Efficacy of mucosal vaccination using a protozoan parasite as a vehicle for antigen delivery: IgG and neutralizing response after rectal administration of LeCoVax-2, a candidate vaccine against COVID-19.

Mucosal vaccination is regarded as a promising alternative to classical, intramuscular vaccine delivery. However, only a limited number of vaccines have been licensed for mucosal administration in humans. Here we propose Leishmania tarentolae, a protozoan parasite, as a potential antigen vehicle for mucosal vaccination, for administration via the rectal or oral routes. To test this hypothesis, we exploited L. tarentolae for the production and delivery of SARS-CoV-2 antigens. Two antigens were assayed in BALB/c mice: Lt-spike, a L. tarentolae clone engineered for the surface expression of the SARS-CoV-2 spike protein; RBD-SD1, a purified portion of the spike protein, produced by another engineered clone of the protozoon. Immune response parameters were then determined at different time points. Both antigens, administered either separately or in combination (Lt-spike + RBD-SD1, hereafter LeCoVax-2), determined significant IgG seroconversion and production of neutralizing antibodies after subcutaneous administration, but only in the presence of adjuvants. After rectal administration, the purified RBD-SD1 antigen did not induce any detectable immune response, in comparison with the intense response observed after administration of LeCoVax-2 or Lt-spike alone. In rectal administration, LeCoVax-2 was also effective when administered without adjuvant. Our results show that L. tarentolae is an efficient and safe scaffold for production and delivery of viral antigens, to be used as vaccines. In addition, rectal vaccination experiments prove that L. tarentolae is suitable as a vaccine vehicle and adjuvant for enteral vaccination. Finally, the combined preparation LeCoVax-2 can be considered as a promising candidate vaccine against SARS-CoV-2, worthy of further investigation.

A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae.

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

The non-pathogenic protozoon Leishmania tarentolae interferes with the activation of NLRP3 inflammasome in human cells: new perspectives in the control of inflammation.

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns. Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation. Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species. Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome.

Rectal Administration of Leishmania Cells Elicits a Specific, Th1-Associated IgG2a Response in Mice: New Perspectives for Mucosal Vaccination against Leishmaniasis, after the Repurposing of a Study on an Anti-Viral Vaccine Candidate.

The mucosal immune system plays a pivotal role in the control of infections, as it represents the first line of defense against most pathogens, from respiratory viruses to intestinal parasites. Mucosal vaccination is thus regarded as a promising strategy to protect animals, including humans, from infections that are acquired by ingestion, inhalation or through the urogenital system. In addition, antigens delivered at the mucosal level can also elicit systemic immune responses. Therefore, mucosal vaccination is potentially effective also against systemic infections acquired through non-mucosal routes, for example, through the bite of hematophagous insects, as in the case of leishmaniasis, a widespread disease that affects humans and dogs. Here, we explored the potential of antigen rectal administration for the generation of anti-Leishmania immunity. Mice were immunized through rectal administration of whole cells of the model parasite Leishmania tarentolae (using a clone engineered to express the spike protein of the SARS-CoV-2 virus generated in a previous study). A specific anti-Leishmania IgG antibody response was detected. In addition, the recorded IgG2a/IgG1 ratio was higher than that of animals injected subcutaneously; therefore, suggesting a shift to a Th1-biased immune response. Considering the importance of a Th1 polarization as a protective response against Leishmania infections, we suggest that further investigation should be focused on the development of novel types of vaccines against these parasites based on rectal immunization.

Intracellular persistence of Leishmania tarentolae in primary canine macrophage cells.

Leishmania tarentolae is a non-pathogenic species first isolated from geckoes in the Mediterranean basin. The finding that dogs test positive against both Leishmania infantum and L. tarentolae raises questions regarding the ability of the latter species to persist and adapt to new hosts. This study aimed to evaluate in vitro the capability of L. tarentolae to colonize, survive and persist in canine primary monocyte-derived mononuclear cells. Monocytes were isolated from dog whole blood samples and placed in 24-well plates for differentiation into macrophages and for incubation with L. tarentolae field-isolated strains (RI-325 and SF-178) and laboratory (LEM-124) strain; the parasite burden was assessed at different time points post-infection. The L. infantum laboratory strain (MON-1) was used as control. Infection parameters were evaluated by microscopy, counting the number of amastigotes/200 infected cells, and by duplex real-time PCR from supernatants and detached cells. Similar to L. infantum, L. tarentolae strains developed into round-shaped amastigote-like forms, with higher infection rates detected at 4 h followed by an overall decrease until 48 h. RI-325 presented also a higher infection rate at 72 h. Data showed that L. tarentolae strains infect and persist inside in vitro primary canine mononuclear cells, opening new perspectives for further laboratory studies.

Leishmania tarentolae as an Antigen Delivery Platform: Dendritic Cell Maturation after Infection with a Clone Engineered to Express the SARS-CoV-2 Spike Protein.

BACKGROUND: Protozoa of the genus Leishmania are characterized by their capacity to target macrophages and Dendritic Cells (DCs). These microorganisms could thus be exploited for the delivery of antigens to immune cells. Leishmania tarentolae is regarded as a non-pathogenic species; it was previously used as a biofactory for protein production and has been considered as a candidate vaccine or as an antigen delivery platform. However, results on the type of immune polarization determined by L. tarentolae are still inconclusive. METHODS: DCs were derived from human monocytes and exposed to live L. tarentolae, using both the non-engineered P10 strain, and the same strain engineered for expression of the spike protein from SARS-CoV-2. We then determined: (i) parasite internalization in the DCs; and (ii) the capacity of the assayed strains to activate DCs and the type of immune polarization. RESULTS: Protozoan parasites from both strains were effectively engulfed by DCs, which displayed a full pattern of maturation, in terms of MHC class II and costimulatory molecule expression. In addition, after parasite infection, a limited release of Th1 cytokines was observed. CONCLUSIONS: Our results indicate that L. tarentolae could be used as a vehicle for antigen delivery to DCs and to induce the maturation of these cells. The limited cytokine release suggests L. tarentolae as a neutral vaccine vehicle that could be administered in association with appropriate immune-modulating molecules.