Enhanced HPV16 E6/E7+ tumor eradication via induction of tumor-specific T cells by therapeutic vaccination with virosomes presenting synthetic long peptides.

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

A polyvalent virosomal influenza vaccine induces broad cellular and humoral immunity in pigs.

BACKGROUND: Influenza A virus (IAV) is endemic in pigs globally and co-circulation of genetically and antigenically diverse virus lineages of subtypes H1N1, H1N2 and H3N2 is a challenge for the development of effective vaccines. Virosomes are virus-like particles that mimic virus infection and have proven to be a successful vaccine platform against several animal and human viruses. METHODS: This study evaluated the immunogenicity of a virosome-based influenza vaccine containing the surface glycoproteins of H1N1 pandemic, H1N2 and H3N2 in pigs. RESULTS: A robust humoral and cellular immune response was induced against the three IAV subtypes in pigs after two vaccine doses. The influenza virosome vaccine elicited hemagglutinin-specific antibodies and virus-neutralizing activity. Furthermore, it induced a significant maturation of macrophages, and proliferation of B lymphocytes, effector and central memory CD4+ and CD8+ T cells, and CD8+ T lymphocytes producing interferon-γ. Also, the vaccine demonstrated potential to confer long-lasting immunity until the market age of pigs and proved to be safe and non-cytotoxic to pigs. CONCLUSIONS: This virosome platform allows flexibility to adjust the vaccine content to reflect the diversity of circulating IAVs in swine in Brazil. The vaccination of pigs may reduce the impact of the disease on swine production and the risk of swine-to-human transmission.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine.

BACKGROUND: Influenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility. METHODS: This study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated. RESULTS: Only intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens. CONCLUSIONS: All findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Innovative Phospholipid Carriers: A Viable Strategy to Counteract Antimicrobial Resistance.

The overuse and misuse of antibiotics have led to the emergence and spread of multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) bacteria strains, usually associated with poorer patient outcomes and higher costs. In order to preserve the usefulness of these life-saving drugs, it is crucial to use them appropriately, as also recommended by the WHO. Moreover, innovative, safe, and more effective approaches are being investigated, aiming to revise drug treatments to improve their pharmacokinetics and distribution and to reduce the onset of drug resistance. Globally, to reduce the burden of antimicrobial resistance (AMR), guidelines and indications have been developed over time, aimed at narrowing the use and diminishing the environmental spread of these life-saving molecules by optimizing prescriptions, dosage, and times of use, as well as investing resources into obtaining innovative formulations with better pharmacokinetics, pharmacodynamics, and therapeutic results. This has led to the development of new nano-formulations as drug delivery vehicles, characterized by unique structural properties, biocompatible natures, and targeted activities such as state-of-the-art phospholipid particles generally grouped as liposomes, virosomes, and functionalized exosomes, which represent an attractive and innovative delivery approach. Liposomes and virosomes are chemically synthesized carriers that utilize phospholipids whose nature is predetermined based on their use, with a long track record as drug delivery systems. Exosomes are vesicles naturally released by cells, which utilize the lipids present in their cellular membranes only, and therefore, are highly biocompatible, with investigations as a delivery system having a more recent origin. This review will summarize the state of the art on microvesicle research, liposomes, virosomes, and exosomes, as useful and effective tools to tackle the threat of antibiotic resistance.

Erythro-Magneto-HA-Virosome: A Bio-Inspired Drug Delivery System for Active Targeting of Drugs in the Lungs.

Over the past few decades, finding more efficient and selective administration routes has gained significant attention due to its crucial role in the bioavailability, absorption rate and pharmacokinetics of therapeutic substances. The pulmonary delivery of drugs has become an attractive target of scientific and biomedical interest in the health care research area, as the lung, thanks to its high permeability and large absorptive surface area and good blood supply, is capable of absorbing pharmaceuticals either for local deposition or for systemic delivery. Nevertheless, the pulmonary drug delivery is relatively complex, and strategies to mitigate the effects of mechanical, chemical and immunological barriers are required. Herein, engineered erythrocytes, the Erythro-Magneto-Hemagglutinin (HA)-virosomes (EMHVs), are used as a novel strategy for efficiently delivering drugs to the lungs. EMHV bio-based carriers exploit the physical properties of magnetic nanoparticles to achieve effective targeting after their intravenous injection thanks to an external magnetic field. In addition, the presence of hemagglutinin fusion proteins on EMHVs' membrane allows the DDS to anchor and fuse with the target tissue and locally release the therapeutic compound. Our results on the biomechanical and biophysical properties of EMHVs, such as the membrane robustness and deformability and the high magnetic susceptibility, as well as their in vivo biodistribution, highlight that this bio-inspired DDS is a promising platform for the controlled and lung-targeting delivery of drugs, and represents a valuable alternative to inhalation therapy to fulfill unmet clinical needs.

Niosomal virosome derived by vesicular stomatitis virus glycoprotein as a new gene carrier.

Virosomes as membranous vesicles with viral fusion protein in their membrane are versatile vehicles for cargo delivery. The vesicular stomatitis virus glycoprotein (VSV-G) is a common fusogenic protein used in virosome preparation. This glycoprotein has been used in liposomal systems so far, but in this study, we have tried to use the niosomal form instead of liposome for. Niosomes are vesicular systems composed of non-ionic surfactants. Niosomes were constructed by the thin-film hydration method. VSV-G gene in pMD2.G plasmid was expressed in the HEK293T cell line and then was reconstituted in the niosome bilayer. The formation of niosomal virosomes was confirmed with different methods such as SDS-PAGE gel, western blotting, and transmission electron microscopy (TEM). The efficiency of niosomal virosome was investigated with the pmCherry reporter gene. SDS-PAGE and western blotting proved the expression and successful insertion of protein into the bilayer. The TEM images showed the spike projection of VSV-G on the surface of niosomes. The transfection results showed high efficiency of niosomal virosomes as a novel carrier. This report has verified that niosome could be used as an efficient bilayer instead of liposome to construct virosomes.

Bet v 1 contiguous overlapping peptides anchored to virosomes with TLR4 agonist enhance immunotherapy efficacy in mice.

BACKGROUND: Whereas sublingual allergen immunotherapy (AIT) is routinely performed without any adjuvant or delivery system, there is a strong scientific rationale to better target the allergen(s) to oral dendritic cells known to support regulatory immune responses by using appropriate presentation platforms. OBJECTIVE: To identify a safe presentation platform able to enhance allergen-specific tolerance induction. METHODS: Virosomes with membrane-integrated contiguous overlapping peptides (COPs) of Bet v 1 and TLR4 or TLR2/TLR7 agonists were assessed for induction of Bet v 1-specific IgG1, IgG2a and IgE antibodies, hypersensitivity reactions and body temperature drop following subcutaneous injection in naive CD-1 mice. The most promising candidate, Bet v 1 COPs anchored to virosomes with membrane-incorporated TLR4 agonist (Vir.A-Bet v 1 COPs), was further evaluated by the sublingual route in a therapeutic setting in BALB/c mice with birch pollen-induced allergic asthma. Airway hyperresponsiveness, pro-inflammatory cells in bronchoalveolar lavages and polarization of Th cells in the lungs and spleen were then assessed. RESULTS: Both types of adjuvanted virosomes coupled to Bet v 1 COPs triggered a boosted Th1 immunity. Given a more favourable safety profile, Vir.A-Bet v 1 COPs were further evaluated and shown to able to fully reverse asthma symptoms and lung inflammation in a sublingual therapeutic model of birch pollen allergy. CONCLUSIONS AND CLINICAL RELEVANCE: We report herein for the first time on the capacity of a novel and safe presentation platform, that is virosomes with membrane-integrated TLR4 agonist, to improve dramatically sublingual AIT efficacy in a murine model due to its intrinsic dual properties of targeting and stimulating to further promote anti-allergic immune responses. As such, our study paves the ground for further clinical development of this allergen presentation platform for patients suffering from respiratory allergies.

Correlative imaging using super-resolution fluorescence microscopy and soft X-ray tomography at cryogenic temperatures provides a new way to assess virosome solutions for vaccine development.

Active virosomes (AVs) are derivatives of viruses, broadly similar to 'parent' pathogens, with an outer envelope that contains a bespoke genome coding for four to five viral proteins capable of eliciting an antigenic response. AVs are essentially novel vaccine formulations that present on their surface selected viral proteins as antigens. Once administered, they elicit an initial 'anti-viral' immune response. AVs are also internalised by host cells where their cargo viral genes are used to express viral antigen(s) intracellularly. These can then be transported to the host cell surface resulting in a second wave of antigen exposure and a more potent immuno-stimulation. A new 3D correlative microscopy approach is used here to provide a robust analytical method for characterisation of Zika- and Chikungunya-derivatised AV populations including vesicle size distribution and variations in antigen loading. Manufactured batches were compared to assess the extent and nature of batch-to-batch variations. We also show preliminary results that verify antigen expression on the surface of host cells. We present here a reliable and efficient high-resolution 3D imaging regime that allows the evaluation of the microstructure and biochemistry of novel vaccine formulations such as AVs.

A novel combination of microscopies involving X-ray and laser light has been developed at the correlative cryo-imaging beamline B24 of the UK synchrotron which can be used to analyse across- and within-batch variability of active virosome vaccine formulations. We use 3D fluorescence imaging to localise viral components within vaccine vesicles and soft X-ray tomography to characterise sample variability and impact upon delivery to cells. Moreover, we offer the next step in automation of data processing and evaluation to further enable rapid assessment of exosome-based vaccines. Active virosome vaccines are suspensions of membrane-bounded vesicles that carry antigens and genetic material from select viral pathogens. These elicit both an initial immune response through their introduction and a subsequent sustained antigenic potential via gene expression in host cells. In this case, as in all novel vaccine formulations, rapid assessment and batch standardisation are of paramount importance for the medical community and the methods described here provide a robust way of quick and efficient assessment and validation of formulations during research and development and at the production stages.

Protease-Activated Receptor 2 Agonist as Adjuvant: Augmenting Development of Protective Memory CD8 T Cell Responses Induced by Influenza Virosomes.

Protease-activated receptor 2 (PAR-2) is expressed in various tissues, including lung, and when activated, promotes inflammation, differentiation, and migration of dendritic cells. We found that combining influenza virosomes containing hemagglutinin and neuraminidase with a PAR-2 agonist peptide (PAR-2AP) in an intranasal prime boost approach increased survival of mice challenged weeks later with lethal influenza virus over that by virosome or PAR-2AP prime boost alone. No weight loss occurred from influenza challenge after virosome-plus-PAR-2AP prime boost compared with either virosomes or PAR-2AP alone. Thus, virosomes plus PAR-2AP prevented morbidity as well as mortality. Through adoptive transfer, CD8+ lung T cells but not CD4+ T cells from virosomes plus PAR-2AP-primed mice protected from lethal influenza virus challenge and enhanced survival with less weight loss and faster recovery. Virosome-plus-PAR-2AP prime boost resulted in greater percentages of T effector memory phenotype cells (Tem) in lung, and higher frequencies of CD8 Tem and T central memory cells displayed effector functions in response to virus challenge in vivo. Virosome-plus-PAR-2AP prime boost also resulted in greater percentages of Ag-specific CD8+ T cells, both Tem and T central memory cells, in lungs of animals subsequently challenged with live influenza virus. Our findings indicate that PAR-2AP, a short peptide, may be a new and useful mucosal adjuvant.

Comparison of Virosome vs. Liposome as drug delivery vehicle using HepG2 and CaCo2 cell lines.

AIM: The present work involves encapsulation of herbal drug nanocurcumin into the virosomes and compared with a liposome in terms of their in vitro anti-proliferative, anti-inflammatory, and anti-migratory efficacy. METHODS: The anti-proliferative, anti-inflammatory, and anti-migratory efficacy of virosome and liposome were compared in HepG2 and CaCo2 cells by using MTT, Nitric oxide scavenging, and Wound healing assay, respectively. RESULTS: Size of the optimised NC-Virosome and NC-Liposome was 70.06 ± 1.63 and 265.80 ± 1.64 nm, respectively. The prepared NC-Virosome can be stored at -4 °C up to six months. The drug encapsulation efficiency of NC-Virosome and NC-Liposome was found to be 84.66 ± 1.67 and 62.15 ± 1.75% (w/w). The evaluated minimum inhibitory concentration (IC50 value) for NC-Virosome was 102.7 µg/ml and 108.1 µg/ml, while NC-Liposome showed 129.2 µg/ml and 160.1 µg/ml for HepG2 and CaCo2 cells, respectively. Morphological examination depicts detachment of the cells from substratum after exposure to NC-Virosome for 48 h. CONCLUSION: The prepared NC-Virosome provides remarkable in vitro efficacy in both the cell lines with site-specific drug-targeting potential as compared to the liposome, results proved its potential as a drug delivery vehicle for future therapy with reduced toxicity.

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.

Development of pepper vein banding virus chimeric virus-like particles for potential diagnostic and therapeutic applications.

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

Nanoparticles in dermatologic surgery.

Nanotechnology is an emerging branch of science that involves the engineering of functional systems on the nanoscale (1-100 nm). Nanotechnology has been used in biomedical and therapeutic agents with the aim of providing novel treatment solutions where small molecule size may be beneficial for modulation of biologic function. Recent investigation in nanomedicine has become increasingly important to cutaneous pathophysiology, such as functional designs directed towards skin cancers and wound healing. This review outlines the application of nanoparticles relevant to dermatologic surgery.

Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.

Dense Array of Spikes on HIV-1 Virion Particles.

HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ∼7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (P = 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of "cell factory" selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV spikes were screened on the basis of high binding by bnAbs and low binding by nonneutralizing antibodies. Levels of spikes on cells correlated well with those on progeny virions. Importantly, high-Env virus-like particles (hVLPs) were produced with a manifest array of well-defined spikes, and these were shown to be superior in activating desirable B cells. Our study describes HIV particles that are densely coated with functional spikes, which should facilitate the study of HIV spikes and their development as immunogens.

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions.

Human papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells.IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

Virus-Like Vesicles of Kaposi's Sarcoma-Associated Herpesvirus Activate Lytic Replication by Triggering Differentiation Signaling.

Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication.IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the small capsid protein. We quantitatively analyzed proteins and microRNAs in VLVs and characterized the roles of VLVs in manipulating host cells and facilitating viral infection. More importantly, we demonstrated that by upregulating PRDM1 expression, VLVs triggered differentiation signaling in targeted cells and facilitated viral lytic infection via activation of the RTA promoter. Our study not only demonstrates a new strategy for isolating VLVs but also shows the important roles of KSHV-associated VLVs in intercellular communication and the viral life cycle.

Characterization of a Norovirus-specific monoclonal antibody that exhibits wide spectrum binding activities.

Noroviruses (NoVs) are increasingly recognized as the leading cause of acute non-bacterial gastroenteritis worldwide. To screen for NoV-specific monoclonal antibodies (mAbs) with wide spectrum binding activities that could be used for the development of NoV-related detection reagents, we immunized mice with a combination of virus like particles (VLPs) derived from eight different genotypes (two from genogroup I and six from genogroup II), of which two (GI.7 and GII.2) were newly produced VLPs. Indirect enzyme-linked immunosorbent assay (ELISA) confirmed that two mAbs (8D8 and 10B11) bound to all eight major capsid proteins (VP1) with varied binding abilities. Epitope mapping using short peptides covering the N-terminal half of GII.3 VP1 indicated that the binding site of mAb 8D8 was located between amino acid 31 and 60. Multiple amino acid sequence alignment of VP1 suggested that this site harbors conservative sequences across all genogroups. Indirect and sandwich ELISA indicated that mAb 8D8 was unable bind intact VLPs. In summary, we successfully produced GI.7 and GII.2 VLPs using recombinant baculovirus expression system and a cross-reactive mAb by immunizing mice with eight different VLPs that might be useful in the studying and detecting NoVs.

Cargo Retention inside P22 Virus-Like Particles.

Viral protein cages, with their regular and programmable architectures, are excellent platforms for the development of functional nanomaterials. The ability to transform a virus into a material with intended structure and function relies on the existence of a well-understood model system, a noninfectious virus-like particle (VLP) counterpart. Here, we study the factors important to the ability of P22 VLP to retain or release various protein cargo molecules depending on the nature of the cargo, the capsid morphology, and the environmental conditions. Because the interaction between the internalized scaffold protein (SP) and the capsid coat protein (CP) is noncovalent, we have studied the efficiency with which a range of SP variants can dissociate from the interior of different P22 VLP morphologies and exit by traversing the porous capsid. Understanding the types of cargos that are either retained or released from the P22 VLP will aid in the rational design of functional nanomaterials.