Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

Overexpression of c-Myc enhances recombinant protein production in High Five cells after baculovirus infection

Due to their numerous advantages, baculovirus expression vector systems (BEVS) have been widely used to express recombinant proteins for different purposes. Different strategies have been adopted to increase recombinant protein production. In this study, we transiently or stably expressed mousec-Mycin High Five cells using a commercial pIB/V5 vector. Under the control of theOpIE2promoter, this vector could enhance recombinant protein production. We found that transient expression ofc-Mycin High Five cells improved recombinant protein production. Furthermore, we established two stable cell lines, High Five-c-Myc #1 and High Five-c-Myc #2, that stably expressed mousec-Myc. We further found that the expression level of the recombinant protein was increased in these stable cell lines compared to control cell lines. These data indicate that overexpressingc-Mycin cells is a promising way to improve recombinant protein production in BEVS.

Two esterases from the genus Spodoptera degrade sex pheromones and plant volatiles

In moths, high temporal sensitivity in perception of sex pheromones and host plant volatiles suggests the existence of mechanisms acting to maintain antennal sensitivity. The antennal enzymes have been long hypothesized to play a central role in the mechanisms, by rapid metabolism of the odorants soon after the fulfillment of the sensillum receptor activation. In the present study, two putative homologous esterases, SexiCXE13 and SlituCXE13, were cloned by RT–PCR and RACE procedures from Spodoptera exigua and Spodoptera litura, respectively. The phylogenetic tree assigned the two genes into the same group with two previously identified male antennal-specific pheromone-degrading enzymes. SexiCXE13 and SlituCXE13 were expressed in High Five cells, and the enzymatic characteristics and substrate specificity were investigated using the purified recombinant enzymes. Both esterases showed high activity to a variety of acetate substrates, including the sex pheromones, their analogs, and some common plant odorants. Our study, for the first time, provides direct biochemical and molecular evidence that the ubiquitously expressed enzyme has the ability to degrade sex pheromones and plant volatiles, and thus this adds new knowledge to the mechanism underlying the sensitivity of moth olfaction.

Critical Amino Acids in a Conserved Region of the Spacer Domain Contributing to the Binding of Anti-ADAMTS13 Antibodies

Thrombotic thrombocytopenic purpura (TTP) is a severe disorder characterized by the absence or dysfunction of the von Willebrand factor cleaving protease ADAMTS13. In plasma of the majority of patients with TTP autoantibodies directed towards the metalloprotease ADAMTS13 are present. We have previously shown that the spacer domain of ADAMTS13 contains a major binding site for antibodies that develop in patients with TTP. More detailed analysis revealed that residues Tyr657-Tyr666 within the spacer domain comprise part of a common antigenic core that is critical for binding of anti-ADAMTS13 antibodies. Here, we determined individual amino acids within region Tyr657-Tyr666 are involved in binding of a panel of human monoclonal and polyclonal anti-ADAMTS13 antibodies. A series of human monoclonal antibodies reactive with the spacer domain has been isolated from the immunoglobulin repertoire of patients with acquired TTP using phage display. Two human monoclonal antibodies, designated I-9 and II-1 that were both directed against the spacer domain were included in this analysis. Individual amino acids within region Tyr657-Tyr666 were introduced and expressed in the context of an ADAMTS13 variant truncated after the spacer domain. Reactivity of human monoclonal antibodies I-9 and II-1 with the spacer domain variants was determined by immunoprecipation using conditioned medium of stably transfected High Five cells expressing the different variants. Replacement of Arg660, Tyr661 or Tyr665 resulted in a loss of reactivity of the corresponding spacer domain variants with antibody I-9 and II-1. Reactivity of variants in which Gly662 or Glu664 were replaced by an alanine resulted in a reduced binding to antibody I-9 whereas binding to antibody II-1 was not affected by these amino acid replacements. We subsequently addressed the reactivity of polyclonal IgG present in plasma of six patients with acquired TTP with the spacer domain variants described above. First, we showed that polyclonal IgG derived from these patients did react exclusively with the spacer domain and not with other domains on ADAMTS13. Polyclonal antibodies derived from plasma of patients with acquired TTP were subsequently evaluated for binding to the spacer domain variants. For only one of six patients, binding of polyclonal IgG to ADAMTS13 was lost when either Arg660, Tyr661 or Tyr665 was replaced by an alanine. For the other patient plasma’s a reduced reactivity with some of the spacer domain variants was observed; however binding was never completely abolished. These findings suggest that multiple residues within Tyr658-Tyr665 are involved in the binding of polyclonal anti-ADAMTS13 antibodies. To further explore this possibility we generated variants in which double and triple combinations of residues Arg660, Tyr661 or Tyr665 were replaced by an alanine. The resulting variants designated RY1 (both Arg660 and Tyr661 replaced for Ala), RY2 (both Arg660 and Tyr665 replaced for an Ala), YY (both Tyr661 and Tyr665 replaced for an Ala) and RYY (both Arg660, Tyr661 and Tyr665 replaced for an Ala) were evaluated for binding to polyclonal IgG present in plasma. Plasma of two patients showed a lack of binding to variants RY1 and RY2. Three patients showed a lack of reactivity with the YY and the RYY variants. A reduced binding of the three other patients to the RYY variant was observed. These data suggest that Arg660, Tyr661 and Tyr665 contribute to an antigenic surface present on the spacer domain of ADAMTS13. Based on our findings we speculate that an oligoclonal population of anti-ADAMTS13 antibodies targets residues present within this antigenic surface in the spacer domain.