Surface Of Virus-like Particles Research Articles

Chimeric hepatitis B surface antigen virus-like particles expressing the strep A epitope p*17 elicit a humoral immune response in mice

To optimize immunogenicity, bacterial epitopes in putative vaccine constructs can be presented to immune cells as multiple repeated structures on a defined nanoparticle. Virus-like particles (VLPs) are viral capsid proteins that self-assemble to form compact and highly ordered nanoparticles that are within the optimal size range for uptake by dendritic cells. VLPs mimic the live virus in size and form but contain no viral genetic material, are therefore noninfective and are the basis of safe and effective vaccines against hepatitis B virus (HBV) and human papillomavirus (HPV). Due to their particulate nature, molecular stability, and expression of high density and repetitive antigen displays, recombinant cell culture-derived VLPs are ideal platforms for the delivery of small molecules, including bacterial epitopes. We developed a putative vaccine by expressing a minimal epitope from the bacterium Streptococcus pyogenes (Strep A) on the surface of a recombinant VLP comprising multiple copies of HBV small envelope protein (HBsAg-S). Strep A is responsible for a wide spectrum of human infections and postinfectious diseases that disproportionately affect children and young adults living in resource-poor communities. No vaccine is currently available to offer sufficiently broad protection from the numerous and diverse strains of Strep A endemic in these at-risk populations. The Strep A antigen targeted by our vaccine construct is p*17, a cryptic epitope from a highly conserved region of the Strep A M-protein with demonstrated enhanced immunogenicity and broad protective potential against Strep A. To ensure surface expression and optimal immunogenicity, we expressed p*17 within the immunodominant “a” determinant of HBsAg-S. The recombinant VLPs (VLP-p*17) expressed in HEK293T cells spontaneously formed 22 nm particles and induced the production of high titers of p*17-specific IgG in BALB/c mice immunized with three 0.5 μg doses of VLP-p*17 formulated with adjuvant.

Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection

BackgroundApplication of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP’s encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses.ResultsWe constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5–10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template.ConclusionMrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.

Plant and insect virus-like particles: emerging nanoparticles for agricultural pest management.

Virus-like particles (VLPs) represent a biodegradable, biocompatible nanomaterial made from viral coat proteins that can improve the delivery of antigens, drugs, nucleic acids, and other substances, with most applications in human and veterinary medicine. Regarding agricultural viruses, many insect and plant virus coat proteins have been shown to assemble into VLPs accurately. In addition, some plant virus-based VLPs have been used in medical studies. However, to our knowledge, the potential application of plant/insect virus-based VLPs in agriculture remains largely underexplored. This review focuses on why and how to engineer coat proteins of plant/insect viruses as functionalized VLPs, and how to exploit VLPs in agricultural pest control. The first part of the review describes four different engineering strategies for loading cargo at the inner or the outer surface of VLPs depending on the type of cargo and purpose. Second, the literature on plant and insect viruses the coat proteins of which have been confirmed to self-assemble into VLPs is reviewed. These VLPs are good candidates for developing VLP-based agricultural pest control strategies. Lastly, the concepts of plant/insect virus-based VLPs for delivering insecticidal and antiviral components (e.g., double-stranded RNA, peptides, and chemicals) are discussed, which provides future prospects of VLP application in agricultural pest control. In addition, some concerns are raised about VLP production on a large scale and the short-term resistance of hosts to VLP uptake. Overall, this review is expected to stimulate interest and research exploring plant/insect virus-based VLP applications in agricultural pest management. © 2023 Society of Chemical Industry.

An engineered HIV-1 Gag-based VLP displaying high antigen density induces strong antibody-dependent functional immune responses

Antigen display on the surface of Virus-Like Particles (VLPs) improves immunogenicity compared to soluble proteins. We hypothesised that immune responses can be further improved by increasing the antigen density on the surface of VLPs. In this work, we report an HIV-1 Gag-based VLP platform engineered to maximise the presence of antigen on the VLP surface. An HIV-1 gp41-derived protein (Min), including the C-terminal part of gp41 and the transmembrane domain, was fused to HIV-1 Gag. This resulted in high-density MinGag-VLPs. These VLPs demonstrated to be highly immunogenic in animal models using either a homologous (VLP) or heterologous (DNA/VLP) vaccination regimen, with the latter yielding 10-fold higher anti-Gag and anti-Min antibody titres. Despite these strong humoral responses, immunisation with MinGag-VLPs did not induce neutralising antibodies. Nevertheless, antibodies were predominantly of an IgG2b/IgG2c profile and could efficiently bind CD16-2. Furthermore, we demonstrated that MinGag-VLP vaccination could mediate a functional effect and halt the progression of a Min-expressing tumour cell line in an in vivo mouse model.

Long-time scale simulations of virus-like particles from three human-norovirus strains.

The dynamics of the virus like particles (VLPs) corresponding to the GII.4 Houston, GII.2 SMV, and GI.1 Norwalk strains of human noroviruses (HuNoV) that cause gastroenteritis was investigated by means of long-time (about 30 μs in the laboratory timescale) molecular dynamics simulations with the coarse-grained UNRES force field. The main motion of VLP units turned out to be the bending at the junction between the P1 subdomain (that sits in the VLP shell) and the P2 subdomain (that protrudes outside) of the major VP1 protein, this resulting in a correlated wagging motion of the P2 subdomains with respect to the VLP surface. The fluctuations of the P2 subdomain were found to be more pronounced and the P2 domain made a greater angle with the normal to the VLP surface for the GII.2 strain, which could explain the inability of this strain to bind the histo-blood group antigens (HBGAs).

Display of multiple proteins on engineered canine parvovirus-like particles expressed in cultured silkworm cells and silkworm larvae.

Recent progress has been made dramatically in decorating virus-like particles (VLPs) on the surface or inside with functional molecules, such as antigens or nucleic acids. However, it is still challenging to display multiple antigens on the surface of VLP to meet the requirement as a practical vaccine candidate. Herein this study, we focus on the expression and engineering of the capsid protein VP2 of canine parvovirus for VLP display in the silkworm-expression system. The chemistry of the SpyTag/SpyCatcher (SpT/SpC) and SnoopTag/SnoopCatcher (SnT/SnC) are efficient protein covalent ligation systems to modify VP2 genetically, where SpyTag/SnoopTag are inserted into the N-terminus or two distinct loop regions (Lx and L2) of VP2. The SpC-EGFP and SnC-mCherry are employed as model proteins to evaluate their binding and display on six SnT/SnC-modified VP2 variants. From a series of protein binding assays between indicated protein partners, we showed that the VP2 variant with SpT inserted at the L2 region significantly enhanced VLP display to 80% compared to 5.4% from N-terminal SpT-fused VP2-derived VLPs. In contrast, the VP2 variant with SpT at the Lx region failed to form VLPs. Moreover, the SpT (Lx)/SnT (L2) double-engineered chimeric VP2 variants showed covalent conjugation capacity to both SpC/SnC protein partners. The orthogonal ligations between those binding partners were confirmed by both mixing purified proteins and co-infecting cultured silkworm cells or larvae with desired recombinant viruses. Our results indicate that a convenient VLP display platform was successfully developed for multiple antigen displays on demand. Further verifications can be performed to assess its capacity for displaying desirable antigens and inducing a robust immune response to targeted pathogens.

Virus-like particle - mediated delivery of the RIG-I agonist M8 induces a type I interferon response and protects cells against viral infection.

Virus-Like Particles (VLPs) are nanostructures that share conformation and self-assembly properties with viruses, but lack a viral genome and therefore the infectious capacity. In this study, we produced VLPs by co-expression of VSV glycoprotein (VSV-G) and HIV structural proteins (Gag, Pol) that incorporated a strong sequence-optimized 5'ppp-RNA RIG-I agonist, termed M8. Treatment of target cells with VLPs-M8 generated an antiviral state that conferred resistance against multiple viruses. Interestingly, treatment with VLPs-M8 also elicited a therapeutic effect by inhibiting ongoing viral replication in previously infected cells. Finally, the expression of SARS-CoV-2 Spike glycoprotein on the VLP surface retargeted VLPs to ACE2 expressing cells, thus selectively blocking viral infection in permissive cells. These results highlight the potential of VLPs-M8 as a therapeutic and prophylactic vaccine platform. Overall, these observations indicate that the modification of VLP surface glycoproteins and the incorporation of nucleic acids or therapeutic drugs, will permit modulation of particle tropism, direct specific innate and adaptive immune responses in target tissues, and boost immunogenicity while minimizing off-target effects.

Improvement of Modular Protein Display Efficiency in SpyTag-Implemented Norovirus-like Particles.

Genetic fusion and chemical conjugation are the most common approaches for displaying a foreign protein on the surface of virus-like particles (VLPs); however, these methods may negatively affect the formation and stability of VLPs. Here, we aimed to develop a modular display platform for protein decoration on norovirus-like particles (NoV-LPs) by combining the NoV-LP scaffold with the SpyTag/SpyCatcher bioconjugation system, as the NoV-LP is an attractive protein nanoparticle to carry foreign proteins for various applications. The SpyTagged-NoV-LPs were prepared by introducing SpyTag peptide into the C-terminus of the norovirus VP1 protein. To increase surface exposure of the SpyTag peptide on the NoV-LPs, two or three repeated extension linkers (EAAAK) were inserted between the SpyTag peptide and VP1 protein. Fluorescence proteins, EGFP and mCherry, were fused to SpyCatcher and employed as SpyTag conjugation partners. These VP1-SpyTag variants and SpyCatcher-fused EGFP and mCherry were separately expressed in silkworm fat bodies and purified. This study reveals that adding an extension linker did not disrupt the VLP formation; instead, it increased the particle size by 4-6 nm. The conjugation efficiency of the VP1-SpyTag variants with the extended linker improved from ∼15-35 to ∼50-63% based on the densitometric analysis, while it was up to 77% based on an optical quantification of EGFP and mCherry. Results indicate that the linker causes the SpyTag peptides to be positioned further away from the C-termini of VP1 and potentially increases the exposure of the SpyTag to the outer surface of the NoV-LPs, allowing more SpyTag/SpyCatcher complex formation on the VLP surface. Our study provides a strategy for enhancing the conjugation efficiency of NoV-LP and demonstrates the platform's utility for developing vaccines or functional nanoparticles.

Display of Streptococcus iniae α-Enolase on the Surface of Virus-Like Particles (VLPs) of Nervous Necrosis Virus (NNV) Using SpyTag/SpyCatcher.

Virus-like particle (VLP)-based vaccines are promising candidates for overcoming the safety problems of live vaccines and weak immunogenicity of subunit vaccines. VLPs can be used as a platform for the development of combined vaccines by expressing foreign antigens, and foreign antigens can be displayed on the surface of VLPs by conjugation. In the present study, to use nervous necrosis virus (NNV) VLPs as a delivery tool for Streptococcus iniae α-enolase by displaying on the VLP's surface, the split-intein (SpyTag/SpyCatcher) conjugation system was used. NNV capsid protein fused to SpyTag (Capsid-SpyTag) and S. iniae α-enolase fused to SpyCatcher (α-enolase-SpyCatcher) were recombinantly produced, then mixed in various ratios. A ratio of Capsid-SpyTag to α-enolase-SpyCatcher of 1 to 1.5 showed the highest coupling efficiency corresponding to 83-92% of coupled capsid protein dimer and 32-52% of coupled capsid protein monomer. In TEM observation, VLP of Capsid-SpyTag had a regular shape and size of about 40 nm, while VLP fused with α-enolase-SpyCatcher showed an irregular shape and size of about 40-50 nm in diameter. In preliminary immunization experiments, olive flounder (Paralichthys olivaceus) and zebrafish (Danio rerio) immunized with VLP fused with α-enolase-SpyCatcher showed the lowest cumulative mortality against S. iniae infection.

Development of a synthetic nanoparticle vaccine presenting the HIV-1 envelope glycoprotein

Two-component self-assembling virus-like particles (VLPs) are promising scaffolds for achieving high-density display of HIV-1 envelope (gp140) trimers, which can improve the induction of neutralising antibodies (NAbs). In this study gp140 was displayed on the surface of VLPs formed by the AP205 phage coat protein. The CAP256 SU gp140 antigen was selected as the patient who this virus was isolated from developed broadly neutralising antibodies (bNAbs) shortly after superinfection with this virus. The CAP256 SU envelope is also sensitive to several bNAbs and has shown enhanced reactivity for certain bNAb precursors. A fusion protein comprising the HIV-1 CAP256 SU gp140 and the SpyTag (ST) (gp140-ST) was produced in HEK293 cells, and trimers were purified to homogeneity using gel filtration. SpyCatcher (SC)-AP205 VLPs were produced in Escherichia coli and purified by ultracentrifugation. The gp140-ST trimers and the SC-AP205 VLPs were mixed in varying molar ratios to generate VLPs displaying the glycoprotein (AP205-gp140-ST particles). Dynamic light scattering, negative stain electron microscopy and 2D classification indicated that gp140-ST was successfully bound to the VLPs, although not all potential binding sites were occupied. The immunogenicity of the coupled VLPs was evaluated in a pilot study in rabbits. One group was injected four times with coupled VLPs, and the second group was primed with DNA vaccines expressing Env and a mosaic Gag, followed by modified vaccinia Ankara expressing the same antigens. The animals were then boosted twice with coupled VLPs. Encouragingly, gp140-ST displayed on SC-AP205 VLPs was an effective boost to heterologously primed rabbits, leading to induction of autologous Tier 2 neutralising antibodies in 2/5 rabbits. However, four inoculations of coupled VLPs alone failed to elicit any Tier 2 antibodies. These results demonstrate that the native-like structure of HIV-1 envelope trimers and selection of a geometrically-suitable nanoparticle scaffold to achieve a high-density display of the trimers are important considerations that could improve the effect of nanoparticle-displayed gp140.

Virus-like Particles: Fundamentals and Biomedical Applications

Nanotechnology is a fast-evolving field focused on fabricating nanoscale objects for industrial, cosmetic, and therapeutic applications. Virus-like particles (VLPs) are self-assembled nanoparticles whose intrinsic properties, such as heterogeneity, and highly ordered structural organization are exploited to prepare vaccines; imaging agents; construct nanobioreactors; cancer treatment approaches; or deliver drugs, genes, and enzymes. However, depending upon the intrinsic features of the native virus from which they are produced, the therapeutic performance of VLPs can vary. This review compiles the recent scientific literature about the fundamentals of VLPs with biomedical applications. We consulted different databases to present a general scenario about viruses and how VLPs are produced in eukaryotic and prokaryotic cell lines to entrap therapeutic cargo. Moreover, the structural classification, morphology, and methods to functionalize the surface of VLPs are discussed. Finally, different characterization techniques required to examine the size, charge, aggregation, and composition of VLPs are described.

Bioanalytics for Influenza Virus-Like Particle Characterization and Process Monitoring.

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs’ unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies — nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs’ thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90–115 nm for NTA and 129–141 nm for TRPS), surface charges (average of −20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.

Bioengineering of virus-like particles as dynamic nanocarriers for in vivo delivery and targeting to solid tumours.

Virus-like particles (VLPs) are known as self-assembled, non-replicative and non-infectious protein particles, which imitate the formation and structure of original wild type viruses, however, lack the viral genome and/or their fragments. The capacity of VLPs to encompass small molecules like nucleic acids and others has made them as novel vessels of nanocarriers for drug delivery applications. In addition, VLPs surface have the capacity to achieve variation of the surface display via several modification strategies including genetic modification, chemical modification, and non-covalent modification. Among the VLPs nanocarriers, Hepatitis B virus core (HBc) particles have been the most encouraging candidate. HBc particles are hollow nanoparticles in the range of 30-34nm in diameter and 7nm thick envelopes, consisting of 180 or 240 copies of identical polypeptide monomer. They also employ a distinctive position among the VLPs carriers due to the high-level synthesis, which serves as a strong protective capsid shell and efficient self-assembly properties. This review highlights on the bioengineering of HBc particles as dynamic nanocarriers for in vivo delivery and specific targeting to solid tumours.

Antigenicity and immunogenicity of HA2 and M2e influenza virus antigens conjugated to norovirus-like, VP1 capsid-based particles by the SpyTag/SpyCatcher technology

Virus-like particles (VLPs) modified through different molecular technologies are employed as delivery vehicles or platforms for heterologous antigen display. We have recently created a norovirus (NoV) VLP platform, where two influenza antigens, the extracellular domain of matrix protein M2 (M2e) or the stem domain of the major envelope glycoprotein hemagglutinin (HA2) are displayed on the surface of the NoV VLPs by SpyTag/SpyCatcher conjugation. To demonstrate the feasibility of the platform to deliver foreign antigens, this study examined potential interference of the conjugation with induction of antibodies against conjugated M2e peptide, HA2, and NoV VLP carrier. High antibody response was induced by HA2 but not M2e decorated VLPs. Furthermore, HA2-elicited antibodies did not neutralize the homologous influenza virus in vitro. Conjugated NoV VLPs retained intact receptor binding capacity and self-immunogenicity. The results demonstrate that NoV VLPs could be simultaneously used as a platform to deliver foreign antigens and a NoV vaccine.

Next-Generation Computationally Designed Influenza Hemagglutinin Vaccines Protect against H5Nx Virus Infections.

H5N1 COBRA hemagglutinin (HA) sequences, termed human COBRA-2 HA, were constructed through layering of HA sequences from viruses isolated from humans collected between 2004–2007 using only clade 2 strains. These COBRA HA proteins, when expressed on the surface of virus-like particles (VLP), elicited protective immune responses in mice, ferrets, and non-human primates. However, these vaccines were not as effective at inducing neutralizing antibodies against newly circulating viruses. Therefore, COBRA HA-based vaccines were updated in order to elicit protective antibodies against the current circulating clades of H5Nx viruses. Next-generation COBRA HA vaccines were designed to encompass the newly emerging viruses circulating in wild avian populations. HA amino acid sequences from avian and human H5 influenza viruses isolated between 2011–2017 were downloaded from the GISAID (Global Initiative on Sharing All Influenza Data). Mice were vaccinated with H5 COBRA rHA that elicited antibodies with hemagglutinin inhibition (HAI) activity against H5Nx viruses from five clades. The H5 COBRA rHA vaccine, termed IAN8, elicited protective immune responses against mice challenged with A/Sichuan/26621/2014 and A/Vietnam/1203/2004. This vaccine elicited antibodies with HAI activity against viruses from clades 2.2, 2.3.2.1, 2.3.4.2, 2.2.1 and 2.2.2. Lungs from vaccinated mice had decreased viral titers and the levels of cellular infiltration in mice vaccinated with IAN-8 rHA were similar to mice vaccinated with wild-type HA comparator vaccines or mock vaccinated controls. Overall, these next-generation H5 COBRA HA vaccines elicited protective antibodies against both historical H5Nx influenza viruses, as well as currently circulating clades of H5N1, H5N6, and H5N8 influenza viruses.

Neutralization of MERS coronavirus through a scalable nanoparticle vaccine

MERS-CoV continues to cause human outbreaks, so far in 27 countries worldwide following the first registered epidemic in Saudi Arabia in 2012. In this study, we produced a nanovaccine based on virus-like particles (VLPs). VLPs are safe vaccine platforms as they lack any replication-competent genetic material, and are used since many years against hepatitis B virus (HBV), hepatitis E virus (HEV) and human papilloma virus (HPV). In order to produce a vaccine that is readily scalable, we genetically fused the receptor-binding motif (RBM) of MERS-CoV spike protein into the surface of cucumber-mosaic virus VLPs. The employed CuMVTT-VLPs represent a new immunologically optimized vaccine platform incorporating a universal T cell epitope derived from tetanus toxin (TT). The resultant vaccine candidate (mCuMVTT-MERS) is a mosaic particle and consists of unmodified wild type monomers and genetically modified monomers displaying RBM, co-assembling within E. coli upon expression. mCuMVTT-MERS vaccine is self-adjuvanted with ssRNA, a TLR7/8 ligand which is spontaneously packaged during the bacterial expression process. The developed vaccine candidate induced high anti-RBD and anti-spike antibodies in a murine model, showing high binding avidity and an ability to completely neutralize MERS-CoV/EMC/2012 isolate, demonstrating the protective potential of the vaccine candidate for dromedaries and humans.

Hemagglutination Inhibition (HAI) antibody landscapes after vaccination with H7Nx virus like particles.

A systemic evaluation of the antigenic differences of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic differences of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-binding antibodies against co-circulating H7 influenza strains. A panel of eight H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013. VLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the selected H7 HAs, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. A putative glycosylation due to an A169T substitution in antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA of the A/Anhui/1/2013 (H7N9) strain. This study identified key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.

Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

Escherichia Coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles.

Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs.