Antigen-specific Antibody Isotype Research Articles

Antibody response in children with multisystem inflammatory syndrome related to COVID-19 (MIS-C) compared to children with uncomplicated COVID-19.

To comprehensively analyze the quality of the antibody response between children with Multisystem inflammatory syndrome (MIS-C) and age-matched controls at one month after SARS-CoV-2 exposure, and infected in the same time-period. Serum from 20 MIS-C children at admission, and 14 control children were analyzed. Antigen specific antibody isotypes and subclasses directed against various antigens of SARS-CoV-2 as well as against human common coronavirus (HCoVs) and commensal or pathogenic microorganisms were assessed by a bead-based multiplexed serological assay and by ELISA. The functionality of these antibodies was also assessed using a plaque reduction neutralization test, a RBD-specific avidity assay, a complement deposition assay and an antibody-dependent neutrophil phagocytosis (ADNP) assay. Children with MIS-C developed a stronger IgA antibody response in comparison to children with uncomplicated COVID-19, while IgG and IgM responses are largely similar in both groups. We found a typical class-switched antibody profile with high level of IgG and IgA titers and a measurable low IgM due to relatively recent SARS-CoV-2 infection (one month). SARS-CoV-2-specific IgG antibodies of MIS-C children had higher functional properties (higher neutralization activity, avidity and complement binding) as compared to children with uncomplicated COVID-19. There was no difference in the response to common endemic coronaviruses between both groups. However, MIS-C children had a moderate increase against mucosal commensal and pathogenic strains, reflecting a potential association between a disruption of the mucosal barrier with the disease. Even if it is still unclear why some children develop a MIS-C, we show here that MIS-C children produce higher titers of IgA antibodies, and IgG antibodies with higher functionality, which could reflect the local gastro-intestinal mucosal inflammation potentially induced by a sustained SARS-CoV-2 gut infection leading to continuous release of SARS-CoV-2 antigens.

Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes.

Antibody characterization is essential for understanding the immune system and development of diagnostics and therapeutics. Current technologies are mainly focusing on the detection of antigen-specific immunoglobulin G (IgG) using bulk singleplex measurements, which lack information on other isotypes and specificity of individual antibodies. Digital immunoassays based on nucleic acid amplification have demonstrated superior performance by allowing the detection of single molecules in a multiplex and sensitive manner. In this study, we demonstrate for the first time an immuno-rolling circle amplification (immuno-RCA) assay for the multiplex detection of three antigen-specific antibody isotypes (IgG, IgA, and IgM) and its integration with microengraving. To validate this approach, we used the autoimmune disease immune-mediated thrombotic thrombocytopenic purpura (iTTP) as the model disease with anti-ADAMTS13 autoantibodies as the diagnostic target molecules. To identify the anti-ADAMTS13 autoantibody isotypes, we designed a pool of three unique antibody-oligonucleotide conjugates for identification and subsequent amplification and visualization via RCA. To validate this approach, we first confirmed an assay specificity of >88% and a low limit of detection of 0.3 ng/mL in the spiked buffer. Subsequently, we performed a dilution series of an iTTP plasma sample for the multiplex detection of the three isotypes with higher sensitivity compared to an enzyme-linked immunosorbent assay. Finally, we demonstrated single-cell analysis of human B cells and hybridoma cells for the detection of secreted antibodies using microengraving and achieved a detection of 23.3 pg/mL secreted antibodies per hour. This approach could help to improve the understanding of antibody isotype distributions and their roles in various diseases.

The Thomsen-Friedenreich Antigen-Specific Antibody Signatures in Patients with Breast Cancer.

Alterations in the glycosylation of serum total immunoglobulins show these antibodies to have a diagnostic potential for cancer but the disease-related Abs to the tumor-associated antigens, including glycans, have still poorly been investigated in this respect. We analysed serum samples from patients with breast carcinoma (n = 196) and controls (n = 64) for the level of Thomsen-Friedenreich (TF) antigen-specific antibody isotypes, their sialylation, interrelationships, and the avidity by using ELISA with the synthetic TF-polyacrylamide conjugate as an antigen and the sialic acid-specific Sambucus nigra agglutinin (SNA) and ammonium thiocyanate as a chaotrope. An increased sialylation of IgG and IgM, but a lower SNA reactivity of IgA TF antibodies, and a higher level and avidity of the TF-specific IgA were found in cancer patients. Other cancer-related signatures were the highly significant increase of the IgG/IgA ratio and the very low SNA/IgA index in cancer, including patients with an early stage of the disease. These changes showed a good diagnostic potential with about 80% accuracy. Thus, the level of naturally occurring anti-TF antigen antibodies, their sialylation profile, isotype distribution, and avidity displayed cancer-specific changes that could serve as novel noninvasive Ab-based biomarkers for early breast cancer.

Replicon Particle Expressing the E2 Glycoprotein of Bovine Viral Diarrhea Virus Immunization and Evaluation of Antibody Response.

The objective of this study was to develop a new antigen delivery system using an alphavirus replicon particle (RP) to induce humoral antibody responses against bovine viral diarrhea virus (BVDV) recombinant antigen produced from envelope glycoprotein E2. An alphavirus RP expressing the E2 glycoprotein of BVDV was used for immunization of pigs. A fluorescent microsphere immunoassay (FMIA) has been applied to detect BVDV E2 antigen-specific antibody isotype in pig immunized with alphavirus RP. Full-length BVDV E2 (aa 1-375) was cleaved into several pieces, eight E2 DNA fragments, including full-length DNA, were cloned into expression vector pHUE, and the recombinant proteins expressed in BL-21 (DE3) Escherichia coli. After successful conjugation of purified proteins with microsphere beads, a multiplex FMIA platform was constructed, and BVDV E2 alphavirus-based RP-immunized animal serum samples were tested in the presence of bead-bound antigen targets. The results were represented as mean fluorescence intensity (MFI); the MFI values were converted to sample value/positive value (S/P) ratios. BVDV E2 (aa 1-183) showed the highest MFI values of eight recombinant E2 fragments when the specific activity of each fragment was tested. In immunized animals, data for BVDV E2-specific IgA, IgG, and IgM in serum and only IgG and IgA in oral fluids were recorded. The MFI values for the positive serum sample showed a 100-fold increase compared with the negative serum sample. Antibody isotype to BVDV E2 antigens showed that IgG > IgM > IgA in serum, whereas IgG > IgA > IgM in oral fluids. The data presented in this study suggested that boosting with the same doses of alphavirus RP in 3-week intervals may potentially enhance antibody response. The experimental results demonstrate that alphavirus RP-expressing BVDV E2 antigen induces antibody response in pig.

Construction and Preliminary Immunobiological Characterization of a Novel, Non-Reverting, Intranasal Live Attenuated Whooping Cough Vaccine Candidate

We describe the construction and immunobiological properties of a novel whooping cough vaccine candidate, in which the aroQ gene, encoding 3-dehydroquinase, was deleted by insertional inactivation using the kanamycin resistance gene cassette and allelic exchange using a Bordetella suicide vector. The aroQ B. pertussis mutant required supplementation of media to grow but failed to grow on an unsupplemented medium. The aroQ B. pertussis mutant was undetectable in the trachea and lungs of mice at days 6 and 12 post-infection, respectively. Antigen-specific antibody isotypes IgG1 and IgG2a, were produced, and cell-mediated immunity [CMI], using interleukin-2 and interferon-gamma as indirect indicators, was induced in mice vaccinated with the aroQ B. pertussis vaccine candidate, which were substantially enhanced upon second exposure to virulent B. pertussis. Interleukin- 12 was also produced in the aroQ B. pertussis-vaccinated mice. On the other hand, neither IgG2a nor CMI-indicator cytokines were produced in DTaP-vaccinated mice, although the CMI-indicator cytokines became detectable post-challenge with virulent B. pertussis. Intranasal immunization with one dose of the aroQ B. pertussis mutant protected vaccinated mice against an intranasal challenge infection, with no pathogen being detected in the lungs of immunized mice by day 7 post-challenge. B. pertussis aroQ thus constitutes a safe, non-reverting, metabolite-deficient vaccine candidate that induces both humoral and cellmediated immune responses with potential for use as a single-dose vaccine in adolescents and adults, in the first instance, with a view to disrupting the transmission cycle of whooping cough to infants and the community.

Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae)

Antigen-specific antibody responses, T cell subsets and cytokine profiles were studied in 7 heifers naturally infested by Hypoderma sp. in Northwestern Spain. Immunoglobulin G (IgG) levels increased significantly at the end of the endogenous life cycle of the parasite (Mr). Similarly, IgG1 subclass increased considerably when first instars (L1) started their migration towards the back (Nv-Ja), whereas IgG2 increased earlier, coinciding with the arrival of L1 to the resting sites (Jn-Jl). Both subclasses decreased significantly when L3 began to leave the host. IgM levels and CD4 and CD8 profiles hardly oscillated throughout the life cycle of the parasite into the host. The CD4/CD8 ratio showed helper T cell predominance. Serum interferon-γ (IFN-γ) concentrations decreased from October to the end of the study. Interleukin 4 (IL-4) concentrations decreased in January and increased in February and May. There were a significant positive relationship between IL-4 and IgG2 subclass and a negative correlation between IFN-γ, IgG and IgG1 and also between IgM and CD2 and CD8 counts. These results suggest that in the early phases of natural primoinfestations by Hypoderma there is a slight predominance of a Th1 response, characterized by high IgG2 and IFN-γ levels, which is followed by a Th2 response with a clear predominance of IgG1 and IL-4.

TSCOT + Thymic Epithelial Cell-Mediated Sensitive CD4 Tolerance by Direct Presentation

Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.

Autoantigen arrays for multiplex analysis of antibody isotypes

We describe here a microarray-based method for multiplexed, antigen-specific assessment of immunoglobulin (Ig) subclasses. We used 1152-feature arrays composed of 140 antigens or antigen fragments to detect isotype-specific mAb, to quantitatively monitor changes in isotype mAb concentration, and to profile antigen-specific antibody isotype production in a murine model of autoimmunity. This platform can be easily adapted to a variety of applications, and has the potential to elucidate mechanisms that govern development and evolution of antibody responses in in vivo and in vitro systems.

Complex pattern of Th1 and Th2 activation with a preferential increase of autoreactive Th1 cells in BALB/c mice with proteoglycan (aggrecan)-induced arthritis.

The central role of CD4+ T cells and the balance between T helper (Th) subpopulations in the pathogenesis of autoimmune diseases have been extensively studied. Proteoglycan (aggrecan)-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA), which is characterized by a Th1 dominance at the onset of the disease. In addition to CD4+ T cells, antigen-presenting B cells and autoantibodies seem to play an important role in the development and regulation of PGIA. To identify proteoglycan-specific CD4+ T cell subsets and Th1- and Th2-supported antibody isotypes during the progression of PGIA, spleen cells of proteoglycan-immunized BALB/c mice were harvested at different times of immunization, and at different stages of the disease, and their cytokine production and antigen-specific antibody isotype profiles were determined by enzyme-linked immunospot (ELISPOT) assays. Both Th1 and Th2 cytokine-producing cells, with the predominance of IL-4/IL-5-secreting cells, were detected during the prearthritic stage, and a shift toward a Th1 dominance was observed at the time of onset of arthritis. Tissue homogenates of acutely inflamed joints contained significantly higher levels of interferon-gamma than IL-4. The prearthritic period and both the acute and chronic phases of joint inflammation were characterized by IgG1 dominance in the sera and this correlated with the number of IgG1-secreting B cells in the spleen. However, the ratio of autoreactive IgG1/IgG2a-secreting cells decreased in arthritic animals. These results indicate the activation and possible regulatory roles of both Th1 and Th2 subsets in the autoimmune process, with the necessity of a relative increase of autoreactive Th1 cells for the induction of joint inflammation.

Plasmodium falciparum: A Major Role for IgG3 in Antibody-Dependent Monocyte-Mediated Cellular Inhibition of Parasite Growth in Vitro

Tebo, A. E., Kremsner, P. G., and Luty, A. J. F. 2001. Plasmodium falciparum: A major role for IgG3 in antibody-dependent monocyte-mediated cellular inhibition of parasite growth in Vitro. Experimental Parasitology98, 20–28. In an attempt to identify parasite antigen-specific antibody isotype(s) mediating inhibition of growth in vitro, we tested unfractionated sera and their corresponding purified antibody isotype-containing fractions in in vitro assays with asexual-stage parasites of Plasmodium falciparum in the presence or absence of monocytes. Using affinity purification techniques we fractionated individual and pooled serum samples from semi-immune Gabonese adults, to obtain samples containing either IgG1, 2, 3, and 4, IgG1, 2, and 4, or IgG3 alone, and a non-IgG fraction. Antibodies were quantified spectrophotometrically and the presence of different isotypes in individual fractions was confirmed by protein gel electrophoresis. In the absence of monocytes, we observed inhibition of parasite growth with whole serum and varying levels of either growth enhancement or inhibition with purified Ig-containing fractions. When used in a standardized assay of antibody-dependent cellular inhibition (ADCI) with a monocyte:infected erythrocyte ratio of 1:1, seven of eight serum samples inhibited growth to a mean level of 42%, and the different Ig-containing fractions displayed varying mean levels of inhibition: IgG3, 44%; IgG1-4, 22%; IgG1, 2, and 4, 10%; and non-IgG, - 10%. The results suggest that, among the different isotypes present in the serum of semi-immune individuals, parasite antigen-specific IgG3 in particular may play an important role in controlling parasitemia via an ADCI mechanism involving monocyte- derived mediators.

Chlamydia trachomatis Mouse Pneumonitis Lung Infection in IL-18 and IL-12 Knockout Mice: IL-12 Is Dominant over IL-18 for Protective Immunity

Interferon (IFN)-gamma is a key to protective immunity against a variety of intracellular bacterial infections, including Chlamydia trachomatis. Interleukin (IL)-18, a recently identified Th1 cytokine, together with IL-12 is a strong stimulator for IFN-gamma production. We investigated the relative roles of IL-18 and IL- 12 in protective immunity to C. trachomatis mouse pneumonitis (MoPn) infection using gene knockout (KO) and wild-type (WT) mice. Mice were intranasally infected with C. trachomatis MoPn and protective immunity was assessed among groups of mice by daily body weight changes, lung growth of MoPn, and histopathological appearances at day 10 postinfection. The corresponding immune responses for each group of mice at the same postinfection time point were evaluated by measuring antigen-specific antibody isotype responses and cytokine profiles. Our results showed that IL-18 deficiency had little or no influence on clearance of MoPn from the lung, although KO mice exhibited slightly more severe inflammatory reactions in lung tissues, as well as reduced systemic and local IFN-gamma production, compared with WT mice. Results with IL-18 KO mice were in sharp contrast to those observed with IL-12 KO mice that showed substantially reduced clearance of MoPn from the lungs, substantial reductions of antigen-specific systemic and lung IFN-gamma production, decreased ratio of MoPn-specific immunoglobulin G (IgG)2a/IgG1, and severe pathological changes in the lung with extensive polymorphonuclear, instead of mononuclear, cell infiltration. Exogenous IL-12 or IL-18 was able to increase IFN-gamma production in IL-18 KO mice; whereas, only exogenous IL-12, but not IL-18, enhanced IFN-gamma production in IL-12 KO mice. Caspase-1 is the key protease for activation of IL-18 precursor into the bioactive form, and caspase-1 KO mice also displayed similar bacterial clearance and body weight loss to that in WT mice at early stages of MoPn infection. This further confirmed that IL-18 was not essential for host defense against chlamydia infection. These results suggest that IL-12, rather than IL-18, plays the dominant role in the development of protective immunity against chlamydia lung infection, although both cytokines are involved in the in vivo regulation of IFN-gamma production.

Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene

Equine influenza virus infection remains one of the most important infectious diseases of the horse, yet current vaccines offer only limited protection. The equine immune response to natural influenza virus infection results in long-term protective immunity, and is characterized by mucosal IgA and serum IgGa and IgGb antibody responses. DNA vaccination offers a radical alternative to conventional vaccines, with the potential to generate the same protective immune responses seen following viral infection. Antigen-specific antibody isotype responses in serum and mucosal secretions were studied in ponies following particle-mediated delivery of hemagglutinin (HA)-DNA vaccination on three occasions at approximately 63-day intervals. One group of four ponies were vaccinated at skin and mucosal sites and the another group were vaccinated at skin sites only. All ponies were subjected to a challenge infection 30 days after the third vaccination. Skin and mucosal vaccination provided complete protection from clinical signs of infection, while skin vaccination provided partial protection; DNA vaccination provided partial protection from viral shedding. DNA vaccination generated only IgGa and IgGb antibody responses, which occurred with a higher frequency in the skin and mucosa vaccinated ponies. No mucosal IgA response was generated prior to challenge infection and IgA responses were only detected in those ponies which shed virus postchallenge. These results demonstrate that HA-DNA vaccination induces IgG(a) and IgG(b) antibody responses which are associated with protection in the absence of mucosal IgA responses. In addition, additional DNA vaccinations of mucosal sites increased protection and the frequency of seroconversion in ponies.

Systemic levels of cytokines and GAD-specific autoantibodies isotypes in Chinese IDDM patients

It is not clear if a Th1/Th2 imbalance in Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) would lead to a particular antigen-specific IgG subclass dominant as had been shown in the mouse model. In new-onset Type 1 diabetics, an autoantibody response to glutamate decarboxylase (GADab) is frequently observed but the GADab subclass repertoire is not well-established. We determined the systemic levels of representative Th1 and Th2 cytokines and the GADab IgG subclass distribution in 41 Chinese IDDM patients of whom 26 were recently diagnosed (≤1 year) and 32 had GADab, to ascertain a likely association of antigen-specific antibody isotype and the Th1/Th2 dichotomy. With high-sensitivity ELISA systems that measure sub-picogram cytokine concentrations, 26 of the 41 patients (63.4%) had at least one of the pro-inflammatory Th1 cytokines (TNF-α, IFN-γ and IL-12) detected. Fewer patients (4/41) had the anti-inflammatory Th2 cytokine IL-4 detected. For IL-10, all subjects had measurable quantities but only three diabetics had levels above the upper limit for healthy subjects ( n=20). Grouped according to the profile of detectable cytokines, there were 24 Th1, 2 Th2 and 2 Th0 patterns. GAD-specific IgG1 antibody was more frequently expressed; 22 of 32 GADab[+] patients. The rank order for the GADab subclasses was IgG1>4>3>2; IgG2 was found in 11 GADab[+] patients. Recent-onset diabetics have a similar ranking of the GAD-specific IgG subclasses. In human Type 1 diabetes, a predominance of GAD-specific IgG1 antibody response is observed together with a dominant Th1 cytokine pattern.

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Antibody to Mycobacterium tuberculosis 65 kDa heat shock protein in patients with rheumatoid arthritis--a survey of antigen-specific antibody isotypes and subclasses in an endemic area of previous tuberculosis infection.

To clarify the significance of the humoral immune response triggered by the Mycobacterium tuberculosis (M.tb) 65 kDa heat shock protein (hsp) in the pathogenesis of rheumatoid arthritis (RA). M.tb 65 kDa hsp-specific IgG, IgA, IgM, and IgG subclass antibodies in serum or synovial fluid (SF) of RA and other disease patients were determined by enzyme linked immunosorbent assay (ELISA). RA patients did not show any characteristic increase in mycobacterial 65 kDa hsp-specific antibodies compared with healthy individuals. In contrast, antigen-specific IgG and IgG2 antibody titres in the serum of RA patients were significantly lower than those of patients with tuberculosis and normal controls. In addition, there was also no significant difference in antibody titre between the serum and SF of RA patients, nor was any significant difference found between the SF of RA and Reiter's patients. The failure to detect a significant increase in IgG anti-M.tb 65 kDa hsp antibodies in RA patients does not exclude the possibility of microbial immunity in the aetiology of RA. Nevertheless, anti-M.tb 65 kDa hsp antibodies clearly do not appear to be the disease specific markers for RA and their relatively reduced concentrations may argue against their playing a major role in the disease pathogenesis.

Induction of oral tolerance to S-antigen induced experimental autoimmune uveitis by a uveitogenic 20mer peptide

Experimental autoimmune uveitis (EAU) in Lewis rats is a T-cell dependent disease, which is induced by immunization with retinal S-Antigen (S-Ag) or its unveitogenic peptides. We have recently reported that the oral administration of S-Ag prior to the uveitogenic challenge results in suppression of the disease and of the cellular responses. We examined the effect of oral administration of a recently described uveitogenic peptide (Peptide 35) on the development of EAU induced with the whole protein. The severity of inflammation and retinal destruction, as well as the cellular proliferative responses, were suppressed compared to controls fed with Keyhole Limpet Hemocyanin (KLH). In addition, the levels of antigenspecific antibody isotypes in the serum of gavaged rats were determined. Although (IgA) levels were reduced in rats gavaged with S-Ag or the uveitogenic peptide, IgM and IgG levels were unaltered. Thus, the oral administration of Peptide 35 approaches the state of unresponsiveness seen in S-Ag feeding. In addition, the unresponsiveness can be demonstrated on both the cellular and humoral level.