Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 https://doi.org/10.1186/s13071-020-04165-4 Parasites & Vectors RESEARCH Open Access Sirolimus enhances the protection achieved by a DNA vaccine against Leishmania infantum Alba Martínez‑Flórez1, Clara Martori1, Paula L. Monteagudo2,3, Fernando Rodriguez2, Jordi Alberola1† and Alhelí Rodríguez‑Cortés1*† Abstract Background: Leishmaniases are a group of neglected tropical parasitic diseases, mainly affecting vulnerable populations of countries with poor socioeconomic status. Development of efficient vaccines is a priority due to the increasing incidence of drug resistance and toxicity to current treatments. In the search for a safe and efficient protective vaccine for human and dog visceral leishmaniases, we analyzed the suitability of the immunomodulatory drug sirolimus (SIR) to boost a preventive DNA vaccine against leishmaniasis. SIR is an already marketed drug that has been described to boost immune protection against different disease models and has also emerged as a promising therapeutic drug against L. major. Methods: Syrian hamsters were treated with SIR concomitantly with the administration of a DNA vaccine formula‑ tion consisting in four plasmids carrying the Leishmania genes LACK, TRYP, PAPLE22 and KMPII, respectively. Two weeks after the last vaccination, the animals were infected intraperitoneally with L. infantum parasites. Five weeks post- infection the parasite load was measured by real-time PCR in target tissues and immune response was evaluated by determining anti-Leishmania specific antibodies in combination with cytokine expression in the spleen. Results: Our results show that the DNA vaccine itself efficiently reduced the burden of parasites in the skin (P = 0.0004) and lymph nodes (P = 0.0452). SIR administration also enhanced the protection by reducing the parasite load in the spleen (P = 0.0004). Vaccinated animals with or without SIR co-treatment showed lower IFN-γ expression levels than those found in the spleen of control animals. mRNA expression levels of NOS2 and IL-10 were found to be signifi‑ cantly higher in the vaccinated plus SIR treated group. Conclusions: Co-administration of SIR enhances a DNA vaccination regimen against L. infantum, improving the reduction of parasite load in skin, lymph node and spleen. The analysis of immune markers in the spleen after chal‑ lenge suggests that the trend to recover naïve levels of IFN-γ and IL-10, and the concurrent higher expression of NOS2, may be responsible for the protection induced by our vaccine co-administered with SIR. Keywords: Leishmania, Vaccine, Visceral leishmaniasis, Hamster, Sirolimus, mTOR, DNA vaccine Background Transmitted by infected female sand fly vectors, Leish- Leishmaniasis is a neglected disease caused by pro- mania spp. are responsible for over 1,500,000 human tozoan parasites belonging to the genus Leishmania. cases, resulting in more than 20,000 deaths per year, with the highest impact on the poorest people in developing countries [1]. Visceral leishmaniasis (VL), also known as *Correspondence: Alheli.Rodriguez@uab.cat † kala-azar, is the most severe clinical form, resulting in a Jordi Alberola and Alhelí Rodríguez-Cortés contributed equally to this work fatal outcome if left untreated [2]. Leishmania infantum 1 Departament de Farmacologia, de Terapèutica i de Toxicologia, is the causative agent of zoonotic VL in the Neotropical Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain zone and in the Mediterranean Basin, with dogs playing Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 2 of 12 an important role as peridomestic reservoirs and increas- SIR acts by binding and constraining the activity of the ing the risk of infection of susceptible human populations mechanistic (mammalian) target of rapamycin (mTOR), co-habiting in the same area [3]. a highly conserved pathway among eukaryotic organ- Protective immunity against Leishmania is medi- isms that orchestrates cell adaptations to environmental ated by both innate and adaptive immune responses. changes by controlling cell survival strategies, cell prolif- Thus, today we know that the effective activation of eration and metabolic adaptations at the transcriptional macrophages, dendritic cells (DCs), and antigen-spe- and translational levels [23]. Recent studies have opened cific CD4+ and CD8+ T cells are all needed to control a whole new field of research by showing that SIR admin- the spread of Leishmania [4]. The immune response istration during the expansion and contraction phase of induced by Leishmania has been fully described in a primary immune response results in an increased mag- the mouse model, with a CD4+ Th1 response (IFN-γ nitude and quality of CD8+ T memory cells [24]. SIR and TNF-α secretion) leading to the resolution of the administration has shown promising adjuvant results on disease and protection, and a CD4+ Th2 responses different vaccine trials against viruses [25, 26], cancer (IL-10 and IL-4) being associated with disease pathol- models [27, 28] and even against intracellular mycobac- ogy [5]. CD4+ Th1 cells activate CD8+ T cells which teria infection [29]. The protection achieved using SIR are required for optimal resistance, providing ben- in these studies of vaccination was mediated by the gen- eficial cytokines such as IFN-γ and specifically killing eration of memory effector CD8+ T and the concurrent parasite-infected cells [6, 7]. Unlike experimental VL increase in IFN-γ production. In regards to Leishmania, a in mice, most infected people develop asymptomatic recent study using SIR as a therapeutic agent on a mouse infections, which correlates with the activation of spe- model of cutaneous leishmaniasis showed promising cific cell-mediated immunity and a Th1-proinflamma- results, pointing that this molecule could be a potential tory immune response [8]. The active disease, however, candidate for further research in the field of leishmaniasis has been linked to high antibody levels and a progres- [30]. sive Th2-deactivating immune response in the presence We have previously studied the effect of a DNA vaccine of a strong inflammatory reaction [9]. carrying the Leishmania genes LACK, TRYP, PAPLE22 In spite of several efficient drugs currently available, in and KMPII in a hamster model of leishmaniasis. When many occasions these drugs are of limited access to the administered alone, it was poorly immunogenic but affected population. On the other hand, the increasing inci- induced partial protection when followed by a protein dence of toxicity cases and drug-resistant leishmaniasis, vaccine boost [31]. The aim of the present study was to require the development of safe and efficient preventive vac- test in hamsters if the co-administration of SIR would cines [10, 11]. Currently, there are three licensed and com- enhance the protection afforded by a L. infantum DNA vaccine. ­CaniLeish®, Leish-Tech® and ­ Letifend® [12–14]. Despite mercialized vaccines for canine leishmaniases prevention, several efforts made using experimental models [15], there Methods is no licensed vaccine against human leishmaniasis yet. Parasites DNA vaccines are a suitable approach for several rea- The L. infantum strain MCAN/ES/92/BCN83 (zym- sons, including the ability of plasmid DNA to stimulate Th1 odeme MON-1) was kindly provided by Dr Montserrat responses by the presence of CpG motifs [16]. In addition, Portús, Universitat de Barcelona, Spain. It was obtained DNA vaccines are safe, have lower production costs, and from a naturally infected and untreated dog and main- are easy to store because a cold-chain is not required [17]. tained through hamster passages to retain its full viru- Several DNA vaccination approaches against Leishmania lence. Hamsters were intraperitoneally infected with have been developed to date, reaching different degrees of stationary promastigotes, and at 3 months post-infection, protection in mice but achieving less immunogenicity in parasites were isolated from spleen samples incubated in more complex models [18]. Thus, the co-administration R15 medium (RPMI 1640 medium (Gibco, Billings, USA) of immunomodulatory compounds may be of paramount supplemented with 15% heat-inactivated fetal calf serum importance to boost the immune response induced by (Gibco), 1 M 2% HEPES (Gibco) and 1% of total volume DNA vaccines. For this purpose, many adjuvants have been of 10,000 U/ml penicillin, and 10,000 μg/ml streptomycin used in different Leishmania-vaccine trials [19], but phys- (Gibco)). icochemical incompatibilities and undesirable adverse reac- Promastigotes were maintained at 26 °C in R15 tions remain a major problem (reviewed in [20]). medium, and weekly passages were performed. A second Sirolimus (SIR), also known as rapamycin, is an immu- passage of Leishmania infantum cultures at stationary nosuppressant drug which is licensed and marketed pri- growth (6-day culture) was used for infection to assure marily to prevent organ transplant rejection [21, 22]. than > 85% of promastigotes were metacyclic and that Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 3 of 12 virulence was retained [32]. Stationary promastigotes for Immunization infection were washed and resuspended in PBS at 1 × ­107 The open reading frames (ORF) from L. infantum genes parasites/ml. TRYP (GenBank: AF044679), PAPLE22 (GenBank: AF123892), LACK (GenBank: U49695) and KMPII (Gen- Animals Bank: X95267) were individually cloned into a pVAX 1 Thirty-four male, 7-week-old, Syrian golden hamsters vector (Invitrogen, Waltham, USA) as described else- (Mesocricetus auratus, strain RjHAN-AURA) were where [18, 31]. Each DNA construction was purified obtained from Centre d’Elevage René Janvier (Le Genest- using EndoFree Plasmid Maxi Kit (Qiagen, Venlo, Neth- Saint-Isle, France). The animals were kept individually erlands) according to the manufacturer’s instructions. in plastic microfilter cages under BSL2 conditions at the The immunogenicity of these plasmids was previously Servei d’Estabulari of Universitat Autònoma de Barcelona tested [31]. (Barcelona, Spain), and food and water were provided ad Each hamster received three doses (2 weeks apart) of libitum. The study comprised a large experiment with 100 µg of each plasmid administered intramuscularly into 30 hamsters randomly distributed into 3 experimental their hind limb (V and S + V groups). To maintain the groups of 10 animals each: treated with SIR and DNA same handling conditions, the C group was injected with vaccinated group (S + V); DNA-vaccinated group (V); physiological saline solution. and PBS-inoculated control group (C). The 30 hamsters were further infected with Leishmania infantum (see Infection below) and four additional hamsters were used as a non- Two weeks after the administration of the last vaccine infected control group (NI). dose, all animals but the NI group were intraperitoneally inoculated with 1 ml of PBS containing 1 × ­107 L. infan- Rapamune® (sirolimus 1 mg/ml) was purchased from Sirolimus treatment tum metacyclic promastigotes. Animal weight and typical VL clinical signs in the rodent model, i.e. hair and weight Pfizer (New York, USA) and kept under conditions speci- loss, and cutaneous lesions, were weekly evaluated. fied by the manufacturer. Treatment doses of 0.075 mg/ kg in 500 μl of PBS were prepared daily under asep- Necropsy and sample collection tic conditions [24]. The S + V group received a daily All animals (n = 34) were euthanized at 5 weeks post- intraperitoneal dose from day − 1 (one day prior to the challenge (Fig. 1). Hamsters were anesthetized with a 100 first vaccination) for 6 consecutive weeks. The V and C μl intramuscular injection containing ketamine (42.8 mg/ groups of hamsters were handled under the same condi- kg), acepromazine (1.4 mg/kg) and xylazine (9.5 mg/kg). tions and were PBS-inoculated. An intracardiac puncture was performed to obtain 5 ml Fig. 1 Graphical representation of the experimental assay. The study comprised a large experiment with a total of 34 Syrian golden hamsters randomly distributed in 4 groups: NI (non-infected group, n = 4), C (PBS-inoculated control group, n = 10), V (DNA vaccinated group, n = 10), and S + V (sirolimus-treated and DNA vaccinated group, n = 10). Hamsters received sirolimus or a PBS solution daily from one day prior to the first vaccination for 6 consecutive weeks. Three vaccine doses were administered, leaving a 2-week period between them. C, V and S + V animals were challenged two weeks after receiving the last vaccination dose and all hamsters were sacrificed 5 weeks later Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 4 of 12 of blood sample from each animal, and the animals were Table 1 Sequence of primers and probe used for parasite then euthanized in a ­CO2 chamber. Samples of spleen, detection [35] liver, submandibular lymph nodes (LN) and healthy pin- Gene Primer sequence (5ʹ-3ʹ) nae skin from each hamster were aseptically obtained and kept on ice to avoid tissue degradation. Kinetoplastid minicircle DNA F AAC​T TT​TCT​GGT​CCT​CCG​GGTAG​ R ACC​CCC​AGT​T TC​CCGCC​ Evaluation of humoral immune response: total hamster P FAM–AAA​AAT​GGG​TGC​AGA​AAT​–MGB IgG anti‑L. infantum Abbreviations: F, forward primer; R, reverse primer; P, probe hematology crystal tubes (BD ­Vacutainer®; BD, New Jer- Blood samples were collected at the end of the study in TaqMan-MGB probes were used as formerly described sey, USA) and centrifuged at 2000×g for 15 min to obtain [35] (Table 1). Triplicates of 25 ng of DNA of each sam- serum samples and were frozen until analyzed. ELISA ple, a negative control using nuclease-free water (Sigma- plates (Costar high binding transparent flat bottom; Fisher in an Applied Biosystems StepOnePlus™ PCR instrument Aldrich) instead of DNA, and a standard curve were run Scientific, Waltham, USA) were coated overnight at 4 °C with 2 µg per well of Crude Total Leishmania Antigen (Applied Biosystems, Waltham, USA) following a thermal (CTLA) in 100 µl of carbonate-bicarbonate coating buffer cycling profile of 50 °C for 2 min, 95 °C for 10 min, 40 (0.1 M N­ aCO3-H2CO3 pH 9.6). CTLA was provided by Dr data were analyzed using StepOnePlus™ software v2.3 cycles at 95 °C for 15 s, and 60 °C for 1 min. The resulting Cristina Riera, Universitat de Barcelona, Spain. Samples were diluted 1:400 in PBS with 0.05% Tween 20 and 1% (Applied Biosystems). The number of parasites per µg of skimmed milk powder (PBSTM) and serially diluted in the DNA was calculated by interpolation to a standard curve pre-coated plate. Plates were incubated at 37 °C in a humid generated by using a 10-fold serial dilution from 1 ­ 03 to atmosphere for 1 h and were then washed 3 times with −3 ­10 promastigotes of a L. infantum culture (y = − 3.38x PBST and once with PBS. Plates were then incubated for + 28.43). The median qPCR efficiency (99%), and slope 1 h at 37 °C with goat anti-hamster IgG-HRPO (Bio-Rad, (− 3.38) were calculated from three different replicates. Hercules, USA) diluted 1:1000 in PBSTM. Antibody excess Parasite quantification was linear between ­103 and ­10−2 was removed with another cycle of washes as described parasites per sample (R2 = 0.993). above. Then, 100 µl of the peroxidase substrate tetrameth- ylbenzidine (TMB) (Sigma-Aldrich, St. Louis, USA) was Cytokine determination added to each well, and the plates were left to develop for Cytokine expression of all groups (n = 34) was stud- 5 min. The reaction was stopped by the addition of 50 µl of ied in spleen tissue. Samples maintained in RNAlater 1 M ­H2SO4, and the absorbance was recorded at 450 nm (Ambion, Austin, USA) were homogenized with TRI (Anthos 2001 reader; Anthos Labtech instruments, Wals, Reagent (Ambion) and kept frozen until RNA extrac- Austria). The cut-off value was set as the average opti- tion. Total RNA was extracted by using the RiboPure cal densities (ODs) of NI serum samples plus 3 times the kit (Ambion) following the manufacturer’s instruc- standard deviation (cut-off = 0.015 OD). tions, and samples were then treated with DNases TURBO-DNA free kit (Ambion). Clean RNA samples DNA extraction and real‑time PCR absolute parasite were stored at − 80 °C until analyzed. Retrotranscrip- quantification tion was carried out by using the High-Capacity cDNA The efficacy of the vaccination strategies was evaluated Reverse Transcription kit (Applied Biosystems) fol- by measuring the parasite reduction in target organs lowing a thermal profile of 25 °C for 10 min, 37 °C for [33, 34]. Total genomic DNA was obtained from 50 mg 120 min, and 95 °C for 5 min. Levels of IFN-γ, FoxP3, of spleen, liver, skin and LN samples of the study ani- TNF-α, IL-10, IL-21, NOS2 and TGF-β expression were mals by using the High Pure Template Preparation Kit determined in addition to RPL18 as a control gene to (Roche, Basel, Switzerland) following the mouse tail pro- calculate relative gene expression levels [36]. To ensure tocol provided in the manufacturer’s guidelines. DNA amplification of cDNA sequences derived from the ret- concentration was measured using a NanoDrop 2000 rotranscription of the mRNAs of interest, primers were Spectrophotometer (Isogen Life Science, De Meern, The designed including exon boundary sequences based on Netherlands). All DNA samples were kept at − 20 °C basic primer information extracted and modified from until the qPCR was performed. Zivcec et al. [36] and Espitia et al. [37] (Table 2). Rat Primers targeting conserved DNA regions of the (Rattus norvergicus) sequences were used for exon- kinetoplast minicircle DNA from L. infantum and boundary area determination, as hamster sequences are Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 5 of 12 Table 2 Sequences of primers used for expressed cytokines tests included in the WRS2 package v0.4-0 [40, 41]. The detection results were considered statistically significant when P Gene Primer sequence (5ʹ-3ʹ) ≤ 0.05. RPL18 F GTC​CCT​GTC​CCG​GAT​GAT​C Results R GAC​AGT​CCC​CAC​AAC​CAC​G Parasitological protection IFN-γ F CTA​TGT​C TG​GCT​GCT​ACT​GCCA​ Animals from group V showed a reduction in the num- R TTC​ACG​ACA​TCT​AAG​C TA​C TT​GAG​T TAA​ ber of parasites compared to group C in the skin (mean TNF-α F CCA​GAC​ACT​CAC​ACT​CAG​ATC​ATC​T T reduction of 60%; ψ̂ = 94.14, 95% Confidence Interval (CI): R CAC​T TG​GTG​GTT​TGC​TAC​AACGT​ 36.41–180.64, P = 0.0004) and LN (mean reduction of 83%; TGF-β F CTA​CCA​CGC​CAA​C TT​C TG​TCTG​ ψ̂ = 1.04, 95% CI: 0.13–2.05, P = 0.0452). Non-significant R TGT​AGA​GGG​CAA​GGA​CCT​TACTG​ differences were observed in the liver (mean reduction of FoxP3 F CAA​GTG​GCC​TGG​T TG​TGA​GA 78%; ψ̂ = 886.28, 95% CI: − 6.26–1902.01, P = 0.1072) R TGA​TCT​GCT​TGG​CAG​TGC​T ’ and spleen (mean reduction of 4%; ψ̂ = 48.41, 95% CI: IL-21 F CCA​GCT​CCA​CAA​GAT​GTA​AAGGA​ − 222.59–286.74, P = 0.8372). R GCT​GAC​T TG​AGT​T TG​GCC​T TCT​ Compared to group C, animals in the S + V group also IL-10 F CAT​CGA​T TT​C TC​CCC​TGT​GAA​ had reduced numbers of parasites in skin (mean reduc- R GAC​GCC​T TT​C TC​T TG​GAG​C TT​ tion of 79%; ψ̂ = 80.75, 95% CI: 11.03–152.33, P = 0.0400) NOS2 F CCA​GAA​CAG​AGG​GCT​CAA​AGG​ and LN (mean reduction of 83%; ψ̂ = 0.95, 95% CI: 0.09– R CCT​GCA​TCT​C TT​CCC​GAT​AGAG​ 2.03, P = 0.0489), together with a significant protection in Note: Data extracted and modified from Espitia et al. [37] and Zivcec et al. [36] the spleen (mean reduction of 32%; ψ̂ = 369.79, 95% CI: Abbreviations: F, forward primer; R, reverse primer 180.09–561.06, P = 0.0004), with parasite burdens concur- rently lower than group V (mean reduction of 57%; ψ̂ = 321.37, 95% CI: 125.36–551.69, P = 0.0080). Although non- not available for ENSEMBL analysis [38], and previous statistically significant (ψ̂ = 936.50, 95% CI: 44.63–1945.62, BLAST and CLUSTALW analyses confirmed sequence P = 0.0598), there was also a noticeable trend of burden identity. Amplification of each sample was carried reduction in the livers of hamsters of the S + V group com- out in triplicate using SYBR select RT-PCR Master pared to group C (mean reduction of 81%) (Fig. 2). Applied Biosystems StepOnePlus™ PCR instrument Mix Reagents (Applied Biosystems) with the aid of an and StepOnePlus™ Software v2.3 (Applied Biosystems). Analysis of the immune response Anti‑Leishmania IgG production The thermal cycling profile was 10 min at 50 °C, fol- All infected hamsters seroconverted and had IgG antibod- lowed by 40 cycles of 95 °C for 10 min, 95 °C for 15 s, ies against CTLA at the time of sacrifice, 5 weeks post- and 60 °C for 1 min. Melting curves assessed the speci- infection (IgG cut-off = 0.015 OD). Leishmania-specific ficity of our amplification products. Cytokine mRNA IgG production was reduced in group V animals when expression levels were normalized to the expression compared to group C (ψ̂ = 0.12, 95% CI: 0.02–0.22, P = levels of the housekeeping gene RPL18. Fold induction 0.0475), and S + V (ψ̂ = − 0.12, 95% CI: − 0.22–0.04, P levels were calculated by using the median values of the = 0.0180). In contrast, similar levels of Leishmania-specific C group. antibodies were detected between groups S + V and C (Fig. 3). Statistical analysis Cytokine and immune markers production Exploratory data analysis and inference were carried Expression of cytokines (IFN-γ, TNF-α, IL-10, IL-21 and out by using R statistical software v3.2.5 [39]. Differ- TGF-β) plus the Treg immune marker FoxP3, and the ences among groups were analyzed using robust one- NOS2 enzyme was studied and detected by the end of the way ANOVA with corresponding pairwise post-hoc (See figure on next page.) Fig. 2 Leishmania infantum DNA load. qPCR analyses showing the number of parasites detected per μg of DNA extracted from the spleen (a), liver (b), lymph node (c) and skin (d) comparing the three Leishmania-infected studied groups: C (PBS-inoculated control group, n =10), V (DNA vaccinated group, n = 10), and S + V (sirolimus-treated and DNA vaccinated group, n = 10). Parasite number was calculated by interpolation to a standard curve constructed with a L. infantum promastigote culture (e). Abbreviations: Cq, quantification cycle. ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001 Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 6 of 12 Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 7 of 12 higher expression levels of NOS2, IL-10, FoxP3, TGF-β and TNF-α, with a concurrent reduction of IFN-γ when compared to group C (Fig. 4). Statistical analysis showed that the S + V group expressed higher NOS2 (2.12-fold increase; ψ̂ = − 1.04, 95% CI: − 1.74–0.38, P = 0.016) and IL-10 levels (1.8-fold increase; ψ̂ = − 0.87, 95% CI: − 1.33–0.42, P = 0.010) compared with group C. The DNA vaccine alone induced an increase in NOS2 and IL-10 expression, although not significant, when com- pared with group C animals. No differences in TNF-α, TGF-β and FoxP3 expression were found between any vaccinated group and the group C hamsters in the spleen. There were trends (ψ̂ = − 1.04, 95% CI: − 2.51–0.43, P = 0.0552) for both vaccinated groups (V and S + V) in reducing IFN-γ expression (0.588- and 0.609-fold, respectively) compared to group C (Fig. 4). Discussion Previous studies have highlighted the need for finding Fig. 3 Comparison of anti-Leishmania IgG levels in group C (PBS-inoculated control group), V (DNA vaccinated group), and new boosting strategies to potentiate protection induced S + V (sirolimus-treated and DNA vaccinated group) at 5 weeks by DNA immunization. Although mTOR inhibitors have post-challenge. Antibody levels were measured by ELISA against been used for enhancing protection against some bacte- crude total L. infantum antigen (n = 30). Cut-off value (mean + 3 SD) rial and viral infections, and very recently in the treat- was set at 0.015 OD. ANOVA; * P < 0.05 ment of cutaneous leishmaniasis [30], nothing is known about their effect in a vaccination protocol against VL. To the best of our knowledge, the present study repre- study in spleen samples of all experimental groups (n = 34). sents the first use of the immunosuppressive drug SIR in The results are shown in Fig. 4. a vaccination strategy against VL with the aim of improv- First, we compared the non-infected hamsters (NI ing the immune response against this parasitic infection. group) versus the PBS control infected animals (group C). Naked DNA vaccination reduced L. infantum para- Leishmania infantum infection (group C) significantly site loads in the lymph nodes and skin. Our results show increased the expression of IFN-γ mRNAs (ψ̂ = 80.75, that administering SIR during immunization along with 95% CI: 11.03–152.33, P = 0.0400), whereas it significantly the naked DNA vaccine significantly reduces the parasite reduced the expression of TNF-α (ψ̂ = − 842.43, 95% CI: load even in the spleen, the main Leishmania target organ, − 1466.92–217.95, P = 0.0041), IL-10 (ψ̂ = 321.38, 95% CI: indicating that SIR treatment can enhance the protection 125.36–551.69, P = 0.0080), FoxP3 (ψ̂ = − 0.34, 95% CI: provided by the naked DNA vaccine itself. Recently, the − 0.47–0.19, P < 0.0001), and NOS2 (ψ̂ = − 1.04, 95% CI: utilization of SIR as a treatment for L. major cutaneous − 1.74–0.38, P < 0.0001) mRNAs in the spleen (Fig. 4). The leishmaniasis in mice led to a significant reduction of para- transcription profile of V and S + V groups were similar to site burden in draining lymph nodes [30], also confirming that of group C when they were compared to NI animals. that the use of mTOR inhibitors could be a new strategy to IFN-γ was the only exception with levels in vaccinated ani- limit the growth of different Leishmania species. mals not statistically different from those of the NI group. Administering SIR during vaccination results in accelera- Both vaccinated groups (V and S + V) expressed simi- tion of the CD8+ T cell differentiation program towards lar transcription patterns for the studied molecules with memory cells [24], thus promoting enhanced specific immune responses, such as IFN-γ production. Due to the (See figure on next page.) Fig. 4 Cytokine expression in spleen samples at the end of the study. IFN-γ, TNF-α, NOS2, IL-10, IL-21, FOXP3, and TGF-β expression was determined by RT-qPCR using SYBR Green as the detection chemistry. NI: non-infected group (n = 4); C: PBS control group (n = 10); V: DNA vaccinated group (n = 10); S + V: sirolimus-treated and DNA vaccinated group (n = 10). NI group was taken as a reference group, with a value of 1. ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001**** P < 0.0001 Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 8 of 12 Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 9 of 12 lack of reagents available for the hamster model, analy- Consistently, in our study, vaccinated animals presented sis of isolated CD8+ T cells was not possible. Expression lower IFN-γ expression levels in spleen samples than in analyses of some relevant cytokines involved in VL con- control hamsters, suggesting that the combined activity of trol and pathogenesis in spleen tissue were performed to sirolimus with our DNA vaccine regulated the overexpres- characterize the immune response triggered in this organ sion of IFN-γ, and possibly its counterproductive effect. after challenge. Unfortunately, immune response in other Further studies are needed to investigate the cell source target organs was not evaluated in the present study and of IFN-γ and its role during disease in L. infantum infec- future experiments will show if parasite reduction in dif- tion. In addition, measurement of the expression of other ferent tissues caused by SIR and DNA vaccine depends on important cytokines for Leishmania control, such as IL-12, the same immune mechanisms as in the spleen. The pro- could contribute to a better understanding of the protec- tection observed in S + V animals was associated with a tive effect generated by our vaccine protocol. significant increase in NOS2 expression in the spleen, an Parasitological protection in the spleen of SIR-vacci- indication of adequate anti-parasite effector function. In nated animals took place even under high expression accordance with our results, some studies have shown that levels of IL-10. A key role in the pathogenesis of murine inhibition of mTORC1 by rapamycin in macrophages acti- and human VL is usually assigned to IL-10 [52, 53]. How- vated the M1 phenotype, characterized by the secretion of ever, it is worth noting that other authors have shown proinflammatory cytokines, effective parasite killing, and that IL-10 signaling neither influences Th1 responses increased expression of the NOS2 enzyme [42–44]. Levels nor is responsible for vaccine failure in a L. major mouse of NO production and NOS2 expression in hamster mac- model [54]. Conversely, it may play a pivotal role by lim- rophages have been associated with Leishmania vaccine iting excess inflammation during infection, as suggested protection in other studies [45, 46]. Despite the relative for other intracellular pathogens [55, 56]. In our model, unresponsiveness of NOS2 transcription to IFN-γ in ham- L. infantum infection (group C) caused a significant sters [47], other cytokines, such as TNF-α, might induce decrease of this cytokine production with regard to the NO production in macrophages [48]. However, we did not non-infected controls (group NI); in contrast, vaccinated detect a significant increase in TNF-α expression in the animals (V/S + V) showed a slight recovery of IL-10 lev- S + V group, though IL-1, IFN-α, or IFN-β could also be els. Other vaccine attempts with L. infantum antigens in implicated in NOS2 expression. dogs have also shown mixed pro- and anti-inflammatory Surprisingly, despite the parasitological protection cytokine environments, with higher IL-10 production observed in the spleen in SIR-treated hamsters, we found induced in vaccinated animals [57, 58]. These results sug- a decreasing trend in IFN-γ production in the same tissue, gest that IL-10 might play a beneficial immune regulatory suggesting that the mediated protection in our model may role in L. infantum infection. not be due to the expected SIR effect (increase of IFN-γ Other immunoregulatory markers such as IL-21, FoxP3 production), or at least we could not detect it by analyz- and TGF-β showed no significant expression variations ing the whole spleen at five weeks post-infection. Analy- between vaccinated and infected control group (group C) sis of immune response before challenge would have been although a tendency of being dysregulated during infec- very useful to characterize the SIR and vaccination effect tion was detected. In our study, the FoxP3 marker was and future studies will address this important question. downregulated during infection and presented a ten- Another explanation for the unexpected decrease in IFN-γ dency to increase in the S + V group. FoxP3+ regulatory production could be that mTORC1 inhibition enhances T cells (Tregs) produce TGF-beta and IL-10 but their CD8+ T cell memory development but diminishes CD8+ role in this parasitic disease is still not clear [53]. In this T cell effector function with reduced IFN-γ production regard, IL-21 has an important role amplifying IL-10 pro- [49]. SIR administration against L. major infection induced duction by type 1 regulatory T cells (Tr1) in VL patients enhanced specific IFN-γ production, but the treatment [59]. However, in our study, L. infantum-infected ham- schedule and SIR dose were different than those used in sters expressed high levels of IL-21 cytokine that did the present study [30]. Although IFN-γ is a cytokine widely not seem correlated with the changes observed in IL-10 accepted to be involved in Leishmania control, it is also expression. More information is needed to elucidate the known to steadily increase during early stages of infection actual role of Treg and Tr1 cells in VL protection, which without a clear contribution to parasite control [47, 50]. In is becoming essential to the understanding which cells this line, our control infected animals showed higher lev- are responsible for the observed differences. els of this cytokine when compared to non-infected con- Plasmid DNA vaccines are also known to pro- trols. Moreover, recent studies have also shown that IFN-γ mote antibody production [16, 60]. However, serol- has a paradoxical effect in promoting Leishmania growth ogy levels in our study revealed a significant decrease in macrophages from chronically infected hamsters [51]. of anti-Leishmania-specific IgG antibodies five weeks Martínez‑Flórez et al. Parasites Vectors (2020) 13:294 Page 10 of 12 post-challenge in hamsters receiving the vaccine alone Funding This study was supported by the Spanish Ministry of Economy and Competi‑ when compared to the group C. These reduced anti- tiveness grants BES-2011-046954 to AMF, AGL2010-16678 to JA, and AGL2014- body levels have already been observed in a previous 56427-P to JA and ARC. experimental trial of this vaccine and may be explained Availability of data and materials by the capacity of the vaccine to control the aberrant B All data generated or analyzed during this study are included in this published cell differentiation widely associated with disease pro- article. gression [31]. Surprisingly, adding SIR to the immu- Ethics approval and consent to participate nization protocol translated into a significant increase All procedures involving animals were approved by the Universitat Autònoma in Leishmania-specific IgG levels compared to animals de Barcelona Animal Care Committee following the principles of animal pro‑ receiving the DNA vaccine alone, which were close to tection according to the Directive 2010/63/UE (Protocol Number 2277). the control group. An association between increased Consent for publication antibody responses due to polyclonal B cell activation, Not applicable. elevated parasite burden, and the absence of prolifera- Competing interests tive responses, has been described in the VL hamster The authors declare that they have no competing interests. model [61]. However, the increase in Leishmania-spe- cific IgG levels in animals treated with SIR observed in Author details 1 Departament de Farmacologia, de Terapèutica i de Toxicologia, Universitat our study was not accompanied by an increase in para- Autònoma de Barcelona, Bellaterra, Barcelona, Spain. 2 Centre de Recerca site burden. SIR administration during an immuniza- En Sanitat Animal (CReSA), Institut de Recerca i Tecnologia Agroalimentàries tion assay with influenza virus did not alter specific IgG (IRTA), Campus UAB, Bellaterra, 08193 Barcelona, Spain. 3 Department of Micro‑ biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. antibody levels compared to infected non-protected controls [25]. Detection of specific antibodies against Received: 3 December 2019 Accepted: 2 June 2020 each one of the antigen vaccine candidates would be needed to further characterize the SIR response. References 1. Pigott DM, Bhatt S, Golding N, Duda KA, Battle KE, Brady OJ, et al. Global Conclusions distribution maps of the leishmaniases. Elife. 2014;3:e02851. The co-administration of SIR potentiated the protec- 2. Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, et al. 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