RESUMEN
Naturally occurring T cells that recognize microbial peptides via HLA-E, a nonpolymorphic HLA class Ib molecule, could provide the foundation for new universal immunotherapeutics. However, confidence in the biological relevance of putative ligands is crucial, given that the mechanisms by which pathogen-derived peptides can access the HLA-E presentation pathway are poorly understood. We systematically interrogated the HIV proteome using immunopeptidomic and bioinformatic approaches, coupled with biochemical and cellular assays. No HIV HLA-E peptides were identified by tandem mass spectrometry analysis of HIV-infected cells. In addition, all bioinformatically predicted HIV peptide ligands (>80) were characterized by poor complex stability. Furthermore, infected cell elimination assays using an affinity-enhanced T cell receptor bispecific targeted to a previously reported HIV Gag HLA-E epitope demonstrated inconsistent presentation of the peptide, despite normal HLA-E expression on HIV-infected cells. This work highlights the instability of the HIV HLA-E peptidome as a major challenge for drug development.
Asunto(s)
Infecciones por VIH , Antígenos HLA-E , Humanos , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Epítopos , Infecciones por VIH/terapia , Péptidos/metabolismoRESUMEN
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.
Asunto(s)
Antivirales/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Péptidos/metabolismo , Virión/inmunología , Antivirales/farmacología , HumanosRESUMEN
Development of effective malaria vaccines requires delivery platforms to enhance the immunogenicity and efficacy of the target antigens. This is particularly challenging for transmission-blocking malaria vaccines (TBVs), and specifically for those based on the Pfs25 antigen, that need to elicit very high antibody titers to stop the parasite development in the mosquito host and its transmission. Presenting antigens to the immune system on virus-like particles (VLPs) is an efficient way to improve the quantity and quality of the immune response generated. Here we introduce for the first time a new VLP vaccine platform, based on the well-established hepatitis B surface antigen (HBsAg) fused to the SpyCatcher protein, so that the antigen of interest, linked to the SpyTag peptide, can be easily displayed on it (Plug-and-Display technology). As little as 10% of the SpyCatcher::HBsAg VLPs decorated with Pfs25::SpyTag (molar ratio) induces a higher antibody response and transmission-reducing activity in mice compared to the soluble protein, with 50 and 90% of the VLP coupled to the antigen further enhancing the response. Importantly, using this carrier that is a vaccine antigen itself could be beneficial, as we show that anti-HBsAg IgG antibodies are induced without interfering with the Pfs25-specific immune response generated. Furthermore, pre-existing anti-HBsAg immunity does not affect the antigen-specific response to Pfs25::SpyTag-SpyCatcher::HBsAg, suggesting that these VLPs can have a broad use as a vaccine platform.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra la Malaria/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Antígenos de Protozoos/inmunología , Proteínas Protozoarias/inmunología , Vacunas de Partículas Similares a Virus/ultraestructuraRESUMEN
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 Qß 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.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Anticuerpos Antiprotozoarios/inmunología , Vacunas contra la Malaria/inmunología , Nanopartículas , Proteínas Protozoarias/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Femenino , Vacunas contra la Malaria/farmacología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/farmacología , Vacunas de Partículas Similares a Virus/farmacologíaRESUMEN
A persistent goal of vaccine development is the enhancement of the immunogenicity of antigens while maintaining safety. One strategy involves alteration of the presentation of the antigen by combining antigens with a multimeric scaffold. Multi-antigen vaccines are under development, and there are presently far more candidate antigens than antigen scaffolding strategies. This is potentially problematic, since prior immunity to a scaffold may inhibit immune responses to the antigen-scaffold combination. In this study, a series of domains from S. aureus which have been shown to crystallise into multimeric structures have been examined for their scaffolding potential. Of these domains, SAR1376, a 62 amino acid member of the 4-oxalocrotonate tautomerase (4-OT) family, was pro-immunogenic in mice when fused to a range of pathogen antigens from both S. aureus and P. falciparum, and delivered by either DNA vaccination, viral vector vaccines or as protein-in-adjuvant formulations. The adjuvant effect did not depend on enzymatic activity, but was abrogated by mutations disrupting the hexameric structure of the protein. We therefore propose that SAR1376, and perhaps other members of the 4-OT protein family, represent very small domains which can be fused to a wide range of antigens, enhancing immune responses against them.
Asunto(s)
Antígenos Bacterianos/inmunología , Inmunidad , Isomerasas/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Staphylococcus aureus/enzimología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Activación Enzimática/efectos de los fármacos , Vectores Genéticos/metabolismo , Humanos , Isomerasas/química , Ratones Endogámicos BALB C , Proteínas Mutantes/metabolismoRESUMEN
FK506 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.
Asunto(s)
Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/genética , Sirolimus/farmacología , Proteínas de Unión a Tacrolimus/genética , Tacrolimus/farmacología , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/genética , Anticuerpos Antiprotozoarios/biosíntesis , Inmunogenicidad Vacunal , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Culicidae/efectos de los fármacos , Culicidae/parasitología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/inmunología , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Humanos , Insectos Vectores/efectos de los fármacos , Insectos Vectores/parasitología , Estadios del Ciclo de Vida/efectos de los fármacos , Estadios del Ciclo de Vida/inmunología , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos BALB C , Pichia/genética , Pichia/metabolismo , Plásmidos/química , Plásmidos/inmunología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/inmunología , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunación , Vacunas SintéticasRESUMEN
Virus-like particles (VLPs) are non-infectious self-assembling nanoparticles, useful in medicine and nanotechnology. Their repetitive molecularly-defined architecture is attractive for engineering multivalency, notably for vaccination. However, decorating VLPs with target-antigens by genetic fusion or chemical modification is time-consuming and often leads to capsid misassembly or antigen misfolding, hindering generation of protective immunity. Here we establish a platform for irreversibly decorating VLPs simply by mixing with protein antigen. SpyCatcher is a genetically-encoded protein designed to spontaneously form a covalent bond to its peptide-partner SpyTag. We expressed in E. coli VLPs from the bacteriophage AP205 genetically fused to SpyCatcher. We demonstrated quantitative covalent coupling to SpyCatcher-VLPs after mixing with SpyTag-linked to malaria antigens, including CIDR and Pfs25. In addition, we showed coupling to the VLPs for peptides relevant to cancer from epidermal growth factor receptor and telomerase. Injecting SpyCatcher-VLPs decorated with a malarial antigen efficiently induced antibody responses after only a single immunization. This simple, efficient and modular decoration of nanoparticles should accelerate vaccine development, as well as other applications of nanoparticle devices.