Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

Abstract

Background: The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry.

Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases

“…This same strategy (use of (1 PFU/c of each baculovirus) can also be used for the production of biolocals to overcome the drawbacks of the use of high MOIs for infecting the production cultures. This strategy allows a small portion of the cell population to become initially infected, which serves to amplify the baculoviruses to numbers that allow the rest of the culture to become infected in a second wave of infection (Hu and Bentley 2000;Mena et al, 2007). Zhang and Merchuk (2004) estimated that in a cell culture infected at an MOI of 0.1 PFU/cell, the first release of budded baculovirus starts after 8 h p.i.…”

Rotavirus VP6 preparations as a non-replicating vaccine candidates

“…It has been shown by several studies that the structural proteins (VP2, VP6, VP7, and VP4) of RV can be expressed in different prokaryotic and eukaryotic expression systems and form SLPs (VP2), DLPs (VP2 and VP6), and TLPs (VP2, VP6, and VP7 or VP2, VP6, VP7, and VP4) [132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148]. Furthermore, expressing VP6 alone in vitro [14,29] can form spherical, tubular, and trimer structures similar to VP6 purified from the RVs [31].…”

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

“…The innermost layer contains 60 dimers of VP2 (102.7 kDa); the middle shell is formed by 260 trimers of VP6 (44.9 kDa); and the third, outer layer is composed by 260 trimers of the glycoprotein VP7 (37.2 kDa). RLPs are usually produced by co-infection of insect cells with baculoviruses coding for VP2 and VP6 (double-layered particles, DLP) or VP2, VP6, and VP7 (triple-layered particles, TLP) (Mena et al, 2007;Vieira et al, 2005).…”