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Persistence of HIV through integration into host DNA in CD4(+) T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8(+) T cells are triggered to kill infected CD4(+) T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial'' HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A*02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8(+) T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4(+) T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.

Persistence of HIV through integration into host DNA in CD4

Persistence of HIV through integration into host DNA in CD4+ T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8+ T cells are triggered to kill infected CD4+ T cells through recognition of HLA class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with ‘beneficial’ HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtained proof of early presentation of a virion-derived HLA-A*02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag spacer peptide 2 (SP2). FH9-specific CD8+ T cell responses were detectable in individuals with primary HIV infection and eliminated HIV-infected CD4+ T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target.

Vaccine development efforts have recently focused on enabling strong immune responses to poorly immunogenic antigens, via display on multimerisation scaffolds or virus like particles (VLPs). Typically such studies demonstrate improved antibody titer comparing monomeric and nano-arrayed antigen. There are many such studies and scaffold technologies, but minimal side-by-side evaluation of platforms for both the amount and efficacy of antibodies induced. Here we present direct comparison of three leading platforms displaying the promising malaria transmission-blocking vaccine (TBV) target Pfs25. These platforms encompass the three important routes to antigen-scaffold linkage: genetic fusion, chemical cross-linking and plug-and-display SpyTag/SpyCatcher conjugation. We demonstrate that chemically-conjugated Qβ VLPs elicited the highest quantity of antibodies, while SpyCatcher-AP205-VLPs elicited the highest quality anti-Pfs25 antibodies for transmission blocking upon mosquito feeding. These quantative and qualitative features will guide future nanoassembly optimisation, as well as the development of the new generation of malaria vaccines targeting transmission.

SUMMARYFK506 and rapamycin (Rap) are immunosuppressive drugs that act principally on T-lymphocytes. The receptors for both drugs are FK506-binding proteins (FKBPs), but the molecular mechanisms of immunosuppression differ. An FK506–FKBP complex inhibits the protein phosphatase calcineurin, blocking a key step in T-cell activation, while the Rap –FKBP complex binds to the protein kinase target of rapamycin (TOR), which is involved in a subsequent signalling pathway. Both drugs, and certain non-immunosuppressive compounds related to FK506, have potent antimalarial activity. There is however conflicting evidence on the involvement of Plasmodium calcineurin in the action of FK506, and the parasite lacks an apparent TOR homologue. We therefore set out to establish whether inhibition of the Plasmodium falciparum FKBP PfFKBP35 itself might be responsible for the antimalarial effects of FK506 and Rap. Similarities in the antiparasitic actions of FK506 and Rap would constitute indirect evidence for this hypothesis. FK506 and Rap acted indistinguishably on: (i) specificity for different intra-erythrocytic stages in culture, (ii) kinetics of killing or irreversible growth arrest of parasites and (iii) interactions with other antimalarial agents. Furthermore, PfFKBP35's inhibitory effect on calcineurin was independent of FK506 under a range of conditions, suggesting that calcineurin is unlikely to be involved in the antimalarial action of FK506.

Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine.

SUMMARYImmunophilins comprise two protein families, cyclophilins (CYPs) and FK506-binding proteins (FKBPs), and are the major receptors for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (tacrolimus), respectively. Most eukaryotic species have at least one immunophilin and some of them have been associated with pathogenesis of infectious or parasitic diseases or the action of antiparasitic drugs. The human malarial parasitePlasmodium falciparumhas 13 immunophilin or immunophilin-like genes but the functions of their products are unknown. We set out to identify the parasite proteins that interact with the major CYPs, PfCYP19A and PfCYP19B, and the FKBP, PfFKBP35, using a combination of co-immunoprecipitation and yeast two-hybrid screening. We identified a cohort of putative interacting partners and further investigation of some of these revealed potentially novel roles in parasite biology. We demonstrated that (i)P. falciparumCYPs interacted with the heat shock protein 70, (ii) treatment of parasites with CYP ligands disrupted transport of the rhoptry-associated protein 1, and (iii) PfFKBP35 interacted with parasite histones in a way that might modulate gene expression. These findings begin to elucidate the functions of immunophilins in malaria. Furthermore, the known antimalarial effects of CsA, FK506 and non-immunosuppressive derivatives of these immunophilin ligands could be mediated through these partner proteins.

The purpose of this study was to investigate the blood stage of the malaria causing parasite, Plasmodium falciparum, to predict potential protein interactions between the parasite merozoite and the host erythrocyte and design peptides that could interrupt these predicted interactions. We screened the P. falciparum and human proteomes for computationally predicted short linear motifs (SLiMs) in cytoplasmic portions of transmembrane proteins that could play roles in the invasion of the erythrocyte by the merozoite, an essential step in malarial pathogenesis. We tested thirteen peptides predicted to contain SLiMs, twelve of them palmitoylated to enhance membrane targeting, and found three that blocked parasite growth in culture by inhibiting the initiation of new infections in erythrocytes. Scrambled peptides for two of the most promising peptides suggested that their activity may be reflective of amino acid properties, in particular, positive charge. However, one peptide showed effects which were stronger than those of scrambled peptides. This was derived from human red blood cell glycophorin-B. We concluded that proteome-wide computational screening of the intracellular regions of both host and pathogen adhesion proteins provides potential lead peptides for the development of anti-malarial compounds. Irish Research Council Science Foundation Ireland