Adjuvants play an important role in the efficacy of vaccines, contributing to increase the potency and kinetics of the immune response and to direct the type of this response. In the induction of immune responses, protein-carbohydrate interactions are essential since they represent cell-to-cell contacts, leading to crosstalk among cells and consequently their activation . Lectins can play this role given that it may bind carbohydrates and function as pattern recognition receptors (PRRs) in host cells . Several plant lectins have shown adjuvant or immunostimulatory effects in different infections [25, 27, 29, 37]. Among them, the lectin from the S. carinatum latex (ScLL) has been recently studied for its peculiar roles in different experimental models, having an adjuvant effect in cutaneous leishmaniasis  or immunoregulatory action in asthma murine models .
Given that currently there is no effective treatment for neosporosis, control methods are necessary. Among them, the removal of infected animals is the only way to reduce incidence of the disease in herds. However, given the high prevalence of N. caninum and the low occurrence of abortion in herds (less than 5% of cows abort), it does not make sense to cull infected animals. Usually animals are culled due to poor reproductive success, which Neospora infection may be one reason. Thus, vaccination may be a viable method of reducing the incidence of neosporosis and improving the reproductive performance in herds . In this context, several studies have been conducted to obtain reliable formulations in vaccination strategies in order to protect against N. caninum infection [38–40], but studies using plant lectins as adjuvants in neosporosis are scarce in the literature. Recently, we evaluated the role of lectins (ArtinM and Jacalin) from the jackfruit (Artocarpus integrifolia) and showed that ArtinM, a D-mannose binding lectin, presented stronger immunostimulatory and adjuvant effect than Jacalin, a D-galactose binding lectin, in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge .
In the present study, we investigated if another D-galactose binding lectin, ScLL obtained from the Synadenium carinatum latex, could play immunostimulatory role and adjuvant effect in mouse immunization against N. caninum infection as already reported in other infection models. First, we evaluated the in vitro cytokine production by dendritic cells stimulated with antigen, lectin or by the combination of both. We found that NLA, ScLL or NLA + ScLL produced the highest levels of all cytokines investigated (TNF-α, IL-6, IL-10 and IL-12), showing a typical profile of a dose-dependent response, when tested in their greatest concentrations. Also, it was clearly evident the synergistic effect of NLA and ScLL, since cells stimulated with NLA + ScLL secreted the highest levels of all cytokines. These findings indicate that ScLL was able to increase the immunogenicity of the antigen, demonstrating its adjuvant role in the in vitro experiments. In addition, as dendritic cells are professional APCs and have critical functions in modulation of the primary immune response to intracellular parasites, this can be particularly important in vaccination strategies for N. caninum. In this context, the TLR2 innate immune receptor is decisive for dendritic cell activation during N. caninum infection, by using the TLR2/MyD88 recognition pathway with posterior expression of costimulatory and antigen-presentation molecules, T lymphocyte programming, and pro-inflammatory cytokine production, associated with an appropriate IgG synthesis against the parasite . Furthermore, ScLL itself has already shown to increase the expression of IL-12, IL-1 and TNF-α mRNAs in macrophages pretreated with the lectin (10 μg/mL) and infected with L. amazonensis.
The kinetics of the humoral immune response after immunization of mice with NLA, ScLL or both revealed a distinct profile between the IgG1 and IgG2a isotypes, with predominance of IgG1 in all time points analyzed for the NLA + ScLL group, thus reflecting its higher production of IgG in relation to other groups. Before parasite challenge (60 d.a.i.), the NLA group reached similar IgG1 levels to the NLA + ScLL group, but significantly higher than IgG2a levels, demonstrating that NLA alone is also able to induce a Th2-biased humoral immune response. After parasite challenge, there was no significant increase in the levels of IgG1 in NLA-immunized groups, even though they maintained higher than IgG2a in all experimental groups. Regarding the control groups, seroconversion was observed in response to challenge with predominance of the IgG1 isotype, particularly evident in the ScLL group, indicating that this lectin alone is able to direct a Th2-biased humoral immune response. In contrast, IgG2a levels increased after parasite challenge in all groups, and this may represent a shift from Th2-biased immune response to a more protective Th1-biased immune response. Our previous study also showed higher levels of IgG1 than IgG2a after challenge in all groups of mice immunized with NLA associated or not with lectins ArtinM or Jacalin, indicating that this Th2 type of humoral immune response seems to be dependent on the antigen rather than the adjuvant . Indeed, our previous study has shown a considerable increment in both antibody isotypes after challenge in NLA-immunized groups associated or not with CpG, suggesting that the parasite was able to induce both Th2 and Th1 immune responses, although a Th2-biased humoral response was more evident .
In order to observe the immunization-induced cytokine profile before parasite challenge, we evaluated the ex vivo cytokine production by spleen cells from mice immunized with NLA associated or not with ScLL. After antigenic stimulation, both NLA + ScLL and NLA groups showed similar and increased IFN-γ production, but a distinct pattern was found for IL-10, with increased cytokine production in the groups immunized with antigen or lectin alone. When the IFN-γ/IL-10 ratio was analyzed in an attempt to verify the balance between pro-inflammatory and anti-inflammatory cytokines, the highest ratio was found for the NLA + ScLL group and the lowest for the ScLL alone. These results suggest that the ScLL lectin alone is able to induce higher levels of IL-10, leading to an anti-inflammatory or regulatory immune response pattern. It is noteworthy that spleen cells from the ScLL group stimulated with mitogen also showed the highest IL-10 production. In our previous study  mice immunized with NLA alone also showed high IL-10 levels, reinforcing the role of the NLA antigen in inducing an anti-inflammatory or immunoregulatory response. In addition, mice immunized with NLA plus Jacalin or Jacalin alone exhibited the lowest ratio IFN-γ/IL-10 ratio whereas those immunized with NLA + ArtinM or ArtinM alone presented the highest ratio, suggesting that the lectin adjuvant, but not the antigen, was able to modulate the cytokine production, leading to a Th1 type-biased pro-inflammatory immune response [25, 27, 37].
In the present study, the combination of NLA plus ScLL was also able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, indicating a potential adjuvant effect. In our previous studies, however, the ScLL lectin has shown contrasting roles depending on the study model. In immunization of mice against L. amazonensis, ScLL induced increased levels of IgG2a and overexpression of IL-12 and TNF-α mRNAs, leading to a Th1-biased immune response that supported the protective function of this lectin against cutaneous leishmaniasis . Primed macrophages when treated with ScLL showed no significant nitrite oxide (NO) production after infection with L. amazonensis, although they were able to reduce parasite intracellular proliferation . Additionally, oral administration of ScLL reduced IL-4 and IL-5 levels in murine models of acute and chronic inflammation and lead to increased IFN-γ and IL-10 production in asthma inflammatory model, directing a shift from Th2- to Th1-biased immune response .
It is well known that a protective immune response against N. caninum should be a non-exacerbated Th1-type response . Accordingly, in a previous study we demonstrated that a strong cellular immune response associated with increased IFN-γ production and inflammation induced by vaccination with N. caninum excreted-secreted antigen (NcESA) alone or CpG-adjuvanted NcESA rendered mice more susceptible to parasite challenge . In the present study, mice immunized with NLA plus ScLL or ScLL alone presented the highest survival rate (70-80%) and the lowest brain parasite burden, with no significant differences in morbidity scores and body weight changes from baseline. Therefore, the ScLL lectin associated or not with NLA is able to confer protection against parasite challenge regardless of inflammation. Accordingly, a previous study suggests that ScLL inhibits IκBα degradation, a negative regulator of pro-inflammatory NF-κB . In our previous study, mice immunized with ArtinM combined with NLA presented a higher survival rate (86%), even though with high brain tissue inflammation scores, whereas animals immunized with Jacalin alone or associated with NLA had the lowest inflammation scores, but with intermediate survival rates (50–62%) . Therefore, the induction of a pro-inflammatory immune response by ArtinM plus NLA seems to be more favorable than detrimental to the host to control neosporosis. This has been reported for the control of leishmaniasis, where ArtinM plus soluble Leishmania antigen induced a potent pro-inflammatory response with reduction of the parasite load, but without decreasing the lesion size . In the present study, however, mice immunized with NLA alone presented a low survival rate associated with a high brain parasite burden, similarly to the PBS control group, suggesting that NLA alone stimulated a non-protective Th2-biased immune response .
Recent studies have shown that different administration routes and delivery strategies in vaccination against neosporosis in murine models may play a key role in directing the immune response towards a more protective pattern [39, 42, 43]. In this framework, innate responses are crucial for stimulating the Th1-type antibody-and cell-mediated immune responses and lectins play an important role in the protein-carbohydrate binding that mediates the parasite-host cell interactions.
Altogether, the results of the present study showed that ScLL, a D-galactose-binding lectin, when combined with NLA antigen can be used as powerful adjuvant in immunization procedures against neosporosis, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. In addition, the D-galactose-binding seems not be essential for these effects, since another D-galactose binding lectin, Jacalin, showed to be a poor adjuvant in immunization against neosporosis . Future studies should be conducted to evaluate if the immunostimulatory and adjuvant effects of the ScLL lectin may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.