Baculoviral DNA Research Articles

Safety pharmacology of human endogenous retrovirus-enveloped baculoviral DNA vaccines against SARS-CoV-2 in Sprague-Dawley rats and beagle dogs

The coronavirus disease 2019 (COVID-19) emerged as a major global health crisis, posing significant health, economic, and social challenges. Vaccine development has been a crucial response to the severe-acute-respiratory-syndrome-related coronavirus-2 pandemic owing to the critical role of immunization in controlling infectious diseases, leading to the expedited development of several effective vaccines. Although mRNA platform-based COVID-19 vaccines authorized under emergency-use authorization have been administered globally, concerns regarding the vaccines have increased owing to the occurrence of various side effects. The present study aimed to evaluate the safety of a non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) vaccine encoding SARS-CoV-2 antigens. Owing to the limited number of existing safety pharmacology studies on AcHERV as a viral vector vaccine, we conducted neurobehavior (Modified Irwin’s Test), body temperature, and respiratory function studies in rats and cardiovascular system studies in male beagle dogs, which were administered the AcHERV-COVID-19 vaccine using telemetry. The safety assessment revealed no significant toxicological alterations. However, in rats, both sexes administered with the AcHERV-COVID-19 vaccine exhibited a temporary increase in body temperature, which normalized or showed signs of recovery. In conclusion, AcHERV-COVID-19 demonstrates a sufficient safety profile that supports its potential evaluation in future clinical trials.

An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing.

CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery of genetically encoded CRISPR components remains challenging and sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its DNA cargo, can be used to package and deliver proteins to human cells. Using protein-loaded baculovirus (pBV), we demonstrate delivery of Cas9 or base editors proteins, leading to efficient genome and base editing in human cells. By implementing a reversible, chemically inducible heterodimerization system, we show that protein cargoes can selectively and more efficiently be loaded into pBVs (spBVs). Using spBVs we achieved high levels of multiplexed genome editing in a panel of human cell lines. Importantly, spBVs maintain high editing efficiencies in absence of detectable off-targets events. Finally, by exploiting Cas9 protein and template DNA co-delivery, we demonstrate up to 5% site-specific targeted integration of a 1.8 kb heterologous DNA payload using a single spBV in a panel of human cell lines. In summary, we demonstrate that spBVs represent a versatile, efficient and potentially safer alternative for CRISPR applications requiring co-delivery of DNA and protein cargoes.

Baculoviral COVID-19 Delta DNA vaccine cross-protects against SARS-CoV2 variants in K18-ACE2 transgenic mice.

After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronavirus's intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50% survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). The AcHERV-COVID19D induced higher neutralizing antibodies against the Delta variant than the prototype or Omicron variant. On the other hand, cellular immunity was similarly high for all three SARS-CoV2 viruses. Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100% survival upon challenge with Delta and Omicron variants and 71.4% survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants.

HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system

The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.

Human endogenous retrovirus-enveloped baculoviral DNA vaccines against MERS-CoV and SARS-CoV2

Here we report a recombinant baculoviral vector-based DNA vaccine system against Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2). A non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) was constructed as a DNA vaccine vector for gene delivery into human cells. For MERS-CoV vaccine construction, DNA encoding MERS-CoV S-full, S1 subunit, or receptor-binding domain (RBD) was inserted into the genome of AcHERV. For COVID19 vaccine construction, DNA encoding SARS-CoV2 S-full or S1 or a MERS-CoV NTD domain-fused SARS-CoV2 RBD was inserted into the genome of AcHERV. AcHERV-DNA vaccines induce high humoral and cell-mediated immunity in animal models. In challenge tests, twice immunized AcHERV-MERS-S1 and AcHERV-COVID19-S showed complete protection against MERS-CoV and SARS-CoV2, respectively. Unlike AcHERV-MERS vaccines, AcHERV-COVID19-S provided the greatest protection against SARS-CoV2 challenge. These results support the feasibility of AcHERV-MERS or AcHERV-COVID19 vaccines in preventing pandemic spreads of viral infections.

Bioreactor-Based Antigen Production Process Using the Baculovirus Expression Vector System.

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the BEVS in a stirred tank reactor.

Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway.

The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-β) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

Porcine endogenous retrovirus envelope coated baculoviral DNA vaccine against porcine reproductive and respiratory syndrome virus

PERV is a major virus concerning xenotransplantation study. However, the interesting part is that PERV is present in all kinds of pigs without pathogenicity and immune response. Furthermore, since pig cells have receptors for PERV, the gene delivery system using PERV envelope is highly likely to develop into an excellent viral vector in pigs. We developed a recombinant baculovirus with a modified surface for expressing the porcine endogenous retrovirus (PERV) envelope. Porcine reproductive and respiratory syndrome virus (PRRSV) infection is a severe concern in the porcine industry due to reproduction failure and respiratory symptoms. GP5 and M proteins are major immunogenic proteins of PRRSV. Using PERV-modified baculovirus (Ac mPERV) as a delivery vector, we constructed a dual antigen (GP5 and M)-encoding DNA vaccine system, Ac mPERV-C5/C6. Intramuscular immunization in mice and pigs, Ac mPERV-C5/C6 induced comparative high humoral and cellular immune responses. Our results support further development of Ac mPERV-C5/C6 as a potential PRRSV vaccine in the porcine industry. In addition, the Ac mPERV system may be applied to the generation of other effective DNA vaccines against porcine viral diseases.

Baculovirus as a Tool for Gene Delivery and Gene Therapy.

Based on its ability to express high levels of protein, baculovirus has been widely used for recombinant protein production in insect cells for more than thirty years with continued technical improvements. In addition, baculovirus has been successfully applied for foreign gene delivery into mammalian cells without any viral replication. However, several CpG motifs are present throughout baculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production of pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent or -independent signaling pathway, and ultimately limiting the efficiency of transgene expression. On the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses encoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient cells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells, are more susceptible to baculovirus infection than normal cells and are selectively eliminated by the apoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus as a gene expression vector and the mechanism of the host innate immune response induced by baculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors.

Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells.

Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (≤0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.

Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1.

An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA). However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV) envelope-coated, baculovirus-based, virus-like-particle (VLP)–forming DNA vaccine (termed AcHERV-VLP) against pandemic influenza A/California/04/2009 (pH1N1). BALB/c mice immunized with AcHERV-VLP (1×107 FFU AcHERV-VLP, i.m.) and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8), elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines.

Characterization and immunogenicity of norovirus capsid-derived virus-like particles purified by anion exchange chromatography

Recombinant baculovirus (BV) expression systems are widely applied in the production of viral capsid proteins and virus-like particles (VLPs) for use as immunogens and vaccine candidates. Traditional density gradient purification of VLPs does not enable complete elimination of BV-derived impurities, including live viruses, envelope glycoprotein gp64 and baculoviral DNA. We used an additional purification system based on ionic strength to purify norovirus (NoV) GII-4 capsid-derived VLPs. The anion exchange chromatography purification led to highly purified VLPs free from BV impurities with intact morphology. In addition, highly purified VLPs induced strong NoV-specific antibody responses in BALB/c mice. Here, we describe a method for NoV VLP purification and several methods for determining their purity, including quantitative PCR for BV DNA detection.

New baculovirus expression tools for recombinant protein complex production

Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these molecular machines are only present in low amounts in their native hosts, which impede purification from source material. Unraveling their structure and function at high resolution will often depend on heterologous overproduction. Recombinant expression of multiprotein complexes for structural studies can entail considerable, sometimes inhibitory, investment in both labor and materials, in particular if altering and diversifying of the individual subunits are necessary for successful structure determination. Our laboratory has addressed this challenge by developing technologies that streamline the complex production and diversification process. Here, we review several of these developments for recombinant multiprotein complex production using the MultiBac baculovirus/insect cell expression system which we created. We also addressed parallelization and automation of gene assembly for multiprotein complex expression by developing robotic routines for multigene vector generation. In this contribution, we focus on several improvements of baculovirus expression system performance which we introduced: the modifications of the transfer plasmids, the methods for generation of composite multigene baculoviral DNA, and the simplified and standardized expression procedures which we delineated using our MultiBac system.

Heterogeneous Nuclear Ribonucleoprotein R Enhances Transcription from the Naturally Configured c-fos Promoter in Vitro

Transcription of a proto-oncogene c-fos is induced rapidly to high levels by various extracellular stimuli. To explore the molecular mechanism of c-fos gene induction, we established a defined in vitro transcription system for the c-fos promoter that consists of purified activators (SRF, Elk-1, cAMP-responsive element-binding protein, and ATF1), general transcription factors, and RNA polymerase II. In this reconstituted transcription system, activation of c-fos transcription was highly dependent upon coactivators such as PC4 and Mediator, indicating a very weak activation potential of the activators in the context of an unaltered promoter structure. This heightened coactivator dependence, however, allowed us to identify from HeLa nuclear extract a coactivator-like activity termed transcriptional regulator of c-fos (TREF) that enhanced c-fos transcription but not GAL4-VP16-dependent transcription. TREF cooperated with Mediator to enhance c-fos transcription by approximately 60-fold over its basal level and, like Mediator, stimulated activator-independent (basal) transcription as well. Further purification of TREF revealed that it consists of at least three distinct components, one of which was purified to near homogeneity and identified as heterogeneous nuclear ribonucleoprotein R. Recombinant heterogeneous nuclear ribonucleoprotein R enhanced transcription from the c-fos promoter and displayed cooperativity with PC4 and Mediator, thus demonstrating its direct transcriptional activity.

Persistence of naturally occurring and genetically modified Choristoneura fumiferana nucleopolyhedroviruses in outdoor aquatic microcosms

To assess the persistence of genetically modified and naturally occurring baculoviruses in an aquatic environment, replicate (three) outdoor, aquatic microcosms were spiked with spruce budworm viruses [Ireland strain of Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and the recombinant CfMNPVegt(-)/lacZ(+)] at a rate of 1.86 x 10(10) occlusion bodies (OBs) m(-2) of surface area. The presence of virus in water samples collected at various times after inoculation was determined by PCR amplification of baculoviral DNA extracted from OBs. Although UV radiation rapidly degrades baculoviruses under natural conditions, both viruses persisted above the level of detection (>100 OBs 450 microL(-1) of natural pond water) for at least 1 year post-inoculation, with little difference between the viruses in their patterns of persistence. The present microcosm study suggests that occlusion bodies of baculoviruses can persist in the flocculent layer of natural ponds. On disturbance, OBs could re-enter the main water column and thus be available for transport to new locations. Implications for environmental risk assessment are discussed.

A simplified method for the extraction of baculoviral DNA for PCR analysis: A practical application

There are two major strategies to genetically modify baculoviruses. One uses a bacmid-based system which replicates in Escherichia coli using a bacterial origin of replication. The other employs a transfer vector and viral DNA which are co-transfected into insect cells and utilise host enzyme-mediated homologous recombination. Putative recombinants are then typically screened by plaque assay. The bacmid system is more convenient, but it requires a number of complex construction and isolation steps to obtain the correct bacmid genome. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells. Briefly, viral DNA was isolated in three steps: SDS lysis, chloroform extraction and ethanol precipitation. The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR. No prior amplification of the virus by passage in tissue culture was necessary. The major advantage of this method was a reduction in assay times from a few days to a few hours. Moreover, this method is very convenient for detecting baculoviruses in cell culture: cross-contamination within viral stocks, monitoring mixed viral infection and confirmation of viral genomic integrity.

Baculovirus Vector for Gene Delivery and Vaccine Development

The baculovirus Autographa californica multiple nucleopolyhedrovirus has been widely used not only to acheive a high level of foreign gene expression in insect cells, but also for efficient gene transduction into mammalian cells. Recombinant and pseudotyped baculoviruses possessing chimeric or foreign ligands have been constructed to improve the efficiency of gene transduction and to confer specificity for gene delivery into mammalian cells, respectively. Baculoviral DNA CpG motifs induce proinflammatory cytokines through a Toll-like receptor (TLR9)/MyD88-dependent signaling pathway. Other baculovirus components produce type I interferons via a TLR-independent pathway. Baculovirus exhibits a strong adjuvant property and recombinant baculoviruses encoding microbial antigens elicit antibodies to the antigens and provide protective immunity in mice. This review deals with recent progress in the application of baculovirus vectors to gene delivery and vaccine development, and discusses the future prospects of baculovirus vectors.

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle

We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene-based cartilage tissue engineering. However, baculovirus-mediated gene expression in the chondrocytes is transient. In this study, we attempted to prolong the expression by supertransduction, but uncovered that after long-term culture the chondrocytes became more refractory to baculovirus transduction. Therefore, the correlation between baculovirus-mediated enhanced green fluorescent protein (EGFP) expression and cell cycle was investigated by comparing the cycling chondrocytes and chondrocytes rich in quiescent cells, in terms of EGFP expression, virus uptake, cell cycle distribution, nuclear import and methylation of viral DNA. We demonstrated, for the first time, that baculovirus-mediated transduction of chondrocytes is correlated with the cell cycle. The chondrocytes predominantly in G2/M phase were approximately twice as efficient in EGFP expression as the cycling cells, while the cells in S and G1 phases expressed EGFP as efficiently as the cycling cells. Notably, the chondrocyte populations rich in quiescent cells resulted in efficient virus uptake, but less effective nuclear transport of baculoviral DNA and higher degree of methylation, and hence poorer transgene expression. These findings unravel the practical limitations when employing baculovirus in cartilage tissue engineering. The implications and possible solutions are discussed.

C-terminal Src Kinase-homologous Kinase (CHK), a Unique Inhibitor Inactivating Multiple Active Conformations of Src Family Tyrosine Kinases

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

Expression of Recombinant Flavin-Containing Monooxygenases in a Baculovirus/Insect Cell System.

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodoptera frugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.