Enterovirus D68 (EV-D68) is a picornavirus that causes biennial outbreaks of respiratory disease in young children that can progress to rare but severe complications, including acute flaccid myelitis (AFM). EV-D68 virus-like particles (VLPs) elicit potent neutralizing antibodies that are protective in animal models. Here, we report the isolation and characterization of monoclonal antibodies elicited by EV-D68 VLPs in nonhuman primates (NHPs). We identified five potently neutralizing mAbs targeting overlapping epitopes near the capsid fivefold axis of symmetry formed by pentamers of viral protein 1. Cryo-electron microscopy structures of mAbs 1E11 and 5H03 in complex with VLP revealed epitopes on the capsid that bridge the sialic acid binding site and the proteinaceous entry receptor major facilitator superfamily domain-containing protein 6 binding site. We further characterized the mechanisms by which these mAbs neutralize EV-D68 and found that mAbs can disrupt multiple steps in the EV-D68 life cycle, including promoting premature uncoating. Antibodies elicited by VLP immunization of NHP protected as well as a best-in-class human mAb in a mouse challenge model, although single-amino acid mutations in the capsid enabled viral escape. Our results demonstrate that EV-D68 VLP-elicited mAbs target major viral sites of vulnerability, provide insight into their mechanisms of neutralization, and reinforce the potential for VLP-based vaccines as a countermeasure for EV-D68.

Viral entry glycoproteins are often shielded from immune recognition by dense N-linked glycans that limit antibody access to protein epitopes. While glycan-reactive antibodies with unusual architectures have been described, how canonical Y-shaped antibodies engage these glycan-rich surfaces remains poorly defined. Here, we characterize two human antibodies, VRC35 and VRC36, isolated from an HIV-1-infected donor, that recognize diverse glycosylated viral glycoproteins. Cryo-electron microscopy structural analyses of these antibodies in complex with viral entry glycoproteins, including HIV-1 envelope, influenza hemagglutinin, SARS-CoV-2 spike, and the Lassa virus glycoprotein complex, reveal adaptive Fab stoichiometries ranging from single-Fab binding to dimeric and higher-order assemblies are mediated by intra- and inter-IgG interactions that depend on local glycan organization. Dense glycan clustering on HIV-1 and influenza glycoproteins supports multivalent Fab assemblies and correlates with neutralization activity, whereas sparse glycan environments on SARS-CoV-2 and Lassa virus favor weak or heterogeneous engagement without neutralization. Structural and mutational analyses further demonstrate that homotypic Fab-Fab interactions stabilize multivalent engagement and contribute to neutralizing activity. Together, these findings define a structural framework in which viral glycan organization constrains antibody valency and engagement, while somatic hypermutation contributes to the acquisition of homotypic Fab-Fab interactions that facilitate multivalent recognition of viral glycan shields.

Background/Objectives: Vaccine elicitation of antibodies with high HIV-1 neutralization breadth is a long-standing goal. Recently, the induction of such antibodies has been achieved at the fusion peptide site of vulnerability. Questions remain, however, as to how much anti-fusion peptide antibodies can be improved and whether their neutralization breadth and potency are sufficient to prevent HIV-1 infection. Methods: Here, we use yeast display coupled with deep mutational screening and biochemical and structural analyses to study the improvement of the best fusion peptide-directed, vaccine-elicited antibody, DFPH_a.01, with an initial 59% breadth. Results: Yeast display identified both single and double mutations that improved recognition of HIV-1 envelope trimers. We characterized two paratope-distal light chain (LC) mutations, S10R and S59P, which together increased breadth to 63%. Biochemical analysis demonstrated DFPH-a.01_10R59P-LC, and its component mutations, to have increased affinity and stability. Cryo-EM structural analysis revealed elbow-angle influencing by S10R-LC and isosteric positioning by S59P-LC as explanations for enhanced breadth, affinity, and stability. Conclusions: These results, along with another antibody with enhanced performance (DFPH-a.01_1G10A56K-LC with 64% breadth), suggest that mutations improving DFPH_a.01 are plentiful, an important vaccine insight.

Snake envenomation is a neglected tropical disease, with 600 species causing over 100,000 deaths and 300,000 permanent disabilities in humans annually. Broadly neutralizing antibodies and broad chemical inhibitors have been proposed as solutions, but how to develop a therapeutically effective cocktail and the number of required components have been unclear. To address this gap, we iteratively recovered two broadly neutralizing antivenom antibodies from the memory B cells of a hyperimmune human donor with extensive snake venom exposure. The antibodies recognized conserved neutralizing epitopes on prevalent long and short snake neurotoxins, with crystal structures revealing antibody mimicry of the interfaces between these neurotoxins and their host target, the nicotinic acetylcholine receptor. We combined and tested these antibodies and the phospholipase inhibitor varespladib. A 3-component cocktail rescued animals from whole-venom challenge of all species in a 19-member WHO Category 1 and Category 2 elapid diversity set, with complete protection against most snakes observed.

Enterovirus D68 (EV-D68), a member of the Picornaviridae family, causes respiratory illness and can lead to acute flaccid myelitis in children. No specific treatment or vaccine is available. Here, we determine cryo-EM structures of EV-D68 virus-like particles (VLPs), inactivated virus particles (InVPs), and altered virus particles (A-particles) from B3 and A2 subclades. The B3 VLP is a current vaccine candidate, which we show closely resembles its InVP counterpart, particularly at the 5-fold axis of symmetry, the target of potent neutralizing antibodies. Similar structural conservation was observed in the A2 subclade. Sequence variation between B3 and A2 mainly occurred in flexible loops displayed on the particle surface. A canyon-filling pocket factor was present in B3 InVP but absent in A2 InVP. A-particles were predominant in β-propiolactone-inactivated virus at longer but not shorter incubation. Overall, our findings highlight EV-D68 similarities and subclade-specific differences, offering structural insights that relate to vaccine development.

Venezuelan equine encephalitis virus (VEEV) is an arbovirus that causes a disease in which 4%-14% of individuals can develop neurological symptoms. Prior to 1970, VEEV was developed as a biological threat agent due to its stability and high morbidity when administered by aerosol. Currently, no FDA-licensed vaccines nor therapeutics for VEEV exist. Single-domain antibodies (sdAbs) may provide a therapeutic option due to their small size and ability to bind recessed epitopes not recognized by conventional antibodies. This study identified two bivalent sdAbs that were able to protect mice from a lethal challenge against both epizootic and enzootic subtypes of VEEV. Cryo-EM structures of sdAb-VEEV complexes revealed the sdAbs that comprised the bivalent sdAbs to recognize a mixture of conserved and non-conserved regions of the VEEV envelope proteins. While all three of the cryo-EM-characterized epitopes were unique in terms of their recognized VEEV residues, two sdAbs, V2B3 and V2C3, overlapped sterically, explaining why only their combinations with the non-sterically overlapping sdAb V3A8f, which composed the bivalent sdAbs described here, were so particularly effective. Binding and neutralization studies found that the bivalent sdAbs have the potential to be broad-spectrum anti-alphavirus therapeutics as they cross-neutralize multiple alphaviruses.IMPORTANCEAlphaviruses are no longer geographically constrained to one region of the world but are expanding to be of global concern. In many regions of the world, multiple alphaviruses co-circulate; therefore, having a therapeutic that is pan-alphavirus is important. A cocktail of multiple pan-alphavirus binding/neutralizing antibodies (Abs) may provide optimal coverage against alphaviruses while decreasing the prevalence of viral escape mutants, which could cause the therapeutic to no longer be efficacious. Structures of these Abs, defining their recognition, could assist in identifying optimal combinations. A bivalent pan-alphavirus single-domain antibody could be used in a cocktail with already identified alphavirus IgG antibodies.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] similar to 100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50] < 50 mu g/mL) and total lineage-concentrations estimates of 50-200 mu g/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

Applying rational design, we developed 17 kDa cyanobacteriochrome-based near-infrared (NIR-I) fluorescent protein, miRFP718nano. miRFP718nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues. miRFP718nano has maximal emission at 718 nm and an emission tail in the short-wave infrared (SWIR) region, allowing deep-penetrating off-peak fluorescence imaging in vivo. The miRFP718nano structure reveals the molecular basis of its red shift. We demonstrate superiority of miRFP718nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract, mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.66 ns) and exceptional fluorescence brightness, was designed for FLIM experiments. Here, we present the X-ray structure and discuss the structure-functional relations of BrUSLEE. Its development from the EGFP (enhanced green fluorescent proteins) precursor (FL~2.83 ns) resulted in a change of the chromophore microenvironment due to a significant alteration in the side chain conformations. To get further insight into molecular details explaining the observed differences in the photophysical properties of these proteins, we studied their structural, dynamic, and electric properties by all-atom molecular-dynamics simulations in an aqueous solution. It has been shown that compared to BrUSLEE, the mobility of the chromophore in the EGFP is noticeably limited by nonbonded interactions (mainly H-bonds) with the neighboring residues.

We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities.

Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. By exploring miRFP670nano3 as an internal tag, we engineered 32 kDa NIR fluorescent nanobodies, termed NIR-Fbs, whose stability and fluorescence strongly depend on the presence of specific intracellular antigens. NIR-Fbs allowed background-free visualization of endogenous proteins, detection of viral antigens, labeling of cells expressing target molecules and identification of double-positive cell populations with bispecific NIR-Fbs against two antigens. Applying NIR-Fbs as destabilizing fusion partners, we developed molecular tools for directed degradation of targeted proteins, controllable protein expression and modulation of enzymatic activities. Altogether, NIR-Fbs enable the detection and manipulation of a variety of cellular processes based on the intracellular protein profile.

Snake envenomation is a neglected tropical disease, causing >100,000 deaths and 300,000 permanent disabilities in humans annually. Could monoclonal antibody technology provide a solution? Here, we recover Centi-3FTX-D09, a potent broadly neutralizing antivenom antibody from the B-cell memory of a human subject with snake venom exposure. Centi-3FTX-D09 recognized a conserved neutralizing epitope on long 3-finger toxins (3FTXs), a dominant snake neurotoxin. Crystal structures of Centi-3FTX-D09 in complex with 3FTXs from mamba, taipan, krait, and cobra revealed epitope mimicry of the interface between these neurotoxins and their host target, the nicotinic acetylcholine receptor. Centi-3FTX-D09 provided in-vivo protection against diverse recombinant long 3FTXs, in-vivo rescue from whole venom challenge from cobras, black mamba, and king cobra, and, when combined with the phospholipase inhibitor varespladib, in-vivo protection extending to a majority of tested elapid venoms. Thus, a single antibody can broadly neutralize long neurotoxins and contribute to broad protection from envenomation.

For the whole GFP family, a few cases, when a single mutation in the chromophore environment strongly inhibits maturation, were described. Here we study EYFP-F165G - a variant of the enhanced yellow fluorescent protein - obtained by a single F165G replacement, and demonstrated multiple fluorescent states represented by the minor emission peaks in blue and yellow ranges (similar to 470 and similar to 530 nm), and the major peak at similar to 330 nm. The latter has been assigned to tryptophan fluorescence, quenched due to excitation energy transfer to the mature chromophore in the parental EYFP protein. EYFP-F165G crystal structure revealed two general independent routes of post-translational chemistry, resulting in two main states of the polypeptide chain with the intact chromophore forming triad (similar to 85%) and mature chromophore (similar to 15%). Our experiments thus highlighted important stereochemical role of the 165th position strongly affecting spectral characteristics of the protein. On the basis of the determined EYFP-F165G three-dimensional structure, new variants with similar to 2-fold improved brightness were engineered. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

The crystal structure of monomeric red fluorescent protein FusionRed (λex/λem 580/608 mn) has been determined at 1.09 Å resolution and revealed two alternative routes of post-translational chemistry, resulting in distinctly different products. The refinement occupancies suggest the 60:40 ratio of the mature Met63-Tyr64-Gly65 chromophore and uncyclized chromophore-forming tripeptide with the protein backbone cleaved between Met63 and the preceding Phe62 and oxidized Cα-Cβ bond of Tyr64. We analyzed the structures of FusionRed and several related red fluorescent proteins, identified structural elements causing hydrolysis of the peptide bond, and verified their impact by single point mutagenesis. These findings advance the understanding of the post-translational chemistry of GFP-like fluorescent proteins beyond the canonical cyclization-dehydration-oxidation mechanism. They also show that impaired cyclization does not prevent chromophore-forming tripeptide from further transformations enabled by the same set of catalytic residues. Our mutagenesis efforts resulted in inhibition of the peptide backbone cleavage, and a FusionRed variant with ~30% improved effective brightness.

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.

Super-resolution fluorescent imaging in living cells remains technically challenging, largely due to the photodecomposition of fluorescent tags. The recently suggested protein-PAINT is the only super-resolution technique available for prolonged imaging of proteins in living cells. It is realized with complexes of fluorogen-activating proteins, expressed as fusions, and solvatochromic synthetic dyes. Once photobleached, the dye in the complex is replaced with a fresh fluorogen available in the sample. With suitable kinetics, this replacement creates fluorescence blinking required for attaining super-resolution and overcomes photobleaching associated with the loss of an irreplaceable fluorophore. Here we report on the rational design of two protein-PAINT tags based on the 1.58 Å crystal structure of the DiB1:M739 complex, an improved green-emitting DiB3/F74V:M739 and a new orange-emitting DiB3/F53L:M739. They outperform previously reported DiB-based tags to become best in class biomarkers for protein-PAINT. The new tags advance protein-PAINT from the proof-of-concept to a reliable tool suitable for prolonged super-resolution imaging of intracellular proteins in fixed and living cells and two-color PAINT-like nanoscopy with a single fluorogen.

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35–Wat–Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis.

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.