Search

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., (© 2024 Excellergy Inc and The Author(s). Immunological Reviews published by John Wiley & Sons Ltd.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Declaration of interests J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, GlaxoSmithKline, and BTG International Inc. He is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols), and founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study. T.S.J. has served as a consultant for Pfizer. Vanderbilt University has applied for patents for some of the antibodies in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s).)

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.

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, FcεRI, 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:FcεRI 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., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Herpesviruses are ubiquitous, double-stranded DNA, enveloped viruses that establish lifelong infections and cause a range of diseases. Entry into host cells requires binding of the virus to specific receptors, followed by the coordinated action of multiple viral entry glycoproteins to trigger membrane fusion. Although the core fusion machinery is conserved for all herpesviruses, each species uses distinct receptors and receptor-binding glycoproteins. Structural studies of the prototypical herpesviruses herpes simplex virus 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) entry glycoproteins have defined the interaction sites for glycoprotein complexes and receptors, and have revealed conformational changes that occur on receptor binding. Recent crystallography and electron microscopy studies have refined our model of herpesvirus entry into cells, clarifying both the conserved features and the unique features. In this Review, we discuss recent insights into herpesvirus entry by analysing the structures of entry glycoproteins, including the diverse receptor-binding glycoproteins (HSV-1 glycoprotein D (gD), EBV glycoprotein 42 (gp42) and HCMV gH-gL-gO trimer and gH-gL-UL128-UL130-UL131A pentamer), as well gH-gL and the fusion protein gB, which are conserved in all herpesviruses.

Paramyxovirus membrane fusion requires an attachment protein that binds to a host cell receptor and a fusion protein that merges the viral and host membranes. For Nipah virus (NiV), the G attachment protein binds ephrinB2/B3 receptors and activates F-mediated fusion. To visualize dynamic events of these proteins at the membrane interface, we reconstituted NiV fusion activation by overlaying F- and G-expressing cells onto ephrinB2-functionalized supported lipid bilayers and used TIRF microscopy to follow F, G, and ephrinB2. We found that G and ephrinB2 form clusters and that oligomerization of ephrinB2 is necessary for F activation. Single-molecule tracking of F particles revealed accumulation of an immobilized intermediate upon activation. We found no evidence for stable F-G protein complexes before or after activation. These observations lead to a revised model for NiV fusion activation and provide a foundation for investigating other multicomponent viral fusion systems., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

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 μg 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 and pattern recognition receptor 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.

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., (Copyright © 2020, American Association for the Advancement of Science.)

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., Summary: Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Both Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses and are important in a variety of malignancies. Eph family receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV and EBV. Previous studies identified five conserved residues (ELEFN 50-54 ) in the N-terminal domain of KSHV gH that are critical for Eph binding and KSHV infection. However, the specific domains of EBV gH/gL important for EphA2 binding are not well described. We found that the KSHV gH (ELEFN 50-54 ) motif is important for higher KSHV fusion and that EBV gH/gL does not utilize a similar motif for fusion activity. We previously identified that an EBV gL N-glycosylation mutant (gL-N 69 L/S 71 V) was hyperfusogenic in epithelial cells but not in B cells. To determine whether this glycosylation site may be the binding region for EphA2, we compared the EphA2 binding activity of EBV gH/gL and the EBV gH/gL-N 69 L/S 71 V mutant. We found that EBV gH/gL-N 69 L/S 71 V had higher binding affinity for EphA2, indicating that the EBV gL N-glycosylation site might be responsible for inhibiting the binding of gH/gL to EphA2. Loss of N-glycosylation at this site may remove steric hindrance that reduces EBV gH/gL binding to EphA2. In addition, the mutations located in the large groove of EBV gH/gL (R 152 A and G 49 C) also have decreased binding with EphA2. Taken together, our data indicate that the binding site of EphA2 on EBV gH/gL is at least in part proximal to the EBV gL glycosylation site, which in part accounts for differences in EphA2 binding affinity by KSHV. IMPORTANCE 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., (Copyright © 2020 American Society for Microbiology.)

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., Competing Interests: Declaration of Interests A.T.S. is a scientific founder of Immunai and receives research funding from Arsenal Biosciences not related to this study. M.H. is an employee of ATUM. S.D.B. has consulted for Regeneron, Sanofi, and Novartis on topics unrelated to this study. S.D.B., K.R., P.S.K., and A.E.P. have filed provisional patent applications related to serological tests for SARS-CoV-2 antibodies. K.C.N. reports grants from National Institute of Allergy and Infectious Diseases (NIAID), Food Allergy Research & Education (FARE), and End Allergies Together (EAT); National Heart, Lung, and Blood Institute (NHLBI), and National Institute of Environmental Health Sciences (NIEHS), and is the director of FARE and World Allergy Organization (WAO) Center of Excellence at Stanford; advisor at Cour Pharma; co-founder of Before Brands, Alladapt, Latitude, and IgGenix; National Scientific Committee member at Immune Tolerance Network (ITN) and National Institutes of Health (NIH); a recipient of a Research Sponsorship from Nestle; Consultant and Advisory Board Member at Before Brands, Alladapt, Iggenix, NHLBI, and Probio; Data and Safety Monitoring Board member at NHLBI; and has US patents for basophil testing, multifood immunotherapy and prevention, monoclonal antibodies from plasmablasts, and devices for diagnostics. The remaining authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor is an important mediator of nociception and its expression is enriched in nociceptive neurons. TRPV1 signaling has been implicated in bladder pain and is a potential analgesic target. Resiniferatoxin is the most potent known agonist of TRPV1. Acute exposure of the rat bladder to resiniferatoxin has been demonstrated to result in pain-related freezing and licking behaviors that are alleviated by virally encoded IL-4. The interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE) is a powerful inducer of IL-4 secretion, and is also known to alter host cell transcription through a nuclear localization sequence-based mechanism. We previously reported that IPSE ameliorates ifosfamide-induced bladder pain in an IL-4- and nuclear localization sequence-dependent manner. We hypothesized that pre-administration of IPSE to resiniferatoxin-challenged mice would dampen pain-related behaviors. IPSE indeed lessened resiniferatoxin-triggered freezing behaviors in mice. This was a nuclear localization sequence-dependent phenomenon, since administration of a nuclear localization sequence mutant version of IPSE abrogated IPSE's analgesic effect. In contrast, IPSE's analgesic effect did not seem IL-4-dependent, since use of anti-IL-4 antibody in mice given both IPSE and resiniferatoxin did not significantly affect freezing behaviors. RNA-Seq analysis of resiniferatoxin- and IPSE-exposed bladders revealed differential expression of TNF/NF-κb-related signaling pathway genes. In vitro testing of IPSE uptake by urothelial cells and TRPV1-expressing neuronal cells showed uptake by both cell types. Thus, IPSE's nuclear localization sequence-dependent therapeutic effects on TRPV1-mediated bladder pain may act on TRPV1-expressing neurons and/or may rely upon urothelial mechanisms.

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis hematobium -derived protein H-IPSE H06 (IL-4-inducing principle from Schistosoma mansoni eggs) is superior to three doses of MESNA in alleviating hemorrhagic cystitis (Mbanefo EC, Le L, Pennington LF, Odegaard JI, Jardetzky TS, Alouffi A, Falcone FH, Hsieh MH. FASEB J 32: 4408-4419, 2018). Based on prior reports by others on S. mansoni IPSE (M-IPSE) and additional work by our group, we reasoned that H-IPSE mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. hematobium IPSE ortholog IPSE H03 , hereafter called H-IPSE H03 , might be more efficacious in preventing hemorrhagic cystitis compared with systemic administration of IPSE H06 . We report that H-IPSE H03 , like M-IPSE and H-IPSE H06 , activates IgE-bearing basophils in a nuclear factor of activated T-cells reporter assay, indicating activation of the cytokine pathway. Furthermore, H-IPSE H03 attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSE H03 relieves hemorrhagic cystitis-associated allodynia and modulates voiding patterns in mice. Finally, H-IPSE H03 drives increased urothelial cell proliferation, suggesting that IPSE induces bladder repair mechanisms. Taken together, H-IPSE H03 may be a potential novel therapeutic to treat hemorrhagic cystitis by basophil activation, attenuation of allodynia, and promotion of urothelial cell proliferation.

Human cytomegalovirus (HCMV) causes substantial disease in transplant patients and harms the development of the nervous system in babies infected in utero. Thus, there is a major focus on developing safe and effective HCMV vaccines. Evidence has been presented that a major target of neutralizing antibodies (NAbs) is the HCMV pentamer glycoprotein gH/gL/UL128-131. In some studies, most of the NAbs in animal or human sera were found to recognize the pentamer, which mediates HCMV entry into endothelial and epithelial cells. It was also reported that pentamer-specific antibodies correlate with protection against transmission from mothers to babies. One problem with the studies on pentamer-specific NAbs to date has been that the studies did not compare the pentamer to the other major form of gH/gL, the gH/gL/gO trimer, which is essential for entry into all cell types. Here, we demonstrate that both trimer and pentamer NAbs are frequently found in human transplant patients' and pregnant mothers' sera. Depletion of human sera with trimer caused reductions in NAbs similar to that observed following depletion with the pentamer. The trimer- and pentamer-specific antibodies acted in a synergistic fashion to neutralize HCMV and also to prevent virus cell-to-cell spread. Importantly, there was no correlation between the titers of trimer- and pentamer-specific NAbs and transmission of HCMV from mothers to babies. Therefore, both the trimer and pentamer are important targets of NAbs. Nevertheless, these antibodies do not protect against transmission of HCMV from mothers to babies., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with the development of Kaposi's sarcoma (KS). KSHV target cells include endothelial cells, B cells, monocytes, epithelial cells, dendritic cells, macrophages, and fibroblasts. KSHV entry into target cells is a complex multistep process and is initiated by the binding and interaction of viral envelope glycoproteins with the cellular receptors. In the current studies, we have found that EphA4 promotes KSHV glycoprotein H/glycoprotein L (gH/gL)-mediated fusion and infection better than does ephrin A2 (EphA2) in HEK293T cells, indicating that EphA4 is a new KSHV entry receptor. To confirm that epithelial cells express EphA2 and EphA4, we analyzed the expression of EphA2 and EphA4 in epithelial cells, endothelial cells, B cells, monocytes, fibroblasts using RNA sequencing (RNA-seq) data analysis of existing data sets. We found that these cell types broadly express both EphA2 and EphA4, with the exception of monocytes and B cells. To confirm EphA4 is important for KSHV fusion and infection, we generated EphA2 and EphA4 single- and double-knockout cells. We found that both EphA2 and EphA4 play a role in KSHV fusion and infection, since EphA2-EphA4 double-knockout cells had the greatest decrease in fusion activity and infection compared to single-knockout cells. Fusion and infection of KSHV were rescued in the EphA2-EphA4 double-knockout cells upon overexpression of EphA2 and/or EphA4. EphA2 binds to both Epstein-Barr virus (EBV) and KSHV gH/gL; however, EphA4 binds only to KSHV gH/gL. Taken together, our results identify EphA4 as a new entry receptor for KSHV. IMPORTANCE The overall entry mechanism for herpesviruses is not completely known, including those for the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). To fully understand the herpesvirus entry process, functional receptors need to be identified. In the current study, we found that EphA4 can also function for a KSHV entry receptor along with EphA2. Interestingly, we found that EphA4 does not function as an entry receptor for EBV, whereas EphA2 does. The discovery of EphA4 as a KSHV entry receptor has important implications for KSHV pathogenesis in humans, may prove useful in understanding the unique pathogenesis of KSHV infection in humans, and may uncover new potential targets that can be used for the development of novel interventional strategies., (Copyright © 2019 Chen et al.)

Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications caused by acrolein metabolites. We previously showed that a single dose of IPSE (Interleukin-4-inducing principle from Schistosoma eggs), a schistosome-derived host modulatory protein, can ameliorate ifosfamide-related cystitis; however, the mechanisms underlying this urotoxicity and its prevention are not fully understood. To provide insights into IPSE's protective mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without pretreatment with IPSE or IPSE-NLS (a mutant of IPSE lacking nuclear localization sequence). Ifosfamide treatment upregulated a range of proinflammatory genes. The IL-1β-TNFα-IL-6 proinflammatory cascade via NFκB and STAT3 pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress response pathway, which regulates heme homoeostasis and expression of antioxidant enzymes, was highly activated. Anti-inflammatory cascades, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE drove significant downregulation of major proinflammatory pathways including the IL-1β-TNFα-IL-6 pathways, interferon signaling, and reduction in oxidative stress. IPSE-NLS reduced inflammation but not oxidative stress. Taken together, we have identified signatures of acute-phase inflammation and oxidative stress in ifosfamide-injured bladder, which are reversed by pretreatment with IPSE. This work revealed several pathways that could be therapeutically targeted to prevent ifosfamide-induced hemorrhagic cystitis.

Human cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells, but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRα, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes. IMPORTANCE HCMV infects nearly 80% of the world's population and causes significant morbidity and mortality. The current antiviral agents used to treat HCMV infections are prone to resistance and can be toxic to patients, and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics., (Copyright © 2018 American Society for Microbiology.)

Chemotherapy-induced hemorrhagic cystitis (CHC) can be difficult to manage. Prior work suggests that IL-4 alleviates ifosfamide-induced hemorrhagic cystitis (IHC), but systemically administered IL-4 causes significant side effects. We hypothesized that the Schistosoma hematobium homolog of IL-4-inducing principle from Schistosoma mansoni eggs (H-IPSE), would reduce IHC and associated bladder pathology. IPSE binds IgE on basophils and mast cells, triggering IL-4 secretion by these cells. IPSE is also an "infiltrin," translocating into the host nucleus to modulate gene transcription. Mice were administered IL-4, H-IPSE protein or its nuclear localization sequence (NLS) mutant, with or without neutralizing anti-IL-4 antibody, or 2-mercaptoethane sulfonate sodium (MESNA; a drug used to prevent IHC), followed by ifosfamide. Bladder tissue damage and hemoglobin content were measured. Spontaneous and evoked pain, urinary frequency, and bladdergene expression analysis were assessed. Pain behaviors were interpreted in a blinded fashion. One dose of H-IPSE was superior to MESNA and IL-4 in suppressing bladder hemorrhage in an IL-4-dependent fashion and comparable with MESNA in dampening ifosfamide-triggered pain behaviors in an NLS-dependent manner. H-IPSE also accelerated urothelial repair following IHC. Our work represents the first therapeutic exploitation of a uropathogen-derived host modulatory molecule in a clinically relevant bladder disease model and indicates that IPSE may be an alternative to MESNA for mitigating CHC.-Mbanefo, E. C., Le, L., Pennington, L. F., Odegaard, J. I., Jardetzky, T. S., Alouffi, A., Falcone, F. H., Hsieh, M. H. Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis.

Human cytomegalovirus (HCMV) replicates in many diverse cell types in vivo , and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα). HCMV entry into biologically relevant epithelial and endothelial cells and monocyte-macrophages also requires a pentamer, gH/gL complexed with UL128, UL130, and UL131, and there is evidence that the pentamer binds unidentified receptors. We screened an epithelial cell cDNA library and identified the cell surface protein CD147, which increased entry of pentamer-expressing HCMV into HeLa cells but not entry of HCMV that lacked the pentamer. A panel of CD147-specific monoclonal antibodies inhibited HCMV entry into epithelial and endothelial cells, but not entry into fibroblasts. shRNA silencing of CD147 in endothelial cells inhibited HCMV entry but not entry into fibroblasts. CD147 colocalized with HCMV particles on cell surfaces and in endosomes. CD147 also promoted cell-cell fusion induced by expression of pentamer and gB in epithelial cells. However, soluble CD147 did not block HCMV entry and trimer and pentamer did not bind directly to CD147, supporting the hypothesis that CD147 acts indirectly through other proteins. CD147 represents the first HCMV entry mediator that specifically functions to promote entry of pentamer-expressing HCMV into epithelial and endothelial cells. IMPORTANCE Human cytomegalovirus infects nearly 80% of the world's population and causes significant morbidity and mortality. The current method of treatment involves the use of antiviral agents that are prone to resistance and can be highly toxic to patients; currently, there is no vaccine against HCMV available. HCMV infections involve virus dissemination throughout the body, infecting a wide variety of tissues; however, the mechanism of spread is not well understood, particularly with regard to which cellular proteins are utilized by HCMV to establish infection. This report describes the characterization of a newly identified cellular molecule that affects HCMV entry into epithelial and endothelial cells. These results will lead to a better understanding of HCMV pathogenesis and have implications for the development of future therapeutics., (Copyright © 2018 Vanarsdall et al.)

Epstein-Barr virus (EBV) is an oncogenic virus that infects more than 90% of the world's population 1 . EBV predominantly infects human B cells and epithelial cells, which is initiated by fusion of the viral envelope with a host cellular membrane 2 . The mechanism of EBV entry into B cells has been well characterized 3 . However, the mechanism for epithelial cell entry remains elusive. Here, we show that the integrins αvβ5, αvβ6 and αvβ8 do not function as entry and fusion receptors for epithelial cells, whereas Ephrin receptor tyrosine kinase A2 (EphA2) functions well for both. EphA2 overexpression significantly increased EBV infection of HEK293 cells. Using a virus-free cell-cell fusion assay, we found that EphA2 dramatically promoted EBV but not herpes simplex virus (HSV) fusion with HEK293 cells. EphA2 silencing using small hairpin RNA (shRNA) or knockout by CRISPR-Cas9 blocked fusion with epithelial cells. This inhibitory effect was rescued by the expression of EphA2. Antibody against EphA2 blocked epithelial cell infection. Using label-free surface plasmon resonance binding studies, we confirmed that EphA2 but not EphA4 specifically bound to EBV gHgL and this interaction is through the EphA2 extracellular domain (ECD). The discovery of EphA2 as an EBV epithelial cell receptor has important implications for EBV pathogenesis and may uncover new potential targets that can be used for the development of novel intervention strategies.

Urogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium , affects over 112 million people worldwide. As with Schistosoma mansoni infections, the pathology of urogenital schistosomiasis is related mainly to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related organism S. mansoni , which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (here M-IPSE), a major protein secreted from S. mansoni eggs, can infiltrate host cells. Although the function of M-IPSE is unknown, its ability to translocate to the nuclei of host cells and bind DNA suggests a possible role in immune modulation of host cell tissues. Whether IPSE homologs are expressed in other schistosome species has not been investigated. Here, we describe the cloning of two paralog genes, H03-IPSE and H06-IPSE, which are orthologs of M-IPSE, from egg cDNA of S. haematobium Using PCR and immunodetection, we confirmed that the expression of these genes is restricted to the egg stage and female adult worms, while the H-IPSE protein is detectable only in mature eggs and not adults. We show that both H03-IPSE and H06-IPSE proteins can infiltrate HTB-9 bladder cells when added exogenously to culture medium. Monopartite C-terminal nuclear localization sequence (NLS) motifs conserved in H03-IPSE, SKRRRKY, and H06-IPSE SKRGRKY, are responsible for targeting the proteins to the nucleus of HTB-9 cells, as demonstrated by site-directed mutagenesis and green fluorescent protein (GFP) tagging. Thus, S. haematobium eggs express IPSE homologs that appear to perform similar functions in infiltrating host cells., (Copyright © 2017 Pennington et al.)

Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion. IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses., (Copyright © 2017 American Society for Microbiology.)

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studied the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms., Competing Interests: The authors declare no conflict of interest.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry.

Enveloped viruses have evolved diverse transmembrane proteins and protein complexes to enable host cell entry by regulating and activating membrane fusion in a target cell-specific manner. In general terms, the entry process requires a receptor binding step, an activation step and a membrane fusion step, which can be encoded within a single viral protein or distributed among multiple viral proteins. HIV and influenza virus, for example, encode all of these functions in a single trimeric glycoprotein, HIV env or influenza virus hemagglutinin (HA). In contrast, herpesviruses have the host receptor binding, activation and fusogenic roles distributed among multiple envelope glycoproteins (ranging from three to six), which must coordinate their functions at the site of fusion. Despite the apparent complexity in the number of viral entry proteins, herpesvirus entry is fundamentally built around two core glycoprotein entities: the gHgL complex, which appears to act as an 'activator' of entry, and the gB protein, which is thought to act as the membrane 'fusogen'. Both are required for all herpesvirus fusion and entry. In many herpesviruses, gHgL either binds host receptors directly or assembles into larger complexes with additional viral proteins that bind host receptors, conferring specificity to the cells that are targeted for infection. These gHgL entry complexes (ECs) are centrally important to activating gB-mediated membrane fusion and establishing viral tropism, forming membrane bridging intermediates before gB triggering. Here we review recent structural and functional studies of Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) gHgL complexes that provide a framework for understanding the role of gHgL in herpesvirus entry. Furthermore, a recently determined EM model of Herpes Simplex virus (HSV) gB embedded in exosomes highlights how gB conformational changes may promote viral and cellular membrane fusion., (Copyright © 2017. Published by Elsevier B.V.)

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two closely related viruses that cause bronchiolitis and pneumonia in infants and the elderly 1 , with a significant health burden 2-6 . There are no licensed vaccines or small-molecule antiviral treatments specific to these two viruses at present. A humanized murine monoclonal antibody (palivizumab) is approved to treat high-risk infants for RSV infection 7,8 , but other treatments, as well as vaccines, for both viruses are still in development. Recent epidemiological modelling suggests that cross-immunity between RSV, HMPV and human parainfluenzaviruses may contribute to their periodic outbreaks 9 , suggesting that a deeper understanding of host immunity to these viruses may lead to enhanced strategies for their control. Cross-reactive neutralizing antibodies to the RSV and HMPV fusion (F) proteins have been identified 10,11 . Here, we examine the structural basis for cross-reactive antibody binding to RSV and HMPV F protein by two related, independently isolated antibodies, MPE8 and 25P13. We solved the structure of the MPE8 antibody bound to RSV F protein and identified the 25P13 antibody from an independent blood donor. Our results indicate that both antibodies use germline residues to interact with a conserved surface on F protein that could guide the emergence of cross-reactivity. The induction of similar cross-reactive neutralizing antibodies using structural vaccinology approaches could enhance intrinsic cross-immunity to these paramyxoviruses and approaches to controlling recurring outbreaks.

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein-Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, here we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator of EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. These observations clarify key determinants of EBV host cell tropism.

Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD). In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706) of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain., Importance: Infection with Epstein-Barr virus (EBV) causes diseases ranging from the fairly benign infectious mononucleosis to life-threatening cancer. Entry into target cells is the first step for viral infection and is important for EBV to cause disease. Understanding the EBV entry mechanism is useful for the development of infection inhibitors and developing EBV vaccine approaches. Epithelial and B cells are the main target cells for EBV infection. The essential glycoproteins for EBV entry include gB, gH/gL, and gp42. We characterized the function of the EBV gH C-terminal cytoplasmic tail domain (CTD) in fusion using a panel of gH CTD truncation or substitution mutants. We found that the gH CTD regulates fusion by altering gp42 and epithelial cell attachment. Our studies may lead to a better understanding of EBV fusion and entry, which may result in novel therapies that target the EBV entry step., (Copyright © 2016 Chen et al.)

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies., Competing Interests: W.R.S. is a founder of Compuvax, Inc. J.E.C. is a member of the Scientific Advisory Boards of Compuvax and Meissa Vaccines.

Unlabelled: The Paramyxoviridae comprise a large family of enveloped, negative-sense, single-stranded RNA viruses with significant economic and public health implications. For nearly all paramyxoviruses, infection is initiated by fusion of the viral and host cell plasma membranes in a pH-independent fashion. Fusion is orchestrated by the receptor binding protein hemagglutinin-neuraminidase (HN; also called H or G depending on the virus type) protein and a fusion (F) protein, the latter undergoing a major refolding process to merge the two membranes. Mechanistic details regarding the coupling of receptor binding to F activation are not fully understood. Here, we have identified the flexible loop region connecting the bulky enzymatically active head and the four-helix bundle stalk to be essential for fusion promotion. Proline substitution in this region of HN of parainfluenza virus 5 (PIV5) and Newcastle disease virus HN abolishes cell-cell fusion, whereas HN retains receptor binding and neuraminidase activity. By using reverse genetics, we engineered recombinant PIV5-EGFP viruses with mutations in the head-stalk linker region of HN. Mutations in this region abolished virus recovery and infectivity. In sum, our data suggest that the loop region acts as a "hinge" around which the bulky head of HN swings to-and-fro to facilitate timely HN-mediate F-triggering, a notion consistent with the stalk-mediated activation model of paramyxovirus fusion., Importance: Paramyxovirus fusion with the host cell plasma membrane is essential for virus infection. Membrane fusion is orchestrated via interaction of the receptor binding protein (HN, H, or G) with the viral fusion glycoprotein (F). Two distinct models have been suggested to describe the mechanism of fusion: these include "the clamp" and the "provocateur" model of activation. By using biochemical and reverse genetics tools, we have obtained strong evidence in favor of the HN stalk-mediated activation of paramyxovirus fusion. Specifically, our data strongly support the notion that the short linker between the head and stalk plays a role in "conformational switching" of the head group to facilitate F-HN interaction and triggering., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering., Importance: Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F) glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1) is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections.

Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab-Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.

Epstein-Barr virus (EBV) is the prototypical γ-herpesvirus and an obligate human pathogen that infects mainly epithelial cells and B cells, which can result in malignancies. EBV infects these target cells by fusing with the viral and cellular lipid bilayer membranes using multiple viral factors and host receptor(s) thus exhibiting a unique complexity in its entry machinery. To enter epithelial cells, EBV requires minimally the conserved core fusion machinery comprised of the glycoproteins gH/gL acting as the receptor-binding complex and gB as the fusogen. EBV can enter B cells using gp42, which binds tightly to gH/gL and interacts with host HLA class II, activating fusion. Previously, we published the individual crystal structures of EBV entry factors, such as gH/gL and gp42, the EBV/host receptor complex, gp42/HLA-DR1, and the fusion protein EBV gB in a postfusion conformation, which allowed us to identify structural determinants and regions critical for receptor-binding and membrane fusion. Recently, we reported different low resolution models of the EBV B cell entry triggering complex (gHgL/gp42/HLA class II) in "open" and "closed" states based on negative-stain single particle electron microscopy, which provide further mechanistic insights. This review summarizes the current knowledge of these key players in EBV entry and how their structures impact receptor-binding and the triggering of gB-mediated fusion.

Hendra virus (HeV) is one of the two prototypical members of the Henipavirus genus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.

Unlabelled: Herpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry., Importance: The alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The Paramyxoviridae include some of the great and ubiquitous disease-causing viruses of humans and animals. In most paramyxoviruses, two viral membrane glycoproteins, fusion protein (F) and receptor binding protein (HN, H or G) mediate a concerted process of recognition of host cell surface molecules followed by fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. The interactions between the F and HN, H or G viral glycoproteins and host molecules are critical in determining host range, virulence and spread of these viruses. Recently, atomic structures, together with biochemical and biophysical studies, have provided major insights into how these two viral glycoproteins successfully interact with host receptors on cellular membranes and initiate the membrane fusion process to gain entry into cells. These studies highlight the conserved core mechanisms of paramyxovirus entry that provide the fundamental basis for rational anti-viral drug design and vaccine development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: We recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, comprising the C-type lectin domain (CTLD), away from the membrane and distant from where the membrane-bound form of gp42 might be tethered. gp42 is a type II membrane glycoprotein, with functional gp42 formed by cleavage near the gp42 amino-terminal transmembrane domain. This cleavage results in an approximately 50-amino-acid unstructured region that is responsible for binding gH/gL with nanomolar affinity. Our previous studies had shown that membrane-bound gp42 is not functional in B cell fusion. To investigate whether we could restore gp42 function by extending it from the membrane, we introduced one, two, and four structured immunoglobulin-like domains from muscle protein titin into a membrane-bound form of gp42 and tested function in binding to gHgL and HLA class II and function in fusion. We hypothesized that cleavage of gp42 generates a soluble functional form that relieves steric hindrance imposed on gHgL by membrane-bound gp42. All of the linker mutants had a dominant-negative effect on gp42 function, indicating that gp42 fusion function could not be restored simply by the addition of one to four titin domains., Importance: Epstein-Barr virus (EBV) is associated with numerous diseases from benign mononucleosis to Burkitt's and Hodgkin's lymphoma, nasopharyngeal and gastric carcinoma, and lymphoproliferative disorders in patients with immune dysfunction resulting from immune suppression. Among the glycoproteins important for fusion, gp42, along with gH/gL, determines EBV tropism between epithelial and B cells. The function of gp42 is dependent on N-terminal cleavage, since membrane-bound gp42 cannot mediate fusion. We further investigated whether insertion of a linker into membrane-bound gp42 would relieve steric hindrance imposed on membrane-bound gp42 and restore fusion function. However, adding one, two, or four structured immunoglobulin-like domains to membrane gp42 did not restore fusion activity, indicating that the architecture and membrane orientation of the B cell fusion-triggering complex of EBV may be easily perturbed and that gp42 cleavage is essential for B cell fusion., (Copyright © 2015 Rowe et al.)

Unlabelled: Epstein-Barr virus (EBV) infects target cells via fusion with cellular membranes. For entry into epithelial cells, EBV requires the herpesvirus conserved core fusion machinery, composed of glycoprotein B (gB) and gH/gL. In contrast, for B cell fusion it requires gB and gH/gL with gp42 serving as a cell tropism switch. The available crystal structures for gH/gL allow the targeted analysis of structural determinants of gH to identify functional regions critical for membrane fusion. Domain II of EBV gH contains two disulfide bonds (DBs). The first is unique for EBV and closely related gammaherpesviruses. The second is conserved across the beta- and gammaherpesviruses and is positioned to stabilize a putative syntaxin-like bundle motif. To analyze the role of these DBs in membrane fusion, gH was mutated by amino acid substitution of the DB cysteines. Mutation of the EBV-specific DB resulted in diminished gH/gL cell surface expression that correlated with diminished B cell and epithelial cell fusion. In contrast, mutation of the conserved DB resulted in wild-type-like B cell fusion, whereas epithelial cell fusion was greatly reduced. The gH mutants bound well to gp42 but had diminished binding to epithelial cells. Tyrosine 336, located adjacent to cysteine 335 of the conserved DB, also was found to be important for DB stabilization and gH/gL function. We conclude that the conserved DB has a cell type-specific function, since it is important for the binding of gH to epithelial cells initiating epithelial cell fusion but not for fusion with B cells and gp42 binding., Importance: EBV predominantly infects epithelial and B cells in humans, which can result in EBV-associated cancers, such as Burkitt and Hodgkin lymphoma, as well as nasopharyngeal carcinoma. EBV is also associated with a variety of lymphoproliferative disorders, typically of B cell origin, observed in immunosuppressed individuals, such as posttransplant or HIV/AIDS patients. The gH/gL complex plays an essential but still poorly characterized role as an important determinant for EBV cell tropism. In the current studies, we found that mutants in the DB C278/C335 and the neighboring tyrosine 336 have cell type-specific functional deficits with selective decreases in epithelial cell, but not B cell, binding and fusion. The present study brings new insights into the gH function as a determinant for epithelial cell tropism during herpesvirus-induced membrane fusion and highlights a specific gH motif required for epithelial cell fusion., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)