out and wild-type mice have similar numbers of leptospires in the blood, kidney or liver during acute infection.Nucleotide-binding oligomerization domain (NOD) receptors are present in the cytosol of phagocytic and epithelial cells where they sense the presence of bacterial peptidoglycan fragments, leading to the activation of nucleoprotein Nf-κb which increases the expression of inflammatory cytokines. 55Ratet et al 56 have demonstrated that neither NOD1 nor NOD2 receptors interfere with in vivo or in vitro Leptospira infection.
Neutrophil is one of the most important cells in the innate immune response against microorganisms.Besides phagocytosing and releasing microbicidal molecules, these cells extrude neutrophil extracellular traps (NETs) 57 that contain DNA, histone and proteases such as myeloperoxidase.NETs prevent the spread of pathogens by immobilizing and killing them, 58 independent of degranulation and bacteria internalization.NETs are often present at the site of infection, however, they have also been observed in blood vessels, where bacteria can be found in sepsis. 59In ex vivo experiments, it was demonstrated that Leptospira could induce NETosis from human neutrophils, suggesting that this mechanism is relevant for the early acute phase. 57Furthermore, saprophytic species were less effective in eliciting NETosis in human neutrophils and DNA degradation 57 when compared to pathogenic Leptospira.The pathogenic ones express proteins on their surface that act as nucleases and can degrade the NETs, evading the immune response as a result. 60,61he inflammatory infiltrate is predominantly made up of macrophages plus lower numbers of lymphocytes, plasma cells and monocytic cells.These cells release proinflammatory cytokines TNF-alpha and IL-6 that are associated with higher mortality in the most severe cases of leptospirosis. 9,13,62,63eptospira is an extracellular bacterium and consequently the production of specific antibodies is of utmost importance for controlling this infection in different animal species, including humans. 6During infection, the majority of circulating specific antibodies are anti-LPS, which poses challenges in our search for a universal anti-Leptospira vaccine that could provide immune protection against antigenically unrelated serovars.
Cell-mediated immunity is an important defence mechanism, although its role against Leptospira infection is still poorly understood.Some pathogenic Leptospira serovars escape from phagolysosomes into the cytosol in the human macrophage cell line acting as an intracellular pathogen. 64Thus, peptides from this pathogen can be complexed to MHC class I molecules and presented to CD8 + T-lymphocytes as reported in human patients, in which the presence of CD8 + T-lymphocytes specific for LigA-derived peptides was detected. 65,66

| Immune evasion
Pathogenic leptospires have developed several strategies to evade the immune system, thus allowing their survival in the host, causing several complications.The main mechanisms of evasion are recruitment of host regulatory proteins of the complement system 33,42,[67][68][69][70][71][72] ; secretion of metalloproteinases, 28,31,73 biofilm formation, 74 changes in LPS composition and expression of catalase. 75Pathogenic Leptospira also binds in vitro to plasminogen, laminin, fibronectin, thrombin, fibrinogen, extracellular matrix components and certain proteins of the Coagulation System. 76Consequently, these molecules collaborate to the establishment of Leptospira infection in the host.Plasminogen, for example, contributes to the negative feedback of complement system activation by generating plasmin, which may cleave C3b, C4b and C5, interfering in the activation of the three pathways of the complement system. 32,76In addition, the binding of leptospires with fibronectin, which is present in serum and extracellular molecules matrix (ECM), and with laminin, present in basement membranes, contributes to interactions directly with the ECM and to adhesion on cell surfaces. 68,77

LEPTOSPIROSIS VACCINES
Given the importance of this zoonosis, the development of prophylactic measures is highly desirable, with vaccination being the most viable strategy.Commercially available anti-leptospirosis vaccines are composed of whole inactivated Leptospira (bacterin), and are permitted for human use only in some countries such as Japan, Cuba, France and China. 78However, these vaccines only induce serovarspecific protection and short-term immunity, thus requiring annual immunization.In addition, serious side effects are observed after immunization with these vaccines. 79he search for a safe and effective immunizer is an important goal and several approaches have been investigated, such as (1) attenuated vaccines, which can mobilize both cellular and humoral immune responses; (2) recombinant protein and DNA vaccines, based on immunogenic proteins encoded by conserved genes in pathogenic species; and (3) lipopolysaccharide (LPS) vaccines, which activate the innate immune system and triggers a strong immunogenic response.
Thus, the aim of this review was to highlight several promising candidates for vaccine development against leptospirosis, taking into account characteristics such as immunogenicity, the conserved nature of their genes among pathogenic leptospires, their role in virulence or evasion of the immune response and other factors.Before describing the different leptospirosis vaccines, the animal models and the use of different adjuvants will first be discussed.

| Animal models for leptospirosis vaccines
Animal models are important for a better understanding of the biology of infections including transmission, colonization, pathogenesis and the immune response of leptospirosis.Golden Syrian hamsters are preferably the most used animal model to vaccine studies due to their susceptibility to this infection, and they exhibit clinical features of acute leptospirosis similar to those that are observed in patients.Hamsters are also chosen for maintenance of Leptospira strains virulence, determination of strain infectivity and infection routes and analysis of renal pathology. 802][83] The limited availability of commercial reagents to study the immune response in these animals is a major problem for most research laboratories.While reagents to study the immune response in mice are more easily obtained, rodents are generally resistant to Leptospira.One possible strategy is to use certain immunodeficient mouse strains, such as C3H/HeJ which are TLR4 deficient and consequently do not respond to E. coli LPS, often present as a contaminant in recombinant protein preparations used in experimental vaccines.5][86] Recently, C3H/ HeJ mice were used to evaluate the immune response after immunization with recombinant exotoxins (VM proteins) derived from L. interrogans serovar Lai, prepared with glucopyranoside lipid A/squalene as adjuvant.Once challenged, the immunized mice were protected from lethal challenge infection with L. interrogans serovar Canicola and presented lower leptospiral load in the kidney and liver.

| Adjuvants
Adjuvants are present in most inactivated vaccines and can amplify, guide or accelerate the immune response, enhancing protection against infection. 88,89Adjuvants can delay antigen release and induce cytokine secretion, including certain chemokines that contribute to the recruitment of immune cells like antigen-presenting cells (APCs).APCs are important for triggering the cellular response and interacting with B lymphocytes, increasing the production of specific antibodies.
The development of vaccines requires the search for adjuvants that enhance immunogenicity. 90Currently, the number of adjuvants licensed for human use is limited, among them, aluminium salts (Alum), emulsions (MF59, AS03 and Montanide), adjuvant systems AS04 and AS01 and virosomes (Common Ingredients in U.S. Licensed Vaccines|FDA).
Aluminium salt-based adjuvants are safe and widely used; it is known that in humans they are capable to induce humoral and CD4 + T cell helper cell response. 91In hamsters, this adjuvant in combination with Leptospira antigen subunit is able to induce mainly a humoral response and a limited T-cell response. 92,93In addition, aluminium salts are also used as vehicles for TLR agonist molecules.
AS04 is an adjuvant composed of alum and 3-O-desacyl-4′-monophosphoryl lipid A (MPL), a TLR4 agonist, and can lead to cytokine production and T lymphocyte recruitment, improving antigen presentation by dendritic cells, antibody production and memory B cells. 94,95The use of AS04 could be of interest since leptospires escape from TLR4 recognition by human macrophages. 24,25il-in-water emulsion-based adjuvants, on the other hand, are formulated with completely metabolized oils.When compared to aluminium salts, emulsions are stronger adjuvants as they promote local cellular activation and collaborate with antigen uptake by dendritic cells, leading to mononuclear cell recruitment. 90MF59 promotes an immunostimulatory environment that expresses several cytokines that activate dendritic cells by TLR-independent mechanisms, macrophages, neutrophils and eosinophils, inducing a humoral and Th1 response. 96S03 is also an oil-in-water emulsion; however, it contains vitamin E, an immunostimulant, which is associated with increased antigen uptake by monocytes, cytokine expression, recruitment of granulocytes and humoral response. 97In humans, AS03 activates an innate immune response and enhances CD4 + T cell response. 98,9901 is formed by a combination of immunostimulants, namely MPL and saponin QS21 that activates CD4 + T cells and improves the antibody response. 100This adjuvant is interesting because MPL induces an innate immune response by TLR4 while QS21 activates caspase-1 in subcapsular sinus macrophages, stimulating the innate and acquired immune response. 100,101AS01 can be drained to the lymph node where it can activate a larger repertoire of APCs and induce Th1 type immunity in mice. 102irus-like particles (VLPs) can easily reach lymphatic vessels and accumulate in lymph nodes where they stimulate the activation of B and T cells. 103,104For this reason, VLPs are being exploited to deliver adjuvants and antigens by binding them on the surface or inside the molecule.Among the possible cargoes that VLPs can carry are ligands of TLRs, activators of dendritic cells and antigens. 105n the last few years, the use of nanomaterials as adjuvants has been explored because of favourable biocompatibility. 106Nanomaterials as adjuvants are employed for antigen and vaccine delivery and among their actions is sustained and controlled antigen release, which contributes to increased uptake by APCs. 107,108In addition, the antigens when carried in nanoparticles are protected from proteolytic degradation that occurs in vivo, demanding lower antigen dosage in the formulation of immunizers.Given that leptospires can escape from TLR4 recognition by human macrophages, the use of adjuvants that stimulate the Th1 and Th2 responses and increase the production of memory cells is ideal.

| Prime-boost protocols
Most vaccines use the same formulation when the protocol involves the application of two or more doses (known as homologous prime-boost).On the other hand, protocols employing different formulations have been used to boost the immune response (known as the heterologous primeboost regimen).This strategy may be especially efficient to increase the immunogenicity of DNA vaccines that activate both the humoral and cellular immunity. 79,109,110eterologous doses can differ by the amount of protein, presence or change of adjuvant, vector and use of different antigens.This strategy increases the production of neutralizing antibodies and activation of cytotoxic TCD8 + T lymphocytes when compared to homologous prime-boost regimens. 111,112 prime-boost regimen for the application of an experimental leptospirosis vaccine was first used by Feng et al 113 when they demonstrated that a heterologous protocol (primed first with DNA and later boosted with protein) resulted in better protection when compared to homologous protocols (primed with DNA/boosted with DNA or primed with protein/boosted with the same protein (Table 1).Similar results were reported by other groups.79,[113][114][115] However, Buaklin et al observed better protection against Leptospira using a homologous prime-boost regimen.  Thirst anti-leptospirosis vaccine was developed over a century ago and were composed of inactivated Leptospira preparations, known as bacterins.116 So far, only bacterins have been licensed for veterinary use and, with restrictions, in humans.Bacterins are composed of one (monovalent) or more (polyvalent) pathogenic Leptospira serovars chemically inactivated using ethanol, formaldehyde, phenol or by physical methods such as heat, irradiation or freezing.
These vaccines induce mostly a humoral immune response based on the production of specific antibodies against Leptospira LPS.Considering that LPS is a T lymphocyte-independent antigen, clonal expansion of B lymphocytes, high-affinity antibodies and memory cells are not produced in robust amounts.Consequently, these bacterins induce only short-term immune protection, require annual boosters and confer protection restricted only to the Leptospira serovars that are present in their composition, due to the variability of the leptospiral LPS. 4 Besides the presence of LPS, other components in Leptospira bacterins may cause serious adverse effects, such as fever, nausea and pain.
The use of bacterins in humans is restricted to only a few countries, such as France, Japan, China and Cuba.In these regions, vaccine application is more directed to the populations at risk, for example, mineworkers, sewage workers, agricultural workers (especially in rice plantations), veterinarians and communities without adequate sanitation. 78n France, the use of a vaccine composed of formaldehyde-inactivated Leptospira serogroup Icterohaemorrhagiae has been allowed since 2007 and clinical trial results showed a seroconversion rate of 96%-100% after receiving two doses, requiring a repeat procedure every 1-2 years. 117wo vaccines are currently available for use in China, a phenol-inactivated polyvalent whole-cell vaccine and a bivalent outer membrane vaccine. 118,119The bivalent inactivated vaccine is composed of the envelope antigens of Leptospira serogroups Icterohaemorrhagiae and Hebdomadis, and has been used since 1993 due to trial results demonstrating 95%-100% seroconversion with only one subcutaneous dose annually. 118The polyvalent vaccine, on the other hand, is composed of Leptospira serogroups Lai, Linhai, Autumnalis, Canicola, Pomona, Australis and phenol inactivated Hebdomadis serovars and has been used in China since 2004 after exhibiting a 95%-100% seroconversion rate after two doses and repeated annually. 119imilarly, a polyvalent vaccine was used in Japan, composed of Leptospira Australis, Autumnalis, Hebdomadis and Copenhageni serovars inactivated by formalin. 120hile in Cuba, a trivalent vaccine has been used in the population since 1998, being formulated by Leptospira Canicola, Copenhageni and Mozdok serovars inactivated with formaldehyde and absorbed with aluminium hydroxide.In this case, the immunization depends on the administration of two intramuscular doses with an interval of 6 weeks. 121Even with complete vaccination, the risk of new outbreaks remains since bacterins confer short-term protection and are restricted to the serovars present in their formulation.In addition, high manufacturing costs and low reproducibility of results have interfered with vaccine success. 122Bacterins are widely used to immunize dogs, pigs and cattle, mainly to control leptospirosis dissemination in animal production, but their efficacy is only partial even when formulated by local serovar isolates. 123ecently, the efficacy of five currently commercial leptospirosis vaccines for pigs and cattle was evaluated using hamsters as animal model.These animals were split into groups, and each received one of the vaccines and after 15 days the animals were challenged with a local strain of Leptospira.The results showed that only two out of five vaccines were effective in protecting the animals. 124imilar results were observed in a second study, in which two out of nine commercial vaccines were effective in protecting Brazilian dogs against leptospirosis. 125n cattle, the induced immune response was evaluated based on three different anti-leptospirosis vaccines, two of them being monovalent vaccines formulated from Hardjo serovar and a pentavalent vaccine also containing this serovar.It was observed that monovalent vaccines were able to induce protection against Hardjo serovar infection, while pentavalent was ineffective.Apparently, according to the results, immunity against leptospirosis in cattle depends on a cell-mediated immune response, since the monovalent vaccines induced lymphocyte proliferation, IFNγ and IgG antibody production. 126n conclusion, besides the widespread use in domestic animals and in humans in few countries, bacterins do not confer adequate efficacy and safety even when they are able to induce high titres of circulating anti-LPS antibodies.

| Attenuated vaccines
Live attenuated vaccines are one of the most widespread immunization approaches to combat important diseases such as influenza, tuberculosis and salmonellosis. 127onsiderable advances have been made towards an attenuated Leptospira vaccine, however, managing the attenuation while maintaining immunogenicity remains a major challenge since the genetic mechanisms responsible for the aetiopathogenesis of leptospirosis are poorly understood. 128ttenuated vaccines mimic a natural infection, triggering activation of strong cellular and humoral immune responses, and inducing long-term immunity with few doses.Leptospira can be attenuated after repeated passages in culture medium or by genetic manipulation.An example of this latter strategy is the use of Manilae mutant strain M1352, which carries altered LPS and does not cause leptospirosis disease in hamsters. 129The immunization with these attenuated bacteria was able to protect animals against challenging doses of serovar Manilae or Pomona better than those immunized with bacterins.A protective response against other serovars different from Manilae M1352 (heterologous challenge) was observed even in the absence of specific anti-LPS antibodies. 129hese results also demonstrated that a single dose of M1352 vaccine was sufficient to protect all animals, preventing kidney colonization and reducing lung damage, while a single dose of bacterin failed to protect them. 129e same attenuated vaccine was capable of protecting against Grippotyphosa, Canicola and Australis serovars but not against Autumnalis serovar. 130nother example of an attenuated vaccine used a mutant L. interrogans serovar Copenhageni deficient in flagellar-coiling protein A (fcpA).This deficiency impairs translational motility and consequently the ability to penetrate mucous membranes, decreasing renal colonization and lethality in animal models, while still causing transient bacteraemia during the first days post-inoculation. 53 single dose of the vaccine was sufficient to confer protection to hamsters against heterologous and homologous challenges, whereas a single dose of bacterins conferred only partial protection against homologous challenge.Furthermore, the inhibition of renal colonization occurs in 80% of those animals challenged with the same serovar. 131ttenuated vaccines have proved to be promising since they can confer some protection against multiple serovars and promote antibody production against conserved proteins.However, even being more immunogenic, inducing higher antibody titres and inducing longer term immunity when compared to bacterins, these vaccines have some limitations, such as the maintenance of viability when stored and the potential for reversion to a virulent bacterial state, and failure to confer renal sterilizing status.The objective of a low-LPS whole-cell vaccine is to target a response against conserved surface membrane protein antigens and so promote long term and broad protection against serovars. 132A recent study developed protocols to produce low LPS vaccines by treating L. interrogans serovar Copenhageni and Canicola with butanol, or butanol followed by inactivation with formaldehyde.These treatments resulted in a 45% reduction of LPS on the bacteria surface. 132The resulting bivalent (containing both serovars) or monovalent (containing one serovar) vaccines had their immunizing ability evaluated after administering two doses to a group of hamsters, which later were challenged with the same (homologous) or different (heterologous) serovars.The results showed that the vaccines containing the Canicola serovar treated with butanol and formaldehyde had an 80% protection rate in heterologous challenges.However, the same protection rate was not observed when Copenhageni was used to immunize followed by challenge with Canicola, thus demonstrating the variability of serovars in eliciting cross-protection. 132urthermore, it was observed that immune serum recognized several conserved proteins present in pathogenic species, suggesting that their cross-protective capacity is possibly due to surface proteins. 132Thus, while the use of bivalent vaccines composed of virulent serovars with low LPS is promising, further studies are required to identify the proteins responsible for this immune protection.

| Recombinant protein vaccines
Recombinant protein vaccines contain a target protein and usually an adjuvant to boost the immune response.These vaccines are effective in protecting against many infectious diseases such as hepatitis B and HPV. 133,134he search for vaccines based on conserved antigens among serovars and with less adverse effects is an important issue in leptospirosis research. 109A list of several recombinant proteins including LipL21, LipL32, LipL41, Loa22, Lsa proteins, LRR proteins, Len proteins, LemA and Lig proteins among others, which have been investigated as potential vaccine candidates is given in Table 2.
The first experimental vaccine prepared with recombinant proteins was developed in 1999 based on the lipoprotein LipL41 and the porin OmpL1, 135 both abundant outer membrane proteins that can be easily removed from the Leptospira membrane.The results revealed that the recombinant vaccine was able to promote homologous protection in one of the three experiments. 135owever, subsequent studies re-evaluated the protective ability of LipL41 and OmpL1, and the results were not conclusive about the ability of these proteins to induce cross-protection or sterilizing immunity, when used together or separately. 136ulti-epitope vaccines developed with recombinant proteins are an interesting approach.Since LipL21, LipL32 and LipL41 are the most abundant lipoproteins on the Leptospira surface, it was investigated whether these proteins alone or in combination might provide protection in guinea pigs against challenge. 82Animals immunized with rLipL32 had a protection rate of about 50%-67%, while animals vaccinated with only rLipL21 or rLipL41 had survival rates at or below 42%. 82Those inoculations formulated with more than one recombinant protein showed greater protection when compared to those prepared with only one lipoprotein.A total of 83.3% of the guinea pigs that received rLipL21 + rLipL32 were protected.The combination of rLipL21 + rLipL41 resulted in 58.3% survival while the combination of rLipL32 + rLipL41 resulted in 67%-75% survival. 82In addition, the animals that received immunizations formulated with the combination of the three lipoproteins had a 91.7% protection rate, being the most promising among the recombinant vaccines tested.However, none of these immunizers induced sterilizing immunity in these animals. 82Since LipL32 induced the highest rates of protection, several research groups have explored the combination of LipL32 with other virulence factors like Lig proteins.
Lig proteins have been considered interesting vaccine candidates for leptospirosis since they are expressed only in virulent strains.Moreover, these proteins can induce specific antibody production in infected patients. 137LigA and LigB are able to bind to extracellular matrix components such as fibronectin and Complement regulatory proteins like Factor H 33 and C4b binding protein. 71Recombinant vaccines prepared with different fragments of LigB and a C-terminal fragment of LipL32 protected against acute Leptospira infection and lung damage and generated a good antibody response with reduced mortality. 138nother study investigated the protective capacity of a recombinant vaccine prepared with LipL32 and LigA non-identical fragment (LigANI; domains 7-13), together or separately.Despite the fact that these proteins induce a humoral response, neither of them was protective against serovar Manilae. 139This contrast with another study, where LigANI in the presence of Freund's complete and incomplete adjuvant conferred 67%-100% immunoprotection in challenges against serovar Copenhageni, despite not conferring sterilizing immunity. 137 recombinant subunit vaccine developed from LigB conserved region (amino acids 131-645) was able to protect hamsters after challenge, and promoted sterile immunity in surviving animals, and induced significant IgG antibody titres. 140Based on this, a subsequent study protected all animals against the challenge.However, neither of the immunizers were capable of conferring sterilizing immunity. 141he C-terminal or variable region of LigA (LAV), mainly domains 10-13, was enough to induce protection against leptospirosis in hamsters. 137,142In a study by Varma et al, 92 LAV was tested with alum (LAV-Alum), AS04 (LAV-AS04) or Montanide ISA720VG (LAV-M) to evaluate the immune response in mice after being immunized.Only LAV-AS04 and LAV-M induced high levels of LAV-specific antibodies, Th1/Th2 responses with significant levels of IgG1/IL-4 and IgG2/IFN-γ, cytotoxic T and B-and T cell memory cells in C57BL/6 mice, while LAV-Alum was only capable of induces Th2 response by IL-4. 92Similar results were observed with hamsters immunized with LAV-AS04 and LAV-M.LAV-M and LAV-AS04 protected 83% and 67%, respectively, while LAV-Alum group only 50%. 92A recent study evaluated the protective capacity of LigANI (domains 7-13) using new adjuvants: LMQ (neutral liposomes containing monophosphoryl lipid A and Quillaja saponaria derived QS21 saponin), LQ (neutral liposomes containing QS21 saponin), LQuil (neutral liposomes containing Quillaja saponaria derived QS21 saponin) or SQuil (squalenein-water emulsion with QuilA saponin). 143Hamsters immunized with LigANI and any of these adjuvants presented 50%-62.5% protection when challenged with L. interrogans serovar Pomona. 143urthermore, reverse vaccinology (see below in item 'Reverse vaccinology') have revealed new potential vaccine targets like LenA, LcpA and OmpA-like proteins.A vaccine containing recombinant proteins LenA and LcpA was able to protect 80% of the animals challenged with serovar Copenhageni and induced high IgG titres. 144In addition, increased plasma levels of IFN-γ, IL-4 and IL-17 were observed, suggesting activation of the cellular immune response. 144mpA-like proteins, such as Loa22, are interesting vaccine candidates because of their role in virulence in several bacteria, like Escherichia coli.Three outer membrane proteins containing the OmpA domain, Lp4337, Lp3685 and Lp0222 were able to minimally protect hamsters against challenging doses of serovar Pomona. 145In addition, Lp4337 induced a strong immune response, which provoked increased serum levels of IFN-γ, IL-4 and IL-10, indicating a cellular immune response.Despite these positive results, those vaccines were unable to provide sterilizing immunity. 145ecombinant vaccines containing Lsa46 and Lsa77 proteins associated with alum were tested in hamsters. 93he animals immunized either with Lsa46 or Lsa77 developed an intense humoral response with a Th1 and Th2 cellular profile, and respectively, 44% and 46% protection. 93ence, the development of recombinant vaccines is a promising field to combat leptospirosis, since the administration of protein antigens activates T-dependent signalling, which leads to clonal expansion of B cells and production of more specific antibodies.Due to this, several surface proteins have been identified and are being studied as possible vaccine candidates by using reverse vaccinology technologies and have been shown to be immunoprotective in some cases.However, different groups, as pointed below obtained divergent results, probably due to different challenge doses, variable virulence of leptospiral strains and improper folding of recombinant proteins.

DNA vaccines
When compared to bacterins, DNA vaccines have many advantages, such as the ability to induce cellular and humoral immune responses without the risk of microorganism replication, low-cost and large-scale production, and the storage stability.The construction of vectors that code for several antigens in a single vaccine may be advantageous for leptospirosis since they have membrane proteins conserved among serovars.These vaccines are composed of plasmids, which contains the genetic sequences of one or more target proteins and the regulatory elements that allow the transcription and translation of these sequences.DNA vaccines are usually administered intramuscularly, which contributes to the inflammatory response and helps to trigger an immune response against the antigens as reviewed in 79.
Over the past few years, scientists have been working to identify promising candidates to develop a DNA vaccine against leptospirosis.Forster et al 146 evaluated the immunoprotective capacity of a DNA vaccine formulated with the commonly conserved region of LigA and LigB, and the results demonstrated that 83.3% of the animals, which received one dose of the DNA vaccine and one dose of the recombinant proteins with adjuvant survived.In addition, the immunization triggered sterilizing immunity and the hamsters had less pathological tissue damage. 146A more recent study used a recombinant Lig chimera (rLC) formulated with LigA (domains 11-13) and LigB (amino acids 131-645) and was tested with different adjuvants. 147The results showed that immunization with rLC/alum or rLC/Montanide protected 100% of hamsters when challenged with L. interrogans serovar Copenhageni L1-130. 147Furthermore, when a DNA vaccine containing the LC gene was used, only 25% of the animals survived after two doses.Finally, all the animals immunized with a heterologous prime-boost protocol (DNA/protein) survived; however, this regimen did not confer sterilizing immunity. 147NA vaccines coding only the lipoprotein gene LipL32 did not result in significant immunoprotection, 83 however, when associated with Loa22 protein, high immunogenicity was observed in mice. 114A DNA vaccine developed from a plasmid coexpressing Loa22 and LipL32 genes was administered to mice and the results indicated higher antibody production compared to animals that received the DNA vaccine expressing each gene on a separate plasmid.In addition, this approach of coexpression is more practical, due to the shorter preparation time and cost.In the same study, the immune response was also increased when the second dose of immunization was composed of recombinant proteins and adjuvant. 114 DNA vaccine containing the whole LipL21 gene was tested in guinea pigs.Specific antibody production and an increase in spleen lymphocytes proliferation rates were detected in the immunized group.81 When the animals were challenged with L. interrogans serovar Lai all survived, including the control group.The immunized animals presented only mild lesions in the liver and kidney compared to the controls.81 Thus, the development of DNA vaccines against leptospirosis is a promising field that remains to be addressed, since this vaccination strategy has several advantages.Among them, these vaccines can induce robust immune responses without Leptospira replication risk, and stimulate cellular and humoral responses, efficient large-scale production at low cost, and finally offer the possibility of constructing a vector containing the gene for several antigens.79

Reverse vaccinology
Since the advent of high-throughput genome sequencing technology, and the subsequent cost reduction, databases containing genomic information from various species have increased dramatically, leading to a demand for improved bioinformatics analysis tools. 110,136At the beginning of the twenty-first century, for the first time, the entire genome of a pathogenic L. interrogans serovar Lai serogroup Icterohaemorrhagiae was sequenced by Ren et al and L. interrogans serovar Copenhageni by Nascimento et al, since then, other serovars and saprophytic leptospires had their DNA sequenced. 148,149everse vaccinology has been an interesting approach to search for prospective vaccine antigens, and is based on bioinformatics techniques, genomic sequences and data analysis.However, for reverse vaccinology to be successful, it is necessary to develop a high-throughput cloning strategy for the selected proteins.As expected, the immunogenicity of these candidates must be first confirmed by in vitro and in vivo assays before evaluating if they confer protection in immunized animals when challenged with pathogenic Leptospira serovars.
The lipoprotein LemA was firstly identified by Hartwig et al 150 using reverse vaccinology and the recombinant protein was able to induce the production of specific antibodies in mice.Anti-LemA antibodies were detected in the serum of severe leptospirosis patients.Later, the same group demonstrated that a prime-boost DNA vaccine resulted in better protection (87.5%) than the recombinant (rLemA+ Alum -50%) and DNA (pTARGET-lemA-62.5%)group when the animals were challenged with L. interrogans Icterohaemorrhagiae. 151 A subsequent study used nanoparticles (halloysite clay nanotubes; HNTs) and amine-functionalized multi-walled carbon nanotubes (NH2-MWCNTs) to improve the immunogenicity of a homologous prime-boost DNA vaccine (pTARGET/lemA). 152wo doses were administered 3 weeks apart, and the results showed that 83.3% of hamsters vaccinated with NH2-MWCNTs-pTARGET/lemA survived after challenge with a lethal dose while 66.7% of the animals that received the HNTs-pTARGET/lemA vaccine were protected and only 50% of the animals treated with pTARGET/lemA survived. 152rassman et al 153 identified 26 potential candidates related to pathogenesis, outer-membrane proteins and LPS composition by reverse vaccinology.A total of 22 candidates were tested in hamsters.Most of them induced significant production of specific IgG antibodies, however, only four proteins induced significant protection in two out of four experiments, but none provided protection in all of them. 154The authors concluded that hamsters might not represent the best animal choice for these studies due to the scarcity of species-specific commercial reagents required to monitor the immune response.They suggested the use of C3H/HeJ mice, since these animals are quite susceptible to leptospirosis.In addition, C3H/HeJ mice are TLR4 deficient and therefore would not respond to the possible presence of contaminant E. coli LPS in recombinant protein preparations.Moreover, there are plenty of options of commercial reagents to monitor the immune response against Leptospira during vaccination in murine models.
In silico studies, two Leptospira LRR proteins, rKU_ Sej_LRR_2012M (LRR2012) and rKU_Sej_LRR_2271 (LRR2271) from L. borgpetersenii were identified and subsequently tested in a hamster model. 155The study observed that 50% of animals immunized with LRR2012 and 75% of animals immunized with LRR2271 recombinant proteins were protected when challenged with Pomona serovar.In addition, the LRR2271 protein induced a strong humoral response and activated the expression of cytokine genes related to Th1 and Th2 responses. 155

| CONCLUDING REMARKS
Ideal vaccines against leptospirosis must confer sterilizing protection, due to a robust humoral immune response and an efficient activation of cellular immunity.These vaccines should provide a lasting and effective immunological memory, generate antibodies with better affinity and protect against most serovars.Another important requirement is a relatively low cost to be available to the most affected populations, which in general are from countries with severe economic restrictions and lack of adequate sanitary conditions.
At this moment, the use of commercially available antileptospirosis vaccines is restricted to bacterins that induce a short-term immunity, with side effects and are serovarspecific.New approaches are under investigation, using Leptospira recombinant proteins, DNA, LPS depleted and attenuated vaccines.
We believe that promising vaccine candidates against leptospirosis will include surface-exposed proteins that are highly conserved among pathogenic Leptospira serovars and capable of activating a strong T cell immune response.In this way, multi-antigenic vaccines could trigger better protection.
Over the past several years, among the most studied candidates, LigA produced results that are more promising.Since LigA is not present in all pathogenic serovars, it would be wise to continue investigating this recombinant protein complexed with different antigens.
The currently available genomic and proteomic sequencing data of leptospires and the use of bioinformatics approaches, especially reverse vaccinology, have led to the identification of new potential vaccine candidates that still need to be tested for their immunogenic capacity in vivo.
In addition, the use of new adjuvants, nanomaterials and prime boost have contributed to increase the efficacy of experimental vaccines against leptospirosis.Further studies are essential to understand the pathogenicity and immunogenicity of leptospires, which will contribute to the development of a universal vaccine.Furthermore, many technological advances in vaccine production have been made in response to the SARS-CoV-2 pandemic, such as mRNA and DNA vaccines.These new vaccine production platforms should be considered in the near future for development of other experimental leptospirosis vaccines.

F I G U R E 2
Leptospira membrane architecture.A double membrane forms the cell wall of Leptospira.The outer membrane is composed of lipopolysaccharides (LPS), surface-exposed proteins (LigA, LigB, LcpA, LenA, LenB, Loa22, LipL41, LipL32, LipL21, Lsa and LRR proteins), porin L1 (OmpL1), among others.The inner membrane (IM) is closely associated with the peptidoglycan cell wall, which is inside the periplasm space (PS).The flagellar hook is responsible for Leptospira motility and is linked to the IM and located inside the PS.Pathogenic Leptospira secrete proteins into the medium.Figure based on 156,157.This figure was created using Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported Licence: https://smart.servier.com, and Canva (https://canva.com).

T A B L E 1
Strategies and antigens used in the development of leptospirosis vaccines.
All authors (I.R.A.; T.A.A.; and L.I.) wrote the article and approved the final revision of the text.ACKNOWLEDGEMENTS This study was supported by research funding from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2017/12924-3 (L.I.) and 2017/10208-9 (T.A.A.), 2020/16104-3 (I.R.A).We thank Dr. Angela Silva Barbosa from Butantan Institute, Prof. Dr. Shaker Chuck Farah from Institute of Chemistry, University of Sao Paulo and Milena Carvalho Carneiro from Institute of Biomedical Sciences for the critical reading of this text.
Vaccine candidates against leptospirosis.
T A B L E 2