Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody.

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase 1 clinical trial investigating a ferritin nanoparticle vaccine displaying H2 hemagglutinin (HA) in H2-naive and H2-exposed adults enabled us to perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited. We temporally map the epitopes targeted by serum antibodies after vaccine prime and boost, revealing that previous H2 exposure results in higher responses to the variable HA head domain. In contrast, initial responses in H2-naive participants are dominated by antibodies targeting conserved epitopes. We use cryoelectron microscopy and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the HA head, including the receptor-binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses post-vaccination.

Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase.

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

C1q enables influenza hemagglutinin stem binding antibodies to block viral attachment and broadens the antibody escape repertoire.

Antigenic drift, the gradual accumulation of amino acid substitutions in the influenza virus hemagglutinin (HA) receptor protein, enables viral immune evasion. Antibodies (Abs) specific for the drift-resistant HA stem region are a promising universal influenza vaccine target. Although anti-stem Abs are not believed to block viral attachment, here we show that complement component 1q (C1q), a 460-kilodalton protein with six Ab Fc-binding domains, confers attachment inhibition to anti-stem Abs and enhances their fusion and neuraminidase inhibition. As a result, virus neutralization activity in vitro is boosted up to 30-fold, and in vivo protection from influenza PR8 infection in mice is enhanced. These effects reflect increased steric hindrance and not increased Ab avidity. C1q greatly expands the anti-stem Ab viral escape repertoire to include residues throughout the HA, some of which cause antigenic alterations in the globular region or modulate HA receptor avidity. We also show that C1q enhances the neutralization activity of non-receptor binding domain anti-SARS-CoV-2 spike Abs, an effect dependent on spike density on the virion surface. These findings demonstrate that C1q can greatly expand Ab function and thereby contribute to viral evolution and immune escape.

Adjuvanted SARS-CoV-2 spike protein vaccination elicits long-lived plasma cells in nonhuman primates.

Durable humoral immunity is mediated by long-lived plasma cells (LLPCs) that reside in the bone marrow. It remains unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein vaccination is able to elicit and maintain LLPCs. Here, we describe a sensitive method to identify and isolate antigen-specific LLPCs by tethering antibodies secreted by these cells onto the cell surface. Using this method, we found that two doses of adjuvanted SARS-CoV-2 spike protein vaccination are able to induce spike protein-specific LLPC reservoirs enriched for receptor binding domain specificities in the bone marrow of nonhuman primates that are detectable for several months after vaccination. Immunoglobulin gene sequencing confirmed that several of these LLPCs were clones of memory B cells elicited 2 weeks after boost that had undergone further somatic hypermutation. Many of the antibodies secreted by these LLPCs also exhibited improved neutralization and cross-reactivity compared with earlier time points. These findings establish our method as a means to sensitively and reliably detect rare antigen-specific LLPCs and demonstrate that adjuvanted SARS-CoV-2 spike protein vaccination establishes spike protein-specific LLPC reservoirs.

Between-hospital care referrals for severe alcohol-related liver disease during the COVID-19 pandemic, 2020 to 2022.

Increased alcohol consumption during the coronavirus disease 2019 pandemic is projected to impact alcohol-related liver disease (ALD) morbidity and mortality. Inter-hospital escalation-of-care referral requests to our tertiary-care hepatology unit were analyzed from January 2020 through December 2022. Most requests to our center were for ALD with an increase in requests from intermediate care units, suggestive of higher acuity illness.

High frequency of HIV precursor-target-specific B cells in sub-Saharan populations.

HIV gp120 engineered outer domain germline-targeting version 8 (eOD-GT8) was designed specifically to engage naive B cell precursors of VRC01-class antibodies. However, the frequency and affinity of naive B cell precursors able to recognize eOD-GT8 have been evaluated only in U.S. populations. HIV infection is disproportionally concentrated in sub-Saharan Africa, so we seek to characterize naive B cells able to recognize eOD-GT8 in sub-Saharan cohorts. We demonstrate that people from sub-Saharan Africa have a higher or equivalent frequency of naive B cells able to engage eOD-GT8 compared with people from the U.S. Genetically, the higher frequency of eOD-GT8-positive cells is accompanied by a higher level of naive B cells with gene signatures characteristic of the VRC01 class, as well as other CD4bs-directed antibodies. Our study demonstrates that vaccination with eOD-GT8 in sub-Saharan Africa could be successful at expanding and establishing a pool of CD4bs-directed memory B cells from naive precursors.

Immune memory shapes human polyclonal antibody responses to H2N2 vaccination.

Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naïve and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naïve participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses.

Designed nanoparticles elicit cross-reactive antibody responses to conserved influenza virus hemagglutinin stem epitopes.

Despite the availability of seasonal vaccines and antiviral medications, influenza virus continues to be a major health concern and pandemic threat due to the continually changing antigenic regions of the major surface glycoprotein, hemagglutinin (HA). One emerging strategy for the development of more efficacious seasonal and universal influenza vaccines is structure-guided design of nanoparticles that display conserved regions of HA, such as the stem. Using the H1 HA subtype to establish proof of concept, we found that tandem copies of an alpha-helical fragment from the conserved stem region (helix-A) can be displayed on the protruding spikes structures of a capsid scaffold. The stem region of HA on these designed chimeric nanoparticles is immunogenic and the nanoparticles are biochemically robust in that heat exposure did not destroy the particles and immunogenicity was retained. Furthermore, mice vaccinated with H1-nanoparticles were protected from lethal challenge with H1N1 influenza virus. By using a nanoparticle library approach with this helix-A nanoparticle design, we show that this vaccine nanoparticle construct design could be applicable to different influenza HA subtypes. Importantly, antibodies elicited by H1, H5, and H7 nanoparticles demonstrated homosubtypic and heterosubtypic cross-reactivity binding to different HA subtypes. Also, helix-A nanoparticle immunizations were used to isolate mouse monoclonal antibodies that demonstrated heterosubtypic cross-reactivity and provided protection to mice from viral challenge via passive-transfer. This tandem helix-A nanoparticle construct represents a novel design to display several hundred copies of non-trimeric conserved HA stem epitopes on vaccine nanoparticles. This design concept provides a new approach to universal influenza vaccine development strategies and opens opportunities for the development of nanoparticles with broad coverage over many antigenically diverse influenza HA subtypes.

Definition and measurement of alcohol-associated insight in early liver transplantation for acute alcohol-associated hepatitis: A systematic review.

BACKGROUND: Alcohol accounts for a large disease burden in hepatology and liver transplantation (LT) and across the globe. Clinical evaluations and decisions about LT candidacy are challenging because they rely on detailed psychosocial assessments and interpretations of psychiatric and substance use disorder data, which often must occur rapidly according to the acuity of end-stage liver disease. Such difficulties commonly occur during the process of candidate selection and liver allocation, particularly during early LT (eLT) in patients with acute alcohol-associated hepatitis (AAH). Patients with AAH commonly have very recent or active substance use, high short-term mortality, psychiatric comorbidities, and compressed evaluation and treatment timetables. LT clinicians report that patients' alcohol-associated insight (AAI) is among the most relevant psychosocial data in this population, yet no studies exist examining how LT teams define and use AAI in eLT or its effect on clinical outcomes. In April 2022, we searched Ovid MEDLINE, Elsevier Embase, EBSCOhost PsycInfo and CINAHL, and Wiley Cochrane Central Register of Controlled Trials for reports describing AAH populations who underwent eLT, which also described psychosocial evaluation parameters. The searches retrieved 1603 unique reports. After eligibility screening, 8 were included in the qualitative analysis. This systematic review reveals that AAI is a poorly defined construct that is not measured in a standardized way. Yet it is a commonly cited parameter in articles that describe the psychosocial evaluation and decision-making of patients undergoing eLT for AAH. This article also discusses the general challenges of assessing AAI during eLT for AAH, existing AAI definitions and rating scales, how AAI has been used to date in the broader hepatology and LT literature, and future areas for clinical and research progress.

ACLP Best Practice Guidance: Evaluation and Treatment of Depression in Solid Organ Transplant Recipients.

We present Academy of Consultation Liaison Psychiatry best practice guidance on depression in solid organ transplant (SOT) recipients, which resulted from the collaboration of Academy of Consultation Liaison Psychiatry's transplant psychiatry special interest group and Guidelines and Evidence-Based Medicine Subcommittee. Depression (which in the transplant setting may designate depressive symptoms or depressive disorders) is a frequent problem among SOT recipients. Following a structured literature review and consensus process, the Academy of Consultation Liaison Psychiatry transplant psychiatry special interest group proposes recommendations for practice: all organ transplant recipients should be screened routinely for depression. When applicable, positive screening should prompt communication with the mental health treating provider or a clinical evaluation. If the evaluation leads to a diagnosis of depressive disorder, treatment should be recommended and offered. The recommendation for psychotherapy should consider the physical and cognitive ability of the patient to maximize benefit. The first-line antidepressants of choice are escitalopram, sertraline, and mirtazapine. Treating depressive disorders prior to transplantation is recommended to prevent posttransplant depression. Future research should address the mechanism by which transplant patients develop depressive disorders, the efficacy and feasibility of treatment interventions (both pharmacological and psychotherapeutic, in person and via telemedicine), and the resources available to transplant patients for mental health care.

An influenza H1 hemagglutinin stem-only immunogen elicits a broadly cross-reactive B cell response in humans.

Current yearly seasonal influenza vaccines primarily induce an antibody response directed against the immunodominant but continually diversifying hemagglutinin (HA) head region. These antibody responses provide protection against the vaccinating strain but little cross-protection against other influenza strains or subtypes. To focus the immune response on subdominant but more conserved epitopes on the HA stem that might protect against a broad range of influenza strains, we developed a stabilized H1 stem immunogen lacking the immunodominant head displayed on a ferritin nanoparticle (H1ssF). Here, we evaluated the B cell response to H1ssF in healthy adults ages 18 to 70 in a phase 1 clinical trial (NCT03814720). We observed both a strong plasmablast response and sustained elicitation of cross-reactive HA stem-specific memory B cells after vaccination with H1ssF in individuals of all ages. The B cell response was focused on two conserved epitopes on the H1 stem, with a highly restricted immunoglobulin repertoire unique to each epitope. On average, two-thirds of the B cell and serological antibody response recognized a central epitope on the H1 stem and exhibited broad neutralization across group 1 influenza virus subtypes. The remaining third recognized an epitope near the viral membrane anchor and was largely limited to H1 strains. Together, we demonstrate that an H1 HA immunogen lacking the immunodominant HA head produces a robust and broadly neutralizing HA stem-directed B cell response.

An influenza hemagglutinin stem nanoparticle vaccine induces cross-group 1 neutralizing antibodies in healthy adults.

Influenza vaccines could be improved by platforms inducing cross-reactive immunity. Immunodominance of the influenza hemagglutinin (HA) head in currently licensed vaccines impedes induction of cross-reactive neutralizing stem-directed antibodies. A vaccine without the variable HA head domain has the potential to focus the immune response on the conserved HA stem. This first-in-human dose-escalation open-label phase 1 clinical trial (NCT03814720) tested an HA stabilized stem ferritin nanoparticle vaccine (H1ssF) based on the H1 HA stem of A/New Caledonia/20/1999. Fifty-two healthy adults aged 18 to 70 years old enrolled to receive either 20 µg of H1ssF once (n = 5) or 60 µg of H1ssF twice (n = 47) with a prime-boost interval of 16 weeks. Thirty-five (74%) 60-µg dose participants received the boost, whereas 11 (23%) boost vaccinations were missed because of public health restrictions in the early stages of the COVID-19 pandemic. The primary objective of this trial was to evaluate the safety and tolerability of H1ssF, and the secondary objective was to evaluate antibody responses after vaccination. H1ssF was safe and well tolerated, with mild solicited local and systemic reactogenicity. The most common symptoms included pain or tenderness at the injection site (n = 10, 19%), headache (n = 10, 19%), and malaise (n = 6, 12%). We found that H1ssF elicited cross-reactive neutralizing antibodies against the conserved HA stem of group 1 influenza viruses, despite previous H1 subtype head-specific immunity. These responses were durable, with neutralizing antibodies observed more than 1 year after vaccination. Our results support this platform as a step forward in the development of a universal influenza vaccine.

Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19.

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood1-4. Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8+ T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner.

Harnessing low dimensionality to visualize the antibody-virus landscape for influenza.

Antibodies constitute a key line of defense against the diverse pathogens we encounter in our lives. Although the interactions between a single antibody and a single virus are routinely characterized in exquisite detail, the inherent tradeoffs between attributes such as potency and breadth remain unclear. Moreover, there is a wide gap between the discrete interactions of single antibodies and the collective behavior of antibody mixtures. Here we develop a form of antigenic cartography called a 'neutralization landscape' that visualizes and quantifies antibody-virus interactions for antibodies targeting the influenza hemagglutinin stem. This landscape transforms the potency-breadth tradeoff into a readily solvable geometry problem. With it, we decompose the collective neutralization from multiple antibodies to characterize the composition and functional properties of the stem antibodies within. Looking forward, this framework can leverage the serological assays routinely performed for influenza surveillance to analyze how an individual's antibody repertoire evolves after vaccination or infection.

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Epstein-Barr virus gH/gL has multiple sites of vulnerability for virus neutralization and fusion inhibition.

Epstein-Barr virus (EBV) is nearly ubiquitous in adults. EBV causes infectious mononucleosis and is associated with B cell lymphomas, epithelial cell malignancies, and multiple sclerosis. The EBV gH/gL glycoprotein complex facilitates fusion of virus membrane with host cells and is a target of neutralizing antibodies. Here, we examined the sites of vulnerability for virus neutralization and fusion inhibition within EBV gH/gL. We developed a panel of human monoclonal antibodies (mAbs) that targeted five distinct antigenic sites on EBV gH/gL and prevented infection of epithelial and B cells. Structural analyses using X-ray crystallography and electron microscopy revealed multiple sites of vulnerability and defined the antigenic landscape of EBV gH/gL. One mAb provided near-complete protection against viremia and lymphoma in a humanized mouse EBV challenge model. Our findings provide structural and antigenic knowledge of the viral fusion machinery, yield a potential therapeutic antibody to prevent EBV disease, and emphasize gH/gL as a target for herpesvirus vaccines and therapeutics.

Sociodemographic Risk Factors for Depression in Patients With Chronic Liver Disease.

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