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.

Humoral immunity depends on a dynamic interplay between circulating polyclonal IgG antibodies and long-lived memory B cells (MBC). To elucidate this relationship, we integrated B-cell receptor sequencing with plasma IgG immunoproteomics to identify and quantify the recall response (plasma IgG and MBC) to inactivated poliovirus vaccine (IPV) in eight adults, 24–55 years after their childhood immunization. IPV immunization elicited highly skewed plasma IgG recall repertoires, dominated by a small subset (~8%) of total antibody lineages that were detectable prior to boosting (i.e., "pre-existing"). These IgG lineages spiked logarithmically in abundance (~102-104 fold-increase) and potently cross-neutralized multiple PV serotypes in vitro (titers ≥ 1:105). Notably, these dominant plasma IgG lineages were clonally linked to class-switched MBCs that circulated in blood 21 days post-boost and bore heavily mutated VH and VL variable region genes. Importantly, IPV-primed ex vivo B-cell cultures (MIMIC, Modular IMmune In vitro Construct) yielded clonal expansions that mirrored the dominant post-boost plasma IgG lineages in vivo , providing an in vitro mechanistic correlate of recall immunity. Together these findings demonstrate a decades-long interconnectivity between persistent MBC clones and the serological IgG repertoire. They further highlight the central role of immunological imprinting, whereby cross-reactive MBC clones—originally primed in childhood—can be reactivated to dominate the circulating IgG recall response more than 50 years after initial exposure. ### Competing Interest Statement The authors have declared no competing interest. Defense Threat Reduction Agency, https://ror.org/04tz64554, HDTRA1-12-C-0105

Objective.Currently, there exists very few ways to isolate cognitive processes, historically defined via highly controlled laboratory studies, in more ecologically valid contexts. Specifically, it remains unclear as to what extent patterns of neural activity observed under such constraints actually manifest outside the laboratory in a manner that can be used to make accurate inferences about latent states, associated cognitive processes, or proximal behavior. Improving our understanding of when and how specific patterns of neural activity manifest in ecologically valid scenarios would provide validation for laboratory-based approaches that study similar neural phenomena in isolation and meaningful insight into the latent states that occur during complex tasks.Approach.Domain generalization methods, borrowed from the work of the brain-computer interface community, have the potential to capture high-dimensional patterns of neural activity in a way that can be reliably applied across experimental datasets in order to address this specific challenge. We previously used such an approach to decode phasic neural responses associated with visual target discrimination. Here, we extend that work to more tonic phenomena such as internal latent states. We use data from two highly controlled laboratory paradigms to train two separate domain-generalized models. We apply the trained models to an ecologically valid paradigm in which participants performed multiple, concurrent driving-related tasks while perched atop a six-degrees-of-freedom ride-motion simulator.Main Results.Using the pretrained models, we estimate latent state and the associated patterns of neural activity. As the patterns of neural activity become more similar to those patterns observed in the training data, we find changes in behavior and task performance that are consistent with the observations from the original, laboratory-based paradigms.Significance.These results lend ecological validity to the original, highly controlled, experimental designs and provide a methodology for understanding the relationship between neural activity and behavior during complex tasks.

PDF file - 128K, The effect of the number of ELP tyrosine residues, ELP MW and concentration on retention radioactivity in FaDu tumor as a function of time post-injection of labeled ELP

The antibody repertoire (Rep-seq) sequencing 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 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-infected horses using NGS (Next generation sequencing). This study 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 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 horse antibodies is widespread, 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 the 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 other species to better understand antibody repertoire characteristics.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galβ1-4GalNAcβ), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.

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.

Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRb:a clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRb:a.

New research and development efforts are highlighting the ways in which electroencephalogram-based (EEG) brain-computer interface (BCI) technology can be used to improve the quality of life for healthy individuals. One such application incorporates cognitive state monitoring into passive BCI (pBCI) systems. Among the challenges facing this development, a significant barrier to adoption is the time-intensive calibration typically needed to tune the system to account for variations in neural activity patterns. An open research question is understanding the relationship between underlying user state and user performance in real-world situations and environments. However, user states are often derived and defined as a function of observed user performance for a particular analysis. Understanding the relationship between user state and user performance ideally requires the definition of user state be independent of observed user performance. This work represents our initial steps towards this goal by using cross-dataset learning, where we define user state from a dataset recorded in a highly-controlled experiment, building a subject-independent pBCI model to predict user state using deep learning approaches, and applying this pBCI model to analyze user performance in a new, unobserved dataset. We show that user performance varies smoothly across a continuum of pBCI model outputs. Our results highlight a promising approach for dealing with one of the major hurdles in the development of BCI systems for healthy users.

SUMMARY Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq 1 ) to the spike ectodomain (S-ECD 2 ) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro , we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 Å 2 ). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning 3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

Antibodies and T cell receptors (TCRs) have critical roles in adaptive immunity and thus the determination of their sequences is essential for understanding immune responses in infections or vaccinations, for the discovery of therapeutic antibodies, and for the engineered T cell therapies. Antibodies are composed of variable heavy (VH) and light (VL) chains, and TCRs are composed of variable TCRα/γ and TCRβ/δ chains. Since those chains are encoded by two different transcripts, the determination of functional sequences requires single-cell sequencing. Emulsification of single-cells followed by barcoding of the transcripts or fusion of VH:VL or TCRβ:α transcripts using overlap extension RT-PCR allows high-throughput single-cell sequencing; however, because cell lysate in emulsion inhibits the RT-PCR reaction, these methods require complex microfluidics devices to bypass the inhibition which most of biomedical laboratories cannot access. We discovered recently engineered xenopolymerase, RTX, has an exceptional cell lysate resistance that can overcome this limitation. By employing this unique feature of RTX, we have developed a one-step emulsion-based ultra-high-throughput sequencing method for paired antibody VH:VL and TCRβ:α repertoires not requiring microfluidics or any other specialized equipment. Using this methodology, we obtained high yields (5,000 to >20,000/sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we applied the methodology to the peripheral blood plasmablasts and T follicular helper (TFH) cells from a seasonal influenza vaccine recipient and discovered high-affinity influenza-specific antibodies and TCRβ:α.

Humans are repeatedly exposed to influenza virus via infections and vaccinations. Understanding how multiple exposures and pre-existing immunity impact antibody responses is essential for vaccine development. Given the recent prevalence of influenza H1N1 A/California/7/2009 (CA09), we examined the clonal composition and dynamics of CA09 hemagglutinin (HA)-reactive IgG repertoire over 5 years in a donor with multiple influenza exposures. The anti-CA09 HA polyclonal response in this donor comprised 24 persistent antibody clonotypes, accounting for 72.6% ± 10.0% of the anti-CA09 HA repertoire over 5 years. These persistent antibodies displayed higher somatic hypermutation relative to transient serum antibodies detected at one time point. Additionally, persistent antibodies predominantly demonstrated cross-reactivity and potent neutralization toward a phylogenetically distant H5N1 A/Vietnam/1203/2004 (VT04) strain, a feature correlated with HA stem recognition. This analysis reveals how “serological imprinting” impacts responses to influenza and suggests that once elicited, cross-reactive antibodies targeting the HA stem can persist for years.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire—pre- and post-vaccination—and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.

Advances in next-generation sequencing have allowed insights into the enormous diversity of the human T cell receptor (TCR). Earlier studies have suggested that ~108 unique TRB genes exist at the periphery. In contrast, the factors that determine the pairing of TCRα and TCRβ chains have remained elusive due to the lack of high-throughput single-cell sequencing technology. We have developed a high-throughput single-cell sequencing technology that enables identification of >3×104 paired TRA:TRB sequences per sample and have conducted sequencing of naïve and memory CD4+ and of total T cells from 15 healthy individuals, including six pairs of monozygotic twins. We found that the frequencies of TRAV:TRBV pairing are essentially determined by the frequencies of the individual gene segments, suggesting the absence of structural constraints in the pairing of the α and β chains. Twins are more similar in the frequencies of TRAV and TRBV combinations than are unrelated individuals, but this difference was explained by a genetic influence on AV and BV gene segment frequencies. Twins and unrelated individuals were compared for the sharing of identical TCR sequences. TRA sequence sharing among twins was comparable to that among unrelated individuals, but TRB sharing was significantly higher in twins. Sharing of the entire TCR was regularly found in twins but not detected for naïve CD4+ T cells from unrelated individuals. Results were similar for naïve and memory T cells, with the genetic influence more prominent in memory T cells, suggesting the additional influence of peripheral selection. In support of this interpretation, the sharing of identical TCR sequences in unrelated individuals correlated with the number of shared MHC class II alleles.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The T cell repertoire in each individual includes T cell receptors (TCRs) of enormous sequence diversity through the pairing of diverse TCR α- and β-chains, each generated by somatic recombination of paralogous gene segments. Whether the TCR repertoire contributes to susceptibility to infectious or autoimmune diseases in concert with disease-associated major histocompatibility complex (MHC) polymorphisms is unknown. Due to a lack in high-throughput technologies to sequence TCR α–β pairs, current studies on whether the TCR repertoire is shaped by host genetics have so far relied only on single-chain analysis. Using a high-throughput single T cell sequencing technology, we obtained the largest paired TCRαβ dataset so far, comprising 965,523 clonotypes from 15 healthy individuals including 6 monozygotic twin pairs. Public TCR α- and, to a lesser extent, TCR β-chain sequences were common in all individuals. In contrast, sharing of entirely identical TCRαβ amino acid sequences was very infrequent in unrelated individuals, but highly increased in twins, in particular in CD4 memory T cells. Based on nucleotide sequence identity, a subset of these shared clonotypes appeared to be the progeny of T cells that had been generated during fetal development and had persisted for more than 50 y. Additional shared TCRαβ in twins were encoded by different nucleotide sequences, implying that genetic determinants impose structural constraints on thymic selection that favor the selection of TCR α–β pairs with entire sequence identities.

Deep convolutional neural networks (CNN) have previously been shown to be useful tools for signal decoding and analysis in a variety of complex domains, such as image processing and speech recognition. By learning from large amounts of data, the representations encoded by these deep networks are often invariant to moderate changes in the underlying feature spaces. Recently, we proposed a CNN architecture that could be applied to electroencephalogram (EEG) decoding and analysis. In this article, we train our CNN model using data from prior experiments in order to later decode the P300 evoked response from an unseen, hold-out experiment. We analyze the CNN output as a function of the underlying variability in the P300 response and demonstrate that the CNN output is sensitive to the experiment-induced changes in the neural response. We then assess the utility of our approach as a means of improving the overall signal-to-noise ratio in the EEG record. Finally, we show an example of how CNN-based decoding can be applied to the analysis of complex data.

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.