Abstract Bispecific antibodies (bsAbs) have unlocked new therapeutic modalities by enabling dual antigen targeting, offering transformative potential in oncology and autoimmune diseases. However, the efficient production of full-length IgG-like bsAbs remains hindered by mispairing of heavy and light chains, yielding heterogeneous, low-purity products. Here, we present an Fc engineering strategy, termed StrandLock, that enables the controlled assembly of bsAbs through asymmetric β-strand redistribution at the CH3 dimer interface, in which the β-strand segment (residues N390–S400) is duplicated in one heavy chain and deleted from the partner. This structural asymmetry, combined with targeted point mutations, promotes obligate heterodimerization while suppressing homodimer formation. Importantly, these modifications enable the correct assembly of bsAbs with purities approaching 99% and allow streamlined manufacturing via a single-step affinity purification process comparable to that used for monoclonal antibodies. The engineered bsAbs retain key Fc effector functions and exhibit comparable in vivo pharmacokinetics to native IgG. Structural analysis confirms preservation of the CH3 fold and native-like interfacial packing. Despite a modest reduction in thermal stability, the molecules remain aggregation-resistant and functionally robust under accelerated stress conditions. We further demonstrate the versatility of the StrandLock design across multiple antibody pairs underscoring its utility as a strategy for bsAb production.

The membrane-proximal external region (MPER), located in the carboxy-terminal section of HIV’s envelope glycoprotein (Env) ectodomain, which is essential for viral entry into host cells, has gained considerable attention as a target for HIV vaccine development due to the exceptional neutralization breadth of antibodies against MPER epitopes. A distinctive feature of broadly neutralizing antibodies (bnAbs) targeting MPER is their requirement to accommodate the viral membrane into the surface of the antigen-binding fragment, or Fab moiety, to optimize antigen recognition. In this study, we sought to elucidate the molecular mechanism behind this interaction and its relevance to the antiviral function of bnAb 10E8. We conducted all-atom molecular dynamics simulations of three systems: (i) Fab 10E8 positioned on the surface of a viral-like lipid bilayer (VL-LB), (ii) Fab 10E8 in complex with an MPER helix anchored to the VL-LB via the Env glycoprotein transmembrane domain (TMD), and (iii) a Fab/MPER-TMD complex similarly embedded in the VL-LB but with a chemically optimized Fab 10E8 variant showing enhanced potency. Comparing these systems enabled us to derive atomic-scale Fab-membrane accommodation profiles pertinent to 10E8’s neutralizing function. Our findings support that Fab adaptation to the viral membrane interface following epitope binding is crucial for developing MPER-targeted neutralizing activity. This analysis also provides insights into pathways for strengthening lipid interactions, which may prove valuable in designing MPER-based biologics and vaccines to prevent or treat HIV infection.

Surfaces loaded with microbes can contribute to the propagation of a wide variety of pathogens. In this work, the antimicrobial potential of a hard coating (CrN) doped with different proportions of a biocide element (copper) has been studied. The coatings have been deposited by reactive high power impulse magnetron sputtering (HiPIMS) technology, on stainless steel and glass, materials used profusely in high human traffic environments. The microstructural and mechanical properties of the coatings have been evaluated, their antiviral (VSV-G) and antibacterial (S. Aureus) activities, and the durability of the antimicrobial activity, simulating tribologically two years of standard cleaning processes.It has been found that adding Cu to CrN caused some reduction in the coating hardness and Young’s modulus, but an increase in the coating adhesion. The effect on surface wettability depended on the Cu content, which caused a reduction for low Cu values (1.1 and 4.4 at. %), but and increase for larger values (11.5% at. %). All in all, the CrN-Cu coatings studied showed both antiviral and antimicrobial activity, even for low Cu content (1.1% at. %). The antimicrobial activity actually rose when the surfaces were subjected to repeated cleaning processes, indication of the robust durability in relevant practical environments.

Pore-forming peptides (PFPs) hold strong anti-tumor potential but require delivery systems to ensure stability and prevent off-target effects. In this study, we develop a strategy to transport a PFP in a biologically latent, precursor-like state. Specifically, based on the therapeutic antibody atezolizumab, which targets the tumor-associated programmed death-ligand 1, we engineered a single-chain IgG (scIgG) construct with flexible linkers embedding the melittin sequence. To further enable tumor-specific activation, melittin was flanked by regions cleavable by matriptase/ST14 (MT), a protease overexpressed in carcinomas. Initial constructs failed due to cytotoxicity during expression, prompting the redesign of melittin into a variant named Pmod2-2. This peptide retained potent pore-forming ability and was compatible with IgG fusion. The resulting scIgG-Pmod2-2 hybrid preserved Fab and Fc functionalities of the atezolizumab IgG, displayed favorable pharmacokinetics and released the active peptide in response to MT. These results highlight the potential of integrating cytolytic PFPs into antibody-based therapeutics.

Preventing microbiological surface contamination in public spaces is nowadays of high priority. The proliferation of a microbial infection may arise through air, water, or direct contact with infected surfaces. Chemical sanitization is one of the most effective approaches to avoid the proliferation of microorganisms. However, extended contact with chemicals for cleaning purposes such as chlorine, hydrogen peroxide or ethanol may lead to long-term diseases as well as drowsiness or respiratory issues, not to mention environmental issues associated to their use. As a potentially safer alternative, in the present work, the efficacy and endurance of the antimicrobial activity of different sol-gel coatings were studied, where one or two biocides were added to the coating matrix resulting on active groups exposed on the surface. Specifically, the coating formulations were synthesized by the sol-gel method. Using the alkoxide route with acid catalysis a hybrid silica-titania-methacrylate matrix was obtained where aromatic liquid eugenol was added with a double function: as a complexing agent for the chelation of the reaction precursor titanium isopropoxide, and as a biocide. In addition, 2-Phenylphenol, ECHA approved biocide, has also been incorporated to the coating matrix. The antibacterial effect of these coatings was confirmed on Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). Additionally, the coatings were non cyto-toxic and displayed virucidal activity. The coating chemical composition was characterized by 29Si NMR, and ATR-FTIR. Furthermore, the thickness and the mechanical properties were characterized by profilometry and nanoindentation, respectively. Finally, the durability of the coatings was studied with tribology tests. Overall, our data support the efficacy of the tested sol-gel coatings and suggest that added features may be required to improve endurance of the antimicrobial effects on operational conditions.

Establishment of interactions with the envelope lipids is a cardinal feature of broadly neutralizing antibodies (bnAbs) that recognize the Env membrane-proximal external region (MPER) of HIV. The lipid envelope constitutes a relevant component of the full "quinary" MPER epitope, and thus antibodies may be optimized through engineering their capacity to interact with lipids. However, the role of the chemically complex lipid nanoenvironment in the mechanism of MPER molecular recognition and viral neutralization remains poorly understood. To approach this issue, we computationally and experimentally investigated lipid interactions of broadly neutralizing antibody 10E8 and optimized versions engineered to enhance their epitope and membrane affinity by grafting bulky aromatic compounds. Our data revealed a correlation between neutralization potency and the establishment of favorable interactions with small headgroup lipids cholesterol and phosphatidylethanolamine, evolving after specific engagement with MPER. Molecular dynamics simulations of chemically modified Fabs in complex with an MPER-Transmembrane Domain helix supported the generation of a nanoenvironment causing localized deformation of the thick, rigid viral membrane and identified sphingomyelin preferentially occupying a phospholipid-binding site of 10E8. Together, these interactions appear to facilitate insertion of the Fabs through their engagement with the MPER epitope. These findings implicate individual lipid molecules in the neutralization function of MPER bnAbs, validate targeted chemical modification as a method to optimize MPER antibodies, and suggest pathways for MPER peptide-liposome vaccine development.

Direct contact with a contaminated surface or cross contamination between surfaces can contribute to the propagation of a wide variety of pathogens. The use of disinfectants often rises concerns due to the presence of hazardous chemicals, ineffectiveness against specific pathogens or induced degradation of the materials.In this work, the disinfection potential of a hard coating (CrN) doped with different proportions of a biocide element (copper) has been studied. The coatings have been deposited by reactive high power impulse magnetron sputtering (hipims) technology. The microstructural and mechanical properties of the coatings have been evaluated, as well as their antiviral (VSV-G) and antibacterial (S. Aureus) activities, and durability, simulating two years of standard cleaning processes.It has been found that adding Cu to CrN caused some reduction in the coating hardness and fracture toughness, but an increase in the coating adhesion. The effect on surface wettability depended on the Cu content, which caused a reduction for low Cu values (1.1 and 4.4 at.%), but and increase for larger values (11.5% at.%). All in all, the CrN-Cu coatings studied showed both antiviral and antimicrobial activity, even for low Cu content (1.1% at.%). Not only this, but the antimicrobial activity actually rose when the surfaces were subjected to repeated cleaning processes.

The fusion peptide (FP) and the Trp-rich membrane proximal external region (MPER) display membrane activity during HIV-1 fusion. These domains are highly conserved in the envelope glycoprotein (Env) and, consequently, antibodies targeting these regions block entry of divergent HIV strains and isolates into target cells. With the aim of recovering concurrent responses against the membrane-active Env domains, we have produced hybrid peptides that connect FP and MPER sequences via flexible aminohexanoic acid tethers, and tested their potential as immunogens. We demonstrate that liposome-based formulations containing FP-MPER hybrid peptides could elicit in rabbits, antibodies with the binding sequence specificity of neutralizing antibodies that engage with the N-terminal MPER sub-region. Determination of the thermodynamic parameters of binding using the Fab 2F5 as an N-terminal MPER antibody model, revealed that the hydrophobic interaction surface for epitope engagement appears to be optimal in the FP-MPER hybrid. In general, our data support: i) the use of liposomes as carriers for membrane active peptides; ii) the capacity of these liposome-based vaccines to focus humoral responses to N-terminal MPER epitopes; and iii) the need to include lipid membranes in immunogens to elicit such specific responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has been responsible for a global pandemic. Monoclonal antibodies (mAbs) have been used as antiviral therapeutics; however, these therapeutics have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs) and in deployment by the need for high doses. In this study, we leveraged the multi-specific, multi-affinity antibody (Multabody, MB) platform, derived from the human apoferritin protomer, to enable the multimerization of antibody fragments. MBs were shown to be highly potent, neutralizing SARS-CoV-2 at lower concentrations than their corresponding mAb counterparts. In mice infected with SARS-CoV-2, a tri-specific MB targeting three regions within the SARS-CoV-2 receptor binding domain was protective at a 30-fold lower dose than a cocktail of the corresponding mAbs. Furthermore, we showed in vitro that mono-specific MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding mAbs lose their ability to neutralize potently, and that tri-specific MBs expanded the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Subunit vaccines typically require co-administration with an adjuvant to elicit protective immunity, adding development hurdles that can impede rapid pandemic responses. To circumvent the need for adjuvant in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine, we engineer a thermostable immunotargeting vaccine (ITV) that leverages the pan-HLA-DR monoclonal antibody 44H10 to deliver the viral spike protein receptor-binding domain (RBD) to antigen-presenting cells. X-ray crystallography shows that 44H10 binds to a conserved epitope on HLA-DR, providing the basis for its broad HLA-DR reactivity. Adjuvant-free ITV immunization in rabbits and ferrets induces robust anti-RBD antibody responses that neutralize SARS-CoV-2 variants of concern and protect recipients from SARS-CoV-2 challenge. We demonstrate that the modular nature of the ITV scaffold with respect to helper T cell epitopes and diverse RBD antigens facilitates broad sarbecovirus neutralization. Our findings support anti-HLA-DR immunotargeting as an effective means to induce strong antibody responses to subunit antigens without requiring an adjuvant.

Significance The high genetic diversity of HIV-1 continues to be a major barrier to the development of therapeutics for prevention and treatment. Here, we describe the design of an antibody platform that allows assembly of a highly avid, multispecific molecule that targets, simultaneously, the most conserved epitopes on the HIV-1 envelope glycoprotein. The combined multivalency and multispecificity translates into extraordinary neutralization potency and pan-neutralization of HIV-1 strains, surpassing that of the most potent anti-HIV broadly neutralizing antibody cocktails.

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using non-specific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but have been limited in efficacy by viral sequence variability in emerging variants of concern (VOCs), and in deployment by the need for high doses. In this study, we leverage the MULTI-specific, multi-Affinity antiBODY (Multabody, MB) platform, derived from the human apoferritin protomer, to drive the multimerization of antibody fragments and generate exceptionally potent and broad SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity for the core of these engineered antibody-like molecules at 2.1 Å resolution. We demonstrate that neutralization potency improvements of the MB over corresponding IgGs translates into superior in vivo protection: in the SARS-CoV-2 mouse challenge model, comparable in vivo protection was achieved for the MB delivered at 30x lower dose compared to the corresponding IgGs. Furthermore, we show how MBs potently neutralize SARS-CoV-2 VOCs by leveraging augmented avidity, even when corresponding IgGs lose their ability to neutralize potently. Multiple mAb specificities could also be combined into a single MB molecule to expand the neutralization breadth beyond SARS-CoV-2 to other sarbecoviruses. Our work demonstrates how avidity and multi-specificity combined can be leveraged to confer protection and resilience against viral diversity that exceeds that of traditional monoclonal antibody therapies.

Antibodies against the carboxy-terminal section of the membrane-proximal external region (C-MPER) of the HIV-1 envelope glycoprotein (Env) are considered as nearly pan-neutralizing. Development of vaccines capable of producing analogous broadly neutralizing antibodies requires deep understanding of the mechanism that underlies C-MPER recognition in membranes. Here, we use the archetypic 10E8 antibody and a variety of biophysical techniques including single-molecule approaches to study the molecular recognition of C-MPER in membrane mimetics. In contrast to the assumption that an interfacial MPER helix embodies the entire C-MPER epitope recognized by 10E8, our data indicate that transmembrane domain (TMD) residues contribute to binding affinity and specificity. Moreover, anchoring to membrane the helical C-MPER epitope through the TMD augments antibody binding affinity and relieves the effects exerted by the interfacial MPER helix on the mechanical stability of the lipid bilayer. These observations support that addition of TMD residues may result in more efficient and stable anti-MPER vaccines.

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

The COVID-19 pandemic has spurred interest in potent and thermostable SARS-CoV-2 vaccines. Here, we assess low-dose immunization with lyophilized nanoparticles decorated with recombinant SARS-CoV-2 antigens. The SARS-CoV-2 Spike glycoprotein or its receptor-binding domain (RBD; mouse vaccine dose, 0.1 μg) was displayed on liposomes incorporating a particle-inducing lipid, cobalt porphyrin-phospholipid (dose, 0.4 μg), along with monophosphoryl lipid A (dose, 0.16 μg) and QS-21 (dose, 0.16 μg). Following optimization of lyophilization conditions, Spike or RBD-decorated liposomes were effectively reconstituted and maintained conformational capacity for binding human angiotensin-converting enzyme 2 (hACE2) for at least a week when stored at 60°C in lyophilized but not liquid format. Prime-boost intramuscular vaccination of hACE2-transgenic mice with the reconstituted vaccine formulations induced effective antibody responses that inhibited RBD binding to hACE2 and neutralized pseudotyped and live SARS-CoV-2. Two days following viral challenge, immunized transgenic mice cleared the virus and were fully protected from lethal disease.

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC 50 ) values as low as 9 × 10 − 14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.

Broadly neutralizing antibodies (bnAbs) against HIV-1 are frequently associated with the presence of autoreactivity/polyreactivity, a property that can limit their use as therapeutic agents. The bnAb 4E10, targeting the conserved Membrane proximal external region (MPER) of HIV-1, displays almost pan-neutralizing activity across globally circulating HIV-1 strains but exhibits nonspecific off-target interactions with lipid membranes. The hydrophobic apex of the third complementarity-determining region of the heavy chain (CDRH3) loop, which is essential for viral neutralization, critically contributes to this detrimental effect. Here, we have replaced the aromatic/hydrophobic residues from the apex of the CDRH3 of 4E10 with a single aromatic molecule through chemical modification to generate a variant that preserves the neutralization potency and breadth of 4E10 but with reduced autoreactivity. Collectively, our study suggests that the localized accumulation of aromaticity by chemical modification provides a pathway to ameliorate the adverse effects triggered by the CDRH3 of anti-HIV-1 MPER bnAbs.

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.