Baculoviruses are arthropod-specific large circular double-stranded DNA viruses successfully used for the control of multiple insect pests. In addition to their application in pest control, baculoviruses have become a versatile and powerful eukaryotic vector for the production of large quantities of recombinant proteins for research and biomedical purposes. Since the first recombinant protein was expressed in 1983 using the baculovirus expression system (BEVS), different strategies have been developed for the generation of recombinant viruses and to increase the stability, yield, and posttranslational modifications of recombinant proteins. In this review, we summarize the main methods and elements playing a role in the BEVS emphasizing recent progresses and future developments with respect to the main aspects involved in protein production using the BEVS.

No abstract

In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

No abstract

Baculoviruses are insect pathogens widely used as biotechnological tools in different fields of life sciences and technologies. The particular biology of these entities (biosafety viruses 1; large circular double-stranded DNA genomes, infective per se; generally of narrow host range on insect larvae; many of the latter being pests in agriculture) and the availability of molecular-biology procedures (e.g., genetic engineering to edit their genomes) and cellular resources (availability of cell lines that grow under in vitro culture conditions) have enabled the application of baculoviruses as active ingredients in pest control, as systems for the expression of recombinant proteins (Baculovirus Expression Vector Systems-BEVS) and as viral vectors for gene delivery in mammals or to display antigenic proteins (Baculoviruses applied on mammals-BacMam). Accordingly, BEVS and BacMam technologies have been introduced in academia because of their availability as commercial systems and ease of use and have also reached the human pharmaceutical industry, as incomparable tools in the development of biological products such as diagnostic kits, vaccines, protein therapies, and-though still in the conceptual stage involving animal models-gene therapies. Among all the baculovirus species, the Autographa californica multiple nucleopolyhedrovirus has been the most highly exploited in the above utilities for the human-biotechnology field. This review highlights the main achievements (in their different stages of development) of the use of BEVS and BacMam technologies for the generation of products for infectious and noninfectious human diseases. Key points• Baculoviruses can assist as biotechnological tools in human health problems.• Vaccines and diagnosis reagents produced in the baculovirus platform are described.• The use of recombinant baculovirus for gene therapy-based treatment is reviewed.

The clustered regularly interspaced short palindromic repeats (CRISPR) system–associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9‐single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock‐ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock‐ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.

Serology testing for COVID‐19 is important in evaluating active immune response against SARS‐CoV‐2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus‐exposed individuals and vaccine‐mediated immunity. In this study, recombinant S protein of SARS‐CoV‐2 was expressed in Rachiplusia nu , an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS‐CoV‐2 S protein that showed immunoreactivity for anti‐SARS‐CoV‐2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost‐effective platform for large‐scale S protein production, and the scale‐up is linear, thus avoiding the use of complex equipment like bioreactors.

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.