Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Comparative Sensitivity and Specificity of the 7SL sRNA Diagnostic Test for Animal Trypanosomiasis.

Animal trypanosomiasis (AT) is a significant livestock disease, affecting millions of animals across Sub-Saharan Africa, Central and South America, and Asia, and is caused by the protozoan parasites Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense, with the largest economic impact in cattle. There is over-reliance on presumptive chemotherapy due to inadequate existing diagnostic tests, highlighting the need for improved AT diagnostics. A small RNA species, the 7SL sRNA, is excreted/secreted by trypanosomes in infected animals, and has been previously shown to reliably diagnose active infection. We sought to explore key properties of 7SL sRNA RT-qPCR assays; namely, assessing the potential for cross-reaction with the widespread and benign Trypanosoma theileri, directly comparing assay performance against currently available diagnostic methods, quantitatively assessing specificity and sensitivity, and assessing the rate of decay of 7SL sRNA post-treatment. Results showed that the 7SL sRNA RT-qPCR assays specific for T. brucei, T. vivax, and T. congolense performed better than microscopy and DNA PCR in detecting infection. The 7SL sRNA signal was undetectable or significantly reduced by 96-h post treatment; at 1 × curative dose there was no detectable signal in 5/5 cattle infected with T. congolense, and in 3/5 cattle infected with T. vivax, with the signal being reduced 14,630-fold in the remaining two T. vivax cattle. Additionally, the assays did not cross-react with T. theileri. Finally, by using a large panel of validated infected and uninfected samples, the species-specific assays are shown to be highly sensitive and specific by receiver operating characteristic (ROC) analysis, with 100% sensitivity (95% CI, 96.44-100%) and 100% specificity (95% CI, 96.53-100%), 96.73% (95% CI, 95.54-99.96%) and 99.19% specificity (95% CI, 92.58-99.60%), and 93.42% (95% CI, 85.51-97.16% %) and 82.43% specificity (95% CI, 72.23-89.44% %) for the T brucei, T. congolense and T. vivax assays, respectively, under the conditions used. These findings indicate that the 7SL sRNA has many attributes that would be required for a potential diagnostic marker of AT: no cross-reaction with T. theileri, high specificity and sensitivity, early infection detection, continued signal even in the absence of detectable parasitaemia in blood, and clear discrimination between infected and treated animals.

The genomic basis of host and vector specificity in non-pathogenic trypanosomatids.

Trypanosoma theileri, a non-pathogenic parasite of bovines, has a predicted surface protein architecture that likely aids survival in its mammalian host. Their surface proteins are encoded by genes which account for ∼10% of their genome. A non-pathogenic parasite of sheep, Trypanosoma melophagium, is transmitted by the sheep ked and is closely related to T. theileri. To explore host and vector specificity between these species, we sequenced the T. melophagium genome and transcriptome and an annotated draft genome was assembled. T. melophagium was compared to 43 kinetoplastid genomes, including T. theileri. T. melophagium and T. theileri have an AT biased genome, the greatest bias of publicly available trypanosomatids. This trend may result from selection acting to decrease the genomic nucleotide cost. The T. melophagium genome is 6.3Mb smaller than T. theileri and large families of proteins, characteristic of the predicted surface of T. theileri, were found to be absent or greatly reduced in T. melophagium. Instead, T. melophagium has modestly expanded protein families associated with the avoidance of complement-mediated lysis. We propose that the contrasting genomic features of these species is linked to their mode of transmission from their insect vector to their mammalian host. This article has an associated First Person interview with the first author of the paper.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Production of functional active human growth factors in insects used as living biofactories.

Growth factors (GFs) are naturally signalling proteins, which bind to specific receptors on the cell surface. Numerous families of GFs have already been identified and remarkable progresses have been made in understanding the pathways that these proteins use to activate/regulate the complex signalling network involved in cell proliferation or wound healing processes. The bottleneck for a wider clinical and commercial application of these factors relay on their scalable cost-efficient production as bioactive molecules. The present work describes the capacity of Trichoplusia ni insect larvae used as living bioreactors in combination with the baculovirus vector expression system to produce three fully functional human GFs, the human epidermal growth factor (huEGF), the human fibroblast growth factor 2 (huFGF2) and the human keratinocyte growth factor 1 (huKGF1). The expression levels obtained per g of insect biomass were of 9.1, 2.6 and 3mg for huEGF, huFGF2 and huKGF1, respectively. Attempts to increase the productivity of the insect/baculovirus system we have used different modifications to optimize their production. Additionally, recombinant proteins were expressed fused to different tags to facilitate their purification. Interestingly, the expression of huKGF1 was significantly improved when expressed fused to the fragment crystallizable region (Fc) of the human antibody IgG. The insect-derived recombinant GFs were finally characterized in terms of biological activity in keratinocytes and fibroblasts. The present work opens the possibility of a cost-efficient and scalable production of these highly valuable molecules in a system that favours its wide use in therapeutic or cosmetic applications.

Significant productivity improvement of the baculovirus expression vector system by engineering a novel expression cassette.

Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS.

Characterization of a Trichoplusia ni hexamerin-derived promoter in the AcMNPV baculovirus vector.

The promoter sequences of the encoding genes for the three most abundant hexamerins of the Lepidoptera Trichoplusia ni were isolated and cloned into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-derived baculovirus expression vector. From the sequences analyzed, the DNA region driving the expression of the Basic juvenile hormone-suppressible protein 2 (BJHSP-2), denominated pB2, presented the highest promoter strength in the context of the baculovirus vector in Sf21 insect cells. This promoter activity occurred earlier in baculovirus-infected cells than that achieved by a conventional polyhedrin promoter (polh), but surprisingly stopped at 48h post-infection. A mapping of pB2 essential promoter elements determined that a region of about 400bp, denominated pB29, retained and even increased the transcriptional activity with respect to the parental full-length sequence. Finally, several chimeric combinations of the insect-derived pB2 with the virus-derived conventional polh or p10 promoters were constructed and incorporated into an AcMNPV baculovirus vector. The pB2-p10 combination showed increased recombinant protein expression at early times post-infection and similar expression levels at very late times post-infection in Sf21 cells with respect to conventional late promoters. To the best of our knowledge, pB2 is the first promoter isolated from the Lepidoptera T. ni, the natural host of AcMNPV, to be assayed in a baculovirus expression vector.