RESUMEN
Tetanus toxin (TeNT) is one of the most toxic proteins. Neutralizing antibodies against TeNT are effective in prevention and treatment. In this study, 14 anti-tetanus nanobodies were obtained from a phage display nanobody library by immunizing a camel with the C-terminal receptor-binding domain of TeNT (TeNT-Hc) as the antigen. After fusion with the human Fc fragment, 11 chimeric heavy-chain antibodies demonstrated nanomolar binding toward TeNT-Hc. The results of toxin neutralization experiments showed that T83-7, T83-8, and T83-13 completely protected mice against 20 × the median lethal dose (LD50) at a low concentration. The neutralizing potency of T83-7, T83-8, and T83-13 against TeNT is 0.4 IU/mg, 0.4 IU/mg and 0.2 IU/mg, respectively. In the prophylactic setting, we found that 5 mg/kg of T83-13 provided the mice with full protection from tetanus, even when they were injected 14 days before exposure to 20 × LD50 TeNT. T83-7 and T83-8 were less effective, being fully protective only when challenged 7 or 10 days before exposure, respectively. In the therapeutic setting, 12 h after exposure to TeNT, 1 ~ 5 mg/kg of T83-7, and T83-8 could provide complete protection for mice against 5 × LD50 TeNT, while 1 mg/kg T83-13 could provide complete protection 24 h after exposure to 5 × LD50 TeNT. Our results suggested that these antibodies represent prophylactic and therapeutic activities against TeNT in a mouse model. The T83-7, T83-8, and T83-13 could form the basis for the subsequent development of drugs to treat TeNT toxicity.
Asunto(s)
Anticuerpos Neutralizantes , Cadenas Pesadas de Inmunoglobulina , Anticuerpos de Dominio Único , Toxina Tetánica , Tétanos , Animales , Toxina Tetánica/inmunología , Tétanos/prevención & control , Tétanos/inmunología , Anticuerpos Neutralizantes/inmunología , Ratones , Anticuerpos de Dominio Único/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Femenino , Camelus/inmunología , Humanos , Anticuerpos Antibacterianos/inmunología , Ratones Endogámicos BALB CRESUMEN
Respirovirus 3 is a leading cause of severe acute respiratory infections in vulnerable human populations. Entry into host cells is facilitated by the attachment glycoprotein and the fusion glycoprotein (F). Because of its crucial role, F represents an attractive therapeutic target. Here, we identify 13 F-directed heavy-chain-only antibody fragments that neutralize recombinant respirovirus 3. High-resolution cryo-EM structures of antibody fragments bound to the prefusion conformation of F reveal three distinct, previously uncharacterized epitopes. All three antibody fragments bind quaternary epitopes on F, suggesting mechanisms for neutralization that may include stabilization of the prefusion conformation. Studies in cotton rats demonstrate the prophylactic efficacy of these antibody fragments in reducing viral load in the lungs and nasal passages. These data highlight the potential of heavy-chain-only antibody fragments as effective interventions against respirovirus 3 infection and identify neutralizing epitopes that can be targeted for therapeutic development.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Microscopía por Crioelectrón , Epítopos , Animales , Anticuerpos Neutralizantes/inmunología , Humanos , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Sigmodontinae , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/química , Femenino , Camelus/inmunología , Camelus/virologíaRESUMEN
Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were â¼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.
Asunto(s)
Anticuerpos Neutralizantes , Proteínas de la Cápside , Virus de la Hepatitis E , Hepatitis E , Anticuerpos de Dominio Único , Virus de la Hepatitis E/inmunología , Animales , Proteínas de la Cápside/inmunología , Anticuerpos de Dominio Único/inmunología , Humanos , Anticuerpos Neutralizantes/inmunología , Hepatitis E/inmunología , Camelus/inmunología , Epítopos/inmunología , Células Hep G2 , Reacciones Cruzadas/inmunología , Genotipo , Especificidad de Anticuerpos/inmunologíaRESUMEN
OBJECTIVE: To screen and obtain specific anti-lymphocyte activation gene-3 (LAG3) nanobody sequences, purify and express recombinant anti-LAG3 nanobody, and verify its effect on promoting T cells to kill tumor cells. METHODS: Based on the camel derived natural nanobody phage display library constructed by the research group, the biotinylated LAG3 antigen was used as the target, and the anti-LAG3 nanobody sequences were screened by biotin-streptavidin liquid phase screening, phage-ELISA and sequencing. The sequence-conjµgated human IgG1 Fc fragment was obtained, the recombinant anti-LAG3 nanobody expression vector was constructed, the expression of the recombinant anti-LAG3 nanobody was induced by IPTG and purified, and the characteristics and functions of the recombinant anti-LAG3 nanobody were verified by SDS-PAGE, Western blot, cytotoxicity assay, etc. RESULTS: One anti-LAG3 nanobody sequence was successfully screened, and the corresponding recombinant anti-LAG3 nanobody-expressing bacteria were constructed. The results of SDS-PAGE, Western blot and cytotoxicity assay showed that the recombinant anti-LAG3 nanobody was successfully expressed, which was specific, and it could promote the killing ability of T cells against tumor cells, and the optimal concentration was 200 µg/mL. CONCLUSION: The recombinant anti-LAG3 nanobody screened and expressed has specific and auxiliary anti-tumor cell effects, which lays a foundation for its subsequent application.
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Proteína del Gen 3 de Activación de Linfocitos , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/farmacología , Humanos , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos CD/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/química , Animales , Biblioteca de Péptidos , Camelus/inmunología , Camelus/genética , Línea Celular Tumoral , Escherichia coli/genética , Linfocitos T/inmunología , Expresión GénicaRESUMEN
Bactrian camels inhabiting desert and semi-desert regions of China are valuable animal models for studying adaptation to desert environments and heat stress. In this study, 16S rRNA technology was employed to investigate the distribution characteristics and differences of mucosal microorganisms in the anterior gland area, posterior gland area, third gland area, cardia gland area, gastric fundic gland area and pyloric gland area of 5-peak adult healthy Bactrian camels. We aimed to explore the possible reasons for the observed microbial distribution from the aspects of histological structure and mucosal immunity. Bacteroides and Fibrobacteria accounted for 59.54% and 3.22% in the gland area, respectively, and 52.37% and 1.49% in the wrinkled stomach gland area, respectively. The gland area showed higher abundance of Bacteroides and Fibrobacteria than the wrinkled stomach gland area. Additionally, the anterior gland area, posterior gland area, third gland area, and cardia gland area of Bactrian camels mainly secreted acidic mucus, while the gastric fundic gland area mainly secreted neutral mucus and the pyloric region mainly secreted a mixture of acidic and neutral mucus. The results of immunohistochemistry techniques demonstrated that the number of IgA+ cells in the anterior glandular area, posterior glandular area, third glandular area, and cardia gland area was significantly higher than that in the fundic and pyloric gland area (p < 0.05), and the difference in IgA+ between the fundic and pyloric gland area was not significant (p > 0.05). The study revealed a large number of bacteria that can digest and degrade cellulose on the mucosa of the gastric gland area of Bactrian camels. The distribution of IgA+ cells, the structure of the mucosal tissue in the glandular region, and the composition of the mucus secreted on its surface may have a crucial influence on microbial fixation and differential distribution.
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Camelus , Mucosa Gástrica , Inmunidad Mucosa , ARN Ribosómico 16S , Animales , Camelus/microbiología , Camelus/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/inmunología , Mucosa Gástrica/metabolismo , ARN Ribosómico 16S/genética , Bacterias/clasificación , Inmunoglobulina A/metabolismo , MasculinoRESUMEN
Food-borne biotoxins from microbes, plants, or animals contaminate unclean, spoiled, and rotten foods, posing significant health risks. Neutralizing such toxins is vital for human health, especially after food poisoning. Nanobodies (Nbs), a type of single-domain antibodies derived from the genetic cloning of a variable domain of heavy chain antibodies (VHHs) in camels, offer unique advantages in toxin neutralization. Their small size, high stability, and precise binding enable effective neutralization. The use of Nbs in neutralizing food-borne biotoxins offers numerous benefits, and their genetic malleability allows tailored optimization for diverse toxins. As nanotechnology continues to evolve and improve, Nbs are poised to become increasingly efficient and safer tools for toxin neutralization, playing a pivotal role in safeguarding human health and environmental safety. This review not only highlights the efficacy of these agents in neutralizing toxins but also proposes innovative solutions to address their current challenges. It lays a solid foundation for their further development in this crucial field and propels their commercial application, thereby contributing significantly to advancements in this domain.
Asunto(s)
Anticuerpos de Dominio Único , Animales , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Humanos , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Anticuerpos Neutralizantes/inmunología , Toxinas Biológicas/inmunología , Enfermedades Transmitidas por los Alimentos/prevención & control , Enfermedades Transmitidas por los Alimentos/inmunología , Camelus/inmunologíaRESUMEN
The pharyngeal tonsil, located in the nasopharynx, can effectively defend against pathogens invading the body from the upper respiratory tract and play a crucial role in mucosal immunity of the respiratory tract. Immunoglobulin A (IgA) and Immunoglobulin G (IgG) serve as key effector molecules in mucosal immunity, exhibiting multiple immune functions. This study aimed to investigate the distribution patterns and age-related alterations of IgA and IgG antibody-secreting cells (ASCs) in the pharyngeal tonsils of Bactrian camels. Twelve Alashan Bactrian camels were categorized into four age groups: young (1-2 years, n=3), pubertal (3-5 years, n=3), middle-aged (6-16 years, n=3) and old (17-20 years, n=3). The distribution patterns of IgA and IgG ASCs in the pharyngeal tonsils of Bactrian camels of different ages were meticulously observed, analyzed and compared using immunohistochemical and statistical methods. The results revealed that IgA ASCs in the pharyngeal tonsils of all age groups were primarily clustered or diffusely distributed in the reticular epithelium and its subepithelial regions (region A) and around the glands (region C), scattered in the subepithelial regions of non-reticular epithelium (region B), and sporadically distributed in the interfollicular regions (region D). Interestingly, the distribution pattern of IgG ASCs in the pharyngeal tonsils closely mirrored that of IgA ASCs. The distribution densities of IgA and IgG ASCs in these four regions were significantly decreased in turn (P<0.05). However, IgA ASCs exhibited significantly higher densities than IgG ASCs in the same region (P<0.05). Age-related alterations indicated that the distribution densities of IgA and IgG ASCs in each region of the pharyngeal tonsils exhibited a trend of initially increasing and subsequently decreasing from young to old camels, reaching a peak in the pubertal group. As camels age, there was a significant decrease in the densities of IgA and IgG ASCs in all regions of the pharyngeal tonsils (P<0.05). The results demonstrate that the reticular epithelium and its subepithelial regions in the pharyngeal tonsils of Bactrian camels are the primary regions where IgA and IgG ASCs colonize and exert their immune functions. These regions play a pivotal role in inducing immune responses and defending against pathogen invasions in the pharyngeal tonsils. IgA ASCs may be the principal effector cells of the mucosal immune response in the pharyngeal tonsils of Bactrian camels. Aging significantly reduces the densities of IgA and IgG ASCs, while leaving their distribution patterns unaffected. These findings will provide valuable insights for further investigations into the immunomorphology, immunosenescence, and response mechanisms of the pharyngeal tonsils in Bactrian camels.
Asunto(s)
Células Productoras de Anticuerpos , Camelus , Inmunoglobulina A , Inmunoglobulina G , Animales , Camelus/inmunología , Inmunoglobulina A/análisis , Células Productoras de Anticuerpos/inmunología , Envejecimiento , Factores de Edad , Masculino , Inmunidad Mucosa , Tonsila Faríngea/inmunología , Femenino , Tonsila Palatina/inmunología , Tonsila Palatina/citologíaRESUMEN
Infant formulas based on hydrolysed cow's milk proteins are used when breastfeeding is not feasible in cow's milk allergic infants. Camel milk has been shown to be well-tolerated by the majority of children with cow's milk allergy (CMA) and may be a substitute in management of CMA. Here we aimed to evaluate the impact of processing on immunogenicity, sensitising, antibody-binding and cross-reactive capacity of cow's and camel milk. Cow's and camel milk were processed by means of enzyme hydrolysis or heat treatment. Brown Norway rats were immunised with PBS, non-processed, enzyme hydrolysed or heat-treated cow's or camel milk. In vivo tests were performed for evaluation of clinical signs. Blood and faecal samples were analysed for levels and specificity of antibody responses. Cow's and camel milk showed similar sensitising capacity. Processing decreased the sensitising capacity of cow's milk, yet only enzyme hydrolysis but not heat treatment decreased the sensitising capacity of camel milk. Processing affected the specificity of antibodies raised in the rats, though the effect differed between cow's and camel milk. The study showed a low cross-reactivity between cow's and camel milk, which was decreased with processing, suggesting that processing of camel milk may improve its usefulness in CMA management.
Asunto(s)
Camelus , Reacciones Cruzadas , Hipersensibilidad a la Leche , Proteínas de la Leche , Leche , Animales , Camelus/inmunología , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/prevención & control , Ratas , Bovinos , Leche/química , Leche/inmunología , Proteínas de la Leche/inmunología , Femenino , Ratas Endogámicas BN , Manipulación de Alimentos/métodos , MasculinoRESUMEN
Milk is a whitish liquid that is secreted from mammary glands; and considered as the primary source of nutrition for newborns since they are not able to digest solid food. However, it contains primary nutrients, as well as growth and immune factors. Early weaning is a critical issue that face women and their babies in developing countries. To avoid infant malnutrition, they tend to use other milk types instead of baby formula. Therefore, the present study aimed to evaluate the impact of cow, buffalo, goat or camel milk consumption on oxidative stress, inflammation and immune response in male and female Sprague Dawley rats post weaning time. The amino acids, fatty acids, minerals and vitamins in the tested milk types were evaluated. Animals were divided into 5 groups (control, cow, buffalo, goat and camel milk administrated groups) (10 rats/group); each animal was administrated by 3.4 ml/day. Rats were administered with milk for 6 weeks; at the end of the 5th week, five animals of each group were isolated and the remaining five animals were immunized with sheep red blood cells (SRBCs) and kept for another week to mount immune response. The effect of different milk types on rats' immune response towards SRBCs was evaluated through pro-inflammatory cytokines, antioxidants, ESR and CRP measurement; together, with the histopathological examination of spleen samples and hemagglutination assay. Camel milk consumption reduced oxidative stress and inflammation in spleen that resulted from SRBCs immunization; in addition to, B cell stimulation that was apparent from the high level of anti-SRBCs antibodies. Camel milk is recommended for newborn consumption, due to its high-water content, unsaturated fatty acids, and vitamin C, as well as low lactose and fat content.
Asunto(s)
Búfalos , Camelus , Cabras , Inflamación , Leche , Estrés Oxidativo , Ratas Sprague-Dawley , Destete , Animales , Leche/inmunología , Camelus/inmunología , Búfalos/inmunología , Cabras/inmunología , Femenino , Inflamación/inmunología , Ratas , Masculino , BovinosRESUMEN
We developed an ELISPOT assay for evaluating Middle East respiratory syndrome coronavirus (MERS-CoV)âspecific T-cell responses in dromedary camels. After single modified vaccinia virus Ankara-MERS-S vaccination, seropositive camels showed increased levels of MERS-CoVâspecific T cells and antibodies, indicating suitability of camel vaccinations in disease-endemic areas as a promising approach to control infection.
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Camelus , Infecciones por Coronavirus , Linfocitos T , Vacunas Virales , Animales , Camelus/inmunología , Linfocitos T/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Vacunas Virales/inmunología , Vacunación/veterinaria , Ensayo de Immunospot Ligado a Enzimas , Anticuerpos AntiviralesRESUMEN
Bactrian camels have specific mucosa-associated lymphoid tissue (MALT) throughout the large intestine, with species-unique cystic Peyer's patches (PPS) as the main type of tissue. However, detailed information about the molecular characteristics of PPS remains unclear. This study applied a transcriptomic analysis, untargeted metabolomics, and 16S rDNA sequencing to compare the significant differences between PPS and the adjacent normal intestine tissues (NPPS) during the healthy stage of three young Bactrian camels. The results showed that samples from PPS could be easily differentiated from the NPPS samples based on gene expression profile, metabolites, and microbial composition, separately indicated using dimension reduction methods. A total of 7,568 up-regulated and 1,266 down-regulated differentially expressed genes (DEGs) were detected, and an enrichment analysis found 994 DEGs that participated in immune-related functions, and a co-occurance network analysis identified nine hub genes (BTK, P2RX7, Pax5, DSG1, PTPN2, DOCK11, TBX21, IL10, and HLA-DOB) during multiple immunologic processes. Further, PPS and NPPS both had a similar pattern of most compounds among all profiles of metabolites, and only 113 differentially expressed metabolites (DEMs) were identified, with 101 of these being down-regulated. Deoxycholic acid (DCA; VIP = 37.96, log2FC = -2.97, P = 0), cholic acid (CA; VIP = 13.10, log2FC = -2.10, P = 0.01), and lithocholic acid (LCA; VIP = 12.94, log2FC = -1.63, P = 0.01) were the highest contributors to the significant dissimilarities between groups. PPS had significantly lower species richness (Chao1), while Firmicutes (35.92% ± 19.39%), Bacteroidetes (31.73% ± 6.24%), and Proteobacteria (13.96% ± 16.21%) were the main phyla across all samples. The LEfSe analysis showed that Lysinibacillus, Rikenellaceae_RC9_gut_group, Candidatus_Stoquefichus, Mailhella, Alistipes, and Ruminococcaceae_UCG_005 were biomarkers of the NPPS group, while Escherichia_Shigella, Synergistes, Pyramidobacter, Odoribacter, Methanobrevibacter, Cloacibacillus, Fusobacterium, and Parabacteroides were significantly higher in the PPS group. In the Procrustes analysis, the transcriptome changes between groups showed no significant correlations with metabolites or microbial communities, whereas the alteration of metabolites significantly correlated with the alteration of the microbial community. In the co-occurrence network, seven DEMs (M403T65-neg, M329T119-neg, M309T38-neg, M277T42-2-neg, M473T27-neg, M747T38-1-pos, and M482t187-pos) and 14 genera (e.g., Akkermansia, Candidatus-Stoquefichus, Caproiciproducens, and Erysipelatoclostridium) clustered much more tightly, suggesting dense interactions. The results of this study provide new insights into the understanding of the immune microenvironment of the cystic PPS in the cecum of Bactrian camels.
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Camelus , Ganglios Linfáticos Agregados , Animales , Bacterias , Camelus/inmunología , Camelus/microbiología , Ciego/inmunología , Intestino Grueso/inmunología , Ganglios Linfáticos Agregados/inmunología , MultiómicaRESUMEN
The last two decades have seen the emergence of three highly pathogenic coronaviruses with zoonotic origins, which prompted immediate attention to the underlying cause and prevention of future outbreaks. Intensification of camel husbandry in the Middle East has resulted in increased human-camel interactions, which has led to the spread of potentially zoonotic viruses with human spillover risks like MERS-coronavirus, camelpox virus, etc. Type-I interferons function as the first line of defense against invading viruses and are pivotal for limiting viral replication and immune-mediated pathologies. Seven novel dromedary camel interferon delta genes were identified and cloned. Functional characterization of this novel class of IFNs from the mammalian suborder tylopoda is reported for the first time. The camel interferon-delta proteins resemble the reported mammalian counterparts in sequence similarity, conservation of cysteines, and phylogenetic proximity. Prokaryotically expressed recombinant camel interferon-δ1 induced IFN-stimulated gene expression and also exerted antiviral action against camelpox virus, an endemic zoonotic virus. The pre-treatment of camel kidney cells with recombinant camel IFN-δ1 increased cell survival and reduced camelpox virus in a dose-dependent manner. The identification of novel IFNs from species with zoonotic spillover risk such as camels, and evaluating their antiviral effects in-vitro will play a key role in improving immunotherapies against viruses and expanding the arsenal to combat emerging zoonotic pathogens.
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Camelus , Interferón Tipo I , Animales , Camelus/genética , Camelus/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , FilogeniaRESUMEN
Middle East respiratory syndrome coronavirus (MERS-CoV) is an emergent coronavirus that has caused frequent zoonotic events through camel-to-human spillover. An effective camelid vaccination strategy is probably the best way to reduce human exposure risk. Here, we constructed and evaluated an inactivated rabies virus-vectored MERS-CoV vaccine in mice, camels, and alpacas. Potent antigen-specific antibody and CD8+ T-cell responses were generated in mice; moreover, the vaccination reduced viral replication and accelerated virus clearance in MERS-CoV-infected mice. Besides, protective antibody responses against both MERS-CoV and rabies virus were induced in camels and alpacas. Satisfyingly, the immune sera showed broad cross-neutralizing activity against the three main MERS-CoV clades. For further characterization of the antibody response induced in camelids, MERS-CoV-specific variable domains of heavy-chain-only antibody (VHHs) were isolated from immunized alpacas and showed potent prophylactic and therapeutic efficacies in the Ad5-hDPP4-transduced mouse model. These results highlight the inactivated rabies virus-vectored MERS-CoV vaccine as a promising camelid candidate vaccine.
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Camélidos del Nuevo Mundo/virología , Camelus/virología , Infecciones por Coronavirus/veterinaria , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos/inmunología , Camélidos del Nuevo Mundo/inmunología , Camelus/inmunología , Línea Celular Tumoral , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Cricetinae , Femenino , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Virus de la Rabia/genética , Virus de la Rabia/inmunología , Vacunación , Vacunas Sintéticas/inmunología , Células Vero , Vacunas Virales/genéticaRESUMEN
Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.
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Blastocisto/fisiología , Camelus/inmunología , Camelus/fisiología , Técnicas de Cultivo de Embriones/métodos , Transferencia de Embrión , Técnicas de Transferencia Nuclear , Ultrasonografía/métodos , Animales , Clonación de Organismos/métodos , Embrión de Mamíferos , Desarrollo Embrionario , Femenino , Oocitos/citología , Embarazo , Índice de Embarazo , ReproducciónRESUMEN
BACKGROUND: Recent studies have reported pathogen-species-specific modulating effects on the innate immune system. Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae are important pathogenic bacteria responsible for different infectious diseases in several animal species. In the present study, a whole blood culture with S. aureus, E. coli, or S. agalactiae and flow cytometry were used to investigate, whether stimulation with different bacterial species induces different immunomodulation patterns in camel leukocytes. The expression of different cell surface myeloid markers and cell adhesion molecules on monocytes and neutrophils was investigated. In addition, the capacity of monocytes and neutrophils to produce reactive oxygen species (ROS) was analyzed. RESULTS: Stimulation with either of the bacterial species resulted in the expansion of the camel CD14highMHCIIhigh monocyte subset with a reduced fraction of CD14highMHCIIlow monocytes. For the CD14lowMHCIIhigh monocytes, however, only stimulation with S. aureus or S. agalactiae increased their fractions in blood. Although all bacterial species elicited the upregulation of cell surface MHC class II molecules on granulocytes, the increase was, however, highest on cells stimulated with S. aureus. The expression levels of the two adhesion molecules, CD11a and CD18, on neutrophils and monocytes were differently affected by bacterial stimulation. Functionally, E. coli failed to stimulate ROS production in monocytes, while induced a strong ROS production response in granulocytes. S. agalactiae elicited a week ROS production in granulocytes when compared to the other two pathogens. CONCLUSIONS: The different responsiveness of monocytes and granulocytes toward different bacterial species indicates different host-pathogen interaction mechanisms for the two cell populations. In addition, the phenotypic and functional differences between cells stimulated with E. coli, S. aureus, or S. agalactiae suggests pathogen-species-specific modulating effects of the bacterial pathogens on the camel innate myeloid cells.
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Camelus/sangre , Inmunomodulación , Leucocitos/inmunología , Animales , Camelus/inmunología , Camelus/microbiología , Escherichia coli/fisiología , Interacciones Huésped-Patógeno , Masculino , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/fisiología , Streptococcus agalactiae/fisiologíaRESUMEN
The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.
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Inmunidad Adaptativa/genética , Camelus/virología , Enfermedades Transmisibles Emergentes/inmunología , Infecciones por Coronavirus/inmunología , Inmunidad Innata/genética , Zoonosis/inmunología , Animales , Anticuerpos Antivirales , Bronquios/citología , Bronquios/fisiología , COVID-19/genética , COVID-19/inmunología , COVID-19/virología , Camelus/genética , Camelus/inmunología , Cilios/fisiología , Enfermedades Transmisibles Emergentes/genética , Enfermedades Transmisibles Emergentes/transmisión , Enfermedades Transmisibles Emergentes/virología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/transmisión , Infecciones por Coronavirus/virología , Reservorios de Enfermedades/virología , Femenino , Predisposición Genética a la Enfermedad , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Humanos , Masculino , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/aislamiento & purificación , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Emiratos Árabes Unidos , Replicación Viral/genética , Replicación Viral/inmunología , Zoonosis/genética , Zoonosis/transmisión , Zoonosis/virologíaRESUMEN
The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.
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Anticuerpos Neutralizantes/inmunología , Camelus/inmunología , Anticuerpos de Dominio Único/inmunología , Tenascina/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/farmacología , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Colitis Ulcerosa/inmunología , Colon/inmunología , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/secundario , Unión Proteica , Anticuerpos de Dominio Único/farmacología , Tenascina/administración & dosificación , Tenascina/inmunologíaRESUMEN
BACKGROUND: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. RESULTS: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect antiPCV2 antibodies. The cutoff value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. CONCLUSIONS: In brief, the newly developed cELISA based PCV2-Nb15HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.
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Anticuerpos Antivirales/inmunología , Infecciones por Circoviridae/veterinaria , Circovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de los Porcinos/inmunología , Animales , Anticuerpos Antivirales/sangre , Camelus/inmunología , Infecciones por Circoviridae/sangre , Infecciones por Circoviridae/diagnóstico , Infecciones por Circoviridae/inmunología , Circovirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/economía , Peroxidasa de Rábano Silvestre/inmunología , Inmunización , Masculino , Proteínas Recombinantes de Fusión/inmunología , Sensibilidad y Especificidad , Anticuerpos de Dominio Único/inmunología , Porcinos/sangre , Porcinos/inmunología , Porcinos/virología , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/virología , Factores de TiempoRESUMEN
The development of prophylactic and therapeutic agents for coronavirus disease 2019 (COVID-19) is a current global health priority. Here, we investigated the presence of cross-neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in dromedary camels that were Middle East respiratory syndrome coronavirus (MERS-CoV) seropositive but MERS-CoV free. The tested 229 dromedaries had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins, including the S trimer and M, N, and E proteins. Using SARS-CoV-2 competitive immunofluorescence immunoassays and pseudovirus neutralization assays, we found medium-to-high titers of cross-neutralizing antibodies against SARS-CoV-2 in these animals. Through linear B cell epitope mapping using phage immunoprecipitation sequencing and a SARS-CoV-2 peptide/proteome microarray, we identified a large repertoire of Betacoronavirus cross-reactive antibody specificities in these dromedaries and demonstrated that the SARS-CoV-2-specific VHH antibody repertoire is qualitatively diverse. This analysis revealed not only several SARS-CoV-2 epitopes that are highly immunogenic in humans, including a neutralizing epitope, but also epitopes exclusively targeted by camel antibodies. The identified SARS-CoV-2 cross-neutralizing camel antibodies are not proposed as a potential treatment for COVID-19. Rather, their presence in nonimmunized camels supports the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.
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Anticuerpos Neutralizantes/inmunología , Camelus/inmunología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/farmacología , Animales , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , COVID-19/inmunología , Camelus/virología , Reacciones Cruzadas , Epítopos , Femenino , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunologíaRESUMEN
Tumor necrosis factor (TNF-α) is an important clinically tested cytokine that could induce autoimmune diseases and inflammation. Therefore, the anti-TNF-α therapy strategy was developed and used therapeutically in various diseases, especially in the cytokine storm associated chimeric antigen receptor (CAR) T-cell therapy and antiviral therapy. Compare with other anti-TNF-α inhibitors, anti-TNF-α Nb (nanobody) has many unique advantages. Herein, we reported a novel humanized scaffold for library construction, which could be soluble and expressed in Escherichia coli (E.coli), and the efficiency capacity could reach as high as 2.01 × 109. Meanwhile, an anti-TNF-α Nb was selected for further study after 4 rounds of screening, NT-3, as the optimal Nb could effectively inhibit TNF-mediated cytotoxicity. The IC50 of NT-3 was determined as 0.804 µM, and its apoptosis inhibition rate was 62.47 % in L929 cells. Furthermore, the molecular docking results showed that complementarity-determining regions (CDRs) of NT-3 could connect to TNF for blocking function through strong hydrogen bonds and salt bridges. In general, our study not only provided a good Nb screening platform in vitro without animal immunization, but also generated a series of novel humanized anti-TNF-α Nb candidates with potential applications.