High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

<b><i>Background:</i></b> High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. <b><i>Methods:</i></b> Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of <i>KEL</i>*<i>01</i> and <i>KEL</i>*<i>02</i>, <i>JK</i>*<i>01</i> and <i>JK</i>*<i>02</i>, <i>FY</i>*<i>01</i>, <i>FY</i>*<i>02</i>, and <i>FY</i>*<i>02 N.01</i>, <i>DI</i>*<i>01</i> and <i>DI</i>*<i>02</i>, <i>GYPB</i>*<i>03</i> and <i>GYPB</i>*<i>04</i>, <i>RHCE</i>*<i>E</i> and <i>RHCE</i>*<i>e,</i> and <i>DO</i>*<i>01</i> and <i>DO</i>*<i>02.</i> Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. <b><i>Results:</i></b> The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. <b><i>Conclusions:</i></b> We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

Neutralization Fingerprinting Technology for Characterizing Polyclonal Antibody Responses to Dengue Vaccines

Dengue is a major public health threat. There are four serotypes of dengue virus (DENV), therefore efforts are focused on development of safe and effective tetravalent DENV vaccines. While neutralizing antibodies contribute to protective immunity, there are still important gaps in understanding of immune responses elicited by dengue infection and vaccination, including defining immune correlates of protection. To that end, here we present a computational modeling framework for evaluating the specificities of neutralizing antibodies elicited to tetravalent DENV vaccines, based on the concept of antibody-virus neutralization fingerprints. We developed and applied this framework to samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials, to characterize the effect of prior dengue infection (baseline) on the specificities of vaccine-elicited antibody responses. Our results suggested a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities was predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The analysis framework proposed here can apply to studies of sequential dengue infections and other tetravalent DENV vaccines and can contribute to understanding dengue immune correlates of protection to help guide further vaccine development and optimization.

Structural mapping of antibody landscapes to human betacoronavirus spike proteins

Preexisting immunity against seasonal coronaviruses (CoV) represents an important variable in predicting antibody responses and disease severity to Severe Acute Respiratory Syndrome CoV 2 (SARS-2) infections. We used electron microscopy based polyclonal epitope mapping (EMPEM) to characterize the antibody specificities against β-CoV spike proteins in sera from healthy donors (HDs) or SARS-2 convalescent donors (CDs). We observed that most HDs possessed antibodies specific to seasonal human CoVs (HCoVs) OC43 and HKU1 spike proteins while the CDs showed reactivity across all human β-CoVs. Detailed molecular mapping of spike-antibody complexes revealed epitopes that were differentially targeted by antibodies in preexisting and convalescent serum. Our studies provide an antigenic landscape to β-HCoV spikes in the general population serving as a basis for cross-reactive epitope analyses in SARS-2 -infected individuals.

Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 × 10−14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.

COVID-19 antibody detection and assay performance using red cell agglutination

Red cells can be labelled with peptides from the SARS-CoV-2 spike protein and used for serologic screening of SARS-CoV-2 antibodies. We evaluated 140 convalescent COVID-19 patients and 275 healthy controls using this C19-kodecyte assay. The analytical performance of the new assay was compared with a virus neutralizing assay and 2 commercial chemiluminescent antibody tests (Total assay and IgG assay, Ortho). The C19-kodecyte assay detected SARS-CoV-2 antibodies with a sensitivity of 92.8% and specificity of 96.3%, well within the minimum performance range required by FDA for EUA authorization of serologic tests. The Cohen's kappa coefficient was 0.90 indicating an almost perfect agreement with the Total assay. The Pearson correlation coefficient was 0.20 with the neutralizing assay (0.49 with IgG, and 0.41 with Total assays). The limited correlation in assay reaction strengths suggested that the assays may detect different antibody specificities. Our easily scalable C19-kodecyte assay may vastly improve test capacity in blood typing laboratories using their routine setups for column agglutination technique.

Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of β-1,2 mannotriose protective epitopes

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and β anomery complemented with a synthetic β-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or β linkages, excepted for MAb B6.1 (protective epitope) reacting with β-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients’ sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing β- and α-Man epitopes and detection of antibodies against β-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and β-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/β ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Frequent development of broadly neutralizing antibodies in early life in a large cohort of HIV-infected children

Recent studies conducted in small cohorts of children have indicated that broadly neutralizing antibodies (bnAbs) may develop earlier after HIV infection compared to adults. To define the frequency and kinetics of bnAb responses in a larger pediatric cohort, we evaluated plasma from 212 ART-naïve, children living with HIV aged 1 to 3 years. Neutralization breadth and potency was assessed using a panel of 10 tier-2 viruses and compared to those of adults with chronic HIV. Further, the magnitude, epitope specificity and IgG subclass distribution of Env-specific antibodies were also assessed using a binding antibody multiplex assay. We found that 1-year-old children demonstrated neutralization breadth comparable to that of chronically-infected adults, and breadth continued to increase with age such that the pediatric cohort overall exhibited significantly greater neutralization breadth than adults (p= 0.014). Similarly, binding antibody responses increased with age, and the levels in 2 to 3 year-old children were comparable to those of adults. Overall, there was no significant difference in antibody specificities or IgG subclass distribution between the pediatric and adult cohorts. Interestingly, the neutralization activity was mapped to a single epitope (CD4 binding site, V2 or V3 glycans) in only 5 of 38 pediatric broadly neutralizing samples, suggesting a polyclonal neutralization response may develop in most children. These results contribute to a growing body of evidence suggesting that the early life immune system may present advantages for the development of an effective HIV vaccine.

Membrane Env liposomes facilitate immunization with multivalent full-length HIV spikes

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bnAbs). Such bnAbs target HIV’s trimeric, membrane embedded envelope glycoprotein spikes, (m)Env. Soluble (s)Env trimers have been used as vaccines, but engineering sEnvs for stability, multivalency and desired antigenicity is problematic, and deletes key neutralizing epitopes on glycoprotein (gp)41 while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. MEnv trimers were fixed, purified and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter 133 nm) in correct orientation. These particles were recognized by HIV bnAbs and not non-nAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provide clues on how nAb responses may be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enable rational immunization strategies to elicit HIV bnAbs using multimerized mEnv. Importance A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane embedded envelope glycoprotein spikes (m)Env that HIV uses to enter cells. Due to HIV Env’s low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane embedded targets of therapeutic interest.

Anti-Cancer Auto-Antibodies: Roles, Applications and Open Issues

Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body’s defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community.

Profiling serum antibodies with a pan allergen phage library identifies key wheat allergy epitopes

Allergic reactions occur when IgE molecules become crosslinked by antigens such as food proteins. Here we create the ‘AllerScan’ programmable phage display system to characterize the binding specificities of anti-allergen IgG and IgE antibodies in serum against thousands of allergenic proteins from hundreds of organisms at peptide resolution. Using AllerScan, we identify robust anti-wheat IgE reactivities in wheat allergic individuals but not in wheat-sensitized individuals. Meanwhile, a key wheat epitope in alpha purothionin elicits dominant IgE responses among allergic patients, and frequent IgG responses among sensitized and non-allergic patients. A double-blind, placebo-controlled trial shows that alpha purothionin reactivity, among others, is strongly modulated by oral immunotherapy in tolerized individuals. AllerScan may thus serve as a high-throughput platform for unbiased analysis of anti-allergen antibody specificities.