Inducing Long Lasting B Cell and T Cell Immunity Against Multiple Variants of SARS-CoV-2 Through Mutant Bacteriophage Qß-Receptor Binding Domain Conjugate.

More than 3 years into the global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat to public health. Immunities acquired from infection or current vaccines fail to provide long term protection against subsequent infections, mainly due to their fast-waning nature and the emergence of variants of concerns (VOCs) such as Omicron. To overcome these limitations, SARS-CoV-2 Spike protein receptor binding domain (RBD)-based epitopes are investigated as conjugates with a powerful carrier, the mutant bacteriophage Qß (mQß). The epitope design is critical to eliciting potent antibody responses with the full length RBD being superior to peptide and glycopeptide antigens. The full length RBD conjugated with mQß activates both humoral and cellular immune systems in vivo, inducing broad spectrum, persistent, and comprehensive immune responses effective against multiple VOCs including Delta and Omicron variants, rendering it a promising vaccine candidate.

Synthesis and Immunological Study of N-Glycan-Bacteriophage Qß Conjugates Reveal Dominant Antibody Responses to the Conserved Chitobiose Core.

N-Glycosylation plays an important role in many biological recognition processes. However, very few N-glycan-specific antibodies are available for functional studies and potentially for therapeutic development. In this study, we sought to synthesize bacteriophage Qß conjugates with representative N-glycans and investigate their immunogenicity for raising N-glycan-specific antibodies. An array of Qß glycoconjugates bearing five different human N-glycans and two different chemical linkers were synthesized, and the immunization of the N-glycan-Qß conjugates was performed in mice. We found that the N-glycan-Qß conjugates raised significant IgG antibodies that recognize N-glycans, but, surprisingly, most of the glycan-dependent antibodies were directed to the shared chitobiose core and were nonspecific for respective N-glycan structures. The linker chemistry was found to affect antibody specificity with adipic acid-linked N-glycan-Qß immunogens raising antibodies capable of recognizing both the N-acetylglucosamine (GlcNAc) moieties of the chitobiose core. In contrast, antibodies raised by N-glycan-Qß immunogens with a triazole linker preferentially recognized the innermost N-acetylglucosamine moiety at the reducing end. We also found that sialylation of the N-glycans significantly suppressed the immune response. Furthermore, the N-glycan-Qß immunogens with an adipic acid linker elicited higher glycan-specific antibody titers than the N-glycan-triazole-Qß immunogens. These findings delineate several challenges in eliciting mammalian N-glycan-specific antibodies through the conventional glycoconjugate vaccine design and immunization.

Structure Guided Design of Bacteriophage Qß Mutants as Next Generation Carriers for Conjugate Vaccines.

Vaccines are critical tools to treat and prevent diseases. For an effective conjugate vaccine, the carrier is crucial, but few carriers are available for clinical applications. In addition, a drawback of current protein carriers is that high levels of antibodies against the carrier are induced by the conjugate vaccine, which are known to interfere with the immune responses against the target antigen. To overcome these challenges, we obtained the near atomic resolution crystal structure of an emerging protein carrier, i.e., the bacteriophage Qß virus like particle. On the basis of the detailed structural information, novel mutants of bacteriophage Qß (mQß) have been designed, which upon conjugation with tumor associated carbohydrate antigens (TACAs), a class of important tumor antigens, elicited powerful anti-TACA IgG responses and yet produced lower levels of anticarrier antibodies as compared to those from the wild type Qß-TACA conjugates. In a therapeutic model against an aggressive breast cancer in mice, 100% unimmunized mice succumbed to tumors in just 12 days even with chemotherapy. In contrast, 80% of mice immunized with the mQß-TACA conjugate were completely free from tumors. Besides TACAs, to aid in the development of vaccines to protect against COVID-19, the mQß based conjugate vaccine has been shown to induce high levels of IgG antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating its generality. Thus, mQß is a promising next-generation carrier platform for conjugate vaccines, and structure-based rational design is a powerful strategy to develop new vaccine carriers.

Synthesis and Immunological Evaluation of Disaccharide Bearing MUC-1 Glycopeptide Conjugates with Virus-like Particles.

Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qß carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qß were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qß-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qß-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qß-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.

Lung Tissue Delivery of Virus-Like Particles Mediated by Macrolide Antibiotics.

Macrophage cells are present in high abundance in the lung to intercept invading microorganisms that gain access through airway mucosal surfaces. Several bacterial pathogens have evolved the capacity to evade the innate immune response by establishing infections within pulmonary macrophages upon phagocytosis, leading to prolonged disease. Macrolide antibiotics such as azithromycin and clarithromycin accumulate in phagocytic cells and have been shown to preferentially distribute in tissues where populations of these cells reside. We employed this class of molecules as targeting ligands to direct virus-like particles (VLPs) to lung-resident macrophages. VLP-macrolide conjugates showed enhanced uptake into RAW 264.7 macrophage cells in culture, with azithromycin displaying the greatest effect; distinct differences were also observed for different macrocycle structures and orientations on the particle surface. Activation of macrophage cells was stimulated by particle uptake toward an intermediate activation state, in contrast to previous reports using macrolide-functionalized gold nanorods that stimulated a cytotoxic macrophage response. Attached azithromycin was also able to direct VLPs to the lungs in mice, with significant accumulation within 2 h of systemic injection. These results suggest that this new class of bioconjugate could serve as an effective platform for intracellular drug delivery in the context of pulmonary infections.

Synthetic and immunological studies of Salmonella Enteritidis O-antigen tetrasaccharides as potential anti-Salmonella vaccines.

The first synthetic carbohydrate based potential anti-Salmonella Enteritidis vaccine has been developed by conjugating a synthetic tetrasaccharide antigen with bacteriophage Qß. High levels of specific and long lasting anti-glycan IgG antibodies were induced by the conjugate, which completely protected mice from lethal bacterial challenges in a passive transfer model.

RNA chaperone assisted intramolecular annealing reaction towards oligouridylated RNA detection in cancer cells.

Proximity induced intramolecular nucleotide strand displacement, which can be simply performed in a single tube or in a complex cellular environment, is one of the key mechanisms for the detection of biological targets, especially for significant genetic molecules. The host factor for RNA phage Qb replication (Hfq), with two distinct single stranded RNA binding sites, has excellent properties as an affinity ligand in a proximity induced reaction. In this research, a versatile RNA chaperone-Hfq assisted RNA annealing strategy for the sensitive detection of the intermediate product, oligouridylated RNA, in a genetic regulation process was developed. Benefiting from the high binding affinity of Hfq for the probe and the target, the sensitive determination of oligouridylated RNA in cell lysis and human cervical cancer (HeLa) cells was successfully achieved. This study has also revealed that the Hfq assisted RNA annealing strategy can be further extended and applied in specific microRNA analysis, and RNA related tumorigenicity and disease diagnosis.

Protective Epitope Discovery and Design of MUC1-based Vaccine for Effective Tumor Protections in Immunotolerant Mice.

Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qß carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qß-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qß-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.

Site-Selective Nucleation and Size Control of Gold Nanoparticle Photothermal Antennae on the Pore Structures of a Virus.

In this Article, we show that the surface of the bacteriophage Qß is equipped with natural ligands for the synthesis of small gold nanoparticles (AuNPs). By exploiting disulfides in the protein secondary structure and the geometry formed from the capsid quaternary structure, we find that we can produce regularly arrayed patterns of ∼6 nm AuNPs across the surface of the virus-like particle. Experimental and computational analyses provide insight into the formation and stability of this composite. We further show that the entrapped genetic material can hold upward of 500 molecules of the anticancer drug Doxorubicin without leaking and without interfering with the synthesis of the AuNPs. This direct nucleation of nanoparticles on the capsid allows for exceptional conduction of photothermal energy upon nanosecond laser irradiation. As a proof of principle, we demonstrate that this energy is capable of rapidly releasing the drug from the capsid without heating the bulk solution, allowing for highly targeted cell killing in vitro.

Polyvalent Hybrid Virus-Like Nanoparticles with Displayed Heparin Antagonist Peptides.

The potential applications for nanomaterials continue to grow as new materials are developed and environmental and safety concerns are more adequately addressed. In particular, virus-like particles (VLPs) have myriad applications in medicine and biology, exploiting both the reliable, symmetric self-assembly mechanism and the ability to take advantage of surface functionalities that may be appropriately modified through mutation or bioconjugation. Herein we describe the design and application of hybrid VLPs for use as potent heparin antagonists, providing an alternative to the toxic heparin antidote protamine. A two-plasmid system was utilized to generate VLPs that contain both the wild-type coat protein and a second coat protein with either a C- or N-terminal cationic peptide extension (4-28 amino acids). Incorporation of the modified coat proteins varied from 8 to 31%, while activated partial thromboplastin time (APTT) assays revealed a range of the heparin antagonist activity. Notably, when examined on the basis of the quantity of peptide delivered due to the varied incorporation rates, it appeared that the VLPs largely followed a similar trend, with the quantity of peptide delivered more closely correlating with heparin antagonist activity. The particle with the highest incorporation rate and best antiheparin activity displayed the C-terminal peptide ARK2A2KA, which corresponds to the Cardin-Weintraub consensus sequence for binding to glycosaminoglycans. Analysis of this particle using heparin affinity chromatography with fraction collection revealed that particles eluting at higher salt concentration had a greater proportion of peptide incorporation. Preliminary dual polarization interferometry experiments further support a strong interaction between this particle and heparin.

Regulating the Uptake of Viral Nanoparticles in Macrophage and Cancer Cells via a pH Switch.

Controlling the uptake of nanomaterials into phagocytes is a challenging problem. We describe an approach to inhibit the cellular uptake by macrophages and HeLa cells of nanoparticles derived from bacteriophage Qß by conjugating negatively charged terminal hexanoic acid moieties onto its surface. Additionally, we show hydrazone linkers can be installed between the surface of Qß and the terminal hexanoic acid moieties, resulting in a pH-responsive conjugate that, in acidic conditions, can release the terminal hexanoic acid moiety and allow for the uptake of the Qß nanoparticle. The installation of the "pH switch" did not change the structure-function properties of the hexanoic acid moiety and the uptake of the Qß conjugates by macrophages.

Biodegradable Viral Nanoparticle/Polymer Implants Prepared via Melt-Processing.

Viral nanoparticles have been utilized as a platform for vaccine development and are a versatile system for the display of antigenic epitopes for a variety of disease states. However, the induction of a clinically relevant immune response often requires multiple injections over an extended period of time, limiting patient compliance. Polymeric systems to deliver proteinaceous materials have been extensively researched to provide sustained release, which would limit administration to a single dose. Melt-processing is an emerging manufacturing method that has been utilized to create polymeric materials laden with proteins as an alternative to typical solvent-based production methods. Melt-processing is advantageous because it is continuous, solvent-free, and 100% of the therapeutic protein is encapsulated. In this study, we utilized melt-encapsulation to fabricate viral nanoparticle laden polymeric materials that effectively deliver intact particles and generate carrier specific antibodies in vivo. The effects of initial processing and postprocessing on particle integrity and aggregation were studied to develop processing windows for scale-up and the creation of more complex materials. The dispersion of particles within the PLGA matrix was studied, and the effect of additives and loading level on the release profile was determined. Overall, melt-encapsulation was found to be an effective method to produce composite materials that can deliver viral nanoparticles over an extended period and elicit an immune response comparable to typical administration schedules.

Heparin Binding to an Engineered Virus-like Nanoparticle Antagonist.

The anticoagulant activity of heparin administered during medical interventions must be reversed to restore normal clotting, typically by titrating with protamine. Given the acute toxicity associated with protamine, we endeavored to generate safer heparin antagonists by engineering bacteriophage Qß virus-like particles (VLPs) to display motifs that bind heparin. A particle bearing a single amino acid change from wild-type (T18R) was identified as a promising candidate for heparin antagonism. Surface potential maps generated through molecular modeling reveal that the T18R mutation adds synergistically to adjacent positive charges on the particle surface, resulting in a large solvent-accessible cationic region that is replicated 180 times over the capsid. Chromatography using a heparin-sepharose column confirmed a strong interaction between heparin and the T18R particle. Binding studies using fluorescein-labeled heparin (HepFL) resulted in a concentration-dependent change in fluorescence intensity, which could be perturbed by the addition of unlabeled heparin. Analysis of the fluorescence data yielded a dissociation constant of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP. Dynamic light scattering (DLS) experiments suggested that T18R forms discrete complexes with heparin when the VLP:heparin molar ratios are equivalent, and in vitro clotting assays confirmed the 1:1 binding stoichiometry as full antagonism of heparin is achieved. Biolayer interferometry and backscattering interferometry corroborated the strong interaction of T18R with heparin, yielding Kd ∼ 1-10 nM. These biophysical measurements further validated T18R, and VLPs in general, for potential clinical use as effective, nontoxic heparin antagonists.

Fluorescent Functionalization across Quaternary Structure in a Virus-like Particle.

Proteinaceous nanomaterials and, in particular, virus-like particles (VLPs) have emerged as robust and uniform platforms that are seeing wider use in biomedical research. However, there are a limited number of bioconjugation reactions for functionalizing the capsids, and very few of those involve functionalization across the supramolecular quaternary structure of protein assemblies. In this work, we exploit the recently described dibromomaleimide moiety as part of a bioconjugation strategy on VLP Qß to break and rebridge the exposed and structurally important disulfides in good yields. Not only was the stability of the quaternary structure retained after the reaction, but the newly functionalized particles also became brightly fluorescent and could be tracked in vitro using a commercially available filter set. Consequently, we show that this highly efficient bioconjugation reaction not only introduces a new functional handle "between" the disulfides of VLPs without compromising their thermal stability but also can be used to create a fluorescent probe.

Highly Efficient Virus Rejection with Self-Organized Membranes Based on a Crosslinked Bicontinuous Cubic Liquid Crystal.

To remove viruses from water, the use of self-assembling liquid crystals is presented as a novel method for the synthesis of membranes with a regular pore size (below 1 nm) and controlled pore structures. Nanostructured bicontinuous cubic liquid-crystalline (LC) thin films are photopolymerized onto a polysulfone support layer. It is found that these membranes reject the virus, Qß bacteriophage (≈20 nm diameter) by >99.9999%. Prepressurization of the membrane appears to enhance their virus rejection properties. This is the first example of nanostructured LC membranes that are used for virus rejection, for which they show great potential.

Dual Functionalized Bacteriophage Qß as a Photocaged Drug Carrier.

Proteinatious nanoparticles are emerging as promising materials in biomedical research owing to their many unique properties and our interest focuses on integrating environmental responsivity into these systems. In this work, the use of a virus-like particle (VLP) derived from bacteriophage Qß as a photocaged drug delivery system is investigated. Ideally, a photocaged nanoparticle platform should be harmless and inert without activation by light yet, upon photoirradiation, should cause cell death. Approximately 530 photocleavable doxorubicin complexes are installed initially onto the surface of Qß by CuAAC reaction for photocaging therapy; however, aggregation and precipitation are found to cause cell death at higher concentrations. In order to improve solution stability, thiol-dibromomaleimide chemistry has been developed to orthogonally modify the VLP. This chemistry provides a robust method of incorporating additional functionality at the disulfides on Qß, which was used to increase the stability and solubility of the drug-loaded VLPs. As a result, the dual functionalied VLPs with polyethylene glycol and photocaged doxorubicin show not only negligible cytotoxicity before photoactivation but also highly controllable photorelease and cell killing power.

Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qß, and the Induction of Anticancer Antibodies.

The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qß. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qß, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qß control. In contrast, GM2 immobilized on Qß through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qß is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.

Directed polyvalent display of sulfated ligands on virus nanoparticles elicits heparin-like anticoagulant activity.

Heparin is a sulfated glycosaminoglycan that is widely used as an anticoagulant. It is typically extracted from porcine or bovine sources to yield a heterogeneous mixture that varies both in molecular weight and in degree of sulfation. This heterogeneity, coupled with concern for contamination, has led to widespread interest in developing safer alternatives. Described herein are sulfated bacteriophage Qß virus-like particles (VLPs) that elicit heparin-like anticoagulant activity. Sulfate groups were appended to the VLP by synthesis of single- and triple-sulfated ligands that also contained azide groups. Following conversion of VLP surface lysine groups to alkynes, the sulfated ligands were attached to the VLP via copper-catalyzed azide-alkyne cycloaddition (CuAAC). MALDI-MS analysis of the intermediate alkyne VLP indicated that the majority of the coat proteins contained 5-7 of the alkyne linkers; similar analysis of the intermediate alkyne particles conjugated to a fluorescein azide suggest that nearly the same number of attachment points (3-6) are modified via CuAAC. Analysis by SDS-PAGE with fluorescent staining indicated altered migration patterns for the various constructs: compared to the wild-type nanoparticle, the modified coat proteins appeared to migrate farther toward the positive pole in the gel, with coat proteins displaying the triple-sulfated ligand migrating significantly farther. Clotting activity analyzed by activated partial thrombin time (APTT) assay showed that the sulfated particles were able to perturb coagulation, with VLPs displaying the triple-sulfated ligand approximately as effective as heparin on a per mole basis; this activity could be partially reversed by protamine. ELISA experiments to assess the response of the complement system to the VLPs indicate that sulfating the particles may reduce complement activation.

Changes in protein domains outside the catalytic site of the bacteriophage Qß replicase reduce the mutagenic effect of 5-azacytidine.

UNLABELLED: The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qß replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qß replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCE: In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qß replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qß. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qß is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified.

Engineered virus-like nanoparticle heparin antagonists.

Virus nanoparticles provide a self-assembling, reproducible multivalent platform that can be chemically and genetically manipulated for the presentation of a wide array of epitopes. Presented herein are engineered bacteriophage Qß nanoparticles that function as potent heparin antagonists. Three successful approaches have been used: 1) chemically appending poly-Arg peptides; 2) point mutations to Arg on the virus capsid; 3) incorporation of heparin-binding peptides displayed externally on the virus surface. Each approach generates particles with good heparin antagonist activity with none of the toxic side effects of protamine, the only drug currently FDA-approved for clinical use as a heparin antagonist.