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.

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.

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.

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.

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.

Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission.

UNLABELLED: Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE: Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments.

Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry.

UNLABELLED: ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1(+) endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env. IMPORTANCE: The recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed-embedded in the membrane and present at high density-on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

An Ebola Virus-Like Particle-Based Reporter System Enables Evaluation of Antiviral Drugs In Vivo under Non-Biosafety Level 4 Conditions.

UNLABELLED: Ebola virus (EBOV) is a highly contagious lethal pathogen. As a biosafety level 4 (BSL-4) agent, however, EBOV is restricted to costly BSL-4 laboratories for experimentation, thus significantly impeding the evaluation of EBOV vaccines and drugs. Here, we report an EBOV-like particle (EBOVLP)-based luciferase reporter system that enables the evaluation of anti-EBOV agents in vitro and in vivo outside BSL-4 facilities. Cotransfection of HEK293T cells with four plasmids encoding the proteins VP40, NP, and GP of EBOV and firefly luciferase (Fluc) resulted in the production of Fluc-containing filamentous particles that morphologically resemble authentic EBOV. The reporter EBOVLP was capable of delivering Fluc into various cultured cells in a GP-dependent manner and was recognized by a conformation-dependent anti-EBOV monoclonal antibody (MAb). Significantly, inoculation of mice with the reporter EBOVLP led to the delivery of Fluc protein into target cells and rapid generation of intense bioluminescence signals that could be blocked by the administration of EBOV neutralizing MAbs. This BSL-4-free reporter system should facilitate high-throughput screening for anti-EBOV drugs targeting viral entry and efficacy testing of candidate vaccines. IMPORTANCE: Ebola virus (EBOV) researches have been limited to costly biosafety level 4 (BSL-4) facilities due to the lack of animal models independent of BSL-4 laboratories. In this study, we reveal that a firefly luciferase-bearing EBOV-like particle (EBOVLP) with typical filamentous EBOV morphology is capable of delivering the reporter protein into murine target cells both in vitro and in vivo Moreover, we demonstrate that the reporter delivery can be inhibited both in vitro and in vivo by a known anti-EBOV protective monoclonal antibody, 13C6. Our work provides a BSL-4-free system that can facilitate the in vivo evaluation of anti-EBOV antibodies, drugs, and vaccines. The system may also be useful for mechanistic study of the viral entry process.

Characterization of a hepatitis C virus-like particle vaccine produced in a human hepatocyte-derived cell line.

An effective immune response against hepatitis C virus (HCV) requires the early development of multi-specific class 1 CD8+ and class II CD4+ T-cells together with broad neutralizing antibody responses. We have produced mammalian-cell-derived HCV virus-like particles (VLPs) incorporating core, E1 and E2 of HCV genotype 1a to produce such immune responses. Here we describe the biochemical and morphological characterization of the HCV VLPs and study HCV core-specific T-cell responses to the particles. The E1 and E2 glycoproteins in HCV VLPs formed non-covalent heterodimers and together with core protein assembled into VLPs with a buoyant density of 1.22 to 1.28 g cm-3. The HCV VLPs could be immunoprecipited with anti-ApoE and anti-ApoC. On electron microscopy, the VLPs had a heterogeneous morphology and ranged in size from 40 to 80 nm. The HCV VLPs demonstrated dose-dependent binding to murine-derived dendritic cells and the entry of HCV VLPs into Huh7 cells was blocked by anti-CD81 antibody. Vaccination of BALB/c mice with HCV VLPs purified from iodixanol gradients resulted in the production of neutralizing antibody responses while vaccination of humanized MHC class I transgenic mice resulted in the prodution of HCV core-specific CD8+ T-cell responses. Furthermore, IgG purified from the sera of patients chronically infected with HCV genotypes 1a and 3a blocked the binding and entry of the HCV VLPs into Huh7 cells. These results show that our mammalian-cell-derived HCV VLPs induce humoral and HCV-specific CD8+ T-cell responses and will have important implications for the development of a preventative vaccine for HCV.

Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles.

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone.

Antigenic and Cryo-Electron Microscopy Structure Analysis of a Chimeric Sapovirus Capsid.

UNLABELLED: The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further subdivided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain; consequently, the P domain is divided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67-229) and P1-1 (229-280), P2 (281-447), and P1-2 (448-567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes. IMPORTANCE: We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.

Enhanced enterovirus 71 virus-like particle yield from a new baculovirus design.

Enterovirus 71 (EV71) is responsible for the outbreaks of hand-foot-and-mouth disease in the Asia-Pacific region. To produce the virus-like particle (VLP) vaccine, we previously constructed recombinant baculoviruses to co-express EV71 P1 polypeptide and 3CD protease using the Bac-to-Bac(®) vector system. The recombinant baculoviruses resulted in P1 cleavage by 3CD and subsequent VLP assembly in infected insect cells, but caused either low VLP yield or excessive VLP degradation. To tackle the problems, here we explored various expression cassette designs and flashBAC GOLD™ vector system which was deficient in v-cath and chiA genes. We found that the recombinant baculovirus constructed using the flashBAC GOLD™ system was insufficient to improve the EV71 VLP yield. Nonetheless, BacF-P1-C3CD, a recombinant baculovirus constructed using the flashBAC GOLD(TM) system to express P1 under the polh promoter and 3CD under the CMV promoter, dramatically improved the VLP yield while alleviating the VLP degradation. Infection of High Five(TM) cells with BacF-P1-C3CD enhanced the total and extracellular VLP yield to ≈268 and ≈171 mg/L, respectively, which enabled the release of abundant VLP into the supernatant and simplified the downstream purification. Intramuscular immunization of mice with 5 µg purified VLP induced cross-protective humoral responses and conferred protection against lethal virus challenge. Given the significantly improved extracellular VLP yield (≈171 mg/L) and the potent immunogenicity conferred by 5 µg VLP, one liter High Five(TM) culture produced ≈12,000 doses of purified vaccine, thus rendering the EV71 VLP vaccine economically viable and able to compete with inactivated virus vaccines.

Diagnostic potential and antigenic properties of recombinant tick-borne encephalitis virus subviral particles expressed in mammalian cells from Semliki Forest virus replicons.

The precursor membrane envelope (prME) proteins of all three tick-borne encephalitis virus (TBEV) subtypes were produced based on expression from Semliki Forest virus (SFV) replicons transcribed from recombinant plasmids. Vero E6 cells transfected by these plasmids showed specific reactivities in immunofluorescence and immunoblot assays by monoclonal antibodies against European and Far-Eastern subtype strains of TBEV, indicating proper folding of the expressed glycoproteins. The prME glycoproteins were secreted into the cell culture supernatant, forming TBEV subviral particles of 20 to 30 nm in diameter. IgM µ-capture and IgG monoclonal antibody (MAb)-capture enzyme immunoassays (EIAs) were developed based on prME Karelia-94 (Siberian subtype) particles. Altogether, 140 human serum samples were tested using these assays, and the results were compared to those obtained with a commercial IgM EIA, an in-house µ-capture IgM assay based on baculovirus-expressed antigen, a commercial IgG EIA, and a hemagglutination inhibition test. Compared to reference enzyme-linked immunosorbent assays (ELISAs), the sensitivities of the generated µ-capture IgM SFV-prME and IgG MAb-capture SFV-prME EIAs were 97.4 to 100% and 98.7%, respectively, and the specificities of the two assays were 100%. IgM and IgG immunofluorescence assays (IFAs) were created based on Vero E6 cells transfected with the recombinant plasmid carrying the TBEV Karelia-94 prME glycoproteins. The IgM IFA was 100% concordant with the µ-capture IgM bac-prME ELISA. The IgG IFA sensitivity and specificity were 98.7% and 100%, respectively, compared to those of the commercial ELISA. In conclusion, the tests developed based on SFV replicon-driven expression of TBEV glycoproteins provide safe and robust alternatives for conducting TBEV serology.

Topological analysis of HIV-1 glycoproteins expressed in situ on virus surfaces reveals tighter packing but greater conformational flexibility than for soluble gp120.

In natural infection, antibodies interact with HIV-1 primarily through nonfunctional forms of envelope glycoproteins (Env), including uncleaved (UNC) gp160 and gp41 stumps. These antigens are important to fully characterize, as they may be decoys that promote nonneutralizing responses and may also be targets for nonneutralizing effector responses. In this study, we compared the antigenic properties of Env expressed in situ on pseudovirion virus-like particle (VLP) surfaces and soluble gp120 using harmonized enzyme-linked immunosorbent assays (ELISAs) and a panel of 51 monoclonal antibodies (MAbs). Only 32 of 46 soluble gp120-reactive MAbs recognized the primary UNC gp160 antigen of VLPs. Indeed, many epitopes were poorly exposed (C1, V2, C1-C4, C4, C4-V3, CD4 induced [CD4i], and PGT group 3) or obscured (C2, C5, and C1-C5) on VLPs. In further studies, VLP Env exhibited an increased degree of inter-MAb competition, the epicenter of which was the base of the V3 loop, where PGT, 2G12, V3, and CD4 binding site specificities competed. UNC gp160 also underwent more drastic soluble CD4 (sCD4)-induced conformational changes than soluble gp120, exposing CD4i, C1-C4, and V2 epitopes. A greater propensity of UNC gp160 to undergo conformational changes was also suggested by the induction of CD4i MAb binding to VLPs by a V3 MAb as well as by soluble CD4. The same effect was not observed for soluble gp120. Taken together, our data suggest that membrane-expressed UNC gp160 exists in a less "triggered" conformational state than soluble gp120 and that MAb binding to UNC gp160 tends to have greater conformational consequences.

Subunit and virus-like particle vaccine approaches for respiratory syncytial virus.

Despite its impact on global health, there is no vaccine available for the prevention of respiratory syncytial virus (RSV) infection. Failure to develop a licensed vaccine is not due to lack of effort, as numerous vaccine candidates have been characterized in preclinical and clinical studies spanning five decades. The vaccine candidates thus far explored can be generally divided into four categories: (1) whole inactivated virus, (2) replication competent, attenuated virus including recombinant viruses, (3) gene-based vectors, and (4) subunit and particulate forms of RSV antigens. The first clinically tested RSV vaccine candidate was a formalin-inactivated purified virus preparation administered to infants and children in the late 1960s. Due to the disastrous outcome of these trials and results of animal models investigating the mechanisms involved, there have been no further studies with inactivated RSV vaccines. Rather, efforts have focused on development of other approaches. In this chapter, we review the history and status of purified proteins, peptides, virus-like particles, virosomes, and nanoparticles and discuss their future potential.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

BACKGROUND: The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. METHODS: To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. RESULTS: Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. CONCLUSION: Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

Ultrastructural, immunofluorescence, and RNA evidence support the hypothesis of a "new" virus associated with Kawasaki disease.

BACKGROUND: Intracytoplasmic inclusion bodies (ICI) have been identified in ciliated bronchial epithelium of Kawasaki disease (KD) patients using a synthetic antibody derived from acute KD arterial IgA plasma cells; ICI may derive from the KD etiologic agent. METHODS: Acute KD bronchial epithelium was subjected to immunofluorescence for ICI and cytokeratin, high-throughput sequencing, and transmission electron microscopy (TEM). Interferon pathway gene expression profiling was performed on KD lung. RESULTS: An intermediate filament cytokeratin "cage" was not observed around KD ICI, making it unlikely that ICI are overproduced or misfolded human protein aggregates. Many interferon-stimulated genes were detected in the bronchial epithelium, and significant modulation of the interferon response pathway was observed in the lung tissue of KD patients. No known virus was identified by sequencing. Aggregates of virus-like particles (VLP) were detected by TEM in all 3 acute KD patients from whom nonembedded formalin-fixed lung tissue was available. CONCLUSIONS: KD ICI are most likely virus induced; bronchial cells with ICI contain VLP that share morphologic features among several different RNA viral families. Expedited autopsies and tissue fixation from acute KD fatalities are urgently needed to more clearly ascertain the VLP. These findings are compatible with the hypothesis that the infectious etiologic agent of KD may be a "new" RNA virus.

Assembly and biological and immunological properties of Newcastle disease virus-like particles.

Virus-like particles (VLPs) released from avian cells expressing the Newcastle disease virus (NDV) strain AV proteins NP, M, HN (hemagglutinin-neuraminidase), and F were characterized. The VLP-associated HN and F glycoproteins directed the attachment of VLPs to cell surfaces and fusion of VLP membranes with red blood cell membranes, indicating that they were assembled into VLPs in an authentic conformation. These particles were quantitatively prepared and used as an immunogen, without adjuvant, in BALB/c mice. The resulting immune responses, detected by enzyme-linked immunosorbent assay (ELISA), virus neutralization, and intracellular cytokine staining, were comparable to the responses to equivalent amounts of inactivated NDV vaccine virus. HN and F proteins from another strain of NDV, strain B1, could be incorporated into these VLPs. Foreign peptides were incorporated into these VLPs when fused to the NP or HN protein. The ectodomain of a foreign glycoprotein, the Nipah virus G protein, fused to the NDV HN protein cytoplasmic and transmembrane domains was incorporated into ND VLPs. Thus, ND VLPs are a potential NDV vaccine candidate. They may also serve as a platform to construct vaccines for other pathogens.

Induction of humoral and cellular immune responses against the HIV-1 envelope protein using γ-retroviral virus-like particles.

This study evaluates the immunogenicity of the HIV envelope protein (env) in mice presented either attached to γ-retroviral virus-like-particles (VLPs), associated with cell-derived microsomes or as solubilized recombinant protein (gp160). The magnitude and polyfunctionality of the cellular immune response was enhanced when delivering HIV env in the VLP or microsome form compared to recombinant gp160. Humoral responses measured by antibody titres were comparable across the groups and low levels of antibody neutralization were observed. Lastly, we identified stronger IgG2a class switching in the two particle-delivered antigen vaccinations modalities compared to recombinant gp160.

A novel HPV 16 L1-based chimeric virus-like particle containing E6 and E7 seroreactive epitopes permits highly specific detection of antibodies in patients with CIN 1 and HPV-16 infection.

BACKGROUND: The presence of IgG antibodies to HPV-16 L1-virus like particles (VLPs) in serum has been reported as a result of persistent exposure to the virus and as a marker of disease progression. However, detection of VLP-specific antibodies in sera does not always indicate a malignant lesion as positive results may also be due to a nonmalignant viral infection. Furthermore, malignant lesions are associated with an increased antibody titer for E6 and E7 proteins. The aim of this study was to develop an ELISA using a novel chimeric virus-like particle (cVLP) encoding an L1 protein fused with a string of HPV-16 E6 and E7 seroreactive epitopes to its C-terminus to be used for detection of HPV-16 specific antibodies in patients with cervical intraepithelial lesion grade 1 (CIN 1). RESULTS: The sera of 30 patients with CIN 1 who also tested positive for HPV-16 DNA and of 30 age-matched normal donors negative for HPV infection were tested for the presence of IgG antibodies specific for either VLP-L1 (HPV-16 L1), gVLP (derived from Gardasil), or cVLP by ELISA. The cVLP-reactive sera yielded two distinct groups of results: (H) reactivity levels that presented very strong cVLP-specific titers, and (L) reactivity levels with significantly lower titers similar to those obtained with VLP-L1 and gVLP antigens. Additionally, the sera that presented the higher cVLP titers closely matched those that had significantly stronger reactivity to E6 and E7 epitopes. Interestingly, the samples with the highest titers corresponded to patients with the higher numbers of sexual partners and pregnancies. On the other hand only 4 out of the 12 sera that harbored antibodies with VLP neutralizing ability corresponded to the group with high cVLP antibody titers. CONCLUSION: We report for the first time that chimeric particles containing HPV-16 L1 protein fused with E6 and E7 seroreactive epitopes enable much better detection of IgG antibodies in the sera of CIN 1 patients positive for HPV-16 infection than those obtained with VLPs containing only the HPV-16 L1 protein. We also found that the sera with higher cVLP antibody titers corresponded to patients with more sexual partners and pregnancies, and not always with to those with a high neutralizing activity. This novel assay could help in the development of a tool to evaluate cervical cancer risk.