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We previously reported a protective antibody response in mice immunized with synthetic microparticle vaccines made using layer-by-layer fabrication (LbL-MP) and containing the conserved T1BT* epitopes from the P. falciparum circumsporozoite protein. To further optimize the vaccine candidate, a benchtop tangential flow filtration method (LbL-by-TFF) was developed and utilized to produce vaccine candidates that differed in the status of base layer crosslinking, inclusion of a TLR2 ligand in the antigenic peptide, and substitution of serine or alanine for an unpaired cysteine residue in the T* epitope. Studies in mice revealed consistent superiority of the Pam3Cys-modified candidates and a modest benefit of base layer crosslinking, as evidenced by higher and more persistent antibody titers (up to 18 months post-immunization), a qualitative improvement of T-cell responses toward a Th1 phenotype, and greater protection from live parasite challenges compared to the unmodified prototype candidate. Immunogenicity was also tested in a non-human primate model, the rhesus macaque. Base layer-crosslinked LbL-MP loaded with T1BT* peptide with or without covalently linked Pam3Cys elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFNγ secretion with lower levels of IL-5 secretion. The Pam3Cys-modified construct was more potent, generating antibody responses that neutralized wild-type P. falciparum in an in vitro hepatocyte invasion assay. IgG purified from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected naïve mice from challenges with transgenic P. berghei sporozoites that expressed the full-length PfCS protein, with 50-88% of passively immunized mice parasite-free for ≥15 days. Substitution of serine for an unpaired cysteine in the T* region of the T1BT* subunit did not adversely impact immune potency in the mouse while simplifying the manufacture of the antigenic peptide. In a Good Laboratory Practices compliant rabbit toxicology study, the base layer-crosslinked, Pam3Cys-modified, serine-substituted candidate was shown to be safe and immunogenic, eliciting parasite-neutralizing antibody responses and establishing the dose/route/regimen for a clinical evaluation of this novel synthetic microparticle pre-erythrocytic malaria vaccine candidate.
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INTRODUCTION: Trauma, hemorrhage, and peritonitis have widely varying impacts on endocrine response in the injured patient. We sought to examine cortisol response in established non-human primate models of traumatic hemorrhage and intra-abdominal contamination. METHODS: Cynomologus Macaques were separated into two experimental groups, the polytrauma and hemorrhage model, involving a laparoscopic liver resection with uncontrolled hemorrhage, cecal perforation, and soft tissue excision; and the traumatic hemorrhage model, involving only liver resection and uncontrolled hemorrhage. Cortisol levels were measured pre-operatively, at the time of injury, and at regular intervals until post-operative day 1. RESULTS: Cortisol levels increased 600% from the pre-operative value in the polytrauma and hemorrhage model, with minimal changes (20%) in the hemorrhage only model. CONCLUSION: Cortisol levels increase dramatically in response to polytrauma and intra-abdominal contamination as compared to hemorrhage only. The lack of response in the hemorrhage only group may be due to relative adrenal insufficiency caused by the shock state or lack of enticing stimuli from fecal peritonitis.
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Traumatismos Abdominales/sangre , Hemorragia/sangre , Hidrocortisona/sangre , Peritonitis/sangre , Traumatismos Abdominales/complicaciones , Traumatismos Abdominales/microbiología , Traumatismos Abdominales/patología , Animales , Modelos Animales de Enfermedad , Heces/microbiología , Hematoma/sangre , Hematoma/etiología , Hematoma/microbiología , Hematoma/patología , Hemorragia/etiología , Hemorragia/patología , Hidrocortisona/análisis , Perforación Intestinal/sangre , Perforación Intestinal/etiología , Perforación Intestinal/microbiología , Perforación Intestinal/patología , Macaca fascicularis , Masculino , Traumatismo Múltiple/sangre , Traumatismo Múltiple/complicaciones , Traumatismo Múltiple/microbiología , Traumatismo Múltiple/patología , Peritonitis/etiología , Peritonitis/microbiologíaRESUMEN
INTRODUCTION: Endocrine dysregulation's role in heterotopic ossification (HO) remains unexplored. We sought to examine corticosterone and testosterone in established rat models of ectopic bone formation, and correlate to HO formation with CT analysis. METHODS: Fifteen rats were placed into three groups of traumatic injury patterns: Blast and injury (120 kPa blast, femoral fracture and quadriceps crush), injury only, and blast only. Serum corticosterone and testosterone levels were drawn until post-operative day 40. HO was analyzed using CT. RESULTS: Corticosterone levels peaked in the blast and injury group in the shortest time post injury, followed by injury only and blast only groups. Testosterone levels reached nadir in similar fashion. Volume of HO was highest in the blast and injury group, followed by the injury only group. CONCLUSION: Corticosterone and testosterone's contribution to HO formation requires further characterization, but this study suggests that high peaks in corticosterone and a low nadir in testosterone are associated with higher volumes of HO.
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Amputación Quirúrgica , Traumatismos por Explosión/sangre , Corticosterona/sangre , Osificación Heterotópica/sangre , Testosterona/sangre , Animales , Traumatismos por Explosión/diagnóstico por imagen , Modelos Animales de Enfermedad , Fémur/diagnóstico por imagen , Fémur/cirugía , Imagenología Tridimensional , Masculino , Ratas Sprague-Dawley , Estadística como Asunto , Tomografía Computarizada por Rayos XRESUMEN
Adjuvants have long been critical components of vaccines, but the exact mechanisms of their action and precisely how they alter or enhance vaccine-induced immune responses are often unclear. In this study, we used broad immunoprofiling of antibody, cellular, and cytokine responses, combined with data integration and machine learning to gain insight into the impact of different adjuvant formulations on vaccine-induced immune responses. A Self-Assembling Protein Nanoparticles (SAPN) presenting the malarial circumsporozoite protein (CSP) was used as a model vaccine, adjuvanted with three different liposomal formulations: liposome plus Alum (ALFA), liposome plus QS21 (ALFQ), and both (ALFQA). Using a computational approach to integrate the immunoprofiling data, we identified distinct vaccine-induced immune responses and developed a multivariate model that could predict the adjuvant condition from immune response data alone with 92% accuracy (p = 0.003). The data integration also revealed that commonly used readouts (i.e. serology, frequency of T cells producing IFN-γ, IL2, TNFα) missed important differences between adjuvants. In summary, broad immune-profiling in combination with machine learning methods enabled the reliable and clear definition of immune signatures for different adjuvant formulations, providing a means for quantitatively characterizing the complex roles that adjuvants can play in vaccine-induced immunity. The approach described here provides a powerful tool for identifying potential immune correlates of protection, a prerequisite for the rational pairing of vaccines candidates and adjuvants.
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Adyuvantes Inmunológicos/farmacología , Aprendizaje Automático , Adyuvantes Inmunológicos/administración & dosificación , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/biosíntesis , Citocinas/sangre , Relación Dosis-Respuesta Inmunológica , Inmunidad Celular , Liposomas , Macaca mulatta , Vacunas/administración & dosificación , Vacunas/inmunologíaRESUMEN
The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.
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Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.
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Antimaláricos/administración & dosificación , Malaria Falciparum/tratamiento farmacológico , Malaria Vivax/tratamiento farmacológico , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium vivax/crecimiento & desarrollo , Animales , Modelos Animales de Enfermedad , Hepatocitos/parasitología , Humanos , Hígado/parasitología , Malaria Falciparum/parasitología , Malaria Vivax/parasitología , Ratones , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Esquizontes/efectos de los fármacos , Esquizontes/crecimiento & desarrollo , Esporozoítos/efectos de los fármacos , Esporozoítos/crecimiento & desarrolloRESUMEN
To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014D0D1) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1ß, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.
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Flagelina/inmunología , Inmunogenicidad Vacunal , Malaria Falciparum/prevención & control , Nanopartículas , Plasmodium falciparum/inmunología , Dominios Proteicos/inmunología , Proteínas Protozoarias/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Antiprotozoarios/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Flagelina/química , Flagelina/genética , Inmunización , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Ratones , Modelos Biológicos , Plasmodium falciparum/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Pliegue de Proteína , Multimerización de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes , Receptor Toll-Like 5/agonistasRESUMEN
We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (PfCSP) CD4+ and CD8+ epitopes, universal TH epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the PfCSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5g of drug substance per 100g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length PfCSP.
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Liposomas/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Nanopartículas/administración & dosificación , Plasmodium falciparum/inmunología , Transporte de Proteínas/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Epítopos/inmunología , Femenino , Malaria Falciparum/prevención & control , Ratones , Ratones Endogámicos C57BL , Esporozoítos/inmunologíaRESUMEN
BACKGROUND: A lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg-Pb/PfCSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes (PfCSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described. METHODS: Inbred mice were vaccinated with PfCSP-SAPN in PBS. Serum antibodies were harvested and effects on P. falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively. RESULTS: The results of this work demonstrate that PfCSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P. falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, PfCSP-SAPN-induced antibodies are able to inhibit wild-type P. falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that PfCSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous fashion via transporter associated with antigen processing (TAP) recruitment to the early endosome (EE), and have partially delayed processing through the endoplasmic reticulum, has the potential to induce the long-lived, effector memory CD8+ T-cells as described previously. CONCLUSION: This paper describes the examination of humoral and cellular immune mechanisms induced by PfCSP-SAPN vaccination which result in sterile host protection against a transgenic P. berghei malaria sporozoite expressing the P. falciparum CSP, and which significantly inhibits native P. falciparum sporozoites from invading and developing within cultured human hepatocytes. These results may indicate the type and mode of action of protective antibodies needed to control P. falciparum sporozoites from infecting humans as well as a potential mechanism of induction of protective long-lived effector memory CD8+ T-cells.
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Vacunas contra la Malaria/inmunología , Nanopartículas , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Femenino , Hepatocitos/parasitología , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8(+) and CD4(+) T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. METHODOLOGY/PRINCIPAL FINDINGS: To establish the basis for a SAPN-based vaccine, B- and CD8(+) T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (â¼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ(+), IL-2(+)) long-lived central memory CD8(+) T-cells. Furthermore, we demonstrated that these Ab or CD8(+) T-cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. CONCLUSION: The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.
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Anticuerpos Antiprotozoarios/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Vacunas de ADN/inmunología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/metabolismo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Malaria/inmunología , Malaria/prevención & control , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Modelos Moleculares , Datos de Secuencia Molecular , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , Plasmodium berghei/genética , Plasmodium berghei/inmunología , Plasmodium berghei/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Multimerización de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)(2)D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2(b), H-2(d), and H-2(k) alleles. Immunized mice produced a CD4(+) T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.
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Antígenos de Protozoos/inmunología , Epítopos de Linfocito B/inmunología , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Nanopartículas/uso terapéutico , Péptidos/uso terapéutico , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Afinidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Malaria/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Plasmodium berghei/inmunologíaRESUMEN
Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67deltaSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67deltaSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 microg per dose, which is a major improvement over previous recombinant p67 products.
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Vacunas Bacterianas/inmunología , Proteínas Protozoarias/inmunología , Theileriosis/prevención & control , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Baculoviridae/genética , Bovinos , ADN Bacteriano/análisis , Inmunización , Masculino , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/aislamiento & purificación , Vacunas de Subunidad/inmunologíaRESUMEN
The application of the baculovirus-insect cell expression system for the production of integral membrane and secreted proteins is often more troublesome than for cytoplasmic proteins. One protein expressed at low levels in insect cells is the Theileria parva sporozoite surface protein p67. Theileria parva is a protozoan parasite, which causes the tick-transmitted disease East Coast fever in cattle. Baculovirus vectors were engineered to produce a secreted form of p67 by replacing the signal peptide of p67 with the honeybee mellitin signal sequence and deleting a putative membrane anchor from the C-terminus. Furthermore, the chitinase and v-cathepsin genes were deleted from the baculovirus expression vector in a bacmid setup, allowing broad scale application of this novel vector. Deletion of the chitinase and v-cathepsin gene had a positive effect on the integrity of both the intracellular and secreted recombinant protein.
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Baculoviridae/genética , Catepsinas/genética , Quitinasas/genética , Regulación Viral de la Expresión Génica , Vectores Genéticos , Proteínas Protozoarias/genética , Baculoviridae/enzimología , Meliteno/genética , Meliteno/fisiología , Señales de Clasificación de Proteína , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
East Coast fever (ECF) in cattle is caused by the tick-borne protozoan parasite Theileria parva. The major sporozoite surface antigen of T. parva (p67) is an important candidate for inclusion in a subunit vaccine. Recently, we reported the expression and production of different parts of p67 as fusions to either GFP or to the baculovirus GP64 envelope glycoprotein in insect cells, which resulted in stable proteins recognized by a monoclonal specific for native p67. The immunogenicity of these fusion proteins was examined in out-bred mice and cattle. In mice, the full length p67 molecule without its signal peptide and transmembrane region, but fused to GFP (GFP:p67deltaSS) was the best immunogen followed by the C-terminus of p67 fused to GP64 (GP64:p67C). These two immunogens also provoked a high level of sero-conversion in cattle when formulated in a water-in-oil or saponin-derived adjuvant with only 100 microg of protein and a single booster. The vaccine-elicited antibodies efficiently inhibited the infectivity of T. parva sporozoites in in vitro neutralization assays. This study demonstrated that these new baculovirus-derived p67 vaccines were highly immunogenic, and that in combination with a suitable adjuvant, they have a clear potential to induce protective immunity in cattle.
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Enfermedades de los Bovinos/parasitología , Proteínas Protozoarias/inmunología , Theileria parva/inmunología , Theileriosis/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Antiprotozoarios/sangre , Baculoviridae/genética , Western Blotting/veterinaria , Bovinos , Enfermedades de los Bovinos/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Proteínas Fluorescentes Verdes , Inmunización/métodos , Inmunización/veterinaria , Proteínas Luminiscentes , Ratones , Pruebas de Neutralización/veterinaria , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/genética , Vacunas Antiprotozoos/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Theileriosis/parasitología , Theileriosis/prevención & control , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunologíaRESUMEN
Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded protein. Here, we report the expression of N- and C-terminal domains of p67 fused to the baculovirus envelope glycoprotein GP64 by cloning the appropriate p67 cDNA segments between the signal sequence and the major portion of GP64. To further advance the generation of such recombinants, existing surface display techniques were combined with bacmid technology. Chimeric proteins were present on the surface of budded viruses as judged by immunogold labelling and were exposed on the surface of insect cells, as concluded from immunofluorescence studies of infected, non-fixed insect cells. In non-denaturing dot blot experiments, a strong reaction was obtained between monoclonal TpM12 and baculovirus particles displaying the p67N-GP64 chimeric protein. This antibody, raised against native p67, also specifically recognized the surface of recombinant-infected cells. Apparently, a more native conformation was achieved than when p67 was expressed in E.coli or in conventional baculovirus expression systems. The baculovirus surface expression system, therefore, provides an improved way of expressing this T.parva sporozoite surface protein.