Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.

Paragraph-antibody paratope prediction using graph neural networks with minimal feature vectors.

SUMMARY: The development of new vaccines and antibody therapeutics typically takes several years and requires over $1bn in investment. Accurate knowledge of the paratope (antibody binding site) can speed up and reduce the cost of this process by improving our understanding of antibody-antigen binding. We present Paragraph, a structure-based paratope prediction tool that outperforms current state-of-the-art tools using simpler feature vectors and no antigen information. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at www.github.com/oxpig/Paragraph. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Self-assembling protein nanoparticles and virus like particles correctly display ß-barrel from meningococcal factor H-binding protein through genetic fusion.

Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal ß-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the ß-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.

Structural characterization of a cross-protective natural chimera of factor H binding protein from meningococcal serogroup B strain NL096.

Invasive meningococcal disease can cause fatal sepsis and meningitis and is a global health threat. Factor H binding protein (fHbp) is a protective antigen included in the two currently available vaccines against serogroup B meningococcus (MenB). FHbp is a remarkably variable surface-exposed meningococcal virulence factor with over 1300 different amino acid sequences identified so far. Based on this variability, fHbp has been classified into three variants, two subfamilies or nine modular groups, with low degrees of cross-protective activity. Here, we report the crystal structure of a natural fHbp cross-variant chimera, named variant1-2,3.x expressed by the MenB clinical isolate NL096, at 1.2 Å resolution, the highest resolution of any fHbp structure reported to date. We combined biochemical, site-directed mutagenesis and computational biophysics studies to deeply characterize this rare chimera. We determined the structure to be composed of two adjacent domains deriving from the three variants and determined the molecular basis of its stability, ability to bind Factor H and to adopt the canonical three-dimensional fHbp structure. These studies guided the design of loss-of-function mutations with potential for even greater immunogenicity. Moreover, this study represents a further step in the understanding of the fHbp biological and immunological evolution in nature. The chimeric variant1-2,3.x fHbp protein emerges as an intriguing cross-protective immunogen and suggests that identification of such naturally occurring hybrid proteins may result in stable and cross-protective immunogens when seeking to design and develop vaccines against highly variable pathogens.

Convergent structural features of respiratory syncytial virus neutralizing antibodies and plasticity of the site V epitope on prefusion F.

Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts.

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate.

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ∼70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ∼50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from -80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at -20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.

Biomedical Applications of Lumazine Synthase.

Lumazine synthase (LS) is a family of enzyme involved in the penultimate step in the biosynthesis of riboflavin. Its enzymatic mechanism has been well defined, and many LS structures have been solved using X-ray crystallography or cryoelectron microscopy. LS is composed of homooligomers, which vary in size and subunit number, including pentamers, decamers, and icosahedral sixty-mers, depending on its species of origin. Research on LS has expanded beyond the initial focus on its enzymatic function to properties related to its oligomeric structure and exceptional conformational stability. These attributes of LS systems have now been repurposed for use in various biomedical fields. This review primarily focuses on the applications of LS as a flexible vaccine presentation system. Presentation of antigens on the surface of LS results in a high local concentration of antigens displayed in an ordered array. Such repetitive structures enable the cross-linking of B-cell receptors and result in strong immune responses through an avidity effect. Potential issues with the use of this system and corresponding solutions are also discussed with the objective of improved utilization of the LS system in vaccine development.

Effect of Phosphate Ion on the Structure of Lumazine Synthase, an Antigen Presentation System From Bacillus anthracis.

Lumazine synthase (LS) is an oligomeric enzyme involved in the biosynthesis of riboflavin in microorganisms, fungi, and plants. LS has become of significant interest to biomedical science because of its critical biological role and attractive structural properties for antigen presentation in vaccines. LS derived from Bacillus anthracis (BaLS) consists of 60 identical subunits forming an icosahedron. Its crystal structure has been solved, but its dynamic conformational properties have not yet been studied. We investigated the conformation of BaLS in response to different stress conditions (e.g., chemical denaturants, pH, and temperature) using a variety of biophysical techniques. The physical basis for these thermal transitions was studied, indicating that a molten globular state was present during chemical unfolding by guanidine HCl. In addition, BaLS showed 2 distinct thermal transitions in phosphate-containing buffers. The first transition was due to the dissociation of phosphate ions from BaLS and the second one came from the dissociation and conformational alteration of its icosahedral structure. A small conformational alteration was induced by the binding/dissociation of phosphate ions to BaLS. This work provides a closer view of the conformational behavior of BaLS and provides important information for the formulation of vaccines which use this protein.

Evaluation of lumazine synthase from Bacillus anthracis as a presentation platform for polyvalent antigen display.

Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array-like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers. We then investigated the effects of linker length, linker rigidity and formaldehyde crosslinking on the protein assembly, conformational integrity, thermal stability, in vitro antibody binding, and immunogenicity in mice. Fusion of the PB10 peptide onto LS, with varying linker lengths, did not affect protein assembly, thermal stability or exposure of the epitope, but had a minor impact on protein conformation. Formaldehyde crosslinking considerably improved protein thermal stability with only minor impact on protein conformation. All LS_PB10 constructs, when administered to mice by injection without adjuvant, elicited measurable anti-ricin serum IgG titers, although the titers were not sufficient to confer protection against a 10× lethal dose ricin challenge. This work sheds light on the biophysical properties, immunogenicity and potential feasibility of LS from B. anthracis as a scaffold system for polyvalent antigen display.

Effects of Protein Conformation, Apparent Solubility, and Protein-Protein Interactions on the Rates and Mechanisms of Aggregation for an IgG1Monoclonal Antibody.

Non-native protein aggregation is a key degradation pathway of immunoglobulins. In this work, the aggregation kinetics of an immunoglobulin gamma-1 monoclonal antibody (IgG1 mAb) in different solution environments was monitored over a range of incubation temperatures for up to seven months using size exclusion chromatography. Histidine and citrate buffers with/without sodium chloride were employed to modulate the mAb's conformational stability, solubility (in the presence of polyethylene glycol, PEG), and protein-protein interactions as measured by differential scanning calorimetry, PEG precipitation, and static light scattering, respectively. The effect of these parameters on the mechanism(s) of mAb aggregation during storage at different temperatures was determined using kinetic models, which were used to fit aggregation data to determine rate constants for aggregate nucleation and growth processes. This approach was used to investigate the effects of colloidal protein-protein interactions and solubility values (in PEG solutions) on the mechanisms and rates of IgG1 mAb aggregation as a function of temperature-induced structural perturbations. Aggregate nucleation and growth pathways for this IgG1 mAb were sensitive to temperature and overall conformational stability. Aggregate growth, on the other hand, was also sensitive to conditions affecting the solubility of the mAb, particularly at elevated temperatures.

Formulation Studies During Preclinical Development of Influenza Hemagglutinin and Virus-Like Particle Vaccine Candidates.

A critical element of vaccine formulation studies is the identification of chemical and physical degradation pathways that compromise structural integrity, and which may in turn affect the clinical safety and efficacy, of macromolecular antigens. Formulation development helps optimize and maintain the long-term storage stability and viability of vaccine antigens in pharmaceutically relevant dosage forms. The protocols presented in this manuscript highlight the use of accelerated stability studies for the formulation of influenza vaccine candidates including virus-like particles (VLP) and particle forming hemagglutinin (HA) antigens. Three case studies, each targeting a different facet of preclinical vaccine formulation development, are reviewed: (1) excipient screening experiments to mitigate VLP physical degradation, (2) methods for monitoring a specific chemical perturbation of the recombinant HA antigen and elucidating its effect on in vitro potency, and (3) maintaining HA conformational stability in the presence of freeze-thaw and freeze-drying stresses.

Production of Well-Characterized Virus-like Particles in an Escherichia coli-Based Expression Platform for Preclinical Vaccine Assessments.

In this chapter we demonstrate a method to produce virus-like particles (VLPs) from Escherichia coli. Standard bacterial protocols are used for the cloning, transformation, and expression of the protein subunits. A two-step protein purification method is highlighted: one step based on separating soluble proteins with ion-exchange affinity chromatography and a second polishing step using size-exclusion columns to isolate VLP species. The ensuing VLPs can be characterized with a variety of biophysical techniques including ultraviolet (UV)-visible spectroscopy for protein quantification, dynamic light scattering for size distribution determination, and transmission electron microscopy to ascertain size and morphology.

Novel Ricin Subunit Antigens With Enhanced Capacity to Elicit Toxin-Neutralizing Antibody Responses in Mice.

RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human phase I clinical trials. In this study, we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability-enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that 2 RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5- to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody titers. Increased toxin neutralizing antibody values and seroconversion rates were evident at different antigen dosages and within 7 days after the first booster. Quantitative stability/flexibility relationships analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98-103, which constitutes a known immunodominant neutralizing B-cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity.

Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles.

Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications.

Conformation-specific display of 4E10 and 2F5 epitopes on self-assembling protein nanoparticles as a potential HIV vaccine.

The self-assembling protein nanoparticle (SAPN) is an antigen-presenting system that has been shown to be suitable for use as a vaccine platform. The SAPN scaffold is based on the principles of icosahedral symmetry, beginning from a monomeric chain that self-assembles into an ordered oligomeric state. The monomeric chain contains two covalently linked α-helical coiled-coil domains, an N-terminal de novo-designed pentameric tryptophan zipper and a C-terminal de novo-designed trimeric leucine zipper, which assemble along the internal symmetry axes of an icosahedron. In this study, we incorporated the membrane proximal external region (MPER) of HIV-1 gp41 from HXB2 into the N-terminal pentamer, referred to as MPER-SAPN, attempting to reproduce the α-helical state of the 4E10 epitope while maintaining a structurally less-constrained 2F5 epitope. Sprague-Dawley rats were immunized with MPER-SAPNs, and their sera were analyzed for induced humoral anti-HIV-1 responses. We show that high membrane proximal external region-specific titers can be raised via the repetitive antigen display of MPER on the SAPN without the need for adjuvant. However, none of the sera displayed a detectable neutralizing activity against HIV-1. Thus, 4E10- and 2F5-like neutralizing antibodies could not be elicited by MPER conformationally restrained in the SAPN context.