SciCombinator

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Concept: Virus-like particle

141

Infection with Kaposi sarcoma-associated herpesvirus (KSHV) is estimated to account for over 44,000 new cases of Kaposi sarcoma annually, with 84% occurring in Africa, where the virus is endemic. To date, there is no prophylactic vaccine against KSHV. KSHV gpK8.1, gB, and gH/gL glycoproteins, implicated in the virus entry into host cells, are attractive vaccine targets for eliciting potent neutralizing antibodies (nAbs) against virus infection. We incorporated gpK8.1, gB, or gH/gL on the surface of virus-like particles (VLPs) and characterized these VLPs for their composition, size, and functionality. To determine which viral glycoprotein(s) elicit the most effective serum-nAbs, we immunized BALB/c mice with gpK8.1, gB, or gH/gL VLPs individually or in combination. Neutralizing antibody assay revealed that sera from mice immunized with the VLPs inhibited KSHV infection of HEK-293 cells in a dose-dependent manner. As a single immunogen, gpK8.1 VLPs stimulated comparable nAb activity to that of UV-inactivated KSHV (UV-KSHV). In contrast, UV-KSHV stimulated higher titers of nAb compared to gB (p = 0.0316) or gH/gL (p = 0.0486). Mice immunized with the combination of gB and gH/gL VLPs had a better nAb response than those immunized with either gB (p = 0.0268), or gH/gL (p = 0.0397) as single VLP immunogens. Immunization with any VLP combination stimulated comparable nAb activity to UV-KSHV serum. Our data provide the first evidence that KSHV gpK8.1, gB, and gH/gL glycoproteins can be incorporated onto the surface of VLPs and used as prophylactic vaccine candidates, with potential to prevent KSHV infection.

Concepts: AIDS, Immune system, Antibody, Neutralizing antibody, Vaccine, Antigen, Kaposi's sarcoma-associated herpesvirus, Virus-like particle

141

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

Concepts: Immune system, Antibody, Protein, Microbiology, Vaccine, Vaccination, Antigen, Virus-like particle

77

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.Despite the success of current vaccination against poliomyelitis, safe, cheap and effective vaccines remain sought for continuing eradication effort. Here the authors use plants to express stabilized virus-like particles of type 3 poliovirus that can induce a protective immune response in mice transgenic for the human poliovirus receptor.

Concepts: Immune system, Infectious disease, Vaccine, Vaccination, Poliomyelitis, Polio vaccine, Virus-like particle, Poliovirus

63

Several Zika virus (ZIKV) vaccine candidates have recently been described which use inactivated whole virus, DNA or RNA that express the virus' Envelope (E) glycoprotein as the antigen. These were successful in stimulating production of virus-targeted antibodies that protected animals against ZIKV challenges, but their use potentially will predispose vaccinated individuals to infection by the related Dengue virus (DENV). We have devised a virus like particle (VLP) carrier based on the hepatitis B core antigen (HBcAg) that displays the ZIKV E protein domain III (zDIII), and shown that it can be produced quickly and easily purified in large quantities from Nicotiana benthamiana plants. HBcAg-zDIII VLPs are shown to be highly immunogenic, as two doses elicited potent humoral and cellular responses in mice that exceed the threshold correlated with protective immunity against multiple strains of Zika virus. Notably, HBcAg-zDIII VLPs-elicited antibodies did not enhance the infection of DENV in Fc gamma receptor-expressing cells, offsetting the concern of ZIKV vaccines inducing cross-reactive antibodies and sensitizing people to subsequent DENV infection. Thus, our zDIII-based vaccine offers improved safety and lower cost production than other current alternatives, with equivalent effectiveness.

Concepts: Immune system, Antibody, Bacteria, Virus, Vaccine, Vaccination, Immunology, Virus-like particle

58

Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components.

Concepts: Immune system, Antibody, Bacteria, Microbiology, Vaccine, Antigen, Polysaccharide, Virus-like particle

51

Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunisation programmes using live-attenuated oral (OPV) and/or inactivated PV vaccines (IPV) have systematically reduced its spread and paved the way for eradication. Immunisation will continue post-eradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. These could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines which mimic the ‘empty’ capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert to an alternative conformation unsuitable for vaccine purposes. Stabilised ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we have selected thermally-stable ECs of type-1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild type (wt) virion. We have identified mutations on the capsid surface and internal networks that are responsible for the EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilised VLPs could be used as novel vaccines.

Concepts: Immune system, Infectious disease, Microbiology, Vaccine, Vaccination, Infection, Polio vaccine, Virus-like particle

28

We show that viruslike particles (VLPs) reassembled in vitro with the RNA bacteriophage MS2 coat protein and an RNA conjugate encompassing a siRNA and a known capsid assembly signal can be targeted to HeLa cells by covalent attachment of human transferrin. The siRNA VLPs protect their cargoes from nuclease, have a double-stranded conformation in the capsid and carry multiple drug and targeting ligands. The relative efficiency of VLP reassembly has been assessed, and conditions have been determined for larger scale production. Targeted VLPs have been purified away from unmodified VLPs for the first time allowing improved analysis of the effects of this synthetic virion system. The particles enter cells via receptor-mediated endocytosis and produce siRNA effects at low nanomolar concentrations. Although less effective than a commercial cationic lipid vector at siRNA delivery, the smaller amounts of internalized RNA with VLP delivery had an effect as good as if not better than the lipid transfection route. This implies that the siRNAs delivered by this route are more accessible to the siRNA pathway than identical RNAs delivered in complex lipid aggregates. The data suggest that the MS2 system continues to show many of the features that will be required to create an effective targeted drug delivery system. The fluorescence assays of siRNA effects described here will facilitate the combinatorial analysis of both future formulations and dosing regimes.

Concepts: DNA, Protein, Microbiology, RNA, Small interfering RNA, Virus-like particle, Bacteriophage MS2

25

The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.

Concepts: Gene expression, Vaccine, Chromatography, Analytical chemistry, PH, Hepatitis B, Virus-like particle, Viral structural proteins

24

Human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MWPyV and KIPyV have been discovered between 2007 and 2012. TSPyV causes a rare skin disease trichodysplasia spinulosa in immunocompromised patients, the role of remaining polyomaviruses in human pathology is not clear. In this study, we assessed the occurrence of serum antibodies against above polyomaviruses in healthy blood donors. Serum samples were examined by enzyme-linked immunoassay (ELISA) using virus-like particles (VLPs) based on the major VP1 capsid proteins of these viruses. Overall, serum antibodies against HPyV6, HPyV7, TSPyV, HPyV9, MWPyV and KIPyV were found in 88.2%, 65.7%, 63.2%, 31.6%, 84.4% and 58%, respectively, of this population. The seroprevalence generally increased with age, the highest rise we observed for HPyV9 and KIPyV specific antibodies. The levels of anti-HPyV antibodies remained stable across the age-groups, except for TSPyV and HPyV9, where we saw change with age. ELISAs based on VLP and GST-VP1 gave comparable seroprevalence for HPyV6 antibodies (88.2% vs.85%.3) but not for HPyV7 antibodies (65.7% vs. 77.2%), suggesting some degree of crossreactivity between HPyV6 and HPyV7 VP1 proteins. In conclusion, these results provide evidence that human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MwPyV and KIPyV circulate widely in the Czech population and their seroprevalence is comparable to other countries. This article is protected by copyright. All rights reserved.

Concepts: Immune system, Antibody, Protein, Microbiology, ELISA, All rights reserved, Copyright, Virus-like particle

24

The Costa Rica HPV16/18 Vaccine Trial (CVT) showed that four-year vaccine efficacy against 12-month HPV16/18 persistent infection was similarly high among women who received one, two, or the recommended three doses of the bivalent HPV16/18 L1 virus-like particle (VLP) vaccine. Live-attenuated viral vaccines, but not simple-subunit vaccines, usually induce durable lifelong antibody responses after a single dose. It is unclear whether noninfectious VLP vaccines behave more like live-virus or simple-subunit vaccines in this regard. To explore the likelihood that efficacy will persist longer term, we investigated the magnitude and durability of antibodies to this vaccine by measuring HPV16- and HPV18-specific antibodies by VLP-ELISA using serum from enrollment, vaccination, and annual visits through four years in four vaccinated groups; one-dose (n = 78), two-doses separated by one month (n = 140), two doses separated by six months (n = 52), and three scheduled doses (n = 120, randomly selected). We also tested enrollment sera from n = 113 HPV16- or HPV18 L1-seropositive women prevaccination, presumably from natural infection. At four years, 100% of women in all groups remained HPV16/18 seropositive; both HPV16/18 geometric mean titers (GMT) among the extended two-dose group were non-inferior to the three-dose group, and ELISA titers were highly correlated with neutralization titers in all groups. Compared with the natural infection group, HPV16/18 GMTs were, respectively, at least 24 and 14 times higher among the two-dose and 9 and 5 times higher among one-dose vaccinees. Antibody levels following one-dose remained stable from month 6 through month 48. Results raise the possibility that even a single dose of HPV VLPs will induce long-term protection. Cancer Prev Res; 6(11); 1242-50. ©2013 AACR.

Concepts: Immune system, HPV vaccine, Microbiology, Vaccine, Vaccination, Immunology, Vaccination schedule, Virus-like particle