Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system

Estes, Mary K.; Crawford, Sue E.; Penaranda, Maria E.; Petrie, Betty L.; Burns, John W.; Chan, Wai-Kit; Ericson, Brad L.; Smith, Gale; Summers, Max D.

doi:10.1128/jvi.61.5.1488-1494.1987

Cited by 139 publications

(50 citation statements)

References 26 publications

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“…Spodoptera frugiperda (Sf9) cells were grown and maintained in TNM-FH medium containing 10% fetal bovine serum (FBS) (36). Baculovirus recombinants encoding the following rotavirus proteins were used: VP1 of virus strain RF (pVL941/Rf-1) (9), VP2 of virus strain RF (BacRf2A) (21), VP3 of virus strain SA11 (pVL1393/SA11-3) (23), and VP6 of virus strain SA11 (pAc461/SA11-6) (14).…”

Section: Cells and Virusmentioning

confidence: 99%

“…Each of the rotavirus genes encoding one of the six structural proteins has been cloned into the baculovirus expression system and expressed in insect cells. VP6 alone is capable of forming VP6 oligomers (14), and VP2 alone assembles into VP2 viruslike particles (VLPs) (21,39). Coexpression of VP2 and VP6 results in the formation of VP2/6 double-layered VLPs (21).…”

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confidence: 99%

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Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Zeng

Wentz

Cohen

et al. 1996

J Virol

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Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negativestain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VP1/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VP1, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 singlelayered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

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Section: Cells and Virusmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Zeng

Wentz

Cohen

et al. 1996

J Virol

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“…VP6 forms trimers organized into hexagons and packed into higherorder structural assemblies, e.g. VP6 nanotubes (VP6T) and nanospheres (VP6S), when expressed in vitro [12,[24][25][26][27] under different conditions [25,28]. The rVP6 is highly immunogenic [9,12,29], and it has also been used as a carrier or delivery platform for heterologous protein antigens and genetically fused epitope-based vaccines, with improved response to the foreign antigen [30][31][32][33][34].…”

Section: Introductionmentioning

confidence: 99%

Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs

Malm

Heinimäki

Vesikari

et al. 2017

Clinical and Experimental Immunology

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SummaryA subunit protein vaccine candidate based on norovirus (NoV) virus‐like particles (VLPs) and rotavirus (RV) VP6 protein against acute childhood gastroenteritis has been proposed recently. RV VP6 forms different oligomeric nanostructures, including tubes and spheres when expressed in vitro, which are highly immunogenic in different animal models. We have shown recently that recombinant VP6 nanotubes have an adjuvant effect on immunogenicity of NoV VLPs in mice. In this study, we investigated if the adjuvant effect is dependent upon a VP6 dose or different VP6 structural assemblies. In addition, local and systemic adjuvant effects as well as requirements for antigen co‐delivery and co‐localization were studied. The magnitude and functionality of NoV GII.4‐specific antibodies and T cell responses were tested in mice immunized with GII.4 VLPs alone or different combinations of VLPs and VP6. A VP6 dose‐dependent adjuvant effect on GII.4‐specific antibody responses was observed. The adjuvant effect was found to be strictly dependent upon co‐administration of NoV GII.4 VLPs and VP6 at the same anatomic site and at the same time. However, the adjuvant effect was not dependent on the types of oligomers used, as both nanotubes and nanospheres exerted adjuvant effect on GII.4‐specific antibody generation and, for the first time, T cell immunity. These findings elucidate the mechanisms of VP6 adjuvant effect in vivo and support its use as an adjuvant in a combination NoV and RV vaccine.

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“…A significant correlation was observed between measured IgA antibodies suggesting that the response measured by virus-capture ELISA is mostly VP6-specific. This can be explained by high abundance [Estes and Greenberg, 2013] and highly immunogenic nature [Estes et al, 1987;Ishida et al, 1996;Lappalainen et al, 2013] of VP6 protein.…”

Section: Discussionmentioning

confidence: 99%

Rotavirus vaccination and infection induce VP6‐specific IgA responses

Lappalainen

Blazevic

Malm

et al. 2016

Journal of Medical Virology

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Rotavirus (RV) is the leading cause of severe gastroenteritis (GE) in young children, but RVGE has drastically been reduced with the introduction of live oral RV vaccines into childhood immunization program in many countries. Serum IgA antibody is a marker of clinical protection against severe RVGE after RV infection and vaccination. This study investigated VP6-specificity of anti-RV IgA antibody levels in Finnish children aged 6-23 months before and after introduction of RotaTeq® into national immunization program. Although RV inner capsid protein VP6 is considered as antigenic target in clinical and seroepidemiological studies, at present VP6 protein is not commonly employed as a primary ELISA-antigen. Thus, sera from 20 unvaccinated and 19 vaccinated children were examined in ELISA with recombinant VP6 (rVP6) protein, and the VP6-specific responses were compared to responses observed with human RV Wa and bovine RV WC3 cell culture antigens. Moreover, fecal antibodies were tested with rVP6 and Wa cell culture antigen. Equal levels of serum anti-RV IgA antibodies were detected by the three antigens. Fecal IgA titers against rVP6 and Wa antigen showed a correlation with the corresponding serum levels. The results suggest that the IgA response measured by virus-capture ELISA is mainly directed to VP6 protein, supporting the usage of rVP6 in detection of anti-RV IgA antibodies. Natural RV infections and vaccinations induced similar levels of serum VP6-specific IgA antibodies. Serum IgA antibodies after RotaTeq® vaccination showed sustained levels up to two years of age in line with long term protection. J. Med. Virol. 89:239-245, 2017. © 2016 Wiley Periodicals, Inc.

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Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system

Cited by 139 publications

References 26 publications

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants

Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs

Rotavirus vaccination and infection induce VP6‐specific IgA responses

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