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1.
Infect Immun ; 87(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31308085

RESUMO

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4+ and CD8+ T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4+ T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4+ T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.


Assuntos
Anticorpos Antiprotozoários/biossíntese , Imunoglobulina G/biossíntese , Vacinas Antimaláricas/biossíntese , Malária/prevenção & controle , Plasmodium yoelii/imunologia , Proteínas de Protozoários/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Feminino , Imunização , Imunogenicidade da Vacina , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Malária/genética , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/administração & dosagem , Manitol/administração & dosagem , Manitol/análogos & derivados , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácidos Oleicos/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas de Subunidades
2.
Semin Immunol ; 39: 52-64, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30219621

RESUMO

The availability of an effective and appropriately implemented malaria vaccine would form a crucial cornerstone of public health efforts to fight this disease. Despite many decades of research, however, no malaria vaccine has yet shown satisfactory protective efficacy or been rolled-out. Validated immunological substitute endpoints have the potential to accelerate clinical vaccine development by reducing the required complexity, size, duration and cost of clinical trials. Besides facilitating clinical development of existing vaccine candidates, understanding immunological mechanisms of protection may drive the development of fundamentally new vaccination approaches. In this review we focus on correlates of protection in malaria vaccine development: Does immunogenicity predict malaria vaccine efficacy and why is this question particularly difficult? Have immunological correlates accelerated malaria vaccine development in the past and will they facilitate it in the future? Does Controlled Human Malaria Infection represent a valid model for identifying such immunological correlates, or a correlate of protection against naturally-acquired malaria in itself?


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Citocinas/sangue , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/genética , Biomarcadores/sangue , Citocinas/biossíntese , Determinação de Ponto Final/métodos , Humanos , Imunogenicidade da Vacina , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/classificação , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinação , Potência de Vacina , Vacinas de Subunidades
3.
Protein Expr Purif ; 152: 122-130, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30059744

RESUMO

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30-75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg-1 plant material after purification based on an initial yield of ∼49 mg kg-1 biomass after blanching.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Antiprotozoários/química , Vacinas Antimaláricas/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Tabaco/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Cromatografia de Afinidade/métodos , Clonagem Molecular , Análise Fatorial , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Ligação Proteica , Desnaturação Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sefarose/análogos & derivados , Tabaco/química , Tabaco/metabolismo , Ultrafiltração/métodos
4.
Nat Commun ; 9(1): 2714, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006528

RESUMO

Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Anticorpos Antiprotozoários/biossíntese , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Vacinas Antimaláricas/administração & dosagem , Malária/prevenção & controle , Proteínas de Protozoários/antagonistas & inibidores , Vacinas de DNA/administração & dosagem , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Feminino , Expressão Gênica , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/imunologia , Centro Germinativo/parasitologia , Memória Imunológica/efeitos dos fármacos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA de Protozoário/genética , RNA de Protozoário/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas de DNA/biossíntese
5.
Am J Trop Med Hyg ; 99(1): 43-50, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29848401

RESUMO

Reticulocyte-binding homologues (RH) are a ligand family that mediates merozoite invasion of erythrocytes in Plasmodium falciparum. Among the five members of this family identified so far, only P. falciparum reticulocyte-binding homologue-5 (PfRH5) has been found to be essential for parasite survival across strains that differ in virulence and route of host-cell invasion. Based on its essential role in invasion and early evidence of sequence conservation, PfRH5 has been prioritized for development as a vaccine candidate. However, little is known about the extent of genetic variability of RH5 in the field and the potential impact of such diversity on clinical outcomes or on vaccine evasion. Samples collected during a prospective cohort study of malaria incidence conducted in Kalifabougou, in southwestern Mali, were used to estimate genetic diversity, measure haplotype prevalence, and assess the within-host dynamics of PfRH5 variants over time and in relation to clinical malaria. A total of 10 nonsynonymous polymorphic sites were identified in the Pfrh5 gene, resulting in 13 haplotypes encoding unique protein variants. Four of these variants have not been previously observed. Plasmodium falciparum reticulocyte-binding homologue-5 had low amino acid haplotype (h = 0.58) and nucleotide (π = 0.00061) diversity. By contrast to other leading blood-stage malaria vaccine candidate antigens, amino acid differences were not associated with changes in the risk of febrile malaria in consecutive infections. Conserved B- and T-cell epitopes were identified. These results support the prioritization of PfRH5 for possible inclusion in a broadly cross-protective vaccine.


Assuntos
Proteínas de Transporte/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Haplótipos , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Polimorfismo Genético , Adolescente , Adulto , Sequência de Aminoácidos , Linfócitos B/imunologia , Linfócitos B/parasitologia , Proteínas de Transporte/imunologia , Criança , Pré-Escolar , Epitopos de Linfócito B/genética , Epitopos de Linfócito T/genética , Eritrócitos/parasitologia , Feminino , Expressão Gênica , Humanos , Lactente , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Masculino , Mali/epidemiologia , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Estudos Prospectivos , Linfócitos T/imunologia , Linfócitos T/parasitologia
6.
J Biotechnol ; 266: 111-117, 2018 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-29269249

RESUMO

Malaria is an infectious disease having a large negative impact on economic growth. Vaccines are considered as a novel strategy to reduce the burden of malaria. Malaria parasite has a complex life cycle and attempts are being made to develop vaccines that target each stage of the life cycle. Oral vaccines seem to be more feasible to implement in poor countries, since they are relatively inexpensive, needle-free administrated, mostly stable at non-refrigerated conditions and painless. By using recombinant technology, suitable oral hosts could serve as antigen delivering vehicles in developing oral vaccines. Chlamydomonas reinhardtii offers beneficial attributes as oral recombinant protein expression platform. Moreover, C. reinhardtii chloroplast is an attractive platform for expressing malaria antigens because it is capable of folding complex proteins, including those requiring disulfide bond formation, while lacking the ability to glycosylate proteins; a valuable quality of any malaria protein expression system, since the Plasmodium parasite lacks N-linked glycosylation machinery. As a first step towards developing an oral vaccine candidate against malaria, here, we expressed a fusion protein consisting of PfCelTOS, a candidate for pre-erythrocytic and transmission-blocking vaccines, fused to human interleukin-2 (IL-2) as vaccine adjuvant in the chloroplast of C. reinhardtii. The effect of light and media on recombinant protein production and cell growth was then studied. Results demonstrated that expressed recombinant proteins accumulate as a soluble, properly folded and functional protein within algal chloroplasts. Moreover, results showed that the highest cell density can be achieved using mixotrophy mode. However, protein accumulation appears to be favored by cultivating in TAP medium in low light.


Assuntos
Antígenos de Protozoários , Chlamydomonas reinhardtii , Cloroplastos , Vacinas Antimaláricas , Plasmodium falciparum/genética , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética
7.
Infect Genet Evol ; 53: 239-247, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28600217

RESUMO

Cell traversal protein of Ookinetes and Sporozoites (CelTOS) is a new malaria vaccine candidate antigen. Since one of the main challenges in malaria vaccine development is the extensive antigenic diversity of this parasite, local and global gene diversity analysis is of particular importance. Therefore, in this study, the genetic diversity of pvceltos gene was investigated among Iranian P. vivax isolates (n=46) and compared with available worldwide pvceltos sequences. One synonymous (C109A) and three amino acid replacements (V118L, K178T, and G179R) were observed in Iranian pvceltos sequences in compare with Sal-1 sequence leading to five haplotypes including PvCelt-A (GSVKGL, 13%), PvCelt-B (GSLKGL, 50%), PvCelt-C (GSLTGL, 17.4%), PvCelt-D (GSVTGL, 13%) and PvCelt-E (GSLTRL, 6.5%). However, amino acid replacements were observed in six positions (G10S, S40N, V118L/M, K178T, G179R/D and L181R) in PvCelTOS antigen of global isolates leading to 11 distinct haplotypes. PvCelt-A and PvCelt-B haplotypes were the most common haplotypes in the world. The overall nucleotide diversity for Iranian isolates was 0.00169, while, the level of nucleotide diversity was ranged from 0.00252 for Thailand to 0.00022 for Peru populations in the world. The analysis of SNPs in relation with the predicted immunodominant regions revealed that only K178T and G179R SNPs are located in putative B-cell epitopes. All replacements were located in CD4+ and/or CD8+ T-cell epitopes. However, the majority of epitopes are located in conserved regions. Knowing whether these changes may alter the affinity of the epitopes for antibodies and/or MHC molecules remains to be investigated in experimental studies. In conclusion, the present study showed a very limited genetic diversity in pvceltos gene among the global clinical isolates that can be regarded as a potential candidate antigen to apply for vivax-based malaria vaccine development.


Assuntos
Antígenos de Protozoários/genética , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Variação Genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Expressão Gênica , Haplótipos , Humanos , Irã (Geográfico) , Vacinas Antimaláricas/biossíntese , Malária Vivax/imunologia , Malária Vivax/parasitologia , Malária Vivax/prevenção & controle , Masculino , Pessoa de Meia-Idade , Plasmodium vivax/química , Plasmodium vivax/imunologia , Plasmodium vivax/isolamento & purificação , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Análise de Sequência de DNA , Esporozoítos/química , Esporozoítos/genética , Esporozoítos/imunologia
8.
J Immunol Methods ; 448: 66-73, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28554543

RESUMO

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Antígenos/imunologia , Linfócitos B/imunologia , Células Clonais/imunologia , Clonagem Molecular/métodos , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/isolamento & purificação , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Regiões 5' não Traduzidas , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/administração & dosagem , Linfócitos B/parasitologia , Ligante de CD40/imunologia , Proliferação de Células , Separação Celular/métodos , Células Clonais/parasitologia , Técnicas de Cocultura , Citometria de Fluxo , Células HEK293 , Humanos , Imunização , Ativação Linfocitária , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Proteínas de Protozoários/administração & dosagem , Fluxo de Trabalho
10.
Vaccine ; 35(8): 1140-1147, 2017 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-28131394

RESUMO

The malaria parasite Plasmodium falciparum presents antigens on the infected erythrocyte surface that bind human receptors expressed on the vascular endothelium. The VAR2CSA mediated binding to a distinct chondroitin sulphate A (CSA) is a crucial step in the pathophysiology of placental malaria and the CSA binding region of VAR2CSA has been identified as a promising vaccine target against placental malaria. Here we designed adenovirus encoded virus-like particles (VLP) by co-encoding Simian Immunodeficiency Virus (SIV) gag and VAR2CSA. The VAR2CSA antigen was fused to the transmembrane (TM) and cytoplasmic tail (CT) domains of either the envelope protein of mouse mammary tumour virus (MMTV) or the hemagglutinin (HA) of influenza A. For a non-VLP incorporation control, a third design was made where VAR2CSA was expressed without TM-CT domains. In the primary immunogenicity study in Balb/c mice, VAR2CSA fused to HA TM-CT was significantly superior in inducing ID1-ID2a specific antibodies after the first immunization. A sequential study was performed to include a comparison to the soluble VAR2CSA protein vaccine, which has entered a phase I clinical trial (NCT02647489). The results revealed the induction of higher antibody responses and increased inhibition of parasite binding to CSA using either VAR2CSA HA TM-CT or VAR2CSA MMTV TM-CT as priming vaccines for protein double-boost immunizations, compared to protein prime-double boost regimen. Analysis of pooled serum samples on peptide arrays revealed a unique targeting of several epitopes in mice that had been primed with VAR2CSA HA TM-CT. Consequently, modification of VLP anchors is an important point of optimization in virus-encoded retroviral VLP-based vaccines, and adenovirus VLPs boosted by recombinant proteins offer hope of increasing the levels of protective VAR2CSA specific antibodies.


Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Feminino , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Vetores Genéticos/química , Vetores Genéticos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Vírus do Tumor Mamário do Camundongo , Camundongos , Camundongos Endogâmicos BALB C , Placenta/química , Placenta/imunologia , Placenta/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Gravidez , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vírus da Imunodeficiência Símia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética
11.
Vaccine ; 35(6): 873-881, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28089547

RESUMO

The key targets of protective antibodies against Plasmodium falciparum remain largely unknown. In this study, we determined immunoreactivity to 1827 recombinant proteins derived from 1565 genes representing ∼30% of the entire P. falciparum genome, for identification of novel malaria vaccine candidates. The recombinant proteins were expressed by wheat germ cell-free system, a platform that can synthesize quality plasmodial proteins that elicit biologically active antibodies in animals. Sera were obtained from indigenous residents of a malaria endemic region in Northern Uganda who were enrolled at the start of a rainy season and prospectively monitored for symptomatic malaria episodes for a year. Immunoreactivity to sera was determined by AlphaScreen; a homogeneous high-throughput system that detects protein interactions. Our analysis revealed antibody responses to 128 proteins that significantly associated with protection from symptomatic malaria. From 128 proteins, 53 were down-selected as the most plausible targets of host protective immune response by virtue of having a predicted signal peptide and/or transmembrane domain(s), or confirmed localization on the parasite surface. The 53 proteins comprised of not only previously characterized vaccine candidates but also uncharacterized proteins. Proteins involved in erythrocyte invasion; RON4, RON2 and CLAG3.1 and pre-erythrocytic proteins; SIAP-2, TRAP and CelTOS, were recommended for prioritization for further evaluation as vaccine candidates. The findings clearly demonstrate that generation of the protein library using the wheat germ cell-free system coupled with high throughput immunoscreening with AlphaScreen offers new options for rational discovery and selection of potential malaria vaccine candidates.


Assuntos
Antígenos de Protozoários/imunologia , Resistência à Doença , Genoma de Protozoário/imunologia , Vacinas Antimaláricas/biossíntese , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/química , Antígenos de Protozoários/química , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Criança , Eritrócitos/parasitologia , Feminino , Células Germinativas/química , Células Germinativas/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Soros Imunes/química , Vacinas Antimaláricas/química , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Masculino , Plasmodium falciparum/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Triticum/química , Triticum/genética , Triticum/metabolismo , Uganda , Adulto Jovem
12.
Protein Expr Purif ; 136: 52-57, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26578115

RESUMO

Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.


Assuntos
Antígenos de Protozoários , Imunogenicidade da Vacina , Vacinas Antimaláricas , Plasmodium vivax/genética , Proteínas de Protozoários , Receptores de Superfície Celular , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Humanos , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium vivax/imunologia , Domínios Proteicos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
13.
PLoS One ; 11(10): e0164053, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27695087

RESUMO

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.


Assuntos
Variação Antigênica , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Feminino , Fermentação , Humanos , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Estabilidade Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Controle de Qualidade , Coelhos , Proteínas Recombinantes
14.
Methods Mol Biol ; 1404: 597-619, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27076325

RESUMO

There are currently no vaccines that provide sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates, but none of them have fulfilled expectations. Therefore, combinations of key antigens from different stages of the parasites life cycle may be essential for the development of efficacious malaria vaccines. Following the identification of promising antigens using bioinformatics, proteomics, and/or immunological approaches, it is necessary to express, purify, and characterize these proteins and explore the potential of fusion constructs combining different antigens or antigen domains before committing to expensive and time-consuming clinical development. Here, using malaria vaccine candidates as an example, we describe how Agrobacterium tumefaciens-based transient expression in plants can be combined with a modular and flexible cloning strategy as a robust and versatile tool for the rapid production of candidate antigens during research and development.


Assuntos
Engenharia Genética/métodos , Vacinas Antimaláricas/genética , Tabaco/genética , Agrobacterium tumefaciens/genética , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/isolamento & purificação , Plasmídeos/genética , Fatores de Tempo , Transformação Genética
15.
J Biotechnol ; 213: 83-96, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25736485

RESUMO

An intensification of pharmaceutical protein production processes can be achieved by the integration of unit operations and application of recurring sequences of all biochemical process steps. Within optimization procedures each individual step as well as the overall process has to be in the focus of scientific interest. This paper includes a description of the development of a fully automated production plant, starting with a two step upstream followed by a four step downstream line, including cell clarification, broth cleaning with microfiltration, product concentration with ultrafiltration and purification with column chromatography. Recursive production strategies are developed where a cell breeding, the protein production and the whole downstream is operated in series but also in parallel, each main operation shifted by one day. The quality and reproducibility of the recursive protein expression is monitored on-line by Golden Batch and this is controlled by Model Predictive Multivariate Control (MPMC). As a demonstration process the production of potential Malaria vaccines with Pichia pastoris is under investigation.


Assuntos
Reatores Biológicos , Vacinas Antimaláricas/biossíntese , Pichia/metabolismo , Modelos Teóricos , Reprodutibilidade dos Testes
16.
Biotechnol Bioeng ; 112(4): 659-67, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25335451

RESUMO

We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.


Assuntos
Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Animais , Anticorpos Antiprotozoários/sangue , Sítios de Ligação , Biotecnologia/métodos , Técnica Direta de Fluorescência para Anticorpo , Imunização/métodos , Imunoglobulina G/sangue , Vacinas Antimaláricas/química , Vacinas Antimaláricas/genética , Espectrometria de Massas , Camundongos , Proteínas Mutantes/biossíntese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Pichia/genética , Pichia/metabolismo , Plasmodium falciparum/imunologia , Proteólise , Coelhos , Deleção de Sequência , Tecnologia Farmacêutica/métodos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação
17.
PLoS One ; 9(1): e87198, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489871

RESUMO

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.


Assuntos
Vacinas Antimaláricas/biossíntese , Malária Falciparum/prevenção & controle , Proteína 1 de Superfície de Merozoito/biossíntese , Tetrahymena thermophila/metabolismo , Vacinação , Animais , Animais não Endogâmicos , Anticorpos Antiprotozoários/sangue , Mapeamento de Epitopos , Feminino , Humanos , Vacinas Antimaláricas/imunologia , Malária Falciparum/sangue , Malária Falciparum/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Camundongos , Plasmodium falciparum/imunologia , Potência de Vacina
18.
PLoS One ; 9(1): e86658, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24475165

RESUMO

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.


Assuntos
Reatores Biológicos , Sistemas de Liberação de Medicamentos/métodos , Vacinas Antimaláricas/biossíntese , Pichia/metabolismo , Plasmodium berghei/química , Proteínas de Protozoários/metabolismo , Animais , Descoberta de Drogas , Imunofluorescência , Vacinas Antimaláricas/administração & dosagem , Vírus do Sarampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Nucleoproteínas/metabolismo , Proteínas de Protozoários/isolamento & purificação , Ribonucleoproteínas/biossíntese
19.
J Nanobiotechnology ; 11: 43, 2013 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-24359024

RESUMO

BACKGROUND: Nanosuspensions are an important class of delivery system for vaccine adjuvants and drugs. Previously, we developed a nanosuspension consisting of the synthetic TLR4 ligand glucopyranosyl lipid adjuvant (GLA) and dipalmitoyl phosphatidylcholine (DPPC). This nanosuspension is a clinical vaccine adjuvant known as GLA-AF. We examined the effects of DPPC supplier, buffer composition, and manufacturing process on GLA-AF physicochemical and biological activity characteristics. RESULTS: DPPC from different suppliers had minimal influence on physicochemical and biological effects. In general, buffered compositions resulted in less particle size stability compared to unbuffered GLA-AF. Microfluidization resulted in rapid particle size reduction after only a few passes, and 20,000 or 30,000 psi processing pressures were more effective at reducing particle size and recovering the active component than 10,000 psi. Sonicated and microfluidized batches maintained good particle size and chemical stability over 6 months, without significantly altering in vitro or in vivo bioactivity of GLA-AF when combined with a recombinant malaria vaccine antigen. CONCLUSIONS: Microfluidization, compared to water bath sonication, may be an effective manufacturing process to improve the scalability and reproducibility of GLA-AF as it advances further in the clinical development pathway. Various sources of DPPC are suitable to manufacture GLA-AF, but buffered compositions of GLA-AF do not appear to offer stability advantages over the unbuffered composition.


Assuntos
Adjuvantes Imunológicos/química , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Nanoestruturas/química , Proteínas de Protozoários/imunologia , 1,2-Dipalmitoilfosfatidilcolina/química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/normas , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Tampões (Química) , Citocinas/biossíntese , Citocinas/imunologia , Estabilidade de Medicamentos , Feminino , Lipídeo A/análogos & derivados , Lipídeo A/química , Lipídeo A/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Malária/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/normas , Tamanho da Partícula , Plasmodium berghei/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sonicação , Suspensões , Receptor 4 Toll-Like/imunologia
20.
J Nanobiotechnology ; 11: 30, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24025216

RESUMO

Due to their unusual properties, carbon nanotubes have been extensively employed in electronics, nanotechnology and optics, amongst other. More recently, they have also been used as vehicles for drug and antigen delivery, the latter being a novel immunization strategy against infectious diseases and cancer. Here we discuss the potential of carbon nanotubes as an antigen delivery tool and suggest further directions in the field of vaccination.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Protozoários/imunologia , Sistemas de Liberação de Medicamentos/métodos , Malária Vivax/prevenção & controle , Nanotubos de Carbono/química , Neoplasias/prevenção & controle , Antígenos de Neoplasias/química , Antígenos de Protozoários/química , Linfócitos B/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinação
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