Production Of Specific Antibodies Research Articles

Immunoproteomic identification of OmpA with potential stimulation of serum‐specific antibody in Nile tilapia ( Oreochromis niloticus ) and its ability to protect against Edwardsiella ictaluri infection

Edwardsiella ictaluri is a Gram-negative bacterium that has been reported to be disease causing in several economic fish including Nile tilapia. Here, immunoproteomic identification found that outer membrane protein A (OmpA) could react with serum from fish naturally infected with E. ictaluri. The expression and production of recombinant OmpA protein could be successfully conducted in Escherichia coli strain BL21(DE3) with the molecular mass of 38 kDa after inclusion bodies were successfully solubilized and refolded. Immunization of fish with the recombinant OmpA protein showed that the recombinant proteins could induce the production of specific antibodies in serum of immunized fish (p < 0.05) at 1-week postimmunization (7 dpi) and reached the highest antibody titre at 3 weeks postimmunization (21 dpi). The serum-specific antibody from fish immunized with the recombinant OmpA exhibited bacterial agglutination property. Furthermore, the partial protection of Nile tilapia was observed in OmpA immunized fish with the relative per cent survival (RPS) of 39.81%. In summary, this study explored the immunoproteomic method as a starting point to investigate OmpA of E. ictaluri and might be considered as a candidate protein for recombinant vaccine development in Nile tilapia against this bacterial infection.

Circulating B10 regulatory cells are decreased in severe and critical COVID-19.

The contribution of B cells in COVID‐19 pathogenesis, beyond the production of specific antibodies against SARS‐CoV‐2, is still not well understood. Since one of their most relevant functional roles includes their immune‐suppressive mechanisms, we decided to evaluate one of the most recognized human B regulatory subpopulations: the IL‐10+ B10 cells, during COVID‐19 onset. After stimulation of PBMCs for IL‐10 induction, we employed multiparametric flow cytometry to determine B10 frequencies in severe and critical COVID‐19 patients and then correlated those with clinical and laboratory parameters. Compared with healthy individuals, we detected a significant reduction in the B10 subset in both patient groups, which correlates with some inflammatory parameters that define the disease severity. This evidence suggests an aberrant role of B10 cells in immune responses against SARS‐CoV‐2 that needs to be further explained.

Characterization and Evaluation of a Novel Conserved Membrane Antigen P35 of Mycoplasma synoviae.

Mycoplasma synoviae (MS) is a major avian pathogen that causes respiratory damage, infectious synovitis, and arthritis in chickens and causes serious economic losses to the global poultry industry. Despite its significance, knowledge on pathogenicity and pathogenic mechanism of MS is lacking, especially regarding its antigens. Bioinformatic analysis showed that the known MS proteins are only the tip of the iceberg among many MS membrane proteins. In this study, we identified and expressed a novel MS membrane protein P35. Sequence similarity showed that P35 was conservative and commonly existed among MS strains. Membrane protein extraction and immunofluorescence assay confirmed that P35 was distributed on the surface of MS. The production of specific antibodies after immunization with recombinant protein rP35 suggested its immunogenicity. The antigenicity of P35 was evaluated from two aspects by using polyantiserum against MS and rP35. Furthermore, in assays to identify the immune peptides of P35, all successfully expressed truncated segments could react with positive polyantiserum of MS, suggesting that P35 had more than one immune peptide. In conclusion, our study successfully identified P35 as a conservative antigen of MS, which may act as a potential candidate for the future development of a vaccine against MS.

Transgenerational Effects of Maternal Immune Activation on Specific Antibody Responses in Layer Chickens.

Activation of the maternal immune system may affect innate and adaptive immune responses in the next generation and may therefore have implications for vaccine efficacy and dietary immune modulation by feed additives. However, transgenerational effects on immune responses in chickens have been investigated to a limited extend. The present study investigated effects of intratracheal (i.t) specific and aspecific immune activation of laying hens on specific antibody production in the next generation. In two experiments laying hens received intratracheally an immune stimulus with human serum albumin (HuSA) or lipopolysaccharide (LPS). In experiment 1, hatchlings of the immune activated hens were at 4 weeks i.t. immunized with HuSA or HuSA+LPS. Maternal immune activation with LPS increased HuSA specific IgY and IgM responses in offspring. These results suggest a transgenerational effect of the maternal immune system on the specific antibody response in the next generation. In experiment 2 hatchlings received either β-glucan-enriched feed or control feed and were i.t. immunized with HuSA. Maternal immune activation with LPS decreased IgY anti-HuSA responses after HuSA immunization within hatchlings that received β-glucan enriched feed. The results of Experiment 2 suggest a transgenerational link between the innate immune system of mother and specific antibody responses in offspring. Despite variabilities in the outcomes of the two experiments, the observations of both suggest a link between the maternal innate immune system and the immune system of the offspring. Furthermore, our results may imply that maternal activation of the innate immune system can influence immune modulating dietary interventions and vaccine strategies in the next generation.

Abstract P3-11-02: Evaluation of the effects of anticancer treatments in cancer patients undergoing SARS-COV2 vaccination (VAX-on study: Breast cancer subgroup analysis)

Abstract Background: Since the beginning of the covid19 pandemic, clinical and demographic data showed that cancer patients are at high risk of developing severe consequences of Sars-Cov2 disease. For this reason, vaccination is strongly recommended, especially for patients on active treatment. Nevertheless, the efficacy of the Sars-Cov2 vaccine in cancer patients is not fully investigated. Our trial aim to explore the seroconversion in a large series of vaccinated cancer patients undergoing active treatment. Here we present a subgroup analysis concerning patients affected by breast cancer. Methods: The “VAX-on” is a single-center study that enrolled 366 cancer patients who underwent oncological treatment within the last six months. The study was approved by the ethics committee and all patients had to sign specific informed consent to be enrolled. Subjects were vaccinated against Sars-Cov2 with mRNA vaccine BNT162b2 (Comirnaty) - Pfizer BioNTech. Blood samples were obtained to quantify the production of specific anti-Spike IgG antibodies at day 21 from the first dose and at 6-8 weeks after the second dose. The antibody laboratory title cut-off of 50 U.A./mL defined the seroconversion. Results are shown as Mean and Standard Deviation for continuous variable, percentage (%) for categorical ones. The Mann-Whitney test or Chi-Square test were used to compare continuous or categorical groups, respectively. Results: A total of 100 patients with breast cancer were enrolled. Clinical and demographic data are summarized in Table 1. The median age was 60.5 years and the majority had an ECOG PS of 0 (75%). Almost all were women (97%), with advanced cancer in 60% of cases. In early or advanced setting 46% patients were treated with chemotherapy while 54% were on target therapy (also including monoclonal antibody and CDK4/6 inhibitors). The mean antibody title after the first dose of mRNA Comirnaty vaccine was 2185.03±9303.26 U.A./mL (M±SD), while after the second dose the mean antibody title rise to 6492.10±10425.95 (M±SD). After the first dose 61% of patients were considered as immunized, meanwhile after the second dose 86% of patients resulted immunized (defined as an antibody title &amp;gt;50 U.A./mL). In the 9 patients in treatment with steroids (prednisone &amp;gt; 10mg/die or equivalent), there was a trend to a decreased antibody development compared to patients without chronic use of steroids (p 0.06 and 0.05 after the first and second dose, respectively). Of interest, patients using G-CSF (12%) had a significant reduction in the production of Sars-Cov2 antibody after vaccination compared to patients who did not use them (p 0.02 and &amp;lt;0.001 after the first and second dose, respectively), with only 75% resulted positively immunized after the second dose (p=0.04). No differences were seen when comparing patients in advanced with non-advanced stage. Conclusions: Our study demonstrated 86% seroconversion in cancer patients after the second dose of mRNA vaccine regardless of disease stage or type of cancer treatment received. Further evaluations are needed to define whether the use of corticosteroids and G-CSF have an impact on seroconversion. Table 1.Parameters *Gender (F) (%)97 (97.00)Age (yrs)60.55±11.94Advanced Stage (Yes) (%)60 (60.00)ECOGPS (%)075 (75.00)121 (21.00)24 (4.00)Setting (%)Other Therapy60 (60.00)Adjuvant &amp; Neoadjuvant40 (40.00)Therapy (%)Other46 (46.00)Target54 (54.00)Last cycle &amp;lt;28 days (Yes) (%)81 (81.00)Timing CHT 1st Dose17.75±28.66Timing CHT 2nd Dose15.80±28.44Steroids (&amp;gt;10 mg)/&amp;gt; 7 days9 (9.00)FANS last 7 days (Yes) (%)15 (15.00)G-CSF last 21 days (Yes) (%)12 (12.00)Previous Allergies (Yes) (%)19 (19.00)Antibody Title 1st Dose2185.03±9303.26Antibody Level 1st Dose (&amp;gt;50) (%)61 (62.89)Antibody Title 2nd Dose6492.10±10425.95Antibody Level 2nd Dose (&amp;gt;50) (%)86 (92.47) Citation Format: Agnese Fabbri, Eleonora Marrucci, Fabrizio Nelli, Marco Mazzotta, Angelo Onorato, Primi Francesca, Mario Giovanni Chilelli, Antonella Virtuoso, Carlo Signorelli, Julio R Giron Berrios, Marta Schirripa, Rossella Donghia, Enzo Maria Ruggeri. Evaluation of the effects of anticancer treatments in cancer patients undergoing SARS-COV2 vaccination (VAX-on study: Breast cancer subgroup analysis) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-11-02.

Pregnant Women Develop a Specific Immunological Long-Lived Memory Against SARS-COV-2.

It is well established that pregnancy induces deep changes in the immune system. This is part of the physiological adaptation of the female organism to the pregnancy and the immunological tolerance toward the fetus. Indeed, over the three trimesters, the suppressive T regulatory lymphocytes are progressively more represented, while the expression of co-stimulatory molecules decreases overtime. Such adaptations relate to an increased risk of infections and progression to severe disease in pregnant women, potentially resulting in an altered generation of long-lived specific immunological memory of infection contracted during pregnancy. How potent is the immune response against SARS-CoV-2 in infected pregnant women and how long the specific SARS-CoV-2 immunity might last need to be urgently addressed, especially considering the current vaccinal campaign. To address these questions, we analyzed the long-term immunological response upon SARS-CoV-2 infection in pregnant women from delivery to a six-months follow-up. In particular, we investigated the specific antibody production, T cell memory subsets, and inflammation profile. Results show that 80% developed an anti-SARS-CoV-2-specific IgG response, comparable with the general population. While IgG were present only in 50% of the asymptomatic subjects, the antibody production was elicited by infection in all the mild-to-critical patients. The specific T-cell memory subsets rebalanced over-time, and the pro-inflammatory profile triggered by specific SARS-CoV-2 stimulation faded away. These results shed light on SARS-CoV-2-specific immunity in pregnant women; understanding the immunological dynamics of the immune system in response to SARS-CoV-2 is essential for defining proper obstetric management of pregnant women and fine tune gender-specific vaccinal plans.

Expression of recombinant porcine reproductive and respiratory syndrome virus nucleocapsid protein with antigenicity and immunogenicity study

Porcine respiratory and reproductive syndrome virus (PRRSV) causes significant economic losses in the swine industry worldwide. Nucleocapsid protein (NP) encoded by ORF7 is the most abundant and high immunogenicity. However, there were only few studies on recombinant NP expression with antigenicity and immunogenicity from PRRSV in Thailand. The aim of this study was to express rNP from PRRSV found in Thailand and evaluate its antigenicity and immunogenicity. ORF7 gene was amplified by RT-PCR. The PCR products were cloned into pGEM®-T Easy vector, and subcloned into pET-24a (+) expression vector and transformed into E. coli. The rNP properties were proved by SDS-PAGE and Western blot. The antigenicity of rNP was evaluated by indirect ELISA comparing to a commercial test kit. The immunogenicity of rNP was proved by rabbit immunization. Then, the antiserum was tested with tissues from PRRSV-infected swine by immunohistochemistry (IHC). In addition, amino acid sequence of the rNP was analyzed and compared to PRRSV reference strains. The PCR products of a whole ORF7 gene were 388 bp. The rNP size was 18 kDa. The indirect ELISA assay showed high antigenicity of the rNP with 100% sensitivity and 93.33% specificity at 0.25 O.D. cutoff value. The rNP could induce specific antibody production in the rabbit, which reacted specifically to PRRSV in tissues by IHC. The amino acid sequence of the rNP had similarities to US, HP-PRRSV and EU PRRSV at 95.93-99.19%, 96.75%, and 57.03-61.47%, respectively. Our rNP can be used for PRRSV diagnosis and test kit development.

Oral immunizations with Bacillus subtilis spores expressing MCP protein provide protection against red-spotted grouper nervous necrosis virus (RGNNV) infection in juvenile grouper, Epinephelus coioides

Nervous necrosis virus (NNV) causes severe viral disease in grouper, resulting in major economic losses in the global grouper aquaculture industry as a result of the high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect grouper against NNV infection. To achieve effective oral and bath immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as the vehicle to deliver MCP (major capsid protein of RGNNV) partnered with CotC, one of the coat proteins of B. subtilis, to the gastrointestinal tract. After routine culturing for 8–12 h in LB medium, B. subtilis containing CotC-MCP (B.s-CotC-MCP) was transferred into the sporulation culture medium. Western blot analysis and the growth curve indicated that the best sporulation time for recombinant WB600 was 24–30 h at 37 °C with continuous shaking (250 rpm). The grouper then received surface displaying B·S-CotC-MCP vaccine against RGNNV via either oral or bath immunization and their immune responses were monitored. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly enhanced in juvenile grouper following oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). The levels of specific antibodies against RGNNV and the transcription of immune-related genes (TNF-α, IL-1β, MHCI and IgM) were also significantly enhanced in juvenile grouper flowing oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). However, only the highest dose of bath vaccination induced the production of specific antibodies significantly and up-regulated transcriptions of several immune-related genes (IgM and MHCI). The lower cumulative mortality in vaccinated groups after RGNNV challenge clearly demonstrated that surface-displayed MCP vaccine could protect fish against RGNNV infection. The relative percentage survival (RPS) value in the orally vaccinated group (88.89%) was much higher than the bath group (18.89%), which was consistent with the production of specific serum antibodies, non-specific immune response and immune related gene expression. Therefore, this study demonstrated that the CotC-MCP spores can trigger high levels of mucosal and humoral immunity, and represent a promising candidate vaccine against NNV infection.

Outer Membrane Vesicles Coating Nano-Glycyrrhizic Acid Confers Protection Against Borderella bronchiseptica Through Th1/Th2/Th17 Responses.

PurposeOuter membrane vesicles (OMVs) are spherical nano-sized proteolipids secreted by numerous pathogenic Gram-negative bacteria. Due to the immunostimulatory properties and protective efficacy, OMVs have received increasing attention as a candidate for the vaccine to prevent and treat bacterial infections. However, the immune response remains elusive due to the low structural stability and poor size homogeneity of the vesicles. In this study, OMVs were used to coat self-assembled glycyrrhizic acid nanoparticles (GANs) and obtain a stable OMV vaccine. The immunoprotective effects and anti-infection efficacy were evaluated in vivo and in vitro.MethodsThe OMVs were prepared by ultrafiltration method and fused with GAN through mechanical extrusion. The characteristics, including morphology, hydrodynamic size, zeta potential, and stability were evaluated. The in vitro immunological function of GAN-OMV on the macrophages and in vivo immune efficacy and anti-infection effect were examined and compared.ResultsThe results showed that the GAN-OMV were homogenous with a size of 130 nm and a stable core-shell structure. Micropinocytosis-dependent and clathrin-mediated endocytotic pathways effectively internalized the GAN-OMV into the macrophages and promoted cell proliferation, cytokine secretion, and M1 polarization. Furthermore, subcutaneous GAN-OMV vaccination contributed to significantly higher Borderella bronchiseptica (Bb)-specific antibody production and lymphocyte proliferation. The splenic lymphocytes of mice immunized with GAN-OMVs displayed a higher ratio of CD4+/CD8+ T cells and CD19+ B cells and produced significantly higher levels of Th1/Th2/Th17 cytokines. GAN-OMV also effectively prevented Bb reinfection.ConclusionIn this study, GAN-OMV was developed successfully to stimulate Th1/Th2/Th17 immune responses against Bb and provide a promising strategy for novel vaccine development against the microbial pathogen.

An Update on Human Papilloma Virus Vaccines: History, Types, Protection, and Efficacy.

Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide. Early prevention with HPV vaccination is a safe and effective method against this disease. HPV vaccines provided more protection against several oncogenic HPV strains. Three prophylactic HPV vaccines have been approved to target high-risk HPV types and protect against HPV-related disorders. These existing vaccines are based on the recombinant DNA technology and purified L1 protein that is assembled to form HPV empty shells. The prophylactic vaccines are highly immunogenic and can induce production of specific neutralizing antibodies. However, therapeutic vaccines are different from these prophylactic vaccines. They induced cell-mediated immunity against transformed cells, instead of neutralizing antibodies. The second generation of prophylactic HPV vaccines, made from alternative viral components using cost-effective production strategies, is undergoing clinical evaluation. The purpose of this review is to provide a complete and up-to-date review of the types of HPV vaccines and the efficiency of each of them for readers.

A Patient with Juvenile Recurrent Respiratory Papillomatosis Complicated by Laryngeal Stenosis after Laryngeal Microsurgery: A Clinical Case

BACKGROUND: The relevance of the problem of juvenile respiratory papillomatosis lies in the recurrent course of the disease in young children. This can lead to the development of respiratory stenosis, requiring repeated, in some cases, monthly surgical treatments for endolaryngeal removal of papillomas, which sometimes leads to persistent scar stenosis of the larynx. We present clinical case of achieving remission of recurrent juvenile papillomatosis of the larynx. CLINICAL CASE: Patient, 5 years old, who applied in February 2016, was hospitalized in the ENT department with severe dyspnea, laryngoscopy revealed laryngeal papillomatosis with 3–4 grade stenosis. Over the past 10 months, the patient has undergone laryngeal surgery 6 times using cold instruments and a CO2 laser due to papillomatosis. Taking into account the recurrent nature of the course of the disease, the patient is administered to consult an allergist-immunologist. Based on the anamnesis and the results of the tests, the immunologist-allergist diagnosed: Primary immunodeficiency, unspecified (insufficiency in the production of specific antibodies), persistent allergic rhinitis, in the stage of incomplete remission and prescribed treatment. The patient was operated twice from 2017 to 2018 for a developed post-operative complication – cicatricial stenosis of the larynx. With these surgical repairs, no recurrence of laryngeal papillomatosis was detected. Furthermore, in 2021, according to endovideolaryngoscopy, remission was confirmed. R In this clinical case, we managed to achieve remission of laryngeal papillomatosis in a patient after surgery and treatment by an immunologist. Allergen elimination and immunocorrection helped us achieve deep remission. Surgical treatment eliminated laryngeal stenosis, recovering breathing and voice quality. Furthermore, this clinical case demonstrates the importance of studying the immune status in patients with laryngeal papillomatosis. CONCLUSION: The results obtained by us show the need for the use of immunomodulators in these patients. All children with prolonged hoarseness should undergo laryngoscopy, which will allow diagnosing laryngeal papillomatosis at earlier uncomplicated stages of the disease. Treatment of laryngeal papillomatosis should be comprehensive and in addition to surgical treatment should include differential immune correction. RESULTS AND DISCUSSION: In the above clinical case, we managed to achieve remission of laryngeal papillomatosis in a patient after surgery and treatment by an immunologist. Allergen elimination and immunocorrection helped us achieve deep remission. Surgical treatment eliminated laryngeal stenosis, recovering breathing and voice quality. Furthermore, this clinical case demonstrates the importance of studying the immune status in patients with laryngeal papillomatosis. The results obtained by us show the need for the use of immunomodulators in these patients. All children with prolonged hoarseness should undergo laryngoscopy, which will allow diagnosing laryngeal papillomatosis at earlier uncomplicated stages of the disease. Treatment of laryngeal papillomatosis should be comprehensive and in addition to surgical treatment should include differential immune correction.

The β-NGF/TrkA Signalling Pathway Is Associated With the Production of Anti-Nucleoprotein IgG in Convalescent COVID-19.

BackgroundThe presentation of SARS-CoV-2 infection varies from asymptomatic to severe COVID-19. Similarly, high variability in the presence, titre and duration of specific antibodies has been reported. While some host factors determining these differences, such as age and ethnicity have been identified, the underlying molecular mechanisms underpinning these differences remain poorly defined.MethodsWe analysed serum and PBMC from 17 subjects with a previous PCR-confirmed SARS-CoV-2 infection and 10 unexposed volunteers following the first wave of the pandemic, in the UK. Anti-NP IgG and neutralising antibodies were measured, as well as a panel of infection and inflammation related cytokines. The virus-specific T cell response was determined by IFN-γ ELISPOT and flow cytometry after overnight incubation of PBMCs with pools of selected SARS-CoV-2 specific peptides.ResultsSeven of 17 convalescent subjects had undetectable levels of anti-NP IgG, and a positive correlation was shown between anti-NP IgG levels and the titre of neutralising antibodies (IC50). In contrast, a discrepancy was noted between antibody levels and T cell IFN-γ production by ELISpot following stimulation with specific peptides. Among the analysed cytokines, β-NGF and IL-1α levels were significantly different between anti-NP positive and negative subjects, and only β-NGF significantly correlated with anti-NP positivity. Interestingly, CD4+ T cells of anti-NP negative subjects expressed lower amounts of the β-NGF-specific receptor TrkA.ConclusionsOur results suggest that the β-NGF/TrkA signalling pathway is associated with the production of anti-NP specific antibody in mild SARS-CoV-2 infection and the mechanistic regulation of this pathway in COVID-19 requires further investigation.

Place of residence, gender, month of birth and age influence on the concentration of specific IgE antibodies in serum. The Epidemiology of Allergic Diseases in Poland (ECAP survey): part three.

IntroductionSpecific immunoglobulins E (sIgE) are important parameters to estimate the severity of allergic diseases.AimTo determine the influence of gender, age and place of residence on the concentration of specific IgE antibodies in serum.Material and methodsThe concentration of sIgE antibodies against allergens of Dermatophagoides pteronyssinus, cat dander, timothy grass, Alternaria alternata were determined in serum of 4077 respondents randomly selected from 8 regions (ECAP study). The positive results of sIgE (≥ 0.35 IU/ml) were correlated with answers to ECAP questionnaires.ResultsIgE antibodies are less frequently detected in respondents living in the country (p < 0.05 to p < 0.005); and they are less frequently detected in respondents living in cities of hardly industrialised regions than in respondents living in cities of industrialised regions (D. pteronyssinus p < 0.05, cat dander p < 0.01). IgE antibodies are more frequently detected in men (p < 0.005 to p < 0.001). IgE antibodies are more frequently detected in respondents born in May-July (timothy grass p < 0.005, Alternaria alternata p < 0.005) and less frequently detected in respondents born in August-October (timothy grass p < 0.005, Alternaria alternata p < 0.05).ConclusionsCharacteristics of the urban environment, non-specific factors and gender affect the concentration of specific IgE antibodies in serum. Influence of seasonal allergens within the first months of life and/or in the course of the foetal period alters the production of specific IgE antibodies.

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants.

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund's complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16–19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.

Generation of Bacillus subtilis displaying alpha-toxin HlaH35LH48L fused with CotB and CotG, and studying the immune response in mice

Introduction: The Bacillus subtilis spore is considered to be a powerful vehicle for surface display and antigen delivery. Being safe and widely used for the purpose of oral bacteriotherapy in humans and animals, B. subtilis spore has potential in the domain of needle-free vaccine development. The spores themselves also behave as an adjuvant. This study aims to generate B. subtilis spores expressing mutant staphylococcal alpha-toxin HlaH35LH48L on the surface and to study their ability to evoke a specific immune response in mice. Methods: Vectors carrying the hlaH35LH48L gene fused with the coding genes of anchor proteins, cotB and cotG, were cloned in E. coli before being transformed into B. subtilis. The generation of the B. subtilis new strains via chromosomal integration was confirmed by PCR. Sporulation was observed under the microscope. The expression of the target protein on the spore surface was determined by sporeELISA. The level of IgG in the serum and IgA in the feces of Swiss mice were analyzed using ELISA to learn about the immune response against the B. subtilis spore administrated via the oral route. Results: The PCR products of an expected size on agarose gel electrophoresis showed successful integration, resulting in the construction of new B. subtilis strains BsHT2331 and BsHT2334. sporeELISA analysis detected a significant HlaH35LH48L expression on the spore surface. The BsHT2331 spores (CotB-HlaH35LH48L) triggered a constant increase in the IgG and IgA levels in mice after three doses as 5.5-fold and 2.5-fold higher than the pre-immunization, respectively (p-value &lt; 0.0001). Meanwhile, the group of mice orally administrated with BsHT2334 (CotG-HlaH35LH48L) showed a notable IgG titer but minor IgA response. Conclusion: The results concluded that the construction of two new strains BsHT2331 and BsHT2334 that used spore coat proteins CotB and CotG to display mutant alpha-toxin HlaH35LH48L on the B. subtilis spore surface was successful. It provided an assessment of the ability of B. subtilis spores to stimulate specific antibody production in mice.

Toxoplasma gondii GRA15 DNA Vaccine with a Liposomal Nanocarrier Composed of an SS-Cleavable and pH-Activated Lipid-like Material Induces Protective Immunity against Toxoplasmosis in Mice.

Toxoplasma gondii affects the health of humans and livestock and causes severe illness in the fetus and immunocompromised individuals. Because of the high incidence and severe consequences of T. gondii infection, a safe and suitable vaccine is needed. We found that lipid nanoparticles (LNPs) consisting of a series of functional materials prepared with vitamin E, such as SS-cleavable and pH-activated lipid-like materials (ssPalmE), were a safe and efficient way to develop next-generation DNA vaccines. In this study, we prepared ssPalmE-LNP to encapsulate pCpG-free-T. gondii dense granule protein 15 DNA (ssPalmE-LNPTgGRA15). Following a challenge infection with avirulent PLK strain of T. gondii, the mice immunized with ssPalmE-LNPTgGRA15 had a significantly higher survival rate and lower clinical scores compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice. Immunization of mice with the ssPalmE-LNPTgGRA15 led to a significantly higher production of specific IgG1 and IG2c antibodies compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice, while there was no statistically significant difference in the concentration of serum interferon-gamma at the acute stage of the infection. These findings indicate that ssPalmE-LNP is an effective cargo for the transportation of DNA vaccines for protozoan infections. To explore the mechanism of protective immunity induced by ssPalmE-LNPTgGRA15, further immunological study is needed in the future.

Evaluation of Cumulative Effect of Standard Triple Immunosuppression on Prevention of De Novo Donor Specific Antibodies (dnDSA) Production in Children after Kidney Transplantation-A Retrospective and Prospective Study.

De novo Donor Specific Antibodies (dnDSA) are associated with inferior graft outcomes. Standard immunosuppression is expected to prevent dnDSA production in low-risk patients. We have evaluated a cumulative effect of a triple immunosuppression (CNI/MMF/Pred), as well as TAC concentration and coefficient of variation on the incidence of dnDSA production. Overall, 67 transplanted patients were evaluated in retrospective (dnDSA for-cause; n = 29) and prospective (dnDSA by protocol; n = 38) groups. In the retrospective group, the eGFR value at first dnDSA detection (median interval—4.0 years post-transplant) was 41 mL/min/1.73 m2; 55% of patients presented biopsy-proven cAMR, and 41% lost the graft within next 2.4 years. Patients from the prospective group presented 97% graft survival and eGFR of 76 mL/min/1.73 m2 at 2 years follow-up, an overall incidence of 21% of dnDSA and 18% of acute (T cell) rejection. None of the patients from the prospective group developed cAMR. Median value of Vasudev score within 2 years of follow-up was not significantly higher in dsDSA negative patients, while median value of TAC C0 > 1–24 months post-transplant was 7.9 in dnDSA negative vs. 7.1 ng/mL in dnDSA positive patients (p = 0.008). Conclusion: dnDSA-negative patients presented a higher exposure to tacrolimus, while not to the combined immunosuppression.

Effects of Lactobacillus salivarius supplementation on the growth performance, liver function, meat quality, immune responses and Salmonella Pullorum infection resistance of broilers challenged with Aflatoxin B1

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins. It has been reported that dietary exposure to AFB1 is related to the low growth performance, immunosuppression, and high susceptibility to infectious diseases of chickens. The aim of the present study was to evaluate the protective effects of Lactobacillus salivarius on broiler chickens challenged with AFB1. First, AFB1 degradation ability of Lactobacillus salivarius was measured by a high-performance liquid chromatography (HPLC) method. Then, the Arbor Acres broiler chickens were randomly assigned to experimental groups. The effects of Lactobacillus salivarius supplementation on the growth performance, liver function, and meat quality were measured, and immune response was also determined after vaccination with attenuated infectious bursal disease virus (IBDV) vaccine of broilers challenged with AFB1. Besides, resistance to Salmonella Pullorum infection along with AFB1 exposure was determined in broilers. The results showed that Lactobacillus salivarius could effectively degrade AFB1. Lactobacillus salivarius supplementation improved growth performance, liver function, and meat quality of broilers challenged with AFB1. In addition, Lactobacillus salivarius supplementation resulted in enhanced specific antibody and IFN-γ production, and lymphocyte proliferation in broilers challenged with AFB1 after IBDV vaccine immunization. Furthermore, Lactobacillus salivarius supplementation enhanced Salmonella Pullorum infection resistance in broilers challenged with AFB1. Our results revealed a tremendous potential of Lactobacillus salivarius as feed additive to degrading AFB1 and increasing broilers production performance in poultry production.

Double Labeling Fluorescent Immunocytochemistry.

Fluorescent immunocytochemistry is a powerful technique based on detecting antigens. It leads to discoveries in cell composition and structure as well as its functioning by expanding knowledge on colocalization between its components. The potency of this method is based on findings in the areas of specific antibodies production, fluorescent labels, and microscopy. Since it merges different fields, it requires basic knowledge on all the steps that are needed in the procedure planning and implementation to be used properly and produce reliable results. Here we describe a protocol of LN-229 human glioblastoma cells double labeling of LC3 and IRS-1 proteins, highlighting the importance of some steps of the procedure and possible variables.

Severity of SARS-CoV-2 infection is linked to double-negative (CD27- IgD-) B cell subset numbers.

ObjectivesThe role of B cells in COVID‐19, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Here, we describe the novel landscape of circulating double-negative (DN) CD27− IgD− B cells in COVID‐19 patients, representing a group of atypical and neglected subpopulations of this cell lineage.MethodsUsing multiparametric flow cytometry, we determined DN B cell subset amounts from 91 COVID-19 patients, correlated those with cytokines, clinical and laboratory parameters, and segregated them by principal components analysis.ResultsWe detected significant increments in the DN2 and DN3 B cell subsets, while we found a relevant decrease in the DN1 B cell subpopulation, according to disease severity and patient outcomes. These DN cell numbers also appeared to correlate with pro- or anti-inflammatory signatures, respectively, and contributed to the segregation of the patients into disease severity groups.ConclusionThis study provides insights into DN B cell subsets’ potential role in immune responses against SARS‐CoV‐2, particularly linked to the severity of COVID‐19.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-021-01525-3.