Advances in the field of aluminum-based adjuvants in sheep: clinicopathological and immunological studies and interaction with small ruminant lentiviruses

Las vacunas son elementos clave en medicina humana y veterinaria y suponen uno de los mayores logros en salud pública y animal de los últimos siglos. Las vacunas intentan reproducir los aspectos inmunológicos provocados por las infecciones naturales para proporcionar protección a largo plazo contra ellas. En veterinaria, las vacunas tienen un impacto positivo en el bienestar de los animales y el rendimiento económico de las explotaciones, ya que reducen drásticamente la morbilidad y la mortalidad debidas a infecciones espontáneas.La intensificación de la producción animal ha aumentado drásticamente durante las últimas décadas, favoreciendo el contacto estrecho entre animales y el contagio de infecciones. Las buenas prácticas de manejo y una estrategia de vacunación adecuada son la piedra angular de cualquier plan sanitario de éxito. En ganadería ovina, los protocolos de vacunación difieren en función de múltiples factores, como el sistema de producción, la localización geográfica, el clima y/o la prevalencia de enfermedades. Además, los protocolos de vacunación pueden verse modificados por campañas de vacunación globales para luchar contra epizootias emergentes o reemergentes. Un ejemplo reciente fue la campaña de vacunación obligatoria contra el virus de la lengua azul (BTV) que tuvo lugar en la mayoría de los países europeos durante la primera década del siglo XXI. La vacunación masiva controló con éxito la infección en rumiantes, pero ocasionó diversos efectos adversos en varios países europeos. En España se describió un síndrome caquectizante asociado a signos neurológicos que fue englobado dentro del síndrome autoinmune/autoinflamatorio inducido por adyuvantes (síndrome ASIA).El tipo de activación del sistema inmune tras la vacunación depende en gran medida del tipo de vacuna empleada. La mayoría de las vacunas ovinas se basan en patógenos inactivados o proteínas recombinantes que a menudo son poco inmunogénicas per se, por lo que necesitan adyuvantes para aumentar la respuesta inmunitaria. Los adyuvantes a base de Aluminio (ABAs) se han utilizado en vacunas humanas y animales durante más de 70 años. Sin embargo, se desconocen gran parte del mecanismo de acción de estos adyuvantes. La inoculación de ABAs induce el reclutamiento de células inflamatorias en el punto de inyección que evoluciona hacia la formación de un nódulo inflamatorio crónico, el llamado granuloma postvacunal, que está compuesto principalmente por macrófagos activados. Estos agregados de macrófagos cargados con Al pueden migrar hacia el linfonodo regional y acumularse en él. Los lentivirus de pequeños rumiantes (SRLV) causan infecciones crónicas en ovejas y cabras en todo el mundo con una alta prevalencia. Los SRLV son retrovirus de ARN monocatenario con una alta heterogeneidad genética. De hecho, se han caracterizado cuatro genotipos principales (A, B, C y E) y más de 35 subtipos. Los SRLV tienen tropismo por el sistema mononuclear-fagocítico e inducen una inflamación lenta, crónica y persistente en cuatro órganos diana principales: pulmón, articulaciones, sistema nervioso y glándula mamaria. El tropismo por cada uno de estos tejidos, así como la severidad de las lesiones, dependen de factores asociados a la estirpe del virus y a la respuesta inmune del hospedador. En la forma articular, el principal signo clínico es artritis en la articulación del carpo, la cual puede ser unilateral o bilateral y en ovinos es causada principalmente por estirpes del genotipo B2. El objetivo de esta Tesis Doctoral es estudiar los efectos de la inoculación repetida de oxihidróxido de aluminio (Al) en ovinos y su relación con la patogenia del SRLV. El Experimento 1 incluyó 84 corderos repartidos en cuatro rebaños (Rebaños 1-4) de 21 animales cada uno. En cada Rebaño, los corderos se dividieron en tres grupos de tratamiento de 7 animales cada uno: Grupo Vaccine, inoculado con vacunas comerciales; grupo Adjuvant-only, que recibió la dosis equivalente de oxihidróxido de Al, y el grupo Control, al que se le inyectó solución salina tamponada con fosfato. El experimento duró 15 meses a lo largo de los cuales los corderos recibieron 19 inoculaciones subcutáneas, reproduciendo la cantidad de Al que pueden recibir estos animales a lo largo de su vida productiva. El Experimento 2 se basó en 15 ovejas hembras adultas (> 4 años) obtenidas de rebaños comerciales, infectadas de forma natural por SRLV y con signos clínicos de artritis bilateral. De manera similar al Experimento 1, los animales fueron dividieron en 3 grupos de tratamiento (Vaccine, Adjuvant-only y Control). Seis animales fueron excluidos tras el período de cuarentena por razones de bienestar animal y el número final de animales en cada grupo fue: grupo Vaccine (n=4), grupo de Adjuvant-only (n=3) y grupo Control (n=2). El experimento duró 75 días a lo largo de los cuales los animales recibieron un total de ocho inoculaciones subcutáneas.Para complementar los resultados moleculares y serológicos del Experimento 2, un grupo adicional (grupo Vaccine-extra; n=2) fue inoculado con 4 dosis de vacunas comerciales a lo largo de 40 días.El Capítulo 1 emplea animales de los Rebaños 1-4 del Experimento 1 y proporciona un análisis de los índices productivos y parámetros clinicopatológicos asociados a inoculación repetida de oxihidróxido de Al en corderos, ya sea solo o como parte de una vacuna. Se observaron ligeras diferencias en la ganancia media diaria y el índice de engrasamiento, lo que indica una reducción en la tasa de crecimiento en los animales vacunados, probablemente relacionado con breves episodios de pirexia y anorexia en los días posteriores a la inoculación. Los parámetros clínicos y hematológicos permanecieron dentro de los rangos normales. La histología no mostró diferencias significativas entre los grupos tratados, aunque se observó una tendencia a presentar una mayor frecuencia de neuronas hipercromáticas (i.e., dark neurons) en la médula espinal lumbar en el grupo Adjuvant-only. Aunque el oxihidróxido de Al se ha asociado con la aparición granulomas en el sitio de inyección y con cambios etológicos en ovejas, los resultados del Capítulo 1 indican que el oxihidróxido de Al inyectado por vía subcutánea no es suficiente para reproducir completamente la clínica caquectizante del síndrome ASIA ovino. Otros factores como el sexo, la raza, la edad, el acervo genético, el sistema de producción, la dieta o las condiciones climáticas podrían influir en el desarrollo de este síndrome.El Capítulo 2 emplea animales del Rebaño 1 del Experimento 1 para la detección de Al en la médula espinal lumbar tras la inoculación repetida de oxihidróxido de Al, ya sea solo o como parte de una vacuna. Los depósitos de Al fueron significativamente más abundantes en la médula espinal lumbar en comparación con el lóbulo parietal en el grupo Adjuvant-only y mostraron una tendencia estadística en el grupo Vaccine. En la médula espinal lumbar, los depósitos de Al fueron más numerosos en los grupos Vaccine y Adjuvant-only en comparación con el grupo Control. En el lóbulo parietal, los depósitos de Al fueron mayores en el grupo de Vaccine en comparación con el grupo Control y el grupo Adjuvant-only. En la médula espinal lumbar, los depósitos de Al fueron más abundantes en la sustancia gris que en la sustancia blanca en los grupos Vaccine y Adjuvant-only y fundamentalmente se encontraban asociados a células de tipo glial. En el lóbulo parietal se encontraron pocos depósitos de Al, en ocasiones relacionados con vasos saguíneos. Los resultados del Capítulo 2 indican que, en ovejas, el oxihidróxido de Al inoculado en el tejido subcutáneo se acumulan selectivamente en la médula espinal lumbar.El Capítulo 3 emplea animales del Rebaño 1 del Experimento 1 y proporciona un profundo análisis de las vías de señalización inflamatoria e inmunitaria en los granulomas postvacunales, los ganglios linfáticos regionales y el bazo de corderos tras la inoculación repetida de oxihidróxido de Al, ya sea solo o como parte de una vacuna. La activación inmunológica fue más evidente en los órganos linfoides secundarios (linfonodo regional y bazo) e implicó la sobreexpresión de toll-like receptors, citoquinas pro-inflamatorias, moléculas co-estimuladoras y proteínas antivirales. Se encontraron pocas diferencias en los granulomas probablemente debido a la falta de un tejido control adecuado. Sin embargo, se observó una reducción en la expresión de interleucina 10 (IL-10) y un aumento de IL-6 que pueden indicar una activación inmune constante en los granulomas de animales vacunados. Los resultados del Capítulo 3 indican que, en ovejas, las vías de señalización pro-inflamatoria e inmunitarias se estimulan tras la inoculación de Al-oxihidróxido, siendo esta estimulación mayor cuando el Al-oxihidróxido forma parte de una vacuna.El Capítulo 4 emplea animales de los Rebaños 2 y 4 del Experimento 1 y animales del Experimento 2. Este capítulo aborda el impacto de los granulomas inducidos por oxihidróxido de Al en la respuesta de anticuerpos, la carga viral y la evolución de las lesiones de SRLV. En los Rebaños 2 y 4 del Experimento 1, el número de nuevos animales seropositivos frente a SRLV fue mayor en los grupos Vaccine y Adjuvant-only que en el grupo Control. En consecuencia, las ovejas artríticas mostraron un aumento en el título de anticuerpos a lo largo del experimento en los grupos Vaccine, Vaccine-extra y Adjuvant-only, pero no en el grupo de control. La cantidad de ADN viral en sangre aumentó ligeramente en los animales del grupo Control, mientras que los grupos Vaccine, Vaccine-extra y Adjuvant-only mostraron una marcada disminución. Además, el análisis de radiografías y termografías mostró una progresión acelerada de la artritis en los grupos Vaccine y Adjuvant-only. Estos animales presentaban granulomas en punto de inoculación en los que se demostró la presencia de SRLV en el citoplasma de los macrófagos. Estos granulomas infectados por SRLV son probablemente los responsables del aumento de los títulos de anticuerpos en los grupos Vaccine, Vaccine-extra y Adjuvant-only. Los resultados del Capítulo 4 indican que la respuesta serológica contra SRLV, así como la dinámica viral y la patología asociada pueden verse afectadas en animales inoculados con ABA, lo que subraya la importancia de los granulomas inducidos por Al en la patogenia de los virus con tropismo por macrófagos.El Capítulo 5 desarrolla una revisión sistemática y un meta-análisis de las publicaciones científicas en el campo de los SRLV durante los últimos 40 años (1981-2020), para evidenciar su prevalencia mundial en ovinos. La mayoría de los artículos utilizaron una sola prueba diagnóstica para estimar la prevalencia de SRLV, mientras que los artículos que utilizaron 3 o más pruebas fueron escasos. La técnica ELISA ha reemplazado progresivamente a AGID a lo largo de las últimas décadas. La infección por SRLV en ovejas está muy extendida en todo el mundo, y Europa es el continente con mayor prevalencia individual (40,9 %). Europa es también el área geográfica en la que se han realizado más estudios. África, Asia y América del Norte muestran valores que oscilan entre el 16,7 % y el 21,8 % a nivel individual. América del Sur y Central muestran la prevalencia individual de SRLV más baja (1,7%). Hubo una fuerte correlación positiva entre la prevalencia individual y prevalencia colectiva. Los resultados del Capítulo 5 demostraron que, a pesar de la importancia mundial de la ganadería ovina, el conocimiento sobre la prevalencia de SRLV es irregular e inconsistente. Es necesario un método gold standard de diagnóstico y una estrategia de muestreo definida entre países y continentes.Esta Tesis Doctoral proporciona un avance sustancial en la comprensión del mecanismo de acción de los ABAs y su interacción con los SRLV. El enfoque multidisciplinar que supone el análisis integrado de datos productivos, bioquímicos, histopatológicos, serológicos, moleculares y epidemiológicos permiten poner de manifiesto la importancia de los ABA, los SRLV y la interacción entre ellos. La persistencia de granulomas en el punto de inyección, la translocación de ABA al sistema nervioso central y la replicación de SRLV dentro de macrófagos cargados con Al exigen el diseño de nuevos adyuvantes vacunales que permitan el desarrollo de vacunas más seguras en ovejas.Vaccines are key elements in human and veterinary medicine and one of the greatest public and animal health achievements of the last centuries. Vaccines try to mimic the immunogenic aspects elicited by natural infections in order to provide long term protection against them. Veterinary vaccines have a positive impact on animal welfare and economics achieving a drastic reduction in the morbidity and mortality induced by spontaneous infections. Intensification in domestic animal production has drastically increased during the last decades favoring animal overcrowding and infection spread. Good management practices and appropriate vaccination strategies to prevent diseases are cornerstone of any successful health plan. In sheep husbandry, vaccination protocols differ depending on a variety of factors such as production system, geographical location, climate, and/or disease prevalence. These protocols can be further modified by compulsory vaccination campaigns to fight against emerging or re-emerging epizootics. A recent example was the compulsory vaccination campaign against bluetongue virus (BTV) that took place in most European countries during the first decade of the 21st century. The mass vaccination successfully controlled the infection in ruminants but raised several sanitary concerns in different European countries. In Spain, a wasting syndrome associated with neurological signs was reported and classified under the umbrella of the autoimmune/autoinflammatory syndrome induced by adjuvants (ASIA syndrome). Immune activation after vaccination highly depends on the type of vaccine employed. Most ovine vaccines are based on inactivated pathogens or recombinant proteins that are often poorly immunogenic as they need adjuvants to strengthen the immune response. Aluminum-based adjuvants (ABAs) have been used in human and veterinary vaccines for more than 70 years. However, precise mechanisms of action of these adjuvants are just partially elucidated. Inoculation of ABAs induce the recruitment of inflammatory cells to the injection site and evolves towards the formation of a chronic inflammatory nodule, the so-called injection-site granuloma as it is mainly composed of activated macrophages. Aggregates of Al-laden macrophages can reach and accumulate in the regional lymph node. Small ruminant lentiviruses (SRLV) cause highly prevalent chronic infections in sheep and goats worldwide. SRLV are highly heterogenic single-stranded RNA retroviruses. Indeed, four main genotypes (A, B, C and E) and more than 35 subgroups have been already characterized. SRLV have tropism for the mononuclear-phagocyte system and induce slow, chronic and persistent inflammation in four main target organs: lung, joints, nervous system and mammary gland. The occurrence of each clinical form and the severity of the lesions depend on viral factors as well as the host immune response. In the articular form, the main clinical sign is unilateral or bilateral carpal arthritis caused by genotype B2 strains in sheep. The aim of this PhD Thesis is to study the effects of the repeated inoculation of aluminum (Al) oxyhydroxide in sheep and its relationship with the SRLV pathogenesis. Experiment 1 included 84 lambs that were divided into four flocks (Flocks 1-4) of 21 animals each. In each Flock, lambs were divided into three treatment groups of 7 animals each: Vaccine group, inoculated with commercial vaccines; Adjuvant-only group, which received the equivalent dose of Al-oxyhydroxide, and Control group, injected with phosphate-buffered saline. The experiment lasted 15 months and lambs received 19 subcutaneous inoculations, mimicking the amount of Al that animals can receive during their productive lifespan. Experiment 2 was based on 15 adult (>4 year-old) female commercial sheep naturally infected by SRLV and showing bilateral arthritis. Similarly to Experiment 1, sheep were divided into 3 treatment groups (Vaccine, Adjuvant-only and Control). Six animals were excluded after the quarantine period for animal welfare reasons and the final number of animals in each group was: Vaccine group (n=4), Adjuvant-only group (n=3) and Control group (n=2). The experiment lasted 75 days and animals received a total of eight subcutaneous inoculations. To complement molecular and serologic results of Experiment 2, an additional group (Vaccine-extra group; n=2) was inoculated with 4 doses of commercial vaccines along 40 days. Chapter 1 is based of animals of Flocks 1-4 of the Experiment 1 and provides an analysis of the growth performance and clinicopathological analyses in lambs repetitively inoculated with Al-oxyhydroxide containing vaccines or Al oxyhydroxide alone. Mild differences in average daily gain and fattening index were observed, indicating a reduced growth performance in Vaccine groups likely related to short-term episodes of pyrexia and anorexia. Clinical and hematological parameters remained within normal limits. Histology showed no significant differences between groups, although there was a tendency to present higher frequency of hyperchromatic, shrunken neurons in the lumbar spinal cord in the Adjuvant-only group. Although Al-oxyhydroxide was previously linked to granulomas at the injection site and with the production of ethological changes in sheep, results of Chapter 1 indicate that injected Al oxyhydroxide is not enough to fully reproduce the wasting presentation of the ovine ASIA syndrome. Other factors such as sex, breed, age, production system, genetic background, diet or climate conditions could play a role. Chapter 2 is based on animals of Flock 1 of the Experiment 1 and report the detection of Al in lumbar spinal cord of lambs repetitively inoculated with Al oxyhydroxide containing vaccines or Al-oxyhydroxide alone. Deposits were significantly more abundant in the lumbar spinal cord than in the parietal lobe in the Adjuvant-only group and they showed a marked statistic trend in the Vaccine group. In the lumbar spinal cord, Al deposits were higher in both the Vaccine and Adjuvant-only groups compared with the Control group. In the parietal lobe, Al deposits were higher in the Vaccine group compared with Control group and Adjuvant-only group. In the lumbar spinal cord, lumogallion reactive Al deposits were more abundant in the gray matter than in the white matter in both Vaccine and Adjuvant-only groups and Al deposits were mostly associated with glial-like cells. In the parietal lobe, few Al deposits, which were sometimes related to vessels, were found. Results of Chapter 2 indicate that in sheep, Al-oxyhydroxide adjuvants inoculated in the subcutaneous tissue selectively accumulates in the lumbar spinal cord. Chapter 3 is based on animals of Flock 1 of the Experiment 1 and provides a deep analysis of the the inflammatory and immune signaling pathways at injection-site granulomas, regional lymph node and spleen of lambs repetitively inoculated with Al-oxyhydroxide containing vaccines or Al-oxyhydroxide alone. Immunological activation was more evident in secondary lymphoid organs (i.e., lymph node and spleen) and involved the overexpression of toll-like receptors, pro inflammatory cytokines, costimulatory molecules and antiviral proteins. Few differences were found in granulomas likely due to the unavailability of proper control tissue. However, reduction in IL-10 expression and the increase of IL-6 may indicate a constant immune activation in granulomas vaccinated animals. Results of Chapter 3 indicate that in sheep, pro-inflammatory and immune signaling pathways are upregulated after the inoculation of Al-oxyhydroxide, being this upregulation higher when Al oxyhydroxide is part of a vaccine. Chapter 4 is based on animals of Flocks 2 and 4 of the Experiment 1 and animals of the Experiment 2. This chapter addresses the impact of Al-induced granulomas on serological response and virus kinetics in SRLV infected animals. In Flock 2 and 4 of Experiment 1, new SRLV-seropositive animals were more abundant in lambs from Vaccine and Adjuvant-only groups compared to Control group. Accordingly, arthritic sheep reported an increase in antibody titers along the experiment in Vaccine, Vaccine-extra and Adjuvant-only groups but not in Control group. SRLV viral DNA copies in blood slightly increased in Control animals, whereas Vaccine, Vaccine-extra and Adjuvant-only groups showed a marked decrease. Moreover, radiographic and thermographic analyses showed an accelerated, significant progression of articular lesions in Vaccine and Adjuvant-only groups. These animals presented injection-site granulomas that evinced granular, intracytoplasmic SRLV immunolabelling in macrophages. These SRLV containing granulomas are likely responsible of the increased antibody titers in Vaccine, Vaccine-extra and Adjuvant-only groups along the experiment. Results of Chapter 4 indicate that serological response against SRLV as well as virological and pathological features may be affected in animals inoculated with ABAs, underscoring the importance of Al-induced granulomas in the pathogenesis of macrophage-tropic viruses. Chapter 5 develops a systematic review and meta-analysis of the scientific publications of the last 40 years (1981-2020) in the SRLV field owing to address worldwide prevalence in sheep. Most articles used a single diagnostic test to estimate SRLV prevalenc

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

[EN] Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validationSIThis research was funded by Spanish Ministry of Science, Innovation, and Universities, grant number RTI2018-096172-B-C31. The APC was funded by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). I.E. was funded by Universidad Pública de Navarra. Hugo Ramírez was financially supported by the “Programa de apoyos para la superación del personal académico (PASPA)” scholarship from DGAPA-UNA

Characterization of Ovine A3Z1 Restriction Properties against Small Ruminant Lentiviruses (SRLVs)

Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motifWe sincerely acknowledge Sandra Hervás-Stubbs from CIMA for her fruitful help. We also acknowledge Greg Towers, University College London for plasmids and protocols. Funded by CICYT (AGL2010-22341-C04-01) and Navarra’s Government (IIQ010449.RI1, IIQ14064.RI1 and PI042-LENTIMOL). Ramsés Reina was supported by the Spanish Ministry of Science and Innovation “Ramón y Cajal” contract. We acknowledge support of the publication fee by the Public University of Navarra and CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Small ruminant macrophage polarization may play a pivotal role on lentiviral infection

Small ruminant lentiviruses (SRLV) infect the monocyte/macrophage lineage inducing a long-lasting infection affecting body condition, production and welfare of sheep and goats all over the world. Macrophages play a pivotal role on the host's innate and adaptative immune responses against parasites by becoming differentially activated. Macrophage heterogeneity can tentatively be classified into classically differentiated macrophages (M1) through stimulation with IFN-gamma displaying an inflammatory profile, or can be alternatively differentiated by stimulation with IL-4/IL-13 into M2 macrophages with homeostatic functions. Since infection by SRLV can modulate macrophage functions we explored here whether ovine and caprine macrophages can be segregated into M1 and M2 populations and whether this differential polarization represents differential susceptibility to SRLV infection. We found that like in human and mouse systems, ovine and caprine macrophages can be differentiated with particular stimuli into M1/M2 subpopulations displaying specific markers. In addition, small ruminant macrophages are plastic since M1 differentiated macrophages can express M2 markers when the stimulus changes from IFN-gamma to IL-4. SRLV replication was restricted in M1 macrophages and increased in M2 differentiated macrophages respectively according to viral production. Identification of the infection pathways in macrophage populations may provide new targets for eliciting appropriate immune responses against SRLV infection.This work was funded by grants from CICYT (no. AGL2010-22341-C04-01), and the Government of Navarra (no. IIQ14064.RI1). We acknowledge the Public University of Navarra and CSIC for fellowships and the JAE-contract (HC and RR)

Diagnosing infection with small ruminant lentiviruses of genotypes A and B by combining synthetic peptides in ELISA

The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status.The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping.Funded by CICYT (AGL2010-22341-C04-01 and AGL2013-49137-C3-1R) and Navarra's Government (IIQ010449.RI1 and IIQ14064.RI1). L. Sanjosé was a FPI-fellow of the Spanish MINECO and R. Reina had a contract of the Public University of Navarra.Peer Reviewe

Identification of the ovine mannose receptor and its possible role in Visna/Maedi virus infection

This study aims to characterize the mannose receptor (MR) gene in sheep and its role in ovine visna/maedi virus (VMV) infection. The deduced amino acid sequence of ovine MR was compatible with a transmembrane protein having a cysteine-rich ricin-type amino-terminal region, a fibronectin type II repeat, eight tandem C-type lectin carbohydrate-recognition domains (CRD), a transmembrane region, and a cytoplasmic carboxy-terminal tail. The ovine and bovine MR sequences were closer to each other compared to human or swine MR. Concanavalin A (ConA) inhibited VMV productive infection, which was restored by mannan totally in ovine skin fibroblasts (OSF) and partially in blood monocyte-derived macrophages (BMDM), suggesting the involvement of mannosylated residues of the VMV ENV protein in the process. ConA impaired also syncytium formation in OSF transfected with an ENV-encoding pN3-plasmid. MR transcripts were found in two common SRLV targets, BMDM and synovial membrane (GSM) cells, but not in OSF. Viral infection of BMDM and especially GSM cells was inhibited by mannan, strongly suggesting that in these cells the MR is an important route of infection involving VMV Env mannosylated residues. Thus, at least three patterns of viral entry into SRLV-target cells can be proposed, involving mainly MR in GSM cells (target in SRLV-induced arthritis), MR in addition to an alternative route in BMDM (target in SRLV infections), and an alternative route excluding MR in OSF (target in cell culture). Different routes of SRLV infection may thus coexist related to the involvement of MR differential expression

Molecular Signature of Aluminum Hydroxide Adjuvant in Ovine PBMCs by Integrated mRNA and microRNA Transcriptome Sequencing

There have been few in vivo studies on the effect of aluminum hydroxide adjuvant and its influence on the immune response to vaccination. In this study, lambs received a parallel subcutaneous treatment with either commercial vaccines containing aluminum hydroxide or an equivalent dose of this compound only with the aim of identifying the activated molecular signature. Blood samples were taken from each animal at the beginning and at the end of the experiment and PBMCs isolated. Total RNA and miRNA libraries were prepared and sequenced. After alignment to the Oar3.1 reference genome and differential expression with 3 programs, gene enrichment modeling was performed. For miRNAs, miRBase and RNAcentral databases were used for detection and characterization. Three expression comparisons were made: vaccinated animals at the beginning and at the end of the treatment, adjuvanted animals at the same times, and animals of both treatments at the end of the experiment. After exposure to both treatments, a total of 2,473; 2,980 and 429 differentially expressed genes were identified in vaccinated animals, adjuvanted animals and animals at the end of both treatments, respectively. In both adjuvant and vaccine treated animals the NF-kappa B signaling pathway was enriched. On the other hand, it can be observed a downregulation of cytokines and cytokine receptors in the adjuvanted group compared to the vaccinated group at the final time, suggesting a milder induction of the immune response when the adjuvant is alone. As for the miRNA analysis, 95 miRNAs were detected: 64 previously annotated in Ovis aries, 11 annotated in Bos taurus and 20 newly described. Interestingly, 6 miRNAs were differentially expressed in adjuvant treated animals, and 3 and 1 in the other two comparisons. Lastly, an integrated miRNA-mRNA expression profile was developed, in which a miRNA-mediated regulation of genes related to DNA damage stimulus was observed. In brief, it seems that aluminum-containing adjuvants are not simple delivery vehicles for antigens, but also induce endogenous danger signals that can stimulate the immune system. Whether this contributes to long-lasting immune activation or to the overstimulation of the immune system remains to be elucidated.This work was supported by a MINECO project grant (AGL2013-49137-C3-3-R to BJ and AGL2013-49137-C3-2-R to LL and AGL2013-49137-C3-1 to DdA), a predoctoral fellowship from the UPV/EHU to EV-M (PIF15/361) and a postdoctoral fellowship from the UPV/EHU to NA (ESPDOC16/43)