Introduction

Infectious diseases are a serious and growing threat to public health. Re-emerging and new pathogens often appear due to the transmission of animal viruses to humans and can potentially pose significant public health risks. Over the last 40 years, at least 30 new pathogens that threaten human health have been identified, most of which were zoonotic1,2.

The emergence of new pathogens frequently occurs in countries with low socioeconomic status, but it is also crucial to consider how easily these pathogens can spread globally1. The recent COVID-19 pandemic demonstrated this widespread transmission, with health, social and economic effects that will be felt for a long time. The pandemic also underscored the importance of responding to emerging threats and ensuring universal access to vaccines3,4,5. Outbreaks of other infectious diseases, such as the Zika and Ebola virus epidemics, have similarly highlighted the need to respond swiftly to new emerging pathogens.

In recent years, climate change has contributed to an increase in epidemiological risk. The extended activity period of insect vectors (e.g., mosquitos and ticks) and the expansion of their habitats may lead to a rise in diseases caused by arboviruses, such as chikungunya virus, dengue virus, tick-borne encephalitis virus (TBEV) and West Nile virus6,7. The benefits of widespread preventive vaccination are undeniable. Vaccination has led to the eradication of smallpox in humans and rinderpest in cattle. Additionally, thanks to vaccination, poliovirus is close to being eradicated, and many other infectious diseases have been brought under control. The widespread use of vaccines offers enormous health and socioeconomic benefits1,8,9.

Historically, antiviral vaccines were often based on inactivated or attenuated viruses, but these approaches carried risks such as potential reversion to virulence or incomplete inactivation. While these drawbacks are no longer major concerns, they were among the factors driving the development of new vaccine strategies. Consequently, contemporary research has focused on developing recombinant vaccines based on safer and more precisely engineered viral components, thereby minimizing the risk of adverse outcomes and increasing vaccine efficacy. Vaccine development remains time-consuming and costly; therefore, alternative expression systems that can offer cost-effective and faster production processes, thereby increasing accessibility and frequency of use, deserve special attention. One such achievement is an expression system based on Leishmania tarentolae, which has been gaining popularity in recent years10,11.

L. tarentolae as an unconventional expression system

The main expression systems used to produce recombinant vaccine antigens are based on mammalian and insect cells, yeast, or bacteria. A comparison of these systems regarding production costs, growth kinetics and yields are present in Table 1. Since no single expression system is universally applicable, the development of new systems facilitates the efficient production of antigens.

Table 1 Comparison of the main characteristics of different expression systems
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L. tarentolae is a single-cell, flagellated protozoan isolated from the lizards Tarentolae annularis and Tarentolae mauritanica in 1921 in Sudan and in 1934 in Algeria, respectively12,13. This protozoan was first used as an expression system by Breitling et al. in 2002 to obtain EGFP and human erythropoietin (hEPO), resulting in the efficient production of both proteins, including posttranslational modifications. To ensure that hEPO was secreted from cells and properly glycosylated, its signal peptide was replaced with a signal peptide from secreted acid phosphatase (SAP1) of Leishmania mexicana14. SAP1 has been well characterized, and its signal peptide is often used for the production of heterologous proteins in L. tarentolae (Fig. 1)15,16.

Fig. 1: Map of a standard expression vector used for protein production in L. tarentolae.
Fig. 1: Map of a standard expression vector used for protein production in L. tarentolae.
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Standard vector comprises of untranslated regions (utr1, utr2, utr3), 5’ and 3’ small subunit of L. tarentolae rRNA gene (3’ ssu, 5’ ssu) and antibiotic resistance gene. The gene of interest may be preceded by a sequence for the signal peptide (mainly a signal peptide from the secreted acid phosphatase SAP1 of Leishmania mexicana) for secreted proteins, followed by the protein tag sequence if needed. Origin of replication from E. coli (E. coli ori) and β-lactomase (bla) are required for plasmid propagation in bacteria. Created with BioRender.com.

L. tarentolae is a biosafety level 1 organism; therefore, it does not require special containment, contributing to its low production costs. This protozoan is relatively easy to cultivate in liquid, aerated culture in a simple incubator at 26 °C. Unlike mammalian cells, it does not require incubators with specific CO2 levels. It is also suitable for scale-up and cultivation in bioreactors (Fig. 2), with a high growth rate of 6–8 h for generation and the ability to reach high cell densities (>108 cells/mL). Additionally, as a lizard parasite that is nonpathogenic to mammals, there is no risk of preexisting immunity, which is often a problem with some systems. These factors make L. tarentolae a good candidate for producing human vaccines or veterinary vaccines for domestic and farm animals17.

Fig. 2: The production of recombinant proteins in L. tarentolae.
Fig. 2: The production of recombinant proteins in L. tarentolae.
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Stages required to produce recombinant proteins in L. tarentolae expression system has been shown in the figure above. Created with BioRender.com.

Currently, a commercially available LEXSY system can be used for producing recombinant proteins by L. tarentolae. This system, developed by the German company Jena Bioscience (“LEXSY - Eukaryotic protein expression in Leishmania tarentolae.” https://www.jenabioscience.com/lexsy-expression) (Fig. 2), allows for the production of proteins, both intracellularly and secreted into the medium, which facilitates easier purification. L. tarentolae secretes only a small amount of endogenous proteins, and the cultivation medium does not contain many proteins; therefore, the recombinant protein constitutes the majority of the proteins in the medium18,19. In recent years, the protein expression system in L. tarentolae has been continuously improved, increasing its utility in this field20,21. For viral antigens produced in L. tarentolae, the production efficiency varied from 0.5 to 20 mg/L of culture depending on the antigen (Table 2). The low costs associated with the L. tarentolae system are mainly due to minimal cultivation requirements and inexpensive culture media. L. tarentolae can be cultivated in brain-heart infusion-based (BHI) or yeast-soybean-based media supplemented with hemin. Although L. tarentolae typically requires animal components, these can be replaced by recombinant nutriments10,18,22,23. For the production of viral antigens discussed in this publication, BHI supplemented with porcine hemin was the most commonly used medium (Table 2). Since both components are of animal origin, their use in vaccine production may incur additional costs due to the need for stringent testing. Other media used for producing viral antigens were SDM-79 and M199. These media based on a mixture of inorganic salts, amino acids, vitamins and other additives, reduce the need for animal components, but were sometimes supplemented with hemin or inactivated bovine serum (FBS). Notably, one study comparing BHI and M199 media reported a higher yield of antigen production with the synthetic M199 medium24. This suggests that the production of other antigens could also be optimized to minimize or eliminate the use of animal components. Additionally, yeast extract medium has been found suitable for Lesihmania culture, and resulted in higher cell densities than for BHI medium22. Since avoiding animal components is required whenever possible in vaccine production, especially for human vaccines, this aspect of vaccine antigens production in L. tarentolae still requires improvement. The challenge of eliminating animal-derived elements from the medium remains a significant hurdle that must be overcome to ensure the safety and efficacy of the produced vaccines before the L. tarentolae system can be applied to clinical-grade material production.

Table 2 The use of L. tarentolae for the production of viral antigens
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Stable cells lines of L. tarentolae can be efficiently obtained with electroporation-mediated transfection with various expression vectors. A schematic representation of a standard vector used for protein expression in L. tarentolae is shown in Fig. 1. Depending on the system, heterologous protein production can be constitutive or inducible18,25. Due to these conditions and rapid growth, protein production costs in Leishmania tarentolae are generally lower than in mammalian cells, though not necessarily cheaper than in yeast or bacteria (Table 1).

L. tarentolae can be used for producing proteins that require posttranslational modifications. This organism can produce glycoproteins with glycosylation patterns highly similar to those in mammalian cells, likely due to its parasitic lifestyle (Fig. 3). This is a notable advantages of the L. tarentolae system, distinguishing it from other systems such as yeast or bacteria. The N-glycosylation profile of L. tarentolae was first investigated in recombinant hEPO, which contained biantennary N-glycans similar to those found in higher eukaryotes but lacking sialylation14,19. Since glycosylation can influence protein immunogenicity, it is a significant feature of L. tarentolae that makes it a promising potential expression system for the production of vaccines against viral targets26,27. O-glycosylation, present in viral proteins, has also been reported in Leishmania28,29. Disulfide bridges, important for protein functionality, have been shown to form in L. tarentolae. Functional proteins requiring this modification, such as human laminin-332 or the hepatitis C virus (HCV) E1E2 glycoprotein complex, have been successfully obtained using this system30,31. For proteins lacking a signal peptide, cleavage of N-terminal methionine might also be important. The presence of methionine aminopeptidase in Leishmania cells allows for similar N-terminal methionine cleavage as in mammalian cells32. The Leishmania tarentolae expression system is distinguished by its ease of cultivation, rapid growth rate, extensive genetic manipulation tools, and ability to express proteins from higher eukaryotes, making it highly advantageous for a wide range of biotechnological research and applications. The numerous benefits of L. tarentolae in vaccine production have been explored in several studies, which will be discussed in this publication (Table 2). Several different strategies based on this protozoan system have been utilized (Fig. 4).

Fig. 3: N-glycosylation patterns in different expression systems.
Fig. 3: N-glycosylation patterns in different expression systems.
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Schematic representation of typical glycan residues in different expression systems has been shown above. Created with BioRender.com.

Fig. 4: Scheme of different approaches to the production of antiviral vaccines with the use of L. tarentolae.
Fig. 4: Scheme of different approaches to the production of antiviral vaccines with the use of L. tarentolae.
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L. tarentolae can be used as an expression system (subunit vaccines) or serve as a whole-cell vaccines. Created with BioRender.com.

Subunit vaccine candidates against viral pathogens produced in L. tarentolae

There are various strategies used in vaccine development. To increase global vaccination coverage, factors such vaccine cost and storage conditions must be considered. For instance, vaccines based on recombinant proteins or virus-like particles (VLPs) may be more accessible compared to mRNA-based vaccines, which require transport and storage at −80 °C. Generally, vaccines based on recombinant proteins or VLPs are stable in 4 °C for months or even years, and can tolerate higher temperatures for shorter periods33,34. Another crucial aspect of vaccine development is the production of veterinary vaccines. These vaccines are essential for protecting both animal and human health by preventing the spread of zoonotic diseases that can be transmitted between animals and humans. Recombinant subunit protein vaccines are particularly promising in veterinary medicine due to their ability to provide targeted protection against specific pathogens, reduce the risk of zoonotic disease transmission, and enhance overall animal and public health.

The first viral protein produced in L. tarentolae was influenza hemagglutinin (HA), the major antigenic determinant of the influenza virus. HA is a membrane protein containing disulfide bridges and N-glycosylation sites35. Despite available seasonal vaccines, influenza pandemics continue to emerge. Each year, annual epidemics result in approximately 3 to 5 million cases of severe illness and cause around 290,000 to 650,000 deaths related to respiratory issues (“World Health Organization. Influenza (Seasonal).” https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal)). Complications from influenza, such as bronchitis, pneumonia, cardiovascular issues, acute respiratory failure, and secondary bacterial infections, can be severe and even fatal, especially in immunocompromised individuals, the elderly, and children36. Studies have shown that vaccination is the most effective way to prevent influenza outbreaks37. However, widespread vaccine distribution in a short time is essential. Challenges with influenza vaccination include the virus’s variability, which necessitates the production of vaccines for different subtypes each year. Current reliance on egg-based production could be supplemented by recombinant antigens, potentially increasing the availability of influenza vaccines38,39.

Pion et al. successfully produced properly glycosylated HA using the L. tarentolae system. The glycosylation of HA is crucial for proper folding and affects immunogenicity. However, it is important that the protein is not hyperglycosylated, which is a common issue in the yeast system, as oligosaccharides in inappropriate positions can mask antigenic sites. The HA protein produced in L. tarentolae also induced an immune response in mice, although the use of an adjuvant was necessary35.

Hepatitis C virus (HCV) is another pathogen for which the protozoal expression system was used to produce a vaccine antigen. HCV infections cause around 64,000 deaths annually, with around 58 million people chronically infected worldwide. Transmission mainly occurs through infected blood, often via shared needles or unsafe medical procedures. Untreated HCV can lead to chronic liver disease, cirrhosis, and increased risk of liver cancer. Despite extensive research, no vaccine has been approved against this virus40. The LEXSY system was first used to produce HCV vaccine antigens in a study published by Grzyb et al.31. The HCV envelope glycoproteins E1 and E2 were successfully produced and found to be highly immunogenic in mice. These glycoproteins are key components of the viral envelope, facilitating viral entry into host cells and playing a crucial role in the virus’s ability to infect hepatocytes.

Another study demonstrated that this system could also be employed to produce HCV VLPs, composed of the E1 and E2 glycoproteins along with core nucleocapsid proteins. The VLPs were used for immunization studies and compared to DNA-based vaccines. The findings revealed that immunization with VLPs induced both humoral and cellular responses in a mouse model and was more effective than DNA-based immunization41.

Additionally, different studies have proposed the development of a bivalent vaccine against hepatitis B virus (HBV) and HCV. Subviral particles built from small surface antigens (sHBsAgs) of HBV, produced in L. tarentolae, were used as a platform to present several epitopes of the HCV E2 glycoprotein. HBV particles composed of sHBsAg have been previously proven to be highly immunogenic and are currently used as anti-HBV vaccines, making them promising candidates for a chimeric, bivalent HBV-HCV vaccine. The studies confirmed that chimeric HBV-HCV antigens are highly immunogenic, providing both cellular and humoral responses against HBV, as well as cross-reactive humoral responses against HCV42,43.

An important aspect of the emergence of infectious diseases is the potential for viral pathogens to transfer from animals to humans. This applies not only to viruses that may pose a threat in the future but also to known pathogens such as tick-borne encephalitis virus. TBEV is transmitted to humans through tick bites in wooded areas of Europe and Asia and through unpasteurized dairy products from infected animals. TBEV causes tick-borne encephalitis, which leads to symptoms like fever, headache, and fatigue. In severe cases, it can cause encephalitis or meningitis, leading to potential long-term neurological complications. Currently, five vaccines against TBEV are based on inactivated virus. In the EU, two vaccines are available: Pfizer’s FSME-Immun® and Bavarian Nordic’s Encepur®, both of which use formaldehyde-inactivated whole virus particles. Widespread vaccination of farm animals could reduce the number of human infections and lower the reservoir of TBEV in the environment. However, the high cost of these inactivated TBEV vaccines limits their use in livestock, making cheaper alternatives needed44,45,46.

A recent study described a vaccine candidate based on TBEV VLPs produced in L. tarentolae. Two TBEV structural proteins, prM and E, were efficiently produced in glycosylated form and assembled into VLPs, which were recognized by virus-neutralizing antibodies. Notably, these VLPs were enveloped but have been secreted and purified from the culture medium, indicating that VLP budding was possible, although the mechanism remains unknown. The VLPs were purified and used in animal models, where they were proven to be highly effective in protecting mice from TBEV infection in a challenge experiment. Therefore, TBEV VLPs produced in L. tarentolae could be a promising candidate for a low-cost human vaccine, as well as a veterinary vaccine47.

Increasing vaccine availability is crucial for diseases like cervical cancer, which is primarily caused by human papilloma virus (HPV). HPV is mainly transmitted through sexual contact and direct skin-to-skin contact, with over 80% of sexually active individuals expected to contract the virus at some point. The high cost of HPV vaccines limits their accessibility in developing countries, where 85% of cervical cancer cases occur48. Increased vaccination rates could significantly reduce the number of cervical cancer cases49,50. Currently, HPV vaccines based on VLPs are produced in yeast (Gardasil®) and insect cells (Cervarix®)51.

Bolhassani et al. proposed an alternative HPV vaccine, also based on HPV VLPs, but produced using the L. tarentolae system. The particles were successfully obtained, and their efficacy as vaccine antigens was confirmed in studies with mice. The results showed that the antibody levels were comparable to those in animals immunized with VLPs produced in insect cells, indicating a high degree of similarity between the produced particles. These VLPs were also the first produced using the L. tarentolae system24.

The production of VLPs using the L. tarentolae system has also been explored in the development of a vaccine for norovirus (NoV). NoVs are highly infectious and are the leading cause of gastroenteritis worldwide. While infections are usually self-limiting, they can persist in vulnerable populations, potentially leading to fatal outcomes. Human transmission occurs mainly via oral-fecal and vomit-oral routes, with no other known reservoirs52,53. NoVs cause approximately 685 million cases of acute gastroenteritis and over 200,000 deaths annually, primarily in developing nations. The global economic burden of NoV infections exceeds $60 billion due to healthcare costs and lost productivity. Despite these impacts, there is currently no available vaccine for NoV, making vaccine development a priority54.

A VLP-based vaccine consisting of the NoV capsid VP1 protein produced in the LEXSY system, as proposed by Panasiuk et al., could be a good candidate. The study demonstrated that the VLPs were properly folded and stimulated the production of neutralizing antibodies55. Additionally, these VLPs were also used as a platform to present the tumor-associated mucin 1 (MUC1) epitope. Immunization with chimeric NoV VP1-MUC1 VLPs led to the production of significant levels of MUC1-specific IgG antibodies, indicating that these chimeric VLPs hold promise for cancer immunotherapy56.

L. tarentolae as a live vector vaccine

Another approach to developing vaccines against viral pathogens by L. tarentolae is the use of live vector vaccines that specifically target dendritic cells (DCs) and lymphoid organs (Fig. 4). Such vaccine antigens stimulate the production of interferon gamma (IFN-γ) and elicit a strong T-cell response. Unlike viral vectors, L. tarentolae cannot multiply in DCs and is completely cleared from murine hosts within a few days, eliminating the need for additional modifications in the genome to prevent replication57,58. Additionally, L. tarentolae offers the advantage of inserting long fragments of exogenous DNA, making it a promising vaccine vector19.

The first antiviral vaccine candidate developed using L. tarentolae was a live vaccine expressing the human immunodeficiency virus 1 (HIV-1) structural protein. HIV is a retrovirus that targets CD4 cells, compromising the immune system. Transmission occurs mainly through unprotected sex, sharing needles, and from mother to child during childbirth or breastfeeding. By the end of 2022, around 39 million people were infected globally, with sub-Saharan Africa being the most affected region. Although no vaccine is available, antiretroviral therapy helps to manage the disease59,60. In this vaccine, the HIV-1 Gag protein was successfully expressed in L. tarentolae and underwent proper proteolytic processing. The antigen’s ability to stimulate an antiviral response was tested in mice and human lymphoid tissue cultures, where it triggered a protective immune response57.

This strategy was similarly applied to vaccines for HPV and HCV. For HPV, L. tarentolae carrying the gene for the nonstructural E7 protein induced high levels of both humoral and cellular responses. It also acted as an immunostimulatory adjuvant, showing comparable efficacy to DNA vaccines and inhibiting tumor growth in animal models61,62. A strong antibody response was also observed when the E7 gene was replaced with the gene encoding HPV L1 structural protein63. In the case of HCV, mice immunized with L. tarentolae carrying HCV epitopes fused with the N-terminal domain of the heat shock protein gp96 demonstrated a strong cellular and antibody response. The live recombinant L. tarentolae vaccine was effective both with and without an adjuvant, and the immune response was long-lasting64.

L. tarentolae as an inactivated delivery platform

Another approach used L. tarentolae as a carrier of SARS-CoV-2 antigens in mucosal vaccination (Fig. 4). This strategy can elicit a different immune response profile to the more common intramuscular or subcutaneous administration. A vaccine administered via mucosal routes could complement current vaccines and potentially provide a longer-lasting immune response. In this study, parasites expressing the SARS-CoV-2 spike protein or a fragment of the spike RBD domain were inactivated with formalin and administered rectally. The immune responses were compared to those administered the purified antigen without a carrier. Notably, rectal administration of the antigen alone did not elicit any detectable immune response, but when the antigen was delivered using the L. tarentolae carrier, it triggered a strong immune response, even without the need for an adjuvant. This demonstrated that L. tarentolae can function as an efficient and safe delivery platform, and possibly an adjuvant for viral antigens65.

L. tarentolae – a tool for the production of viral diagnostic antigens

Viral proteins produced in the LEXSY system can also be used as diagnostic antigens. The COVID-19 pandemic underscored the importance of the rapidly developing diagnostic tools for emerging pathogens, as quick infection detection is critical for containing infectious diseases66. While SARS-CoV-2 diagnostic tests typically use antigens produced in mammalian cells due to the need for glycosylation and proper folding, antigens produced in L. tarentolae have also proven effective in diagnostic assays, yielding comparable results. For instance, the SARS-CoV-2 spike protein receptor-binding domain was efficiently produced in L. tarentolae and used to measure antibody levels in human serum samples67. Additionally, the hepatitis E virus (HEV) capsid protein was successfully produced in this system with high purity, and the developed ELISA test showed 92.4% agreement with a commercial HEV test, demonstrating the strong potential of Leishmania-derived antigens68. Furthermore, L. tarentolae was used to produce the Hendra virus (HeV) attachment glycoprotein, which could aid in developing diagnostic assays for HeV69.

While mammalian cells are recommended for producing diagnostic antigens for viral infections in humans and other mammals, this approach has limitations, such as high production costs due to slow cell growth, and expensive media and culture requirements. The LEXSY system offers a cost-effective alternative, as demonstrated by its ability to produce antigens that can be as effective as those from mammalian cells. Thus, L. tarentolae has the potential to be utilized in the future development of serological tests70,71.

Discussion

Vaccines are the best line of defense against infectious diseases. Due to the growing risk of emerging infectious diseases, it is important to develop technologies that enable the rapid development of new vaccine antigens. It is also essential to ensure the widest possible availability of vaccines globally, not only in high-income countries. Vaccine platforms are not universally applicable to all pathogens; therefore, having a variety of expression systems and other vaccine solutions increases the likelihood of developing effective vaccines.

In recent years, interest in L. tarentolae as an expression system for the production of vaccine antigens, both for human and veterinary purposes, has grown. The low cost of vaccine production using L. tarentolae makes it particularly attractive in this regard. The use of vaccines based on selected virus antigens, rather than the whole virus (attenuated or inactivated), is also important in the context of applying the DIVA (Differentiating Infected from Vaccinated Animals) strategy.

A major advantage of this system is its combination of rapid proliferation with good production efficiency and appropriate post-translational modifications, while avoiding drawbacks such as hyperglycosylation (as seen in yeast) or endotoxin contamination (as seen in bacteria). However, there are currently no approved vaccines produced using this system, and further studies are needed before they can reach the market. Nonetheless, the studies cited here demonstrate the high potential of the L. tarentolae system in this area. Considering all the above factors, it is increasingly likely that this protozoan-based expression system will become an important component of future recombinant vaccines.