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Chimeric recombinant rotavirus-like particles as a vehicle for the display of heterologous epitopes.

ABSTRACT: In order to improve the presentation and immunogenicity of single epitopes, virus-like particles (VLPs) are being used as platforms for the display of foreing epitopes on their surface. The rotavirus major capsid protein VP6 has the ability to self-assemble into empty non-infectious VLPs. In the present study, we analyzed the use of double layered VLPs (made up of VP2 and VP6 rotavirus proteins) as carriers to display a 14 amino acid epitope fused to three different aminoacidic regions of VP6 exposed on the surface of VLPs. Although all chimeric protein were correctly expressed in insect cells, only one of them resulted in spontaneous assembly of VLPs displaying the heterologous epitope on their surface, confirmed by sandwich ELISA and electron microscopy. Furthermore, the injection of chimeric VLPs into mice elicited higher antibody titers than the monomeric chimeric protein. Our results identify an specific amino acid region of VP6 which allows the insertion of at least a 14 amino acid heterolgous epitope and demonstrate its potential as immunogenic carrier.

SUBMITTER: Peralta A

PROVIDER: S-EPMC2777876 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Chimeric recombinant rotavirus-like particles as a vehicle for the display of heterologous epitopes.

Peralta Andrea A Molinari Paula P Taboga Oscar O

Virology journal 20091106

In order to improve the presentation and immunogenicity of single epitopes, virus-like particles (VLPs) are being used as platforms for the display of foreing epitopes on their surface. The rotavirus major capsid protein VP6 has the ability to self-assemble into empty non-infectious VLPs. In the present study, we analyzed the use of double layered VLPs (made up of VP2 and VP6 rotavirus proteins) as carriers to display a 14 amino acid epitope fused to three different aminoacidic regions of VP6 ex ...[more]

PMID: 19891790

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Project description:Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.

Project description:Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.

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Chimeric recombinant rotavirus-like particles as a vehicle for the display of heterologous epitopes.

Publications

Chimeric recombinant rotavirus-like particles as a vehicle for the display of heterologous epitopes.

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