IgG Antibodies In Mice Research Articles

Identification and Characterization of Antigenic Properties of Schistosoma japonicum Heat Shock Protein 90α Derived Peptides.

It is known that schistosome-derived antigens induce innate and adaptive immune responses that are essential for the formation of hepatic immunopathology. Here, we screened and synthesized four peptides derived from Schistosoma japonicum (S. japonicum) heat shock protein 90α (Sjp90α-1, -2, -3, and -4), which is widely expressed in adults and eggs of the genus S. japonicum and induces remarkable immune reactions. To define the antigenicity of these peptides, we stimulated splenocytes with peptides, and the results showed that only the Sjp90α-1 peptide could predominately induce the activation of dendritic cells (DCs) and macrophages as well as alter the proportion of follicular helper T (Tfh) cells. Next, CD4+ T cells were purified and cocultured with mouse bone-marrow-derived DCs (BMDCs) with or without Sjp90α-1 peptide stimulation in vitro, and the results showed that Sjp90α-1-stimulated BMDCs can significantly induce CD4+ T-cell differentiation into Tfh cells, while the direct stimulation of CD4+ T cells with Sjp90α-1 did not induce Tfh cells, indicating that the Sjp90α-1 peptide promotes Tfh cell differentiation depending on the presence of DCs. Furthermore, we selected and prepared an Sjp90α-1-peptide-based antibody and illustrated that it has excellent reactivity with the immunizing peptide and detects a single band of 29 kDa corresponding to the Sjp90α protein. The immunolocalization results showed that the protein recognized by this Sjp90α-1-peptide-based antibody is present in the mature eggs and the tegument of adults, implying that the parasite-derived peptide has a potential interaction with the host immune system. Finally, we evaluated antipeptide IgG antibodies and revealed a significantly higher level of anti-Sjp90α-1 peptide IgG antibodies in mice 3 weeks after S. japonicum infection. In conclusion, we illustrate that these synthetic peptides warrant further investigation by evaluating their antigen-specific immune response and their ability to efficiently induce Tfh cells. Moreover, they may constitute a potentially helpful method for the laboratory diagnosis of schistosomiasis japonica.

Towards the development of an epitope-focused vaccine for SARS-CoV-2

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions.Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

Role of TLR2/MyD88 in the production of specific IgM and IgG antibodies during the immunization of mice against Neospora caninum

Neospora caninum is a parasite relevant to the veterinary field. Innate and adaptive responses against N. caninum induce effector mechanisms that limit parasite replication, but little is known about their role in humoral response. Our work aimed to verify whether key molecules in the TLR2/MyD88-mediated response would impact the production of specific IgM and IgG antibodies in mice during immunization with soluble antigens of N. caninum. We observed that lack of IFN-gamma did not negatively affect the production of specific antibodies. However, mice genetically deficient in Toll-like receptor 2, Myeloid differentiation factor 88, Interleukin 12 and inducible nitric oxide synthase presented significant decrease in antibody levels against N. caninum antigens, which also reflected in the diversity of the antigen recognized by their serum. In that sense, we show here that molecules within this innate recognition pathway may present a direct impact in the induction of an antibody response against N. caninum.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide–CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar KD values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

Different serotypes of Escherichia coli flagellin exert identical adjuvant effects

Bacterial flagellin is a potent powerful adjuvant, which exerts its adjuvant activity by activating the Toll-like receptor 5 (TLR5) signaling pathway to induce host pro-inflammatory responses. Flagellin of Salmonella typhimurium (S. typhimurium) has shown strong adjuvant effects for a variety of vaccine candidates, however, the adjuvanticity of different serotypes of Escherichia coli (E. coli) flagellin (FliC) is unclear. To explore the adjuvant activity of different serotypes of E. coli flagellin, FliCH1, FliCH7, and FliCH19 recombinant flagellins were prokaryotically-expressed and purified. The adjuvanticity of three recombinant flagellins was evaluated by analyzing their abilities to induce the IL-8 production in human colorectal adenocarcinoma (Caco-2) cells and the immune responses to co-administrated FaeG antigen in mice. Sequence analysis showed that the N-and C-terminal regions are highly conserved, whereas the central region is hypervariable. The TLR5 recognized site is identical among these three serotypes of flagellins. Coomassie blue staining SDS-PAGE showed the molecular mass of FliCH1, FliCH7, and FliCH19 recombinant flagellin are 66 kDa, 64 kDa, and 68 kDa, which can be recognized by anti-FliCH1, FliCH7, and FliCH19 serum, respectively. Moreover, the flagellin serotypes induced similar levels of IL-8 and TNF-α production in Caco-2 cells, anti-FaeG specific IgG antibodies in mice, and IL-4 production in mice spleen cells. Our results indicated that E. coli flagellins can be an adjuvant for vaccine candidates and that different serotypes of E. coli flagellins possess identical adjuvant effects.

Dynamics of Whole Virus and Non-Structural Protein 1 (NS1) IgG Response in Mice Immunized with Two Commercial Tick-Borne Encephalitis Vaccines.

The presence of a non-structural protein 1 (NS1) in tick-borne encephalitis (TBE) vaccines and the possible induction of an NS1-specific immune response in vaccinated individuals remains a somewhat controversial topic. Previously, we detected the presence of NS1 in the Encepur TBE vaccine by mass spectrometry and found the induction of NS1-specific IgG antibodies in mice vaccinated with the FSME-Immun TBE vaccine. Here, in this follow-up study, we examined the dynamics and extent of the NS1-specific IgG response in mice vaccinated with these two vaccines in more detail and compared it with the IgG response to the whole virus (WV). Mice were vaccinated at two-week intervals with a total of six doses of each vaccine, and levels of IgG antibodies to TBE virus WV and NS1 were measured by ELISA after each dose. Both vaccines elicited a robust anti-WV IgG response after two doses. The Encepur vaccine did not elicit NS1-specific IgG even after all six doses. In contrast, the FSME-Immun vaccine triggered the production of NS1-specific IgG after four doses. The results indicate that FSME-Immun is the only vaccine that elicits an NS1-specific antibody response in mice. However, compared to WV-specific IgG, the NS1-specific response is weaker, and a higher number of doses is required to induce detectable levels of NS1-specific IgG antibodies.

The AJ072 full-length IgG antibody labels the surface and early endosomes of HEK cells by immunofluorescence

The AJ072 full-length mouse IgG antibody, against the human transferrin receptor, labels the plasma membrane and early endosomes of HEK cells by immunofluorescence.

Gut microbiota-reactive IgG regulates gut microbiota development and immunity against enteric pathogens in early life

Abstract The gut microbiota elicits antigen-specific IgG at steady state that cross-reacts to pathogenic Gram-negative Enterobacteriaceae to confer protection against systemic infection. The role of these IgG antibodies in the development of gut microbiome and immunity against enteric pathogens in early life, however, remains largely undefined. In this study, we demonstrate that gut microbiota-induced maternal IgG is transferred to the neonatal gut through maternal milk via the neonatal Fc receptor and confers critical protection by inhibiting colonization and attachment of C. rodentium to the mucosa in the neonatal gut. Enhanced neonatal immunity against oral C. rodentium infection was observed following maternal immunization with a gut microbiota-derived IgG antigen, outer-membrane protein A (OMP-A), or induction of protective IgG antibodies in germ-free mice after reconstitution with IgG-inducing gut bacteria. Furthermore, we generated a mouse model with complete deficiency in all IgG isotypes. IgG-deficient neonatal mice exhibited increased susceptibility to C. rodentium infection, altered gut microbiome and increased numbers of intestinal IL-17-producing γδ T cells driven by the gut microbiome at steady state. The altered gut microbiome persisted into adulthood in IgG KO mice and contributed to increased disease severity in DSS-induced colitis. Taken together, our studies have defined critical roles for gut microbiome-induced IgG antibodies in shaping the gut microbiome and immunity against Gram-negative enteric pathogens from early life to adulthood.

Induction of Broadly Cross-Reactive Antibodies by Displaying Receptor Binding Domains of SARS-CoV-2 on Virus-like Particles.

The impact of the COVID-19 pandemic has been reduced since the application of vaccination programs, mostly shown in the reduction of hospitalized patients. However, the emerging variants, in particular Omicron, have caused a steep increase in the number of infections; this increase is, nevertheless, not matched by an increase in hospitalization. Therefore, a vaccine that induces cross-reactive antibodies against most or all variants is a potential solution for the issue of emerging new variants. Here, we present a vaccine candidate which displays receptor-binding domain (RBD) of SARS-CoV-2 on virus-like particles (VLP) that, in mice, not only induce strong antibody responses against RBD but also bind RBDs from other variants of concern (VOCs). The antibodies induced by wild-type (wt) RBD displayed on immunologically optimized Cucumber mosaic virus incorporated tetanus toxin (CuMVTT) VLPs bind to wt as well as RBDs of VOCs with high avidities, indicating induction of strongly cross-reactive IgG antibodies. Interestingly, similar cross-reactive IgA antibodies were induced in immunized mice. Furthermore, these cross-reactive antibodies demonstrated efficacy in neutralizing wt (Wuhan) as well as SARS-CoV-2 VOCs (Beta, Delta, and Gamma). In summary, RBDs displayed on VLPs are capable of inducing protective cross-reactive IgG and IgA antibodies in mice, indicating that it may be possible to cover emerging VOCs with a single vaccine based on wt RBD.

Low-Molecular-Weight Heparin Enhanced Therapeutic Effects of Human Adipose-Derived Stem Cell Administration in a Mouse Model of Lupus Nephritis.

BackgroundLupus nephritis is a life-threatening complication in systemic lupus erythematosus (SLE), but the efficiency of current therapies involving corticosteroids, immunosuppressants, and biological agents is limited. Adipose-derived mesenchymal stem cells (ASCs) are gaining attention as a novel treatment for inflammation in SLE. Low-molecular-weight heparin (LMWH) exhibits multiple functions including anti-inflammatory, anti-fibrotic, and cell function-promoting effects. LMWH stimulation is expected to increase the therapeutic effect of ASCs by promoting cellular functions. In this study, we investigated the effects of LMWH on ASC functions and the therapeutic effect of LMWH-activated human-ASCs (hep-hASCs) in an SLE mouse model.MethodsThe cellular functions of human-derived ASCs stimulated with different LMWH concentrations were observed, and the optimum LMWH dose was selected. The mice were assigned to control, human-ASC, and hep-hASC groups; treatments were performed on week 20. Twenty-six week-old mice were sacrificed, and urine protein score, serum blood urea nitrogen, creatinine (Cr), anti-ds DNA IgG antibody, and serum IL-6 levels were analyzed in each group. Mice kidneys were evaluated via histological examination, immunohistochemical staining, and gene expression levels.ResultsLMWH significantly promoted ASC migration and proliferation and hepatocyte growth factor production and upregulated immunomodulatory factors in vitro. Hep-hASC administration resulted in significant disease activity improvement including proteinuria, serum Cr and IL-6 levels, anti-ds DNA IgG antibody, glomerulonephritis, and immune complex in mice. Inflammation and fibrosis in kidneys was significantly suppressed in the hep-hASC group; the gene expression levels of TNF-alpha, TIMP-2, and MMP-2 was significantly downregulated in the hep-hASC group compared with the control group.ConclusionsHep-hASC exhibited higher anti-inflammatory and anti-fibrotic effects than hASCs and may be a candidate tool for SLE treatment in future.

Preclinical evaluation of a candidate naked plasmid DNA vaccine against SARS-CoV-2

New generation plasmid DNA vaccines may be a safe, fast and simple emergency vaccine platform for preparedness against emerging viral pathogens. Applying platform optimization strategies, we tested the pre-clinical immunogenicity and protective effect of a candidate DNA plasmid vaccine specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The DNA vaccine induced spike-specific binding IgG and neutralizing antibodies in mice, rabbits, and rhesus macaques together with robust Th1 dominant cellular responses in small animals. Intradermal and intramuscular needle-free administration of the DNA vaccine yielded comparable immune responses. In a vaccination-challenge study of rhesus macaques, the vaccine demonstrated protection from viral replication in the lungs following intranasal and intratracheal inoculation with SARS-CoV-2. In conclusion, the candidate plasmid DNA vaccine encoding the SARS-CoV-2 spike protein is immunogenic in different models and confers protection against lung infection in nonhuman primates. Further evaluation of this DNA vaccine candidate in clinical trials is warranted.

Development of a Recombinant RBD Subunit Vaccine for SARS-CoV-2.

The novel coronavirus pneumonia (COVID-19) pandemic is a great threat to human society and now is still spreading. Although several vaccines have been authorized for emergency use, only one recombinant subunit vaccine has been permitted for widespread use. More subunit vaccines for COVID-19 should be developed in the future. The receptor binding domain (RBD), located at the S protein of SARS-CoV-2, contains most of the neutralizing epitopes. However, the immunogenicity of RBD monomers is not strong enough. In this study, we fused the RBD-monomer with a modified Fc fragment of human IgG1 to form an RBD-Fc fusion protein. The recombinant vaccine candidate based on the RBD-Fc protein could induce high levels of IgG and neutralizing antibody in mice, and these could last for at least three months. The secretion of IFN-γ, IL-2 and IL-10 in the RBD-stimulated splenocytes of immunized mice also increased significantly. Our results first showed that the RBD-Fc vaccine could induce both humoral and cellular immune responses and might be an optional strategy to control COVID-19.

Cellular and Humoral Immunogenicity of a Candidate DNA Vaccine Expressing SARS-CoV-2 Spike Subunit 1.

The urgent need for effective, safe and equitably accessible vaccines to tackle the ongoing spread of COVID-19 led researchers to generate vaccine candidates targeting varieties of immunogens of SARS-CoV-2. Because of its crucial role in mediating binding and entry to host cell and its proven safety profile, the subunit 1 (S1) of the spike protein represents an attractive immunogen for vaccine development. Here, we developed and assessed the immunogenicity of a DNA vaccine encoding the SARS-CoV-2 S1. Following in vitro confirmation and characterization, the humoral and cellular immune responses of our vaccine candidate (pVAX-S1) was evaluated in BALB/c mice using two different doses, 25 µg and 50 µg. Our data showed high levels of SARS-CoV-2 specific IgG and neutralizing antibodies in mice immunized with three doses of pVAX-S1. Analysis of the induced IgG subclasses showed a Th1-polarized immune response, as demonstrated by the significant elevation of spike-specific IgG2a and IgG2b, compared to IgG1. Furthermore, we found that the immunization of mice with three doses of 50 µg of pVAX-S1 could elicit significant memory CD4+ and CD8+ T cell responses. Taken together, our data indicate that pVAX-S1 is immunogenic and safe in mice and is worthy of further preclinical and clinical evaluation.

Distinct differences in analytical performance of two commercially available assays for specific IgE to egg white and house dust mite allergens

BackgroundMeasurements of allergen-specific IgE antibodies with different manufacturers’ assays show modest or poor agreement. This study compares analytical performance of specific IgE tests for whole allergen extracts and individual allergen components of two assay systems, IMMULITE and ImmunoCAP, using human sera as well as monoclonal antibodies.MethodsComparisons were performed for specific IgE to house dust mite (HDM, n = 44), egg white (EW, n = 36) and the allergen components rDer p 1, rDer p 2, nGal d 1, nGal d 2 and nGal d 4 in human sera and with monoclonal mouse/human chimeric IgE antibodies specific for the same allergen components. Competitive interference with IgE measurement was investigated using allergen-specific monoclonal IgG and IgG4 antibodies.ResultsMeasurements of IgE to HDM and EW in serial dilutions of human sera revealed weaker dilution linearity with IMMULITE than with ImmunoCAP. Analysis of five different monoclonal IgE antibodies with total and specific IgE assays, expected to return similar levels, gave an average specific/total IgE ratio of 0.96 (range 0.71–1.14) with ImmunoCAP and 1.89 (range 0.76–2.85) with IMMULITE, indicating overestimation of specific IgE by IMMULITE. With the EW IgE tests of both assay systems, measurements of a chimeric anti-Gal d 2 IgE antibody were unaffected by a competing mouse IgG antibody. While the same was true for measurement of a chimeric anti-Der p 1 IgE antibody using the HDM test in ImmunoCAP, a suppression of measured concentrations by up to 42% was observed in IMMULITE. Similarly, measurement of HDM-specific IgE in human sera by ImmunoCAP was unaffected by a competing monoclonal anti-Der p 2 IgG4 antibody while IMMULITE displayed a reduction of HDM-specific IgE values by up to 30%.ConclusionsIn this evaluation of analytical performance of two widely used assay systems, ImmunoCAP showed higher accuracy in quantitation of specific IgE and greater resistance against competing allergen-specific non-IgE antibodies which may arise through natural or dietary exposure, or as a result of allergen immunotherapy treatment.

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Abstract 1885: Potent tumoricidal activity of a FGFR4 CART in rhabdomyosarcoma

Abstract Despite decades of multi-module therapies, RMS remains incurable once it has metastasized, thus new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 10% of RMS, and directly activated by PAX3-FOXO1 fusion protein which makes it a tractable target for immunotherapy. We generated fifteen binders against FGFR4 and characterized them further as candidate molecules for immune therapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and made into scFvFc format (mouse Fv and Human IgG1 Fc). The chimeric form of 3A11 antibody was successfully produced in vitro and retained its FGFR4 specificity with an observed binding affinity at nanomolar range. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 scFvFc showed dose dependent binding to FGFR4 only. We then made 3A11 into a second generation CAR. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out and FGFR4 negative cells. Our in vivo testing also found that 3A11 CART was able to eliminate RMS cells in murine xenograft metastatic models. Here we report the successful generation of binders specific to human FGFR4. FGFR4 CAR developed from 3A11 was able to kill FGFR4 positive target cells both in-vivo and in-vitro. Thus, 3A11 CAR T cells targeting FGFR4 may provide effective immune therapies for rhabdomyosarcoma and other FGFR4 expressing cancers and clinical trials are planned. Citation Format: Adam Cheuk, Meijie Tian, Jeetendra Kumar, Peter Azorsa, Nityashree Shivaprasad, Dina Schneider, Berkley Gryder, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Joon-Yong Chung, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent tumoricidal activity of a FGFR4 CART in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1885.

Influence of Caudovirales Phages on Humoral Immunity in Mice.

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses

BackgroundH7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection.MethodsThe H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared.ResultsThe analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples.ConclusionThe McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.