Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8⁺T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

Toxoplasmosis is a disease of major medical importance. Toxoplasma gondii causes congenital infections responsible for stillbirths and spontaneous abortions[1–5]. In addition, it causes neurologic disorders, uveitis, and systemic infections in immune-compromised patients. Toxoplasmic encephalitis is a cause of morbidity and mortality in those with congenital disease and persons with AIDS[6]. T. gondii is acquired by consumption of lightly cooked meat, especially lamb and pork contaminated with bradyzoites[7, 8] or by ingestion of food or water contaminated with oocysts containing sporozoites, which are the product of a sexual cycle in the intestine of the cat [8, 9].

Development of an effective vaccine would prevent this disease. Attenuated T. gondii tachyzoites (e.g., S48, TS-4, T-203, ΔRPS13)have been employed for live vaccinations of non-human animals[10–15]. Parasites attenuated by knockout of gene transcription recently have been proposed as a new type of attenuated vaccine candidate[13–15]. However, safety considerations may limit vaccination of humans with live organisms. Development of peptide-based vaccines created with epitopes which elicit IFN-γ production by CD8⁺ T cells[16] is a promising strategy to mobilize the immune system against T. gondii in humans[16–20]. Thus, an effort to create an immunosense epitope-based vaccine was initiated.

Recently, three peptide epitopes, KSFKDILPK (SAG1_224-232), AMLTAFFLR (GRA6_164-172), and RSFKDLLKK (GRA7_134-142), were found by our group to elicit IFN-γ from peripheral blood mononuclear leukocytes (PBMCs) from T. gondii seropositive HLA-A03 supertype humans but not from PBMCs of T. gondii seronegative HLA-A03 supertype humans[18]. Herein, initially we designed 4 CD8⁺ T cell epitope containing vaccine formulations comprised of lipopeptides that incorporate PADRE as well as a novel oil-in-water emulsion that includes a specially formulated synthetic MLA derivative called GLA-SE[21–24]. First, efficacy of various vaccine formulations consisting of the three previously identified CD8⁺ T cell peptides eliciting HLA-A*1101-restricted, CD8⁺T cell-mediated IFN-γ production in vitro from HLA-A*1101 mice was examined.

Then, in order to identify additional peptides from T. gondii tachyzoites, bradyzoites and sporozoites of Type I and Type II strains that are effective in eliciting IFN-γ from HLA-A03 supertype restricted CD8⁺ T cells, the first step was to select proteins with biologic properties, such as being secreted, compatible with MHC Class I processing. Then, bioinformatic algorithms to identify HLA-A03 supertype bound peptides were utilized to find additional, novel T. gondii-derived, potential CD8⁺ T cell eliciting epitopes restricted by the HLA-A03 supertype. We screened peptides from tachyzoite, bradyzoite and sporozoite proteins (GRA10, GRA15, SAG2C, SAG2D, SAG2X, SAG3, SRS9, BSR4, SPA, MIC) of the type II T. gondii strain, ME49, searching for those with high binding scores in a bioinformatic analysis (IC₅₀ < 50 nM) and then in binding assays. In addition, peripheral blood mononuclear cells from seropositive and seronegative persons were tested for response to these peptides using an IFN-γ ELISpot assay. These latter studies were performed to attempt to identify other peptides that would be promising candidates for inclusion in a multi-epitope, next generation immunosense vaccine. Then, information obtained from testing the first three peptides studied was used to guide formulation and administration of a peptide pool. This pool included the first three peptides identified earlier and tested in the initial experiments combined with the newly identified peptides that elicited IFN-γ from human HLA-A03 supertype restricted CD8⁺ T cells. All the peptides in this pool were tested with a universal T helper epitope called PADRE and a new, promising adjuvant called GLA-SE with Pam₂Cys in HLA-A*1101 transgenic mice. Capacity to induce IFN-γ production by spleen CD8⁺ T cells, and to protect against parasite burden following subsequent challenge were determined. For parasite challenge, a luciferase transfected Type II Prugneaud parasite was administered, followed by luciferin administration and imaging with a Xenogen camera system a week later. This allows detection and quantization of bioluminescent parasites as a biomarker to assess efficacy of immunizations in protection.

In this study, we first evaluated immunization of HLA-A*1101 transgenic mice with either mixtures of peptides or lipopeptides derived from three identified T. gondii specific HLA-A*1101 restricted CD8⁺ T cell epitopes emulsified in 3-deacylated monophosphoryl lipid A(GLA-SE) adjuvant. Immunizations of transgenic mice with a mixture of CD8⁺ epitope peptide pools plus PADRE and adjuvants were able to induce splenocyte to produce IFN-γ and to protect against challenge with high numbers of Type II parasites.

Conjugation of CD8⁺ T cell determinants to lipid groups is known to enhance specific cell-mediated responses to target antigens in experimental animals and humans[25–29], although mechanisms whereby immunity is achieved remains poorly understood. Lipopeptides hold several advantages over other conventional vaccine formulations; for instance, they are self-adjuvanting and display none of the toxicity-associated side effects of other Th1-inducing adjuvant systems. In our work, transgenic mice that were immunized with three short lipopeptide vaccines had T cells that produced IFN-γ. Among them the lipopeptide vaccine formulated with KS9 or AM9 stimulated higher IFN-γ production than the lipopeptide vaccine formulated with RS9. Unexplained and variable responses have been observed to high affinity binding peptides in other models, e.g., studies of Livingstone, Alexander, Sette et al with Lassa fever virus. It will be of interest to better understand possible mechanisms for such lack of response in future studies. However, the lipopeptides with three epitope peptides linked together with alanine spacers did not stimulate an IFN-γ response by splenocytes from immunized mice when the splenocytes subsequently were exposed to each of the peptides in vitro. The reason why the lipopeptides with the three linked peptides did not work well in the transgenic mice might be related to a frame shift caused by the linkers that altered the response to the original peptides rather than the alanines functioning for the intended purpose of introducing a cleavage motif. The three linker "AAA'' between the peptides had previously been demonstrated in other systems to result in sensitization to each linked peptide. However, surprisingly, it did not appear to work well herein.

Because the three linked peptides in the lipopeptide formulation were not effective and we had found that a mixture of the components with a single peptide was as or more robust than the lipopeptide, we tried this approach with the three peptides that had been included in the linked lipopeptide with the universal helper CD4⁺ T cell peptide, PADRE, and adjuvants as described below. The response was robust both in vitro and in vivo (Figures 4 and 5).

Some studies have shown palmitoylated lipopeptide constructs to elicit long-lived, protective cellular responses against a variety of pathogens, including Hepatitis B virus (HBV), influenza virus, and Plasmodium falciparum [25–29]. Our work herein shows that mice immunized with mixture of CD8⁺ and CD4⁺ eliciting peptides and lipid Pam₂Cys emulsified in GLA-SE elicited higher IFN-γ production than mice immunized with lipopeptides constructed with the same components of CD4⁺ and CD8⁺ eliciting peptides, and Pam₂Cys. The approach using cocktails of non-covalently linked lipid mixed to helper T lymphocytes(HTL) and CD8⁺T cell (cytolytic T lymphcyte[CTL] and IFN-γ eliciting) epitopes for simultaneous induction of multiple CD8⁺T specificities would have significant advantages in terms of ease of vaccine development.

HTL responses are crucial for the development of CD8⁺T responses, at least in the case of lipidated covalently or non-covalently linked HTL-CTL epitope constructs formulated in PBS. Several previous studies have illustrated a role for CD4⁺ responses for development of CD8⁺CTL responses, both in humans and in experimental animals[30–35]. The inclusion of PADRE, a synthetic peptide that binds promiscuously to variants of the human MHC class II molecule DR and is effective in mice, also augmented CD8⁺ T cell effector functions by inducing CD4⁺ T helper cells[30–35]. Both CD4⁺ and CD8⁺ epitopes were targeted in order to drive a protective immune response[34, 35].

Adjuvanting antigens contributes to the success of vaccination. An example herein is that 3-deacylated monophosphoryl lipid A(GLA-SE), a detoxified derivative of the lipopolysaccharide (LPS) from Salmonella minnesota R595 was a potent adjuvant. This GLA-SE is a novel adjuvant which was formulated in an emulsion[21–24]. This is a Toll-like receptor 4 (TLR4) agonist that is a potent activator of Th1 responses [21–24]. It has been used as an adjuvant in human vaccine trials for several infectious disease and malignancy indications. It has been very effective as an adjuvant providing CD4⁺ T cell help for immunizations against other protozoan infections such as leishmaniasis [21–24]. In our study, a robust response was observed when GLA-SE was included in preparation for immunization of mice. Pam₂Cys (S-[2,3-bis(palmitoyloxy)propyl]cysteine) is a lipid component of macrophage-activating lipopeptide. Pam₂Cys binds to and activates dendritic cells by engagement of Toll-like receptor 2 (TLR-2)[24]. Toll-like receptors (TLRs) function as pattern-recognition receptors in mammals[36]. We have found that both TLR2 and TLR4 receptors participate in human host defense against T. gondii infection through their activation by GPIs and GIPLs(Melo, Hargrave, Miller, Blackwell, Gazinelli, McLeod et al, in preparation, 2010). TLR2 and TLR4 likely work together with other MyD88-dependent receptors, including other TLRs, to elicit an effective host response against T. gondii infection[36]. In our study, there was a slightly more robust response observed when Pam₂Cys was co-administered for some peptides, but not all of them.

The goal of the present study was to identify HLA-restricted epitopes from T. gondii and evaluate whether they could provide protection against parasite challenge measured as protection against a luciferase producing Type ll parasite using a Xenogen camera system. In the future, additional more detailed studies involving analyses over longer times, other strains of the parasite and challenge with life cycle stages, evaluation of multiple organs including eye and brain, studies of protection in congenital infections, comparisons of delivery of these peptides as DNA encoding them versus other formulations. This future work will follow up and extend these intitial studies of reduction of parasite burden seen in Figures 5 and 7.

Various peptide-based approaches to induction of IFN-γ responses were evaluated as part of ongoing efforts to develop immunosense vaccines for use in humans with each of the supermotifs which would in total include more that 99% of the human population worldwide. Robust protection was achieved in the HLA-A*1101 transgenic mice challenged with Type II parasites following immunizations. In order to identify additional peptides from T. gondii that were present in tachyzoites or bradyzoites[37, 38] or sporozoites of Type I and II strains and elicited IFN-γ from HLA-A03⁺ supertype (which includes the HLA*1011 allele) restricted CD8⁺ T cells, bioinformatic algorithms were utilized to identify novel, T. gondii-derived, epitopes restricted by the HLA-A03 supertype. Then PBMC cells were tested to determine whether the peptides elicited IFN-γ from human CD8⁺ T cells from seropositive persons. This was intended to collectively provide broad coverage for the human population with HLA-A03 supertype worldwide. The additional peptides we identified as immunogenic for human peripheral blood cells were also robust in eliciting IFN-γ from splenocytes of HLA-A*1101 mice and protection when used to immunize these mice. These findings will facilitate development of an immunosense epitope-based vaccine for human use.

A human immunome-based and parasite genome based bioinformatices approach was used to define candidate HLA-A03 supertype restricted peptides. Immunogenicity of a group of T. gondii HLA-A03 supertype restricted peptides, and therefore the proteins from which they are derived, for immune humans and HLA-A*1101 transgenic mice was demonstrated. These peptides elicit interferon-γ production by human CD8⁺T cells. They also elicit interferon γ production by mouse splenocytes when utilized to immunize HLA-A*1101 transgenic mice with a lipopeptide with a universal CD4⁺ T cell eliciting epitope, PADRE, or in peptide pools with PADRE, Pam₂Cys and GLA-SE, a novel adjuvant. Immunization studies demonstrate the need for and the efficacy of adjuvants in immunization of these HLA transgenic mice. Immunogenic peptides included KSFKDILPK (SAG1_224-232); AMLTAFFLR (GRA6_164-172); and RSFKDLLKK (GRA7_134-142); STFWBCLLR (SAG2C_13-21); SSAYVFSVK (SPA_250-258); and AVVSLLRLLK (SPA_89-98). The studies herein provide a foundation for immunosense based vaccines to prevent toxoplasmosis in those with the HLA-A03 supertype and information about how they can be adjuvanted.