Chimaeric Virus-Like Particles Derived from Consensus Genome Sequences of Human Rotavirus Strains Co-Circulating in Africa

<div><p>Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a>, P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> or P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting <i>Spodoptera frugiperda</i> 9 and <i>Trichoplusia ni</i> cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.</p></div

A description of the pFastBACquad constructs prepared in this study from genomic data obtained from human stool samples containing human African rotavirus strains.

<p>All codon-optimised ORFs were inserted into a commercial pUC57 transport plasmid at GeneArt (Life Technologies, New York, NY) and GenScript (GenScript USA Inc. New Jersey, NJ).</p>a<p><b>pFBqG2, pFBqG8 and pFBqG12</b>: Generated by ligating coding regions for G2, G8 and G12 VP7 proteins excised from pUC57G2, pUC57G2 and pUC57G8 plasmids to pFBq vector DNA, respectively. Both insert and vector DNA were prepared by double-digestion with <i>Bam</i> HI and <i>Not</i> I.</p>¥<p>VP4 and VP7 outer capsid proteins expressed by baculoviruses prepared from these expression cassettes were used to generate RV-VLPs in the current study. The VP2 and VP6 proteins that were prepared from strain RVA/Human-wt/ZAF/GR10924/1999/G9P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> formed the scaffolds on to which the outer capsid proteins were assembled.</p>b<p><b>pFBqP</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a><b> and pFBqP</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>: Generated by ligating coding regions for P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> VP4 proteins excised from pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> plasmids to pFBq vector DNA. The vector and insert DNA were prepared by digestion with <i>Sma</i> I and <i>Spe</i> I.</p>c<p><b>pFBqG2P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a><b>, pFBqG2P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>: Generated by ligating the coding regions for P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> VP4 proteins excised from pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> plasmids to recombinant pFBqG2 expression cassettes, respectively. Both insert and vector DNA were prepared by double-digestion with <i>Sma</i> I and <i>Spe</i> I.</p>d<p><b>pFBqG8P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a><b>, pFBqG8P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>: Generated by ligating coding regions for P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> VP4 proteins excised with <i>Sma</i> I and <i>Spe</i> I from pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> plasmids to the recombinant pFBqG8 expression cassettes, respectively. The vector was also prepared by digesting with <i>Sma</i> I and <i>Spe</i> I.</p>e<p><b>pFBqG12P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a><b>, pFBqG12P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>: Generated by ligating the coding regions for P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> VP4 proteins excised from pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and pUC57P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> plasmids to the recombinant pFBqG12 expression vector. Both the insert and vector were double-digested with <i>Sma</i> I and <i>Spe</i> I.</p>f<p><b>FBqG9P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a><b>, pFBqG9P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a><b>, pFBqG2P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a><b>, pFBqG8P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a><b>, pFBqG12P</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>: Double-digestion of pFBqG9P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> with <i>Sma</i> I and <i>Spe</i> I resulted in pFBqG9_<i>Sma</i>I/<i>Spe</i>I and P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>_<i>Sma</i>I/<i>Spe</i>I DNA fragments. Double digesting pFBqG9P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> with <i>Bam</i> HI and <i>Not</i> I resulted in pFBqP<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a><i>_Bam</i>HI/<i>Not</i>I and G9_<i>Bam</i>HI/<i>Not</i>I fragments. Recombinant pFBqG9P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a>, pFBqG9P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>, pFBqG2P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> expression cassettes were engineered by ligating coding regions for P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> and P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> VP4 proteins to the recombinant pFBqG9_<i>Sma</i>I/<i>Spe</i>I vector. Ligating G2_<i>Bam</i>HI/<i>Not</i>I, G8_<i>Bam</i>HI/<i>Not</i>I and G12_<i>Bam</i>HI/<i>Not</i>I fragments to recombinant pFBqP<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>_<i>Bam</i>HI/<i>Not</i>I vector resulted in pFBqG2P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>, pFBqG8P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>, pFBqG12P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> expression cassettes, respectively.</p><p>A description of the pFastBACquad constructs prepared in this study from genomic data obtained from human stool samples containing human African rotavirus strains.</p

Rotavirus virus-like particles produced in insect cells by using dsRNA of wild-type strains.

<p>(<b>I</b>) dRV-VLPs produced by infecting High Five cells with recombinant baculoviruses containing ORFs coding for VP2 (C2) and VP6 (I2) proteins. Scale bar 200 nm. (<b>II</b>) tRV-VLPs produced by infecting Sf9 cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>), VP6 (I2) and VP7 (G9) proteins. Scale bar 200 nm. (<b>III</b>) Chimaeric tRV-VLPs produced by infecting High Five cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a>), VP6 (I2) and VP7 (G8) proteins. Scale bar 200 nm. (<b>IV</b>) Chimaeric tRV-VLPs produced by infecting Sf9 cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a>), VP6 (I2) and VP7 (G2) proteins. Scale bar 200 nm. <b>V</b>) Chimaeric tRV-VLPs produced by infecting High Five cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>), VP6 (I2) and VP7 (G8) proteins. Scale bar 200 nm. <b>VI</b>) Chimaeric tRV-VLPs produced by infecting High Five cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a>), VP6 (I2) and VP7 (G9) proteins. Scale bar 100 nm. <b>VII</b>) Chimaeric tRV-VLPs produced by infecting Sf9 cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a>), VP6 (I2) and VP7 (G12) proteins. Scale bar 100 nm. <b>VIII</b>) Chimaeric tRV-VLPs produced by infecting High Five cells with recombinant baculoviruses containing ORFs coding for VP2 (C2), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a>) and VP6 (I2) proteins. Scale bar 200 nm. tRV-VLPs with a smooth outer ring are shown with arrows.</p

Schematic presentation of the baculovirus expression strategies used to generate RV-VLPs.

<p>The donor plasmids contains ORFs coding for specific rotavirus proteins (labelled downstream to the promoters regulating their expression as described in the text) that were transposed into bacmids which were subsequently used to generate baculoviruses. The restriction sites used for construction of the recombinant transfer plasmids are not indicated on the pFBq plasmids maps above, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167.s001" target="_blank">Fig. S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone-0105167-t001" target="_blank">Table 1</a> for more details. (<b>I</b>) pFBq plasmid construct used to generate recombinant dualcistronic baculoviruses that was used to prepare dRV-VLPs (VP2/6) through single infection of insect cells. (<b>II</b>) pFBq plasmid constructs used to generate recombinant dualcistronic and monocistronic baculoviruses that were used to prepare tRV-VLPs (<b>VP2/6/7</b>) through simultaneous infection of insect cells. (<b>III</b>) pFBq plasmid constructs used to generate recombinant dualcistronic and monocistronic baculoviruses that were used to prepare tRV-VLPs (<b>VP2/6/4</b>) through simultaneous infection of insect cells. (<b>IV</b>) pFBq plasmid constructs used to generate recombinant dualcistronic baculoviruses that were used to prepare tRV-VLPs (<b>VP2/6/7/4</b>) through simultaneous infection of insect cells. (<b>V</b>) pFBq plasmid constructs used to generate recombinant dualcistronic and monocistronic baculoviruses that were used to prepare tRV-VLPs (<b>VP2/6/7/4</b>) through step-wise co-infection strategy. Insect cells were initially infected with dualcistronic baculoviruses confirmed to express VP2/6 and recombinant monocistronic baculoviruses confirmed to express VP4. This was followed by infection with recombinant monocistronic baculoviruses confirmed to express VP7 12 hours post initial infection (hpi).</p

Rotavirus strains and restriction endonucleases used to clone selected VP4 and VP7 encoding ORFs into the pFBq donor plasmid.

a<p>The ORFs coding for the rotavirus proteins were inserted downstream of these promoters as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone-0105167-g001" target="_blank">Fig.1</a>.</p><p>Rotavirus strains and restriction endonucleases used to clone selected VP4 and VP7 encoding ORFs into the pFBq donor plasmid.</p

SDS-PAGE and western blot analysis of RV-VLP gradient fractions.

<p>Electrophoretic separation of selected gradient fractions, which contained structural proteins, out of the 18 fractions of 220 ul collected for each sample is depicted. (<b>I</b>) Gradient fractions for RV-VLPs (dRV-VLP) prepared with baculoviruses confirmed to express VP2 (C2 genotype) and VP6 (I2 genotype). (<b>II</b>) Gradient fractions for RV-VLPs (tRV-VLPs) prepared with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> genotype) and VP6 (I2 genotype). (<b>III</b>) Gradient fractions for RV-VLPs (tRV-VLP) prepared with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> genotype), VP6 (I2 genotype) and VP7 (G2 genotype). (<b>IV</b>) Detection of VP6 (I2 genotype) and VP7 (G8 genotype) with anti-rotavirus IgG antibodies using western blot in selected gradient fractions in which other structural proteins (VP2, VP5* or VP6) were detected using SDS-PAGE. (<b>V</b>) Gradient fractions for RV-VLPs on SDS-PAGE prepared by step-wise co-infection with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> genotype), VP6 (I2 genotype) and VP7 (G2 genotype). (<b>VI</b>) Gradient fractions for RV-VLPs on SDS-PAGE (A) and nitrocellulose membrane, western blot (B) prepared by step-wise co-infection with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> genotype), VP6 (I2 genotype) and VP7 (G2 genotype). An approximately 72 kDa non-specific band was consistently present in almost all sucrose gradient fractions #1 to #8 for each sample. The antibody used was weakly reactive against the inner VP2 capsid proteins in the RV-VLPs hence the absence of VP2 band in panel IV. Lane 1, Ladder, PageRuler Plus Prestain Protein Ladder (Fermentas UAB, Vilnius, Lithuania).</p