Total Antibody Production Research Articles

Autophagy and Akt-Stimulated Cellular Proliferation Synergistically Improve Antibody Production in CHO Cells.

Over the past decade, engineered producer cell lines have led 10-fold increases in antibody yield, based on an improved understanding of the cellular machinery influencing cell health and protein production. With prospects for further production improvements, increased antibody production would enable a significant cost reduction for life-saving therapies. In this study, we strategized methods to increase cell viability and the resulting cell culture duration to improve production lifetimes. By overexpressing the cell surface adenosine A2A receptor (A2AR), the Akt pathway was activated, resulting in improved cellular proliferation. Alternatively, by inducing autophagy through temperature downshift, we were able to significantly enhance cellular-specific productivity, with up to a three-fold increase in total antibody production as well as three-fold higher cell-specific productivity. Interestingly, the expression levels of the autophagy pathway protein Beclin-1 appeared to correlate best with the total antibody production, of autophagy-related proteins examined. Thus, during cell clonal development Beclin-1 levels may serve as a marker to screen for conditions that optimize antibody titer.

In silico and in vivo Investigations of the Immunoreactivity of Klebsiella pneumoniae OmpA Protein as a Vaccine Candidate

The growing threat of antibiotic resistance and Klebsiella pneumoniae infection in healthcare settings highlights the urgent need for innovative solutions, such as vaccines, to address these challenges. This study sought to assess the potential of using K. pneumoniae outer membrane protein A (OmpA) as a vaccine candidate through both in silico and in vivo analyses. The study examined the OmpA protein sequence for subcellular localization, antigenicity, allergenicity, similarity to the human proteome, physicochemical properties, B-cell epitopes, MHC binding sites, tertiary structure predictions, molecular docking, and immune response simulations. The ompA gene was cloned into the pET-28a (+) vector, expressed, purified and confirmed using Western blotting analysis. IgG levels in the serum of the immunized mice were measured using ELISA with dilutions ranging from 1:100 to 1:6400, targeting recombinant outer membrane protein A (rOmpA) and K. pneumoniae ATCC 13883. The sensitivity and specificity of the ELISA method were also assessed. The bioinformatics analysis identified rOmpA as a promising vaccine candidate. The immunized group demonstrated significant production of specific total IgG antibodies against rOmpA and K. pneumoniae ATCC1 13883, as compared to the control group (p < 0.0001). The titers of antibodies produced in response to bacterial exposure did not show any significant difference when compared to the anti-rOmpA antibodies (p > 0.05). The ELISA test sensitivity was 1:3200, and the antibodies in the serum could accurately recognize K. pneumoniae cells. This study is a significant advancement in the development of a potential vaccine against K. pneumoniae that relies on OmpA. Nevertheless, additional experimental analyses are required.

Synthetic saponin derivatives act as functional subunit vaccine adjuvants through an NLRP3-inflammasome-independent mechanism

Abstract Quillaja saponaria 21 (QS-21) is a saponin compound extracted from the bark of a South American evergreen tree. The ability of QS-21 to induce robust cellular and humoral immunity as an adjuvant led to it being evaluated in multiple clinical vaccine trials. However, mechanistic and toxicity questions following its purification hinder its potential use and led to the search for suitable alternative adjuvants. A group of 14 novel synthetic compounds have been chemically derived from a less toxic saponin, lablaboside F, to be tested as suitable alternatives to QS-21. When administered with an OVA peptide antigen intramuscularly into C57BL/6 mice using a prime-boost strategy, compound JG-151 elicited increased production of OVA-specific total IgG antibodies 2 weeks post-boost compared to control mice. When analyzed in vitro, JG-151 displayed decreased cellular toxicity 24 hrs post-treatment compared to QS-21. However, JG-151 neither activated NF-kB alone nor induced significant production of the pro-inflammatory cytokine IL-1ß with MPLA 24 hrs post-treatment. These results indicate JG-151 does not activate the NLRP3 inflammasome, in contrast to QS-21. Our results demonstrate that novel saponin derivatives, such as JG-151, safely elicit humoral immunity through an NLRP3 inflammasome-independent mechanism and could serve as suitable subunit vaccine adjuvant alternatives to QS-21.

Immunological response in health care workers post COVID-19 vaccination

To curb SARS-CoV-2, various vaccines received Emergency Authorization Use by copious agencies. These vaccines are administered to health-care workers, front-line workers, elderly individuals and many others. Health care workers were the first to be administered with vaccines, thus, evaluating the antibody response helps to portray a picture of the burden of SARS-CoV-2 infection amid Health Care Workers. The present study aimed to investigate the production of total antibodies against SARS-CoV-2 before and after vaccination (2 doses) among Health Care Workers along with associated factors. The present study was conducted on 60 healthcare workers as cases who got vaccinated for COVID-19. The semi-quantitative detection of anti-SARS-CoV-2 IgG Ab in the samples was detected using Erba Lisa COVID-19 total antibody indirect ELISA kit using standard protocol as described by the manufacturer. In the present study, co-morbidities such as hypertension was presented in only 8 (12.3%) individuals, diabetes in 4 (6.1%) and Coronary Artery Disease in 1 (1.5%). After first dose of administration, 86.67% HCWs were found seropositive for the anti-SARS-CoV-2 Ab while 13.33% were seronegative, however, after second dose 100% seroprevalence was observed amid HCWs. The statistical analysis depicted a significant difference (p&amp;#60;0.0001) in antibody titres among individuals before and after vaccination. The mean of total antibody levels detected at zero week was (4.1), 4th week, (39.09), 8th week (79.39) and 12th week (105). The present study provides important information on the detectable levels of antibodies induced by COVISHIELD against the SARS-CoV-2 which was independent of gender and age.

Characterization of immune development of fetal and early-life of minipigs

Fetal and child’s immune systems differ from those of adults. Developing immune systems exhibit increased or decreased sensitivity to drugs, infection, or toxicants compared to adult immune systems. Understanding fetal and neonatal immune systems will help predict toxicity or the pathogenesis or prognosis of diseases. In this study, we evaluated whether the innate and adaptive immune system of fetal and young minipigs could respond to external stimuli compared to a medium-treated group and analyzed several immunological parameters for developmental immunotoxicity according to developmental stages. We performed a hematological analysis of fetal cord bloods and the bloods of neonatal and 4-week-old piglets. Splenocytes were isolated at each developmental stage and treated with lipopolysaccharide (LPS), R848, and concanavalin A (ConA). Various cytokines were measured in the cell supernatants. Total antibody production was also evaluated in serum. The percentage of lymphocytes was dominant in gestational weeks (GW) 10 and 12 and started to decrease from postnatal day (PND) 0. From PND0, the percentage of neutrophils increased. Interleukin (IL)-1β, IL-6, and interferon (IFN)-α were induced from GW10 in response to LPS and R848 stimulation. Th1 cytokine induction was detected from PND0 upon ConA stimulation, whereas Th2 cytokine release was observed from GW10. IgM and IgG production was sustained at low levels at fetal stages and was significantly increased after birth. This study reconfirmed that the fetal immune system could respond to external stimuli and that hematological analysis, cytokine evaluation, and antibody subclass measurement can be useful parameters for developmental immunotoxicity using minipigs.

Immune response induced by recombinant pres2/S-protein and a pres2-S-protein fused with a core 18-27 antigen fragment of hepatitis B virus compared to conventional HBV vaccine.

Although comprehensive vaccination is the cornerstone of public health programs to control hepatitis B virus (HBV) infections, 5% of people who receive the existing vaccine do not develop proper immunity against HBV. To overcome this challenge, researchers have tried using various protein fragments encoded by the virus genome to achieve better immunization rates. An important antigenic component of HBsAg called the preS2/S or M protein has also received much attention in this area. The gene sequences of preS2/S and Core18-27 peptide were extracted from the GenBank (NCBI). Final gene synthesis was conducted with pET28. Groups of BALB/c mice were immunized with 10μg/ml of recombinant proteins and 1μg/ml CPG7909 adjuvant. Serum levels of IF-γ, TNF-α, IL-2, IL-4, and IL-10 were measured by ELISA assay method on spleen cell cultures on day 45, and IgG1, IgG2a, and total IgG titers obtained from mice serum were quantified on days 14 and 45. Statistical analysis did not show any significant difference between the groups regarding IF-γ level. There were, however, significant differences in terms of IL-2 and IL-4 levels between the groups receiving preS2/S-C18-27 with and without adjuvant and the groups receiving both preS2/S and preS2/S-C18-27 (Plus Recomb-Plus Recomb: the group of mice that received both preS2/S and preS2/S-C18-27 simultaneously). The strongest total antibody production was induced by immunization with both recombinant proteins without CPG adjuvant. The groups that received both preS2/S and preS2/S-C18-27, whether with or without adjuvant, were significantly different from those that received the conventional vaccine considering most abundant interleukins. This difference suggested that higher levels of efficacy can be achieved by the use of multiple virus antigen fragments rather than using a single fragment.

Severe fever with thrombocytopenia syndrome virus aerosol infection in C57/BL6 mice

Although secondary cases have become infected with the SFTSV after being in the same space without direct contact with the index case, it has not been experimentally determined if the SFTSV can be transmitted through aerosols. Here, this study aimed to verify if the SFTSV could be transmitted by aerosols. Firstly, we demonstrated that the SFTSV can infect BEAS-2B cells, and SFTSV genomes can be isolate from mild patient's sputum, which provided a foundation for the existence of SFTSV aerosol transmission. Then, we evaluated total antibody production in serum and viral load in tissue of mice infected with SFTSV by aerosols. The results showed that the presence of antibodies is related to the dose of virus infection and the SFTSV preferentially replicates in the lungs of mice following an aerosol exposure. Our study will help update the prevention and treatment guidelines for SFTSV and prevent the spread of the SFTSV in hospitals.

Peptide vaccines designed with the aid of immunoinformatic against Caseous Lymphadenitis promotes humoral and cellular response induction in mice.

Caseous Lymphadenitis (CLA) is a chronic disease that affects also small ruminants. CLA is caused by Corynebacterium pseudotuberculosis and is responsible for high economic losses due to the formation of superficial and visceral granulomas, the latter is considered as asymptomatic CLA causing high levels of dissemination. Several vaccination strategies, in which the use of synthetic peptides stands out. Thus, this work aimed to evaluate the protective potential of peptide vaccines designed to determine the immunodominant epitopes of CP40 against CLA in mice. The animals were divided into eight groups separated in controls (G1—PBS, G2—Saponin and G9—rCP40) and experimental (G3—pep1, G4- pep2, G5-pep3, G6-pep4, G7-pep5 and G8-pep6), these were vaccinated on days 0 and 15 by a subcutaneous route. 60 days after the first immunization, all animals were challenged with C. pseudotuberculosis. On days 0, 15, 60, and 120 after the first immunization, blood samples were taken to measure immunoglobulins. On the same day of the challenge, the splenocytes were isolated and assayed for the production of IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, and IL-10. After vaccinations, the animals were challenged and all of them were affected by the disease which led to their death. The G6 and G8 groups provided 10% protection and the G7 provided 20%. The G3 and G4 groups provided 30% and 40% protection respectively. The peptides showed the production of Total IgG antibodies and cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α), indicating a possible activation of the Th1 type response. However, groups G3, G5, G6, and G8 showed production of IL-17. None of the study groups showed IL-10 production. The immunogenicity of the peptides was not enough to protect these animals and it is believed that the use of adjuvants based on PAMPs may improve the immune response offered by these peptides.

Co-administration of silymarin elevates the therapeutic effect of praziquantel through modulation of specific antibody profiles, Th1/Th2/Tregs cytokines and down-regulation of fibrogenesis in mice with Mesocestoides vogae (Cestoda) infection

Silymarin (SIL) represents a natural mixture of polyphenols showing an array of health benefits. The present study, carried out on a model cestode infection induced by Mesocestoides vogae tetrathyridia in the ICR strain of mice, was aimed at investigating the impact of SIL as adjunct therapy on the activity of praziquantel (PZQ) in relation to parasite burden, immunity and liver fibrosis within 20 days post-therapy. In comparison with PZQ alone, co-administration of SIL and PZQ stimulated production of total IgG antibodies to somatic and excretory-secretory antigens of metacestodes and modified the expression patterns of immunogenic molecules in both antigenic preparations. The combined therapy resulted in the elevation of IFN-γ and a decline of TNF-α and TGF-β1 in serum as compared to untreated group; however, SIL attenuated significantly the effect of PZQ on IL-4 and stimulated PZQ-suppressed phagocytosis of peritoneal macrophages. In the liver, SIL boosted the effect of PZQ on gene expression of the same cytokines in a similar way as was found in serum, except for down-regulation of PZQ-stimulated TNF-α. Compared to PZQ therapy, the infiltration of mast cells into liver after SIL co-administration was nearly abolished and correlated with suppressed activities of genes for collagen I, collagen III and α-SMA. In conclusion, co-administration of SIL modified the effects of PZQ therapy on antigenic stimulation of the immune system and modulated Th1/Th2/Tregs cytokines. In liver this was accompanied by reduced fibrosis, which correlated with significantly higher reduction of total numbers of tetrathyridia after combined therapy as compared with PZQ treatment.

The potential of a DIVA-like recombinant vaccine composed by rNcSAG1 and rAtHsp81.2 against vertical transmission in a mouse model of congenital neosporosis

Neospora caninum is the etiological agent of neosporosis, a worldwide infectious disease recognized as the major cause of abortions and reproductive failures in livestock, responsible for significant economic losses in cattle industries. Currently, there are not cost-effective control options for this pathology, and the development of a vaccine involving new and integrated approaches is highly recommended. In this study, we evaluated the immunogenic and protective efficacy, as well as the potential DIVA (Differentiation of Infected from Vaccinated Animals) character of a recombinant subunit vaccine composed by the major surface antigen from N. caninum (NcSAG1) and the carrier/adjuvant heat shock protein 81.2 from Arabidopsis thaliana (AtHsp81.2) in a mouse model of congenital neosporosis. BALB/c female mice were intraperitoneal (i.p.) immunized with a mixture of equimolar quantities of rNcSAG1 and rAtHSP81.2 or each protein alone (rNcSAG1 or rAtHsp81.2). The vaccine containing a mixture of rNcSAG1 and rAtHsp81.2 significantly enhanced the production of specific anti-rNcSAG1 total IgG (tIgG), IgG1 and IgG2a antibodies in immunized mice when compared to control groups (non-vaccinated and rAtHsp81.2 immunized mice) as well as to the group of mice immunized only with the antigen (rNcSAG1). In addition, partial protection against vertical transmission and improvement of the offspring survival time was observed in this group. On the other hand, rAtHsp81.2 induced the production of specific anti-rAtHsp81.2 tIgG, allowing us to differentiate vaccinated from infected mice. Despite further experiments have to be made in cattle to test the capability of this vaccine formulation to differentiate vaccinated from infected animals in the field, our results suggest that the formulation composed by rNcSAG1 and rAtHsp81.2 could serve as a basis for the development of a new vaccine approach against bovine neosporosis.

ERdj5 in Innate Immune Cells Is a Crucial Factor for the Mucosal Adjuvanticity of Cholera Toxin.

Cholera toxin (CT) is one of most strong mucosal adjuvants, but it cannot be clinically used owing to its toxicity. The cytosolic A1 subunit of CT (CTA1) is the molecule responsible for its immunostimulatory activity, which increases the concentration of cyclic AMP and causes the induction of pro-inflammatory cytokines in innate immune cells. However, the importance of endoplasmic reticulum (ER) molecules involved in CTA1 retro-translocation to induce immune responses remained to be investigated. ERdj5 is an ER protein which is expected to transfer CTA1 to the Hrd1 complex for the retro-translocation of CTA1. In this study, we investigated the physiological relevance of ERdj5 in immune stimulation by CT. ERdj5-knockout (ERdj5 KO) mice had decreased production of antigen-specific IgG in the serum and IgA in the mucosal secretion after intranasal immunization with Ag and CT. Especially, IgG2c isotypes were specifically reduced in the absence of ERdj5. ERdj5 KO dendritic cells (DCs) failed to full activation with decreased expression of costimulatory molecules, such as MHC class II, CD80, and CD 86. In ERdj5 KO DCs, secretion of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, was reduced. The cytokine signatures of several helper T cells were reduced in ERdj5 KO mice following intranasal CT immunization. The absence of ERdj5 affects the immunostimulatory properties of CT but does not affect the response to the CTB pentamer, the response to alum, total antibody production, or cytokine release from DCs exposed to CpG. Interestingly, CT enhanced the expression of ER stress proteins in ERdj5 KO innate immune cells. These results suggested that ERdj5 contributed as a decisive factor to the immunostimulatory capacity of CT via CTA1 retro-translocation.

Kv1.3 Channel Blockade Modulates the Effector Function of B Cells in Granulomatosis with Polyangiitis.

B cells are central to the pathogenesis of granulomatosis with polyangiitis (GPA), exhibiting both (auto)antibody-dependent and -independent properties. Class-switched memory B cells in particular are a major source of pathogenic autoantibodies. These cells are characterized by high expression levels of Kv1.3 potassium channels, which may offer therapeutic potential for Kv1.3 blockade. In this study, we investigated the effect of the highly potent Kv1.3 blocker ShK-186 on B cell properties in GPA in vitro. Circulating B cell subsets were determined from 33 GPA patients and 17 healthy controls (HCs). Peripheral blood mononuclear cells (PBMCs) from GPA patients, and HCs were stimulated in vitro in the presence and absence of ShK-186. The production of total and antineutrophil cytoplasmic antibodies targeting proteinase 3 (PR3-ANCA) IgG was analyzed by enzyme-linked immunosorbent assay and Phadia EliA, respectively. In addition, effects of ShK-186 on B cell proliferation and cytokine production were determined by flow cytometry. The frequency of circulating switched and unswitched memory B cells was decreased in GPA patients as compared to HC. ShK-186 suppressed the production of both total and PR3-ANCA IgG in stimulated PBMCs. A strong decrease in production of tumor necrosis factor alpha (TNFα), interleukin (IL)-2, and interferon gamma was observed upon ShK-186 treatment, while effects on IL-10 production were less pronounced. As such, ShK-186 modulated the TNFα/IL-10 ratio among B cells, resulting in a relative increase in the regulatory B cell pool. ShK-186 modulates the effector functions of B cells in vitro by decreasing autoantibody and pro-inflammatory cytokine production. Kv1.3 channel blockade may hold promise as a novel therapeutic strategy in GPA and other B cell-mediated autoimmune disorders.

Identification of immunogenic proteins and evaluation of four recombinant proteins as potential vaccine antigens from Vibrio anguillarum in flounder (Paralichthys olivaceus)

Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection.

Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V.anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V.anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V.anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg+cells in lymphocytes and induce production of total antibodies, specific antibodies against V.anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V.anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V.anguillarum and could be vaccine candidates against V.anguillarum.

Lymphotoxin-Dependent B Cell-FRC Crosstalk Promotes De Novo Follicle Formation and Antibody Production following Intestinal Helminth Infection.

Secondary lymphoid tissues provide specialized niches for the initiation of adaptive immune responses and undergo a remarkable expansion in response to inflammatory stimuli. Although the formation of B cell follicles was previously thought to be restricted to the postnatal period, we observed that the draining mesenteric lymph nodes (mLN) of helminth-infected mice form an extensive number of new, centrally located, B cell follicles in response to IL-4Rα-dependent inflammation. IL-4Rα signaling promoted LTα1β2 (lymphotoxin) expression by Bcells, which then interacted with CCL19 positive stromal cells to promote lymphoid enlargement and the formation of germinal center containing B cell follicles. Importantly, de novo follicle formation functioned to promote both total and parasite-specific antibody production. These data reveal a role for type 2 inflammation in promoting stromal cell remodeling and de novo follicle formation by promoting B cell-stromal cell crosstalk.

Antibody and cellular immune responses of naïve mares to repeated vaccination with an inactivated equine herpesvirus vaccine

Equine herpesvirus type 1 (EHV-1) continues to cause severe outbreaks of abortions or myeloencephalopathy in horses despite widely used vaccination. The aim of this work was to determine the effects of frequent vaccination with an inactivated EHV vaccine on immune development in horses. Fifteen EHV-1 naive mares were vaccinated a total of 5 times over a period of 8 months with intervals of 20, 60, 90 and 60 days between vaccine administrations. Total antibody and antibody isotype responses were evaluated with a new sensitive EHV-1 Multiplex assay to glycoprotein C (gC) and gD for up to 14 months after initial vaccination. Antibodies peaked after the first two vaccine doses and then declined despite a third administration of the vaccine. The fourth vaccine dose was given at 6 months and the gC and gD antibody titers increased again. Mixed responses with increasing gC but decreasing gD antibody values were observed after the fifth vaccination at 8 months. IgG4/7 isotype responses mimicked the total Ig antibody production to vaccination most closely. Vaccination also induced short-lasting IgG1 antibodies to gC, but not to gD. EHV-1-specific cellular immunity induced by vaccination developed slower than antibodies, was dominated by IFN-γ producing T-helper 1 (Th1) cells, and was significantly increased compared to pre-vaccination values after administration of 3 vaccine doses. Decreased IFN-γ production and reduced Th1-cell induction were also observed after the second and fourth vaccination. Overall, repeated EHV vaccine administration did not always result in increasing immunity. The adverse effects on antibody and cellular immunity that were observed here when the EHV vaccine was given in short intervals might in part explain why EHV-1 outbreaks are observed worldwide despite widely used vaccination. The findings warrant further evaluation of immune responses to EHV vaccines to optimize vaccination protocols for different vaccines and horse groups at risk.

The Effect of High Carbohydrate Consumption on Glucose Levels and Antibody Production in Nonobese Diabetic Mice

The aim of this study was to evaluate the effect of chronic treatment with diets rich in carbohydrates on the IgM and IgG antibody production and the seric glucose concentration in diabetes. Nonobese diabetic (NOD) mice received, ad libitum, by oral route, the diet consisting of an aqueous extract (20 mg/mL) of the following flours: babassu mesocarp, manioc, corn or rice, during 120 days. The diet intake was monitored throughout this period. At the end, the weight variation, blood glucose, serum IgG and IgM antibody and IgM anti-insulin titers, were determined. The babassu and manioc flour extracts altered Purina chow intake and these animals also presented a significant increase in body weight. In contrast, treatment with rice flour resulted in a significant weight loss. Moderate to severe hyperglycemia was observed in the groups receiving rice and manioc, whereas treatment with babassu mesocarp flour and cornmeal resulted in hypoglycemia. The extracts did not alter the IgG concentration. On the other hand, the cornmeal extract caused a marked reduction in both total IgM and anti-insulin IgM antibody production. Although babassu mesocarp flour, cornmeal and manioc flour caused important variations in the parameters studied, only treatment with the rice flour extract anticipated the onset of diabetes in male mice genetically predisposed to the disease.

Antibody and cellular immune responses of naïve mares to repeated vaccination with an inactivated equine herpesvirus vaccine

Equine herpesvirus type 1 (EHV-1) continues to cause severe outbreaks of abortions or myeloencephalopathy in horses despite widely used vaccination. The aim of this work was to determine the effects of frequent vaccination with an inactivated EHV vaccine on immune development in horses. Fifteen EHV-1 naïve mares were vaccinated a total of 5 times over a period of 8 months with intervals of 20, 60, 90 and 60 days between vaccine administrations. Total antibody and antibody isotype responses were evaluated with a new sensitive EHV-1 Multiplex assay to glycoprotein C (gC) and gD for up to 14 months after initial vaccination. Antibodies peaked after the first two vaccine doses and then declined despite a third administration of the vaccine. The fourth vaccine dose was given at 6 months and the gC and gD antibody titers increased again. Mixed responses with increasing gC but decreasing gD antibody values were observed after the fifth vaccination at 8 months. IgG4/7 isotype responses mimicked the total Ig antibody production to vaccination most closely. Vaccination also induced short-lasting IgG1 antibodies to gC, but not to gD. EHV-1-specific cellular immunity induced by vaccination developed slower than antibodies, was dominated by IFN-γ producing T-helper 1 (Th1) cells, and was significantly increased compared to pre-vaccination values after administration of 3 vaccine doses. Decreased IFN-γ production and reduced Th1-cell induction were also observed after the second and fourth vaccination. Overall, repeated EHV vaccine administration did not always result in increasing immunity. The adverse effects on antibody and cellular immunity that were observed here when the EHV vaccine was given in short intervals might in part explain why EHV-1 outbreaks are observed worldwide despite widely used vaccination. The findings warrant further evaluation of immune responses to EHV vaccines to optimize vaccination protocols for different vaccines and horse groups at risk.

Immunostimulatory activities of Haberlea rhodopensis leaf extract on the specific antibody response: protective effects against γ-radiation-induced immunosuppression

Immunomodulatory and radioprotective properties of Haberlea rhodopensis extract (HRE) in relation to the primary and secondary specific antibody production were evaluated in New Zealand white rabbits. Rabbits were immunised with keyhole limpet hemocyanin (KLH) and were treated with HRE before irradiation with single dose of 2Gy. Control group without irradiation were also treated with HRE. Rabbits were reimmunised and treated again with HRE on Day 36. KLH-specific total IgM, G, A and KLH-specific IgG antibody production were determined during primary and secondary antibody response by enzyme-linked immunosorbent assay (ELISA). We observed a well-defined γ-radiation-induced immunosuppression on the total primary antibody production. Application of HRE restored primary total KLH-specific antibody as well as significantly increased primary KLH-specific IgG antibody response. We also established that HRE strongly enhanced KLH-specific antibody production after reimmunisation. We concluded that HRE exhibits immunostimulatory properties and could play protective role against γ-irradiation damage of immune response.

Abstract A11: The function of B cells in non-small cell lung cancer patients.

Abstract Lung cancer is the leading cause of cancer death in both men and women. Although novel therapies are emerging, we are interested in boosting tumor-specific immune responses in infiltrating lymphocytes. In a flow cytometric analysis of the immune infiltrate in non-small cell lung cancer (NSCLC) tumors (n=25), we found that the total number of infiltrating B cells (TIL-Bs) in the tumor versus the matched tumor-adjacent tissue was increased compared to other immune subsets, specifically T cells. Most NSCLC TIL-Bs were activated (CD69+ and CD86+) and had a memory phenotype (CD27+). Proliferation, antigen presentation, and antibody production studies were done to assess the range of function in TIL-Bs. Positively isolated TIL-Bs that were stimulated for three days through the antigen receptor had varied proliferation when compared to B cells from the tumor adjacent tissue, which suggests a spectrum of function in TIL-Bs at the site of the tumor. For antigen presentation, TIL-Bs stimulated CD4 tumor infiltrating lymphocytes (CD4 TILs) to proliferate without the addition of tumor lysate compared to the negative and positive controls (T cells alone and T cells activated through the T cell receptor, respectively). This suggests that TIL-Bs can present endogenous tumor antigens to CD4 TILs; however, antigen presentation is not as robust with TIL-Bs from other patient tumors, which is another example of a spectrum in TIL-B function. Finally, we assayed total antibody production (IgG) by TIL-Bs to determine if this is a function at the site of the tumor. Tumor and tumor adjacent B cells produce antibody in comparison to B cells from the periphery of a normal donor; this function is uniform unlike proliferation and antigen presentation. We predict that TIL-Bs have a range of function in their response against tumors. Our overall hypothesis is that tumor immunity in NSCLC is influenced by the molecular signature of TIL-Bs, which directs proliferation, presentation of tumor antigens to T cells, and production of anti-tumor antibodies. We are determining if proliferation, antigen presentation, and antibody production are tumor antigen-specific, particularly for the lung cancer-testis antigen XAGE-1b. This antigen was the most highly expressed in our patient tumor cells when analyzed by real time PCR and has been shown to play a role in NSCLC. We are performing experiments with TIL-Bs to test XAGE-1b specific proliferation and antigen presentation. Further, we generated a XAGE-1b ELISA to test the specificity of the antibodies generated by the TIL-Bs. Three out of twelve patient sera were positive for XAGE-1b-specific antibodies indicating that the XAGE-1b ELISA is working for the detection of XAGE-1b antibodies. Further, we are isolating cells from tumor, tumor adjacent, and normal (cancer-free) lung tissue to analyze the molecular signature of TIL-Bs to identify a pathway upstream of B cell signaling that may be altered and influencing TIL-B function. Results from this study will advance the understanding of the function of TIL-Bs in solid tumors, and which functions can be targeted. Ultimately, this project will provide a platform for a therapeutic vaccine for lung cancer patients, and more importantly, will help us understand why some patients do not respond well to immunotherapy. Such results may lead to the development of a prognostic tool to screen patients that will respond to vaccine. Citation Format: Tullia C. Bruno, Daniel Munson, Brandon Moore, Katherine Waugh, Jeffrey Kern, Jill E. Slansky. The function of B cells in non-small cell lung cancer patients. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A11.