IgG2a Antibodies Research Articles

Comparison of immunogenical potency of Leishmania major (MRHO/IR/75/ER) antigens prepared by 3 different methods in conjunction with Alum-Naltrexone adjuvant in BALB/c mice

ObjectivesTo evaluate the immunization ability of three Leishmania major antigens including formaline killed promastigotes (FKP), autoclaved Leishmania major (ALM) and soluble Leishmania antigen (SLA), they were used to immunize BALB/c mice in association with a mixture of alum and naltrexone (Alum-Nalt) as adjuvant. MethodsEach mouse from any of three groups received FKP, ALM or SLA antigens. Three additional groups were injected with same antigenes plus Alum-Nalt. One more group was injected with PBS to be the control group. Booster injections were given at 14th and 28th days. Two weeks after the last immunization, seven mice from each group were exposed to live promastigotes subcutaneously, and skin lesion formation among each mouse was monitored and recorded for 60 days. One week later, cellular and humoral immune responses of other immunized mice were evaluated by measuring the serum levels of anti-ALM-specific IgG1, IgG2a and total IgG antibodies, splenic anti-ALM-specific IFN-γ and IL-5 production and splenic lymphocyte proliferation after adding ALM. ResultsIn the challenge test, all mice immunized with ALM antigen and Alum-Nalt were prevented from formation of skin ulcer. Also, the mice from the same group showed higher IFN-γ production and splenic lymphocyte proliferation and higher anti-ALM-specific IgG2a production.

Glucocorticoid insensitivity by staphylococcal enterotoxin B in keratinocytes of allergic dermatitis is associated with impaired nuclear translocation of the Glucocorticoid Receptor α

Backgroundstaphylococcal enterotoxin plays an important role in patients with glucocorticoid (GC)-resistant atopic dermatitis (AD), but the exact mechanism is not fully understood. ObjectiveThe aim of this study was to investigate the mechanisms underlying the ability of staphylococcal enterotoxin B (SEB) to induce steroid insensitivity through impaired nuclear translocation of GRα in keratinocytes. MethodsThe steroid-resistant AD induced by SEB was assessed by analyzing dermatitis score, dermal thickness, scratching behavior, infiltrating cells/HPF, levels of SEB-specific IgE and IgG2a antibody. In addition, dexamethasone (DEX)-induced GRα nuclear translocation and keratinocyte-derived cytokines and chemokines were analyzed in the lesional keratinocytes of AD and in HaCaT cells. Furthermore, the expressions of immunophilins FKBP51, FKBP52 and HSP90 responsive to GC in HaCaT cells were determined in the presence of SEB. ResultsSEB dose-dependently diminished the inhibitory effect of DEX on dermatitis score, dermal thickness, scratching behavior, infiltrating cells/HPF, keratinocyte-derived cytokines and chemokines such as RANTES, MCP-1, TSLP and GM-CSF. In vivo and in vitro data showed that in the presence of DEX, SEB dose-dependently caused a marked decrease of GRα nuclear translocation in lesional keratinocytes of AD and in HaCaT cells. Importantly, in the presence of DEX, SEB increased the expression of FKBP51 and the product of keratinocyte-derived cytokines and chemokines in HaCaT cells. ConclusionThese results demonstrate that GC insensitivity by SEB in keratinocytes of AD is associated with impaired nuclear translocation of the GRα. Increased DEX-induced FKBP51 by SEB may contribute to accumulation of the GRα in cytoplasm of keratinocytes.

Mouse Chow Composition Influences Immune Responses and Food Allergy Development in a Mouse Model.

Our diet is known to substantially influence the immune response not only by support of mucosal barriers but also via direct impact on immune cells. Thus, it was of great interest to compare the immunological effect of two mouse chows with substantial differences regarding micro-, macronutrient, lipid and vitamin content on the food allergic response in our previously established mouse model. As the two mouse chows of interest, we used a soy containing feed with lower fatty acid (FA) amount (soy-containing feed) and compared it to a soy free mouse chow (soy-free feed) in an established protocol of oral immunizations with Ovalbumin (OVA) under gastric acid suppression. In the animals receiving soy-containing feed, OVA-specific IgE, IgG1, IgG2a antibody levels were significantly elevated and food allergy was evidenced by a drop of body temperature after oral immunizations. In contrast, mice on soy-free diet had significantly higher levels of IL-10 and were protected from food allergy development. In conclusion, soy-containing feed was auxiliary during sensitizations, while soy-free feed supported oral tolerance development and food allergy prevention.

Immunization with pneumococcal elongation factor Tu enhances serotype-independent protection against Streptococcus pneumoniae infection

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2 years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4+ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.

Structural effects and lymphocyte activation properties of self-assembled polysaccharide nanogels for effective antigen delivery

The success of immunotherapeutic vaccines is often limited by their inability to activate the cytotoxic T lymphocyte (CTL)-inducing Th1 pathway. We investigated the ability of self-assembled nanogels (CHP or CH-CDex) to activate this pathway, and characterised them chemically and biologically. Once loaded with antigen (ovalbumin, OVA) their OVA encapsulation and dissociation rates suggested the possibility of effective antigen delivery. The DC2.4 dendritic cell line took up either vaccine time-dependently, but both vaccines required CpG DNA for class I MHC presentation. The nanogel vaccines interacted with RAW264.7, a Balb/c mouse-derived macrophage cell line, and co-localised with lysosomes, suggesting their endocytotic internalization in RAW264.7. Both vaccines activated CTLs better than OVA alone. Unlike OVA alone, the nanogel vaccines induced IgG2a antibody production in mice, whereas the former induced IgG1 antibodies. OVA-nanogel delivery to the draining lymph nodes (DLNs) was higher than that for OVA alone, reaching a deeper medullary area. Furthermore, Langerin+ CD103+ DCs interacted with the nanogel vaccines effectively, which is a subset of cross-presentation DC, in the DLNs. The nanogel vaccines each had good anti-tumour efficacy in OVA tumour-bearing mice compared with the OVA alone. Thus, CHP and CH-CDex nanogels should be investigated further because of the great potential they offer for immunotherapy.

Robust mucosal and systemic responses against HTLV-1 by delivery of multi-epitope vaccine in PLGA nanoparticles

In this investigation, the immunogenicity of HTLV-1 fusion epitope-loaded PLGA nanoparticles (NPs) was assessed in the absence or presence of co-encapsulated CpG ODN adjuvant, in a mice model. For this purpose, the multi-epitope chimera including Tax, env, and gag immunodominant HTLV-1 epitopes was encapsulated in biodegradable PLGA NPs with or without CpG adjuvant. PLGA nanospheres produced by a double emulsion method had a size of <200 nm, and encapsulation efficiency of chimera antigen was 85%. The release profile of radiolabeled chimera indicated that only 17.4% and 20.1% of chimera were released from PLGA NPs without or with co-encapsulated CPG ODN during one month, respectively. The PLGA formulations significantly elevated titers of IgG1, IgG2a, and sIgA antibodies, as well as IL-10, and IFN-γ cytokines and also reduced the amount of TGF-β1 production relative to the other vaccines. Additionally, co-delivery of chimera and CpG ODN in PLGA NPs significantly promoted cellular and mucosal responses compared to the incorporation of CpG and chimera antigen. In summary, these results revealed that the sustained release of chimera from PLGA as an efficient polymeric system elicited potent cell-mediated and mucosal immunity without inflammatory responses against HTLV-1. Therefore, the proper design of vaccine formulation and immunization strategy are crucial factors to construct an efficient vaccine.

IL-17A promotes the neuroinflammation and cognitive function in sevoflurane anesthetized aged rats via activation of NF-\u03baB signaling pathway

BackgroundTo investigate the role of IL-17A in the neuroinflammation and cognitive function of aged rats anaesthetized with sevoflurane through NF-κB pathway.MethodThe aged and young adult rats were randomly divided into Control (inhale oxygen only), Sevoflurane (inhale oxygen and sevoflurane), Sevo (Sevoflurane) + anti-IL-17A (injected with IL-17A antibody, inhale oxygen and sevoflurane), and Sevo + NC groups (injected with IgG2a antibody, inhale oxygen and sevoflurane). Cognitive function was evaluated by Morris water maze and contextual fear conditioning tests. Tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6 and monocyte chemoattractant protein (MCP)-1 expressions in the hippocampus of rats were detected by ELISA (enzyme-linked immunosorbent assay) assay, and Nuclear factor (NF)-κB pathway-related proteins by Western blot.ResultsSevoflurane anaesthetized aged rats showed longer escape latency and swimming distance, fewer platform crossing times, shortened stay time in the platform quadrant compared to Control rats; In addition, increased levels in hippocampal expression of malondialdehyde (MDA), IL-17A, NF-κB p65, inducible nitric oxide synthase (iNOS) and COX-2, as well as a reduced level of superoxide dismutase (SOD) were also observed in these animals. However, the sevoflurane anesthetized aged rats treated with anti-IL-17A presented a completely opposite tendency concerning the above factors (all P < 0.05). Nevertheless, there was no significant difference in the acquisition of learning or memory, neuroinflammation and oxidative stress of young adult rats in all groups (all P > 0.05).ConclusionAnti-IL-17A may alleviate neuroinflammation and oxidative stress via inhibiting NF-κB pathway, thereby attenuating post-operative cognitive dysfunction (POCD) in aged rats anaesthetized with sevoflurane.

Evaluation and characteristics of immunological adjuvant activity of purified fraction of Albizia julibrissin saponins

ABSTRACTPurpose: The purified fraction of Albizia julibrissin saponins (AJSAF) was evaluated and characterized for the adjuvant activity on porcine reproductive and respiratory syndrome virus (PRRSV) vaccine.Methods: The effects of AJSAF on serum PRRSV N protein-specific antibody titers, splenocyte proliferation, natural killer (NK) cell activity, mRNA expression of cytokines and transcription factors, secretion of cytokines, T cells response in splenocytes, as well as delayed type hypersensitivity (DTH) in the mice immunized PRRSV vaccine were determined by ELISA, MTT assay, flow cytometry and quantitative real-time PCR (qRT-PCR).Results: AJSAF not only significantly enhanced the serum PRRSV N protein-specific IgG, IgG1, IgG2a and IgG2b antibody titers in the mice immunized with PRRSV CH-1R modified live vaccine (CH-1R MLV), inactivated vaccine (CH-1R IAV), and highly pathogenic JXA1-R modified live vaccine (JXA1-R MLV), but promoted the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and PRRSV N protein-stimulated splenocyte proliferation, the activities of NK cells and delayed type hypersensitivity (DTH) in the mice immunized CH-1R MLV. AJSAF also remarkably induced the production of both Th1 (IFN-γ) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 cytokines (IFN-γ and IL-2) and transcription factors (T-bet and STAT4) as well as Th2 cytokines (IL-4 and IL-10) and transcription factors (GATA-3 and STAT6) in splenocytes from the CH-1R MLV-immunized mice. Furthermore, AJSAF markedly increased the frequencies of PRRSV N protein-specific Th1 (INF-γ+ and IL-2+) and Th2 (IL-4+ and IL-10+) CD4 T cells as well as Tc1 (INF-γ+ and IL-2+) and Tc2 (IL-4+ and IL-10+) CD8 T cells in splenocytes from the CH-1R MLV-immunized mice.Conclusions: Our results demonstrated that AJSAF had a potential to enhance and improve immune responses and elicit both Th1/Th2 and Tc1/Tc2 response to PRRSV vaccine, and that AJSAF would be a promising adjuvant candidate for PRRSV vaccine.

A Pluronic® F127-based polymeric micelle system containing an antileishmanial molecule is immunotherapeutic and effective in the treatment against Leishmania amazonensis infection

Clioquinol (5-chloro-7-iodoquinolin-8-ol or ICHQ) was recently showed to presents an in vitro effective antileishmanial action, causing changes in membrane permeability, mitochondrial functionality, and parasite morphology. In the present study, ICHQ was incorporated into a Poloxamer 407-based polymeric micelles system (ICHQ/M), and its antileishmanial activity was in vivo evaluated in L. amazonensis-infected BALB/c mice. Amphotericin B (AmpB) and its liposomal formulation (Ambisome®) were used as controls. Parasitological and immunological evaluations were performed 30 days after the treatment. Results indicated more significant reductions in the average lesion diameter and parasite burden in ICHQ or ICHQ/M-treated mice, which were associated with the development of a polarized Th1 immune response, based on production of high levels of IFN-γ, IL-12, TNF-α, GM-CSF, and antileishmanial IgG2a antibody. Control groups´ mice produced high levels of IL-4, IL-10, and IgG1 isotype antibody. No organic toxicity was found by using ICHQ or ICHQ/M to treat the animals, although those receiving AmpB and Ambisome® have presented higher levels of renal and hepatic damage markers. In conclusion, results suggested that the ICHQ/M composition can be considered as an antileishmanial candidate to be tested against human leishmaniasis.

The essential role of complement in antibody-mediated resistance to Salmonella.

SummaryVaccines can serve as essential tools to prevent bacterial diseases via the induction of long‐lasting IgG responses. The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc‐gamma receptors (FcγRs) can potentially play a crucial role in IgG‐mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene‐targeted mice lacking either Fcγ RI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody‐mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O‐antigen in gene‐targeted mice, we demonstrate that the complement system is essential for the IgG‐mediated reduction of bacterial numbers in the tissues.

Assessment of Th1/Th2 Bias of STING Agonists Coated on Microneedles for Possible Use in Skin Allergen Immunotherapy.

Microneedle-based skin allergen-specific immunotherapy (AIT) can benefit from adjuvants that can stimulate a stronger Th1 response against the allergen. We evaluated two stimulator of interferon genes (STING) agonists, namely, cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP), as skin adjuvants using coated microneedles (MNs). For comparison, the approved subcutaneous (SC) hypodermic injection containing alum was used. Ovalbumin (Ova) was used as a model allergen. Ova-specific IgG2a antibody in serum, which is a surrogate marker for Th1 type immune response was significantly higher when STING agonists were used with coated MNs as compared to SC injection of Ova+alum in mice. In contrast, IgG1 antibody, a surrogate marker for Th2 type immune response, was at comparable levels in the MN and SC groups. Restimulation of splenocytes with Ova produced higher levels of Th1 cytokines (IFN-γ and IL-2) in the STING agonists MN groups as compared to the SC group. In conclusion, delivery of STING agonists into the skin using coated MNs activated the Th1 pathway better than SC- and MN-based delivery of alum. Thus, STING agonists could fulfill the role of adjuvants for skin AIT and even for infectious disease vaccines, where stimulation of the Th1 pathway is of interest.

Immune Stimulation of RAP domain binding protein (rTgRA15) from Toxoplasma gondii

ABSTRACTToxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG1 and IgG2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.

Therapeutic potential of artesunate in experimental autoimmune myasthenia gravis by upregulated T regulatory cells and regulation of Th1/Th2 cytokines.

Artesunate is a semi-synthetic derivative of a Chinese herb named Artemisia annua L. that is commonly used as an antimalarial agent in the history of traditional Chinese medicine. Many studies have reported artesunate possesses anti-inflammatory and immunoregulation properties. The present study was conducted to explore whether artesunate was effective in experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. Our data showed that artesunate could improve the clinical symptoms and suppress the development of EAMG. Artesunate exerted its immunomodulatory effects by inhibiting lymphocyte proliferation and the expression of costimulatory molecules CD86, modulating Th1/Th2 cytokine expression levels, and enhancing the level of Treg cells. The final result of administration of artesunate was the decreased synthesis of anti-R97-116 IgG, IgG2a, and IgG2b antibodies. The treatment effect of artesunate was more obvious at dose of 10 mg/kg. These date suggest that artesunate might be a potential drug for the treatment of human myasthenia gravis (MG).

A Leishmania hypothetical protein-containing liposome-based formulation is highly immunogenic and induces protection against visceral leishmaniasis

Leishmania proteins have been evaluated as vaccine candidates against leishmaniasis; however, most antigens present low immunogenicity and need to be added with immune adjuvants. A low number of licensed adjuvants exist on the market today; therefore, research conducted to produce new products is desirable. The present study sought to evaluate the immunogenicity and protective efficacy of a recombinant Leishmania hypothetical protein, namely LiHyR, administered with saponin or liposomes in BALB/c mice. Immunological and parasitological parameters were evaluated, and results showed significant protection against Leishmania infantum infection produced by both compositions in the immunized animals; however, this was not identified when the antigen was used alone. In addition, the liposomal formulation was more effective in inducing a polarized Th1 response in the vaccinated animals, which was maintained after challenge and reflected by lower parasitism found in all evaluated organs when the limiting dilution technique and RT-PCR assay were employed. The protected animals showed higher levels of protein and parasite-specific IFN-γ IL-2, IL-12, GM-CSF, and TNF-α, which were evaluated by capture ELISA and flow cytometry, in addition to a higher production of anti-protein and anti-parasite IgG2a antibodies, both before and after challenge. The Lip/rLiHyR combination induced higher IFN-γ production through both CD4+ and CD8+ T cell subtypes. Results indicate the possibility of using the LiHyR, containing a liposomal formulation, as a vaccine candidate against visceral leishmaniasis.

Immunization with a recombinant fusion protein protects mice against Helicobacter pylori infection

More than 50% of the world's population is infected with the bacterium Helicobacter pylori. If left untreated, infection with H. pylori can cause chronic gastritis and peptic ulcer disease, which may progress into gastric cancer. Owing to the limited efficacy of anti-H. pylori antibiotic therapy in clinical practice, the development of a protective vaccine to combat this pathogen has been a tempting goal for several years. In this study, a chimeric gene coding for the antigenic parts of H. pylori FliD, UreB, VacA, and CagL was generated and expressed in bacteria and the potential of the resulting fusion protein (rFUVL) to induce humoral and cellular immune responses and to provide protection against H. pylori infection was evaluated in mice. Three different immunization adjuvants were tested along with rFUVL: CpG oligodeoxynucleotides (CpG ODN), Addavax, and Cholera toxin subunit B. Compared to the control group that had received PBS, vaccinated mice showed significantly higher cellular recall responses and antigen-specific IgG2a, IgG1, and gastric IgA antibody titers. Importantly, rFUVL immunized mice exhibited a reduction of about three orders of magnitude in their stomach bacterial loads. Thus, adjuvanted rFUVL might be considered as a promising vaccine candidate for the control of H. pylori infection.

Evaluating the Vaccine Potential of a Tetravalent Fusion Protein (rBmHAXT) Vaccine Antigen Against Lymphatic Filariasis in a Mouse Model.

Lymphatic filariasis (LF) is a tropical parasitic infection of human transmitted by mosquitoes. Chronic infection results in severe physical disability in the infected patients. Although several potential vaccine antigens were identified by several groups, there are no licensed prophylactic vaccine to date against this infection in the human. Previous attempts from our laboratory to develop a trivalent prophylactic vaccine against LF showed that >90% protection could be achieved in rodent models. However, this trivalent vaccine gave only 35% protection in non-human primates. The major focus of this study was to develop a tetravalent prophylactic vaccine (rBmHAXT) and test the vaccine potential in a mouse model. We evaluated three different adjuvant formulations; alum, glucopyranosyl lipid adjuvant in stable emulsion (GLA/SE) alum (AL019), and mannosylated chitosan (MCA) to determine the optimum adjuvant formulation for rBmHAXT. Results presented in this study show that rBmHAXT + AL019 gave the highest rate of protection (>88%) against challenge infection, compared to rBmHAXT + AL007 (79%), rBmHAXT + MCA (79%) and controls. Analysis of the immune correlates of protection showed that all three adjuvants elicited high titer of antigen-specific IgG1, IgG2a, and IgG2b antibodies. High number of IFN-γ-producing antigen-specific memory cells were generated in the vaccinated animals irrespective of the adjuvants used. Similarly, spleen cells from rBmHAXT-vaccinated animals secreted IL-4, IL-10, and IFN-γ in response to rBmHAXT suggesting the generation of a balanced Th1/Th2 response. There was also an increase in IL-17-secreting cells in rBmHAXT-vaccinated animals. These findings thus suggest that rBmHAXT + AL019 is a better prophylactic formulation for LF.

Abstract 5761: Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions

Abstract Several agonistic antibodies against human CD40 are currently being explored as immune therapy for cancer patients. Previously, anti-CD40 antibody therapy showed some clinical efficacy, but significant toxicity at the efficacious dose limited further development. To develop an anti-CD40 agonistic antibody with an improved clinical therapeutic index, we focused on modifications to the heavy chain isotype that would impact interactions with specific Fc receptors. In mouse models, anti-CD40 murine IgG1 demonstrated stronger immune stimulatory activities than murine IgG2a. The CD40 murine IgG2a antibody induced acute cytokine release within hours of dosing and led to mortality weeks later. To generate human antibodies with an Fc structure that functions similarly to murine IgG1, human IgG1 Fc mutations with reduced binding on human CD16, enhanced binding on CD32A/B, and maintained binding on CD64 were identified through a proprietary mammalian cell surface display technology. A panel of anti-human CD40 antibodies carrying different Fc regions were created. In a CD40 transfected HEK293 NFκB reporter cell assay, where agonistic activity was not related to Fcγ receptor interactions, anti-CD40 human IgG2 demonstrated stronger agonistic activity than human IgG1 antibodies with either wild type or mutated Fc. This result confirmed the reported role of human IgG2 hinge in impacting the agonistic activity of anti-CD40. In assays using primary B cells or monocyte-derived dendritic cells, which constitutively express Fcγ receptors, a subset of anti-CD40 human IgG1 antibodies showed stronger potency than the corresponding human IgG2, due to the better Fcγ receptor crosslinking for IgG1 antibodies. Swapping the human IgG2 hinge into IgG1 Fc did not further enhance the agonistic activity of anti-CD40 antibodies in the primary cell assays, suggesting that the Fc-crosslinking plays a dominant role in mediating the agonistic activity of these anti-CD40 antibodies in biological systems with cells expressing Fcγ receptor. Engineering the proprietary human IgG1 Fc mutations to these anti-CD40 antibodies further enhanced agonistic activity compared to wild type IgG1 in primary cell assays. In addition to the potent agonistic activity, anti-CD40 with the IgG1 Fc mutations demonstrated lower ADCC and reduced cytokine production driven by Fc receptor activation in a co-culture system. Finally, an Fc modified anti-CD40 agonistic antibody demonstrated an acceptable safety profile in cynomolgous monkey toxicity studies. These data suggest anti-CD40 with the proprietary IgG1 Fc mutations represents a newer generation of CD40 agonists for oncology immune therapy. Citation Format: Shiming Ye, Diane Cohen, Donghee Choi, Siu sze Tan, Joanna Xiong, Yoshiko Akamatsu, Debra Chao, Diane Hollenbaugh, Fiona Harding. Identification of Fc mutations that optimize agonistic activity of human anti-CD40 monoclonal antibodies by enhancing appropriate Fcg receptor interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5761.

Thinking outside the Laboratory: Analyses of Antibody Structure and Dynamics within Different Solvent Environments in Molecular Dynamics (MD) Simulations

Monoclonal antibodies (mAbs) have revolutionized the biomedical field, directly influencing therapeutics and diagnostics in the biopharmaceutical industry, while continuing advances in computational efficiency have enabled molecular dynamics (MD) simulations to provide atomistic insight into the structure and function of mAbs. Despite the success of MD tools, further optimizations are still required to enhance the computational efficiency of complex mAb simulations. This issue can be tackled by changing the way the solvent system is modelled to reduce the number of atoms to be tracked but must be done without compromising the accuracy of the simulations. In this work, the structure of the IgG2a antibody was analyzed in three solvent systems: explicit water and ions, implicit water and ions, and implicit water and explicit ions. Root-mean-square distance (RMSD), root-mean-square fluctuations (RMSF), and interchain angles were used to quantify structural changes. The explicit system provides the most atomistic detail but is ~6 times slower in its exploration of configurational space and required ~4 times more computational time on our supercomputer than the implicit simulations. Overall, the behavior of the implicit and explicit simulations is quantifiably similar, with the inclusion of explicit ions in the implicit simulation stabilizing the antibody to reproduce well the statistical fluctuations of the fully explicit system. Therefore, this approach holds promise to maximize the use of computational resources to explore antibody behavior.

Expression and immunogenicity analysis of EsxA protein of Staphylococcus aureus isolated from cow milk

To study the immunogenicity of EsxA protein of Staphylococcus aureus, the EsxA-pET-28a recombinant plasmid was constructed and the expression product was analyzed by SDS-PAGE and Western blotting after the positive recombinant plasmid was induced by IPTG. Mice were immunized with purified EsxA protein and then the IgG, IgG1 and IgG2a antibody were detected with indirect ELISA. Then the histopathological examination, bacteria loading and immune protection of immunized mice were studied after challenge with S. aureus. The recombinant protein EsxA was successfully induced and expressed. After immunization the EsxA specific antibody titer could reach 1:900. Bacteria loading and pathological damage of liver, spleen and kidney were reduced after immunization with EsxA in the immunized mice. The protection rate of immunized mice was 75%. In conclusion, EsxA protein has good immunogenicity.

Unexpected involvement of IL-13 signalling via a STAT6 independent mechanism during murine IgG2a development following viral vaccination.

In this study, recombinant pox viral vaccination was shown to induce highly elevated IgG2a and low IgG1 antibody expression in mice lacking IL-4 or STAT6, whilst IL-13-/- mice exhibited elevated IgG1, but very low IgG2a. These findings revealed that IL-13 and IL-4 differentially regulated antibody development. To understand this further, when STAT6-/- mice were given a vaccine co-expressing IL-13Rα2 that temporarily sequestered IL-13, significantly reduced IgG2a expression, was detected. These findings for the first time demonstrated that IL-13 regulated IgG2a differentiation utilising an alternative IL-13R signalling pathway independent of STAT6 (IL-13Rα2 pathway). This was further corroborated by the (i) elevated IL-13Rα2 expression detected on STAT6-/- lung MHCII+ CD11c+ cells 24 h post IL-13 inhibitor vaccination and ii) significant up-regulation of IL-13Rα2 expression on spleen and lung derived MHCII+ CD11c+ following inhibition of STAT6 signalling in vitro, or vaccination with IL-4R/STAT6 antagonist in vivo. When T follicular helper (Tfh) cells which regulate antibody differentiation were assessed post vaccination, although no difference in IL-4 expression was observed, greatly reduced IFN-γ expression was detected in IL-13-/- and STAT6-/- mice compared to wild-type. These findings support the notion that the balance of IL-13 level at the vaccination site can differentially regulate T and B-cell immune outcomes.