Production Of Specific Antibodies Research Articles

Production of Specific Polyclonal Antibody against the Larvae of Corallivorous Gastropod Drupella fragum for Prediction of Outbreaks

The term corallivorous gastropod refers to a group of snails that feeds on coral and inhabits coral communities worldwide. Outbreaks of these species cause serious damage to coral communities. There are various reasons behind the outbreaks; however, further clarifications are needed. It may be possible to predict outbreaks by measuring the number of floating larvae of corallivorous gastropods in seawater. Drupella fragum is the most damaging species in Japan, so we produced antibodies against D. fragum larvae in order to easily detect this species in the field. Antibody specificity analysis in aquarium-hatched corallivorous gastropods showed a higher specificity against D. fragum compared to D. cornus. A field study using the antibody showed that many D. fragum larvae were detected from June to November at all stations. The larvae at the Shirigai station were collected in June and July in large numbers compared to the other stations. Large groups of D. fragum were collected around the sampling point in Shirigai in September 2016. Our results imply that there is a possibility that outbreaks could be predicted using this antibody.

Generation and characterisation of a semi-synthetic siderophore-immunogen conjugate and a derivative recombinant triacetylfusarinine C–specific monoclonal antibody with fungal diagnostic application

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.

Two allergens from Scylla paramamosain share common epitopes showed different allergenic potential in Balb/c mice

Filamin C (FLN c) and triosephosphate isomerase (TIM) are novel allergens of crab (Scylla paramamosain) which are sharing common epitopes. This work aimed to assess their contributions to the induction and elicitation of allergenic responses. Balb/c mice were sensitized by intraperitoneal injections and challenged by intragastric gavage with purified proteins. Upon oral challenge, FLN c triggered more severe anaphylactic symptoms, higher levels of specific antibodies and histamine in serum than TIM, while TIM was a more active promotor of early specific antibody production and stimulated stronger Th2-biased responses. Combined with the results of in vitro assays, the data demonstrated that though with common epitopes, the two allergens showed a different allergenicity, TIM favored Th2 polarization in sensitization stage, while FLN c had a better ability to stimulate B cells and is highly immunogenic in oral challenge stage. The findings can help with the better understanding of allergenicity of crab allergens.

Correlation between the natural infection by Corynebacterium pseudotuberculosis in goats and serum progesterone levels and corpus luteum development

Caseous Lymphadenitis (CLA) is an infectious disease that affects small ruminants, and the bacterium Corynebacterium pseudotuberculosis is its etiologic agent. This disease presents a high morbidity and a great economic impact on goat farming, leading to reduced milk and meat production and reproductive losses. The available data about the influence of C. pseudotuberculosis on the reproductive system were obtained after experimental inoculations. In this way, this study aimed to evaluate the influence of the natural infection by C. pseudotuberculosis on the luteal function and serum progesterone (P4) levels in goats. Sixteen female goats were diagnosed for the presence of C. pseudotuberculosis specific antibodies and divided into 2 groups: CLA positive and negative animals. They were submitted to estrous synchronization, followed by controlled mating. Corpus luteum and serum levels of P4 were evaluated on the 7th and 20th days after mating (D7 and D20). The ultrasonographic results were not statistically different between the 2 groups, as well as serum P4 levels on D7. However, a significant increase in serum P4 levels on the CLA positive group was identified on D20, along with a significant correlation between C. pseudotuberculosis specific antibody production, and P4 serum levels. The result of this study indicates that the infection by C. pseudotuberculosis may influence the reproductive status of female goats through an enhanced production of progesterone.

A multi‐epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E.ictaluri immunogenic proteins previously reported. The 49.32kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E.ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E.ictaluri infections.

Production and persistence of specific antibodies in COVID-19 patients with hematologic malignancies: role of rituximab

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.

Immunogenicity of engineered Lactobacillus plantarum expressing porcine epidemic diarrhea virus S1 gene

To investigate whether the engineered Lactobacillus plantarum expressing the porcine epidemic diarrhea virus (PEDV) S1 gene can protect animals against PEDV, guinea pigs were fed with recombinant L. plantarum containing plasmid PVE5523-S1, with a dose of 2×10⁸ CFU/piece, three times a day, at 14 days intervals. Guinea pigs fed with wild type L. plantarum and the engineered L. plantarum containing empty plasmid pVE5523 were used as negative controls. For positive control, another group of guinea pigs were injected with live vaccine for porcine epidemic diarrhea and porcine infectious gastroenteritis (HB08+ZJ08) by intramuscular injection, with a dose of 0.2 mL/piece, three times a day, at 14 days intervals. Blood samples were collected from the hearts of the four groups of guinea pigs at 0 d, 7 d, 14 d, 24 d, 31 d, 41 d and 48 d, respectively, and serum samples were isolated for antibody detection and neutralization test analysis by enzyme-linked immunosorbent assay (ELISA). The spleens of guinea pigs were also aseptically collected to perform spleen cells proliferation assay. The results showed that the engineered bacteria could stimulate the production of secretory antibody sIgA and specific neutralizing antibody, and stimulate the increase of IL-4 and IFN-γ, as well as the proliferation of spleen cells. These results indicated that the engineered L. plantarum containing PEDV S1 induced specific immunity toward PEDV in guinea pigs, which laid a foundation for subsequent oral vaccine development.

New ways of specific therapy for tuberculosis of skin and mucous membranes. Wісhmаnn (Arch. f. Dermal, u. Syph., 1922, Bd. 139)

Proceeding from the assumption that the lymph glands of our body serve as a place of production and accumulation of specific antibodies at tuberculosis infection, the author suggests using for therapeutic purposes injections of extracts or glands from the same patient or extracted from another. He considers the most suitable glands in the stage of swelling and edema; he cautions against using extracts from softening and lumpy glands.

Influence of SARS-CoV-2 vaccination on the epidemiological and clinical characteristics of imported COVID-19 cases in Chengdu

Objective: To analyze the epidemiological and clinical characteristics of imported COVID-19 cases after SARS-CoV-2 vaccination and to provide evidence for the prevention and control of COVID-19. Methods: The imported COVID-19 cases in Chengdu as of April 15, 2021 were divided into the vaccinated group and unvaccinated group according to the history of SARS-CoV-2 vaccination. The epidemiological and clinical data of the cases were collected retrospectively, and the differences in epidemiological and clinical characteristics of the two groups were compared. Laboratory tests consisted of nucleic acid test, clinical index test, serum antibody test and lymphocyte test. Software WPS2019 was used for data management and software R 4.0.3 was used for statistical analysis. Results: A total of 75 COVID-19 cases were included in the analysis, in which 20 had received SARS-CoV-2 vaccination and only 4 with clinical symptoms, 55 patients did not receive SARS-CoV-2 vaccination, and 16 had clinical symptoms. In vaccinated group, the first injection time of vaccination ranged from July to November 2020, and 10 cases received two doses of vaccine simultaneously and 10 cases received two doses of vaccine at intervals of 14-57 days. The intervals between the completion of vaccination and the onset ranged from 87 days to 224 days. The differences in classification and clinical type between the two groups were significant. Significant differences were observed in case classification and clinical type between vaccinated group and unvaccinated group (P<0.05). The vaccinated group had a relatively high proportion of asymptomatic infections (40.00%, 8/20), while mild infections were mainly observed in the unvaccinated group(76.36%,42/55). The differences in Ct values (ORF1ab gene and N gene) at the diagnosis were not significant between vaccinated group and unvaccinated group (P>0.05), similar results were also observed in lymphocyte subtypes, procalcitonin and C-reactive protein level comparisons. Serum amyloid A level was higher in unvaccinated group than in vaccinated group (P<0.05). However, the SARS-CoV-2 related serum antibody of IgM, IgG and total antibody levels were significantly higher in vaccinated group (P<0.05). Conclusions: Risk of infection still exists with SARS-CoV-2 after vaccination, which can facilitate the production of specific serum antibody of IgM and IgG when people are exposed to the virus. It has a certain protective effect on SARS-CoV-2 infected persons. Vaccination can reduce the clinical symptoms and mitigate disease severity.

AeMOPE-1, a Novel Salivary Peptide From Aedes aegypti, Selectively Modulates Activation of Murine Macrophages and Ameliorates Experimental Colitis.

The sialotranscriptomes of Aedes aegypti revealed a transcript overexpressed in female salivary glands that codes a mature 7.8 kDa peptide. The peptide, specific to the Aedes genus, has a unique sequence, presents a putative secretory nature and its function is unknown. Here, we confirmed that the peptide is highly expressed in the salivary glands of female mosquitoes when compared to the salivary glands of males, and its secretion in mosquito saliva is able to sensitize the vertebrate host by inducing the production of specific antibodies. The synthetic version of the peptide downmodulated nitric oxide production by activated peritoneal murine macrophages. The fractionation of a Ae. aegypti salivary preparation revealed that the fractions containing the naturally secreted peptide reproduced the nitric oxide downmodulation. The synthetic peptide also selectively interfered with cytokine production by murine macrophages, inhibiting the production of IL-6, IL-12p40 and CCL2 without affecting TNF-α or IL-10 production. Likewise, intracellular proteins associated with macrophage activation were also distinctively modulated: while iNOS and NF-κB p65 expression were diminished, IκBα and p38 MAPK expression did not change in the presence of the peptide. The anti-inflammatory properties of the synthetic peptide were tested in vivo on a dextran sulfate sodium-induced colitis model. The therapeutic administration of the Ae. aegypti peptide reduced the leukocytosis, macrophage activity and nitric oxide levels in the gut, as well as the expression of cytokines associated with the disease, resulting in amelioration of its clinical signs. Given its biological properties in vitro and in vivo, the molecule was termed Ae des-specific MOdulatory PEptide (AeMOPE-1). Thus, AeMOPE-1 is a novel mosquito-derived immunobiologic with potential to treat immune-mediated disorders.

PpSP32-like protein as a marker of human exposure to Phlebotomus argentipes in Leishmania donovani foci in Bangladesh

Phlebotomus argentipes is a predominant vector of Leishmania donovani, the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes, sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti-P. argentipes saliva IgG responses. The anti-rPagSP06 and anti-P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti-P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area.

PD-1 Inhibition Enhances Blinatumomab Response in a UCB/PDX Model of B-Cell Acute Lymphoblastic Leukemia

IL-2Ry immune deficient mice engrafted with human umbilical cord blood (UCB) develop human immune systems with mixed B, T, NK, and myeloid populations. We have previously modified NOD/SCID/Gamma (NSG) and NOD/RAG/Gamma (NRG) mice to include transgenic expression of human cytokines SCF, GM-CSF, and IL-3 (NSGS/NRGS). Relative to NSG/NRG, NSGS/NRGS mice have increased myeloid and NK fractions, more complete B cell differentiation, and faster T cell reconstitution. Additionally, both innate and adaptive immune function is also improved as measured by DTH assay and human antigen specific antibody production in response to immunization. NSGS and NRGS mice also more readily engraft leukemia samples, particularly in the absence of conditioning. We took advantage of the improved nature of hematopoietic xenografts in NRGS mice to determine whether or not humanized mice can serve as an in vivo model to test immune therapies against human leukemia.Busulfan conditioned NRGS mice were humanized by engraftment with red blood cell depleted UCB. Engraftment was monitored by the appearance of significant levels of human CD45+CD3+ T cells in the peripheral blood (PB). Once mice were reconstituted with >25% T cell graft in PB, we injected an aggressive, relapsed/refractory t(4;11) patient sample to induce fatal ALL in the humanized NRGS mice. All control humanized NRGS mice succumbed to disease between 40 and 60 days after leukemia injection with rapidly expanding B-ALL in the PB, bone marrow, and spleen. Mice treated with several courses of the bi-specific T cell engager (BiTE) molecule, blinatumomab, were partially protected from disease, and only half of these mice developed detectable B-ALL at the end of the experiment (day 90 post-injection). Strikingly, the combination of blinatumomab and the PD-1 antibody, pembrolizumab, fully protected the mice from B-ALL disease. All blinatumomab/pembrolizumab treated mice survived until the end of the experiment at which time there was no evidence of disease in PB, bone marrow, or spleen preparations. Importantly, additional experiments showed that this blinatumomab effect was dependent on the presence of the human immune system. Blinatumomab did not affect initiation (early treatment) or progression (late treatment) of B-ALL in non-humanized mice that were engrafted with the same patient sample.T cell levels were monitored during the experiment and showed a slight decline upon blinatumomab treatment. In contrast, T cell levels were significantly higher in mice that received both blinatumomab and pembrolizumab, implying augmented T cell activation in vivo. This may be a relevant finding because it is likely that some patients fail blinatumomab therapy due to sub-optimal T cell levels. The addition of pembrolizumab may offer means to combat this problem. Nevertheless, mice with less than 20% CD45+CD3+ T cells in the PB failed to respond to either blinatumomab or combined blinatumomab/pembrolizumab treatment, indicating there is a lower limit to potentiating the T cell response during BiTE therapy.These results demonstrate the potential utility of dual UCB/PDX models for examining novel combinations of immune therapies in vivo and suggest that additional studies combining BiTEs with immune checkpoint inhibitors are warranted. DisclosuresNo relevant conflicts of interest to declare.

Experience of Using a Complex Antigenic Preparation of the Plague Microbe to Assess the Severity of a Specific Anti-Plague Response

Background. Improving the methodology of immunological monitoring in natural foci of plague in the Russian Federation and adjacent territories to increase the effectiveness of epidemiological surveillance of plague is an urgent line of research. The lack of correlation between the production of specific antibodies to the capsular antigen (F1) ofthe plague microbe with other indicators of the state of cellular defense reactivity indicates the need to search for new informative and accessible markers for assessing anti-plague immunity.Objective: to evaluate possibility of using the complex preparation (F1 and cell membranes) evaluate the possibilities of using an artificial antigenic complex based on F1 and cell membranes (CM) of the plague microbe in antigen-specific tests in vitro in people vaccinated against plague.resu. The study involved 153 volunteers living in the territory enzootic for plague (the village of Khandagayty ofthe Ovyur kozhuun of the Tyva Republic and the village of Kosh-Agach of the Kosh-Agach district of the Altai Republic). The study included the determination of spontaneous and mitogen-induced production of cytokines (IFN-γ, IL-4, TNF-α) by blood cells, titers of specific IgG antibodies to the capsular antigen F1 of the plague microbe and concentrations ofthe main classes of immunoglobulins (IgM, IgG, IgA and IgE) in blood serum, as well as immunophenotyping of blood lymphocytes (CD3, CD4, CD8, CD16, CD19).Results. Comparative assessment of the level of cytokines (TNF-α, IFN-γ and IL-4) in spontaneous/induced F1+CM Y. pestis tests revealed a statistically significant increase in the production of cytokines TNF-α and IFN-γ in the antigeninduced tests compared with spontaneous (p &lt; 0.01).Conclusion. Thus, the effectiveness of the use of artificial antigenic complex based on F1 and cell membranes ofthe plague microbe has been shown to assess the production of cytokines in antigen-specific cell tests in vitro, which justifies the need for further research.

Covid-19 vaccination in patients with multiple myeloma: Focus on immune response.

Covid-19 vaccination in patients with multiple myeloma: Focus on immune response.

HER2-antigen-specific humoral immune response in breast cancer lymphocytes transplanted in hu-PBL hIL-4 NOG mice

The status of humoral immunity of cancer patients is not clear compared to cellular immunity because the ability of specific antibody production is difficult to analyze in vitro. We previously developed a humanized mouse model to evaluate antigen-specific antibody production by transplanting human peripheral blood mononuclear cells (PBMCs) into NOG-hIL-4-Tg mice (hu-PBL hIL-4 NOG). In this study, these mice were transplanted with PBMCs derived from breast cancer patients (BC) and immunized with a human epidermal growth factor receptor 2 (HER2) peptide, CH401MAP, to analyze humoral immunity of BCs. The hu-PBL hIL-4 NOG mice recapitulated immune environment of BCs as the ratio of CD8+/CD4+T cells was lower and that of PD-1 + T cells was higher compared to healthy donors (HDs). Diverse clusters were detected in BC-mouse (BC-M) plasma components involving immunoglobulins and complements unlike HD-M, and there was a significant diversity in CH401MAP-specific IgG titers in BC-M. The number of B cell clones producing high CH401MAP-specific IgG was not increased by immunization in BC-M unlike HD-M. These results demonstrated that the humoral immunity of BCs appeared as diverse phenotypes different from HDs in hu-PBL hIL-4 NOG mice, which may provide important information for the study of personalized medicine.

Evaluation of Gilthead Seabream (Sparus aurata) Immune Response after LCDV-Sa DNA Vaccination.

Simple SummaryLymphocystis disease is the main viral pathology in gilthead seabream aquaculture. Currently, there are no treatments or vaccines to control this disease, thus our main goal was to construct a DNA vaccine that can be used in the future to stop the spread of this pathology in sea farms. The vaccine consisted of a plasmid DNA that contains a known viral gene. Once it was established that the vaccine drives the expression of the antigenic viral protein in fish, vaccination experiments were conducted to determine if the vaccinated fish become protected against the viral infection. In addition, the immune response triggered by the vaccine was also evaluated in order to understand the mechanisms underlying such protection. The obtained results showed that in vaccinated fish an activation of several genes relating to both the inflammatory process and the mucosal immunity were produced, as well as specific anti-viral antibodies. Although limited, our results deserve further investigation to assess the efficacy of the vaccine in bigger fish populations and to confirm the mode of action of the vaccine.Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was to develop a DNA vaccine, and to evaluate both the protection conferred against LCDV-Sa infection and the immune response in vaccinated fish. The vaccine was constructed by cloning the mcp gene (ORF LCDVSa062R) into pcDNA3.1/NT-GFP-TOPO. Two independent vaccination trials were conducted. In the first one, 5–7 g fish were intramuscularly injected with the vaccine (pcDNA-MCP) or the empty-plasmid, and the distribution and expression of the vaccine was investigated. Furthermore, vaccinated fish were challenged with LCDV-Sa in order to access the protective capacity of the vaccine. In the second trial, 70–100 g fish were vaccinated as specified, and the immune response was evaluated analyzing the expression of 23 immune-related genes and the production of specific antibodies. The results showed that the vaccine triggers an immune response characterized by the overexpression of genes relating to the inflammatory process, but not the innate antiviral immunity relating to type I IFN (interferon), and also induces the production of specific neutralizing antibodies, which could explain the protection against LCDV-Sa in vaccinated fish.

The healing myocardium mobilizes a distinct B-cell subset through a CXCL13-CXCR5-dependent mechanism.

Recent studies have revealed that B cells and antibodies can influence inflammation and remodelling following a myocardial infarction (MI) and culminating in heart failure-but the mechanisms underlying these observations remain elusive. We therefore conducted in mice a deep phenotyping of the post-MI B-cell responses in infarcted hearts and mediastinal lymph nodes, which drain the myocardium. Thereby, we sought to dissect the mechanisms controlling B-cell mobilization and activity in situ. Histological, flow cytometry, and single-cell RNA-sequencing (scRNA-seq) analyses revealed a rapid accumulation of diverse B-cell subsets in infarcted murine hearts, paralleled by mild clonal expansion of germinal centre B cells in the mediastinal lymph nodes. The repertoire of cardiac B cells was largely polyclonal and showed no sign of antigen-driven clonal expansion. Instead, it included a distinct subset exclusively found in the heart, herein termed 'heart-associated B cells' (hB) that expressed high levels of Cd69 as an activation marker, C-C-chemokine receptor type 7 (Ccr7), CXC-chemokine receptor type 5 (Cxcr5), and transforming growth factor beta 1 (Tgfb1). This distinct signature was not shared with any other cell population in the healing myocardium. Moreover, we detected a myocardial gradient of CXC-motif chemokine ligand 13 (CXCL13, the ligand of CXCR5) on Days 1 and 5 post-MI. When compared with wild-type controls, mice treated with a neutralizing CXCL13-specific antibody as well as CXCR5-deficient mice showed reduced post-MI infiltration of B cells and reduced local Tgfb1 expression but no differences in contractile function nor myocardial morphology were observed between groups. Our study reveals that polyclonal B cells showing no sign of antigen-specificity readily infiltrate the heart after MI via the CXCL13-CXCR5 axis and contribute to local TGF-ß1 production. The local B-cell responses are paralleled by mild antigen-driven germinal centre reactions in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies.

Production of polyclonal antibody against the recombinant PirBvp protein of Vibrio parahaemolyticus

BackgroundAcute hepatopancreatic necrosis disease (AHPND) is caused by toxin-producing strains of Vibrio parahaemolyticus which contain deadly binary toxins PirAvp and PirBvp encoded in pVA1 plasmid. The polyclonal antibodies against PirBvp protein could be used to develop immunochromatographic test strip for in-field diagnosis of AHPND. ResultsIn this study, PirBvp gene was amplified, cloned, and expressed in E. coli. The expressed protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot probed with 6xHis antibodies. Then, the recombinant PirBvp (rPirBvp) was purified using Ni-Sepharose column. Rabbits were immunized with the purified rPirBvp, and produced antibodies were analyzed using Ouchterlony double immunodiffusion. The antibody titration and antibody purification were performed by ELISA and affinity chromatography, respectively. Finally, antibody specificity and sensitivity were evaluated by dot blotting. The present study showed a high titer of polyclonal antibodies in rabbit serum after immunization and the titer increased steadily during the immunization schedule. The highest titer of antibody reached up to 2,560,000 with LOD of 0.1 ng/mL. The purified antibodies showed no cross-reactivity with proteins from other Vibrio species, and the detection threshold ranged from 6.25 to 12.5 ng toxin/dot. ConclusionThis study highlights the production of high titer and specific polyclonal antibodies as an initial material towards the development of lateral-flow strip test.

BEI Inactivated Vaccine Induces Innate and Adaptive Responses and Elicits Partial Protection upon Reassortant Betanodavirus Infection in Senegalese Sole.

Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducing the mid-term viral load and avoiding the down-regulation of immune response post-challenge. On the other hand, the formalin-inactivated vaccine improved the survival of fish upon infection without inducing the production of IgM-NNV antibodies and only stimulating the expression of herc4 and mhcii genes (in head-kidney and brain, respectively) during the vaccination period; this suggests that other immune-related pathways may be involved in the partial protection provoked. Although these vaccines against NNV showed encouraging results, further studies are needed to improve sole protection and to fully understand the underlying immune mechanism.

Oral multivalent epitope vaccine, based on UreB, HpaA, CAT, and LTB, for prevention and treatment of Helicobacter pylori infection in C57BL / 6 mice.

As the resistance of Helicobacter pylori to traditional triple therapy is gradually revealed, an increasing number of people are focusing on vaccine treatments for H.pylori infection. Epitope vaccines are a promising strategy for the treatment of H.pylori infection, and multivalent vaccines will be more effective than monovalent vaccines. In this study, we designed a multivalent vaccine named LHUC, which consists of the adjuvant LTB as well as three Th cell epitopes (HpaA154-171 , UreB237-251, and UreB546-561 ) and five B-cell epitopes (UreB349-363 , UreB327-334 , CAT394-405 , CAT387-397, and HpaA132-141 ) from UreB, HpaA, and catalase. In BALB/c mice, the specificity and immunogenicity of the fusion peptide LHUC and the neutralization of H.pylori urease and catalase by the specific IgG elicited by LHUC were evaluated. The preventive and therapeutic effects of LHUC were evaluated in C57BL/6 mice infected with H.pylori. The results showed that compared with LTB and PBS, LHUC induced specific IgG and IgA antibody production in mice, and IgG antibodies significantly inhibited the H.pylori urease and catalase activities in vitro. Additionally, by detecting the levels of IFN-γ, IL-4, and IL-17 in lymphocyte supernatants, we proved that LHUC could activate Th1, Th2, and Th17 mixed T-cell immune responses in vivo. Finally, a C57BL/6 mouse model of gastric infection with H.pylori was established. The results showed that compared with the effects of LTB and PBS, the prevention and treatment effects of oral inoculation with LHUC significantly inhibited bacterial colonization. In conclusion, LHUC, a multivalent vaccine based on multiple H.pylori antigens, is a promising and safe vaccine that can effectively reduce the colonization of H.pylori in the stomach.