Recent progress in vaccine development targeting pre-clinical human toxoplasmosis

Molecular properties of DNA vaccines

Using nucleic acids for eliciting immune responses in hosts was first reported in the early 1990s by Tang et al. [13]. Since its discovery, DNA vaccines rapidly emerged into the scientific limelight and were actively researched. Structurally, DNA vaccines are composed of a bacteria-derived plasmid encoding a specific antigen of interest whose expression is controlled by a strong viral promoter for optimal gene expression in vivo, such as the cytomegalovirus (CMV) or the simian virus 40 (SV40). The precise mechanism underlying how these DNA vaccines induce cellular and humoral immune responses despite their low expression levels in hosts remains largely unknown. However, 3 possible mechanisms describe how these vaccines may facilitate antigen presentation [14]. First, upon delivery into hosts by the parenteral route of immunization, the plasmid DNA encoding the antigen is internalized by somatic cells in the vicinity, such as myocytes or keratinocytes. The antigens are transcribed within these cells and eventually presented to CD8⁺ T cells via the membrane histocompatibility complex (MHC) class I. Second, the antigen-presenting cells (APCs), such as dendritic cells, are recruited to the injection site. These cells become transfected by the plasmid DNA and present the expressed antigen of interest via MHC I and II. Last, the plasmid-infected somatocytes are phagocytosed by APCs to enable cross-priming and antigen presentation to CD4⁺ and CD8⁺ T cell subsets.

There are several factors favoring DNA vaccines over traditional vaccines. The production costs for DNA vaccines are relatively lower than traditional vaccines. Also, because infectious pathogens are not being introduced into the host, this vaccination approach is safe for use [15]. While DNA vaccines appear promising, there are safety concerns even if an infectious pathogen is not used for immunization. For instance, the possibility of antigen-encoding plasmid DNA integration into the host chromosome is one such consequence [16], though later studies revealed that the probability of genetic integration is extremely low [17]. Furthermore, DNA vaccines are weakly immunogenic. Specifically, suboptimal vaccine efficacies were reported from DNA vaccine studies conducted in non-human primates, as indicated by the low levels of antibody responses [15]. Based on these profiles, several DNA vaccines against various infectious diseases have undergone clinical evaluations but a clinical DNA vaccine trial for toxoplasmosis remains unreported.

Current progress in T. gondii DNA vaccine development

DNA vaccines are the most prevalent vaccine platforms being investigated throughout the world. The sheer amount of DNA vaccine-based publications skyrocketed in the early 2000s, and its popularity has remained unchanged [18]. This research trend is no exception for T. gondii vaccines, as most studies revolve around DNA vaccines. Despite the extensive research, most DNA vaccine results were suboptimal, while a few studies reported exceptional findings. Different T. gondii antigens conferred differing degrees of protection in mice. For example, DNA vaccines expressing the dense granule (GRA) 39 antigen prolonged the survival of Kunming mice by 20 days, but none could survive the challenge infection with the RH strain. Cyst burden reduction upon challenge infection with 10 cysts of PRU strain was suboptimal, as not even 50% cyst burden reduction was observed [19]. Contrastingly, GRA24-expressing DNA vaccines prolonged the survival of RH-infected BALB/c mice up to 30 days post-infection [20]. This was also the case with T. gondii Myc regulation 1 (MYR1)-expressing vaccine, which significantly prolonged the survival of immunized mice against RH challenge infection [21].

Several strategies improved the protective efficacy but were only marginally effective. Adjuvanting DNA vaccines had a minor effect on the vaccine’s protective efficacy. Although supplementing the T. gondii GRA7 DNA vaccine with the calcium phosphate nanoparticle adjuvant prolonged the survival of immunized mice, it was only 2 days longer than the unadjuvanted control group [22]. Conflicting results were observed from multi-antigenic vaccines. Combined immunization with DNA vaccines expressing the microneme proteins (MIC) 5 and 16 as antigens reduced the brain cyst burden by half in mice challenged with the PRU strain but, as with other vaccines, failed to confer prolonged protection against the RH strain [23]. On the contrary, multi-antigenic DNA vaccines expressing the SAG2, rhoptry protein (ROP) 9, and MIC3 ensured that immunized mice survived the challenge infection with the highly virulent RH strain regardless of the infection doses [24]. Protection induced by these DNA vaccines was tabulated and briefly described (Table 1).

Table 1

Protective efficacy of DNA vaccines expressing various T. gondii antigens

Molecular properties of protein subunit vaccines

Protein subunit vaccines use a small fraction of a pathogenic agent’s antigenic component to elicit immune responses in vaccinees. Like the traditional inactivated whole-organism vaccines, protein subunit vaccines are incapable of replicating in hosts and are safe but possess low immunogenicity. Therefore, protein subunit vaccines often require multiple immunization doses or adjuvant incorporation to achieve long-lasting immunity [25]. With the introduction of recombinant DNA technology and advancements in molecular biology, mass-producing foreign genes of interest in various expression systems has become feasible. Bacterial expression systems are frequently used to produce large quantities of protein of interest at a low cost. However, given the nature of prokaryotic organisms, proteins are misfolded, and post-translational modifications (PTMs) observed in mammals are lacking [26]. The need for downstream purification for endotoxin removal and processing of expressed antigens further hampers this. Like the bacterial expression system, yeast and insect cells can rapidly produce significant amounts of proteins of interest. While PTMs occur in these organisms, glycosylation patterns are not identical to those observed in mammalian cells [27]. Based on PTM, mammalian cells would be ideal for antigenic protein production. However, improvements are needed as mammalian cell-derived antigen yields are relatively lower than antigens produced in the aforementioned expression systems.

Current progress in T. gondii recombinant protein subunit vaccine development

Like DNA vaccines, much progress has been made using recombinant subunit vaccines. Subunit protein vaccines are safe, but their immunogenicity pales in comparison to other vaccine platforms [28]. While some protein-based T. gondii vaccines are protective, others failed to elicit desirable protection. The latter was predominantly observed in studies that utilized ubiquitous eukaryotic proteins as antigens. Vaccines expressing the T. gondii aspartic protease 3 (ASP3) prolonged the survival duration by 11 days against RH challenge infection [29]. T. gondii peroxiredoxin 1 (PRX1) vaccine failed to confer complete protection against the moderately virulent type II PLK strain [30]. However, as with DNA vaccines, conflicting protection results were observed from subunit vaccines (Table 2). Cocktail subunit vaccines conferred protection against types I and II T. gondii lineages. Intramuscular immunization with subunit proteins T. gondii macrophage migration inhibitory factor, calcium-dependent protein kinase 3, and the 14-3-3 protein resulted in complete protection against RH tachyzoite and PRU strains [31]. Given this circumstance, more research on improving these vaccines’ protective efficacy is required.

Table 2

Efficacy of T. gondii vaccines based on protein subunit

Molecular properties of VLP vaccines

Although VLPs appear similar to protein subunit vaccines, they are not necessarily the same and should be categorized differently. VLPs are highly immunogenic self-assembled particles that mimic the structural aspects of native virions. However, these particles are inherently safe due to the lack of genetic material. Molecular and structural factors that contribute to the high immunogenicity of VLP-based vaccines have been described in detail [32], and as such, these aspects will be briefly described. In VLPs, antigens of interest are repetitively presented in a dense array which is critical to mounting efficient immune responses against the target antigen [33,34].

Furthermore, because the size of VLPs is less than 200 nm, they are rapidly trafficked into the lymph nodes [35]. The surface charge is another structural property of VLPs that improves immunogenicity compared to protein subunit vaccines. For example, particle-based vaccines possess charged surfaces that enhance their interaction with professional APCs, which may not be accurate for solubilized antigens [36]. Nonetheless, there are limitations to VLP vaccine technology, such as production costs. Like protein subunit vaccines, PTM must be considered during VLP vaccine assembly.

Current progress in T. gondii VLP vaccine development

To date, all VLP-based vaccine studies reported are chimeric, expressing parasitic antigens on the surface of influenza virus matrix protein 1. Surprisingly, VLP immunization elicited considerable protection against virulent type I and moderately virulent type II strains in mice, such as those expressing MIC8 [37] and ROP13 [38] as surface antigens (Table 3). In a comparative study, VLPs expressing ROP18 antigens were more efficacious than those expressing ROP4 [39]. Similar to DNA and subunit vaccines, a multi-antigenic vaccine approach enhanced the protective efficacy of VLP vaccines. While ROP4 and ROP13 VLPs were protective and ensured 100% survival [40], VLPs co-expressing ROP4 and ROP13 antigens led to brain cyst burden reduction compared to VLPs expressing either antigen alone in BALB/c mice [41]. VLPs co-expressing MIC8 and ROP18 reduced the parasite burden following challenge infection with the T. gondii GT1 strain [42]. Further supplementing this vaccine with the inner membrane complex subcompartment protein 3 (IMC) conferred partial protection against the virulent GT1 strain but elicited complete protection against ME49 [43,44].

Table 3

Protective efficacy of T. gondii vaccines based on virus-like particle

A research group demonstrated that chimeric hepatitis B virus-based VLPs expressing CD8 and CD4 T cell epitopes prolonged the survival of immunized mice upon challenge infection with T. gondii RH strain [45]. The impact of immunization regimen and adjuvant use was also evaluated. Herein, increasing the number of immunizations and supplementing adjuvants did not decrease brain cyst size but significantly reduced the cyst burden in ME49-infected mice [46,47]. More research on these T. gondii VLP vaccines is required, especially against the highly virulent strains, which are lethal even at small infection doses. However, the outlook for this vaccine platform appears promising. Furthermore, as all VLP-based vaccine studies were conducted in mice, evaluating their protective efficacy in higher-order eukaryotic organisms should be considered.