Antibody Subclasses Research Articles

Single-chain antibodies from alpaca for the detection of Fasciola hepatica antigens

To produce recombinant single-chain antibodies from alpaca that will bind to the excreted-secreted (ES) Fasciola hepatica antigen with high affinity and specificity, so as to develop new diagnostic technologies of human and animal fascioliasis. A gene bank of DNA of the variable dominions of heavy single-chain antibodies (VHH) has been created, based on mononuclear cells of peripheral blood of an alpaca immunized with the ES antigen of F. hepatica. The gene bank was screened with the ES antigen by differential phage display, selecting ten VHH that bind specifically to ES. The anti-ES VHH was cloned in an expression vector, the recombinant protein (VHH-ES1) of 15.3 kDa was produced by fermentation in E. coli and purified to homogeneity by affinity chromatography. The binding of VHH-ES1 to the ES antigen was evaluated by ELISA using VHH-ES1 as capture antibody, policlonal anti-ES serum of rabbit and conjugated rabbit anti IgG with radish peroxidase. A VHH that binds to the ES antigen (VHH-ES1) has been identified through differential phage display and produced by fermentation in E. coli; this corresponds to an antibody of the long-hinge IgG2 subclass. The binding of the VHH-ES1 antibody to the antigen shows linearity with respect to the concentration of ES in the 50-5,000 ng/mL range and the limit of detection value of the antigen is in the 30-170 ng/mL range of ES (R2=0.99). The VHH-ES1 binds with affinity and specificity to the ES antigen of F. hepatica and is a promissory antibody to be assessed for the development of new fascioliasis diagnostic technologies.

Antibodies prevalence against Haemophilus influenzae type b in Jeddah population, Saudi Arabia. II. Antibodies subclasses.

Our previous reported that Jeddah, Saudi Arabia population have a comparable and protective herd immunity represented by IgG, IgA and IgM against Haemophilus influenzae type b (Hib), by using indirect ELISA test was used to evaluate Haemophilus influenzae type b (Hib) anti-polyribosyl-ribitol phosphate (PRP) total antibodies (IgM, IgG, IgA) in 1,003sera samples. Current report was evaluated the IgG and IgA subclasses responsible about this protection using same methodology. IgG, IgA, and their subclasses are responsible about this circulating protection. Our study will consider the first report evaluate the levels of IgA subclasses and its relation to ages, as well as the relations between anti-Hib antibody subclass and age. The current results demonstrated that the highest levels concentrated in IgG1 and IgG2, while IgG3 and IgG4 showed the lowest levels. So, their concentrations were arranged in IgG1 > IgG2 > IgG3 > IgG4. The results indicated that the age and gender have no effect on both IgG or IgA subclasses in healthy immunized individuals enrolled. While, IgA1 concentrations were significantly higher than IgA2 in all age categories regardless of gender. It seem that the IgG1, IgG2 and IgA1 subclasses were the main constituent of Jeddah herd immunity against Hib. Finally, to the best of our knowledge, there were no previous reports that focusing on the levels of IgA subclasses and its relation to ages, so our study considers the first worldwide.

Data Coming out of the Human Inhibitor PUP Study (HIPS) Reveal 4 Subgroups of Patients with Distinct Antibody Signatures

Data Coming out of the Human Inhibitor PUP Study (HIPS) Reveal 4 Subgroups of Patients with Distinct Antibody Signatures

Antibodies against recombinant alpha-galactosidase A in Fabry disease: Subclass analysis and impact on response to treatment

BackgroundTreatment of Fabry disease (FD) with recombinant alpha-galactosidase A (r-αGAL A) is complicated by the formation of anti-drug antibodies in the majority of male patients with the classical disease phenotype. Detailed information regarding antibody subtypes, onset and persistence of antibody development and their effect on treatment efficacy is sparse. MethodsA retrospective study was carried out in 39 male patients with classical FD, treated with either agalsidase-alfa or agalsidase-beta (mean follow up of 10 years). With six to twelve months intervals plasma-induced in vitro inhibition of enzyme activity, lysoglobotriaosylsphingosine (lysoGb3) levels and renal function were assessed. In a subset of 12 patients, additionally anti- r-αGAL A IgM, IgA and IgG1, 2, 3 and 4 levels were analyzed. ResultsIn 23 out of 39 patients, plasma-induced in vitro inhibition of r-αGAL A activity was observed (inhibition-positive). The inhibition titer was strongly negatively correlated to the decrease in lysoGb3: agalsidase-alfa (FElog10(inhibition) = −10.3, P ≤.001), agalsidase-beta (FElog10(inhibition) = −4.7, P ≤.001). Inhibition-positive patients had an accelerated decline in renal function (FE = 1.21, p = .042). During treatment IgG1 anti-r-αGAL A levels increased only in inhibition-positive patients (p = .0045). IgG4 anti-r-αGAL A antibodies developed in 7 out of 9 inhibition-positive patients. Other antibody subclasses were either not present or too low to quantify. ConclusionDevelopment of inhibiting antibodies against r-αGAL A negatively affects the biochemical response to ERT and resulted in an accelerated decline in renal function. The presence of IgG1 and IgG4 anti-r-αGAL A antibodies is associated with in vitro αGAL A activity inhibition.

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.

The resistance against Trichinella spiralis infection induced by primary infection with respiratory syncytial virus.

Human infections with Trichinella spiralis and respiratory syncytial virus (RSV) are common, as T. spiralis infections are re-emerging in various parts of the world and RSV infections remain a threat for infants. Yet, studies investigating the relationship pertaining to the two are severely lacking. In particular, immune response induction via RSV and T. spiralis remain largely elusive. Here, we investigated the resistance against T. spiralis infection induced upon primary infection with RSV. RSV, notorious for causing severe inflammatory reaction in the lungs, were intranasally infected, followed with a T. spiralis infection in mice. Our results revealed that primary RSV infection in mice significantly raised T. spiralis-specific and total IgE, IgG and its subclass antibody responses upon T. spiralis challenge infection (RSV-Ts). Blood eosinophil levels were decreased in RSV-Ts, accompanied with significant increase in both Th1 and Th2 cytokines. Antibodies generated against RSV in RSV-infected mice were found to react with T. spiralis excretory/secretory antigen, showing several bands determined through immunoblotting. RSV-Ts also had a marked reduction of T. spiralis worm burden in diaphragm. These results indicate that immune responses induced by RSV infection contribute to resistance against subsequent T. spiralis infection.

Baseline exposure, antibody subclass, and hepatitis B response differentially affect malaria protective immunity following RTS,S/AS01E vaccination in African children

BackgroundThe RTS,S/AS01E vaccine provides partial protection against malaria in African children, but immune responses have only been partially characterized and do not reliably predict protective efficacy. We aimed to evaluate comprehensively the immunogenicity of the vaccine at peak response, the factors affecting it, and the antibodies associated with protection against clinical malaria in young African children participating in the multicenter phase 3 trial for licensure.MethodsWe measured total IgM, IgG, and IgG1–4 subclass antibodies to three constructs of the Plasmodium falciparum circumsporozoite protein (CSP) and hepatitis B surface antigen (HBsAg) that are part of the RTS,S vaccine, by quantitative suspension array technology. Plasma and serum samples were analyzed in 195 infants and children from two sites in Ghana (Kintampo) and Mozambique (Manhiça) with different transmission intensities using a case-control study design. We applied regression models and machine learning techniques to analyze immunogenicity, correlates of protection, and factors affecting them.ResultsRTS,S/AS01E induced IgM and IgG, predominantly IgG1 and IgG3, but also IgG2 and IgG4, subclass responses. Age, site, previous malaria episodes, and baseline characteristics including antibodies to CSP and other antigens reflecting malaria exposure and maternal IgGs, nutritional status, and hemoglobin concentration, significantly affected vaccine immunogenicity. We identified distinct signatures of malaria protection and risk in RTS,S/AS01E but not in comparator vaccinees. IgG2 and IgG4 responses to RTS,S antigens post-vaccination, and anti-CSP and anti-P. falciparum antibody levels pre-vaccination, were associated with malaria risk over 1-year follow-up. In contrast, antibody responses to HBsAg (all isotypes, subclasses, and timepoints) and post-vaccination IgG1 and IgG3 to CSP C-terminus and NANP were associated with protection. Age and site affected the relative contribution of responses in the correlates identified.ConclusionsCytophilic IgG responses to the C-terminal and NANP repeat regions of CSP and anti-HBsAg antibodies induced by RTS,S/AS01E vaccination were associated with malaria protection. In contrast, higher malaria exposure at baseline and non-cytophilic IgG responses to CSP were associated with disease risk. Data provide new correlates of vaccine success and failure in African children and reveal key insights into the mode of action that can guide development of more efficacious next-generation vaccines.

Meningococcal PorB induces a robust and diverse antigen specific T cell response as a vaccine adjuvant

Vaccines formulated with adjuvant have been effective against numerous infectious diseases, almost always due to induction of functional antibodies that recognizes the pathogen of interest. There is an unmet clinical need for vaccine adjuvants that induce T cells responses to potentially enhance protection against malignancies and intracellular pathogens, where a humoral response, alone, may not be adequate for protection. In this study, we demonstrate that a TLR2 ligand-based adjuvant, meningococcal PorB, has broad immunostimulatory activity with the ability to induce a robust and diverse vaccine antigen specific T cell response. We demonstrate that a vaccine formulated with PorB admixed with ovalbumin induces a wide variety of antigen specific antibody subclasses and effector molecules (MIG, MCP-1, IP-10, MIP-1α, KC & IL-2) with known roles for inducing T cell responses, along with elevated levels of Th1 and Th2 type cytokines upon antigen stimulation. We confirmed production of these cytokines by examining the antigen-specific T cells induced by PorB in vivo. After two immunizations with vaccine formulated with PorB/OVA, antigen-specific CD4 and CD8 T cells were significantly increased in numbers and produced IL-4 or IFN-γ upon ex vivo antigen re-stimulation. Finally, in a Listeria mouse infection model, vaccine formulated with PorB significantly reduced the bacterial burden upon a low dose infection and increased survival upon a high dose infection with recombinant Listeria monocytogenes engineered to express OVA (rLmOVA), a pathogen that requires OVA-antigen specific cytotoxic CD8 T cells for clearance. In summary, PorB is able to induce antigen specific broad B and T cell responses, illustrating its potential as a potent and new vaccine adjuvant.

Establishment of P38Bf, a Core-Fucose-Deficient Mouse-Canine Chimeric Antibody Against Dog Podoplanin

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We have previously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not with lymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mouse-canine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and a mouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Core-fucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that the KD of P38A, P38B, and P38Bf were 1.9 × 10−7, 5.2 × 10−9, and 6.5 × 10−9, respectively. Both P38B and P38Bf revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate for antibody therapy against dPDPN-expressing canine cancers.

Cross-Protective Immune Responses Induced by Sequential Influenza Virus Infection and by Sequential Vaccination With Inactivated Influenza Vaccines.

Sequential infection with antigenically distinct influenza viruses induces cross-protective immune responses against heterologous virus strains in animal models. Here we investigated whether sequential immunization with antigenically distinct influenza vaccines can also provide cross-protection. To this end, we compared immune responses and protective potential against challenge with A(H1N1)pdm09 in mice infected sequentially with seasonal A(H1N1) virus followed by A(H3N2) virus or immunized sequentially with whole inactivated virus (WIV) or subunit (SU) vaccine derived from these viruses. Sequential infection provided solid cross-protection against A(H1N1)pdm09 infection while sequential vaccination with WIV, though not capable of preventing weight loss upon infection completely, protected the mice from reaching the humane endpoint. In contrast, sequential SU vaccination did not prevent rapid and extensive weight loss. Protection correlated with levels of cross-reactive but non-neutralizing antibodies of the IgG2a subclass, general increase of memory T cells and induction of influenza-specific CD4+ and CD8+ T cells. Adoptive serum transfer experiments revealed that despite lacking neutralizing activity, serum antibodies induced by sequential infection protected mice from weight loss and vigorous virus growth in the lungs upon A(H1N1)pdm09 virus challenge. Antibodies induced by WIV vaccination alleviated symptoms but could not control virus growth in the lung. Depletion of T cells prior to challenge revealed that CD8+ T cells, but not CD4+ T cells, contributed to cross-protection. These results imply that sequential immunization with WIV but not SU derived from antigenically distinct viruses could alleviate the severity of infection caused by a pandemic and may improve protection to unpredictable seasonal infection.

Human leukocyte antigen antibody detection technologies in platelet transfusion refractoriness, with special emphasis on functional test

In the clinical settings of transfusion medicine, human leukocyte antigen (HLA) antibody detection and platelet cross-matching are important tests applied in the management of patients with platelet transfusion refractoriness (PTR). Presently, fluorescence beads technologies using extracted and purified HLA antigens are applied for the HLA antibody screening, and highly sensitive methods such as flow cytometry crossmatch (FCXM) and immunocomplex capture fluorescence analysis (ICFA), based on the use of live intact cells, are used for the cross-matching. Such highly sensitive methods have importantly contributed for the prevention of refractoriness caused by low titer antibodies but, on the other hand, they also detect those low titer antibodies or naturally occurring antibodies without or with unknown clinical relevance. Moreover, the use of recombinant antigens has been shown to be associated with specific issues. In addition, although the relationship between the antibody features and the clinical implications, such as the relevance of antibody subclass in platelet clearance, has been investigated in the field of transplantation, presently, few evidence exists in the literature related to blood transfusion. Here, we reviewed on the available HLA antibody detection methods, the issues of the present methods, and present a new method for the evaluation of clinically significant HLA antibodies using in vitro platelet phagocytosis assay.

Correlation of Lyme Disease-Associated IgG4 Autoantibodies With Synovial Pathology in Antibiotic-Refractory Lyme Arthritis.

To determine whether IgG subclasses of Borrelia burgdorferi antibodies differ from those of 3 Lyme disease (LD)-associated autoantibodies. IgG antibody subclasses were determined by enzyme-linked immunosorbent assay in serum samples from 215 patients with features representative of each of the 3 stages of LD. Antibody and cytokine profiles were measured in matched serum and synovial fluid (SF) samples from patients with Lyme arthritis. Synovial tissue from patients with antibiotic-refractory arthritis was examined for histologic features, IgG subclasses of plasma cells, and messenger RNA (mRNA) subclass expression. B burgdorferi antibodies were primarily of the IgG1 and IgG3 subclasses, and the levels increased as the infection progressed. In contrast, LD-associated autoantibodies were mainly of the IgG2 and IgG4 subclasses, and these responses were found primarily in patients with either antibiotic-refractory or antibiotic-responsive arthritis, particularly in SF. However, compared with the responsive group, the inflammatory milieu in SF in the refractory group was enriched for cytokines representative of innate, Th1, Th2, and Th17 responses. Synovial tissue in a subgroup of patients with refractory arthritis showed marked expression of mRNA for IgG4 antibodies and large numbers of IgG4-staining plasma cells. IgG4 autoantibodies in SF to each of the 3 LD-associated autoantigens correlated with the magnitude of obliterative microvascular lesions and fibrosis in the tissue. Our findings indicate that the subclasses of IgG antibodies to B burgdorferi differ from those of LD-associated autoantibodies. Furthermore, the correlation of IgG4 autoantibodies with specific synovial pathology in the refractory group suggests a role for these autoantibodies, either protective or pathologic, in antibiotic-refractory Lyme arthritis.

IgG subclass responses to excreted-secreted antigens of Plasmodium falciparum in a low-transmission malaria area of the Peruvian Amazon

BackgroundMalaria in Peru is concentrated in the Amazon region, especially in Loreto, and transmission is focused in rural and peri-urban communities. The government has approved a malaria elimination plan with a community approach and seeks to reduce the risk of transmission through preventive interventions, but asymptomatic and low-parasite-density infections are challenges for disease control and elimination. IgG antibodies play a critical role in combating infection through their ability to reduce parasitaemia and clinical symptoms. In particular, IgG subclasses have important roles in controlling malaria disease and may provide new insight into the development of malaria control strategies and understanding of malaria transmission. Through the use of excreted-secreted antigens from Plasmodium falciparum, were evaluated the responses of the four IgG subclasses in symptomatic and asymptomatic malarial infections.ResultsHigher levels of whole IgG were observed in asymptomatic carriers (P < 0.05). IgG3 and IgG1 were the most prevalent subclasses and did not show differences in their antibody levels in either type of carrier. All symptomatic carriers were positive for IgG4, and the presence of IgG3 and IgG2 were correlated with protection against parasitaemia. IgG2 showed lower prevalence and antibody titers in comparison to other subclasses.ConclusionsThis is the first study that characterizes the IgG subclass response in the Peruvian Amazon, and these results show that even in populations from regions with low malaria transmission, a certain degree of naturally acquired immunity can develop when the right antibody subclasses are produced. This provides important insight into the potential mechanisms regulating protective immunity.

Anti-HLA Donor-Specific IgG Subclasses and C1q-binding Evolution in Posttransplant Monitoring.

BackgroundThe identification of low-level antibodies by single-antigen bead methodology has brought advancements to risk evaluation of kidney transplant recipients. However, the use of mean fluorescence intensity (MFI) to quantify antibodies and to guide therapy is not enough. Notably, immunoglobulin G (IgG) subclass switching is hypothesized to follow a programmed sequence after an emergency signal from the germinal center. In transplantation this process is not clear yet. In the present study, we sequentially evaluate anti-HLA donor specific antibody (DSA) subclasses, their profile changes, and C1q-binding ability and the influence of those characteristics on antibody mediated rejection (AMR) occurrence and allograft function.MethodsA total of 30 DSA-positive patients were tested for IgG subclass content and C1q-binding in sequential serum samples.ResultsTwenty-one patients were DSA-positive before transplant; patients sensitized only by transfusion or pregnancies had IgG1 and/or IgG3, and patients sensitized by both transfusion and pregnancies or previous transplant showed a broader range of IgG subclasses. C1q binding was detected in high MFI made up of IgG1 or multiple IgG subclasses. Only 4 patients were positive for C1q posttransplantation and 3 of these showed an increase in MFI, changes in subclasses patterns, AMR, and allograft dysfunction.ConclusionsPosttransplant evaluation of DSA subclasses and the ability to bind C1q may be informative for both AMR occurrence and allograft dysfunction. Monitoring these events may help to better define risk and interventional time points.

PPD-specific IgG1 antibody subclass upregulate tumour necrosis factor expression in PPD-stimulated monocytes: possible link with disease pathogenesis in tuberculosis.

Cachexia is a prominent feature of advanced tuberculosis, in association with increased expression of the monokine tumour necrosis factor (TNF)-alpha. Monocytes, have high affinity receptors (mannose, complement and Fc gamma1 and gamma111) which mediate antigen uptake and subsequent cytokine activation. Several mycobacterial proteins, including PPD, can stimulate TNF-alpha secretion from monocytes. However, the role of various receptors in stimulating or regulating TNF-alpha secretion is still unclear. We have previously shown selective augmentation of opsonic antibodies (IgG1 and IgG3) in tuberculosis patients with advanced pulmonary disease. We now analyse the role of opsonizing antibodies in modulating TNF-alpha expression in antigen stimulated monocytes. PPD was used as the prototypic mycobacterial antigen to stimulate monocytes from PPD skin test negative donors (n = 7) in the presence of plasma from tuberculosis patients (n = 8), containing known amounts of IgG1 and IgG3 anti-PPD antibodies. TNF-alpha secretion was enhanced in the presence of TB plasma (4/8) but not in the presence of control plasma. Using Spearman Rank analysis (two-tailed Fisher exact test), a significant correlation (rho = 0.762; P = 0. 04) was observed between IgG1 antibodies and enhancement of TNF-alpha secretion. No significant association was observed with IgG2 (rho = 0.310; P = 0.41), IgG3 (rho = 0.089; P = 0.81) or IgG4 (rho = - 0.357; P = 0.347) subclass antibodies. Absorption of IgG1 with protein 'A' removed the enhancement of TNF-alpha secretion activity from the plasma samples. Our results therefore indicate that IgG1 antibodies may enhance the chronic release of TNF-alpha in TB patients with progressive disease and, for the first time, show a direct link between disease pathogenesis and raised antibody levels.

Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime–protein boost regimen against bovine viral diarrhea virus

The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime–protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4+IFN-γ+ T cells and CD8+IFN-γ+ T cells among total CD3+ T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime–protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime–protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime–protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.

Zika convalescent macaques display delayed induction of anamnestic cross-neutralizing antibody responses after dengue infection

Structural similarities between Zika (ZIKV) and dengue virus (DENV) leads to the induction of cross-reactive responses. We have previously demonstrated that ZIKV exposed macaques significantly enhance DENV viremia. Here we show that this enhancement of DENV infection occurred in the presence of high levels of DENV cross-reactive IgG1 subclass of binding antibodies (bAb) with low DENV neutralizing antibody (nAb) activity (<1:10). The DENV-2 nAb titres after ZIKV infection were, however, higher than those induced in DENV-2 only infected animals suggesting that ZIKV induced low titres of cross-nAb against DENV. Surprisingly, DENV-2 infection of animals previously infected with ZIKV was not accompanied by an anamnestic increase in cross-nAb titres till about 1 week after DENV-2 infection. This delay coincided with enhanced DENV-2 viremia indicating that high levels of cross-bAb in the absence of high nAb contributes to enhancement of DENV infection. Serum collected 8 weeks after DENV-2 infection had high levels of nAb and showed delayed antibody dependent enhancement (ADE) of infection (1:100 dilution) as compared with serum that was collected from ZIKV infected animals prior to DENV-2 infection (1:10 dilution). Examination of serum from macaques that were simultaneously infected with both ZIKV and DENV-2 showed high levels of nAb and delayed ADE responses raising the possibility that the low levels of cross-nAb induced by ZIKV infection could be overcome by co-immunization against ZIKV and DENV infection. Taken together, our results provide additional insights into the nature and kinetics of cross-reactive antibody responses and identify a critical correlate that could potentially prevent enhancement of DENV infection during ZIKV convalescence.

Antibody responses to \u03b1-Gal in African children vary with age and site and are associated with malaria protection

Naturally-acquired antibody responses to malaria parasites are not only directed to protein antigens but also to carbohydrates on the surface of Plasmodium protozoa. Immunoglobulin M responses to α-galactose (α-Gal) (Galα1-3Galβ1-4GlcNAc-R)-containing glycoconjugates have been associated with protection from P. falciparum infection and, as a result, these molecules are under consideration as vaccine targets; however there are limited field studies in endemic populations. We assessed a wide breadth of isotype and subclass antibody response to α-Gal in children from Mozambique (South East Africa) and Ghana (West Africa) by quantitative suspension array technology. We showed that anti-α-Gal IgM, IgG and IgG1–4 levels vary mainly depending on the age of the child, and also differ in magnitude in the two sites. At an individual level, the intensity of malaria exposure to P. falciparum and maternally-transferred antibodies affected the magnitude of α-Gal responses. There was evidence for a possible protective role of anti-α-Gal IgG3 and IgG4 antibodies. However, the most consistent findings were that the magnitude of IgM responses to α-Gal was associated with protection against clinical malaria over a one-year follow up period, especially in the first months of life, while IgG levels correlated with malaria risk.

PO-486 Surrogate biomarkers of clinical efficacy in stage IIIB/IV non-small-cell lung cancer patients treated with an optimised EGF-based vaccination schedule

IntroductionIn previous clinical trials were reported that an EGF-based vaccine administration was immunogenic in advanced non-small cell lung cancer (NSCLC) patients and the elicited immune response was associated with patient´s survival. However, the description of additional key features associated with a ‘protective’ humoral response, using an optimised immunisation schedule, remained unknown.Material and methodsA phase III trial was designed using an optimised immunisation schedule. It included higher antigen dose and injections at multiple vaccination sites. Immune response and circulating biomarkers were studied in a subset of patients. EGF-specific antibody titers, IgG subclasses and peptide immunodominance was assessed by ELISA. In vitro EGF-neutralisation capacity of immune sera was evaluated by Western Blot and EGF-IgG binding kinetics by surface plasmon resonance (SPR) technology. Additionally, circulating levels of EGFR ligands (EGF, TGFα, AR) and others NSCLC- associated circulating factors were measured.Results and discussionsEGF vaccine administration elicited antibody titers higher than 1:4000 in 80% of vaccinated patients after 3 months of treatment. The EGF-specific humoral response was directed against the central region of the EGF molecule and was mainly composed by IgG3/IgG4 antibody subclasses. The capacity of post-immune sera to inhibit EGFR phosphorylation increased during the course of the immunisation scheme and was associated with EGF-specific antibody affinity maturation. Basal concentrations of EGF and TGFα in the serum were affected by EGF- based immunisation. The avidity and the EGFR phosphorylation inhibition capacity of elicited polyclonal antibodies was associated with the clinical benefit of treated patients.ConclusionOptimising the vaccination schedule allowed to induce the development of a protective humoral response in a high percent of NSCLC vaccinated patients. Quality of induced anti-EGF polyclonal antibodies was associated with clinical benefit (clinical trial registration number: RPCEC00000161).

Distribution of Anti-ABO Immunoglobulin G Subclass and C1q Antibody in ABO Incompatible Kidney Transplantation

Introduction To investigate the correlation between serum anti-ABO immunoglobulin G (IgG) and IgG subtypes, anti-ABO IgG subclasses were measured by flow cytometry (FCM) in ABO-incompatible (ABO-i) kidney transplant recipients (KTRs). We also evaluated baseline anti-ABO C1q antibody of KTRs. Method Baseline anti-ABO IgG titers were measured with both FCM and column agglutination technique (CAT) methods in 18 ABO-i KTRs. The mean florescence intensity (MFI) ratios of baseline Anti-ABO IgG subclasses were obtained by FCM method and followed up after rituximab (RTX), each plasmapheresis (PP) session and kidney transplantation. Correlation between the values of IgG subclass and total IgG titer were analyzed. Baseline anti-ABO C1q antibody was measured by FCM methods. Result The baseline MFI ratios of total IgG, IgG1, IgG2, IgG3 and IgG4 were 202.46, 62.41, 30.01, 1.04 and 1.13, respectively. The MFI ratios of IgG1, IgG2, and total IgG measured at baseline and pre-PP were positively correlated with the baseline ABO titer measured by CAT (rho, baseline; 0.641, 0.638, 0.663, PP; 0.662, 0.581, 0.662). However, Spearman correlation analysis revealed that baseline ABO titer did not show any correlation with the values after final PP and transplantation. IgG1 and IgG2 as well as total IgG were removed effectively after serial PP. However, anti-ABO C1q antibody was not detected and neither correlated with total IgG and any IgG subclasses. Conclusion Our findings suggest that IgG1 and IgG2 are dominant IgG subtypes in ABO-i KTRs. The mean baseline levels of IgG1 and IgG2 were correlated with baseline total IgG titer. However, anti-ABO C1q antibody was not detected in the present study. Further study is warranted to evaluate the function of each IgG subtype to activate complement pathway.