OcaB, also known as Bob‐1 or Obf‐1, is a transcriptional co‐activator which regulates Igκ gene transcription, recombination and receptor editing; it is required for normal development of transitional ...B cells and for germinal center formation. Here we report that abnormal B cell development in OcaB–/– mice results in a skewed Igκ repertoire including anti‐DNA antibodies, suggesting that OcaB is essential for antibody repertoire selection. To determine whether OcaB is required for BCR‐mediated B cell selection, we introduced a pre‐recombined α hen egg lysozyme (HEL) Ig transgene into OcaB–/– mice. We find that in OcaB–/– mice expressing transgenic αHEL Ig bone marrow B cell development is normal up to the immature B cell stage, but fails to progress to the transitional B cell stage. We conclude that OcaB is required for normal selection of the antibody repertoire in developing B cells.
Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, ...we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.
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•Antibodies against HBsAg from different donors share sequence similarities•Broadly neutralizing antibodies (bNAbs) to HBsAg target 3 non-overlapping epitopes•Co-crystal structure reveals a hairpin loop that explains immune escape mutation•Combination of complementary bNAbs controlled HBV infection and prevented escape mutation
Human antibody immune response against HBV is poorly studied. Wang et al. cloned human anti-S antibodies from selected individuals and identified broadly neutralizing antibodies that can prevent or treat infection in humanized mice. Co-crystal structure explains the necessity of using complementary antibodies to suppress the emergence of escape mutations.
Antiretroviral therapy controls, but does not cure, HIV-1 infection due to a reservoir of rare CD4
T cells harboring latent proviruses. Little is known about the transcriptional program of latent ...cells. Here, we report a strategy to enrich clones of latent cells carrying intact, replication-competent HIV-1 proviruses from blood based on their expression of unique T cell receptors. Latent cell enrichment enabled single-cell transcriptomic analysis of 1,050 CD4
T cells belonging to expanded clones harboring intact HIV-1 proviruses from 6 different individuals. The analysis reveals that most of these cells are T effector memory cells that are enriched for expression of HLA-DR, HLA-DP, CD74, CCL5, granzymes A and K, cystatin F, LYAR, and DUSP2. We conclude that expanded clones of latent cells carrying intact HIV-1 proviruses persist preferentially in a distinct CD4
T cell population, opening possibilities for eradication.
The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct ...functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.
The SARS-CoV-2 pandemic prompted a global vaccination effort and the development of numerous COVID-19 vaccines at an unprecedented scale and pace. As a result, current COVID-19 vaccination regimens ...comprise diverse vaccine modalities, immunogen combinations, and dosing intervals. Here, we compare vaccine-specific antibody and memory B cell responses following two-dose mRNA, single-dose Ad26.COV.2S, and two-dose ChAdOx1, or combination ChAdOx1/mRNA vaccination. Plasma-neutralizing activity, as well as the magnitude, clonal composition, and antibody maturation of the RBD-specific memory B cell compartments, showed substantial differences between the vaccination regimens. While individual monoclonal antibodies derived from memory B cells exhibited similar binding affinities and neutralizing potency against Wuhan-Hu-1 SARS-CoV-2, there were significant differences in epitope specificity and neutralizing breadth against viral variants of concern. Although the ChAdOx1 vaccine was inferior to mRNA and Ad26.COV.2S in several respects, biochemical and structural analyses revealed enrichment in a subgroup of memory B cell neutralizing antibodies with distinct RBD-binding properties resulting in remarkable potency and breadth.
Chromosome translocations between c-
myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations ...frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-
myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-
myc/IgH chromosome translocations induced by IL6.
B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and ...amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.
Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The ...circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti–HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.
The detection of genomic regions unusually rich in a given pattern is an important undertaking in the analysis of next-generation sequencing data. Recent studies of chromosomal translocations in ...activated B lymphocytes have identified regions that are frequently translocated to c-myc oncogene. A quantitative method for the identification of translocation hotspots was crucial to this study. Here we improve this analysis by using a simple probabilistic model and the framework provided by scan statistics to define the number and location of translocation breakpoint hotspots. A key feature of our method is that it provides a global chromosome-wide nominal control level to clustering, as opposed to previous methods based on local criteria. While being motivated by a specific application, the detection of unusual clusters is a widespread problem in bioinformatics. We expect our method to be useful in the analysis of data from other experimental approaches such as of ChIP-seq and 4C-seq.
The analysis of translocations from B lymphocytes with the method described here reveals the presence of longer hotspots when compared with those defined previously. Further, we show that the hotspot size changes substantially in the absence of DNA repair protein 53BP1. When 53BP1 deficiency is combined with overexpression of activation-induced cytidine deaminase, the hotspot length increases even further. These changes are not detected by previous methods that use local significance criteria for clustering. Our method is also able to identify several exclusive translocation hotspots located in genes of known tumor supressors.
The detection of translocation hotspots is done with hot_scan, a program implemented in R and Perl. Source code and documentation are freely available for download at https://github.com/itojal/hot_scan.
The identification and characterization of conserved epitopes on the HIV-1 viral spike that are immunogenic in humans and targeted by neutralizing antibodies is an important step in vaccine design. ...Antibody cloning experiments revealed that 32% of all HIV-neutralizing antibodies expressed by the memory B cells in patients with high titers of broadly neutralizing antibodies recognize one or more "core" epitopes that were not defined. Here, we show that anti-core antibodies recognize a single conserved epitope on the gp120 subunit. Amino acids D474, M475, R476, which are essential for anti-core antibody binding, form an immunodominant triad at the outer domain/inner domain junction of gp120. The mutation of these residues to alanine impairs viral fusion and fitness. Thus, the core epitope, a frequent target of anti-HIV-neutralizing antibodies, including the broadly neutralizing antibody HJ16, is conserved and indispensible for viral infectivity. We conclude that the core epitope should be considered as a target for vaccine design.
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