Jonathan R. McDaniel

The World Health Organization (WHO) recently recommended the programmatic use of the R21/Matrix-M vaccine for Plasmodium falciparum malaria prevention in children living in malaria-endemic areas. To determine its effects on humoral immunity, we conducted a proteomic analysis of polyclonal IgG antibodies directed against the NANP tetrapeptide of the circumsporozoite protein (CSP), which comprises the vaccine’s core immunogen. In 10 malaria-naïve adult volunteers, R21/Matrix-M induced polarized IgG anti-NANP repertoires, heavily skewed for IGHV3-30/3-33 genes bearing minimal somatic mutation, which remained static in composition following a controlled human malaria infection challenge. Notably, these vaccine-generated antibodies cross-reacted with another protective CSP epitope, the N-terminal junction region, despite its absence from the R21 construct. NANP-specific IGHV3-30/3-33 mAbs mined from polyclonal IgG repertoires blocked sporozoite invasion in vitro and prevented parasitemia in vivo. Overall, R21/Matrix-M elicits polarized, minimally mutated, polyclonal IgG responses that can target multiple protective CSP epitopes, offering molecular insight into the serological basis for its demonstrated efficacy against P. falciparum malaria.

The sequencing of the antibody repertoire (Rep-seq) revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse ( Equus caballus ) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis of diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-immunized horses using HTS technology. In this study, we obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 and 82% in IgM and IgG isotypes. We observed that besides horses antibody can use all of the functional IGHV genes, around 80% of their antibodies use only three IGHV gene segments, and around 55% use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horses antibodies is highly frequent, present in more than 90% of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45% of the antibody clones have more than ten nucleotides in both N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and in other species to better understand antibody repertoire characteristics.

Objective To determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD. Methods We combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro. Results Multiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01). Conclusions The composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones.

A public anti-COVID antibody repertoire: Most analyses of the antibody responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have focused on antibodies cloned from memory B cells. This approach has led researchers to conclude that neutralizing antibodies (nAbs) primarily target the receptor-binding domain (RBD) of the virus's spike protein. Voss et al. took a different approach, using proteomic deconvolution of the serum immunoglobulin G antibody repertoire from four COVID-19 convalescent patients. They found that the nAb response was largely directed against epitopes such as the N-terminal domain (NTD), which lie outside the RBD. Several of these nAbs were shared among donors and targeted an NTD epitope that is frequently mutated by variants of concern. Science , abg5268, this issue p. 1108

We report a simple and low-cost one-step high-throughput technology for sequencing the paired antibody/TCR repertoire.

We report the development of drug-encapsulating nanoparticles that bind endogenous albumin upon intravenous injection and evaluate their in vivo performance in a murine as well as canine animal model. The gene encoding a protein-G derived albumin binding domain (ABD) was fused to that of a chimeric polypeptide (CP), and the ABD-CP fusion was recombinantly synthesized by bacterial expression of the gene. Doxorubicin (DOX) was conjugated to the C-terminus of the ABD-CP fusion, and conjugation of multiple copies of the drug to one end of the ABD-CP triggered its self-assembly into ∼100 nm diameter spherical micelles. ABD-decorated micelles exhibited submicromolar binding affinity for albumin and also preserved their spherical morphology in the presence of albumin. In a murine model, albumin-binding micelles exhibited dose-independent pharmacokinetics, whereas naked micelles exhibited dose-dependent pharmacokinetics. In addition, in a canine model, albumin binding micelles resulted in a 3-fold increase in plasma half-life and 6-fold increase in plasma exposure as defined by the area under the curve (AUC) of the drug, compared with naked micelles. Furthermore, in a murine colon carcinoma model, albumin-binding nanoparticles demonstrated lower uptake by the reticuloendothelial system (RES) system organs, the liver and spleen, that are the main target organs of toxicity for nanoparticulate delivery systems and higher uptake by the tumor than naked micelles. The increased uptake by s.c. C26 colon carcinoma tumors in mice translated to a wider therapeutic window of doses ranging from 20 to 60 mg equivalent of DOX per kg body weight (mg DOX equiv·kg^–1 BW) for albumin-binding ABD-CP-DOX micelles, as compared to naked micelles that were only effective at their maximum tolerated dose of 40 mg DOX equiv·kg^–1 BW.

FCRL4, a low-affinity IgA Ab receptor with strong immunoregulatory potential, is an identifying feature of a tissue-based population of memory B cells (Bmem). We used two independent approaches to perform a comparative analysis of the Ag receptor repertoires of FCRL4⁺ and FCRL4⁻ Bmem in human tonsils. We determined that FCRL4⁺ Bmem displayed lower levels of somatic mutations in their Ag receptors compared with FCRL4⁻ Bmem but had similar frequencies of variable gene family usage. Importantly, Abs with reactivity to commensal microbiota were enriched in FCRL4⁺ cells, a phenotype not due to polyreactive binding characteristics. Our study links expression of the immunoregulatory FCRL4 molecule with increased recognition of commensal microbial Ags.

A better understanding of antitumor immune responses is the key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients. Using a synergistic combination of high-throughput B-cell sequencing and quantitative immunoproteomics, we describe the prospective identification of tumor-reactive SN B cells (based on clonal frequency) and also demonstrate an unequivocal link between affinity-matured expanded B-cell clones in the SN and antitumor IgG in the blood. This technology could facilitate the discovery of antitumor antibody therapeutics and conceivably identify novel tumor antigens. Lastly, these findings highlight the unique and specialized niche the SN can fill in the advancement of cancer immunotherapy.