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Background & Aims Human noroviruses are responsible for approximately 200,000 deaths worldwide each year. In 2012, the GII.4 Sydney strain emerged and became the major circulating norovirus strain associated with human disease. Our understanding of the human norovirus-specific antibody response is limited because few human monoclonal antibodies (mAbs) to noroviruses have been described, and there are no functional assays to measure virus neutralization. We studied the antibody-mediated response to the genogroup (G) II.4 strain by isolating mAbs to GII.4 from infected patients and developing virus neutralization assays. Methods We used a robust human hybridoma technique to isolate mAbs from patients previously infected with norovirus and identified mAbs that blocked virus binding to cell receptors, using virus-like particles to test blockade ability. We tested the ability of select mAbs to neutralize live human noroviruses using stem cell–derived human enteroids. Results We isolated a panel of 25 IgG or IgA human mAbs that recognized norovirus GII.4 Sydney 2012 and determined their potential to block virus binding to cell receptors. In competition binding studies, most antibodies recognized 3 major antigenic sites on the GII.4 Sydney 2012 protruding (P) domain. Conclusions We isolated and characterized human mAbs that neutralize live human norovirus GII.4 Sydney 2012—the predominant strain responsible for recent outbreaks. Analyses of these antibodies identified neutralizing epitopes; further studies will provide insight into the human immune response to this deadly virus. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) and Bundibugyo virus (BDBV) belong to the family Filoviridae and cause a severe disease in humans. We previously isolated a large panel of monoclonal antibodies from B cells of human survivors from the 2007 Uganda BDBV outbreak, 16 survivors from the 2014 EBOV outbreak in the Democratic Republic of the Congo, and one survivor from the West African 2013–2016 EBOV epidemic. Here, we demonstrate that EBOV and BDBV are capable of spreading to neighboring cells through intercellular connections in a process that depends upon actin and T cell immunoglobulin and mucin 1 protein. We quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. One of the antibodies, BDBV223, specific to the membrane-proximal external region, induces virus accumulation at the plasma membrane. The inhibiting activity of BDBV223 depends on BST2/tetherin. [Display omitted] • TIM-1 is required for cell-to-cell transmission in ebolavirus infections • Viral spread through intercellular connections can be blocked by antibodies • Inhibition of virus cell-to-cell spread by BDBV223 depends on BST2 • BDBV223 escape mutants can be spread by intercellular connections Santos et al. demonstrate that ebolaviruses spread to neighboring cells through intercellular connections. They develop methodologies to quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. The antibody BDBV223 has the property of inducing virus accumulation at the plasma membrane, dependent on the expression of BST2. [ABSTRACT FROM AUTHOR]

Dengue is a major public health threat. There are four dengue virus (DENV) serotypes; therefore, efforts are focused on developing 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. To that end, here, we develop a computational modeling framework based on the concept of antibody-virus neutralization fingerprints in order to characterize samples from clinical studies of TAK-003, a tetravalent vaccine candidate currently in phase 3 trials. Our results suggest a similarity of neutralizing antibody specificities in baseline-seronegative individuals. In contrast, amplification of pre-existing neutralizing antibody specificities is predicted for baseline-seropositive individuals, thus quantifying the role of immunologic imprinting in driving antibody responses to DENV vaccines. The neutralization fingerprinting analysis framework presented here can contribute to understanding dengue immune correlates of protection and help guide further vaccine development and optimization. [Display omitted] • Development of a computational framework to dissect antibody responses to TAK-003 • Assessment of the role of immunologic imprinting in driving antibody specificities • A generalizable framework for characterizing and optimizing multivalent vaccines Raju et al. describe the development of a computational algorithm to deconvolute the types of antibodies elicited in response to multivalent dengue vaccines. The results highlight the role of prior dengue infection in driving the response to subsequent vaccination. This study presents a generalizable framework for characterizing and optimizing multivalent vaccines. [ABSTRACT FROM AUTHOR]

The E1 and E2 envelope proteins of hepatitis C virus (HCV) form a heterodimer that drives virus-host membrane fusion. Here, we analyze the role of each amino acid in E1E2 function, expressing 545 individual alanine mutants of E1E2 in human cells, incorporating them into infectious viral pseudoparticles, and testing them against 37 different monoclonal antibodies (MAbs) to ascertain full-length translation, folding, heterodimer assembly, CD81 binding, viral pseudoparticle incorporation, and infectivity. We propose a model describing the role of each critical residue in E1E2 functionality and use it to examine how MAbs neutralize infection by exploiting functionally critical sites of vulnerability on E1E2. Our results suggest that E1E2 is a surprisingly fragile protein complex where even a single alanine mutation at 92% of positions disrupts its function. The amino-acid-level targets identified are highly conserved and functionally critical and can be exploited for improved therapies and vaccines. [Display omitted] • Test 545 hepatis C virus (HCV) E1E2 envelope mutants for infectivity, antibody binding • Identify residues important for HCV E1 and E2 folding, E1E2 interaction, infectivity • HCV E1E2 is a fragile protein complex where most mutations compromise function • Functional residues of E1E2 are highly conserved across genotypes Pfaff-Kilgore et al. describe the role of individual amino acids in hepatis C virus E1E2 protein function by generating a comprehensive E1E2 mutation library and testing 545 clones for E1E2 folding, assembly, and infectivity. Their model describes the role of residues in E1E2 functionality and examines how antibodies neutralize infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] New technologies now allow determination of millions/billions of antibody sequences. [•] An unexpectedly high number of somatic variants for each B cell clone is sustained. [•] The size of total expressed antibody repertoires is much smaller than anticipated. [•] Shared features of repertoires between individuals suggest high level regulation. [•] Emerging proteomics and computational methods complement DNA sequencing. [ABSTRACT FROM AUTHOR]

Background & Aims: The loss of parietal cells from the fundic mucosa leads to the emergence of metaplastic lineages associated with an increased susceptibility to neoplastic transformation. Both intestinal metaplasia (IM) and spasmolytic polypeptide (TFF2/SP) expressing metaplasia (SPEM) have been identified in human stomach, but only SPEM is present in most mouse models of gastric metaplasia. We previously determined that loss of amphiregulin (AR) promotes SPEM induced by acute oxyntic atrophy. We have now examined whether SPEM in the AR−/− mouse predisposes the stomach to gastric neoplasia. Methods: Gross pathology of 18-month-old wild-type, AR−/−, and TGF-α−/− mice were examined. Ki-67, β-catenin, Pdx-1, TFF3, and TFF2/SP expression was analyzed by immunohistochemistry. Metaplastic gastric mucosa was analyzed by dual immunostaining for TFF2/SP with MUC2 or TFF3. Results: By 18 months of age, more than 70% of AR−/− mice developed SPEM while 42% showed goblet cell IM labeled with MUC2, TFF3, and Pdx-1. A total of 28% had invasive gastric lesions in the fundus. No antral abnormalities were observed in AR−/− mice. Metaplastic cell lineages in AR−/− mice showed increases in cell proliferation and cytosolic β-catenin expression. Dual staining for TFF2/SP with MUC2 or TFF3 showed glands containing both SPEM and IM with intervening cells expressing both TFF2/SP and MUC2 or TFF2/SP and TFF3. Conclusions: AR−/− mice develop SPEM, which gives rise to goblet cell IM and invasive fundic dysplastic lesions. The AR−/− mouse represents the first mouse model for spontaneous development of fundic SPEM with progression to IM. [Copyright &y& Elsevier]

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs. [Display omitted] • LIBRA-seq identifies 54042-4, a potently neutralizing SARS-CoV-2 antibody • 54042-4 maintains potent neutralization against Alpha, Beta, Gamma, and Delta VOCs • The epitope of 54042-4 is highly conserved among current SARS-CoV-2 isolates Kramer et al. demonstrate that antibody 54042-4 recognizes residues highly conserved across global SARS-CoV-2 isolates. Antibody 54042-4 potently neutralizes all known circulating variants of concern (VOCs) and could be developed as a clinical candidate to treat COVID-19 infection. [ABSTRACT FROM AUTHOR]

Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiV B) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape. [Display omitted] • Antibodies targeting the henipavirus receptor-binding protein (RBP) were isolated • Two potently neutralizing mAbs, HENV-103 and HENV-117, bind to distinct sites on the RPB • HENV-103 and HENV-117 delivered as a cocktail display enhanced protection in hamsters Doyle et al. describe two human monoclonal antibodies that target the henipavirus receptor-binding protein, HENV-103 and HENV-117, that display highly potent activity in vitro and enhanced therapeutic efficacy in vivo when delivered as a cocktail. [ABSTRACT FROM AUTHOR]

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies. [Display omitted] • Glycan cap antibody-mediated GP destabilization correlates with synergy • Cryo-EM structures reveal antibodies target a highly conserved epitope • Antibodies use long CDRH3 loops to displace and mimic portions of the glycan cap • Glycan cap antibodies block cleavage events required for viral entry A rare subset of ebolavirus antibodies targeting the glycan cap are broadly neutralizing. Murin et al. report cryo-EM structures and custom in vitro assays identifying a conserved site of vulnerability in the glycan cap and detail mechanisms of action, including structural mimicry, trimer instability, and blocking cleavage. [ABSTRACT FROM AUTHOR]

The collection of T cell receptors (TCRs) generated by somatic recombination is large but unknown. We generate large TCR repertoire datasets as a resource to facilitate detailed studies of the role of TCR clonotypes and repertoires in health and disease. We estimate the size of individual human recombined and expressed TCRs by sequence analysis and determine the extent of sharing between individual repertoires. Our experiments reveal that each blood sample contains between 5 million and 21 million TCR clonotypes. Three individuals share 8% of TCRβ- or 11% of TCRα-chain clonotypes. Sorting by T cell phenotypes in four individuals shows that 5% of naive CD4+ and 3.5% of naive CD8+ subsets share their TCRβ clonotypes, whereas memory CD4+ and CD8+ subsets share 2.3% and 0.4% of their clonotypes, respectively. We identify the sequences of these shared TCR clonotypes that are of interest for studies of human T cell biology. • TCR clonotype sharing between individuals is higher than expected by chance • High-frequency TCR clonotypes are captured using gDNA and mRNA sequencing methods • Shared TCR clonotypes have a higher probability of being generated • Functional annotation for TCR clonotypes can be obtained using GenBank Soto et al. examine the extent to which five healthy adults share their T cell receptor (TCR) repertoire. Using sequencing and bioinformatics, they show a high prevalence of shared clonotypes even considering different T cell phenotypes. Possible functions for some clonotypes are inferred based on homology with TCRs in GenBank. [ABSTRACT FROM AUTHOR]

Summary Spondweni virus (SPOV) is the flavivirus that is most closely related to Zika virus (ZIKV). Although SPOV causes sporadic human infections in Africa, recently it was found in Culex mosquitoes in Haiti. To investigate the pathogenic spectrum of SPOV, we developed infection models in mice. Although two SPOV strains failed to cause disease in immunocompetent mice, each accumulated in the brain, spleen, eye, testis, and kidney when type I interferon signaling was blocked and unexpectedly caused infection, immune cell infiltration, and swelling in the ankle. In pregnant mice, SPOV replicated in the placenta and fetus but did not cause placental insufficiency or microcephaly. We identified human antibodies from ZIKV or DENV immune subjects that neutralized SPOV infection and protected against lethal challenge. Our experiments describe similarities and differences in clinical syndromes between SPOV and ZIKV and suggest that their serological relatedness has implications for antibody therapeutics and flavivirus vaccine development. Graphical Abstract Highlights • Development of mouse models of SPOV infection • SPOV uniquely infects feet of mice and results in acute joint swelling • Serological relatedness between SPOV, ZIKV, and DENV established • Anti-ZIKV and anti-DENV neutralizing mAbs have protective activity in vivo Salazar et al. show that SPOV, the flavivirus most closely related to ZIKV, infects mice when type I interferon signaling is blocked. SPOV causes ankle swelling and infection in the foot, which is more typical of alphaviruses. Human antibodies from ZIKV or DENV subjects protect against lethal SPOV challenge. [ABSTRACT FROM AUTHOR]

• Dengue virus-sized particles (VSPs) can be obtained from E. coli inclusion bodies. • Centrifugation and affinity chomatography provide 90–99% pure product. • In adequate buffer conditions, VSPs are thermally and storage stable. • VSPs induce a high titer of non-neutralizing antibodies in rabbits. Dengue fever is one of the most wide-spread vector-borne diseases in the world. Although dengue-associated mortality is low, morbidity and economic impact are high. Current licensed vaccines are limited and mediate only partial protection, thus a cost-effective vaccine with improved efficacy is strongly needed. In this work, recombinant dengue serotype 1 E protein was produced in E. coli , inclusion bodies were isolated and the E protein solubilized in urea and purified using an immobilized metal chelate affinity column. The protein was refolded by dialysis in order to obtain virus-like particles (VLPs). Particle assembly was confirmed using size-exclusion chromatography, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy and stimulated emission depletion fluorescence (STED) microscopy. Particle diameter was strongly dependent on temperature, pH, buffer salt composition, and addition of L-arginine. Particles were stable in carbonate buffer at pH 9.5 and higher at 4 °C and did not aggregate during short-term temperature increase up to 55 °C. However, on basis of the above analyses, especially the results of DLS, TEM and STED, it was concluded that the particles obtained did not have an optimal virus-like structure and were therefore designated "virus-sized particles" (VSPs) rather than VLPs. Immunization of rabbits with the particles did not induce neutralizing antibodies, despite the recognition of the native virus by rabbit antibodies. As the titers against the immunogen were much higher than against the (heat-inactivated) virus, it is assumed that the conformation of the particles at the time of immunization was not optimal. Studies are currently underway to improve the quality of the E protein virus-sized particles towards true virus-like particles in order to optimize its potential as a dengue vaccine candidate. [ABSTRACT FROM AUTHOR]

Abstract: Influenza viruses exhibit a fascinating level of antigenic heterogeneity that facilitates re-infection in the human population. The human antibody repertoire also manifests endless capability for variation in the genes that specify the portion of antibody molecules interacting with epitopes. A recent explosion of techniques for isolating human monoclonal antibodies to viruses has led to isolation of new antibodies that allow glimpses into the molecular basis for recognition and escape that underlies the constant antigenic drift in influenza surface proteins. These studies also reveal evidence for lifelong persistence of immunity to some influenza viruses. [Copyright &y& Elsevier]

Respiratory syncytial virus is the second most common cause of infant mortality and a major cause of morbidity and mortality in older adults (aged >60 years). Efforts to develop a respiratory syncytial virus vaccine or immunoprophylaxis remain highly active. 33 respiratory syncytial virus prevention candidates are in clinical development using six different approaches: recombinant vector, subunit, particle-based, live attenuated, chimeric, and nucleic acid vaccines; and monoclonal antibodies. Nine candidates are in phase 3 clinical trials. Understanding the epitopes targeted by highly neutralising antibodies has resulted in a shift from empirical to rational and structure-based vaccine and monoclonal antibody design. An extended half-life monoclonal antibody for all infants is likely to be within 1 year of regulatory approval (from August, 2022) for high-income countries. Live-attenuated vaccines are in development for older infants (aged >6 months). Subunit vaccines are in late-stage trials for pregnant women to protect infants, whereas vector, subunit, and nucleic acid approaches are being developed for older adults. Urgent next steps include ensuring access and affordability of a respiratory syncytial virus vaccine globally. This review gives an overview of respiratory syncytial virus vaccines and monoclonal antibodies in clinical development highlighting different target populations, antigens, and trial results. [ABSTRACT FROM AUTHOR]

Abstract: Antiviral antibody responses in infants are limited in quality. One reason for this finding could be that the majority of B cells in infants are CD5+ cells, a subset of B cells that is thought to contain cells expressing polyreactive, low-affinity B cell receptors. We analyzed the rotavirus (RV)-specific antibody heavy chain variable region (VH) repertoire in CD5+ and CD5− B cells of four RV-infected children between 10 and 19 months of age. We found that the RV-specific B cell repertoire in CD5+ cells was VH3 family biased, in contrast to the VH1/VH4 dominance seen in CD5− B cells. The immunodominant RV-specific gene segment in CD5− B cells was VH1-46, which is the dominant segment used in RV-specific peripheral blood B cells from infants and adults. In contrast, the immunodominant gene segment was VH3-23 in RV-specific CD5+ B cells, which is the dominant gene segment in randomly selected B cells. Both RV-specific CD5+ and RV-specific CD5− B cells from all children studied demonstrated very low frequencies of somatic mutations. In conclusion, CD5+ B cells in infants responding to RV use an antibody gene repertoire that differs from the virus-specific repertoire of CD5− B cells, and both CD5+ and CD5− RV-specific B cells exhibit a low frequency of somatic mutations. [Copyright &y& Elsevier]

The mortality rate triples for the 50% of allogeneic hematopoietic cell transplant (HCT) recipients diagnosed new-onset post-transplant diabetes mellitus (PTDM). Calcineurin inhibitors and corticosteroids are assumed the cause for hyperglycemia, however patients developing PTDM have elevated fasting C-peptide levels before HCT. We tested the hypotheses that an oral glucose tolerance test (OGTT) would predict new-onset PTDM and that PTDM progresses from established insulin resistance present before HCT. Glucose tolerance and insulin sensitivity were quantified with OGTTs and euglycemic hyperinsulinemic clamps before and 90 days after matched related donor peripheral blood HCT in 20 patients without diabetes. PTDM was diagnosed by a weekly fasting BG ≥126mg/dL or random BG ≥200mg/dL from day 0 to day+100. PTDM was diagnosed in 11 (55%) patients at a median of 22 days post-HCT (range, 0-88 days). Table 1 outlines patient characteristics and OGTT results. PTDM preceded grade 2-4 acute GVHD or corticosteroids in 9 (82%) and 11 (100%) patients, respectively. Pre-transplant 2-hour, post-prandial OGTT plasma glucose values did not discriminate between patients developing PTDM and those maintaining euglycemia after HCT. However, fasting pre-transplant glucose and C-peptide levels were elevated in patients developing PTDM. All 5 patients with impaired fasting glucose (100-125mg/dl) compared to 6 out of 15 individuals with normal fasting glucose levels (<100mg/dl) progressed to PTDM after HCT (p= 0.04). In response to insulin stimulation during the clamp, patients developing PTDM had lower whole-body glucose utilization (M) and decreased peripheral/skeletal muscle uptake (Rd) before and after HCT, respectively when compared to non-PTDM patients. PTDM patients also demonstrated decreased whole body and peripheral/skeletal muscle insulin sensitivity after transplant. Estimated 2-year survival was decreased in patients developing PTDM (Figure 1). PTDM risk is dependent on pre-transplant insulin resistance and independent of immunosuppression. Impaired fasting glucose levels identified PTDM susceptibility. Peripheral insulin resistance could be targeted for the prevention/treatment of PTDM. [ABSTRACT FROM AUTHOR]

• The CYD-TDV vaccine displays epitopes targeted by potent serotype-specific and cross-reactive neutralizing human antibodies. • The CYD-2 envelopes display epitopes specific of the mature conformation. • The CYD-TDV has the ability to trigger antibody responses qualitatively similar to those induced by wild-type viruses. The recombinant yellow fever-17D–dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1–4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1–4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these laboratory markers relate to vaccine efficacy and safety. [ABSTRACT FROM AUTHOR]

• Insulin resistance before HCT is a risk factor for PTDM independent of immunosuppression. • Fasting pretransplant glucose levels identified PTDM susceptibility. • Insulin resistance could be targeted for prevention and treatment of PTDM after HCT. New-onset post-transplant diabetes mellitus (PTDM) occurs frequently after allogeneic hematopoietic cell transplant (HCT). Although calcineurin inhibitors and corticosteroids are assumed to be the cause for hyperglycemia, patients developing PTDM have elevated fasting C-peptide levels before HCT and before immunosuppressive medications. To determine if PTDM results from established insulin resistance present before transplant, we performed oral glucose tolerance tests (OGTTs) and measured whole body, peripheral, and hepatic insulin sensitivity with euglycemic hyperinsulinemic clamps before and 90 days after HLA-identical sibling donor HCT in 20 patients without pretransplant diabetes. HCT recipients were prospectively followed for the development of new-onset PTDM defined as a weekly fasting blood glucose ≥ 126 mg/dL or random blood glucose ≥ 200 mg/dL. During the first 100 days all patients received calcineurin inhibitors, and 11 individuals (55%) were prospectively diagnosed with new-onset PTDM. PTDM diagnosis preceded corticosteroid treatment. During the pretransplant OGTT, elevated fasting (87 mg/dL versus 101 mg/dL; P =.005) but not 2-hour postprandial glucose levels predicted PTDM diagnosis (P =.648). In response to insulin infusion during the euglycemic hyperinsulinemic clamp, patients developing PTDM had lower whole body glucose utilization (P =.047) and decreased peripheral/skeletal muscle uptake (P =.031) before and after transplant, respectively, when compared with non-PTDM patients. Hepatic insulin sensitivity did not differ. Survival was decreased in PTDM patients (2-year estimate, 55% versus 100%; P =.039). Insulin resistance before HCT is a risk factor for PTDM independent of immunosuppression. Fasting pretransplant glucose levels identified PTDM susceptibility, and peripheral insulin resistance could be targeted for prevention and treatment of PTDM after HCT. [ABSTRACT FROM AUTHOR]

Abstract: Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150–152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms. [Copyright &y& Elsevier]

Abstract: The human Ab repertoire exhibits restrictions during fetal life characterized by biases of variable gene segment usage and lack of junctional diversity. We tested the hypotheses that Ab repertoire restriction persists in the early postnatal period and contributes to the observed poor quality of specific Ab responses made by neonates to viruses and vaccines. We analyzed the molecular determinants of B cell responses in humans to respiratory syncytial virus (RSV). Analysis of the variable gene segment usage of adult RSV-specific B cells revealed a repertoire profile in these cells similar to that seen in randomly selected B cells, which was VH3-dominant. Four gene segments (VH3–23, VH3–30, VH3–33 and VH4–04) accounted for almost half of the VH genes used. In contrast, very young infant RSV-specific antibodies exhibited a biased repertoire characterized by comparable use of the VH1, VH3, and VH4 families, and less common use of the four immunodominant gene segments. Infants and children older than three months used an antibody repertoire similar to that of adults. Mutational analysis revealed that the antibody variable genes of infants under three months of age also possessed significantly fewer somatic mutations in both framework and complementarity-determining region (CDR) regions than those of adults, even in a child with recurrent RSV infection. These data suggest that neonates use a biased antibody gene repertoire that is less VH3-focused and that possesses a dramatically lower frequency of somatic mutations. These biased features of the RSV-specific repertoire likely contribute to the poor functional Ab response in very young infants. [Copyright &y& Elsevier]

Abstract: Cytotoxic T lymphocytes (CTLs) are critical for the control of respiratory syncytial virus infection (RSV) in humans and mice. Recently, we identified a new H-2Kd-restricted subdominant epitope in the respiratory syncytial virus M2 protein. In this study, we investigated if modification of anchor residues at positions 2 and 9 in the dominant M282–90 epitope in the M2 protein would alter the CTL epitope dominance hierarchy following immunization with plasmid DNA encoding M2 proteins. We showed that immunogenicity of the subdominant epitope M2127–135 was enhanced when the anchor residues of the dominant epitope were mutated, suggesting that the immunodominant epitope induces a suppression of response to the subdominant epitope. [Copyright &y& Elsevier]

Abstract: We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies. [Copyright &y& Elsevier]

Abstract: Memory B cells and the antibodies they encode are important for protective immunity against infectious pathogens. Characterization of naïve and memory B cell antibody repertoires will elucidate the molecular basis for the generation of antibody diversity in human B cells and the optimization of antibody structures that bind microbial antigens. In this study we aimed to investigate the influence of antigenic selection on the antibody genes of the two CD27+ memory B cell subsets, comparing them with the naïve repertoire in CD27− cells. We analyzed and compared the Ig heavy chain gene transcripts in three recently defined circulating naïve and memory B cell subsets (CD19+IgD+CD27− [naïve], CD19+IgD+CD27+ [un-class-switched memory] or CD19+IgD− CD27+ [class-switched memory]) at the single cell level. We found similar biased patterns of variable, diversity and joining heavy chain gene usages in all three groups of cells. CD19+IgD+CD27+ memory B cells harbored as diverse an antibody gene repertoire as CD19+IgD−CD27+ memory B cells. Interestingly, CD19+IgD+CD27+ memory B cells possessed a lower frequency of somatic mutations, a higher incidence of exonuclease activity at the 3′ end of D regions, and a lower frequency of N and P nucleotide additions at both VH–D and D–JH junctions of CDR3 regions compared to CD19+IgD−CD27+ memory B cells. These data suggest distinct functional mechanisms underlying selection of this unique subset of un-class-switched memory B cells. [Copyright &y& Elsevier]

Abstract: Somatic hypermutation (SHM) of immunoglobulin genes requires activation-induced cytidine deaminase (AID). The error-prone DNA polymerases, such as Pol η, Pol ζ, and Pol ι, also have been implicated in the process. Human adult antibodies directed to microbial pathogens are increased in affinity and function compared with those of infants. Adult antibodies achieve this increased affinity through somatic mutations, which are lacking in the B cells of infants. It is unknown if infant B cells are capable of upregulating the cell machinery needed to introduce mutations after stimulation through the antigen receptor. We show here that infant B cells exhibit similar kinetics and magnitude of transcription of AID and pol η genes and only marginally lower levels of pol ι and pol ζ genes after stimulation through the B cell receptor. These data suggest that the ability to upregulate gene transcription of enzymes mediating SHM is not a limiting determinant of the functional quality of infant antibody responses. [Copyright &y& Elsevier]

Abstract: Respiratory viruses (RVs) are known to be major causes of morbidity and mortality in recipients of hematopoietic stem cell transplants (HSCTs), but prospective long-term studies are lacking. We prospectively screened all adult HSCT recipients (172 allogeneic [alloHSCT] and 240 autologous [autoHSCT]) who underwent transplantation during a 4-year period (1999 to 2003) for the development of a first episode of symptomatic upper respiratory tract infections and/or lower respiratory tract infections (LRTI) by an RV. RVs studied were influenza A and B viruses (n = 39), human respiratory syncytial virus (n = 19), human adenoviruses (n = 11), human parainfluenza viruses 1 to 3 (n = 8), human enteroviruses (n = 5), human rhinoviruses (n = 3), and the recently discovered human metapneumoviruses (n = 19). During the study, 51 and 32 cases of RV symptomatic infections were identified of alloHSCT and autoHSCT recipients (2-year incidence, 29% and 14%, respectively). Risk factors for progression of upper respiratory tract infection to LRTI included severe (&#60;0.2 × 109/L) and moderate (&#60;0.2 × 109/L) lymphocytopenia in alloHSCT (P = .02) and autoHSCT (P = .03). Death from LRTI was attributed to an RV in 8 alloHSCT recipients. Symptomatic RV had no effect on 2-year outcomes, with the possible exception of influenza A and B virus infections in autoHSCT: these were associated with nonrelapse mortality (P = .02). In conclusion, this prospective trial allows an estimation of the minimum incidence of a first RV infection in adult HSCT recipients and identifies risk factors for acquisition of an RV infection and progression to LRTI; this should aid in the design of future studies. In addition, human metapneumovirus should be added to the potentially serious causes of RV infections in HSCT. [Copyright &y& Elsevier]

Abstract: Somatic hypermutation of antibody genes is mediated by activation-induced cytidine deaminase and targets primarily hotspot motifs. We tested the hypothesis that the antibody variable genes of virus-specific B cells from infants exhibit a decreased frequency of somatic mutations compared with adults. We also sought to determine whether virus-specific B cells exhibit predominantly hotspot or randomly directed processes. We analyzed somatic mutations in rotavirus (RV)-specific B cells from otherwise healthy but recently RV-infected infants or adults in comparison with B cells from healthy volunteers not recently infected. We compared these antibody variable gene sequences with those derived from RV-specific B cells from an adult patient with X-linked hyper-IgM syndrome (XHIM). We found that the overall mutational frequency within the antibody variable region was lowest in RV-specific B cells from RV-infected infants, followed by randomly selected B cells, followed by RV-specific B cells from the patient with XHIM. RV-specific memory B cells from healthy adults exhibited the highest frequency of mutations. Approximately half of mutations in random or RV-specific B cells from adults or infants occurred at the DGYW/WRCH or WA/TW hotspot motifs. These findings suggest that virus-specific antibodies require both hotspot and randomly-directed processes. [Copyright &y& Elsevier]

The amount of antibody (Ab) variable gene sequence information is expanding rapidly, but our ability to predict the function of Abs from sequence alone is limited. Here, we describe a sequence-to-function prediction method that couples structural data for a single Ab/antigen (Ag) complex with repertoire data. We used a position-specific structure-scoring matrix (P3SM) incorporating structure-prediction scores from Rosetta to identify Ab variable loops that have predicted structural similarity to the influenza virus-specific human Ab CH65. The P3SM approach identified new members of this Ab class. Recombinant Ab expression, crystallography, and virus inhibition assays showed that the HCDR3 loops of the newly identified Abs possessed similar structure and antiviral activity as the comparator CH65. This approach enables discovery of new human Abs with desired structure and function using cDNA repertoires that are obtained readily with current amplicon sequencing techniques. • New position-specific structure-scoring matrix method using Rosetta • Effective sequence-to-function prediction for identifying virus-specific antibodies • Crystallography and virus inhibition assays validated the models Finn et al. describe a sequence-to-function prediction method that couples structural data for a single antibody-antigen complex with large-scale antibody variable gene repertoire data. Using a position-specific structure-scoring matrix incorporating structure-prediction scores from Rosetta, they identified Ab variable loops that have predicted structural similarity to an influenza virus-specific human antibody. [ABSTRACT FROM AUTHOR]

The influenza hemagglutinin (HA) glycoprotein is the target of many broadly neutralizing antibodies. However, influenza viruses can rapidly escape antibody recognition by mutation of hypervariable regions of HA that overlap with the binding epitope. We hypothesized that by designing peptides to mimic antibody loops, we could enhance breadth of binding to HA antigenic variants by reducing contact with hypervariable residues on HA that mediate escape. We designed cyclic peptides that mimic the heavy-chain complementarity-determining region 3 (CDRH3) of anti-influenza broadly neutralizing antibody C05 and show that these peptides bound to HA molecules with <100 nM affinity, comparable with that of the full-length parental C05 IgG. In addition, these peptides exhibited increased breadth of recognition to influenza H4 and H7 subtypes by eliminating clashes between the hypervariable antigenic regions and the antibody CDRH1 loop. This approach can be used to generate antibody-derived peptides against a wide variety of targets. • Cyclic peptides based on antibody loops were modeled and optimized using R osetta • Computationally designed peptides bind with high affinity to diverse influenza strains • Peptides have broad binding activity due to their small footprint on influenza surface Sevy et al. have developed a method for creating cyclic peptides from antibody loops using computational design. These peptides have broad binding activity against seasonal influenza variants and the approach represents a new strategy for peptide discovery. [ABSTRACT FROM AUTHOR]

Summary Dengue virus (DENV) particles are released from cells in different maturation states. Fully immature DENV (immDENV) is generally non-infectious, but can become infectious when complexed with anti-precursor membrane (prM) protein antibodies. It is unknown how anti-prM antibody-coated particles can undergo membrane fusion since the prM caps the envelope (E) protein fusion loop. Here, we determined cryoelectron microscopy (cryo-EM) maps of the immDENV:anti-prM complex at different pH values, mimicking the extracellular (pH 8.0) or endosomal (pH 5.0) environments. At pH 5.0, there are two structural classes with fewer antibodies bound than at pH 8.0. These classes may represent different maturation states. Molecular simulations, together with the measured high-affinity pr:antibody interaction (versus the weak pr:E interaction) and also the low pH cryo-EM structures, suggest how antibody:pr complex can dislodge from the E protein at low pH. This exposes the E protein fusion loop enhancing virus interaction with endosomes. Graphical Abstract Highlights • ImmDENV3 complexed with anti-prM antibody structures at neutral and acidic pH • Two structural classes at acidic pH represent DENV maturation intermediates • Structures show how the antibody helps overcome barrier for immature virus to fuse • MD simulates maturation structural transitions and antibody assisted removal of pr Wirawan et al. present structures of immature dengue virus complexed with anti-prM Fabs at pH 8.0 and pH 5.0. The two structural classes at pH 5.0 showed different maturation stages. These structures also suggest the mechanism on how antibodies stimulate immature dengue virus attachment to liposomes. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are a subset of CD4+ T cells that are characterized by the expression of CD25 and Foxp3 and are capable of suppressing alloimmune responses. We assessed whether high frequencies of circulating skin or gut tissue–specific Tregs at engraftment could predict acute graft-vs-host disease (aGVHD) incidence and survival in a cohort of hematopoietic cell transplant (HCT) recipients. Tregs were analyzed at engraftment in 74 patients receiving HCT. Treg skin-homing (CLA+) or gut-homing (α4β7 +) subsets were identified by flow cytometry, and patients were divided into high CLA+ Tregs or high α4β7 + Tregs groups, using the 75th percentile of tissue-specific Treg percentages as a threshold. At day +100 post-HCT, the cumulative incidence of any stage skin or gut aGVHD was significantly lower in those patients with high CLA+ Tregs or high α4β7 + Tregs at engraftment, respectively (high CLA+ Tregs, 24.0% vs low CLA+ Tregs, 55.1%; p = 0.011 for skin aGVHD or high α4β7 + Tregs, 47.3% vs low α4β7 + Tregs, 74.5%; p = 0.029 for gut aGVHD). The 2-year probabilities of overall survival and nonrelapse mortality were 73.4% and 7.5% among patients with high frequencies of tissue-specific Tregs vs 49.4% and 36.1% for those with both low CLA+ Tregs and low α4β7 + Tregs (p = 0.039, p = 0.010). These results suggest that a threshold value for CLA+ or α4β7 + Tregs could be used to predict important HCT outcomes, and to direct the rationale use of tissue-specific pre-emptive therapies to decrease clinical aGVHD and improve HCT survival. [Copyright &y& Elsevier]

Abstract: There has been an explosion of interest in the human B cell response to HIV infection of late. Recent advances in techniques for isolation of human antibodies and antibody secreting cell lines have facilitated a rapid expansion in the number of antibodies available for study. Early analysis of these repertoires reveals interesting features of the HIV-specific antibody response. HIV-specific repertoires exhibit a high level of clonality in circulating cells, and high levels of somatic mutations within the antibody variable gene segments. It appears that many if not most antibodies in circulation bind to virus envelope conformations that are found only in complex oligomeric structures on virion particles or virus-like particles. The rapid isolation of large panels of novel human neutralizing antibodies promises to reveal new insights into the fundamental principles underlying antibody-mediated neutralization of HIV. [Copyright &y& Elsevier]