Antigen Conformation Research Articles

MHC-independent αβT cells: Lessons learned about thymic selection and MHC-restriction.

Understanding the generation of an MHC-restricted T cell repertoire is the cornerstone of modern T cell immunology. The unique ability of αβT cells to only recognize peptide antigens presented by MHC molecules but not conformational antigens is referred to as MHC restriction. How MHC restriction is imposed on a very large T cell receptor (TCR) repertoire is still heavily debated. We recently proposed the selection model, which posits that newly re-arranged TCRs can structurally recognize a wide variety of antigens, ranging from peptides presented by MHC molecules to native proteins like cell surface markers. However, on a molecular level, the sequestration of the essential tyrosine kinase Lck by the coreceptors CD4 and CD8 allows only MHC-restricted TCRs to signal. In the absence of Lck sequestration, MHC-independent TCRs can signal and instruct the generation of mature αβT cells that can recognize native protein ligands. The selection model thus explains how only MHC-restricted TCRs can signal and survive thymic selection. In this review, we will discuss the genetic evidence that led to our selection model. We will summarize the selection mechanism and structural properties of MHC-independent TCRs and further discuss the various non-MHC ligands we have identified.

Mucosal Delivery of HIV-1 Glycoprotein Vaccine Candidate Enabled by Short Carbon Nanotubes.

The HIV-1 envelope glycoprotein spike is the target of antibodies, and therefore represents the main viral antigen for antibody-based vaccine design. One of the challenges in HIV-1 vaccine development is finding efficient ways for the immune system to recognize and respond to HIV-1 without establishing an infection. Since HIV-1 enters the body at mucosal surfaces, induction of immune response at these sites is a preferred preventive approach. Nasal administration is a very effective route for mucosal immunization since it can stimulate mucosal immune responses both locally and distantly. In this paper, Luna develops a safe, short carbon nanotube (CNT)-based, needle-free delivery platform known as "CNTVac". The size of short CNT was controlled to possess HIV-1 particle-like morphology (100-200 nm) capable of efficiently delivering a broad range of antigens intranasally. PEG-Lipid served as the antigen conformation protector and mucosal barrier penetration enhancer (Schematic Figure) was localized between V1V2 antigens, which caused highly enhanced local IgA and systemic antibody IgG responses in mice and rabbits. The short CNT incorporated with PEG-Lipid could not only serve as efficient delivery system but also reduce the amount of lipid usage in order to balance the vaccine dosage in order to eliminate the potential adverse effect. These data suggest a promising platform technology for vaccine delivery.

Viral vectors expressing group B meningococcal outer membrane proteins induce strong antibody responses but fail to induce functional bactericidal activity

Adenoviral vectored vaccines, with the appropriate gene insert, induce cellular and antibody responses against viruses, parasites and intracellular pathogens such as Mycobacterium tuberculosis. Here we explored their capacity to induce functional antibody responses to meningococcal transmembrane outer membrane proteins. Vectors expressing porin A and ferric enterobactin receptor A antigens were generated, and their immunogenicity assessed in mice using binding and bactericidal assays. The viral vectors expressed the bacterial proteins in an in vitro cell-infection assay and, after immunisation of mice, induced higher titres (>105 end-point titre) and longer lasting (>32 weeks) transgene-specific antibody responses in vivo than did outer membrane vesicles containing the same antigens. However, bactericidal antibodies, which are the primary surrogate of protection against meningococcus, were undetectable, despite different designs to support the presentation of the protective B-cell epitopes. These results demonstrate that, while the transmembrane bacterial proteins expressed by the viral vector induced strong and persistent antigen-specific antibodies, this platform failed to induce bactericidal antibodies. The results suggest that conformation or post-translational modifications of bacterial outer membrane antigens produced in eukaryote cells might not result in presentation of the necessary epitopes for induction of functional antibodies.

An Integrated Platform for Serological Detection and Vaccination of COVID-19.

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is an ongoing pandemic. Detection and vaccination are essential for disease control, but they are distinct and complex operations that require significant improvements. Here, we developed an integrated detection and vaccination system to greatly simplify these efforts. We constructed recombinant baculoviruses to separately display the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2. Insect cells infected by the recombinant baculoviruses were used to generate a cell-based system to accurately detect patient serum. Notably, although well-recognized by our newly developed detection system in which S-displaying insect cells acted as antigen, anti-S antibodies from many patients were barely detectable by Western blot, evidencing that COVID-19 patients primarily produce conformation-dependent anti-S antibodies. Furthermore, the same baculovirus constructs can display N (N-Bac) or S (S-Bac) on the baculovirus envelope and serve as vector vaccines. Animal experiments show that S-Bac or N-Bac immunization in mice elicited a strong and specific antibody response, and S-Bac in particular stimulated effective neutralizing antibodies without the need for adjuvant. Our integrated system maintains antigen conformation and membrane structure to facilitate serum detection and antibody stimulation. Thus, compared with currently available technologies, our system represents a simplified and efficient platform for better SARS-CoV-2 detection and vaccination.

Paratope states in solution improve structure prediction and docking

Structure-based antibody design and accurate predictions of antibody-antigen interactions remain major challenges in computational biology. By using molecular dynamics simulations, we show that a single static X-ray structure is not sufficient to identify determinants of antibody-antigen recognition. Here, we investigate antibodies that undergo substantial conformational changes upon antigen binding and have been classified as difficult cases in an extensive benchmark for antibody-antigen docking. We present thermodynamics and transition kinetics of these conformational rearrangements and show that paratope states can be used to improve antibody-antigen docking. By using the unbound antibody X-ray structure as starting structure for molecular dynamics simulations, we retain a binding competent conformation substantially different to the unbound antibody X-ray structure. We also observe that the kinetically dominant antibody paratope conformations are chosen by the bound antigen conformation with the highest probability. Thus, we show that paratope states in solution can improve antibody-antigen docking and structure prediction.

Visualization of early oligomeric α-synuclein pathology and its impact on the dopaminergic system in the (Thy-1)-h[A30P]α-syn transgenic mouse model.

Aggregation of alpha-synuclein (α-syn) into Lewy bodies and Lewy neurites is a pathological hallmark in the Parkinson´s disease (PD) brain. The formation of α-syn oligomers is believed to be an early pathogenic event and the A30P mutation in the gene encoding α-syn, causing familial PD, has been shown to cause an accelerated oligomerization. Due to the problem of preserving antigen conformation on tissue surfaces, α-syn oligomers are difficult to detect ex vivo using conventional immunohistochemistry with oligomer-selective antibodies. Herein, we have instead employed the previously reported α-syn oligomer proximity ligation assay (ASO-PLA), along with a wide variety of biochemical assays, to discern the pathological progression of α-syn oligomers and their impact on the dopaminergic system in male and female (Thy-1)-h[A30P]α-syn transgenic (A30P-tg) mice. Our results reveal a previously undetected abundance of α-syn oligomers in midbrain of young mice, whereas phosphorylated (pS129) and proteinase k-resistant α-syn species were observed to a larger extent in aged mice. Although we did not detect loss of dopaminergic neurons in A30P-tg mice, a dysregulation in the monoaminergic system was recorded in older mice. Taken together, ASO-PLA should be a useful method for the detection of early changes in α-syn aggregation on brain tissue, from experimental mouse models in addition to post mortem PD cases.

The Identification of the Amino Acids on Platelet Factor 4 That Bind Pathogenic Antibodies in Heparin-Induced Thrombocytopenia

Introduction: Heparin induced thrombocytopenia (HIT) is an adverse drug reaction that occurs when heparin binds to platelet factor 4 (PF4) and forms immunogenic multimolecular complexes. As a result, anti-PF4/heparin IgG antibodies bind to the PF4/heparin complexes, leading to cross-linking of FcγIIa receptors on platelets and FcγRI on monocytes resulting in their activation and an increased risk of thrombosis. Approximately 50% of cardiac surgery patients produce anti-PF4/heparin antibodies, but only a subset (<1%) produces pathogenic HIT antibodies. Current enzyme immunoassays (EIAs) cannot distinguish between pathogenic and non-pathogenic anti-PF4/heparin antibodies and will give a positive test even in the presence of the clinically insignificant non-pathogenic HIT antibodies. Further functional testing, such as the 14C-serotonin release assay (SRA), is required to identify samples containing the pathogenic anti-PF4/heparin antibodies that will lead to HIT. In this study, we hypothesized that different epitopes on PF4 could explain some of the differences between pathogenic and non-pathogenic antibodies.Methods: We used alanine scanning mutagenesis (ASM) to produce 70 mutations of PF4, each with a unique amino acid substitution. Single point mutations were made by mutating non-alanine residues to alanine, and alanine residues to valine. The PF4 mutants were isolated from Escherichia coli and used in an EIA. The binding capacity of the monoclonal pathogenic HIT antibody (KKO) and a non-pathogenic anti-PF4/heparin antibody (RTO) to each of the PF4 mutants, relative to wild-type PF4, was determined. Point mutations of PF4 that resulted in >25% loss of binding were identified. We compared our candidate amino acids to binding sites previously identified in the PF4 and KKO crystal structure. In addition, we screened nine EIA-positive/SRA-positive (pathogenic) and six EIA-positive/SRA-negative (non-pathogenic) patient samples using the PF4 mutants. Student's t-test was used to determine whether the binding capacities of each amino acid was significantly different between the two patient sample groups.Results: We identified 12 amino acid mutations of PF4 that caused ≥25% reduction in KKO binding. Most implicated amino acids cluster in two regions within the PF4 molecule. We identified amino acids on PF4 that were required for KKO binding which were not previously identified based on crystallographic studies. These results are consistent with the previous mutagenesis and crystallographic studies (Cai et al, Nat Commun. 2015;6:8277) of the KKO and PF4 complex but also contradict some previously stated amino acids that were thought to be part of this binding site. ASM of RTO and PF4 revealed only one amino acid that was affected. We used pathogenic and non-pathogenic EIA-positive human sera to further refine the location of the binding sites. We identified 10 amino acids that were required for the binding of human pathogenic HIT sera.Conclusions: Using sequential point mutations, we identified the amino acids that are critical for the binding of pathogenic HIT antibodies to PF4. These amino acids are either directly involved in antibody binding or influence the antigenic conformation of PF4. This library of PF4 mutant proteins can help differentiate pathogenic and non-pathogenic HIT antibodies and this information can improve diagnostic assays.This study was funded by the Canadian Institutes for Health Research. DisclosuresArnold:Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Dova: Consultancy; Novartis: Consultancy, Research Funding; UCB: Consultancy; Rigel: Consultancy.

Obesity is Associated with an Altered Baseline and Post-Vaccination Influenza Antibody Repertoire

As highlighted by the ongoing COVID-19 pandemic, vaccination is critical for infectious disease prevention and control. Obesity is associated with increased morbidity and mortality from respiratory virus infections. While obese individuals respond to influenza vaccination, what is considered a seroprotective response may not fully protect the global obese population. In a cohort vaccinated with the 2010-2011 trivalent inactivated influenza vaccine, baseline immune history and vaccination responses were found to significantly differ in obese individuals compared to healthy controls, especially towards the 2009 pandemic strain of A/H1N1 influenza virus. Young, obese individuals displayed responses skewed towards linear peptides versus conformational antigens, suggesting aberrant obese immune response. Overall, these data have vital implications for the next generation of influenza vaccines, and towards the current SARS-CoV-2 vaccination campaign.Funding: The National Institute for Biotechnology in the Negev (TH) Israel Science Foundation Individual Research Grant NO. 882/17 (TH) The Israel-America Foundation and the Ben-Gurion University Center for Multidisciplinary Research in Aging (TH) National Institute of Allergy and Infectious Diseases under HHS contract HHSN27220140006C (EAK and SSC) National Institute of Allergy and Infectious Diseases grant R01 NIH/NIAID/AI078090 (MAB, TLN and SSW) ALSAC (EAK and SSC)Declaration of Interest: None to declare. Ethical Approval: All procedures were approved by the Biomedical Institutional Review Board at the University of North Carolina at Chapel Hill.

Abstract P-44: SARS-CoV-2 Inactivation by Ultraviolet Light Does Not Violate Virus Morphology, Antigenic and Immunogenic Properties

Background: Vaccination is the most effective tool for reducing the morbidity and mortality of COVID-19. Particular interest is the development of whole-virion inactivated vaccines, since such vaccines include a complete set of viral structural proteins. One of the requirements for the whole-virion vaccines is the guarantee of complete virus inactivation while maintaining the native conformation of protective antigens. The aim of this study is to evaluate the effect of ultraviolet (UV) treatment on the morphology, antigenic and immunogenic properties of the SARS-CoV-2. Methods: SARS-CoV-2, strain "Dubrovka" (GenBank No.: MW514307.1), grown in Vero CCL81 cell culture (ATCC). Viral reproduction was monitored by real-time qRT-PCR, ELISA and virus titration in Vero cells. Virus was inactivated by treating with ultraviolet light for 5 minutes using a standard biosafety cabinet UV irradiator. Virus inactivation control was performed by three "blind" passages in Vero cells. Clarified viral material was concentrated on Amicon MWCO 100 kDa (Millipore) columns. Negatively stained with uranyl acetate viral preparation was examined by transmission electron microscopy (TEM). Mice were immunized with a UV-inactivated virus subcutaneously in two variants (5 animals per group) - with and without Freund's adjuvant, twice with an interval between immunizations of 2 weeks. The titer of virus-neutralizing antibodies in mouse sera was determined in Vero cell culture. Results: Preparation of the SARS-CoV-2 coronavirus with a titer of 7.5 lg TCID50/ml and concentration of viral RNA of 8.5 lg copies/ml was obtained in Vero cells. After UV-treatment the presence in the prepapation of SARS-CoV-2 antigen was confirmed by ELISA with a set of COVID-19 convalescent sera. The particles with coronavirus morphodiagnostic signs were imaged by TEM - rounded shape with characteristic spikes of 12-15 nm on the envelope, the diameter of the virion was 90-110 nm. Neutralizing antibodies were detected in the sera of all immunized mice, whereas in animals of the control group neutralizing antibodies were not detected. Neutralizing antibodies titer was significantly higher in animals immunized by a virus with Freund's adjuvant - on average 448, than without adjuvant - 64 (p&lt;0.01). Conclusion: Treatment of SARS-CoV-2 by UV light completely inactivates its infectivity, while retaining the typical coronavirus virions morphology, antigenic properties, and ability to induce in mice a synthesis of neutralizing antibodies.

Tolerogenic Immunotherapy: Targeting DC Surface Receptors to Induce Antigen-Specific Tolerance.

Dendritic cells (DCs) are well-established as major players in the regulation of immune responses. They either induce inflammatory or tolerogenic responses, depending on the DC-subtype and stimuli they receive from the local environment. This dual capacity of DCs has raised therapeutic interest for their use to modify immune-activation via the generation of tolerogenic DCs (tolDCs). Several compounds such as vitamin D3, retinoic acid, dexamethasone, or IL-10 and TGF-β have shown potency in the induction of tolDCs. However, an increasing interest exists in defining tolerance inducing receptors on DCs for new targeting strategies aimed to develop tolerance inducing immunotherapies, on which we focus particular in this review. Ligation of specific cell surface molecules on DCs can result in antigen presentation to T cells in the presence of inhibitory costimulatory molecules and tolerogenic cytokines, giving rise to regulatory T cells. The combination of factors such as antigen structure and conformation, delivery method, and receptor specificity is of paramount importance. During the last decades, research provided many tools that can specifically target various receptors on DCs to induce a tolerogenic phenotype. Based on advances in the knowledge of pathogen recognition receptor expression profiles in human DC subsets, the most promising cell surface receptors that are currently being explored as possible targets for the induction of tolerance in DCs will be discussed. We also review the different strategies that are being tested to target DC receptors such as antigen-carbohydrate conjugates, antibody-antigen fusion proteins and antigen-adjuvant conjugates.

Challenge Accepted – Paratope States in Solution Improve Structure Prediction and Docking

The rise of antibodies as biotherapeutic proteins has attracted the interest in characterizing and understanding the antibody binding interface. Structure-based antibody design and accurate predictions of antibody-antigen interactions remain major challenges in computational biology. By using molecular dynamics simulations in combination with enhanced sampling techniques, we show that a single static X-ray structure is not sufficient to identify determinants of antibody recognition and to characterize functionally relevant conformational rearrangements. Thus, we suggest that antibodies should be described as ensembles of paratope states in solution, which are defined by a combination of correlated loop movements, which favor distinct interface orientations and elbow angles. In this study, we focus our attention on characterizing paratope states in solution of antibodies which undergo substantial conformational changes upon antigen binding. Because of their conformational changes, they have been classified as “difficult” cases for antibody-antigen docking in the Critical Assessment of PRediction of Interactions (CAPRI) blind docking challenge. Here, we present transition kinetics and thermodynamics of these conformational rearrangements of the paratope and show that kinetically relevant states can be used to improve antibody-antigen docking. We use the Rosetta SnugDock docking algorithm to show that by even using the unbound antibody X-ray structure as starting structures for molecular dynamics simulations, a binding competent conformation substantially different to the unbound antibody X-ray structure, can be retained. We also observe that the kinetically dominant antibody conformations are chosen by the bound antigen conformation with the highest probability. Thus, we show that kinetically relevant antibody conformations obtained by a combination of molecular dynamics simulations and Markov-state models can improve antibody-antigen docking and structure prediction.

Structural Characterization and Modeling of a Respiratory Syncytial Virus Fusion Glycoprotein Nanoparticle Vaccine in Solution.

The respiratory syncytial virus (RSV) fusion (F) protein/polysorbate 80 (PS80) nanoparticle vaccine is the most clinically advanced vaccine for maternal immunization and protection of newborns against RSV infection. It is composed of a near-full-length RSV F glycoprotein, with an intact membrane domain, formulated into a stable nanoparticle with PS80 detergent. To understand the structural basis for the efficacy of the vaccine, a comprehensive study of its structure and hydrodynamic properties in solution was performed. Small-angle neutron scattering experiments indicate that the nanoparticle contains an average of 350 PS80 molecules, which form a cylindrical micellar core structure and five RSV F trimers that are arranged around the long axis of the PS80 core. All-atom models of full-length RSV F trimers were built from crystal structures of the soluble ectodomain and arranged around the long axis of the PS80 core, allowing for the generation of an ensemble of conformations that agree with small-angle neutron and X-ray scattering data as well as transmission electron microscopy (TEM) images. Furthermore, the hydrodynamic size of the RSV F nanoparticle was found to be modulated by the molar ratio of PS80 to protein, suggesting a mechanism for nanoparticle assembly involving addition of RSV F trimers to and growth along the long axis of the PS80 core. This study provides structural details of antigen presentation and conformation in the RSV F nanoparticle vaccine, helping to explain the induction of broad immunity and observed clinical efficacy. Small-angle scattering methods provide a general strategy to visualize surface glycoproteins from other pathogens and to structurally characterize nanoparticle vaccines.

Designs and Characterization of Subunit Ebola GP Vaccine Candidates: Implications for Immunogenicity.

The humoral responses of Ebola virus (EBOV) survivors mainly target the surface glycoprotein GP, and anti-GP neutralizing antibodies have been associated with protection against EBOV infection. In order to elicit protective neutralizing antibodies through vaccination a native-like conformation of the antigen is required. We therefore engineered and expressed in CHO cells several GP variants from EBOV (species Zaire ebolavirus, Mayinga variant), including a soluble GP ΔTM, a mucin-like domain-deleted GP ΔTM-ΔMUC, as well as two GP ΔTM-ΔMUC variants with C-terminal trimerization motifs in order to favor their native trimeric conformation. Inclusion of the trimerization motifs resulted in proteins mimicking GP metastable trimer and showing increased stability. The mucin-like domain appeared not to be critical for the retention of the native conformation of the GP protein, and its removal unmasked several neutralizing epitopes, especially in the trimers. The soluble GP variants inhibited mAbs neutralizing activity in a pseudotype transduction assay, further confirming the proteins’ structural integrity. Interestingly, the trimeric GPs, a native-like GP complex, showed stronger affinity for antibodies raised by natural infection in EBOV disease survivors rather than for antibodies raised in volunteers that received the ChAd3-EBOZ vaccine. These results support our hypothesis that neutralizing antibodies are preferentially induced when using a native-like conformation of the GP antigen. The soluble trimeric recombinant GP proteins we developed represent a novel and promising strategy to develop prophylactic vaccines against EBOV and other filoviruses.

The FixBox: Hardware to Provide on-Orbit Fixation Capabilities to the EMCS on the ISS

Plant biology is an important area for the future of space exploration, but biological spaceflight experiments have been always constrained by the hardware capabilities. The European Modular Cultivation System (EMCS) unit was an incubator for small organisms, such as Arabidopsis thaliana, built by the European Space Agency (ESA) and was decommissioned in 2018. Here, we describe the FixBox system as add-on hardware to provide fixation capabilities to the plant growth cassettes, which, initially, were not designed to be used for that purpose. Tests were performed to ensure the successful use of this device in the EMCS facility. We also evaluate the required adaptations to the hardware, e.g., to guarantee that the reduced fluid motion in microgravity does not cause any bubbles that could impair the quality of fixation. Arabidopsis thaliana seedlings grown during spaceflight were fixed in the FixBox either in glutaraldehyde or formaldehyde. Electron microscopical images and confocal microscopy immunofluorescent localizations showed an excellent preservation of both cell ultrastructure and antigen conformation. Thus, it is possible to modify existing hardware to comply with the scientific requirements to augment the existing capabilities on board the ISS. In addition, it is also possible to reuse culture chambers from predesigned experimental containers into new modular subunits as FixBox. Similarly, we can design new hardware compatible with a novel cultivation chamber on board, such as is available in BIOLAB, to be used later with FixBox. Lessons learned for future space plant biology researchers include how to manage the number of hardware requirements and constraints on how to preserve the biological samples.

Innovative IgG Biomarkers Based on Phage Display Microbial Amyloid Mimotope for State and Stage Diagnosis in Alzheimer's Disease.

An innovative approach to identify new conformational antigens of Aβ1–42 recognized by IgG autoantibodies as biomarkers of state and stage in Alzheimer’s disease (AD) patients is described. In particular, through the use of bioinformatics modeling, conformational similarities between several Aβ1–42 forms and other amyloid-like proteins with F1 capsular antigen (Caf1) of Yersinia pestis were first found. pVIII M13 phage display libraries were then screened against YPF19, anti-Caf1 monoclonal antibody, and IgGs of AD patients, in alternate biopanning cycles of a so-called “double binding” selection. From the selected phage clones, one, termed 12III1, was found to be able to prevent in vitro Aβ1–42-induced cytotoxicity in SH-SY5Y cells, as well as to promote disaggregation of preformed fibrils, to a greater extent with respect to wild-type phage (pC89). IgG levels detected by 12III1 provided a significant level of discrimination between diseased and nondemented subjects, as well as a good correlation with the state progression of the disease. These results give significant impact in AD state and stage diagnosis, paving the way for the development not only for an innovative blood diagnostic assay for AD precise diagnosis, progressive clinical assessment, and screening but also for new effective treatments.

Functional epitopes on hepatitis E virions and recombinant capsids are highly conformation-dependent

ABSTRACT Hepatitis E virus (HEV) is responsible for epidemic and sporadic acute hepatitis cases, especially in developing countries. Hepatitis E has become a vaccine-preventable disease in recent years with the development of a licensed vaccine. Most functional and neutralizing monoclonal antibodies (mAbs) are known to be highly sensitive to antigen conformation. In this study, a similar approach was used to characterize the conformational sensitivity of antibodies in human or mouse serum samples. Interestingly, comparative binding analysis using different antigen forms showed that the antibodies in the sera of naturally infected individuals, of human vaccinees and from mice immunized with the HEV p239 vaccine all exhibited a strong preference to particulate antigens over the monomeric form of the truncated capsid protein. The degree of discriminating the two test antigens is similar for serum samples to that for the well-characterized murine mAbs. A functional assay for assessing the inhibition of subviral particle cell entry by antibodies was used to determine the functional titers of anti-HEV antibodies in mouse sera. A good correlation was observed between the functional and binding titers in mouse sera determined using two different methods. This result supports the continued use of the enzyme-linked immunosorbent assay as the primary serological assay assuming that the coating antigen contains conformational and native-like epitopes, as in the case for HEV p239.

Designing Chimeric Virus-like Particle-based Vaccines for Human Papillomavirus and HIV: Lessons Learned.

Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphology to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human papillomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including production methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLP-based HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens.

Efficient evolved antibody mimetic designed ankyrin repeat proteins against programmed death-ligand 1 on E. coli surface display

Designed ankyrin repeat proteins (DARPins) are a kind of proteins containing the similar characteristics to antibodies. Which can specifically bind the target with high affinity. Here, we described an efficient method to select of DARPins effectively against the programmed death-ligand 1(PD-L1), a prominent target for cancer immune therapy, by E. coli surface display. E. coli surface display combined with magnetic cell sorting (MACS) were easy to manipulate with the advantages of solubility selected, antigen conformation kept and easily unbinding DARPins washed. The selected DARPin bound specifically to PD-L1 overexpressing cell CT26 and had a micromolar affinity for PD-L1. Animal experiments with tumor-bearing mice were demonstrated that the selected DARPin could significantly inhibit the growth rate of PD-L1 overexpressing tumor cells. This selected method of DARPin may provide a quick drug candidate generation method for cancer immunotherapy.

Stabilization of the V2 loop improves the presentation of V2 loop–associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers

A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development.

Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin‐induced thrombocytopenia

Essentials Many patients produce antibodies but few lead to heparin-induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non-pathogenic antibodies. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti-PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. Objectives To identify PF4 amino acids essential for binding pathogenic HIT antibodies. Methods Alanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet-activating murine monoclonal anti-PF4/heparin antibody (KKO) and HIT patient sera (n=9). Results and Conclusions We identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n=45) and non-pathogenic antibodies (EIA-positive but not platelet activating, n=28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4.