Background Omalizumab, a therapeutic mAb targeting IgE, is approved for the treatment of multiple allergic indications. However, its moderate affinity for IgE necessitates frequent high-dose administrations, limiting its therapeutic use and efficacy. Attempts to develop next-generation anti-IgE antibodies with improved affinity, such as ligelizumab or HAE1, have yielded alternatives that are either less safe or not demonstrably superior. Objective We sought to generate optimized omalizumab variants featuring 2 specific molecular enhancements: increased IgE binding affinity while preserving epitope specificity to enhance target neutralization and improved potency to actively dissociate prebound IgE from its high-affinity receptor FcεRI. Methods Using a targeted yeast display selection strategy applied to mutated omalizumab libraries, we identified the anti-IgE clone C03 and engineered 2 flexible variants, C03-H1L2 and C03-H2L2. Results The C03 antibodies demonstrated approximately 10-fold higher IgE binding affinity compared with omalizumab, resulting in superior inhibition of IgE binding to FcεRI. Furthermore, C03-H1L2 and C03-H2L2 exhibited enhanced potency in displacing FcεRI-bound IgE from humanized mouse mast cells and human basophils without triggering spontaneous cell activation. In a systemic anaphylaxis mouse model, single-dose administration of the flexible C03 variants, in contrast to omalizumab, desensitized allergic effector cells within 36 hours, fully preventing antigen-induced anaphylaxis. Conclusions These findings underscore the importance of engineering next-generation anti-IgE therapies with higher affinity and disruptive potency to optimize current treatment approaches.

Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs. Monoclonal antibodies from people after natural human parainfluenza virus type 3 infection can protect from infection in vitro and in vivo by targeting both pre-fusion F and haemagglutinin-neuraminidase HN proteins of the virus.

The etiology of allergy is closely linked to type 2 inflammatory responses ultimately leading to the production of allergen-specific immunoglobulin E (IgE), a key driver of many allergic conditions. At a high level, initial allergen exposure disrupts epithelial integrity, triggering local inflammation via alarmins including IL-25, IL-33, and TSLP, which activate type 2 innate lymphoid cells as well as other immune cells to secrete type 2 cytokines IL-4, IL-5 and IL-13, promoting Th2 cell development and eosinophil recruitment. Th2 cell dependent B cell activation promotes the production of allergen-specific IgE, which stably binds to basophils and mast cells. Rapid degranulation of these cells upon allergen re-exposure leads to allergic symptoms. Recent advances in our understanding of the molecular and cellular mechanisms underlying allergic pathophysiology have significantly shaped the development of therapeutic intervention strategies. In this review, we highlight key therapeutic targets within the allergic cascade with a particular focus on past, current and future treatment approaches using monoclonal antibodies. Specific targeting of alarmins, type 2 cytokines and IgE has shown varying degrees of clinical benefit in different allergic indications including asthma, chronic spontaneous urticaria, atopic dermatitis, chronic rhinosinusitis with nasal polyps, food allergies and eosinophilic esophagitis. While multiple therapeutic antibodies have been approved for clinical use, scientists are still working on ways to improve on current treatment approaches. Here, we provide context to understand therapeutic targeting strategies and their limitations, discussing both knowledge gaps and promising future directions to enhancing clinical efficacy in allergic disease management.

To the Editor, Early in the coronavirus disease 2019 (COVID19) pandemic, age has been recognized as one of the major risk factors for poor clinical outcome.1 Based on hospitalization rates, it has also rapidly become evident that fewer women than men were affected by severe disease manifestation.2 With the primary goal to protect the most vulnerable populations, those older than 65, scientists around the world have successfully developed different vaccines with unprecedented speed.3 Although it is well established that immune responses against infections decline with age,4 it is less clear how vaccineelicited immunity varies between different sex and age groups.5 Given the importance of understanding these biological parameters, which may directly affect translatability of research findings into the clinic, we sought to investigate the immune response against severe acute respiratory syndrome coronavirus 2 (SARSCoV2) in a proteinbased and vesicular stomatitis virus (VSV)vectored vaccination approach in young and aged mice of both sexes. First, we used the recombinant receptorbinding domain (RBD) of the SARSCoV2 spike protein from the original reference strain emulsified in an aluminum hydroxide containing wet gel suspension (i.e. Alum) to subcutaneously immunize C57BL/6 mice (Figure S1). Seven days later, they received a booster injection and the vaccine response was assessed on day 28 (Figure 1A,B). To test the induction of humoral immunity as a function of age, we measured antigenspecific IgG in young (2 months old) and aged (18– 19 months old) mice by ELISA. Consistent with other studies, the systemic RBDspecific IgG response was significantly diminished in aged mice (Figure 1C,D). This agerelated decline in total RBDspecific IgG is primarily due to a loss of IgG1 production since the other subclasses remained barely detectable (Figure S2). To further characterize humoral immunity, we measured the total number of plasma Bcells in spleen by flow cytometry and quantified RBDspecific plasma Bcells in the spleen of immunized mice by ELISpot. While the total number of splenic plasma Bcells was increased in aged mice the RBDspecific IgG positive Bcells were significantly diminished (Figure 1E,F) and correlated with serum IgG levels (Figure 1G), suggesting that the agerelated reduction of RBDspecific plasma cell formation might contribute to the concomitant decrease in antibody titers. Previous studies have reported significant alterations in T follicular helper (Tfh) and regulatory (Tfr) cell numbers in lymphoid organs in aged mice contributing to impaired plasma Bcell generation and defective antibody production.6 Indeed, we measured an agerelated increase in both Tfr and Tfh populations in the spleen as quantified by flow cytometry, while the number of classical T regulatory cells (Tregs) remained unchanged (Figure S3A). Most importantly, the live SARSCoV2 neutralization potency of serum from aged mice was significantly reduced for the original reference strain and different other variants of concern (i.e. alpha, gamma and delta), which is in line with the agerelated decrease in RBDspecific serum antibody titers and plasma Bcells (Figure 1H). Additionally, we evaluated sexspecific differences in vaccination response across age in the same cohorts of immunized C57BL/6 mice (Figure 2A). While RBDspecific IgG and IgG1 responses in serum were higher in young females as compared to young male controls (Figure 2B,C), these differences were no longer apparent in the aged mice, and there were no detectable sexspecific differences in the number of splenic plasma Bcells (Figure 2D). In line with higher RBDspecific antibody titers, young female C57BL/6 mice also showed more potent virus neutralization of the SARSCoV2 reference strain when subcutaneously immunized with a proteinbased vaccine or intramuscularly injected with two VSVvectored COVID19 vaccine candidates (i.e., VSVSD21 and VSVMqSD21) as compared to male controls (Figure 2E and Figure S4A,B). However, neutralization of the other tested variants of concern was diminished and equally weak in both sexes (Figure 2E), indicating that mutations in the RBD domain of these variants were sufficient to escape the vaccineinduced antibody response. To test whether these findings were conserved across different mouse strains, we repeated the same immunization regimen in young BALB/c mice. The observed outcome was essentially the same with females showing a better vaccination response than male mice (Figure S5A– E). The sexspecific differences in humoral immune response of young C57BL/6 mice persisted even after an additional injection with a proteinbased vaccine 21 days after the first boost as assessed on day 42 (Figure 2F– J). Strikingly, we found increased numbers of RBDspecific plasma Bcells in the bone marrow of young female mice in this context. In summary, our data demonstrate significant ageand sexrelated differences in the humoral immune response to different

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections pose a significant health burden. Using pre-fusion conformation fusion (F) proteins, we isolated a panel of anti-F antibodies from a human donor. One antibody (RSV-199) potently cross-neutralized 8 RSV and hMPV strains by recognizing antigenic site III, which is partially conserved in RSV and hMPV F. Next, we determined the cryoelectron microscopy (cryo-EM) structures of RSV-199 bound to RSV F trimers, hMPV F monomers, and an unexpected dimeric form of hMPV F. These structures revealed how RSV-199 engages both RSV and hMPV F proteins through conserved interactions of the antibody heavy-chain variable region and how variability within heavy-chain complementarity-determining region 3 (HCDR3) can be accommodated at the F protein interface in site-III-directed antibodies. Furthermore, RSV-199 offered enhanced protection against RSV A and B strains and hMPV in cotton rats. These findings highlight the mechanisms of broad neutralization and therapeutic potential of RSV-199.

Human cytomegalovirus (HCMV) is a herpesvirus that produces disease in transplant patients and newborn children. Entry of HCMV into cells relies on gH/gL trimer (gHgLgO) and pentamer (gHgLUL128-131) complexes that bind cellular receptors. Here, we studied the structure and interactions of the HCMV trimer, formed by AD169 strain gH and gL and TR strain gO proteins, with the human platelet-derived growth factor receptor alpha (PDGFRα). Three trimer surfaces make extensive contacts with three PDGFRα N-terminal domains, causing PDGFRα to wrap around gO in a structure similar to a human hand, explaining the high-affinity interaction. gO is among the least conserved HCMV proteins, with 8 distinct genotypes. We observed high conservation of residues mediating gO-gL interactions but more extensive gO variability in the PDGFRα interface. Comparisons between our trimer structure and a previously determined structure composed of different subunit genotypes indicate that gO variability is accommodated by adjustments in the gO-PDGFRα interface. We identified two loops within gO that were disordered and apparently glycosylated, which could be deleted without disrupting PDGFRα binding. We also identified four gO residues that contact PDGFRα, which when mutated produced markedly reduced receptor binding. These residues fall within conserved contact sites of gO with PDGFRα and may represent key targets for anti-trimer neutralizing antibodies and HCMV vaccines. Finally, we observe that gO mutations distant from the gL interaction site impact trimer expression, suggesting that the intrinsic folding or stability of gO can impact the efficiency of trimer assembly. IMPORTANCE HCMV is a herpesvirus that infects a large percentage of the adult population and causes significant levels of disease in immunocompromised individuals and birth defects in the developing fetus. The virus encodes a complex protein machinery that coordinates infection of different cell types in the body, including a trimer formed of gH, gL, and gO subunits. Here, we studied the interactions of the HCMV trimer with its receptor on cells, the platelet derived growth factor receptor α (PDGFRα), to better understand how HCMV coordinates virus entry into cells. Our results add to our understanding of HCMV strain-specific differences and identify sites on the trimer that represent potential targets for therapeutic antibodies or vaccine development.

Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated "disruption" of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.

Background: Anaphylaxis represents one of the most severe and fatal forms of allergic reactions. Like most other allergies, it is caused by activation of basophils and mast cells by allergen-mediated cross-linking of IgE bound to its high-affinity receptor, Fc epsilon RI, on the cell surface. The systemic release of soluble mediators induces an inflammatory cascade, rapidly causing symptoms with peak severity in minutes to hours after allergen exposure. Primary treatment for anaphylaxis consists of immediate intramuscular administration of adrenaline. Objective: While adrenaline alleviates life-threatening symptoms of an anaphylactic reaction, there are currently no disease-modifying interventions available. We sought to develop potent and fast-acting IgE inhibitors with the potential to rapidly terminate acute allergic reactions. Methods: Using affinity maturation by yeast display and structure-guided molecular engineering, we generated 3 optimized disruptive IgE inhibitors based on designed ankyrin repeat proteins and assessed their ability to actively remove IgE from allergic effector cells in vitro as well as in vivo in mice. Results: The engineered IgE inhibitors rapidly dissociate preformed IgE:Fc epsilon RI complexes, terminate IgE-mediated signaling in preactivated human blood basophils in vitro, and shut down preinitiated allergic reactions and anaphylaxis in mice in vivo. Conclusions: Fast-acting disruptive IgE inhibitors demonstrate the feasibility of developing kinetically optimized inhibitors for the treatment of anaphylaxis and the rapid desensitization of allergic individuals.

Targeting of immunoglobulin E (IgE) represents an interesting approach for the treatment of allergic disorders. A high-affinity monoclonal anti-IgE antibody, ligelizumab, has recently been developed to overcome some of the limitations associated with the clinical use of the therapeutic anti-IgE antibody, omalizumab. Here, we determine the molecular binding profile and functional modes-of-action of ligelizumab. We solve the crystal structure of ligelizumab bound to IgE, and report epitope differences between ligelizumab and omalizumab that contribute to their qualitatively distinct IgE-receptor inhibition profiles. While ligelizumab shows superior inhibition of IgE binding to FcεRI, basophil activation, IgE production by B cells and passive systemic anaphylaxis in an in vivo mouse model, ligelizumab is less potent in inhibiting IgE:CD23 interactions than omalizumab. Our data thus provide a structural and mechanistic foundation for understanding the efficient suppression of FcεRI-dependent allergic reactions by ligelizumab in vitro as well as in vivo.

Virus entry into target cells is the first step for virus infection. Understanding the overall entry mechanism, including the binding mechanism of specific virus glycoproteins with cellular receptors, can be useful for the design of small molecule inhibitors and vaccine development. Recently, EphA2 was identified as an important entry receptor for both KSHV and EBV. In the present study, we investigated the required binding sites within EphA2 and EBV gH/gL that mediate the interaction of these two proteins allowing entry into epithelial cells and found that it differed in compared to the interaction of KSHV gH/gL with EphA2. Our discoveries may uncover new potential interventional strategies that block EBV and KSHV infection of target epithelial cells.

Background: Serological immunoassays that are able to identify protective immunity against SARS-CoV-2 are urgently required to adapt quarantine measures, assess vaccination responses and evaluate donor plasma. To date, however the utility of such immunoassays remains unclear. In a mixed-design evaluation study, we compared the performance of serological immunoassays that are based on various SARS-CoV-2 proteins and assessed the neutralizing activity of COVID-19 patient sera. Methods: Patients admitted with confirmed SARS-CoV-2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in-house enzyme-linked immunosorbent assay utilizing recombinant receptor binding domain (RBD) of the SARS-CoV-2 spike protein was developed and compared to three commercially available enzyme-linked immunosorbent assays (ELISAs) targeting the nucleocapsid (N), the S1 domain of the spike protein (S1) and a lateral flow immunoassay (LFI) based on full-length spike protein. Neutralisation assays with live SARS-CoV-2 were performed to determine immunity. Findings: With a prevalence of 19.9%, we find 106 COVID-19 positive results among the 534 tested individuals. Time to IgG seroconversion occurred between day 0 and day 21 after the start of symptoms or positive RT-PCR. While the ELISAs showed sensitivities of 84.9% for RBD, 85.9% for S1 and 68.9% for Nprotein, the measured specificity amounted to 94.6% for the RBD, 97.7% for the S1 and 96.3% for the N protein. The LFI performed inferior with 71.4% sensitivity and 95.8% specificity. Out of 54 COVID-19 positive individuals 96.3% showed full neutralisation of live SARS-CoV-2 at serum dilutions ≥1:16, while none of the 6 COVID-19 negative sera revealed neutralizing activity. Interpretation: Our data suggest that ELISAs based on RBD and S1 protein of SARS-CoV-2 are adequate diagnostic predictors of protective immunity. Funding Statement: Development of SARS-CoV-2 reagents was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C. MN is supported by a research grant of the Swiss National Science Foundation (#179334). AE received grant support from the Research Fund of the Swiss Lung Association, Bern and the Uniscientia foundation. Declaration of Interests: All authors declare that there is no conflict of interests. Ethics Approval Statement: The study protocol was approved by the appropriate ethics committee and the authorities of the University Hospital and conducted in accordance with the Declaration of Helsinki.

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

Background: Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted “infiltrin” protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE’s effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium.

B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.

BACKGROUND:Serological immunoassays that can identify protective immunity against SARS-CoV-2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed-design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS-CoV-2 proteins and assessed the neutralizing activity of antibodies in patient sera.METHODS:Consecutive patients admitted with confirmed SARS-CoV-2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in-house enzyme-linked immunosorbent assay utilizing recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was developed and compared to three commercially available enzyme-linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1), and a lateral flow immunoassay (LFI) based on full-length spike protein. Neutralization assays with live SARS-CoV-2 were performed.RESULTS:One thousand four hundred and seventy-seven individuals were included comprising 112 SARS-CoV-2 positives (defined as a positive real-time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS-CoV-2 positive individuals, 96.3% showed full neutralization of live SARS-CoV-2 at serum dilutions ≥ 1:16, while none of the 6 SARS-CoV-2-negative sera revealed neutralizing activity.CONCLUSIONS:ELISAs targeting RBD and S1 protein of SARS-CoV-2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS-CoV-2.

Background: Parasitic infections can increase susceptibility to bacterial co-infections. This may be true for urogenital schistosomiasis and bacterial urinary tract co-infections (UTI). We previously reported that this co-infection is facilitated by S. haematobium eggs triggering interleukin-4 (IL-4) production and sought to dissect the underlying mechanisms. The interleukin-4 inducing principle from Schistosoma mansoni eggs (IPSE) is one of the most abundant schistosome egg-secreted proteins and binds to IgE on the surface of basophils and mast cells to trigger IL-4 release. IPSE can also translocate into host nuclei using a nuclear localization sequence (NLS) to modulate host transcription. We hypothesized that IPSE is the factor responsible for the ability of S. haematobium eggs to worsen UTI pathogenesis. Methods: Mice were intravenously administered a single 25 mg dose of recombinant S. haematobium-derived IPSE, an NLS mutant of IPSE or PBS. Following IPSE exposure, mice were serially weighed, and organs analyzed by histology to assess for toxicity. Twenty-four hours after IPSE administration, mice were challenged with the uropathogenic E. coli strain UTI89 by urethral catheterization. Bacterial cfu were measured using urine. Bladders were examined histologically for UTI-triggered pathogenesis and by PCR for antimicrobial peptide expression.Results: Unexpectedly, IPSE administration did not result in significant differences in urine bacterial CFU. However, IPSE administration did lead to a significant reduction in UTI-induced bladder pathogenesis and the expression of anti-microbial peptides in the bladder. Despite the profound effect of IPSE on UTI-triggered bladder pathogenesis and anti-microbial peptide production, mice did not demonstrate systemic ill effects from IPSE exposure.Conclusions: Our data show that IPSE may play a major role in S. haematobium-associated urinary tract co-infection, albeit in an unexpected fashion. These findings also indicate that IPSE either works in concert with other IL-4-inducing factors to increase susceptibility of S. haematobium-infected hosts to bacterial co-infection, or does not contribute to enhancing vulnerability to this co-infection.