BCG was proposed as a recombinant vaccine vehicle for expressing heterologous antigens a long time ago because of its notable features. It is safe and has been administere billions of individuals with nonspecific immunostimulatory effects. BCG can be administered soon after birth, is highly immunogenic, and has prolonged persistence inside macrophages, thereby inducing long-lasting humoral and cellular immune responses. Moreover, it provides the possibility of generating T cell-mediated immunity against the cloned heterologous antigen [47,48].

Several studies have reported the use of recombinant BCG (rBCG) expressing foreign antigens from diverse pathogens. Seixas et al. (2007) [49] produced and characterized a rBCG-expressing LipL32 as an antigen against leptospirosis. Animals immunized with different constructs of rBCG/LipL32 showed the seroconversion of total anti-LipL32, with a higher titer than wild-type BCG, which was used as a control. Oliveira et al. (2019) [50] used rBCG in combination with a multiepitope protein approach based on the leptospiral antigens LipL32, LemA, and LigA (domains 11–13) to find a way to elicit a protective immune response and prevent renal colonization. Protective immunity induced by chimeric rBCG conferred 80–100% survival; no bacteria were detected in renal cultures, and qPCR data from the cultures were negative. Dorneles et al. (2020) [51] investigated the same antigens used in different chimeric constructs to transform BCG. Recently, Bettin et al. (2022) [52] developed an rBCG vectored vaccine expressing a chimeric antigen based on the TonB-dependent receptor (TBDR) epitopes (LIC10896, LIC10964, and LIC12374) from L. interrogans. Hamsters vaccinated with the rBCG:TBDRchi construct were fully protected from lethal leptospirosis, whereas the same recombinant protein as a subunit vaccine failed to protect animals (44.4% survival, p > 0.05; data not published). In studies performed by Oliveira et al. (2019) [50], Dorneles et al. (2020) [51], and Bettin et al. (2022) [52], it was shown that rBCG constructs were able to induce immune protection and prevent renal colonization against challenge with virulent L. interrogans. The combination of rBCG and chimeric multiepitope proteins appears to be a promising alternative against leptospirosis [53].

Lactobacillus species represent an attractive tool for vaccine production due to their generally regarded as safe (GRAS) status, reported adjuvant properties due to the peptidoglycan layer of some strains [54], and mucoadhesive ability, which is excellent for safe mucosal delivery vehicles of prophylactic and therapeutic molecules [55,56]. In addition, it is characterized by easy genetic manipulation and the availability of well-defined industrial production processes [57]. Different Lactobacillus-based vaccine prototypes have been developed and administered via the mucosal route, leading to both mucosal and systemic immune responses against expressed antigens [58]. A recent study showed that repeated oral administration of L. plantarum, a commensal probiotic and agonist of TLR-2 and NOD2, to C3H/HeJ mice mitigated acute leptospirosis and reduced renal lesions, although it did not prevent renal colonization against intraperitoneal infection with L. interrogans strain Fiocruz L1-130 [59].

Infected mice pretreated with L. plantarum [59] exhibited a 50% reduction in fibrosis and produced fewer transcripts of ColA1 than the negative control group (infected but pretreated with PBS), suggesting that oral treatment may have reduced the accumulation of collagen in the tubulointerstitial spaces, thereby preventing severe kidney pathology. Additionally, pretreatment with L. plantarum also induced the occurrence of mononuclear lymphocyte infiltrates, tubular damage, and higher interstitial nephritis scores than infected controls pretreated with PBS. The authors suggested that the pretreatment with L. plantarum in mice infected with L. interrogans triggers a complex myeloid and T-cell response that manages the deployment of monocytes from lymphoid tissue and the recruitment of neutrophils and macrophages to the kidney. Furthermore, the presence of myeloid cells in the kidney may be associated with a reduction in the observed pathogenesis. The use of L. plantarum as an immune modulator associated with a vaccine strategy against leptospirosis seems to be valuable and deserves further investigation [59].

E. coli can also be used as a delivery system, usually through the oral route. Oral approaches to delivering vaccines offer several advantages over other delivery systems, including convenience, cost-effectiveness, and the ability to induce both local and systemic immune responses, which have been well-described before [60]. Recently, an oral immunization based on a lipidated form of LigA using E. coli as a delivery system revealed a correlation between IgG levels and the survival of immunized hamsters [61]. The vaccine formulation was based on E. coli expressing a fusion of the OspA lipoprotein signal peptide with LigA immunoglobulin-like domains 7–13. The OspA signal peptide resulted in the lipidation of LigA and the incorporation of LigA 7–13 into the E. coli membrane fraction. This lipidation was able to modulate the immune response induced by oral immunization, as observed with the OspA formulation [62], which appeared to be important for overcoming oral tolerance by inducing a Th1/Th2 immune response [62].

Hamsters that were immunized by oral gavage with E. coli expressing the lipidated LigA7-13 antigen and challenged by intraperitoneal and intradermal routes developed a protective immune response to lethal challenges by L. interrogans serovar Copenhageni (37.5% and 62.5%, respectively). However, prevention against renal colonization was not observed [61]. In both experiments, LigA7-13-immunized animals that survived had higher antibody levels after 2 weeks of immunization than control-immunized animals. The natural adjuvant capabilities of E. coli and the lipidation of LigA7-13 may contribute independently to the production of highly immunogenic oral vaccines [61].

Salmonella is an intracellular pathogen that remains restricted to the endosomal compartment of eukaryotic cells and resists nonspecific killing mechanisms [63]. Recombinant antigens expressed in attenuated Salmonella strains can be delivered orally to mucosal surfaces, inducing a protective immune response against various targeted pathogens [64]. The invasive characteristics of Salmonella make it able to elicit B- and T-cell memory responses and confer upon Salmonella the significant potential to elicit long-lasting immunity. Samakchan et al. (2021) [65] evaluated the immune response of a recombinant attenuated Salmonella vaccine (RASV) prototype, NRSL32. This potential model was composed of an in-frame fusion between nucleotides encoding the N-terminal segment of the SspH2 effector protein containing the T3S signal and the leptospiral antigen LipL32. NRSL32 is an interesting candidate for the development of oral bacterial vectors [65].

The antigen delivery platform of RASV is based on the natural infection of intracellular Salmonella, which translocates virulence-effector proteins into host cells through T3SS. NRSL32 has demonstrated the ability to elicit effective immune responses by delivering LipL32 protein using SPI-2 T3SS [65]. In leptospirosis, the humoral immune response is the major protective immune mechanism against infection [66]. This RASV model stimulates adaptive humoral, cell-mediated, and mucosal immune responses. Significant titers of total IgG and IgA against rLipL32 were detected for a long time after vaccination. The stimulated antibodies were capable of specifically binding to LipL32 on the surface of pathogenic Leptospira spp. [65]. Moreover, this platform was capable of stimulating both Th1- and Th2-biased responses, although lethal challenge studies have not yet been conducted.

Viral vectors are based on modified viruses that can deliver genetic code for antigens to the target host. Several viral vectors are available for recombinant vaccine development, with differences in virion type, particle size, transgene capacity, and replicative cycle [67,68]. Viral vectors are characterized by their ability to induce cellular and potent antibody responses, high immunogenicity with intrinsic adjuvant properties, and the possibility of administering a single-dose schedule with long-lasting immunity [67,69]. Nevertheless, concerns about reduced effectiveness caused by previous exposure to the vector and the complexity of design and manufacture are challenges that need to be overcome [68]. Despite the many advantages associated with this system and its broad use for delivering antigens from specific pathogens for more than four decades [69], there is only one record of a study using this platform for leptospirosis vaccine development.

Branger et al. (2001) [70] produced a vectorized vaccine using recombinant adenovirus expressing the LipL32 protein from the serovar autumnalis, which provided 87% protection in gerbils challenged with L. interrogans serovar Canicola. In the same study, a similar OmpL1 adenovirus construct failed to protect animals. Despite the protection observed for the LipL32 adenovirus formulation, the negative control groups showed high survival rates (47–50%), which may indicate a sublethal challenge. It is likely that the limited use of viral platforms in leptospiral antigens relies on the difficulty of designing vector constructs, the high level of biosafety required, and other manufacturing challenges.