Protective efficacy in a hamster model of a multivalent vaccine for human visceral leishmaniasis (Mulevaclin) consisting of the kmp11, leish-f3+, and ljl143 antigens in virosomes, plus gla-se adjuvant

microorganisms Article Protective Efficacy in a Hamster Model of a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin) Consisting of the KMP11, LEISH-F3+, and LJL143 Antigens in Virosomes, Plus GLA-SE Adjuvant Laura Fernández 1,†, Jose Carlos Solana 1,*,† , Carmen Sánchez 1, Mª Ángeles Jiménez 2, Jose M. Requena 3, Rhea Coler 4, Steven G. Reed 5, Jesus G. Valenzuela 6, Shaden Kamhawi 6, Fabiano Oliveira 6, Epifanio Fichera 7, Reinhard Glueck 7,‡, Maria Elena Bottazzi 8, Gaurav Gupta 7, Pedro Cecilio 9,10,11 , Begoña Pérez-Cabezas 9,10, Anabela Cordeiro-da-Silva 9,10,11 , Luigi Gradoni 12 , Eugenia Carrillo 1,§ and Javier Moreno 1,§   Citation: Fernández, L.; Solana, J.C.; Sánchez, C.; Jiménez, M.Á.; Requena, J.M.; Coler, R.; Reed, S.G.; Valenzuela, J.G.; Kamhawi, S.; Oliveira, F.; et al. Protective Efficacy in a Hamster Model of a Multivalent Vaccine for Human Visceral Leishmaniasis (MuLeVaClin) Consisting of the KMP11, LEISH-F3+, and LJL143 Antigens in Virosomes, Plus GLA-SE Adjuvant. Microorganisms 2021,9, 2253. https://doi.org/10.3390/ microorganisms9112253 Academic Editor: Gustavo Dominguez-Bernal Received: 22 September 2021 Accepted: 26 October 2021 Published: 29 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1WHO Collaborating Centre for Leishmaniasis, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Madrid, Spain; [email protected] (L.F.); [email protected] (C.S.); [email protected] (E.C.); javier[email protected] (J.M.) 2 Facultad de Veterinaria, Departamento de Medicina y Cirugía Animal, Universidad Complutense de Madrid, 28040 Madrid, Spain; [email protected] 3Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Madrid, Spain; [email protected] 4Center for Global Infectious Disease Research (CGIDR), Seattle Children’s Research Institute, Seattle, WA 98109, USA; [email protected]g 5HDT Bio Corp, Seattle, WA 98102, USA; [email protected]g 6Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, NIAID, NIH, Rockville, MD 20852, USA; [email protected] (J.G.V.); [email protected] (S.K.); [email protected] (F.O.) 7Etna Biotech S.R.L, 95121 Catania, Italy; [email protected] (E.F.); [email protected] (R.G.); [email protected] (G.G.) 8Department of Pediatrics, Texas Children’s Center for Vaccine Development, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX 77030, USA; [email protected] 9Parasite Disease Group, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, 4200-135 Porto, Portugal; [email protected] (P.C.); [email protected] (B.P.-C.); cordeir[email protected] (A.C.-d.-S.) 10 IBMC-Instituto de Biologia Celular E Molecular, Universidade do Porto, 4150-180 Porto, Portugal 11 Faculdade de Farmácia da, Universidade do Porto, 4099-002 Porto, Portugal 12 Unit of Vector-Borne Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy; [email protected] *Correspondence: [email protected] † Laura Fernández and Jose Carlos Solana are joint first authors. ‡ Deceased. § Eugenia Carrillo and Javier Moreno are joint last authors. Abstract: Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, fatal if untreated. Vaccination is the most cost-effective approach to disease control; however, to date, no vaccines against human VL have been made available. This work examines the efficacy of a novel vaccine consisting of the Leishmania membrane protein KMP11, LEISH-F3+ (a recombinant fusion protein, composed of epitopes of the parasite proteins nucleoside hydrolase, sterol-24-c-methyltransferase, and cysteine protease B), and the sand fly salivary protein LJL143, in two dose ratios. The inclusion of the TLR4 agonist GLA-SE as an adjuvant, and the use of virosomes (VS) as a delivery system, are also examined. In a hamster model of VL, the vaccine elicited antigen-specific immune responses prior to infection with Leishmania infantum. Of note, the responses were greater when higher doses of KMP11 and LEISH-F3+ proteins were administered along with the GLA-SE adjuvant and/or when delivered within VS. Remarkably, hamsters immunized with the complete combination (i.e., all antigens in VS + GLA-SE) showed significantly lower parasite burdens in the spleen compared to those in control animals. This protection was underpinned by a more intense, specific humoral response against the KMP11, LEISH-F3+, and LJL143 antigens in vaccinated animals, but a significantly less intense antibody response to the pool of soluble Leishmania antigens (SLA). Overall, these results indicate Microorganisms 2021,9, 2253. https://doi.org/10.3390/microorganisms9112253 https://www.mdpi.com/journal/microorganisms Microorganisms 2021,9, 2253 2 of 17 that this innovative vaccine formulation confers protection against L. infantum infection, supporting the advancement of the vaccine formulation into process development and manufacturing and the conduction of toxicity studies towards future phase I human clinical trials. Keywords: leishmaniasis; hamster; vaccine; virosomes; KMP11; LEISH-F3; LJL143; GLA-SE 1. Introduction Leishmaniasis is responsible for one of the highest rates of disability-adjusted life years (DALYs) caused by any neglected tropical disease [ 1 ]. It affects 12 million people worldwide, and approximately 350 million people are at risk [ 2 ]. Visceral leishmaniasis (VL), caused by Leishmania infantum and Leishmania donovani, is the most severe form of the disease, leading to death if left untreated [3]. Control of the leishmaniases depends on the identification of clinical cases and reservoir hosts and their treatment. However, most anti-Leishmania drugs available can cause adverse side effects (including toxicity), are difficult to administer, and face the problem of emerging resistance [ 4 , 5 ]. Since cured patients are resistant to re-infection, vaccination is thought to be the most cost-effective way of gaining control over this disease [ 6 ]. Certainly, vaccines against canine leishmaniasis have been developed; these are based either on parasite protein extracts (CaniLeish) [ 7 ]) or recombinant proteins (Leish-Tec [ 8 ] and LetiFend [ 9 ]) and have all been marketed at some point in Brazil or Europe [ 10 , 11 ]. However, there is still no effective vaccine approved for human use [ 12 ]. The present work aimed to assess the protective potential of a new vaccine formulation based on two parasite antigenic elements—the membrane protein KMP11 and the fusion protein LEISH-F3+—plus the salivary protein LJL143 from the sand fly Lutzomyia longipalpis, vehiculated in virosomes and adjuvanted by a strong TLR-4 agonist, against L. infantum infection. KMP11 is a well-known antigen that has already been used in pre-clinical experiments in the form of a DNA vaccine [ 13 ] and chimeric vaccines [ 14 , 15 ], one of which—against VL and post-kala-azar dermal leishmaniasis—is now being tested in the context of phase I clinical trials [ 16 ]. Although the efficacy of vaccines based on KMP-11 depends on the nature of the antigen, most of the studies performed as DNA vaccines were successful against Leishmania [ 14 , 15 ]. LEISH-F3 is a recombinant fusion protein based on the parasite proteins nucleoside hydrolase (NH) and sterol-24-c-methyltransferase (SMT). It conferred protection against Leishmania infection in rodent models and elicited a Th1-like immune response in humans, as per the results of a phase I clinical trial [ 17 ]. In an attempt to increase its antigenic diversity, a new version of this protein was recently developed that includes a third component derived from Leishmania cysteine protease B (CPB). This new protein, LEISH-F3+, shows immunogenic and prophylactic properties that can be superior to those of LEISH-F3 in mouse and hamster models [18,19]. Sand fly saliva is composed of pharmacologically active components (sialogenins) with anti-hemostatic, anti-inflammatory, and immunomodulatory properties that play important roles in Leishmania infection by impairing anti-parasitic immune responses [ 20 , 21 ]. This highlights the need to include vector elements in vaccine strategies [ 20 ]. In fact, some sand fly salivary proteins conferred protection against Leishmania infection—alone [ 22 – 24 ], or in combination with other vaccine elements as a way to generate wider and stronger immune responses [ 25 ]. For example, the Lu. longipalpis salivary protein LJL143 induces humoral responses and a Th1-cytokine profile in mice [26] and dogs [23,27]. Glucopyranolsyl lipid A, when formulated in a stable oil-in-water nanoemulsion (GLA-SE), functions as a TLR4 agonist that induces a Th1-response. Importantly, it has been previously used in human clinical trials as an adjuvant to enhance the immune response against Leishmania and other pathogens [17,28–30]. Microorganisms 2021,9, 2253 3 of 17 Virosomes (VS) are one of the few adjuvant systems approved by regulatory authorities for human use which have carrier capabilities. The technology platform is robust enough to allow for the efficient loading of a variety of antigens such as proteins, peptides, carbohydrates, and nucleic acids; importantly, the production level can be easily scaled up. The above characteristics, together with excellent safety records, make virosomes suitable delivery systems for a vaccine against VL [ 31 – 33 ]. Their main targets are dendritic cells; they enhance antigen uptake and promote cytotoxic T cell-mediated immune responses [34,35]. The first step in the development of any vaccine involves preclinical animal testing in a variety of different species and models. Animal models such as rodents (mice, hamsters), dogs, and primates (non-human) are used to study visceral leishmaniasis. BALB/c mice are the main immunological model of infection used for the pre-clinical evaluation of drugs and vaccines [ 36 – 39 ]. Previous studies undertaken by our consortium (the EU-FP7 MuLeVaClin consortium) have shown that different combinations of the antigens KMP11, LEISH-F3+, and LJL143, in conjunction with GLA-SE and VS, induce a Th1-type immune response in BALB/c mice, which might be protective against subsequent Leishmania infection [ 26 ]. The latter study also showed LJL143 to be the most immunogenic element tested, inducing a strong cellular response elicited via IFN-γand IL-10. On the contrary, hamsters are an excellent model for studying the pathogenesis of VL; in fact, in contrast to the self-resolving mouse model, the hamster model of visceral leishmaniasis parallels human disease [ 40 ]. Therefore, the infection of hamsters with L. infantum and L. donovani is considered the best experimental model available to assess the efficacy of drugs and vaccines against VL [ 40 ]. However, good results in animal models do not guarantee success when applying the same formulation to humans. The present work aimed to test the efficacy of this vaccine formulation in hamsters after intracardiac L. infantum infection, a model suitable for the study of VL and its clinical progression [41]. 2. Materials and Methods 2.1. Ethics Statement All procedures described here were approved by the Committee on Ethics and Animal Welfare of the Instituto de Salud Carlos III (CBA 06_2014) and performed according to the Spanish legislation on the protection of animals for experimentation and other scientific purposes (Royal Decree 53/2013, law 32/2007), which adheres to the European Directive 86/609/EEC. 2.2. Experimental Animals, Parasites, Antigens, and Adjuvant Animals and parasites. The VL model animals used in this work were golden hamsters (Mesocricetus auratus) (Janvier, Le Genest-Saint-Isle, France); the parasite species used was L. infantum (MCAN/ES/98/LLM-724, JPC strain). Soluble Leishmania antigen (SLA). This antigen pool was prepared from L. infantum promastigotes using a freeze–thaw procedure [ 42 ]. Briefly, promastigotes were washed in PBS and centrifuged at 1000 × gfor 20min at 4 ◦ C. They were then suspended in 50 mM of Tris-HCl and 5 mM of EDTA, pH 7, and subjected to freeze–thaw cycles, sonicated, and centrifuged again at 27,000 × gfor 20 min at 4 ◦ C. The supernatant containing the soluble proteins was centrifuged at 100,000 × gfor 4 h at 4 ◦ C before being aliquoted under sterile conditions and stored at − 80 ◦ C. The protein concentration of the extract was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. SLA is not part of the vaccine’s composition; it was only used in immunological assays. Microorganisms 2021,9, 2253 4 of 17 KMP11. The L. infantum gene coding for KMP11 (LinJ.35.2260) was cloned into the vector pET-28b for expression in E. coli BL21, as previously described [ 26 ]. The gene was amplified from Leishmania genomic DNA using the oligonucleotides 5 0 -CCATGGCCACCACGTA CGAGG and 5 0 -GGATCCTTACTTGGACGGGTACTGCG, avoiding the addition of any tag for protein purification purposes in order to fulfill the requirements of good manufacturing practices. The protein was purified by ammonium sulfate precipitation (at least 80% ammonium sulfate saturation) and by anion-exchange chromatography. KMP11 was then eluted in 10 mM of Tris-HCl, pH = 8.5, and 150 mM of NaCl and passed through a polymyxin-B agarose matrix (Sigma-Aldrich, St. Louis, MO, USA) for endotoxin removal. The purity was >95%, as determined by SDS-PAGE and Coomassie staining. LEISH-F3+. The chimeric protein LEISH-F3+ was originated by the fusion of the Leishmania CPB antigen and LEISH-F3 [ 18 ]. The production and purification processes were similar to those previously described [17]. LJL143. The sand fly salivary protein was produced using a mammalian expression system, as explained elsewhere [ 23 ]. Briefly, DNA coding for LJL143 was subcloned into the Pichia pastoris secretory expression vector pPICZ α A (Invitrogen, Carlsbad, CA, USA) between the EcoRI/XbaI restriction sites. The correct insertion sequence was confirmed by double-stranded sequencing using the vector flanking primers α -factor and 3 0 AOX-1 before electroporation into Pichia pastoris X-33. The expression of LJL143 was induced with 0.5% methanol at 30 ◦ C for 72 h. The purity of the expressed protein was confirmed by Coomassie staining (NuPAGE Bis-Tris gel; Invitrogen, Waltham, MA, USA). Adjuvant. The synthetic TLR-4 agonist glucopyranosyl lipid A in stable emulsion (GLA-SE) was produced and provided by the Infectious Disease Research Institute (IDRI; Seattle, WA, USA) [43]. 2.3. Virosome-Based Antigens Virosomes containing each of the antigens individually were prepared, as previously described [ 26 ]. Briefly, a mixture of 1 mg of inactivated influenza virus A/H1N1/California and 32 mg of phosphatidylcholine (Lipoid Ag, Steinhausen, Switzerland) was centrifuged at 100,000 × gfor 30 min, and the supernatant, containing haemagglutinin and neuraminidase, was recovered. These influenza antigens were then used to produce VS that included the vaccine antigens (individually) by detergent removal. The VS were then sterile-filtered and their size distribution was determined using a Zetasizer Nano instrument (Malvern Instruments, Malvern, UK). The parasite-derived and/or virus-derived protein contents of the VS were checked by SDS-PAGE Coomassie staining. 2.4. Immunization and Infection Hamsters were distributed into six groups (9 animals per group) and immunized intramuscularly with 100 µ L of the different vaccine formulations (see below) three times at four-week intervals. The antigens were administered to animals in different groups at different concentrations (except for LJL143, which was limited to 1 µ g to prevent immunodominance), combined or not with the adjuvant GLA-SE. Briefly, the animals in groups PA 1+1+1 and PA 5+5+1 (see legend to Table 1) were immunized with 1 or 5 µ g of KMP11 and LEISH-F3+ proteins, respectively, plus 1 µ g of LJL143 in combination with 1 µ g of the GLA-SE adjuvant. The groups VPA 1+1+1 and VPA 5+5+1 received the same doses as the previous groups, individually within VS (i.e., VS-KMP11, VS-LEISH-F3+, and VS-LJL143 associations). Animals in group P 5+5+1 were immunized with the antigen mix (5 µ g of KMP11 and LEISH-F3+ and 1 µ g of LJL143) without an adjuvant or VS. The control group animals received only PBS (Table 1). Microorganisms 2021,9, 2253 5 of 17 Table 1. Experimental vaccination groups. Group Components PBS PBS 1 P 5+5+1 LEISH-F3 + (5 µg)/KMP11 (5 µg)/LJL143 (1 ug) PA 1+1+1 LEISH-F3 + (1 µg)/KMP11 (1 µg)/LJL143 (1 µg)/GLA-SE (1 µg) PA 5+5+1 LEISH-F3 + (5 µg)/KMP11 (5 µg)/LJL143 (1 µg)/GLA-SE (1 µg) VPA 1+1+1 VSLEISH-F3 + (1 µg)/VS-KMP11 (1 µg)/VS-LJL143 (1 µg)/GLA-SE (1 µg) VPA 5+5+1 VS-LEISH-F3 + (5 µg)/VS-KMP11 (5 µg)/VS-LJL143 (1 µg)/GLA-SE (1 µg) P: proteins; PA: proteins combined with the adjuvant; VS: virosomes containing KMP11, LEISH-F3+, or LJL143; VPA: VS including each protein individually, combined with the adjuvant. Four weeks after administration of the last dose, the animals were infected intracardially with 2 × 10 7 L. infantum promastigotes in the stationary phase [ 44 , 45 ]. One hamster of the VPA 5+5+1 group died after infection; this group, therefore, had eight animals instead of nine. Twelve weeks after infection, all animals were anesthetized with 2% isoflurane (Verflurano; Virbac, Carros, France) and euthanized by cardiac puncture. 2.5. Whole Blood Collection One day before infection, each animal was anesthetized with isoflurane 2% and a small sample of was blood collected from the carotid vein in an EDTA-Ca 2+ tube (Microvette; Sarstedt, Numbrecht, Germany) [ 46 ] to obtain plasma. During euthanasia, approximately 4 mL of blood were extracted from each hamster and collected in lithium heparin tubes (Sarstedt) to obtain plasma and to isolate peripheral blood mononuclear cells (PBMCs). 2.6. Cell Proliferation Assay PBMCs were isolated from blood obtained during euthanasia using a Ficoll–Hypaque density gradient (Lymphocyte Isolation Solution; Rafer, UK) as previously described [ 44 ]. Then, 1 × 10 5 PBMCs/well were plated into 96-well microplates in an RMPI 160 medium supplemented with 2 mM of L-glutamine, 25 mM of Hepes, 100 U/mL of penicillin, 100 µ g/mL of streptomycin (all from Lonza, Basel, Switzerland), and 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA). Cells were stimulated for 5 days (37 ◦ C, 5% CO 2 ) with 10 µ g/mL of the individual vaccine antigens (KMP11, LEISH-F3+, or LJL143) or with SLA. Concanavalin A (5 µ g/mL) (Sigma-Aldrich, St. Louis, MO, USA) and supplemented RPMI were used as positive and negative controls of the assay, respectively. BrdU was added to each well during the last 24 h of culture, to examine lymphocyte proliferation using the ELISA GE Healthcare Cell Proliferation Kit (GE Healthcare Life Sciences, Marlborough, MA, USA). 2.7. Humoral Response Analysis Plasma was obtained after blood centrifugation (500 × gfor 10 min at 4 ◦ C) and stored at − 20 ◦ C until use. The presence and reactivity of IgG antibodies against SLA, KMP11, LEISH-F3+, and LJL143 were determined by ELISA. Briefly, Nunc Maxisorp 96-microtiter well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4 ◦ C with 1 µ g/well of SLA, KMP11, LEISH-F3+, or LJL143 in carbonate buffer (Na 2 CO 3 15 mM, NaHCO 3 39 mM, pH 9). The plates were then washed three times with PBS 0.01% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) and coated with 1% bovine serum albumin (BSA) in PBS (Sigma-Aldrich, St. Louis, MO, USA) (blocking solution) for 1 h at 37 ◦ C. Plasma samples were then added—diluted 1/100 in blocking solution—for 1 h at 37 ◦ C. For IgG detection, the wells were washed and incubated for 1 h at 37 ◦ C with IgG α -hamster peroxidase-conjugated secondary antibodies (Abd Serotec, Oxford, UK) diluted 1/5000 (SLA and LEISH-F3+) or 1/1000 (KMP11 and LJL143). Finally, the plates were washed and incubated with o-phenylenediamine for 15 min. The colorimetric reaction was stopped using H 2 SO 4 1M and the optical density was measured at 490 nm in a spectrophotometer (Thermo Fisher Scientific) to determine reactivity against each antigen. Microorganisms 2021,9, 2253 6 of 17 2.8. Parasite Load Quantification by PCR Following necropsy, liver and spleen samples of each animal were homogenized in supplemented RPMI (see above) using a 40 µ M Falcon Cell Strainer (Thermo Fisher Scientific, Waltham, MA, USA). To obtain DNA, around 3 × 10 6 cells were lysed with 400 µ L of NET10 buffer (NaCl 10 mM, EDTA 10 mM, Tris-HCl 10 mM, pH 8.0), 40 µ L of SDS 10% and 0.1 mg/mL of proteinase K (20 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 70 ◦ C. The DNA was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich) [47] and dissolved in 100 µL of nuclease-free water. The DNA concentration was measured using a NanoDrop ® 1000 spectrophotometer (Thermo Fisher Scientific). The Leishmania DNA was quantified by quantitative real-time PCR (qPCR) in 4 µ L of DNA from the different samples, using a LightCycler 2.0 thermocycler (Roche, Basel, Switzerland), 1000 nM of primer R223, 500 nM of primer R333, 2.5 mM of MgCl 2, and LightCycler FastStart DNA Master SYBR Green I (Roche) [ 44 ]. The parasite numbers in each sample were obtained using a standard curve [ 48 ]. The final results are expressed as relative units (RU) (i.e., with respect to the mean value of the control group) [ 49 ]. In addition, the mean RU of each group was calculated for comparison with that of the control group (considered as 1), and the difference is expressed as the percentage reduction or increase in parasite burden. 2.9. Histopathology Liver samples were collected after euthanasia, fixed in 10% buffered formalin, trimmed transversely along the main lobule, embedded in paraffin blocks, and processed and stained with hematoxylin and eosin following standard laboratory procedures. Two step sections were examined under light microscopy by a trained pathologist (blind to the study). The total number of granulomas in each section was counted and classified according to their maturation status. The efficiency of granuloma maturation and organization has been associated with the host capacity to resist infection in human, murine, and canine models [ 14 , 50 – 52 ]. Moreover, the effectiveness of protozoan vaccines has been related to the capacity to induce sterile granulomas [ 14 ]. Thus, granulomas were classified and counted as immature (developing granulomas), organized (distinct granulomas) or sterile (distinct, organized granulomas with central Kupffer cells and macrophages, surrounded by mixed inflammatory cells with a predominance of lymphocytes), according to previously reported classifications [14,51]. 2.10. Statistical Analysis The normality of the data was examined using the D’Agostino–Pearson test and the experimental groups were consequently compared using the Mann–Whitney U test. Significance was set at p< 0.05. All calculations were made using GraphPad Prism v.8.01 software (GraphPad Software Inc., San Diego, CA, USA). Histopathology results were analyzed using a non-parametric ANOVA Kruskal–Wallis test with SPSS software (IBM Inc. SPSS Inc. statistics). 3. Results 3.1. Antigenicity of the Different Vaccine Formulations Hamsters (9 animals per group) were immunized three times intramuscularly at four-week intervals with PBS or different permutations of the vaccine components. The P 5+5+1 group received the non-formulated and non-adjuvanted proteins KMP11 (5 µ g), LEISH-F3+ (5 µ g), and LJL143 (1 µ g). The PA groups received the proteins and the adjuvant GLA-SE in two different combinations: 1 µ g of each protein (PA 1+1+1) or 1 µ g of LJL143 and 5 µ g of KMP11 and LEISH-F3+ (PA 5+5+1). The VPA groups received the GLA-SEadjuvanted antigens formulated in virosomes using the same dosages as above (VPA 1+1+1 and VPA 5+5+1). Four weeks after the third and final immunization—just before performing infection—blood samples were obtained to evaluate vaccine antigenicity. A Microorganisms 2021,9, 2253 7 of 17 very low IgG response to SLA was detected; of note, no differences were found between the vaccinated and control groups (Figure 1A). However, the immunized animals showed a specific antibody response against the different vaccine antigens. Anti-KMP11 antibodies were found in animals immunized with 5 µ g of KMP11 and LEISH-F3+ proteins (P 5+5+1), but not in animals immunized with 1 µ g (P 1+1+1) or those in the VPA 1+1+1, VPA 5+5+1 groups (Figure 1B). Interestingly, the presence of the adjuvant GLA-SE in the PA 5+5+1 group increased serum reactivity against KMP11 compared to that recorded for the group immunized with proteins alone (p= 0.0133). Humoral response against the chimeric protein LEISH-F3+ was present in nearly all the immunized animals—especially in the PA 5+5+1 and both VPA groups (Figure 1C). The exception was the group PA 1+1+1; the animals showed responses similar to those seen in the control group (p= 0.3450). Additionally, a strong IgG response was also detected against the sand fly salivary protein LJL143 ( Figure 1D ). Interestingly, although all animals were immunized with the same dose of LJL143 (1 µ g), a significant increase (p< 0.05) in reactivity against this protein was observed in the presence of the TLR-4 agonist adjuvant. Microorganisms 2021, 9, x FOR PEER REVIEW 7 of 17 LEISH-F3+ (5 µg), and LJL143 (1 µg). The PA groups received the proteins and the adjuvant GLA-SE in two different combinations: 1 µg of each protein (PA 1 + 1 + 1) or 1 µg of LJL143 and 5 µg of KMP11 and LEISH-F3+ (PA 5 + 5 + 1). The VPA groups received the GLA-SE-adjuvanted antigens formulated in virosomes using the same dosages as above (VPA 1 + 1 + 1 and VPA 5 + 5 + 1). Four weeks after the third and final immunization— just before performing infection—blood samples were obtained to evaluate vaccine antigenicity. A very low IgG response to SLA was detected; of note, no differences were found between the vaccinated and control groups (Figure 1A). However, the immunized animals showed a specific antibody response against the different vaccine antigens. AntiKMP11 antibodies were found in animals immunized with 5 µg of KMP11 and LEISHF3+ proteins (P 5 + 5 + 1), but not in animals immunized with 1 µg (P 1 + 1 + 1) or those in the VPA 1 + 1 + 1, VPA 5 + 5 + 1 groups (Figure 1B). Interestingly, the presence of the adjuvant GLA-SE in the PA 5 + 5 + 1 group increased serum reactivity against KMP11 compared to that recorded for the group immunized with proteins alone (p = 0.0133). Humoral response against the chimeric protein LEISH-F3+ was present in nearly all the immunized animals—especially in the PA 5 + 5 + 1 and both VPA groups (Figure 1C). The exception was the group PA 1 + 1 + 1; the animals showed responses similar to those seen in the control group (p = 0.3450). Additionally, a strong IgG response was also detected against the sand fly salivary protein LJL143 (Figure 1D). Interestingly, although all animals were immunized with the same dose of LJL143 (1 µg), a significant increase (p < 0.05) in reactivity against this protein was observed in the presence of the TLR-4 agonist adjuvant. Figure 1. Humoral responses in immunized animals. Four weeks after the last immunization, IgG reactivity against L. infantum SLA (A), KMP11 (B), LEISH-F3+ (C), and LJL143 (D) was determined by ELISA in plasma from immunized hamsters (n = 9 per group). Results are shown as whisker (min to max) plots. Statistical differences between groups are indicated (Mann–Whitney U test): ** p < 0.01; *** p < 0.001. Figure 1. Humoral responses in immunized animals. Four weeks after the last immunization, IgG reactivity against L. infantum SLA ( A ), KMP11 ( B ), LEISH-F3+ ( C ), and LJL143 ( D ) was determined by ELISA in plasma from immunized hamsters (n= 9 per group). Results are shown as whisker (min to max) plots. Statistical differences between groups are indicated (Mann–Whitney U test): ** p< 0.01 ; *** p< 0.001. 3.2. Immune Responses in Vaccinated Hamsters after L. infantum Infection Four weeks after the last immunization, the hamsters were infected with 2 × 10 7 L. infantum promastigotes, and 12 weeks later the humoral responses were examined. On one hand, a significant reduction was seen in SLA-specific IgG antibodies for the VPA 1+1+1 ( p< 0.0001 ) and VPA 5+5+1 (p= 0.0055) groups compared to those in unvaccinated control hamsters (Figure 2A). Microorganisms 2021,9, 2253 8 of 17 On the other hand, reactivity against the antigens included in the vaccine candidate was generally higher in the vaccinated groups versus the control group after infection. Differences were also observed depending on the antigen dose, the presence of the adjuvant, and the use of VS (Figure 2B–D). Reactivity against KMP11 increased significantly ( p= 0.0133 ) in the animals vaccinated with 5 µ g antigens together with the adjuvant (PA 5+5+1). However, no significant increase was observed for the PA 1+1+1 group compared to the control group ( p= 0.4894 ), although the picture was different when the proteins were included in virosomes (VPA 1+1+1) (significant difference versus the control group, p= 0.0021 ). In fact, animals vaccinated with the complete vaccine formulation (VPA 1+1+1 and VPA 5+5+1), as well as those in the PA 5+5+1 group, showed the highest levels of anti-KMP11 antibodies (no significant differences among these three groups). Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 17 3.2. Immune Responses in Vaccinated Hamsters After L. infantum Infection Four weeks after the last immunization, the hamsters were infected with 2 × 107 L. infantum promastigotes, and 12 weeks later the humoral responses were examined. On one hand, a significant reduction was seen in SLA-specific IgG antibodies for the VPA 1 + 1 + 1 (p < 0.0001) and VPA 5 + 5 + 1 (p = 0.0055) groups compared to those in unvaccinated control hamsters (Figure 2A). On the other hand, reactivity against the antigens included in the vaccine candidate was generally higher in the vaccinated groups versus the control group after infection. Differences were also observed depending on the antigen dose, the presence of the adjuvant, and the use of VS (Figure 2B–D). Reactivity against KMP11 increased significantly (p = 0.0133) in the animals vaccinated with 5 µg antigens together with the adjuvant (PA 5 + 5 + 1). However, no significant increase was observed for the PA 1 + 1 + 1 group compared to the control group (p = 0.4894), although the picture was different when the proteins were included in virosomes (VPA 1 + 1 + 1) (significant difference versus the control group, p = 0.0021). In fact, animals vaccinated with the complete vaccine formulation (VPA 1 + 1 + 1 and VPA 5 + 5 + 1), as well as those in the PA 5 + 5 + 1 group, showed the highest levels of anti-KMP11 antibodies (no significant differences among these three groups). Similarly, with respect to LEISH-F3+, the presence of the adjuvant in the PA 5 + 5 + 1 group helped generate a stronger antibody response than that seen in the P 5 + 5 + 1 group. This response was greater than that recorded for the control group (p = 0.0028) and similar to that seen in the VPA 5 + 5 + 1 group (p = 0.0025). Of note, the antigen doses did not influence the reactivity when proteins were administered in VS (VPA 1 + 1 + 1 and VPA 5 + 5 + 1) (Figure 2C). Importantly, sera from control animals did not react with the LJL143 protein (Figure 2D). In fact, the responses of the immunized and infected animals to this antigen were similar to those observed prior to infection (Figures 1D and 2D). Additionally, as for the other antigens examined, the IgG response against LJL143 was stronger when the adjuvant was present (PA 5 + 5 + 1) (p = 0.0030). Figure 2. Specific IgG responses after L. infantum challenge. One month after the last immunization, the hamsters were infected intracardially with 2 × 107 L. infantum stationary-phase promastigotes. Figure 2. Specific IgG responses after L. infantum challenge. One month after the last immunization, the hamsters were infected intracardially with 2 × 10 7 L. infantum stationary-phase promastigotes. Twelve weeks later, they were euthanized, and plasma was obtained. Specific antibody reactivity against SLA ( A ), and against the specific proteins KMP11 ( B ), LEISH-F3+ ( C ), and LJL143 ( D ), was assessed by ELISA. The results are shown as whisker (min to max) plots for each group (n= 9). Significant differences between groups are indicated (Mann–Whitney U test): * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001. Similarly, with respect to LEISH-F3+, the presence of the adjuvant in the PA 5+5+1 group helped generate a stronger antibody response than that seen in the P 5+5+1 group. This response was greater than that recorded for the control group (p= 0.0028) and similar to that seen in the VPA 5+5+1 group (p= 0.0025). Of note, the antigen doses did not influence the reactivity when proteins were administered in VS (VPA 1+1+1 and VPA 5+5+1) (Figure 2C). Microorganisms 2021,9, 2253 9 of 17 Importantly, sera from control animals did not react with the LJL143 protein (Figure 2D ). In fact, the responses of the immunized and infected animals to this antigen were similar to those observed prior to infection (Figures 1D and 2D). Additionally, as for the other antigens examined, the IgG response against LJL143 was stronger when the adjuvant was present (PA 5+5+1) (p= 0.0030). The absence of cellular immunity against Leishmania is decisive in the progression of infection. Therefore, in line with our previous study [ 26 ], next, the proliferative capacity of hamster PBMCs was examined in response to SLA and the specific vaccine antigens KMP11, LEISH-F3+, or LJL143 (Figure 3). The proliferation of PBMCs in response to SLA was similar in all groups; in contrast to the humoral response, no significant differences due to vaccination were observed (Figure 3A). Interestingly, no proliferation was detected after KMP11 stimulation (Figure 3B). Differently, specific cellular responses to LEISH-F3+ and LJL143 were observed, but of a lower magnitude than that seen in response to SLA ( Figure 3C,D ). It is noteworthy that the specific lymphoproliferation against the vaccine antigens was no different from that seen in the control group. This is surprising in the case of LJL143 since the control animals had no contact with the antigen (Figure 3D). This finding indicates that proliferative PBMC responses against this protein require no immunization step. Microorganisms 2021, 9, x FOR PEER REVIEW 9 of 17 Twelve weeks later, they were euthanized, and plasma was obtained. Specific antibody reactivity against SLA (A), and against the specific proteins KMP11 (B), LEISH-F3+ (C), and LJL143 (D), was assessed by ELISA. The results are shown as whisker (min to max) plots for each group (n = 9). Significant differences between groups are indicated (Mann–Whitney U test): * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The absence of cellular immunity against Leishmania is decisive in the progression of infection. Therefore, in line with our previous study [26], next, the proliferative capacity of hamster PBMCs was examined in response to SLA and the specific vaccine antigens KMP11, LEISH-F3+, or LJL143 (Figure 3). The proliferation of PBMCs in response to SLA was similar in all groups; in contrast to the humoral response, no significant differences due to vaccination were observed (Figure 3A). Interestingly, no proliferation was detected after KMP11 stimulation (Figure 3B). Differently, specific cellular responses to LEISH-F3+ and LJL143 were observed, but of a lower magnitude than that seen in response to SLA (Figure 3C,D). It is noteworthy that the specific lymphoproliferation against the vaccine antigens was no different from that seen in the control group. This is surprising in the case of LJL143 since the control animals had no contact with the antigen (Figure 3D). This finding indicates that proliferative PBMC responses against this protein require no immunization step. Figure 3. Antigen-specific lymphoproliferation assay in hamsters infected with L. infantum. Twelve weeks after infection, PBMCs were isolated from the peripheral blood of euthanized mice (n = 9 hamsters per group) and stimulated with SLA (A), KMP11 (B), LEISH-F3+ (C) or LJL143 (D) (10 µg/mL each) for 5 days at 37 °C. Lymphoproliferation was determined by the measurement of BrdU incorporation by ELISA. The results show the cell stimulation index—calculated as the ratio between the absorbance of antigen-stimulated cells and the result for non-stimulated cells (RPMI control)—of each animal in a scatter plot. Dotted lines represent the stimulation index obtained in the context of three naive hamsters. 3.3. The Complete Vaccine Formulation Confers Protection Against L. infantum Infection in Hamsters To determine the efficacy of the proposed vaccine, the parasite load was measured by qPCR 12 weeks after infection in the target organs of VL. No differences were seen in the context of liver parasite burdens between immunized animals and controls (Figure 4A). Importantly, in the spleen, parasite load in the VPA 5 + 5 + 1 group was significantly lower—some 86% reduction—than in the control group (p = 0.0393) (Figure 4B). Figure 3. Antigen-specific lymphoproliferation assay in hamsters infected with L. infantum. Twelve weeks after infection, PBMCs were isolated from the peripheral blood of euthanized mice ( n= 9 hamsters per group) and stimulated with SLA ( A ), KMP11 ( B ), LEISH-F3+ ( C ) or LJL143 ( D ) (10 µ g/mL each) for 5 days at 37 ◦ C. Lymphoproliferation was determined by the measurement of BrdU incorporation by ELISA. The results show the cell stimulation index—calculated as the ratio between the absorbance of antigen-stimulated cells and the result for non-stimulated cells (RPMI control)—of each animal in a scatter plot. Dotted lines represent the stimulation index obtained in the context of three naive hamsters. 3.3. The Complete Vaccine Formulation Confers Protection against L. infantum Infection in Hamsters To determine the efficacy of the proposed vaccine, the parasite load was measured by qPCR 12 weeks after infection in the target organs of VL. No differences were seen in the context of liver parasite burdens between immunized animals and controls ( Figure 4A) . 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