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Highlights • Older people are more susceptible to poor health outcomes after infection. • Ageing is associated with reduced antibody titres in response to vaccination, limiting vaccine efficacy. • A key contributing factor to the poor humoral immunity in ageing is the reduced size and function of the germinal centre response. • The diminished germinal centre response in ageing can be attributed to changes in many of the cellular players involved in the response., Vaccines are a highly effective intervention for conferring protection against infections and reducing the associated morbidity and mortality in vaccinated individuals. However, ageing is often associated with a functional decline in the immune system that results in poor antibody production in older individuals after vaccination. A key contributing factor of this age-related decline in vaccine efficacy is the reduced size and function of the germinal centre (GC) response. GCs are specialised microstructures where B cells undergo affinity maturation and diversification of their antibody genes, before differentiating into long-lived antibody-secreting plasma cells and memory B cells. The GC response requires the coordinated interaction of many different cell types, including B cells, T follicular helper (Tfh) cells, T follicular regulatory (Tfr) cells and stromal cell subsets like follicular dendritic cells (FDCs). This review discusses how ageing affects different components of the GC reaction that contribute to its limited output and ultimately impaired antibody responses in older individuals after vaccination. An understanding of the mechanisms underpinning the age-related decline in the GC response is crucial in informing strategies to improve vaccine efficacy and extend the healthy lifespan amongst older people.

Toxoplasmosis acquired in early pregnancy is a potentially severe complication for the fetus. Evaluating the risk of transplacental infection in pregnant women accessing the Tuscany Reference Center for Infectious Diseases in Pregnancy during the last 20 years with suspected or confirmed toxoplasmosis acquired in early pregnancy was the aim of the study.We retrospectively enrolled all pregnant women undergoing amniocentesis for toxoplasmosis acquired in the first 16 gestational weeks in the period 1999-2019, comparing patients with certain acute infection (seroconversion occurred in pregnancy, CAIP) with those with suspected acute infection (IgG positive with low/intermediate IgG avidity index, SAIP).237 patients were enrolled, 187 (78.9%) with SAIP and 50 (21.1%) with CAIP. Specific IgM was detected in 47.5% and 76.7% (p-value 0.001), and the mean IgG avidity index was 22.7% and 7.1% (p-value 0.001) in the SAIP and in the CAIP group, respectively. The mean delay from diagnosis to antibiotic initiation was 14.6 in SAIP and 11 days in CAIP group. Toxoplasma DNA was detected in the amniotic fluid in one case in a patient with CAIP. Excluding 24 newborns with not available data, prevalence of congenital infection was 0.47% [1/213 (95% CI 0.08%-2.61%)], 0% [0/178 (95% CI 0%-2.11%)] in SAIP and 2.8% [1/35 (95% CI 0.51%-14.53%)] in CAIP group.Toxoplasmosis acquired in early pregnancy has a low risk of fetal infection. Actively discussing case-by-case amniocentesis indication with patients, especially when a recent toxoplasmosis is not properly confirmed, is desirable.

Factors such as antibody clearance and target affinity can influence antibodies’ effective doses for specific indications. However, these factors vary considerably across antibody classes, precluding direct and quantitative comparisons. Here, we apply a dimensionless metric, the therapeutic exposure affinity ratio (TEAR), which normalizes the therapeutic doses by antibody bioavailability, systemic clearance and target-binding property to enable direct and quantitative comparisons of therapeutic doses. Using TEAR, we revisited and dissected the doses of up to 60 approved antibodies. We failed to detect a significant influence of target baselines, turnovers or anatomical locations on antibody therapeutic doses, challenging the traditional perceptions. We highlight the importance of antibodies’ modes of action for therapeutic doses and dose selections; antibodies that work through neutralizing soluble targets show higher TEARs than those working through other mechanisms. Overall, our analysis provides insights into the factors that influence antibody doses, and the factors that are crucial for antibodies’ pharmacological effects.

Strong quantitative and functional antibody responses to the quadrivalent human papillomavirus (HPV) vaccine were reported in mid-adult aged men, but there are limited data on the avidity of the antibody response and the memory B-cell response following vaccination. Although circulating antibodies induced by vaccination are believed to be the main mediators of protection against infection, evaluation of avidity of antibodies and memory B cell responses are critical for a better understanding of the vaccine immunogenicity mechanisms. Both the modified enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunosorbent spot (ELISpot) assay are tools to measure the humoral and cellular immune responses post vaccination to characterize vaccine immunogenicity. The avidity of HPV-16 and HPV-18 specific IgG in the serum of mid-adult aged men (N = 126) who received three quadrivalent HPV vaccine doses was examined using a modified ELISA. HPV-16 memory B-cell responses were assessed via ELISpot at month 0 (prior to vaccination) and 1-month post-dose three of the vaccine (month 7). The quadrivalent vaccine induced an increase in HPV-16 and HPV-18 antibody avidity at month 7. HPV-18 avidity levels moderately correlated with anti-HPV-18 antibody titers, but no association was observed for HPV-16 antibody titers and avidity levels. The HPV-16-specific memory B-cell response was induced following three vaccine doses, however, no association with anti-HPV-16 antibody avidity was observed. Three doses of quadrivalent HPV vaccine increased antibody affinity maturation for HPV-16/18 and increased the frequency of anti-HPV-16 memory B-cells in mid-adult aged men.

Antibody profiling is a fundamental component of understanding the humoral response in a wide range of disease areas. Most currently used approaches operate by capturing antibodies onto functionalised surfaces. Such measurements of surface binding are governed by an overall antibody titre, while the two fundamental molecular parameters, antibody affinity and antibody concentration, are challenging to determine individually from such approaches. Here, by applying microfluidic diffusional sizing (MDS), we show how we can overcome this challenge and demonstrate reliable quantification of alloantibody binding affinity and concentration of alloantibodies binding to Human Leukocyte Antigens (HLA), an extensively used clinical biomarker in organ transplantation, both in buffer and in crude human serum. Capitalising on the ability to vary both serum and HLA concentrations during MDS, we show that both affinity and concentration of HLA-specific antibodies can be determined directly in serum when neither of these parameters is known. Finally, we provide proof of principle in clinical transplant patient sera that our assay enables differentiation of alloantibody reactivity against HLA proteins of highly similar structure, providing information not attainable through currently available techniques. These results outline a path towards detection and in-depth profiling of humoral immunity and may enable further insights into the clinical relevance of antibody reactivity in clinical transplantation and beyond.

Loop length variation in the complementary determining regions (CDRs) 1 and 2 encoded in germline variable antibody genes provides structural diversity in naïve antibody libraries. In synthetic single framework libraries the parental CDR-1 and CDR-2 length is typically unchanged and alternative lengths are provided only at CDR-3 sites. Based on an analysis of the germline repertoire and structure-solved anti-hapten and anti-peptide antibodies, we introduced combinatorial diversity with alternative loop lengths into the CDR-L1, CDR-L3 and CDR-H2 loops of anti-digoxigenin and anti-microcystin-LR single chain Fv fragments (scFvs) sharing human IGKV3-20/IGHV3-23 frameworks. The libraries were phage display selected for folding and affinity, and analysed by single clone screening and deep sequencing. Among microcystin-LR binders the most frequently encountered alternative loop lengths were one amino acid shorter (6 aa) and four amino acids longer (11 aa) CDR-L1 loops leading up to 17- and 28-fold improved affinity, respectively. Among digoxigenin binders, 2 amino acids longer (10 aa) CDR-H2 loops were strongly enriched, but affinity improved anti-digoxigenin scFvs were also encountered with 7 aa CDR-H2 and 11 aa CDR-L1 loops. Despite the fact that CDR-L3 loop length variants were not specifically enriched in selections, one clone with 22-fold improved digoxigenin binding affinity was identified containing a 2 residues longer (10 aa) CDR-L3 loop. Based on our results the IGKV3-20/IGHV3-23 scaffold tolerates loop length variation, particularly in CDR-L1 and CDR-H2 loops, without compromising antibody stability, laying the foundation for developing novel synthetic antibody libraries with loop length combinations not existing in the natural human Ig gene repertoire.

Objective To establish a model to predict high cytomegalovirus (CMV) immunoglobulin (Ig)G avidity index (AI) using clinical information, to contribute to the mental health of CMV-IgM positive pregnant women. Methods We studied 371 women with IgM positivity at ≤14 w of gestation. Information on the age, parity, occupation, clinical signs, IgM and G values, and IgG AI was collected. The IgG AI cut-off value for diagnosing congenital infection was calculated based on a receiver operating characteristic curve analysis. Between-group differences were assessed using the Mann–Whitney U-test or χ2 analysis. The factors predicting a high IgG AI were determined using multiple logistic regression. Results The women were divided into high or low IgG AI groups based on an IgG AI cut-off value of 31.75. There were significant differences in the IgG and IgM levels, age, clinical signs, and the number of women with one parity between the two groups. In a multiple logistic regression analysis, IgM and the number of women with one parity were independent predictors. This result helped us establish a mathematical model that correctly classified the IgG AI level for 84.6% of women. Conclusion We established a highly effective model for predicting a high IgG AI immediately after demonstrating IgM positivity.

The characteristics of single domain and ease of gene manipulation of the single domain antibody (sdAb) make it suitable for affinity maturation in vitro. Since the affinity of antibodies can influence the immunoassays' sensitivity, a nanobody (Nb), the anti-ochratoxin A sdAb (AOA-sdAb), was herein selected as the model antibody to explore feasible approach for improving its affinity. Homology modeling and molecular docking were used to analyze the interaction between OTA and the AOA-sdAb. After alanine scanning verification, Gly53, Met79, Ser102, and Leu149 were determined as the key amino acids of the AOA-sdAb. Two site-directed saturated mutation libraries were constructed by two-site mutation against those four key amino acids. After biopanning and identification, a mutant Nb-G53Q&S102D was obtained with a half maximal inhibition concentration (IC50) of 0.29 ng/mL and a KD value of 52 nM, which is 1.4-fold and 1.36-fold lower than that of the original sdAb, respectively. The computer simulation analysis indicated that the hydrogen bond, hydrophobic interaction, and side chain steric hindrance of amino acid residues are critical for the binding affinity of the AOA-sdAb. Overall, the techniques shown in this study are effective ways at ‘identifying residues involved in antigen binding’ that can be altered by site-directed mutation.

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n = 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.

Background Dating of primary human cytomegalovirus (HCMV) infection in pregnancy is crucial to define whether infection occurred before or during pregnancy and at which gestational age. Objective The aim of this study was to identify a diagnostic strategy for determination of early, intermediate and late phase of HCMV primary infection during pregnancy. Study design Sequential serum samples from 40 pregnant women with defined onset of HCMV primary infection were tested retrospectively for IgM, IgG and IgG avidity against whole HCMV lysate, along with anti-p52 IgM and anti-gB IgG (Euroimmun AG). Results Anti-HCMV IgM were positive in all samples collected within the first 2 months, then decreased remaining weakly positive in about 40% of samples collected within 6–12 months after infection. Anti-p52 IgM followed similar kinetics but decreased earlier, remaining weakly positive only in 20% of late samples. Anti-HCMV IgG were positive in all samples and showed variable kinetics. Their avidity increased from low levels, observed within 2 months, to intermediate/high levels from 4 months onwards. Anti-gB IgG increased over time following kinetics similar to anti-HCMV IgG avidity. By combining results of anti-HCMV IgM plus IgG avidity, and confirming them with anti-p52 IgM plus anti-gB IgG as second-line assays, the early (within 2–3 months) and late (after 3 months) phases of HCMV infection were satisfactorily defined, whereas the intermediate phase overlapped with the beginning of the late phase. Conclusion Anti-p52 IgM and anti-gB IgG provide additional tools besides classical anti-HCMV IgM, IgG and IgG avidity in dating HCMV primary infections.

Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of VH and DH gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.

High-affinity binding of antibodies provides for increased specificity and usually higher effector functions in vivo. This goal, well documented in cancer immunotherapy, is very relevant to vaccines as well, and has particularly significant application toward glycan antigens. The inability to elicit high-affinity antibodies has limited potential applications of glycan-based immunogens, giving rise to insufficient population coverage due to low titers and short duration of protection. That such vaccines have achieved widespread use in spite of these shortcomings highlights the surpassing importance of glycans as prophylactic immunological targets. New advances in the combination of synthetic chemistry, bioconjugation, and mechanistic immunology offer the possibility to vastly expand the number of potential molecular targets in cancer and infectious diseases by opening a wider world of carbohydrate structures to immunological recognition and high-affinity response.

5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10−11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.

A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48–57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4–7.8 10−7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.

B cell immunoglobulin (Ig) repertoire composition shapes immune responses. The generation of Ig diversity begins with Ig variable region exon assembly from gene segments, random inter-segment junction sequence diversity, and combinations of Ig heavy and light chain. This generates vast preemptive sequence freedom in early developing B lineage cell Ig genes that can anticipate a great diversity of threats. This freedom is met with large restrictions that ultimately define the naïve (i.e. preimmune) Ig repertoire. Activation-induced somatic hypermutation (SHM), which further diversifies Ig V regions, is also met with strong selection that shapes Ig affinity maturation. While individual repertoire features, such as affinity for self and competition for foreign antigen, are known to drive selection, the selection filters themselves may be subject to regulation. Large sequence freedom coupled with strong selection for each diversification process provides flexibility for demand-driven regulation to dynamically balance antigen recognition capacities and associated autoimmune risks according to host needs.

Serological methods to differentiate between recently acquired and established HIV-1 infections are a useful tool in the HIV-surveillance to characterize the epidemic, identify groups at risk and assess HIV-preventive interventions. Therefore, an avidity-based, modified BioRad Genscreen™ HIV-1/2 assay (BRAEUR) was evaluated according to the avidity-based, modified BioRad HIV-1/2 Plus O protocol (BRAUSA). Overall, 692 well defined samples (82.5% B and 17.5% non-B subtypes) from recent (

To gain insights into the current Japanese pertussis immunization schedule, we examined the distributions of antibody titers and avidities to pertussis toxin (PT) and filamentous hemagglutinin (FHA) in 460 Japanese healthy subjects (aged 1-60 years) based on age category. Our avidity enzyme-linked immunosorbent assays revealed that young children aged 1-2 years, which corresponded to ages after receiving primary and/or booster pertussis vaccinations, had relatively high-avidity anti-PT IgG (mean avidity index [AI], 40.5%) compared with other age groups (AI, 26.5-31.9%); however, they had relatively low-avidity anti-FHA IgG (AI, 41.8%). In contrast, children aged 3-6 years had both low-avidity anti-PT IgG (AI, 26.5%) and low-avidity anti-FHA IgG (AI, 40.4%). A significant age-related difference in anti-PT IgG avidity was observed between children aged 1-2 years and 3-6 years (P < 0.05); however, the difference in anti-FHA IgG avidity was not significant. The anti-PT IgG avidity was positively correlated with the antibody titer, especially among children aged 1-15 years (rs = 0.508-0.685; P < 0.01), indicating that the avidity of vaccine-induced anti-PT IgG decreases with decreasing IgG antibody titer to PT. Our findings strongly suggest that vaccine-induced anti-PT IgG avidity rapidly wanes after vaccination, but this is not observed for anti-FHA IgG avidity. Because children aged 3-6 years have both low-quantity and low-quality antibodies against PT, an additional booster vaccination with acellular pertussis vaccines is required for such children in Japan.

The standard membrane-feeding assay (SMFA) is a functional assay that has been used to inform the development of transmission-blocking vaccines (TBV) against Plasmodium falciparum malaria. For Pfs230, a lead target antigen for TBV development, a few studies have tested either a single anti- Pfs230 polyclonal or monoclonal antibody (one antibody per study) at serial dilutions and showed a dose-dependent response. Further, there have been reports that the SMFA activity of anti-Pfs230 polyclonal and monoclonal antibodies were enhanced in the presence of complement. However, no analysis has been performed with multiple samples, and the impact of anti-Pfs230 antibody titers, IgG subclass profile and avidity were evaluated together in relation to transmission-reducing activity (TRA) by SMFA. In this report, a total of 39 unique anti-Pfs230 IgGs from five different mouse immunization studies were assessed for their ELISA units (EU), IgG2/IgG1 ratio and avidity by ELISA, and the functionality (% transmission-reducing activity, %TRA) by SMFA. The mice were immunized with Pfs230 alone, Pfs230 conjugated to CRM(197), or a mixture of unconjugated Pfs230 and CRM(197) proteins using Alhydrogel or Montanide ISA720 adjuvants. In all studies, the Pfs230 antigen was from the same source. There was a significant correlation between EU and %TRA (p < 0.0001 by a Spearman rank test) for the anti-Pfs230 IgGs. Notably, multiple linear regression analyses showed that both IgG2/IgG1 ratio and avidity significantly affected %TRA (p = 0.003 to p = 0.014, depending on the models) after adjusting for EU. The results suggest that in addition to antibody titers, IgG2/IgG1 ratio and avidity should each be evaluated to predict the biological activity of anti-Pfs230 antibodies for future vaccine development.

We have been optimizing the design of a conjugate vaccine for nicotine addiction that employs a peptide-based hapten carrier. This peptide, which is produced by solid-phase protein synthesis, contains B cell and T cell epitope domains and eliminates the non-relevant, but highly immunogenic sequences in microbial carriers. In this report, the amino acid sequences in the T cell domain were optimized for improved vaccine activity and multivalent formulations containing structurally distinct haptens were tested for the induction of additive antibody responses. Trivalent vaccines produced antibody concentrations in mice that were 100 times greater than the amount of nicotine measured in smokers, and significantly reduced acute nicotine toxicity in rats. Two additional features were explored that distinguish the peptide from traditional recombinant carriers. The first is the minimal induction of an anti-carrier response, which can suppress nicotine vaccine activity. The second employs solid-phase synthesis to manufacture haptenated peptide. This approach obviates conventional conjugation chemistries and streamlines production of a more potent vaccine antigen.

Background: Immunity to rubella-virus (RV) is commonly determined by measuring specific IgG (RV-IgG). However, RV-IgG results may be different and even discordant, depending on the assay used. Cell-mediated immunity is not routinely investigated for diagnostic purposes. Objectives: Our aim was to investigate humoral and cellular immunity of women with negative or equivocal RV-IgG before, and after post-partum vaccination. Study design: A total of 186 pregnant women were included in the study. During pregnancy, humoral immunity was investigated with two RV-IgG immunoassays, an immunoblot and a T-cell mediated immunity test. In the post-partum vaccination period, measuring RV-IgM and RV-IgG avidity allowed us to determine whether women raised a primary or a secondary immune response. Results: Before vaccination, 52.2% women, supposed to be susceptible, had positive anti-E1 RV-IgG indicating strong evidence of previous exposure to RV. All (100%) pregant women who had a positive immunoblot before immunization raised a secondary immune response to vaccination, and 96.8% who had a negative immunoblot before immunization, raised a primary immune response to vaccination. All women who raised a primary immune response after vaccination had negative anti-E1 RV-IgG and negative cell-mediated immunity. Discussion: These results indicate that individuals can have evidence of protective immunity against rubella despite negative RV-IgG.

Interleukin‐18 (IL‐18) is a pro-inflammatory cytokine that evokes both innate and acquired immune responses. IL-18 is initially synthesized as an inactive precursor and the cleavage for processing into a mature, active molecule is mediated by pro-inflammatory caspases following the activation of inflammasomes. Two types of monoclonal antibodies were raised: anti-IL-1863−68 antibodies which recognize full-length1−193 and cleaved IL-18; and anti-IL-18 neoepitope antibodies which specifically recognize the new N-terminal 37YFGKLESK44 of IL-18 cleaved by pro-inflammatory caspase-1/4. These mAbs were suitable for Western blotting, capillary Western immunoassay (WES), immunofluorescence, immunoprecipitation, and function-blocking assays. WES analysis of these mAbs allowed visualization of the IL-18 bands and provided a molecular weight corresponding to the pro-inflammatory caspase-1/4 cleaved, active form IL-1837−193, and not to the inactive precursor IL-18, in the serum of patients with adult-onset Still's disease (6/14, 42%) and hemophagocytic activation syndrome (2/6, 33%). These monoclonal antibodies will be very useful in IL-18 and inflammasome biology and for diagnostic and therapeutic strategies for inflammatory diseases.

Graphical abstract, Highlights • Meta-analyses of vaccine data for Teladorsagia shows significant protection (P, Using data from five independent vaccine trials, which employed a subunit cocktail vaccine containing eight recombinant proteins to protect sheep against Teladorsagia circumcincta, a strategy was developed to simplify antigen complexity of the vaccine. A meta-analysis of data from these five trials demonstrated statistically significant reductions in cumulative faecal egg count and worm burden in vaccinated sheep when compared with those which had received adjuvant only (P = 0.009 and P

CMV IgG avidity assays are widely used and can be helpful in pregnant women to date the onset of CMV primary infection; however, these tests are not standardized and sometimes give inconclusive results. We evaluated the performances of Mikrogen recomLine CMV IgG and IgG Avidity compared to the VIDAS CMV IgG Avidity. On a first sample set of 89 sequential sera collected from 40 women with precisely determined onset of CMV primary infection, the combination of Mikrogen recomLine CMV IgG and IgG Avidity showed an accurate interpretation in 83.1% (74/89), an incorrect result in 4.5% (4/89), and an inconclusive result in 12.4% (11/89) and showed a better sensitivity to diagnose infections14 weeks compared to VIDAS (85.9% vs. 76.9%). On a second sample set of 89 sera with an intermediate VIDAS CMV IgG Avidity, the combination of line immunoassays provided additional information on the time of infection in 79% (70/89) of the samples. This combination of line assays is useful as additional confirmatory testing and can help to date more precisely the onset of CMV primary infection.

Subunit vaccines generally require adjuvants to achieve optimal immune responses. Toll-like receptor (TLR) agonists are promising immune potentiators, but rapid diffusion from the injection site reduces their local effective concentration and may cause systemic reactions. In this study, we investigated the potential of aluminum hydroxide adjuvant (AH) to adsorb the TLR3 agonist poly(I:C) and TLR9 agonist CpG and compared the effect of the combination adjuvant on the immune response with either the TLR agonists or AH alone in mice. Poly(I:C) and CpG readily adsorbed onto AH and this combination adjuvant induced a stronger IgG1 and IgG2a immune response with a significant increase of antibody avidity. The combination adjuvant enhanced antigen uptake and activation of dendritic cells in vitro. It induced an inflammatory response at the injection site similar to AH but without eosinophils which are typically observed with AH. A distinctive antigen-containing monocyte/macrophage population with an intermediate level of CD11c expression was identified in the draining lymph nodes after immunization with TLR agonists and the combination adjuvant. Injection of the combination adjuvant did not induce an increase of TNFα and CXCL10 in serum in contrast to the injection of soluble TLR agonists. These results indicate that this combination adjuvant is a promising formulation to solve some of the unmet needs of current vaccines.

Many of the most successful drugs generated in recent years are based upon monoclonal antibodies (mAbs). However, for some therapeutic and diagnostic applications mAbs are far from ideal; for example, while their relatively large size and inherent receptor binding aids their longevity in vivo it can also limit their tissue penetration. Further, their structural complexity makes them expensive to produce and prone to denaturation in non-physiological environments. Thus, researchers have been searching for alternative antigen-binding molecules that can be utilized in situations where mAbs are suboptimal tools. One potential source currently being explored are the shark-derived binding domains known as VNARs. Despite their small size VNARs can bind antigens with high specificity and high affinity. Combined with their propensity to bind epitopes that are inaccessible to conventional mAbs, and their ability to resist denaturation, VNARs are an emerging prospect for use in therapeutic, diagnostic, and biotechnological applications.

Recombinant immunotoxins are chimeric proteins composed of a targeting peptide that binds to a specific tumor antigen and a toxin protein killing target cells. Recombinant immunotoxin exhibits potent cancer inhibiting effects both in vivo and in vitro. However, the non-specific toxicity causes severe syndromes limiting their clinical application. To reduce toxicity caused by recombinant immunotoxins in general, we divided an immunotoxin into two nontoxic segments that may restore toxic bioactivity on tumor cell surface based on the intein mediated trans-splicing reaction. Both split and reconstituted immunotoxins were tested for their biological activities. We found that the reconstituted immunotoxin retained antigen specificity and affinity toward cancer cells overexpressing HER2/neu. After being internalized into HER2/neu positive cells, the reconstituted immunotoxin showed comparable cytotoxicity as the original immunotoxin, while the split immunotoxin fragments showed no toxic activity to cells with or without HER2/neu expression. This approach can potentially be used under clinical settings to reduce non-specific toxicity by administering patients with inactive immunotoxin fragments. Cytotoxic effect only occurs at tumor sites where the inactive fragments bind, trans-splice and become active toxin.

Toxocarosis is a zoonotic disease caused by migration and subsequent localization of nematode larvae of Toxocara spp. in human organs and tissues, which is manifested with development of various non-specific clinical symptoms. Main diagnostic methods are serological and consists in proving the presence of anti-Toxocara IgG antibodies in patient's sera. In humans, anti-Toxocara IgG has been shown to persist in the serum for a long time and cannot be used to distinguish between past and recent infection. Aim of the present work is to investigate the diagnostic significance of the specific IgG avidity level, determined by an immuno enzyme test developed by us, and the presence of anti-Toxocara IgA for distinguishing between acute and chronic toxocarosis. The study included 130 patients with positive results in routine serological ELISA and Western blot tests and with clinical symptoms of visceral and ocular toxocarosis. The results revealed low IgG avidity (≤40%) in nine (7.3%) and presence of anti-Toxocara IgA antibodies in 36 (26.2%) of the subjects. Low avidity of IgG antibodies was found only in the first tests, and a presence of specific IgA for up to 9 months. The results of our study give us reason to believe that determination of the IgG avidity in toxocarosis is of greater diagnostic value than the presence of IgA to establish the stage of the disease.

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.

Highlights • Production of highly specific chicken egg yolk (IgY) monoclonal antibodies using phage display method against SARS CoV-2 will be an effective platform which holds much effective clinical and scientific advantages. • This method holds higher specificity, consistency and reproducibility. • Highly scalable manufacturing and lower cross-reactive due to the absence of invariable regions., The present state of diagnostic and therapeutic developmental race for vaccines against the SARS CoV-2 (nCOVID-19) focuses on prevention and control of this global pandemic which also represents a critical challenge to the global health community. Although development of novel vaccines can prevent the SARS CoV-2 infections, it is still impeded by several other factors and therefore novel approaches towards treatment and management of this disease is the urgent need. Passive immunotherapy plays a vital role as a possible alternative to meet this challenge and among various antibody sources, chicken egg yolk antibodies (IgY) can be used as an alternative to mammalian antibodies which have been previously studied against SARS CoV outbreak in China. In this review, we discuss the strategies for the use of chicken egg yolk (IgY) antibodies in the development of rapid diagnosis and immunotherapy against SARS CoV-2. Also, IgY antibodies have previously been used against various respiratory bacterial and viral infections in humans and animals. Compared to mammalian antibodies (IgG), chicken egg yolk antibodies (IgY) have greater binding affinity to specific antigens, ease of extraction and lower production costs, hence possessing remarkable pathogen-neutralizing activity of pathogens in respiratory and lungs. We provide an overall importance for the use of monoclonal chicken egg yolk antibodies (IgY) using phage display method describing their potential passive immunotherapeutic application for the treatment and prevention of SARS CoV-2 infection which is simple, fast and safe way of approach for treating patients effectively.

Limited knowledge exists in post-partum women regarding durability of SARS-CoV-2 vaccine-induced antibody responses and their neutralising ability against SARS-CoV-2 variants of concern (VOC).We elucidated longitudinal mRNA vaccination-induced antibody profiles of 13 post-partum and 13 non-post-partum women (control).The antibody neutralisation titres against SARS-CoV-2 WA-1 strain were comparable between post-partum and non-post-partum women and these levels were sustained up to four months post-second vaccination in both groups. However, neutralisation titers declined against several VOCs, including Beta and Delta. Higher antibody binding was observed against SARS-CoV-2 receptor-binding domain (RBD) mutants with key VOC amino acids when tested with post-second vaccination plasma from post-partum women compared with controls. Importantly, post-vaccination plasma antibody affinity against VOCs RBDs was significantly higher in post-partum women compared with controls.This study demonstrates that there is a differential vaccination-induced immune responses in post-partum women compared with non-post-partum women, which could help inform future vaccination strategies for these groups.The antibody characterisation work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds.

Antibodies that bind polyethylene glycol (PEG) can be induced by pegylated biomolecules and also exist in a significant fraction of healthy individuals who have never received pegylated medicines. The binding affinity of antibodies against PEG (anti-PEG antibodies) likely varies depending on if they are induced or naturally occurring. Anti-PEG antibodies can accelerate the clearance of pegylated medicines from the circulation, resulting in loss of drug efficacy, but it is unknown how accelerated blood clearance is affected by anti-PEG antibody affinity. We identified a panel of anti-PEG IgG and IgM antibodies with binding avidities ranging over several orders of magnitude to methoxy polyethylene glycol-epoetin beta (PEG-EPO), which is used to treat patients suffering from anemia. Formation of in vitro immune complexes between PEG-EPO and anti-PEG IgG or IgM antibodies was more obvious as antibody affinity increased. Likewise, high affinity anti-PEG antibodies produced greater accelerated blood clearance of PEG-EPO as compared to low affinity antibodies. The molar ratio of anti-PEG antibody to PEG-EPO that accelerates drug clearance in mice correlates with antibody binding avidity. Our study indicates that the bioactivity of PEG-EPO may be reduced due to rapid clearance in patients with either high concentrations of low affinity or low concentrations of high affinity anti-PEG IgG and IgM antibodies.

Summary: Background: Measuring anti-spike protein antibodies in human plasma or serum is commonly used to determine prior exposure to SARS-CoV-2 infection and to assess the anti-viral protection capacity. According to the mass-action law, a lesser concentration of tightly binding antibody can produce the same quantity of antibody-antigen complexes as higher concentrations of lower affinity antibody. Thus, measurements of antibody levels reflect both affinity and concentration. These two fundamental parameters cannot be disentangled in clinical immunoassays, and so produce a bias which depends on the assay format. Methods: To determine the apparent affinity of anti-spike protein antibodies, a small number of antigen-coated magnetic microparticles were imaged by fluorescence microscopy after probing antigen-antibody equilibria directly in patient plasma. Direct and indirect anti-SARS-CoV-2 immunoassays were used to measure antibody levels in the blood of infected and immunised individuals. Findings: We observed affinity maturation of antibodies in convalescent and vaccinated individuals, showing that higher affinities are achieved much faster by vaccination. We demonstrate that direct and indirect immunoassays for measuring anti-spike protein antibodies depend differently on antibody affinity which, in turn, affects accurate interpretation of the results. Interpretation: Direct immunoassays show substantial antibody affinity dependence. This makes them useful for identifying past SARS-CoV-2 exposure. Indirect immunoassays provide more accurate quantifications of anti-viral antibody levels. Funding: The authors are all full-time employees of Abbott Laboratories. Abbott Laboratories provided all operating funds. No external funding sources were used in this study.

Current diagnosis of acute myocardial infarction involves quantification of circulating cTn levels. This work endeavoured to generate and enhance recombinant antibody fragments targeting various epitopes on the N- and C-terminals of the cTnI molecule, thereby facilitating highly sensitive detection of the troponin molecule. From this approach, two anti-cTnI scFv antibodies were successfully selected using either pHage display or structural reformatting of full length anti-cTnI IgG. Their antibody binding affinity was further optimised via chain shuffling and/or site directed mutagenesis, resulting in scFv with heightened sensitivity when compared to the wild-type scFv. If used in conjunction with existing anti-mid fragment cTnI antibodies, these N- and C- terminal-targeting scFvs show high potential for the enhancement of current cTnI detection assays by limiting the effects from cTnI degradation or troponin complex formation.

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in newborns, young children, elderly, and immune-compromised. The RSV fusion (F) glycoprotein is a major focus of vaccine development and the target of palivizumab (Synagis®) which is licensed as an immuno-prophylactic for use in newborn children at high risk of infection. However, clinical use of a narrowly targeted monoclonal antibodies leads to the generation of escape mutant strains that are fully resistant to neutralization by the antibody. Herein, we evaluated the RSV F nanoparticle vaccine (RSV F vaccine), produced as near-full-length, pre-fusogenic F trimers that form stable protein-detergent nanoparticles. The RSV F vaccine induces polyclonal antibodies that bind to antigenic site II as well as other epitopes known to be broadly neutralizing. Cotton rats immunized with the RSV F vaccine produced antibodies that were both neutralizing and protected against wild-type RSV infection, as well as against a palivizumab-resistant mutant virus. Use of aluminum phosphate adjuvant with the RSV F vaccine increased site II antibody avidity 100 to 1000-fold, which correlated with enhanced protection against challenge. The breadth of the vaccine-induced antibody response was demonstrated using competitive binding with monoclonal antibodies targeting antigenic sites Ø, II, IV, and VIII found on pre-fusion and post-fusion conformations of RSV F. In summary, we found the RSV F vaccine induced antibodies that bind to conserved epitopes including those defined as pre-fusion F specific; that use of adjuvant increased antibody avidity that correlated with enhanced protection in the cotton rat challenge model; and the polyclonal, high-avidity antibodies neutralized and protected against both wild-type and palivizumab-resistant mutant virus. These data support the ongoing clinical development of the aluminum phosphate adjuvanted RSV F nanoparticle vaccine.

Due mainly to their high level of affinity and specificity, therapeutic monoclonal antibodies (mAbs) have been frequently selected as treatment for cancer, autoimmune or chronic inflammatory diseases. Despite the increasing number of mAbs and related products in the biopharmaceutical market, they are still expensive, can cause undesired side effects, and eventually cause resistance. Antibody engineering, which emerged to overcome limitations faced by mAb therapy, has supported the development of modified mAbs for immunotherapy. As part of this approach, researchers have invested in obtaining antibody fragments, as well as in Fc region modifications, since interactions with Fc receptors influence an antibody's half-life and mechanism of action. Thus, Fc engineering results in antibodies with more desirable characteristics and functions for which they are intended, creating "fit-for-purpose" antibodies with reduced side effects. Furthermore, aglycosylated antibodies, produced in bacterial cultivation, have been an alternative to create new effector functional human immunotherapeutics, while reducing mAb therapy costs. This review highlights some features that enhance mAb performance, related to the improvement of antibody half-life and effector responses by both Fc-engineering and glycoengineering.

Activating Transcription Factor 2 (ATF2) plays an important role in mammalian cell proliferation, apoptosis and DNA repair. Its activation is dependent on the sequential phosphorylation of residue threonine 71 (T71) followed by threonine 69 (T69) in its transactivation domain. While these modifications can be directed by a variety of kinases, the time to reach full phosphorylation is dependent on which signaling pathway has been activated, which is thought to be important for proper temporal regulation. To explore this phenomenon further, there have been ongoing efforts to generate affinity reagents for monitoring phosphorylation events in cellular assays. While phospho-specific antibodies have been valuable tools for monitoring cell signaling events, those raised against a peptide containing two or more adjacent phosphosites tend to cross-react with that peptide's various phospho-states, rendering such reagents unusable for studying sequential phosphorylation. As an alternative, we have employed the N-terminal Forkhead-associated 1 (FHA1) domain of yeast Rad53p as a scaffold to generate recombinant affinity reagents via phage display and were successful in generating a set of reagents that can distinguish between the dual-phosphorylated epitope, 63-IVADQpTPpTPTRFLK-77, and the mono-phosphorylated epitope, 63-IVADQpTPTPTRFLK-77, in the human ATF2 transactivation domain.

Fibroblast growth factor 21 (FGF21), an endocrine hormone in the FGF family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus. FGF21 functions through a receptor complex that consists of an FGF receptor (FGFR) and a co-receptor β-Klotho. Here, we identify and biochemically and structurally characterize 39F7, a high-affinity agonistic monoclonal antibody (mAb) against β-Klotho that mimics FGF21 function. The co-crystal structure of β-Klotho KL1 domain in complex with 39F7 Fab revealed that the recognition of 39F7 is centered on Trp-295 of β-Klotho in a FGF21 noncompetitive manner. KL1 adopts a (β/α)8 TIM barrel fold which resembles that of β-glycosylceramidase, but lacks molecular features for enzymatic activity, suggesting that KL1 functions as a scaffold protein instead. In vitro characterization demonstrated that, although 39F7 does not compete with FGF21, it is specific for β-Klotho/FGFR1c activation. Furthermore, the agonistic activity of 39F7 required the full IgG molecule to be bivalent, suggesting that 39F7 functions by promoting receptor/co-receptor dimerization. Supported by negative stain EM analysis of full-length β-Klotho, we propose a molecular model wherein the agonistic antibody 39F7 acts in a β-Klotho– and FGFR1c-dependent manner, mimicking FGF21 activity. More importantly, 39F7 offers promising therapeutic potential in the axis of FGF21 signaling as an antibody therapy alternative to FGF21 analogs for treatment of metabolic diseases.

Monoclonal antibodies (MAbs) are a unique and attractive class of biologics and are potential substitutes for post-exposure rabies prophylaxis. The safety, tolerance, and broad neutralization efficiency of a MAb cocktail called CL184, composed of the antibodies CR4098 and CR57, was confirmed in a phase I clinical trial. We have prepared a series of single-chain Fv fragments (scFvs) and leucine zipper Fv fragments (zipFvs) from CR57 and CR4098. In this study, we selected and formed scFv and zipFv cocktails and compared their protective effects against the rabies virus. Mice and hamster challenge models demonstrated the improved protection of the zipFv cocktail compared with scFv cocktail, because of its stronger affinity. The results indicate that zipFv production is a promising novel method for the genetic engineering of antibody fragments and improving affinity through systematic screening may be important when designing small molecule antibodies against RV.

The widespread use of monoclonal antibodies for therapeutic applications has led to intense interest in optimizing several of their natural properties (affinity, specificity, stability, solubility and effector functions) as well as introducing non-natural activities (bispecificity and cytotoxicity mediated by conjugated drugs). A common challenge during antibody optimization is that improvements in one property (e.g., affinity) can lead to deficits in other properties (e.g., stability). Here we review recent advances in understanding trade-offs between different antibody properties, including affinity, specificity, stability and solubility. We also review new approaches for co-optimizing multiple antibody properties and discuss how these methods can be used to rapidly and systematically generate antibodies for a wide range of applications.

A novel label-free protocol was developed for measuring the affinity between ractopamine and its monoclonal antibody (McAb) based on microdialysis (MD) on-line sampling integrated with flow injection chemiluminescent detection. In this study, unbound ractopamine was sampled by MD probe from homogeneous immunoreaction equilibrious systems, and then real-time quantified using flow injection chemiluminescent detection. The quantified concentrations of unbound ractopamine in the immunoreaction equilibrious systems were treated with Scatchard analysis and Klotz analysis to obtain the affinity constant. The mean recovery of MD probe for sampling ractopamine was found to be 24.2%. The affinity constants calculated by Scatchard analysis and Klotz analysis both were 1.0 × 106 M−1, indicating that the investigated ractopamine mouse McAb was a medium-affinity antibody. The result showed good agreement with that obtained from thiocyanate elution test. This protocol for measuring antibody affinity is free of protein conjugation of hapten and enzyme labeling of McAb. Therefore it avoids affinity decrease resulting from steric hindrance, occupancy of the antigenic determinants, and deactivation of antibody, which has been frequently encountered in the reported conventional approaches. It opens up a new pathway for direct measurement of antibody affinity with a facile, rapid, accurate and low-cost approach.

A critical property of a prophylactic HIV vaccine is likely to be its ability to elicit broadly neutralizing antibodies (bnAbs). BnAbs typically have multiple unusual features and are generated in a fraction of HIV-infected individuals through complex pathways. Current vaccine design approaches seek to trigger rare B cell precursors and then steer affinity maturation toward bnAbs in a multi-stage multi-component immunization approach. These vaccine design strategies have been facilitated by molecular descriptions of bnAb interactions with stabilized HIV trimers, the use of an array of sophisticated approaches for immunogen design, the development of novel animal models for immunogen evaluation and advanced technologies to interrogate antibody responses. In this review, we will discuss leading HIV bnAb vaccine immunogens, immunization strategies and future improvements.

Antibody design aims to create new antibodies with biological activity that can be used in therapy and research. Traditional methods for antibody discovery, such as animal immunization and large-scale library screening, generate antibodies that bind to the target of interest, but do not necessarily have the desired functional effect. Computational methods can be utilized as a means to guide the search for biologically relevant antibodies, focusing on specificity and affinity determinants to target a particular region of the antigen. Such an approach would allow for the design of epitope-specific antibodies that will have the desired effect on the function of the targeted protein.

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70 – 89) and young (aged 20 – 29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.

To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction. Finally, the library for anti-VP3 scFv was screened by four rounds of adsorption-elution-enrichment with the purified VP3 protein. The characters of binding ability, specificity and neutralization of soluble antibodies expressed were evaluated by ELISA. The results showed 7 VP3-specific scFvs have been screened and identified with high both sensitivity and specificity for binding DHAV-1. To our knowledge, this is the first report for VP3-specific scFv of DHAV-1 and potentially promising application used in prevention and treatment of duck viral hepatitis.

Pep27 from Streptococcus pneumoniae is reported to initiate pneumococcal autolysis, thereby constituting a major virulence factor. Although a few antisera recognizing Pep27 have been reported, no monoclonal, well-characterized antibody for Pep27 has been developed. Here we screened two single-chain antibody variable fragments (scFv) using a phage display from a large human synthetic scFv library to select clones E2 and F9. Dissociation constants (Kd) of E2 and F9 were 1.1 μM and 0.50 μM, respectively. E2 and F9 did not cross-react with other pneumococcal and unrelated proteins. The epitopes of Pep27 were localized to residues 24, 26 and 27 by alanine scanning. Molecular docking analysis supported the experimentally investigated epitope. The E2 and F9 clones specifically detected Pep27 in an environment mimicking in vivo conditions, demonstrated in human serum. The scFv clones characterized here represent molecular tools for the detection of pneumococcal diseases with potential for further improvement in affinity.

The daily clinical practice frequently force physicians to make adecision of whether a patient is coursing an infectious disease at theacute or the chronic stage, since treatment may be different dependingon it. This is the case of toxoplasmosis, a worldwide disease caused bythe intracellular parasite Toxoplasma gondii. When a pregnant woman isinfected for the first time by T. gondii and is not treated, the parasite cancross the placenta and severely affect the fetus. In case of primary infection,the treatment protocol includes using antiparasitic drugs toprevent serious damages, including death of the unborn baby. However,antiparasitic chemicals have side effects that should be avoided, unlesstreatment is mandatory. That is why discrimination between long-termand primary T. gondii infection turns out to be critical in infectedpregnant women (Montoya and Remington, 2008).In this work we have used rP22a, an acute-phase recombinant protein werecently described (Costa et al., 2017), to assess anti-T. gondii IgG AI,calculated by two new easier approaches, one based on the area underthe avidity curve (AUAC) and the other one based on the E-P titrationmethod with minor variations. We compare our results with those obtainedwhen calculating the AI by the conventional methods abovementioned, and the performance to render a proper sample classificationas compared to that obtained having used a commercial kit asstandard. Fil: Garcia, Valeria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Peverengo, Luz María. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Peretti, Leandro Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: González, Verónica Doris Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Dalla Fontana, Maria L.. Laboratorio Central de la Provincia de Santa Fe; Argentina Fil: Marcipar, Iván Sergio. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lagier, Claudia Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called “stereotypic RFs,” are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2–CH3 elbow in the canonical antigen-binding manner involving a large antigen–antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu(432)–His(435) region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

Current serological methods cannot distinguish active from past infection with Borrelia burgdorferi sensu lato. The aim of this study was to develop an IgG avidity Western blot and assess its potential to differentiate patients with early and late Lyme borreliosis (LB) i.e. active disease, from those infected in the past.An IgG avidity Western blot was developed. Penalized linear discriminant analysis (PLDA) was employed to compare the Western blot/avidity Western blot profiles of an evaluation panel consisting of 75 sera from patients with early (n = 26) and late (n = 24) LB and past infection (n = 25). The PLDA models produced were used to predict infection stage for 20 well characterised sera from the Centers for Disease Control and Prevention (CDC) Lyme disease serum repository and 112 routine seropositive sera (disease stage unknown), to validate and assess the usefulness of the avidity Western blot/avidity Western blot and PLDA approach.PLDA correctly classified 40/51 (78%) of patients when early LB and past infection groups in the evaluation panel were compared. Likewise, when late LB and past infection groups were compared, 34/49 (69%) were correct. The resultant PLDA models correctly predicted infection stage for 18/20 (90%) of the CDC sera, validating the use of the avidity Western blot/avidity Western blot and PLDA approach. When tested with the routine sera, 21/29 (72%) tested with the early LB vs. past infection model were correct but only 32/83 (39%) with the late LB vs. past infection model. Past infection was predicted for 40/112 (35%) of the routine sera, 80% of which correlated with the clinical picture.The Western blot/avidity Western blot with PLDA approach shows exciting potential for being able to predict disease stage in some patients with LB, which could improve patient management.