Self-amplifying RNA (SAM) represents a versatile tool that can be used to develop potent vaccines, potentially able to elicit strong antigen-specific humoral and cellular-mediated immune responses to virtually any infectious disease. To protect the SAM from degradation and achieve efficient delivery, lipid nanoparticles (LNPs), particularly those based on ionizable amino-lipids, are commonly adopted.Herein, we compared commonly available cationic lipids, which have been broadly used in clinical investigations, as an alternative to ionizable lipids. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG) was used. The cationic lipids investigated including 3ß-[N-(N',N'dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDA), 1,2dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) and N-(4-carboxybenzyl)-N,Ndimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ). Whilst all cationic LNP (cLNP) formulations promoting high association with cells in vitro, those formulations containing the fusogenic lipid 1,2dioleoyl-sn-3-phosphoethanolamine (DOPE) in combination with DOTAP or DDA were the most efficient at inducing antigen expression. Therefore, DOTAP and DDA formulations were selected for further in vivo studies and were compared to benchmark ionizable LNPs (iLNPs). Biodistribution studies revealed that DDA-cLNPs remained longer at the injection site compared with DOTAP-cLNPs and iLNPs when administered intramuscularly in mice. However, both the cLNP formulations and the iLNPs induced strong humoral and cellular-mediated immune responses in mice that were not significantly different at a 1.5 µg SAM dose. In summary, cLNPs based on DOTAP and DDA are an efficient alternative to iLNPs to deliver SAM vaccines.

We created and tested multi-epitope DNA or protein vaccines with TLR4 ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) for their ability to protect against Toxoplasma gondii in HLA transgenic mice. Our constructs each included 5 of our best down-selected CD8(+) T cell-eliciting epitopes, a universal CD4(+) helper T lymphocyte epitope (PADRE), and a secretory signal, all arranged for optimal MHC-I presentation. Their capacity to elicit immune and protective responses was studied using immunization of HLA-A*11:01 transgenic mice. These multi-epitope vaccines increased memory CD8(+) T cells that produced IFN-γ and protected mice against parasite burden when challenged with T. gondii. Endocytosis of emulsion-trapped protein and cross presentation of the antigens must account for the immunogenicity of our adjuvanted protein. Thus, our work creates an adjuvanted platform assembly of peptides resulting in cross presentation of CD8(+) T cell-eliciting epitopes in a vaccine that prevents toxoplasmosis

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 μM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

Ability of CD8+ T cells to act as cytolytic effectors and produce IFN-γ was shown to mediate resistance to Toxoplasma gondii in murine models due to recognition of peptides restricted by murine MHC Class I molecules. However, no T. gondii specific HLA-B07 restricted peptides were proven protective against T gondii. Recently, two T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA720–28 (LPQFATAAT) and GRA327–35 (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702, and elicited IFN-γ from PBMCs of seropositive HLA-B*0702 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ producing T cells were identified by ELISPOT and proliferation assays utilizing splenic T lymphocytes from HLA transgenic mice. When HLA-B*0702 mice were immunized with one of the epitopes identified, GRA720–28 in conjunction with a universal CD4+ T cell epitope (PADRE) and adjuvants (CD4+ T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam2Cys for CD8+ T cells), this immunization induced CD8+ T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates feasibility of bioinformatics followed by an empirical approach based on HLA binding to test this biological activity for identifying protective HLA-B*0702 restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.

Micronemal proteins of the thrombospondin-related anonymous Background: protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against , the causative agent Plasmodium falciparum of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in . We performed a Toxoplasma gondii Methods: careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and virulence. We in vivoResults: verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of causes a mic2 slightly delayed infection leading only to mild attenuation of virulence; in vivo, like with wildtype parasites, inoculation with even low numbers of KO mic2 parasites causes lethal disease in mice. However, deletion of causes mic2 delayed host cell egress , possibly via disrupted signal transduction in vitro pathways.We confirm a critical role of MIC2 in parasite Conclusions: attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite. Amendments from Version 1In response to the reviewers comments, we modified figure 5 and 6 (error bars, size bars have been added). We also provide more information regarding the in vivo experiments and tuned down the interpretation regarding virulence of mic2KO parasites. Furthermore, supplementary information for the RNAseq analysis has been added. New data added: RH FPKM, MIC2 KO FPKM and AMA1 FPKM, contain the FPKM of the three triplicates for all Toxoplasma genes for RH, mic2 KO and ama1 KO strains. MIC2KO-RH and AMA1KO-RH total results obtained after Cutdiff analysis between RH and mutants. The addition of these data will allow to access the whole RNA sequencing results and perform independent analysis.