RESUMO
Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.
Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Formação de Anticorpos , Anticorpos Antivirais , Linfócitos T , Antígenos , OrganoidesRESUMO
Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.
Assuntos
Coxiella burnetii , Febre Q , Vacinas , Animais , Camundongos , Febre Q/prevenção & controle , Antígenos de Bactérias , Anticorpos , EpitoposRESUMO
Coxiella burnetii is an obligate intracellular bacterium and the causative agent of Q fever. C. burnetii is considered a potential bioterrorism agent because of its low infectious dose; resistance to heat, drying, and common disinfectants; and lack of prophylactic therapies. Q-Vax, a formalin-inactivated whole-bacteria vaccine, is currently the only prophylactic measure that is protective against C. burnetii infections but is not U.S. Food and Drug Administration approved. To overcome the safety concerns associated with the whole-bacteria vaccine, we sought to generate and evaluate recombinant protein subunit vaccines against C. burnetii To accomplish this, we formulated C. burnetii Ags with a novel TLR triagonist adjuvant platform, which used combinatorial chemistry to link three different TLR agonists together to form one adjuvanting complex. We evaluated the immunomodulatory activity of a panel of TLR triagonist adjuvants and found that they elicited unique Ag-specific immune responses both in vitro and in vivo. We evaluated our top candidates in a live C. burnetii aerosol challenge model in C56BL/6 mice and found that several of our novel vaccine formulations conferred varying levels of protection to the challenged animals compared with sham immunized mice, although none of our candidates were as protective as the commercial vaccine across all protection criteria that were analyzed. Our findings characterize a novel adjuvant platform and offer an alternative approach to generating protective and effective vaccines against C. burnetii.
Assuntos
Vacinas Bacterianas/imunologia , Coxiella burnetii/fisiologia , Febre Q/imunologia , Receptores Toll-Like/agonistas , Adjuvantes Imunológicos , Animais , Vacinas Bacterianas/síntese química , Técnicas de Química Combinatória , Modelos Animais de Doenças , Feminino , Humanos , Imunidade , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos C57BL , Vacinas de Subunidades AntigênicasRESUMO
BACKGROUND: There are limited data regarding immunological correlates of protection for the modified vaccinia Ankara (MVA) smallpox vaccine. METHODS: A total of 523 vaccinia-naive subjects were randomized to receive 2 vaccine doses, as lyophilized MVA given subcutaneously, liquid MVA given subcutaneously (liquid-SC group), or liquid MVA given intradermally (liquid-ID group) 28 days apart. For a subset of subjects, antibody-dependent cellular cytotoxicity (ADCC), interferon-γ release enzyme-linked immunospot (ELISPOT), and protein microarray antibody-binding assays were conducted. Protein microarray responses were assessed for correlations with plaque reduction neutralization titer (PRNT), enzyme-linked immunosorbent assay, ADCC, and ELISPOT results. RESULTS: MVA elicited significant microarray antibody responses to 15 of 224 antigens, mostly virion membrane proteins, at day 28 or 42, particularly WR113/D8L and WR101H3L. In the liquid-SC group, responses to 9 antigens, including WR113/D8L and WR101/H3L, correlated with PRNT results. Three were correlated in the liquid-ID group. No significant correlations were observed with ELISPOT responses. In the liquid-ID group, WR052/F13L, a membrane glycoprotein, correlated with ADCC responses. CONCLUSIONS: MVA elicited antibodies to 15 vaccinia strain antigens representing virion membrane. Antibody responses to 2 proteins strongly increased and significantly correlated with increases in PRNT. Responses to these proteins are potential correlates of protection and may serve as immunogens for future vaccine development. CLINICAL TRIALS REGISTRATION: NCT00914732.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Vacina Antivariólica/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacínia , Vacinas Virais/administração & dosagem , Formação de Anticorpos , Antígenos Virais , Humanos , Imunidade Celular , Imunização , Análise Serial de Proteínas , Vacinas Atenuadas , Vaccinia virus/imunologiaRESUMO
Vaccination has been playing an important role in treating both infectious and cancerous diseases. Nevertheless, many diseases still lack proper vaccines due to the difficulty to generate sufficient amounts of antigen-specific antibodies or T cells. Adjuvants provide an important route to improve and direct immune responses. However, there are few adjuvants approved clinically and many of them lack the clear structure/adjuvanticity relationship. Here, we synthesized and evaluated a series of dendronized polypeptides (denpols) functionalized with varying tryptophan/histidine (W/H) molar ratios of 0/100, 25/75, 50/50, 75/25, and 100/0 as tunable synthetic adjuvants. The denpols showed structure-dependent inflammasome activation in THP1 monocytic cells and structure-related activation and antigen cross-presentation in vitro in bone marrow-derived dendritic cells. We used the denpols with bacterial pathogen Coxiella burnetii antigens in vivo, which showed both high and tunable adjuvating activities, as demonstrated by the antigen-specific antibody and T cell responses. The denpols are easy to make and scalable, biodegradable, and have highly adjustable chemical structures. Taken together, denpols show great potential as a new and versatile adjuvant platform that allows us to adjust adjuvanticity based on structure-activity correlation with the aim to fine-tune the immune response, thus advancing vaccine development.
Assuntos
Vacinas , Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias , Peptídeos/farmacologia , VacinaçãoRESUMO
The development of vaccines against malaria and serodiagnostic tests for detecting recent exposure requires tools for antigen discovery and suitable animal models. The protein microarray is a high-throughput, sample sparing technique, with applications in infectious disease research, clinical diagnostics, epidemiology, and vaccine development. We recently demonstrated Qdot-based indirect immunofluorescence together with portable optical imager ArrayCAM using single isotype detection could replicate data using the conventional laser confocal scanner system. We developed a multiplexing protocol for simultaneous detection of IgG, IgA, and IgM and compared samples from a controlled human malaria infection model with those from controlled malaria infections of Aotus nancymaae, a widely used non-human primate model of human malaria. IgG profiles showed the highest concordance in number of reactive antigens; thus, of the 139 antigens recognized by human IgG antibody, 111 were also recognized by Aotus monkeys. Interestingly, IgA profiles were largely non-overlapping. Finally, on the path toward wider deployment of the portable platform, we show excellent correlations between array data obtained in five independent laboratories around the United States using the multiplexing protocol (R2 : 0.60-0.92). This study supports the use of this platform for wider deployment, particularly in endemic areas where such a tool will have the greatest impact on global human health.
Assuntos
Imunoensaio/métodos , Imunoglobulina G/análise , Malária Falciparum/diagnóstico , Análise Serial de Proteínas/métodos , Proteoma/análise , Animais , Aotidae , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina A/análise , Imunoglobulina M/análise , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Plasmodium falciparum/isolamento & purificação , Pontos QuânticosRESUMO
Background: Chronic asymptomatic Plasmodium falciparum infections are common in endemic areas and are thought to contribute to the maintenance of malaria immunity. Whether treatment of these infections increases the subsequent risk of clinical episodes of malaria is unclear. Methods: In a 3-year study in Mali, asymptomatic individuals with or without P. falciparum infection at the end of the 6-month dry season were identified by polymerase chain reaction (PCR), and clinical malaria risk was compared during the ensuing 6-month malaria transmission season. At the end of the second dry season, 3 groups of asymptomatic children were identified: (1) children infected with P. falciparum as detected by rapid diagnostic testing (RDT) who were treated with antimalarials (n = 104), (2) RDT-negative children whose untreated P. falciparum infections were detected retrospectively by PCR (n = 55), and (3) uninfected children (RDT/PCR negative) (n = 434). Clinical malaria risk during 2 subsequent malaria seasons was compared. Plasmodium falciparum-specific antibody kinetics during the dry season were compared in children who did or did not harbor asymptomatic P. falciparum infections. Results: Chronic asymptomatic P. falciparum infection predicted decreased clinical malaria risk during the subsequent malaria season(s); treatment of these infections did not alter this reduced risk. Plasmodium falciparum-specific antibodies declined similarly in children who did or did not harbor chronic asymptomatic P. falciparum infection during the dry season. Conclusions: These findings challenge the notion that chronic asymptomatic P. falciparum infection maintains malaria immunity and suggest that mass drug administration during the dry season should not increase the subsequent risk of clinical malaria.
Assuntos
Malária Falciparum/epidemiologia , Plasmodium falciparum , Adolescente , Adulto , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Infecções Assintomáticas , Criança , Pré-Escolar , Doença Crônica , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lactente , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Masculino , Mali/epidemiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Vigilância da População , Risco , Estações do Ano , Adulto JovemRESUMO
Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.
Assuntos
Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacina Antivariólica/imunologia , Vaccinia virus/imunologia , Transferência Adotiva , Animais , Especificidade de Anticorpos , Antígenos Virais/metabolismo , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Testes de Neutralização , Vacina Antivariólica/metabolismo , Vacínia/imunologia , Vacínia/prevenção & controle , Vacínia/virologiaRESUMO
Organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. However, the detection equipment is often expensive, fragile, and hard to deploy widely. Quantum dots (Qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. Here, we have evaluated a small benchtop "personal" optical imager (ArrayCAM) developed for quantification of protein arrays probed by Qdot-based indirect immunofluorescence. The aim was to determine if the Qdot imager system provides equivalent data to the conventional organic dye-labeled antibody/laser scanner system. To do this, duplicate proteome microarrays of Vaccinia virus, Brucella melitensis and Plasmodium falciparum were probed with identical samples of immune sera, and IgG, IgA, and IgM profiles visualized using biotinylated secondary antibodies followed by a tertiary reagent of streptavidin coupled to either P3 (an organic cyanine dye typically used for microarrays) or Q800 (Qdot). The data show excellent correlation for all samples tested (R > 0.8) with no significant change of antibody reactivity profiles. We conclude that Qdot detection provides data equivalent to that obtained using conventional organic dye detection. The portable imager offers an economical, more robust, and deployable alternative to conventional laser array scanners.
Assuntos
Diagnóstico por Imagem/métodos , Técnica Indireta de Fluorescência para Anticorpo/métodos , Análise Serial de Proteínas/métodos , Pontos Quânticos , Anticorpos/sangue , Anticorpos/imunologia , Brucella melitensis/imunologia , Brucella melitensis/fisiologia , Brucelose/sangue , Brucelose/imunologia , Brucelose/microbiologia , Corantes Fluorescentes/química , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Malária Falciparum/sangue , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Microscopia Confocal , Plasmodium falciparum/imunologia , Plasmodium falciparum/fisiologia , Reprodutibilidade dos Testes , Vacínia/sangue , Vacínia/imunologia , Vacínia/virologia , Vaccinia virus/imunologia , Vaccinia virus/fisiologiaRESUMO
Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Francisella tularensis/imunologia , Análise em Microsséries , Proteoma/análise , Testes Sorológicos/métodos , Tularemia/diagnóstico , Adulto , Antígenos de Bactérias/análise , Francisella tularensis/química , Humanos , Espanha , Estados UnidosRESUMO
Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen. Proteome-wide Ab profiling studies reveal the humoral response to be strongly biased toward virion-associated Ags, and several membrane proteins induce Ab-mediated protection against VACV challenge in mice. Some studies have indicated that the CD4 response is also skewed toward proteins with virion association, whereas the CD8 response is more biased toward proteins with early expression. In this study, we have leveraged a VACV strain Western Reserve (VACV-WR) plasmid expression library, produced previously for proteome microarrays for Ab profiling, to make a solubilized full VACV-WR proteome for T cell Ag profiling. Splenocytes from VACV-WR-infected mice were assayed without prior expansion against the soluble proteome in assays for Th1 and Th2 signature cytokines. The response to infection was polarized toward a Th1 response, with the distribution of reactive T cell Ags comprising both early and late VACV proteins. Interestingly, the proportions of different functional subsets were similar to that present in the whole proteome. In contrast, the targets of Abs from the same mice were enriched for membrane and other virion components, as described previously. We conclude that a "nonbiasing" approach to T cell Ag discovery reveals a T cell Ag profile in VACV that is broader and less skewed to virion association than the Ab profile. The T cell Ag mapping method developed in the present study should be applicable to other organisms where expressible "ORFeome" libraries are also available, and it is readily scalable for larger pathogens.
Assuntos
Antígenos Virais/imunologia , Proteoma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Vaccinia virus/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Imunidade Humoral , Imunização , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
BACKGROUND: Immunoglobulin G antibodies (Abs) to Plasmodium falciparum antigens have been associated with naturally acquired immunity to symptomatic malaria. METHODS: We probed protein microarrays covering 824 unique P. falciparum protein features with plasma from residents of a community in Kenya monitored for 12 weeks for (re)infection and symptomatic malaria after administration of antimalarial drugs. P. falciparum proteins recognized by Abs from 88 children (aged 1-14 years) and 86 adults (aged ≥ 18 years), measured at the beginning of the observation period, were ranked by Ab signal intensity. RESULTS: Abs from immune adults reacted with a total 163 of 824 P. falciparum proteins. Children gradually acquired Abs to the full repertoire of antigens recognized by adults. Abs to some antigens showed high seroconversion rates, reaching maximal levels early in childhood, whereas others did not reach adult levels until adolescence. No correlation between Ab signal intensity and time to (re)infection was observed. In contrast, Ab levels to 106 antigens were significantly higher in children who were protected from symptomatic malaria compared with those who were not. Abs to antigens predictive of protection included P. falciparum erythrocyte membrane protein 1, merozoite surface protein (MSP) 10, MSP2, liver-stage antigen 3, PF70, MSP7, and Plasmodium helical interspersed subtelomeric domain protein. CONCLUSIONS: Protein microarrays may be useful in the search for malaria antigens associated with protective immunity.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Malária/imunologia , Plasmodium falciparum/imunologia , Análise Serial de Proteínas , Adolescente , Adulto , Idoso , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/sangue , Antimaláricos/uso terapêutico , Criança , Pré-Escolar , Feminino , Humanos , Imunidade Inata , Imunoglobulina G/sangue , Lactente , Quênia , Malária/sangue , Malária/tratamento farmacológico , Proteínas de Membrana/imunologia , Merozoítos/imunologia , Camundongos , Pessoa de Meia-Idade , Plasmodium falciparum/isolamento & purificação , Modelos de Riscos Proporcionais , Proteínas de Protozoários/sangue , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Adulto JovemRESUMO
BACKGROUND: Malaria is a public health problem in parts of Thailand, where Plasmodium falciparum and Plasmodium vivax are the main causes of infection. In the northwestern border province of Tak parasite prevalence is now estimated to be less than 1% by microscopy. Nonetheless, microscopy is insensitive at low-level parasitaemia. The objective of this study was to assess the current epidemiology of falciparum and vivax malaria in Tak using molecular methods to detect exposure to and infection with parasites; in particular, the prevalence of asymptomatic infections and infections with submicroscopic parasite levels. METHODS: Three-hundred microlitres of whole blood from finger-prick were collected into capillary tubes from residents of a sentinel village and from patients at a malaria clinic. Pelleted cellular fractions were screened by quantitative PCR to determine parasite prevalence, while plasma was probed on a protein microarray displaying hundreds of P. falciparum and P. vivax proteins to obtain antibody response profiles in those individuals. RESULTS: Of 219 samples from the village, qPCR detected 25 (11.4%) Plasmodium sp. infections, of which 92% were asymptomatic and 100% were submicroscopic. Of 61 samples from the clinic patients, 27 (44.3%) were positive by qPCR, of which 25.9% had submicroscopic parasite levels. Cryptic mixed infections, misdiagnosed as single-species infections by microscopy, were found in 7 (25.9%) malaria patients. All sample donors, parasitaemic and non-parasitaemic alike, had serological evidence of parasite exposure, with 100% seropositivity to at least 54 antigens. Antigens significantly associated with asymptomatic infections were P. falciparum MSP2, DnaJ protein, putative E1E2 ATPase, and three others. CONCLUSION: These findings suggest that parasite prevalence is higher than currently estimated by local authorities based on the standard light microscopy. As transmission levels drop in Thailand, it may be necessary to employ higher throughput and sensitivity methods for parasite detection in the phase of malaria elimination.
Assuntos
Malária Falciparum/epidemiologia , Malária Vivax/epidemiologia , Plasmodium falciparum , Plasmodium vivax , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antiprotozoários/sangue , Infecções Assintomáticas , Análise por Conglomerados , Estudos Transversais , Humanos , Malária Falciparum/diagnóstico , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Vivax/diagnóstico , Malária Vivax/imunologia , Malária Vivax/parasitologia , Pessoa de Meia-Idade , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Plasmodium vivax/genética , Plasmodium vivax/imunologia , Prevalência , Estudos Soroepidemiológicos , Tailândia/epidemiologia , Adulto JovemRESUMO
Acquisition of acute toxoplasmosis during the first trimester of pregnancy can have catastrophic consequences for the foetus. Diagnosis is routinely based on the detection of maternal Toxoplasma gondii--antibodies using whole parasite extracts as detection antigen. While such assays are sensitive, they show no specificity for the stage of infection. We hypothesized diagnosis might be improved using recombinant antigens for detection, particularly if antibodies to certain antigen(s) were associated with early or late stages of infection. To address this, protein microarrays comprising 1513 T. gondii exon products were probed with well-characterized sera from seronegative ('N') controls, and acute ('A'), chronic/IgM-persisting ('C/M') and chronic ('C') toxoplasmosis cases from Turkey. Three reactive exon products recognized preferentially in A infections, and three recognized preferentially in C/M infections, were expressed in Escherichia coli and tested for discrimination in IgG ELISAs. The best discriminators were exon 1 of TGME49_086450 (GRA5) which detected C/M infections with 70.6% sensitivity and 81.8% specificity, and exon 6 of TGME49_095700 (ubiquitin transferase domain-containing protein) which detected A infections with 84.8% sensitivity and 82.4% specificity. Overall, the data support a recombinant protein approach for the development of improved serodiagnostic tests for toxoplasmosis.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Toxoplasma/metabolismo , Toxoplasmose/sangue , Estudos de Casos e Controles , Humanos , Imunoglobulina G/sangue , Análise Serial de Proteínas , Sensibilidade e Especificidade , Testes Sorológicos , Toxoplasmose/diagnóstico , Turquia/epidemiologiaRESUMO
Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella-specific immunity remain poorly defined. We have previously shown evidence for antibody-mediating protection against invasive Salmonellosis in mice and African children. To generate an overview of antibody targeting in systemic Salmonellosis, a Salmonella proteomic array containing over 2,700 proteins was constructed and probed with immune sera from Salmonella-infected mice and humans. Analysis of multiple inbred mouse strains identified 117 antigens recognized by systemic antibody responses in murine Salmonellosis. Importantly, many of these antigens were independently identified as target antigens using sera from Malawian children with Salmonella bacteremia, validating the study of the murine model. Furthermore, vaccination with SseB, the most prominent antigenic target in Malawian children, provided mice with significant protection against Salmonella infection. Together, these data uncover an overlapping immune signature of disseminated Salmonellosis in mice and humans and provide a foundation for the generation of a protective subunit vaccine.
Assuntos
Salmonelose Animal/imunologia , Infecções por Salmonella/imunologia , Animais , Formação de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Atividade Bactericida do Sangue , Criança , Pré-Escolar , Convalescença , Feminino , Humanos , Lactente , Recém-Nascido , Malaui , Masculino , Camundongos , Camundongos Endogâmicos , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Infecções por Salmonella/sangue , Vacinação , Vacinas Atenuadas/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Little is known concerning immunodominance within the CD4 T-cell response to viral infections and its persistence into long-term memory. We tested CD4 T-cell reactivity against each viral protein in persons immunized with vaccinia virus (VV), either recently or more than 40 years ago, as a model self-limited viral infection. Similar tests were done with persons with herpes simplex virus 1 (HSV-1) infection as a model chronic infection. We used an indirect method capable of counting the CD4 T cells in blood reactive with each individual viral protein. Each person had a clear CD4 T-cell dominance hierarchy. The top four open reading frames accounted for about 40% of CD4 virus-specific T cells. Early and long-term memory CD4 T-cell responses to vaccinia virus were mathematically indistinguishable for antigen breadth and immunodominance. Despite the chronic intermittent presence of HSV-1 antigen, the CD4 T-cell dominance and diversity patterns for HSV-1 were identical to those observed for vaccinia virus. The immunodominant CD4 T-cell antigens included both long proteins abundantly present in virions and shorter, nonstructural proteins. Limited epitope level and direct ex vivo data were also consistent with pronounced CD4 T-cell immunodominance. We conclude that human memory CD4 T-cell responses show a pattern of pronounced immunodominance for both chronic and self-limited viral infections and that this pattern can persist over several decades in the absence of antigen.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Memória Imunológica , Vacina Antivariólica/imunologia , Vaccinia virus/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Humanos , Imunidade Celular , Interferon gama/biossíntese , Interleucina-2/biossíntese , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Vacinas AtenuadasRESUMO
Vaccines aim to efficiently and specifically activate the immune system via a cascade of antigen uptake, processing, and presentation by antigen-presenting cells (APCs) to CD4 and CD8 T cells, which in turn drive humoral and cellular immune responses. The specific formulation of vaccine carriers can not only shield the antigens from premature sequestering before reaching APCs but also favorably promote intracellular antigen presentation and processing. This study compares two different acid-degradable polymeric nanoparticles that are capable of encapsulating a moderately immunogenic antigen, GFP, at nearly full efficacy via electrostatic interactions or molecular affinity between His tag and Ni-NTA-conjugated monomners. This resulted in GFP-encapsulating NPs composed of ketal monomers and crosslinkers (KMX/GFP NPs) and NTA-conjugated ketal monomers and crosslinkers (NKMX/GFP NPs), respectively. Encapsulated GFP was found to be released more rapidly from NKMX/GFP NPs (electrostatic encapsulation) than from KMX/GFP NPs (affinity-driven encapsulation). In vivo vaccination studies demonstrated that while repeated injections of either NP formulation resulted in poorer generation of anti-GFP antibodies than injections of the GFP antigen itself, sequential injections of NPs and GFP as prime and booster vaccines, respectively, restored the humoral response. We proposed that NPs primarily assist APCs in antigen presentation by T cells, and B cells need to be further stimulated by free protein antigens to produce antibodies. The findings of this study suggest that the immune response can be modulated by varying the chemistry of vaccine carriers and the sequences of vaccination with free antigens and antigen-encapsulating NPs.
Assuntos
Antígenos , Nanopartículas , Polímeros , Nanopartículas/química , Animais , Polímeros/química , Camundongos , Antígenos/imunologia , Antígenos/química , Vacinação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/imunologia , Feminino , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Vacinas/imunologia , Vacinas/química , Vacinas/administração & dosagemRESUMO
Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.
Assuntos
Vacinas Antimaláricas , Malária Falciparum , Malária Vivax , Malária , Animais , Humanos , Plasmodium vivax , Esporozoítos , Formação de Anticorpos , Imunização , Vacinação , Malária/prevenção & controle , Malária Falciparum/parasitologia , Malária Vivax/parasitologia , Plasmodium falciparumRESUMO
Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.
Assuntos
Vacinas contra Ebola , Ebolavirus , Doença pelo Vírus Ebola , Polissorbatos , Esqualeno , Animais , Camundongos , Anticorpos Antivirais , Sudão , Filogenia , Anticorpos Neutralizantes , Camundongos Endogâmicos C57BL , Glicoproteínas , Adjuvantes Imunológicos , Linfócitos T , CitocinasRESUMO
Influenza B virus (FLUBV) poses a significant infectious threat, with frequent vaccine mismatch limiting its effectiveness. Our previous work investigated the safety and efficacy of modified live attenuated FLUBV vaccines with rearranged genomes (FluB-RAM and FluB-RANS) or a temperature-sensitive PB1 segment with a C-terminal HA tag (FluB-att). In this study, we compared the immune responses of female and male DBA/2J mice vaccinated with these vaccines, including versions containing a chimeric HA segment with an N-terminal IgA-inducing peptide (IGIP). Importantly, both recombinant viruses with and without IGIP remained genetically stable during egg passage. We found that introducing IGIP strengthened vaccine attenuation, particularly for FluB-RAM/IGIP. Prime-boost vaccination completely protected mice against lethal challenge with a homologous FLUBV strain. Notably, recombinant viruses induced robust neutralizing antibody responses (hemagglutination inhibition titers ≥40) alongside antibodies against NA and NP. Interestingly, female mice displayed a consistent trend of enhanced humoral and cross-reactive IgG and IgA responses against HA, NA, and NP compared to male counterparts, regardless of the vaccine used. However, the presence of IGIP generally led to lower anti-HA responses but higher anti-NA and anti-NP responses, particularly of the IgA isotype. These trends were further reflected in mucosal and serological responses two weeks after challenge, with clear distinctions based on sex, vaccine backbone, and IGIP inclusion. These findings hold significant promise for advancing the development of universal influenza vaccines.