Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Polymers (Basel) ; 15(18)2023 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-37765685

RESUMO

This report details the first systematic screening of free-radical-produced methacrylate oligomer reaction mixtures as alternative vaccine adjuvant components to replace the current benchmark compound squalene, which is unsustainably sourced from shark livers. Homo-/co-oligomer mixtures of methyl, butyl, lauryl, and stearyl methacrylate were successfully synthesized using catalytic chain transfer control, where the use of microwave heating was shown to promote propagation over chain transfer. Controlling the mixture material properties allowed the correct viscosity to be achieved, enabling the mixtures to be effectively used in vaccine formulations. Emulsions of selected oligomers stimulated comparable cytokine levels to squalene emulsion when incubated with human whole blood and elicited an antigen-specific cellular immune response when administered with an inactivated influenza vaccine, indicating the potential utility of the compounds as vaccine adjuvant components. Furthermore, the oligomers' molecular sizes were demonstrated to be large enough to enable greater emulsion stability than squalene, especially at high temperatures, but are predicted to be small enough to allow for rapid clearance from the body.

2.
Nat Commun ; 14(1): 1138, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36878897

RESUMO

Adjuvant-containing subunit vaccines represent a promising approach for protection against tuberculosis (TB), but current candidates require refrigerated storage. Here we present results from a randomized, double-blinded Phase 1 clinical trial (NCT03722472) evaluating the safety, tolerability, and immunogenicity of a thermostable lyophilized single-vial presentation of the ID93 + GLA-SE vaccine candidate compared to the non-thermostable two-vial vaccine presentation in healthy adults. Participants were monitored for primary, secondary, and exploratory endpoints following intramuscular administration of two vaccine doses 56 days apart. Primary endpoints included local and systemic reactogenicity and adverse events. Secondary endpoints included antigen-specific antibody (IgG) and cellular immune responses (cytokine-producing peripheral blood mononuclear cells and T cells). Both vaccine presentations are safe and well tolerated and elicit robust antigen-specific serum antibody and Th1-type cellular immune responses. Compared to the non-thermostable presentation, the thermostable vaccine formulation generates greater serum antibody responses (p < 0.05) and more antibody-secreting cells (p < 0.05). In this work, we show the thermostable ID93 + GLA-SE vaccine candidate is safe and immunogenic in healthy adults.


Assuntos
Imunogenicidade da Vacina , Vacinas contra a Tuberculose , Vacinas de Subunidades Antigênicas , Adulto , Humanos , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Anticorpos/imunologia , Células Produtoras de Anticorpos/imunologia , Leucócitos Mononucleares/imunologia , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/farmacologia , Vacinas contra a Tuberculose/uso terapêutico , Imunogenicidade da Vacina/imunologia , Resultado do Tratamento , Voluntários Saudáveis , Temperatura , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/farmacologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Método Duplo-Cego
3.
NPJ Vaccines ; 8(1): 14, 2023 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-36797262

RESUMO

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.

4.
Org Process Res Dev ; 27(12): 2317-2328, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38524776

RESUMO

Emulsions of the triterpene squalene ((6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, CAS 111-02-4) have been used as adjuvants in influenza vaccines since the 1990s. Traditionally sourced from shark liver oil, the overfishing of sharks and concomitant reduction in the oceanic shark population raises sustainability issues for vaccine adjuvant grade squalene. We report a semisynthetic route to squalene meeting current pharmacopeial specifications for use in vaccines that leverages the ready availability of trans-ß-farnesene ((6E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, CAS 18794-84-8), manufactured from sustainable sugarcane via a yeast fermentation process. The scalability of the proposed route was verified by a kilo-scale GMP synthesis. We also report data demonstrating the synthesized semi-synthetic squalene's physical stability and biological activity when used in a vaccine adjuvant formulation.

5.
NPJ Vaccines ; 7(1): 136, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36323666

RESUMO

mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature. This saRNA vaccine is formulated with a nanostructured lipid carrier (NLC), providing stability, ease of manufacturing, and protection against degradation. In preclinical studies, this saRNA/NLC vaccine induced strong humoral immunity, as demonstrated by high pseudovirus neutralization titers to the Alpha, Beta, and Delta variants of concern and induction of bone marrow-resident antibody-secreting cells. Robust Th1-biased T-cell responses were also observed after prime or homologous prime-boost in mice. Notably, the saRNA/NLC platform demonstrated thermostability when stored lyophilized at room temperature for at least 6 months and at refrigerated temperatures for at least 10 months. Taken together, this saRNA delivered by NLC represents a potential improvement in RNA technology that could allow wider access to RNA vaccines for the current COVID-19 and future pandemics.

7.
Front Immunol ; 9: 2420, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386348

RESUMO

The rapid generation of strong T cell responses is highly desirable and viral vectors can have potent CD8+ T cell-inducing activity. Immunity to leishmaniasis requires selective T cell responses, with immunization schemes that raise either CD4 or CD8 T cell responses being protective in small animal models. We have defined the leishmaniasis vaccine candidate recombinant fusion antigens, LEISH-F2 and LEISH-F3+, that when formulated in a stable emulsion with a Toll-like receptor (TLR) 4 agonist, induce protective CD4+ T cell responses in animal models as well as providing therapeutic efficacy in canine leishmaniasis and in clinical trials in leishmaniasis patients. We used the genetic sequences of these validated vaccine antigens to design RNA vaccine constructs. Immunization of mice with the RNA replicons induced potent, local innate responses that were surprisingly independent of TLR7 and activated antigen-presenting cells (APC) to prime for extremely potent antigen-specific T helper 1 type responses upon heterologous boosting with either of the subunit vaccines (recombinant antigen with second generation glucopyranosyl lipid A in stable oil-in-water emulsion; SLA-SE). Inclusion of RNA in the immunization schedule also generated MHCI-restricted T cell responses. Immunization with LEISH-F2-expressing RNA vaccine followed later by subunit vaccine afforded protection against challenge with Leishmania donovani. Together, these data indicate the utility of heterologous prime-boost immunization schemes for the induction of potent antigen-specific CD4 and CD8 T cell responses for protection against intracellular pathogens.


Assuntos
Imunidade Heteróloga , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Linhagem Celular , Citocinas/metabolismo , Feminino , Humanos , Imunização Secundária , Leishmania donovani/genética , Vacinas contra Leishmaniose/genética , Leishmaniose Visceral/prevenção & controle , Ativação Linfocitária/imunologia , Camundongos , NF-kappa B/metabolismo , Transporte Proteico , Linfócitos T/metabolismo , Receptor 7 Toll-Like/metabolismo , Vacinas de Subunidades Antigênicas/genética , Vacinas Sintéticas/imunologia
8.
BMC Infect Dis ; 18(1): 500, 2018 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-30285653

RESUMO

BACKGROUND: Visceral leishmaniasis (VL), caused by Leishmania donovani complex parasites, is a neglected parasitic disease that is generally fatal if untreated. Despite decades of research to develop a sensitive VL diagnostic test, definitive diagnosis of VL still mainly relies on the visualization of the parasite in aspirates from the spleen, liver or bone marrow, an invasive and dangerous process with variable sensitivity. A sensitive assay that can detect Leishmania antigen from blood samples will help confirm cause, cure or recurrence of VL. METHODS: In this study, rabbit polyclonal antibodies were raised against eight recombinant Leishmania proteins that are highly abundant in Leishmania. The antibodies were purified and labeled with biotin for developing a prototype sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA for the Leishmania 40S ribosomal protein S12 detected target antigen with the highest sensitivity and specificity and could detect 1 pg of purified protein or as few as 60 L. donovani parasites. The 40S ribosomal protein S12 sandwich ELISA could detect the target antigen from Peripheral Blood Mononuclear Cell (PBMC) samples in 68% of VL patients and post-kala-azar dermal leishmaniasis (PKDL) patients, providing an estimation of parasitemia ranging from 15 to 80 amastigotes per ml of blood. CONCLUSION: These results indicate that the 40S ribosomal protein S12 sandwich ELISA warrants further tests with more clinical samples of VL patients and other parasitic diseases. It is hopeful that this ELISA could become a useful tool for confirming VL diagnosis, monitoring treatment progress, disease recurrence and possibly detecting asymptomatic Leishmania infections with a high parasite load.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose Visceral/sangue , Proteínas Ribossômicas/imunologia , Adolescente , Adulto , Animais , Antígenos de Protozoários/imunologia , Infecções Assintomáticas , Estudos de Casos e Controles , Feminino , Humanos , Leishmania/imunologia , Leishmania/patogenicidade , Leishmaniose/sangue , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Leucócitos Mononucleares/parasitologia , Masculino , Pessoa de Meia-Idade , Doenças Negligenciadas , Carga Parasitária , Parasitemia/sangue , Parasitemia/diagnóstico , Coelhos , Proteínas Ribossômicas/genética , Sensibilidade e Especificidade
9.
Vaccine ; 34(25): 2779-86, 2016 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-27142329

RESUMO

Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Animais , Anticorpos Antiprotozoários/sangue , Escherichia coli , Feminino , Humanos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Vacinas Sintéticas/imunologia
10.
J Clin Microbiol ; 54(4): 1025-30, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26842701

RESUMO

Infection with Leishmania donovaniis typically asymptomatic, but a significant number of individuals may progress to visceral leishmaniasis (VL), a deadly disease that threatens 200 million people in areas where it is endemic. While diagnosis of acute VL has been simplified by the use of cost-effective confirmatory serological tests, similar standardized tools are not widely available for detecting asymptomatic infection, which can be 4 to 20 times more prevalent than active disease. A simple and accurate serological test that is capable of detecting asymptomaticL. donovaniinfection will be useful for surveillance programs targeting VL control and elimination. To address this unmet need, we evaluated recombinant antigens for their ability to detect serum antibodies in 104 asymptomaticL. donovani-infected individuals (qualified as positive forL. donovani-specific antibodies by direct agglutination test [DAT]) from the Mymensingh district of Bangladesh where VL is hyperendemic. The novel proteins rKR95 and rTR18 possessed the greatest potential and detected 69% of DAT-positive individuals, with rKR95 being more robust in reactivity. Agreement in results for individuals with high DAT responses, who are more likely to progress to VL disease, was 74%. When considered along with rK39, a gold standard antigen that is used to confirm clinical diagnosis of VL but that is now becoming widely used for surveillance, rKR95 and rTR18 conferred a sensitivity of 84% based on a theoretical combined estimate. Our data indicate that incorporating rKR95 and rTR18 with rK39 in serological tests amenable to rapid or high-throughput screening may enable simple and accurate detection of asymptomatic infection. Such tests will be important tools to measureL. donovaniinfection rates, a primary goal in surveillance and a critical measurement with which to assess elimination programs.


Assuntos
Anticorpos Antiprotozoários/sangue , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Testes Sorológicos/métodos , Antígenos de Protozoários/imunologia , Doenças Assintomáticas , Bangladesh , Humanos , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade
11.
Diagn Microbiol Infect Dis ; 84(3): 191-6, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26658314

RESUMO

We previously reported that tandem repeat (TR) proteins from Trypanosoma cruzi could serve as targets of the antibody response and be useful as diagnostic indicators. To optimize reagents for detecting T. cruzi infection we evaluated individual TR proteins and identified several that were recognized by the majority of Chagas patient's sera collected from individuals form Brazil. We then produced novel, recombinant fusion proteins to combine the reactive TR proteins into a single diagnostic product. Direct comparison of the antibody response of serum samples that were readily detected by the established fusion antigen used in commercial detection of Chagas disease, TcF, revealed strong responses to TcF43 and TcF26 proteins. While the TcF43 and TcF26 antigens enhanced detection and strength of signal, they did not compromise the specificity of detection compared to that obtained with TcF. Finally, it was apparent by testing against a panel of 84 serum samples assembled on the basis of moderate or weak reactivity against TcF (mostly signal:noise <5) that TcF43 and TcF26 could more strongly detected by many of the sera that had low TcF antibody levels. Taken together, these data indicate that TcF43 and TcF26 could be used to enhance the detection of T. cruzi infection as well as supporting a diagnosis of Chagas disease.


Assuntos
Antígenos de Protozoários/imunologia , Doença de Chagas/diagnóstico , Doença de Chagas/imunologia , Epitopos/imunologia , Testes Sorológicos , Trypanosoma cruzi/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Doença de Chagas/parasitologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Protozoários/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/normas , Trypanosoma cruzi/genética , Adulto Jovem
12.
BMC Infect Dis ; 15: 384, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26395447

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) can be fatal without timely diagnosis and treatment. Treatment efficacies vary due to drug resistance, drug toxicity and co-morbidities. It is important to monitor treatment responsiveness to confirm cure and curtail relapse. Currently, microscopy of spleen, bone marrow or lymph node biopsies is the only definitive method to evaluate cure. A less invasive test for treatment success is a high priority for VL management. METHODS: In this study, we describe the development of a capture ELISA based on detecting Leishmania donovani antigens in urine samples and comparison with the Leishmania Antigen ELISA, also developed for the same purpose. Both were developed as prototype kits and tested on patient urine samples from Sudan, Ethiopia, Bangladesh and Brazil, along with appropriate control samples from endemic and non-endemic regions. Sensitivity and specificity were assessed based on accurate detection of patients compared to control samples. One-Way ANOVA was used to assess the discrimination capacity of the tests and Cohen's kappa was used to assess their correlation. RESULTS: The Leishmania Antigen Detect ELISA demonstrated >90% sensitivity on VL patient samples from Sudan, Bangladesh and Ethiopia and 88% on samples from Brazil. The Leishmania Antigen ELISA was comparable in performance except for lower sensitivity on Sudanese samples. Both were highly specific. To confirm utility in monitoring treatment, urine samples were collected from VL patients at days 0, 30 and 180 post-treatment. For the Leishmania Antigen Detect ELISA, positivity was high at day 0 at 95%, falling to 21% at day 30. At day 180, all samples were negative, corresponding well with clinical cure. A similar trend was also seen for the Leishmania Antigen ELISA albeit; with lower positivity of 91% at Day 0 and more patients, remaining positive at Days 30 and 180. DISCUSSION: The Leishmania Antigen Detect and the Leishmania Antigen ELISAs are standardized, user- friendly, quantitative and direct tests to detect Leishmania during acute VL as well as to monitor parasite clearance during treatment. They are a clear improvement over existing options. CONCLUSION: The ELISAs provide a non-invasive method to detect parasite antigens during acute infection and monitor its clearance upon cure, filling an unmet need in VL management. Further refinement of the tests with more samples from endemic regions will define their utility in monitoring treatment.


Assuntos
Antígenos de Protozoários/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Leishmania donovani/imunologia , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Antígenos de Protozoários/urina , Bangladesh , Brasil , Etiópia , Humanos , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/urina , Sensibilidade e Especificidade , Sudão
13.
Clin Vaccine Immunol ; 18(7): 1118-24, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21632891

RESUMO

A subunit vaccine using a defined antigen(s) may be one effective solution for controlling leishmaniasis. Because of genetic diversity in target populations, including both dogs and humans, a multiple-antigen vaccine will likely be essential. However, the cost of a vaccine to be used in developing countries must be considered. We describe herein a multiantigen vaccine candidate comprised of antigens known to be protective in animal models, including dogs, and to be recognized by humans immune to visceral leishmaniasis. The polyprotein (KSAC) formulated with monophosphoryl lipid A, a widely used adjuvant in human vaccines, was found to be immunogenic and capable of inducing protection against Leishmania infantum, responsible for human and canine visceral leishmaniasis, and against L. major, responsible for cutaneous leishmaniasis. The results demonstrate the feasibility of producing a practical, cost-effective leishmaniasis vaccine capable of protecting both humans and dogs against multiple Leishmania species.


Assuntos
Leishmaniose Visceral/prevenção & controle , Poliproteínas/uso terapêutico , Adjuvantes Imunológicos , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários , Cães , Humanos , Leishmania infantum/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/veterinária , Lipídeo A/análogos & derivados , Poliproteínas/imunologia , Vacinas Protozoárias
14.
PLoS Negl Trop Dis ; 4(9)2010 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-20856856

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75-85%, specificity of 70-92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms. METHODOLOGY/PRINCIPAL FINDINGS: Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83-99.61%; K39: 84.30-98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53-98.95%; K39: 85.54-96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46-99.1 in Sudan and 98.1% [95% CI = 89.93-99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25-93.23%] in Sudan and 88.7% [95% CI = 76.97-95.73%] in Bangladesh). CONCLUSIONS/SIGNIFICANCE: Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.


Assuntos
Antígenos de Protozoários , Leishmaniose Visceral/diagnóstico , Parasitologia/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Adulto , Anticorpos Antiprotozoários/sangue , Criança , Feminino , Humanos , Imunoensaio/métodos , Masculino , Proteínas Recombinantes , Sensibilidade e Especificidade , Sudão
15.
J Immunol ; 185(3): 1701-10, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20601594

RESUMO

Therapy of intracellular pathogens can be complicated by drug toxicity, drug resistance, and the need for prolonged treatment regimens. One approach that has shown promise is immunotherapy. Leishmaniasis, a vector-borne disease ranked among the six most important tropical infectious diseases by the World Health Organization, has been treated clinically with crude or defined vaccine preparations or cytokines, such as IFN-gamma and GM-CSF, in combination with chemotherapy. We have attempted to develop an improved and defined immunotherapeutic using a mouse model of cutaneous leishmaniasis. We hypothesized that immunotherapy may be improved by using TLR synergy to enhance the parasite-specific immune response. We formulated L110f, a well-established Leishmania poly-protein vaccine candidate, in conjunction with either monophosphoryl lipid A, a TLR4 agonist, or CpG, a TLR9 agonist, or a combination of these, and evaluated anti-Leishmania immune responses in absence or presence of active disease. Only mice treated with L110f plus monophosphoryl lipid A-CpG were able to induce a strong effective T cell response during disease and subsequently cured lesions and reduced parasite burden when compared with mice treated with L110f and either single adjuvant. Our data help to define a correlate of protection during active infection and indicate TLR synergy to be a potentially valuable tool in treating intracellular infections.


Assuntos
Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/terapia , Receptores Toll-Like/fisiologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Bactérias/administração & dosagem , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/fisiologia , Quimioterapia Combinada , Feminino , Interleucina-12/biossíntese , Leishmaniose Cutânea/microbiologia , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/administração & dosagem , Pirina , Receptores Toll-Like/agonistas
16.
Clin Vaccine Immunol ; 17(2): 298-303, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20016045

RESUMO

Despite the reduction in the number of leprosy cases registered worldwide as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains stable in many countries. This indicates that Mycobacterium leprae, the causative agent of leprosy, is still being transmitted and that, without an earlier diagnosis, transmission will continue and infection will remain a health problem. The current means of diagnosis of leprosy is based on the appearance of clinical symptoms, which in many cases occur after significant and irreversible nerve damage has occurred. Our recent work identified several recombinant antigens that are specifically recognized by leprosy patients. The goal of the present study was to produce and validate the reactivity of a chimeric fusion protein that possesses the antibody binding properties of several of these proteins. The availability of such a chimeric fusion protein will simplify future test development and reduce production costs. We first identified the antibody binding regions within our top five antigen candidates by performing enzyme-linked immunosorbent assays with overlapping peptides representing the amino acid sequences of each protein. Having identified these regions, we generated a fusion construct of these components (protein advances diagnostic of leprosy [PADL]) and demonstrated that the PADL protein retains the antibody reactivity of the component antigens. PADL was able to complement a protein that we previously produced (the leprosy IDRI [Infectious Disease Research Institute] diagnostic 1 [LID-1] protein) to permit the improved diagnosis of multibacillary leprosy and that had a good ability to discriminate patients with multibacillary leprosy from control individuals. A serological diagnostic test consisting of these antigens could be applied within leprosy control programs to reduce transmission and to limit the appearance of leprosy-associated disabilities and stigmatizing deformities by directing treatment.


Assuntos
Antígenos de Bactérias , Técnicas de Laboratório Clínico/métodos , Hanseníase/diagnóstico , Proteínas Recombinantes de Fusão , Adolescente , Adulto , Idoso , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Mapeamento de Epitopos , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoensaio/métodos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Adulto Jovem
17.
J Immunol ; 181(11): 7948-57, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19017986

RESUMO

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-gamma recall responses using PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49 of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-gamma, and TNF. Priority vaccine Ags elicited pluripotent CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T cell Ags suitable to be included in subunit vaccines against tuberculosis.


Assuntos
Antígenos de Bactérias/farmacologia , Linfócitos T CD8-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose Pulmonar/imunologia , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Biologia Computacional , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/imunologia , Interferon gama/imunologia , Camundongos , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/genética , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/prevenção & controle , Fator de Necrose Tumoral alfa/imunologia
18.
Infect Immun ; 74(7): 3939-45, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16790767

RESUMO

Visceral leishmaniasis (VL) is a form of leishmaniasis, which is caused by infection with the protozoan parasite Leishmania, and is often fatal unless it is treated. Rapid and accurate diagnosis of VL is important for effective treatment. Here we report the cloning of previously undescribed tandem repeat (TR) proteins of Leishmania infantum and an evaluation of VL patient antibody responses to the corresponding proteins. By screening an L. infantum expression library with sera from human VL patients or infected hamsters, we identified 43 genes encoding B-cell antigens. Surprisingly, 19 of the 43 genes (44%) were TR proteins, and that percentage was significantly higher than that for genes picked randomly from the database. We then expressed the TR regions of LinJ16.1750, LinJ22.1590, and LinJ33.2870 and the entire LinJ28.2310 protein. These recombinant proteins were all recognized by Sudanese VL patient sera in an enzyme-linked immunosorbent assay. Recombinant LinJ16.1750 (rLinJ16.1750) showed the best performance among these antigens in terms of both sensitivity and specificity. Serological evaluation revealed that 97% (34 of 35) of Sudanese VL patients had significantly elevated antibody levels to rLinJ16.1750. Furthermore, when eight of the patient sera which had low reactivities to rK39 were tested with the novel recombinant antigens, some of the sera showed stronger antibody responses to these antigens than to rK39. Our results suggest that TR regions from the novel L. infantum proteins identified in this study are immunodominant B-cell epitopes and may represent good candidates for serodiagnosis of VL.


Assuntos
Antígenos de Protozoários/sangue , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/sangue , Sequências de Repetição em Tandem , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Clonagem Molecular , Cricetinae , Biblioteca Gênica , Humanos , Leishmania infantum/genética , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Prevalência , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sequências de Repetição em Tandem/genética , Sequências de Repetição em Tandem/imunologia
19.
Clin Vaccine Immunol ; 13(3): 333-40, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16522774

RESUMO

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.


Assuntos
Antígenos de Bactérias/sangue , Proteínas de Bactérias/sangue , Hanseníase/diagnóstico , Mycobacterium leprae/imunologia , Anticorpos Antibacterianos/metabolismo , Reações Antígeno-Anticorpo , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Glicolipídeos , Humanos , Hanseníase/imunologia , Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Análise de Sequência de Proteína
20.
J Clin Invest ; 113(9): 1296-306, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15124021

RESUMO

Chronic intestinal inflammation, as seen in inflammatory bowel disease (IBD), results from an aberrant and poorly understood mucosal immune response to the microbiota of the gastrointestinal tract in genetically susceptible individuals. Here we used serological expression cloning to identify commensal bacterial proteins that could contribute to the pathogenesis of IBD. The dominant antigens identified were flagellins, molecules known to activate innate immunity via Toll-like receptor 5 (TLR5), and critical targets of the acquired immune system in host defense. Multiple strains of colitic mice had elevated serum anti-flagellin IgG2a responses and Th1 T cell responses to flagellin. In addition, flagellin-specific CD4(+) T cells induced severe colitis when adoptively transferred into naive SCID mice. Serum IgG to these flagellins, but not to the dissimilar Salmonella muenchen flagellin, was elevated in patients with Crohn disease, but not in patients with ulcerative colitis or in controls. These results identify flagellins as a class of immunodominant antigens that stimulate pathogenic intestinal immune reactions in genetically diverse hosts and suggest new avenues for the diagnosis and antigen-directed therapy of patients with IBD.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Doença de Crohn/imunologia , Flagelina/imunologia , Doenças Inflamatórias Intestinais/etiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Proteínas de Bactérias/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Ceco/microbiologia , Células Cultivadas , Clonagem Molecular , Doença de Crohn/patologia , Relação Dose-Resposta Imunológica , Escherichia coli/metabolismo , Flagelina/genética , Humanos , Imunoglobulina G/sangue , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Camundongos SCID , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...