RESUMEN

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Asunto(s)

Plasmodium falciparum , Esporozoítos , Animales , Humanos , Ratones , Culicidae/parasitología , Malaria/parasitología , Malaria/prevención & control , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/química , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Esporozoítos/crecimiento & desarrollo , Esporozoítos/patogenicidad , Hepatocitos/parasitología , Hígado/parasitología , Proteína 1 de Superficie de Merozoito , Eritrocitos/parasitología , Técnicas In Vitro

RESUMEN

Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSPRepeat) can protect against malaria. However, it has also been suggested that the CSPRepeat is a decoy that prevents the immune system from mounting responses against other domains of CSP. Here, we show that, following parasite immunization, B cell responses to the CSPRepeat are immunodominant over responses to other CSP domains despite the presence of similar numbers of naive B cells able to bind these regions. We find that this immunodominance is driven by avid binding of the CSPRepeat to cognate B cells that are able to expand at the expense of B cells with other specificities. We further show that mice immunized with repeat-truncated CSP molecules develop responses to subdominant epitopes and are protected against malaria. These data demonstrate that the CSPRepeat functions as a decoy, but truncated CSP molecules may be an approach for malaria vaccination.

Asunto(s)

Anticuerpos Antiprotozoarios/biosíntesis , Inmunización/métodos , Vacunas contra la Malaria/administración & dosificación , Malaria/prevención & control , Péptidos/administración & dosificación , Plasmodium berghei/efectos de los fármacos , Proteínas Protozoarias/genética , Animales , Anopheles/parasitología , Anticuerpos Neutralizantes/biosíntesis , Linfocitos B/inmunología , Linfocitos B/parasitología , Femenino , Expresión Génica , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Péptidos/inmunología , Plasmodium berghei/inmunología , Plasmodium berghei/patogenicidad , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Unión Proteica , Proteínas Protozoarias/inmunología , Esporozoítos/inmunología , Esporozoítos/efectos de la radiación , Transgenes , Vacunas Atenuadas

RESUMEN

The Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines. However, due to its size and complexity, expression of the full-length protein has been difficult, leading to focus on the C-terminal six cysteine domain (6C) with the use of fusion proteins to facilitate expression and folding. In this study, we utilized the baculovirus system to evaluate the expression of three Pfs48/45 proteins including the full-length protein, the 6C domain fragment and the 6C domain mutant to prevent glycosylation. Expression of the recombinant Pfs48/45 proteins was conducted in super Sf9 cells combined with the use of tunicamycin to prevent N-glycosylation. The proteins were then evaluated as immunogens in mice to demonstrate the induction of functionally active polyclonal antibody responses as measured in the standard membrane feeding assay (SMFA). Only the 6C protein was found to exhibit significant transmission-reducing activity. Further characterization of the biologically active 6C protein demonstrated it was homogeneous in terms of size, charge, conformation, absence of glycosylation, and containing proper disulfide bond pairings. This study presents an alternative expression system, without the need of a fusion protein partner, for the Pfs48/45 6C protein fragment including further evaluation as a potential transmission-blocking vaccine candidate.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/biosíntesis , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/biosíntesis , Animales , Baculoviridae/genética , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Proteínas Recombinantes/inmunología

RESUMEN

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

Asunto(s)

Biotecnología , Microbiología Industrial , Lactobacillus/metabolismo , Vacunas contra la Malaria/biosíntesis , Malaria Falciparum/prevención & control , Glicoproteínas de Membrana/biosíntesis , Proteínas Protozoarias/biosíntesis , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Antiprotozoarios/inmunología , Composición de Medicamentos , Inmunización , Inmunogenicidad Vacunal , Lactobacillus/genética , Vacunas contra la Malaria/química , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/farmacología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Ratones , Nanopartículas , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/farmacología , Saponinas/farmacología , Relación Estructura-Actividad , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/farmacología

RESUMEN

BACKGROUND: Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. METHODS: We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. RESULTS: In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90% on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. CONCLUSION: The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

Asunto(s)

Antígenos de Protozoos/metabolismo , Virus de la Hepatitis B del Pato/genética , Vacunas contra la Malaria/biosíntesis , Pichia/metabolismo , Vacunas de Partículas Similares a Virus/biosíntesis , Animales , Anticuerpos Bloqueadores/inmunología , Antígenos de Protozoos/genética , Patos/virología , Humanos , Malaria/prevención & control , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/aislamiento & purificación , Plasmodium falciparum/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/aislamiento & purificación

RESUMEN

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.

Asunto(s)

Anticuerpos Antiprotozoarios/biosíntesis , Inmunoglobulina G/biosíntesis , Vacunas contra la Malaria/biosíntesis , Malaria/prevención & control , Plasmodium yoelii/inmunología , Proteínas Protozoarias/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/parasitología , Femenino , Inmunización , Inmunogenicidad Vacunal , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Malaria/genética , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/administración & dosificación , Manitol/administración & dosificación , Manitol/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ácidos Oléicos/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vacunas de Subunidad

RESUMEN

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?

Asunto(s)

Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Citocinas/sangre , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/genética , Biomarcadores/sangre , Citocinas/biosíntesis , Determinación de Punto Final/métodos , Humanos , Inmunogenicidad Vacunal , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/clasificación , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Vacunación , Potencia de la Vacuna , Vacunas de Subunidad

RESUMEN

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.

Asunto(s)

Inmunidad Adaptativa/efectos de los fármacos , Anticuerpos Antiprotozoarios/biosíntesis , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Vacunas contra la Malaria/administración & dosificación , Malaria/prevención & control , Proteínas Protozoarias/antagonistas & inhibidores , Vacunas de ADN/administración & dosificación , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/parasitología , Femenino , Expresión Génica , Centro Germinal/efectos de los fármacos , Centro Germinal/inmunología , Centro Germinal/parasitología , Memoria Inmunológica/efectos de los fármacos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/inmunología , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/biosíntesis , Ratones , Ratones Endogámicos BALB C , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/genética , Plasmodium berghei/inmunología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , ARN Protozoario/genética , ARN Protozoario/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vacunas de ADN/biosíntesis

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/química , Anticuerpos Antiprotozoarios/química , Vacunas contra la Malaria/aislamiento & purificación , Nicotiana/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antiprotozoarios/metabolismo , Cromatografía de Afinidad/métodos , Clonación Molecular , Análisis Factorial , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Plantas Modificadas Genéticamente , Unión Proteica , Desnaturalización Proteica , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sefarosa/análogos & derivados , Nicotiana/química , Nicotiana/metabolismo , Ultrafiltración/métodos

RESUMEN

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.

Asunto(s)

Proteínas Portadoras/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Haplotipos , Malaria Falciparum/epidemiología , Plasmodium falciparum/genética , Polimorfismo Genético , Adolescente , Adulto , Secuencia de Aminoácidos , Linfocitos B/inmunología , Linfocitos B/parasitología , Proteínas Portadoras/inmunología , Niño , Preescolar , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Eritrocitos/parasitología , Femenino , Expresión Génica , Humanos , Lactante , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Masculino , Malí/epidemiología , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Plasmodium falciparum/inmunología , Estudios Prospectivos , Linfocitos T/inmunología , Linfocitos T/parasitología

RESUMEN

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.

Asunto(s)

Antígenos de Protozoos , Chlamydomonas reinhardtii , Cloroplastos , Vacunas contra la Malaria , Plasmodium falciparum/genética , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética

RESUMEN

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.

Asunto(s)

Antígenos de Protozoos/genética , Epítopos de Linfocito B/química , Epítopos de Linfocito T/química , Variación Genética , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Niño , Preescolar , Mapeo Epitopo , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Expresión Génica , Haplotipos , Humanos , Irán , Vacunas contra la Malaria/biosíntesis , Malaria Vivax/inmunología , Malaria Vivax/parasitología , Malaria Vivax/prevención & control , Masculino , Persona de Mediana Edad , Plasmodium vivax/química , Plasmodium vivax/inmunología , Plasmodium vivax/aislamiento & purificación , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Análisis de Secuencia de ADN , Esporozoítos/química , Esporozoítos/genética , Esporozoítos/inmunología

Asunto(s)

Bacteriemia/tratamiento farmacológico , Farmacorresistencia Bacteriana , Enterocolitis Seudomembranosa/tratamiento farmacológico , Vacunas contra la Malaria/biosíntesis , Vacunas Neumococicas/biosíntesis , Antibacterianos/uso terapéutico , Bacteriemia/microbiología , Huesos/efectos de los fármacos , Huesos/microbiología , Vías de Administración de Medicamentos , Enterocolitis Seudomembranosa/microbiología , Humanos , Articulaciones/efectos de los fármacos , Articulaciones/microbiología , Vacunas contra la Malaria/inmunología , Plásmidos/química , Plásmidos/metabolismo , Vacunas Neumococicas/inmunología , Ensayos Clínicos Controlados Aleatorios como Asunto , Rifampin/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos/inmunología , Linfocitos B/inmunología , Células Clonales/inmunología , Clonación Molecular/métodos , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/aislamiento & purificación , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Regiones no Traducidas 5' , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Formación de Anticuerpos , Especificidad de Anticuerpos , Antígenos/administración & dosificación , Linfocitos B/parasitología , Ligando de CD40/inmunología , Proliferación Celular , Separación Celular/métodos , Células Clonales/parasitología , Técnicas de Cocultivo , Citometría de Flujo , Células HEK293 , Humanos , Inmunización , Activación de Linfocitos , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/administración & dosificación , Flujo de Trabajo

RESUMEN

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.

Asunto(s)

Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/inmunología , Eritrocitos/inmunología , Eritrocitos/parasitología , Femenino , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Vectores Genéticos/química , Vectores Genéticos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Inmunización , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Endogámicos BALB C , Placenta/química , Placenta/inmunología , Placenta/parasitología , Plasmodium falciparum/química , Plasmodium falciparum/efectos de los fármacos , Embarazo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Virus de la Inmunodeficiencia de los Simios , Vacunas de Partículas Similares a Virus/biosíntesis , Vacunas de Partículas Similares a Virus/genética

RESUMEN

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.

Asunto(s)

Antígenos de Protozoos/inmunología , Resistencia a la Enfermedad , Genoma de Protozoos/inmunología , Vacunas contra la Malaria/biosíntesis , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adolescente , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/química , Antígenos de Protozoos/química , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Niño , Eritrocitos/parasitología , Femenino , Células Germinativas/química , Células Germinativas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Sueros Inmunes/química , Vacunas contra la Malaria/química , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Masculino , Plasmodium falciparum/genética , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Triticum/química , Triticum/genética , Triticum/metabolismo , Uganda , Adulto Joven

RESUMEN

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.

Asunto(s)

Antígenos de Protozoos , Inmunogenicidad Vacunal , Vacunas contra la Malaria , Plasmodium vivax/genética , Proteínas Protozoarias , Receptores de Superficie Celular , Animales , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/aislamiento & purificación , Humanos , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Plasmodium vivax/inmunología , Dominios Proteicos , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

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.

Asunto(s)

Variación Antigénica , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/inmunología , Proteínas de la Membrana/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Femenino , Fermentación , Humanos , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/efectos adversos , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Estabilidad Proteica , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Control de Calidad , Conejos , Proteínas Recombinantes

RESUMEN

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.

Asunto(s)

Ingeniería Genética/métodos , Vacunas contra la Malaria/genética , Nicotiana/genética , Agrobacterium tumefaciens/genética , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Vectores Genéticos/genética , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/aislamiento & purificación , Plásmidos/genética , Factores de Tiempo , Transformación Genética

RESUMEN

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.

Asunto(s)

Reactores Biológicos , Vacunas contra la Malaria/biosíntesis , Pichia/metabolismo , Modelos Teóricos , Reproducibilidad de los Resultados