Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disorder characterized by serious physical and cognitive impairments. Recent research underscores the role of immune dysfunction, including the role of autoantibodies, in ME/CFS pathophysiology. Expanding on previous studies, we analyzed 7542 antibody–antigen interactions in ME/CFS patients using two advanced platforms: a 1134 autoantibody Luminex panel from Oncimmune and Augmenta Bioworks, along with Rapid Extracellular Antigen Profiling (REAP), a validated high-throughput method that measures autoantibody reactivity against 6183 extracellular human proteins and 225 human viral pathogen proteins. Unlike earlier reports, our analysis of 172 participants revealed no significant differences in autoantibody reactivities between ME/CFS patients and controls, including against GPCRs such as β-adrenergic receptors. However, subtle trends in autoantibody ratios between male and female ME/CFS subgroups, along with patterns of herpesvirus reactivation, suggest the need for broader and more detailed exploration.

Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy (cryo-EM) analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species. ### Competing Interest Statement The authors have declared no competing interest.

Nanobodies, single-domain antibodies derived from camelid heavy-chain antibodies, are known for their high affinity, stability, and small size, which make them useful in biological research and therapeutic applications. However, traditional nanobody generation methods rely on camelid immunization, which can be costly and time-consuming, restricting their practical feasibility. In this study, we present a phage-displayed synthetic library for nanobody discovery. To validate this approach, we screened nanobodies targeting various Drosophila secreted proteins. The nanobodies identified were suitable for applications such as immunostaining and immunoblotting, supporting the phage-displayed synthetic library as a versatile platform for nanobody development. To address the challenge of limited accessibility to high-quality synthetic libraries, this library is openly available for non-profit use.

Although the role of cellular immunity in checkpoint immunotherapy (CPI) for cancer is well established1,2, the effect of antibody-mediated humoral immunity is comparably underexplored. Here we used rapid extracellular antigen profiling3 to map the autoantibody reactome within a cohort of 374 patients with cancer treated with CPIs and 131 healthy control participants for autoantibodies to 6,172 extracellular and secreted proteins (the 'exoproteome'). Globally, patients with cancer treated with CPIs had diverse autoreactivities that were elevated relative to control individuals but changed minimally with treatment. Autoantibody signatures in patients treated with CPI strikingly distinguished them from healthy individuals. Although associations of specific autoantibodies with immune-related adverse events were sparse, we detected numerous individual autoantibodies that were associated with greatly altered odds ratios for response to therapy. These included autoantibodies to immunomodulatory proteins, such as cytokines, growth factors and immunoreceptors, as well as tumour surface proteins. Functional evaluation of several autoantibody responses indicated that they neutralized the activity of their target proteins, which included type I interferons (IFN-I), IL-6, OSM, TL1A, and BMPR1A and BMPR2. Modelling the effects of autoantibodies to IFN-I and TL1A in preclinical mouse tumour models resulted in enhanced CPI efficacy, consistent with their effects in patients. In conclusion, these findings indicate that autoantibodies to the exoproteome modify CPI responses and highlight therapeutically actionable pathways that can be exploited to augment immunotherapy.

Lyme disease, caused byBorrelia burgdorferi, is the most common tick-borne infection in the United States. Arthritis is a major clinical manifestation of infection, and synovial tissue damage has been attributed to the excessive pro-inflammatory responses. The secretory leukocyte protease inhibitor (SLPI) promotes tissue repair and exerts anti-inflammatory effects. The role of SLPI in the development of Lyme arthritis in C57BL/6 mice, which can be infected withB. burgdorferi, but only develop mild joint inflammation, was therefore examined.SLPI-deficient C57BL/6 mice challenged withB. burgdorferihad a higher infection load in the tibiotarsal joints and marked periarticular swelling, compared to infected wild type control mice. The ankle joint tissues ofB. burgdorferi-infectedSLPI-deficient mice contained significantly higher percentages of infiltrating neutrophils and macrophages.B. burgdorferi-infectedSLPI-deficient mice also exhibited elevated serum levels of IL-6, neutrophil elastase, and MMP-8. Moreover, using a recently developed BASEHIT (BActerial Selection to Elucidate Host-microbe Interactions in high Throughput) library, we found that SLPI directly interacts withB. burgdorferi. These data demonstrate the importance of SLPI in suppressing periarticular joint inflammation in Lyme disease.

Checkpoint inhibitors targeting CTLA-4 and PD-1 revolutionized the treatment of cancer patients, but their use is limited by the emergence of immune-related adverse events (irAEs). We assessed autoreactive B cell frequencies in the blood of cancer patients before and after treatment with checkpoint inhibitors by testing the reactivity of recombinant antibodies cloned from single B cells. We found that anti-PD-1 and anti-CTLA-4 combination therapy induced the emergence of autoreactive mature naive B cells, whereas central B cell tolerance remained functional. In contrast, anti-PD-1 alone did not alter autoreactive B cell counterselection. Anti-CTLA-4 injections in humanized mice also resulted in the production of autoreactive B cells, whereas anti-PD-1 did not. We conclude that CTLA-4 but not PD-1 is required for the removal of developing autoreactive mature naive B cells and that CTLA-4 blockade broadens the peripheral B cell repertoire, which likely contains clones that promote not only irAEs but also antitumor responses.

BACKGROUND:The symptomatic and immune responses to COVID-19 vaccination of people with Long COVID are poorly characterized. METHODS:In this prospective study, we evaluated changes in symptoms and immune responses after COVID-19 vaccination in 16 vaccine-naïve individuals with Long COVID. Surveys were administered before vaccination and at 2, 6, and 12 weeks after receiving the first vaccine dose of the primary series. Simultaneously, SARS-CoV-2-reactive TCR enrichment, SARS-CoV-2-specific antibody responses, antibody responses to other viral and self-antigens, and circulating cytokines were quantified before vaccination and at 6 and 12 weeks after vaccination. RESULTS:At 12 weeks post-vaccination, self-reported improved health is seen in 10 out of 16 participants, 3 have no change, and 3 have worse health although 2 report transient improvement after vaccination. One participant reporting worse health was hospitalized twice with chest pain (after each dose). Symptom outcomes are most associated with plasma biosignatures. Higher baseline sIL-6R is associated with symptom improvement, and stably elevated levels of IFN-β and CNTF are associated with no improvement. Significant elevation in SARS-CoV-2-specific TCRs and spike protein-specific IgG are observed at 6 and 12 weeks after vaccination. No changes in reactivities are observed against herpes viruses and self-antigens. CONCLUSIONS:In this study of 16 people with Long COVID, vaccination is associated with increased SARS-CoV-2 spike protein-specific IgG and T cell expansion in most participants. Specific immune features are associated with symptom change after vaccination and most participants experience improved health or no change following vaccination.

Ixodes scapularis is a primary vector of tick-borne pathogens in North America. Repeated exposure to these ticks can induce a humoral response to tick antigens and acquired tick resistance. However, identifying antigens contributing to this resistance is challenging because of the vast number of I. scapularis proteins secreted during feeding. To address this, we developed I. scapularis rapid extracellular antigen monitoring ( Isc REAM), a technique to detect antibody responses to more than 3000 tick antigens. We validated Isc REAM with immunoglobulin G (IgG) from guinea pigs vaccinated with tick antigens, including a cement antigen cocktail that induced tick resistance. Furthermore, we explored the natural response to tick bites by profiling antigens recognized by IgG isolated from a tick-resistant individual, as well as from others with Lyme disease and tick-bitten guinea pigs and mice, to identify 199 recognized antigens. We observed that several antigens contained histamine-binding domains. This work enhances our understanding of the host immune response to I. scapularis and defines immunogen candidates for future antitick vaccines.

IgE antibodies against the allergen Ara-h2 can cause life-threatening anaphylaxis upon exposure to peanuts. Desensitization strategies aim at inducing IgG responses against Ara-h2 which may compete with anaphylactogenic IgE. Here we assessed anti-Ara-h2 titers in an unselected cohort of 24,536 adult patients admitted to a general hospital for disparate medical reasons. Surprisingly, adult (n=177) and pediatric (n=76) patients with cystic fibrosis (pwCF) had IgG4, but not IgE, against several peanut and soybean allergens, yet did not suffer from peanut allergy. Antibody repertoires were not globally perturbed in pwCF, and heterozygous Cystic Fibrosis Transmembrane Regulator (CFTR) mutation carriers had the same prevalence of food allergies as pwCF. Peanut sensitisation of Cftr-/- mice failed to induce IgE and was associated with elevated IFN-γ. We conclude that CFTR is a key regulator of anaphylactogenic and tolerogenic responses to food allergens. CFTR-controlled cytokine responses including IFN-γ, in combination with a compromised epithelial barrier, may trigger a preferential IgG4 response resulting in tolerance to food allergens.### Competing Interest StatementAA is a member of the clinical and scientific advisory board of Fluidic Analytics and member of the board of directors of Mabylon AG and AB2Bio AG. AA has formerly been a scientific advisor to Mabylon AG. TPJK is a member of the board of directors of Fluidic Analytics. RM and MS are employed by Cellerys AG. All authors declare no competing interests.### Funding StatementGrants of the Swiss Personalized Health Network (Driver Grant 2017DRI17), of the Swiss National Science Foundation (SNF; grant #179040), of the Innovation Fund of the University Hospital Zurich (INOV00096), of the NOMIS Foundation, the Schwyzer Winiker Stiftung, and the Baugarten Stiftung (coordinated by the USZ Foundation, USZF27101) were awarded to AA and ME.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethikkommission Kanton Zuerich of University of Zurich gave ethical approval for this work. The Yale IRB of Yale Medical School gave ethical approval for this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesSmall amounts of the biological samples can be shared if available, upon reasonable request, and if approval by an ethics committee as well as an MTA is in place. This study did not generate new unique reagents. Specific data sets can be shared upon reasonable request and if approval by an ethics committee as well as a data transfer agreement is in place. Code employed in this study will be publicly available on Zenodo in the peer-reviewed version of this manuscript after publication. Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request.

The myriad microorganisms that live in close association with humans have diverse effects on physiology, yet the molecular bases for these impacts remain mostly unknown(1-3). Classical pathogens often invade host tissues and modulate immune responses through interactions with human extracellular and secreted proteins (the 'exoproteome'). Commensal microorganisms may also facilitate niche colonization and shape host biology by engaging host exoproteins; however, direct exoproteome-microbiota interactions remain largely unexplored. Here we developed and validated a novel technology, BASEHIT, that enables proteome-scale assessment of human exoproteome-microbiome interactions. Using BASEHIT, we interrogated more than 1.7 million potential interactions between 519 human-associated bacterial strains from diverse phylogenies and tissues of origin and 3,324 human exoproteins. The resulting interactome revealed an extensive network of transkingdom connectivity consisting of thousands of previously undescribed host-microorganism interactions involving 383 strains and 651 host proteins. Specific binding patterns within this network implied underlying biological logic; for example, conspecific strains exhibited shared exoprotein-binding patterns, and individual tissue isolates uniquely bound tissue-specific exoproteins. Furthermore, we observed dozens of unique and often strain-specific interactions with potential roles in niche colonization, tissue remodelling and immunomodulation, and found that strains with differing host interaction profiles had divergent interactions with host cells in vitro and effects on the host immune system in vivo. Overall, these studies expose a previously unexplored landscape of molecular-level host-microbiota interactions that may underlie causal effects of indigenous microorganisms on human health and disease.

The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina 1 . Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.

Autoantibodies influence a wide range of conditions beyond autoimmune diseases

Background: Teclistamab (tec) is a bispecific antibody (bsAb) which binds BCMA on multiple myeloma (MM) cells and CD3 on T-cells, leading to tumor cell death. Tec is currently approved for relapsed/refractory (R/R) MM based on the MajesTEC-1 trial, which demonstrated an ORR of 63% with median progression-free survival (PFS) of 11.3 months. However, PFS is typically shorter in the real-world setting (Dima et al, TCT 2024, Mohan et al, BCJ 2024). Interleukin 18 (IL-18) is an immunostimulatory cytokine that triggers T-cell proliferation and differentiation into the Th1 phenotype important in anticancer immunity. Recently published work suggests that IL-18-armored CAR T-cells targeting BCMA (but not unarmored cells without paracrine IL-18 secretion) can eliminate myeloma cells even with low BCMA expression (Ng et al, Blood 2024). Trials of recombinant IL-18 in oncology have been disappointing due to its inhibition by IL-18 binding protein, a high-affinity decoy receptor that acts as a cytokine sink. ST-067 is a decoy-resistant IL-18 variant shown to potentiate T-cell activity in pre-clinical models (Minnie et al, Sci Immunol 2022). Given the room for improvement with responses to bsAb therapy in patients with MM, we are conducting a Phase 1b study to investigate the combination of tec and ST-067 in MM after a ST-067 lead-in. The ST-067 monotherapy lead-in allows for safety evaluation as well as potential T-cell ‘priming’ before tec initiation, while then continuing ST-067 in combination with tec may allow for deeper and more durable responses in MM as compared to tec alone. Methods: This is an open-label single-arm Phase 1b trial of ST-067, a decoy-receptor resistant version of IL-18, in R/R MM. Patients will receive ST-067 monotherapy during Cycle 1 (28 days) and then tec + ST-067 from Cycle 2 onwards. Enrollment of 20 patients is planned. Eligibility criteria include MM with measurable disease, eligibility to receive commercial tec (≥4 prior lines, including exposure to a PI, IMID, and CD38 mAb), age ≥18, ECOG PS 0-2, and adequate organ function. Patients with ≥12 months since prior BCMA therapy are also eligible. During Cycle 1, participants will receive ST-067 monotherapy (subcutaneously once per week) at escalating dose levels (DL): DL1 0.03 milligrams per kilogram (mg/kg), DL2 0.06 mg/kg, and DL3 0.12 mg/kg. Patients will be hospitalized for their first dose to evaluate for CRS. During Cycle 2, ST-067 will be administered with full-dose tec following inpatient tec step-up dosing. Doses of ST-067 and tec must be separated ≥48 hours and any CRS must have resolved before administration of the next agent. From Cycle 3 onward, ST-067 will be dosed q2week subcutaneously while tec will be dosed every 1-2 weeks per investigator discretion. For patients who have achieved ≥PR by IMWG criteria and received at least 3 treatment-level tec doses, ST-067 and tec can be dosed on the same day. The primary endpoint is evaluation of the safety profile and optimal biologic dose of ST-067 as monotherapy and with tec; key DLTs include Grade 3+ CRS and Grade 2+ ICANS. Other endpoints include efficacy (including MRD status at Day +28) and exploratory markers of T-cell fitness and tumor burden. Enrollment is expected to begin in the fall of 2024. Discussion: ST-067 can potentiate T-cell function, particularly in effector CD8+ T-cells that are critical to tec's anti-MM activity from the very first dose (Firestone et al, Blood Adv 2024). Additionally, ST-067 may be able to promote stem-like memory precursor T-cells that are likely important to long-term responses. If shown to have a favorable safety and efficacy profile in this and future studies, ST-067 has the potential to become a routine addition to bsAb therapy in MM and other malignancies. Funding: Simcha IL-18, Inc.

The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.

Vector-borne diseases are a leading cause of death worldwide and pose a substantial unmet medical need. Pathogens binding to host extracellular proteins (the “exoproteome”) represents a crucial interface in the etiology of vector-borne disease. Here, we used bacterial selection to elucidate host-microbe interactions in high throughput (BASEHIT)—a technique enabling interrogation of microbial interactions with 3,324 human exoproteins—to profile the interactomes of 82 human-pathogen samples, including 30 strains of arthropod-borne pathogens and 8 strains of related non-vector-borne pathogens. The resulting atlas revealed 1,303 putative interactions, including hundreds of pairings with potential roles in pathogenesis, including cell invasion, tissue colonization, immune evasion, and host sensing. Subsequent functional investigations uncovered that Lyme disease spirochetes recognize epidermal growth factor as an environmental cue of transcriptional regulation and that conserved interactions between intracellular pathogens and thioredoxins facilitate cell invasion. In summary, this interactome atlas provides molecular-level insights into microbial pathogenesis and reveals potential host-directed targets for next-generation therapeutics.

CD47 expression on canine cancer samples.