Affinity gaps among B cells in germinal centers drive the selection of MPER precursors.

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.

Vaccination induces broadly neutralizing antibody precursors to HIV gp41.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses.

Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice.

Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.

Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

SNARE proteins are required for macroautophagy.

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

Multiplexed CRISPR/CAS9-mediated engineering of pre-clinical mouse models bearing native human B cell receptors.

B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.

One-step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock-in mice.

Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30-50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.

Pre-HAART CD4+ T-lymphocytes as biomarkers of post-HAART immune recovery in HIV-infected children with or without TB co-infection.

BACKGROUND: Infection with the Human Immunodeficiency Virus (HIV) dramatically increases the risk of developing active tuberculosis (TB). Several studies have indicated that co-infection with TB increases the risk of HIV progression and death. Sub-Saharan Africa bears the brunt of these dual epidemics, with about 2.4 million HIV-infected people living with TB. The main objective of our study was to assess whether the pre-HAART CD4+ T-lymphocyte counts and percentages could serve as biomarkers for post-HAART treatment immune-recovery in HIV-positive children with and without TB co-infection. METHODS: The data analyzed in this retrospective study were collected from a cohort of 305 HIV-infected children being treated with HAART. A Lehmann family of ROC curves were used to assess the diagnostic performance of pre- HAART treatment CD4+ T-lymphocyte count and percentage as biomarkers for post-HAART immune recovery. The Kaplan-Meier estimator was used to compare differences in post-HAART recovery times between patients with and without TB co-infection. RESULTS: We found that the diagnostic performance of both pre-HARRT treatment CD4+ T-lymphocyte count and percentage was comparable and achieved accuracies as high as 74%. Furthermore, the predictive capability of pre-HAART CD4+ T-lymphocyte count and percentage were slightly better in TB-negative patients. Our analyses also indicate that TB-negative patients have a shorter recovery time compared to the TB-positive patients. CONCLUSIONS: Pre-HAART CD4+ T-lymphocyte count and percentage are stronger predictors of immune recovery in TB-negative pediatric patients, suggesting that TB co-infection complicates the treatment of HIV in this cohort. These findings suggest that the detection and treatment of TB is essential for the effectiveness of HAART in HIV-infected pediatric patients.

Polysaccharide enabled biogenic fabrication of pH sensing fluorescent gold nanoclusters as a biocompatible tumor imaging probe.

A biocompatible natural polysaccharide (PSP001) isolated from the fruit rind of Punica granatum was conjugated with L-cysteine (Y) to be used as a skeleton for the fabrication of fluorescent gold nanoclusters (AuNCs) represented as PSP-Y-AuNCs. With an average size of ~ 6 nm, PSP-Y-AuNCs demonstrated high quantum yield (31%), with a pH-sensitive fluorescence emission behavior. An emission maximum of 520 nm was obtained at acidic pH, which was blue shifted with increasing pH. This feature provides the possibilities for accurate ratiometric pH imaging. The PSP-Y-AuNCs not only demonstrated excellent biocompatibility with cancer cells and isolated peripheral lymphocytes and red blood cells but also demonstrated to be an active molecular imaging probe with appealing cellular uptake efficiency. The investigations with BALB/c mice further confirmed the non-toxic nature and in vivo imaging potential of the AuNCs. Estimation of the bio-distribution on solid tumor bearing syngeneic murine models revealed a tumor-targeted enhanced fluorescence emission pattern which is attributed to the pH responsive fluorescence behavior and the acidic microenvironment of the tumor. These findings were further confirmed with an impressive tumor accumulation pattern displayed in a xenograft of human cancer bearing nude mice. On account of their impressive biocompatibility and photophysical features, PSP-Y-AuNCs can exploited for the real-time fluorescence imaging of cancer tissues. Graphical abstract Fluorescent gold nanoclusters (PSP-Y-AuNCs) fabricated using a non-toxic natural polysaccharide (PSP001) demonstrated pH sensitive fluorescence emission pattern. The increased fluorescence readouts at acidic conditions and excellent biocompatibility made the PSP-Y-AuNCs an appealing candidate for in vivo tumor imaging applications.

Atg29 phosphorylation regulates coordination of the Atg17-Atg31-Atg29 complex with the Atg11 scaffold during autophagy initiation.

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.

Antibody production relies on the tRNA inosine wobble modification to meet biased codon demand.

Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.

mRNA-LNP prime boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Phosphatidylinositol 4-kinases are required for autophagic membrane trafficking.

Macroautophagy (hereafter autophagy) is a degradative cellular pathway that protects eukaryotic cells from stress, starvation, and microbial infection. This process must be tightly controlled because too little or too much autophagy can be deleterious to cellular physiology. The phosphatidylinositol (PtdIns) 3-kinase Vps34 is a lipid kinase that regulates autophagy, but the role of other PtdIns kinases has not been examined. Here we demonstrate a role for PtdIns 4-kinases and PtdIns4P 5-kinases in selective and nonselective types of autophagy in yeast. The PtdIns 4-kinase Pik1 is involved in Atg9 trafficking through the Golgi and is involved in both nonselective and selective types of autophagy, whereas the PtdIns4P 5-kinase Mss4 is specifically involved in mitophagy but not nonselective autophagy. Our data indicate that phosphoinositide kinases have multiple roles in the regulation of autophagic pathways.

Cardiopulmonary bypass surgery-cold alert!

The term "cold agglutinin (CA)" refers to a group of disorders caused by anti-erythrocyte autoantibodies that preferentially bind RBCs at cold temperatures (4°C-18°C). CAs contribute to 10 to 15% of autoimmune hemolytic anemia. We report a case of CAs diagnosed intraoperatively during emergency mitral valve replacement.

Two ZnF-UBP domains in isopeptidase T (USP5).

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.