RESUMEN
Traumatic brain injury (TBI) and Alzheimer's disease (AD) have overlapping mechanisms but it remains unknown if pathophysiological characteristics and cognitive trajectories in AD patients are influenced by TBI history. Here, we studied AD patients (stage MCI or dementia) with TBI history (ADTBI+, n=110), or without (ADTBI-, n=110) and compared baseline CSF concentrations of amyloid beta 1-42 (Aß42), phosphorylated tau181 (pTau181), total tau, neurofilament light chain (NfL), synaptosomal associated protein-25kDa (SNAP25), neurogranin (Ng), neuronal pentraxin-2 (NPTX2) and glutamate receptor-4 (GluR4), as well as differences in cognitive trajectories using linear mixed models. Explorative, analyses were repeated within stratified TBI groups by TBI characteristics (timing, severity, number). We found no differences in baseline CSF biomarker concentrations nor in cognitive trajectories between ADTBI+ and ADTBI- patients. TBI >5 years ago was associated with higher NPTX2 and a tendency for higher SNAP25 concentrations compared to TBI ≤ 5 years ago, suggesting that TBI may be associated with long-term synaptic dysfunction only when occurring before onset or in a pre-clinical disease stage of AD.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Biomarcadores , Lesiones Traumáticas del Encéfalo , Cognición , Proteínas tau , Humanos , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/psicología , Lesiones Traumáticas del Encéfalo/líquido cefalorraquídeo , Lesiones Traumáticas del Encéfalo/complicaciones , Biomarcadores/líquido cefalorraquídeo , Masculino , Femenino , Anciano , Péptidos beta-Amiloides/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo , Neurogranina/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Persona de Mediana Edad , Disfunción Cognitiva/líquido cefalorraquídeo , Disfunción Cognitiva/etiología , Disfunción Cognitiva/diagnóstico , Anciano de 80 o más Años , Proteína 25 Asociada a Sinaptosomas/líquido cefalorraquídeo , Proteína C-Reactiva/líquido cefalorraquídeo , Proteínas del Tejido NerviosoRESUMEN
BACKGROUND: Lack of early molecular biomarkers in sporadic behavioral variants of frontotemporal dementia (bvFTD) and its clinical overlap with primary psychiatric disorders (PPD) hampers its diagnostic distinction. Synaptic dysfunction is an early feature in bvFTD and identification of specific biomarkers might improve its diagnostic accuracy. Our goal was to understand the differential diagnostic potential of cerebrospinal fluid (CSF) synaptic biomarkers in bvFTD versus PPD and their specificity towards bvFTD compared with Alzheimer's disease (AD) and controls. Additionally, we explored the association of CSF synaptic biomarkers with social cognition, cognitive performance, and disease severity in these clinical groups. METHODS: Participants with probable bvFTD (n = 57), PPD (n = 71), AD (n = 60), and cognitively normal controls (n = 39) with available CSF, cognitive tests, and disease severity as frontotemporal lobar degeneration-modified clinical dementia rating scale (FTLD-CDR) were included. In a subset of bvFTD and PPD cases, Ekman 60 faces test scores for social cognition were available. CSF synaptosomal-associated protein 25 (SNAP25), neurogranin (Ng), neuronal pentraxin 2 (NPTX2), and glutamate receptor 4 (GluR4) were measured, along with neurofilament light (NfL), and compared between groups using analysis of covariance (ANCOVA) and logistic regression. Diagnostic accuracy was assessed using ROC analyses, and biomarker panels were selected using Wald's backward selection. Correlations with cognitive measures were performed using Pearson's partial correlation analysis. RESULTS: NPTX2 concentrations were lower in the bvFTD group compared with PPD (p < 0.001) and controls (p = 0.003) but not compared with AD. Concentrations of SNAP25 (p < 0.001) and Ng (p < 0.001) were elevated in patients with AD versus those with bvFTD and controls. The modeled panel for differential diagnosis of bvFTD versus PPD consisted of NfL and NPTX2 (AUC = 0.96, CI: 0.93-0.99, p < 0.001). In bvFTD versus AD, the modeled panel consisted of NfL, SNAP25, Ng, and GluR4 (AUC = 0.86, CI: 0.79-0.92, p < 0.001). In bvFTD, lower NPTX2 (Pearson's r = 0.29, p = 0.036) and GluR4 (Pearson's r = 0.34, p = 0.014) concentrations were weakly associated with worse performance of total cognitive score. Lower GluR4 concentrations were also associated with worse MMSE scores (Pearson's r = 0.41, p = 0.002) as well as with worse executive functioning (Pearson's r = 0.36, p = 0.011) in bvFTD. There were no associations between synaptic markers and social cognition or disease severity in bvFTD. CONCLUSION: Our findings of involvement of NTPX2 in bvFTD but not PPD contribute towards better understanding of bvFTD disease pathology.
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Enfermedad de Alzheimer , Demencia Frontotemporal , Degeneración Lobar Frontotemporal , Humanos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/líquido cefalorraquídeo , Demencia Frontotemporal/patología , Degeneración Lobar Frontotemporal/diagnóstico , Curva ROC , Pruebas Neuropsicológicas , Biomarcadores/líquido cefalorraquídeoRESUMEN
Alzheimer's disease (AD) is heterogenous at the molecular level. Understanding this heterogeneity is critical for AD drug development. Here we define AD molecular subtypes using mass spectrometry proteomics in cerebrospinal fluid, based on 1,058 proteins, with different levels in individuals with AD (n = 419) compared to controls (n = 187). These AD subtypes had alterations in protein levels that were associated with distinct molecular processes: subtype 1 was characterized by proteins related to neuronal hyperplasticity; subtype 2 by innate immune activation; subtype 3 by RNA dysregulation; subtype 4 by choroid plexus dysfunction; and subtype 5 by blood-brain barrier impairment. Each subtype was related to specific AD genetic risk variants, for example, subtype 1 was enriched with TREM2 R47H. Subtypes also differed in clinical outcomes, survival times and anatomical patterns of brain atrophy. These results indicate molecular heterogeneity in AD and highlight the need for personalized medicine.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , ProteómicaRESUMEN
BACKGROUND: Synapse loss is an early event that precedes neuronal death and symptom onset and is considered the best neuropathological correlate of cognitive decline in Alzheimer's disease (AD). Vesicle-associated membrane protein 2 (VAMP-2) has emerged as a promising biomarker of AD-related synapse degeneration in cerebrospinal fluid (CSF). The aim of this study was to explore the CSF profile of VAMP-2 across the AD continuum in relation to core AD biomarkers, other synaptic proteins, neurogranin (Ng) and synaptosomal-associated Protein-25 kDa (SNAP-25) and cognitive performance. METHODS: We developed a digital immunoassay on the Single Molecule Array platform to quantify VAMP-2 in CSF and used existing immunoassays to quantify Ng, SNAP-25 and core CSF AD biomarkers. The clinical study included 62 cognitively unimpaired AD biomarker-negative subjects and 152 participants across the AD continuum from the SPIN cohort (Sant Pau Initiative on Neurodegeneration). Cognitive measures of episodic, semantic, executive and visuospatial domains and global cognition were included. Statistical methods included χ2 tests, spearman correlation, and ANCOVA analyses. RESULTS: The VAMP-2 assay had a good analytical performance (repeatability 8.9%, intermediate precision 10.3%). Assay antibodies detected native VAMP-2 protein in human brain homogenates. CSF concentrations of VAMP-2, neurogranin and SNAP-25 were lower in preclinical AD stage 1 compared to controls and higher at later AD stages compared to AD stage 1 and were associated with core AD biomarkers, particularly total tau (adj. r2 = 0.62 to 0.78, p < 0.001). All three synaptic proteins were associated with all cognitive domains in individuals on the AD continuum (adj. r2 = 0.04 to 0.19, p < 0.05). CONCLUSIONS: Our novel digital immunoassay accurately measures VAMP-2 changes in CSF, which reflect AD biomarkers and cognitive performance across multiple domains.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Disfunción Cognitiva/diagnóstico , Neurogranina/líquido cefalorraquídeo , Vesículas Sinápticas/patología , Proteínas tau/líquido cefalorraquídeo , Proteína 2 de Membrana Asociada a VesículasRESUMEN
BACKGROUND: Loss of synaptic functionality has been recently identified as an early-stage indicator of neurological diseases. Consequently, monitoring changes in synaptic protein levels may be relevant for observing disease evolution or treatment responses in patients. Here, we have studied the relationship between fluid biomarkers of neurodegeneration and synaptic dysfunction in patients with Alzheimer's disease (AD), frontotemporal dementia (FTD), and subjective cognitive decline (SCD). METHODS: The exploratory cohort consisted of cerebrospinal fluid (CSF) samples (n = 60) from patients diagnosed with AD (n = 20), FTD (n = 20), and SCD (n = 20) from the Amsterdam Dementia Cohort. We developed two novel immunoassays for the synaptic proteins synaptosomal-associated protein-25 (SNAP25) and vesicle-associated membrane protein-2 (VAMP2). We measured the levels of these biomarkers in CSF, in addition to neuronal pentraxin-2 (NPTX2), glutamate ionotropic receptor-4 (GluR4), and neurogranin (Ng) for this cohort. All in-house immunoassays were validated and analytically qualified prior to clinical application. CSF neurogranin (Ng) was measured using a commercially available ELISA. RESULTS: This pilot study indicated that SNAP25, VAMP2, and Ng may not be specific biomarkers for AD as their levels were significantly elevated in patients with both AD and FTD compared to SCD. Moreover, the strength of the correlations between synaptic proteins was lower in the AD and FTD clinical groups compared to SCD. SNAP25, VAMP2, and Ng correlated strongly with each other as well as with total Tau (Tau) and phosphorylated Tau (PTau) in all three clinical groups. However, this correlation was weakened or absent with NPTX2 and GluR4. None of the synaptic proteins correlated to neurofilament light (NfL) in any clinical group. CONCLUSION: The correlation of the synaptic biomarkers with CSF Tau and PTau but the lack thereof with NfL implies that distinct pathological pathways may be involved in synaptic versus axonal degeneration. Our results reflect the diversity of synaptic pathology in neurodegenerative dementias.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Demencia Frontotemporal , Humanos , Enfermedad de Alzheimer/líquido cefalorraquídeo , Demencia Frontotemporal/líquido cefalorraquídeo , Neurogranina/líquido cefalorraquídeo , Proyectos Piloto , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Disfunción Cognitiva/líquido cefalorraquídeoRESUMEN
Psychiatric disorders are widely underreported diseases, especially in their early stages. So far, there is no fluid biomarker to confirm the diagnosis of these disorders. Proteomics data suggest the synaptic protein glutamate receptor 4 (GluR4), part of the AMPA receptor, as a potential diagnostic biomarker of major depressive disorder (MDD). A novel sandwich ELISA was established and analytically validated to detect GluR4 in cerebrospinal fluid (CSF) samples. A total of 85 subjects diagnosed with MDD (n = 36), bipolar disorder (BD, n = 12), schizophrenia (SCZ, n = 12) and neurological controls (CON, n = 25) were analysed. The data exhibited a significant correlation (r = 0.74; CI:0.62 to 0.82; p < 0.0001) with the antibody-free multiple reaction monitoring (MRM) mass spectrometry (MS) data. CSF GluR4 levels were lower in MDD (p < 0.002) and BD (p = 0.012) than in CON. Moreover, subjects with SCZ described a trend towards lower levels than CON (p = 0.13). The novel GluR4 ELISA may favour the clinical application of this protein as a potential diagnostic biomarker of psychiatric disorders and may facilitate the understanding of the pathophysiological mechanisms behind these disorders.
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Trastorno Depresivo Mayor , Humanos , Trastorno Depresivo Mayor/diagnóstico , Proteómica , Receptores de GlutamatoRESUMEN
BACKGROUND: We previously identified four Alzheimer's disease (AD) subgroups with increasingly higher cerebrospinal fluid (CSF) levels of tau phosphorylated at threonine 181 (p-tau). These subgroups included individuals across the cognitive spectrum, suggesting p-tau subgroups could reflect distinct biological changes in AD, rather than disease severity. Therefore, in the current study, we further investigated which potential processes may be related with p-tau subgroups, by comparing individuals on CSF markers for presynaptic structure [vesicle-associated membrane protein 2 (VAMP2)], postsynaptic structure [neurogranin (NRGN)], axonal damage [neurofilament light (NfL)], and amyloid production [beta-secretase 1 (BACE1) and amyloid-beta 1-40 (Aß40)]. METHODS: We selected 348 amyloid-positive (A+) individuals (53 preclinical, 102 prodromal, 193 AD dementia) and 112 amyloid-negative (A-) cognitively normal (CN) individuals from the Amsterdam Dementia Cohort (ADC). Individuals were labeled according to their p-tau subgroup (subgroup 1: p-tau ≤ 56 pg/ml; subgroup 2: 57-96 pg/ml; subgroup 3: 97-159 pg/ml; subgroup 4: > 159 pg/ml). CSF protein levels were measured with ELISA (NRGN, BACE1, Aß40, NfL) or single-molecule array (Simoa) (VAMP2). We tested whether protein levels differed between the p-tau subgroups within A+ individuals with linear models corrected for age and sex and whether disease stage influenced these relationships. RESULTS: Among A+ individuals, higher p-tau subgroups showed a higher percentage of AD dementia [subgroup 1: n = 41/94 (44%); subgroup 2: n = 81/147 (55%); subgroup 3: n = 59/89 (66%); subgroup 4: n = 7/11 (64%)]. Relative to controls, subgroup 1 showed reduced CSF levels of BACE1, Aß40, and VAMP2 and higher levels of NfL. Subgroups 2 to 4 showed gradually increased CSF levels of all measured proteins, either across the first three (NfL and Aß40) or across all subgroups (VAMP2, NRGN, BACE1). The associations did not depend on the clinical stage (interaction p-values ranging between 0.19 and 0.87). CONCLUSIONS: The results suggest that biological heterogeneity in p-tau levels in AD is related to amyloid metabolism and synaptic integrity independent of clinical stage. Biomarkers reflecting amyloid metabolism and synaptic integrity may be useful outcome measures in clinical trials targeting tau pathology.
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Enfermedad de Alzheimer , Amiloidosis , Enfermedad de Alzheimer/líquido cefalorraquídeo , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/líquido cefalorraquídeo , Proteínas Amiloidogénicas , Ácido Aspártico Endopeptidasas , Biomarcadores/líquido cefalorraquídeo , Humanos , Neurogranina/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Proteína 2 de Membrana Asociada a Vesículas , Proteínas tau/líquido cefalorraquídeoRESUMEN
Trypanosoma evansi is a widely spread parasite that causes the debilitating disease "surra" in several types of ungulates. This severely challenges livestock rearing and heavily weighs on the socio-economic development in the affected areas, which include countries on five continents. Active case finding requires a sensitive and specific diagnostic test. In this paper, we describe the application of an unbiased immunization strategy to identify potential biomarkers for Nanobody (Nb)-based detection of T. evansi infections. Alpaca immunization with soluble lysates from different T. evansi strains followed by panning against T. evansi secretome resulted in the selection of a single Nb (Nb11). By combining Nb11-mediated immuno-capturing with mass spectrometry, the T. evansi target antigen was identified as the glycolytic enzyme enolase. Four additional anti-enolase binders were subsequently generated by immunizing another alpaca with the recombinant target enzyme. Together with Nb11, these binders were evaluated for their potential use in a heterologous sandwich detection format. Three Nb pairs were identified as candidates for the further development of an antigen-based assay for Nb-mediated diagnosis of T. evansi infection.
RESUMEN
BACKGROUND: Animal trypanosomosis caused by Trypanosoma evansi is known as "surra" and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings. METHODOLOGY/PRINCIPLE FINDINGS: An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool. CONCLUSIONS/SIGNIFICANCE: Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF (TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections.
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Técnicas de Amplificación de Ácido Nucleico/métodos , Recombinasas/metabolismo , Trypanosoma/aislamiento & purificación , Tripanosomiasis/diagnóstico , Animales , ADN Protozoario/genética , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Trypanosoma/genéticaRESUMEN
Trypanosoma are blood-borne parasites and are the causative agents of neglected tropical diseases (NTDs) affecting both humans and animals. These parasites mainly rely on glycolysis for their energy production within the mammalian host, which is why trypanosomal glycolytic enzymes have been pursued as interesting targets for the development of trypanocidal drugs. The structure-function relationships of pyruvate kinases (PYKs) from trypanosomatids (Trypanosoma and Leishmania) have been well-studied within this context. In this paper, we describe the structural and enzymatic characterization of PYK from T. congolense (TcoPYK), the main causative agent of Animal African Trypanosomosis (AAT), by employing a combination of enzymatic assays, thermal unfolding studies and X-ray crystallography.
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Piruvato Quinasa , Trypanosoma congolense/metabolismo , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Humanos , Cinética , Modelos Estructurales , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Piruvato Quinasa/química , Piruvato Quinasa/metabolismo , Tripanosomiasis Africana/parasitologíaRESUMEN
Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as 'test-of-cure' tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.
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Inmunoensayo/métodos , Anticuerpos de Dominio Único/inmunología , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/inmunología , Animales , Antígenos de Protozoos/sangre , Antígenos de Protozoos/inmunología , Bovinos , Ratones , Parasitemia/diagnóstico , Parasitemia/inmunología , Parasitemia/parasitología , Proteínas Protozoarias/inmunología , Piruvato Quinasa/inmunología , Sensibilidad y Especificidad , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Trypanosoma congolense/fisiología , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/parasitologíaRESUMEN
Trypanosoma evansi (T. evansi) is the most widely spread pathogenic trypanosome in the world. The control of trypanosomiasis depends on accurate diagnosis and effective treatment. Focusing on the presence of T. evansi in Asia, we developed a detection assay based on tracing phosphate ions (Pi) generated during LAMP targeting the variant surface glycoprotein (VSG) gene of Rode Trypanozoon antigenic type 1.2 (RoTat 1.2 VSG). The diagnostic potential as well as the use of the assay as a test-of-cure method after berenil treatment, was assessed in mice at different time points of infection. In addition, 67 buffalo blood collected from Tongling county, Anhui province, as well as 42 cattle sera from the Shanghai area, were used to evaluate the diagnostic validity of the test. The detection limit of the novel LAMP assay was determined to be as low as 1 fg of T. evansi DNA, while the reaction time for the test was only 30min. Hence it outperforms both microscopy and PCR. In the test-of-cure assessment, successful berenil mediated cure could be confirmed within 48h after treatment. This offers a tremendous advantage over conventional antibody-based diagnostic tools in which successful cure only can be confirmed after months. In the cattle and buffalo screening, the LAMP was able to detect a false-negative determined sample, wrongly classified in a conventional microscopy and PCR screening. Finally, no cross-reactivity was observed with other zoonotic parasites, such as T. evansi type B, T. congolense, T. brucei, Schistosoma japonicum, Plasmodium falciparum, Leishmania donovani, Toxoplasma gondii and Angiostrongylus cantonensis. We conclude that the novel LAMP assay is sensitive, specific and convenient for field use, particularly in areas where infection incidence has become extremely low. The LAMP assay could be used as a tool for trypanosomiasis control and elimination strategies in areas where T. evansi Type A infections are causing a threat to livestock farming.
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Enfermedades de los Bovinos/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/normas , Trypanosoma/genética , Tripanosomiasis/veterinaria , Medicina Veterinaria/métodos , Animales , Bovinos , China , ADN Protozoario/genética , Límite de Detección , Ratones , Sensibilidad y Especificidad , Tripanosomiasis/diagnóstico , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is a disease of swine, caused by an arterivirus, the PRRS virus (PRRSV). This virus infects pigs worldwide and causes huge economic losses. Due to genetic drift, current vaccines are losing their power. Adaptable vaccines could provide a solution to this problem. This study aims at producing in planta a set of antigens derived from the PRRSV glycoproteins (GPs) to be included in a subunit vaccine. We selected the GP3, GP4 and GP5 and optimized these for production in an Arabidopsis seed platform by removing transmembrane domains (Tm) and/or adding stabilizing protein domains, such as the green fluorescent protein (GFP) and immunoglobulin (IgG) 'Fragment crystallizable' (Fc) chains. Accumulation of the GPs with and without Tm was low, reaching no more than 0.10% of total soluble protein (TSP) in homozygous seed. However, addition of stabilizing domains boosted accumulation up to a maximum of 2.74% of TSP when GFP was used, and albeit less effectively, also the Fc chains of the porcine IgG3 and murine IgG2a increased antigen accumulation, to 0.96% and 1.81% of TSP respectively, while the murine IgG3 Fc chain did not. Antigens with Tm were less susceptible to these manipulations to increase yield. All antigens were produced in the endoplasmic reticulum and accordingly, they carried high-mannose N-glycans. The immunogenicity of several of those antigens was assessed and we show that vaccination with purified antigens did elicit the production of antibodies with virus neutralizing activity in mice but not in pigs.