Suspension culture titration: A simple method for measuring baculovirus titers

Journal of Virological Methods 183 (2012) 201–209

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Suspension culture titration: A simple method for measuring baculovirus titers Leila Matindoost a,b,∗ , Leslie C.L. Chan a , Ying Mei Qi a , Lars K. Nielsen a , Steven Reid a a b

Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4072, Australia Environmental Sciences Institute, Shahid Beheshti University, G. C., Tehran 19839, Iran

a b s t r a c t Article history: Received 28 October 2011 Received in revised form 24 April 2012 Accepted 25 April 2012 Available online 3 May 2012 Keywords: Virus titration Baculovirus Insect cells Suspension culture

The baculovirus-insect cell expression system is an important technology for the production of recombinant proteins and baculovirus-based biopesticides. Budded virus titration is critical when scaling up baculovirus production processes in suspension cultures, to ensure reproducible infections, especially when a low multiplicity of infection (MOI) is applied. In this study, a simple suspension culture titration (SCT) assay was developed that involves accurate measurements of the initial cell densities (ICDs) and peak cell densities (PCDs) of an infected culture, from which the MOI and hence the virus inoculum infectious titer can be estimated, using the established Power–Nielsen baculovirus infection model. The SCT assay was assessed in parallel with two adherent culture-based assays (MTT and AlamarBlue) for the Heliothine baculovirus HaSNPV, and was shown to be more objective, time-efficient and reproducible. The model predicted a linear correlation between log(PCD/ICD) and log(MOI), hence an alternative modelindependent SCT assay was also developed, which relies on a well-replicated standard curve relating suspension culture-derived PCD/ICD ratios with plaque or endpoint assay-derived MOIs. Standard curves with excellent linearity were generated for HaSNPV and the industrially significant rAcMNPV, demonstrating the feasibility of this simple titration approach, especially in terms of its applicability to a wide range of virus infection kinetics. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The baculovirus-insect cell expression system is a very valuable and widely used technology for the production of recombinant eukaryotic proteins, due to its potential for high-level heterologous gene expression, and ability to perform near-authentic post-translational modifications resulting in biologically active proteins (King and Possee, 1992; Kost et al., 2005). The applications of baculovirus expression are expanding and evolving, including virus-like particles (VLPs) for the Cervarix® human papilloma virus vaccine (Senger et al., 2009), hemagglutinin antigens for the FluBlok® influenza vaccine (Baxter et al., 2011), adeno-associated virus (AAV) vectors for gene therapy (Mena et al., 2010), baculovirus display (fusing peptides/proteins on baculovirus particles for use as immunogens), humanized N-glycoproteins, and gene delivery vectors for mammalian cells (Kost et al., 2005). Baculoviruses are also increasingly being reconsidered as viable biopesticides for the control of insect pests of agriculture and forestry (Moscardi, 1999; Moscardi et al., 2011; Szewczyk et al., 2006).

∗ Corresponding author at: Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD 4072, Australia. Tel.: +61 733463147; fax: +61 733463973. E-mail addresses: [email protected] (L. Matindoost), [email protected] (L.C.L. Chan), [email protected] (S. Reid). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.04.015

The scale-up of baculovirus production processes is most efficiently carried out using suspension insect cell cultures, and one of the key prerequisites of reproducible baculovirus infections in such cultures is the ability to apply an accurate and consistent multiplicity of infection (MOI). This is especially important when a low MOI process is prescribed, in order to reduce the amount of virus inoculum required (Wong et al., 1996), and to discourage the selection for non-productive defective interfering particles (DIPs) (Kool et al., 1991). The key measurements required for calculation of the MOI are the cell density at the time of infection or initial cell density (ICD), and the infectious budded virus (BV) titer of the virus inoculum. The BV titer is by far the more challenging one to measure, and much research has been devoted to developing accurate and reproducible methodologies for BV titration (Janakiraman et al., 2006; Kitts and Green, 1999; Lo and Chao, 2004; Mena et al., 2003; Nguyen, 2007; Pouliquen et al., 2006; Roldao et al., 2009; Shen et al., 2002). Adherent culture titration (ACT) assays are the most common approach for quantifying infectious BV. The most established of these is the plaque assay (‘the gold standard’), which provides a direct measurement of the concentration of infectious BV or plaque forming units (PFUs), by counting well-isolated virus plaques formed on an agarose-immobilized cell monolayer (King and Possee, 1992). However, plaque assays are complex to perform, and are difficult to get right without considerable operator experience, hence compromising reproducibility. For example, the

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condition of stock cells, the confluency of the cell monolayer, the quality and temperature of the agarose overlay, and the efficiency of the virus adsorption step, will greatly impact on the number of plaques formed (Dee and Shuler, 1997b; King and Possee, 1992; O’Reilly et al., 1994). Other titration assays only provide an indirect measurement of infectious virus titers, but these are generally less complex to perform. For example, the well-established endpoint dilution ACT assay is conducted in multi-well plates, and depends on the scoring of a large number of negative and positive-infected wells (O’Reilly et al., 1994). However, the endpoint assay is statistically more complex to analyze, as it follows a binomial rather than a Poisson distribution (like plaque assays), but a maximum likelihood estimator can be used to improve its accuracy (Nielsen et al., 1992). The virus titer unit of an endpoint assay, the median tissue culture infectious dose (TCID50 ), can be theoretically converted to the PFU by using the Poisson distribution-derived conversion factor, −ln(0.5) = 0.693 (O’Reilly et al., 1994). Another multi-well plate ACT assay is the immunological assay (Kitts and Green, 1999), which combines the features of plaque and endpoint assays, and involves the use of an antibody to a viral envelope glycoprotein to score foci of infection in each well. Two other multi-well plate ACT assays were developed recently, involving the use of cell viability dyes (MTT or AlamarBlue) to score negative and positive-infected wells (Mena et al., 2003; Pouliquen et al., 2006). Unlike the previous assays, the MTT and AlamarBlue assays rely on the generation of dose–response (sigmoidal) curves to obtain the median tissue culture lethal dose (TCLD50 ), which is then converted to a TCID50 or PFU basis via correlation with endpoint or plaque assays. Suspension culture titration (SCT) assays have also been developed recently, which rely on the measurement of cell size change (Janakiraman et al., 2006), cell growth cessation (Roldao et al., 2009), or cell expression of a reporter gene via flow cytometry (Roldao et al., 2009), to indicate the extent of virus infection. Apart from infectious BV assays, there are other assays that measure the total concentration of virus particles using techniques such as quantitative real-time PCR (Lo and Chao, 2004) and flow cytometry (Ferris et al., 2011; Shen et al., 2002). However, these assays are of limited use for calculating the MOI, if the proportion of non-infectious virus particles in the virus stock is undefined. Unfortunately, the total vs infectious virus particles (T/I) ratio is a difficult number to pinpoint. For example, the T/I ratio was found to be as low as 4 for recombinant Autographa californica nucleopolyhedrovirus (rAcMNPV) (Rosinski et al., 2002) and 3 for Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) (Pedrini et al., 2011) for freshly harvested virus stocks (40–60 h post infection). However, this ratio is temporally dynamic and will increase with increasing time post infection, and is likely to reach very high (>100) levels upon storage (Pedrini et al., 2011; Rosinski et al., 2002), due to virus particle aggregation, which leads to a progressive loss of infectivity (Jorio et al., 2006). Hence there are a wide variety of infectious BV titration assays to choose from, but each has its own specific strengths and weaknesses. In terms of assay duration, the well-established plaque and endpoint assays are quite lengthy (up to 7 days required) (O’Reilly et al., 1994), while the immunological (48 h), AlamarBlue (24 h) and SCT (18–24 h) assays have a speed advantage (Kitts and Green, 1999; Pouliquen et al., 2006; Roldao et al., 2009). A reporter gene will increase the objectivity of the plaque and endpoint assays (although not necessarily essential), while it is not required for most of the other assays, which have their own objective means of identifying an infection event. One major disadvantage of the existing titration assays is procedural complexity, involving the analysis of multiple virus dilutions, and the setup of a large number of well (microplate) or shaker infections just to obtain the titer of one virus sample. Another disadvantage is the fact that these assays

Fig. 1. Correlation between the peak cell density/initial cell density (PCD/ICD) ratio and the multiplicity of infection (MOI) for rAcMNPV–Sf9 and wild-type HaSNPV–HzAM1 infected systems, as predicted by the Power–Nielsen baculovirus infection model. (A) Non-transformed data and (B) Log-transformed data.

are primarily optimized for the titration of rAcMNPV, the most prominent baculovirus expression vector (Jarvis, 2009; Roldao et al., 2009). rAcMNPV exhibits fast infection kinetics (Pedrini et al., 2011; Power et al., 1994), hence assays designed for its titration may not work as efficiently for baculoviruses exhibiting slower kinetics such as HaSNPV (Pedrini et al., 2011). In this study, a new type of SCT assay for infectious BV titration has been developed, with the objective of delivering a less complex, more robust, and more objective methodology that is consistent with a suspension culture-based baculovirus production process, and that is equally effective for baculoviruses with slow or fast kinetics. This assay is informed by the Power–Nielsen baculovirus infection model (Power et al., 1994), a powerful tool that allowed insights to be made on the most efficient means of devising the new methodology. The model predicted that the peak cell density (PCD)/ICD ratio of an infected culture will decline linearly with increasing MOI (after log transformation), which indicated the dose-dependency of cell growth suppression on the amount of infectious virus added. The model showed that such a linear correlation can be obtained from baculovirus-insect cell systems, whether the infection kinetics are slow (HaSNPV–HzAM1) or fast (rAcMNPV–Sf9) (Fig. 1B). Hence, a titration assay can be devised by first generating a one-off standard curve relating suspension culture-derived PCD/ICD ratios with adherent culture-derived

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MOIs from the same virus dilution series. Future virus titrations can then be carried out simply by setting up suspension culture infections at a specific virus inoculum volume, estimating the PCD/ICD ratio and then calculating the MOI applied via the standard curve, and finally converting the MOI to the virus inoculum titer. Apart from an initial intensive effort to generate the standard curve (with adequate replication to reduce the variability of the adherent culture-based assays), this titration approach is very time and resource efficient when compared to the existing assays, requiring only a small number of suspension culture infections to be conducted, and relying on very objective and reproducible means of measuring the titration outputs (conventional cell counts). The first part of this study involved the titration of HaSNPV, in which the efficacy and reproducibility of two existing ACT assays (MTT and AlamarBlue) were statistically compared with those of the newly developed SCT assay. This comparison study was in a similar vein to that reported recently for the titration of rAcMNPV (Roldao et al., 2009). At this stage of the study, the SCT assay was dependent on the Power–Nielsen model, which meant that the suspension-culture derived PCD/ICD ratios were used as inputs for in silico simulations to estimate the MOIs applied. In the second part of this study, a model-independent standard curve-based SCT assay was developed for both the rAcMNPV–Sf9 and HaSNPV–HzAM1 systems, as described previously, to broaden its applicability and user-friendliness, as modelling expertise is not likely to be available in most virology laboratories.

2. Materials and methods 2.1. Cell lines, media and virus stocks 2.1.1. HaSNPV–HzAM1 system The Helicoverpa zea cell line (BCIRL–HZAM1), wild-type H. armigera single-capsid nucleopolyhedrovirus (family Baculoviridae, genus Alphabaculovirus) (HaSNPV, Strain H25EA1), and in-house low-cost serum-free medium (VPM3) used in this study have been described in the PCT patent, WO/2005/045014 (Reid and Lua, 2005). VPM3-adapted HzAM1 stock cells were maintained as 100 mL suspension cultures in 250 mL disposable Erlenmeyer flasks (Corning, Lowell, MA, USA), which were agitated at 120 rpm on an orbital shaker and incubated at 28 ◦ C. These default stock cells were used for both suspension and adherent culture-based titration assays for HaSNPV, with the exception of plaque assays. In the latter case, the HzAM1 cells used were adapted to 2 mL adherent cultures (6-well microplates, Corning), and grown in TNMFH medium (Sigma–Aldrich) supplemented with 10% fetal bovine serum (Gibco® Qualified FBS, Invitrogen, Carlsbad, CA, USA). Passage 1 (P1) HaSNPV virus stock was prepared by extracting occlusion derived virions (ODVs) from caterpillar-derived OBs (Lynn, 1994; Reid and Lua, 2005). Briefly, 40 ␮L of alkaline saline (0.5 M Na2 CO3 , 1.0 M NaCl) was added to 500 ␮L of OB suspension (1010 OBs/mL), which was incubated at 28 ◦ C for 30 min to lyse the OBs. The resulting ODV suspension was neutralized by dilution with 9.5 mL of VPM3 medium, sterilized with 0.2 ␮m syringe filters (Sartorius-Stedim Biotech, Aubagne, France), and then used to infect a 100 mL shaker culture seeded at 5 × 105 cells/mL. The P1 infected culture was incubated for 4 days post infection (d.p.i.), and was then used to establish the P2 infection (5 × 105 cells/mL, 100 mL, 15% (v/v) virus inoculum). The P2 infected culture was incubated for 3 d.p.i. and then centrifuged (1000 × g, 10 min) to retrieve the BV-containing supernatant as the P2 working virus stock, which was aliquoted into three fractions: freshly harvested unstored virus (stock 1) and frozen virus stored at −80 ◦ C (stocks 2 and 3).

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2.1.2. rAcMNPV–Sf9 system The Spodoptera frugiperda clone 9 cell-line (Sf9; ATCC CRL 1711) and recombinant ␤-galactosidase-expressing A. californica multi-capsid nucleopolyhedrovirus (family Baculoviridae, genus Alphabaculovirus) (rAcMNPV) used in this study have been described previously (Haas and Nielsen, 2005). The Sf9 cells were propagated in the commercial serum-free medium Sf-900 II (Invitrogen), in shaker flask cultures as described for HzAM1 cells, and were used for both suspension and adherent culture-based titration assays for rAcMNPV. The rAcMNPV working virus stock was established from a cryopreserved virus stock (Haas and Nielsen, 2005). Briefly, a 100 mL shaker infection (P3) was set up at 1.5 × 106 cells/mL and an MOI of 0.01 plaque forming units (PFU)/cell, which was incubated for 4 d.p.i. and then centrifuged (1000 × g, 10 min) to recover the BV-containing supernatant as the P3 working virus stock (stored at 4 ◦ C). 2.2. Cell density enumeration Cell densities were enumerated using an improved Neubauer hemocytometer (Weber, England) and a phase-contrast microscope (Olympus, Japan). Cell viabilities were estimated using the Trypan Blue exclusion method. A relative error of 15% (95% confidence interval) is obtained by performing the hemocytometer counts in triplicate (Nielsen et al., 1991). In specific cases, total cell densities were also enumerated using a MultisizerTM 4 coulter counter (Beckman Coulter, Fullerton, CA), which provided much reduced count variability (2%). 2.3. Baculovirus titration assays 2.3.1. Plaque assay A plaque assay was used to titrate the HaSNPV–HzAM1 system in this study, which was derived from previous procedures (Chakraborty and Reid, 1999; Pedrini, 2004). Briefly, adherent culture-adapted HzAM1 cells (in TNMFH + 10% FBS medium) at mid-exponential growth phase were pelleted (100 × g, 5 min) and resuspended in serum-free TNMFH medium to a density of 3.4 × 105 cells/mL. Six-well plates (Corning) were then dispensed with 1.75 mL of cell suspension per well, and incubated at 28 ◦ C for 1 h for cell attachment (around 50% confluence). The HaSNPV BV sample was serially diluted with serum-free medium to obtain the 10−4 , 10−5 and 10−6 dilutions, which were then added to the sixwell plates (0.25 mL per well, 6 wells per dilution level). The plates were then centrifuged (1000 × g, 1 h) to promote attachment of BV to the cell monolayer. Serum was not added to the medium in these steps as it may compete with BV for cell binding sites (Dee and Shuler, 1997a). Finally, the supernatant in each well was replaced with an agarose overlay (0.9× TNMFH, 10% FBS, 1% Seaplaque® agarose), and the plates were incubated at 28 ◦ C for 6 days for virus plaque formation. The number of plaques per well were enumerated with the aid of a stereo dissecting microscope (Olympus, Japan). Only wells with 5–50 plaques were counted, and the BV titer was calculated using Eq. (1), where each plaque is referred to as a plaque forming unit (PFU), V is the virus inoculum volume (0.25 mL/well) and D is the dilution factor. BV Titer (Plaque Assay) (PFU/mL) =

Average PFU/well ×D V

(1)

2.3.2. Endpoint dilution assay An endpoint dilution assay was used to titrate the rAcMNPV–Sf9 system in this study, which is based on a modified Reed and Muench methodology (Nielsen et al., 1992; Reed and Muench, 1938) that is well-established in this group (Chan et al., 1998;

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Power et al., 1994; Rosinski et al., 2002; Wong et al., 1996). Briefly, flat-bottomed 96-well microplates were seeded with midexponential phase Sf9 cells (25 ␮L/well, 4 × 105 cells/mL), and 10-fold serial dilutions of the virus sample were prepared in Sf900 II medium. The 10−3 –10−8 virus dilutions were each added to 16 wells (25 ␮L/well), and the microplates were then incubated at 28 ◦ C for 7 days. The microplates were then scored for positive ␤galactosidase production by addition of its chromogenic substrate X-Gal (Sigma–Aldrich) (25 ␮L/well, 0.77 mg/mL final concentration), which was enzymatically cleaved to form a blue product. The number of positive and negative wells for each virus dilution was converted to the undiluted BV concentration (PFU/mL) by means of a Microsoft Excel-based limiting dilution assay analyzer developed by Nielsen et al. (1992). Endpoint titration of virus samples was conducted in triplicate. 2.3.3. MTT colorimetric assay The tetrazolium ring of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) can be cleaved by mitochondrial enzymes in viable cells, resulting in the formation of an insoluble magenta-colored salt (formazan). Hence, a BV titration assay can be developed that relates the reduction in cell viability (indicated by the MTT chromogenic reaction) with the virus dose applied, as described previously for the rAcMNPV–Sf9 system (Mena et al., 2003). In this study, the MTT assay was adapted for the HaSNPV–HzAM1 system. Briefly, flat-bottomed 96-well microplates (Corning) were seeded with mid-exponential phase HzAM1 cells (50 ␮L/well, 5 × 103 cells/mL), and 10-fold serial dilutions of the virus sample were prepared in VPM3 medium (ranging from 100 to 10−7 ). Each virus dilution was then added to 10 wells (at 10 ␮L/well). The infected microplate cultures were incubated at 28 ◦ C for 5 days, after which 10 ␮L of MTT dye (5 g/L, Sigma–Aldrich) was added to each well, and the microplates were then incubated at 28 ◦ C for 2 h. Finally, the microplates were centrifuged (2000 × g, 10 min) to pellet the cells, the supernatants were removed, and 50 ␮L of DMSO (Sigma–Aldrich) was added per well to solubilize the formazan salt. The degree of color formation in each well, and for each virus dilution, was measured by spectrophotometry at an absorbance of 570 nm (Spectramax M5 microplate reader; Molecular Devices, Sunnyvale, CA). A standard curve was then generated by plotting absorbance (y) against dilution factor D (log-scale), and fitting the data-points with a 4-parameter logistic (sigmoidal) regression using SigmaPlot (Systat Software, Chicago, IL, USA) as shown in Eq. (2): y = y0 +

a 1 + (D/D0 )

(2)

b

The key parameter obtained from the curve-fit is D0 , which is the dilution factor at which the response is 50%. The BV titer, expressed in terms of the 50% tissue-culture lethal dose (TCLD50 ), was then calculated using Eq. (3) with a virus inoculum volume V of 0.01 mL/well. The MTT assay was used to titer the HaSNPV–P2 virus stocks 1, 2 and 3, with each titration replicated three times. BV Titer (MTT Assay) (TCLD50 /mL) =

1 D0 V

(3)

2.3.4. AlamarBlue colorimetric assay The AlamarBlue assay is similar to the MTT assay in terms of using a chromogenic reaction as a cell viability indicator, and relating cell viability reduction with the virus inoculum titer. In this case, Resazurin (AlamarBlue® ; Invitrogen) is reduced by the mitochondrial enzymes of viable cells to Resorufin (blue to bright-red fluorescence). An AlamarBlue titration assay was described previously for the rAcMNPV–Sf21 system (Pouliquen et al., 2006),

and an adapted version was used for the HaSNPV–HzAM1 system in this study. Briefly, flat-bottomed 96-well microplates were seeded with mid-exponential phase HzAM1 cells (100 ␮L/well, 3 × 104 cells/mL), and 2-fold serial dilutions of the virus sample were prepared in VPM3 medium (ranging from 21 to 210 ). Each virus dilution (including the virus-free medium control) was added to 5 wells (100 ␮L/well). Then, 20 ␮L/well of AlamarBlue was added to the control wells (9.1% (v/v) final concentration) to establish the initial (fI ) fluorescence reading (530 nm excitation, 590 nm emission, Spectramax M5). The microplate cultures were incubated at 28 ◦ C for 3 days, after which 20 ␮L/well of AlamarBlue was added to the infected wells. The final (fF ) fluorescence readings were then obtained for both infected and control wells. Fluorescence readings were always performed 5 h after dye addition (and incubation at 28 ◦ C) to allow sufficient time for Resorufin formation. The percentage of growth inhibition (GI) was estimated from the initial and final fluorescence measurements using the following expression, for each dilution factor: GI (%) =

fF (infected) − fI (control) fF (control) − fI (control)

(4)

A standard curve was then generated by plotting GI against dilution factor D (log scale), and fitting the data-points with the same sigmoidal regression used for the MTT assay (Eq. (2)). The key parameter D0 was then obtained, and the BV titer (TCLD50 /mL) was calculated using Eq. (3) (V = 0.1 mL/well). The AlamarBlue assay was used to titer the HaSNPV–P2 virus stocks 1, 2 and 3, with each titration replicated three times.

2.3.5. Correlation between the colorimetric and plaque assays The MTT and AlamarBlue titration assays measure virus titers in terms of TCLD50 /mL, hence it is desirable to derive a correlation between TCLD50 and PFU. The correlation experiments were conducted separately for each colorimetric assay. For the MTT assay, a virus dilution series was prepared in VPM3 medium using the HaSNPV–P2 virus stock 3. Each diluted sample was then titered using triplicate MTT and plaque assays, and the resulting TCLD50 and PFU data were log-transformed and plotted against each other to derive a linear correlation using SigmaPlot. The AlamarBlue assay correlation experiment was similarly conducted, except that virus stock 1 was used.

2.3.6. Suspension culture titration assay (model-dependent) The model-dependent SCT assay employed the Power–Nielsen unstructured model of baculovirus infection (Power and Nielsen, 1996) to convert assay outputs into BV titers. This model was originally formulated for the rAcMNPV–Sf9 system in Matlab code (MathWorks, Natick, MA), and was subsequently modified for the HaSNPV–HzAM1 system using separately established virus kinetic parameters (Pedrini et al., 2011). Virus samples (HaSNPV–P2 virus stocks 1, 2 and 3) were first appropriately diluted with medium, then added at a particular virus volumetric fraction (FV ) to a shaker culture (50 mL infected culture volume at an ICD of 5 × 105 cells/mL, in a 125 mL Erlenmeyer flask), so that the PCD of the infection was not more than 50% of the maximum uninfected PCD achievable in the medium. This precaution was followed to avoid nutrient limitation, as indicated by the cell yield concept (Wong et al., 1996). Each SCT assay was performed in triplicate flasks, and total cell densities were estimated at 0, 1, 2, 3 and 4 d.p.i. using hemocytometer counts to obtain the two key titration variables: ICD (cells/mL) and PCD (cells/mL). An in silico simulation was then carried out using the HaSNPV model, to predict the MOI (PFU/cell) applied based on these assay outputs. From

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the model-predicted MOI, the BV titer of the virus inoculum was calculated using Eq. (5): BV Titer (SCT Assay) (PFU/mL) =

ICD × MOI × D FV

(5)

In practice, FV was set at 0.05 (virus volume/infected culture volume), and a preliminary model simulation was conducted using an initial ‘guestimate’ of the virus titer, to determine the virus dilution D required to obtain a PCD/ICD ratio of at least 1.5, and with an upper limit that did not result in nutrient limited cultures. A sizeable difference between the PCD and ICD was desirable as hemocytometer cell counts exhibit significant measurement errors, although such errors were moderated by carrying out each shaker titration in triplicate. If the infected cells had undergrown or overgrown according to the suspension culture assay criteria, the titration was repeated using an improved estimate of the virus dilution required. 2.3.7. Suspension culture titration assay (model-independent): standard curve generation Entirely empirical log(PCD/ICD) vs log(MOI) standard curves were generated for both HaSNPV and rAcMNPV, to establish the model-independent SCT assay. Using the model as a guide (Fig. 1B), and having titered the virus standard, a virus dilution series was designed with sufficient standard points to cover the desired ranges of log(PCD/ICD) and log(MOI), which are dictated by the virus kinetics. Each standard point (virus dilution) was analyzed by both SCT and ACT assays in triplicate. The SCT assay was set up as described previously, but only to obtain the PCD/ICD ratio (in silico simulations were not conducted). One of two ACT assays was used to determine the infectious BV titer of each virus dilution, being either the plaque assay for HaSNPV titration or the endpoint assay for rAcMNPV titration, since these are the most established and reliable in this laboratory. The BV titer of a particular virus dilution was then used to calculate the MOI of the corresponding SCT assay, based on the ICD and FV settings applied. The virus standards used were a separately prepared HaSNPV P2 virus stock (used immediately after harvest without storage, and different from stocks 1, 2 and 3), and the rAcMNPV–P3 working virus stock. Total cell densities were measured using a MultisizerTM 4 coulter counter instead of hemocytometer counts, to improve the accuracy of the PCD/ICD ratio. SigmaPlot was used to generate the linear correlation between log(PCD/ICD) and log(MOI). 2.4. Statistical analysis Statistical analysis was performed using the SPSS Statistical Software, Version 19 (IBM, Armonk, NY, USA). All error estimates were in terms of the 95% confidence interval. A one-way analysis of variance (ANOVA), incorporating Scheffe’s method, was used to determine whether titers measured via different titration methods were significantly different at the 95% confidence level. 3. Results 3.1. Adherent culture colorimetric titration assays for HaSNPV The MTT and AlamarBlue ACT assays required the derivation of a sigmoidal relationship between virus dilution factor and the colorimetric response variable. Examples of MTT and AlamarBlue titration curves are presented in Fig. 2A and B respectively, for the titration of HaSNPV–P2 virus stock 3. The 4-parameter logistic sigmoidal model fitted well with the experimental data, exhibiting an R2 value of 0.9983 and a D0 value of 7.00 (± 1.74) × 10−4 for the MTT assay, and an R2 value of 0.9939 and a D0 value of 2.97 (± 0.59)×10−2 for the AlamarBlue assay. Using Eq. (2),

Fig. 2. Dose response (sigmoidal) curves from two adherent culture colorimetric titration assays for the HaSNPV–HzAM1 system. (A) MTT assay and (B) AlamarBlue assay. GI is the growth inhibition factor. The error bars represent the 95% confidence interval.

the titer of virus stock 3 was estimated to be 1.43 × 105 and 3.37 × 102 TCLD50 /mL from the respective assays, which were obviously very divergent. The TCLD50 -based titers from the colorimetric assays were converted to a PFU basis, by means of separate standard curves relating the plaque assay-derived PFU/mL with the MTT or AlamarBluederived TCLD50 /mL (both variables being log-transformed), as presented in Fig. 3A and B respectively. These linear correlations fitted well with the experimental data, exhibiting R2 values of 0.99 (MTT) and 0.98 (AlamarBlue). It was striking to observe that the TCLD50 estimates were much lower than those for the PFU, particularly for the AlamarBlue case. The linear relationship between PFU and TCLD50 for both colorimetric assays are expressed as follows: log

 PFU  mL

= 0.8685(±0.1196) × log

 TCLD MTT  50 mL

+ 1.9710 (±0.5727)

log

 PFU  mL

= 0.3058(±0.0480) × log + 6.4148(±0.0737)

(6)

 TCLD AlamarBlue  50 mL (7)

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Fig. 3. Standard curves for converting the TCLD50 -based titers of adherent culture colorimetric titration assays to the PFU-based titers of plaque assays, for the HaSNPV–HzAM1 system. (A) MTT assay and (B) AlamarBlue assay. The error bars represent the 95% confidence interval.

The BV titer estimates of HaSNPV–P2 virus stocks 1, 2 and 3 from the MTT and AlamarBlue assays are compared in Table 1 (each assay being triplicated for each virus stock). The TCLD50 -based titers from the colorimetric assays were converted to PFU/mL using Eqs. (6) and (7). In both assays, virus stock 1 (unfrozen) exhibited higher titers than stocks 2 and 3 (frozen), which was expected as freezethawing results in significant virus degradation (Jorio et al., 2006). BV titers were highest when measured by the AlamarBlue assay, but a one-way ANOVA (using Scheffe’s multiple comparison procedure) showed that the titers from both colorimetric assays were not significantly different from each other (P > 0.05), for each of the virus stocks tested (Table 1). Furthermore, both MTT and AlamarBlue assays exhibited poor reproducibilities, since their titer estimates have 95% confidence intervals of 55 and 33% respectively (Table 1). 3.2. Suspension culture titration assay for HaSNPV (model-dependent) The model-dependent SCT assay measured higher BV titers than the ACT assays for HaSNPV–P2 virus stocks 1, 2 and 3 (Table 1). The average titer of the three virus stocks from the SCT assay was

Fig. 4. Standard curves for the model-independent suspension culture titration assay, correlating the suspension culture-derived peak cell density/initial cell density (PCD/ICD) ratio with the adherent culture-derived multiplicity of infection (MOI). (A) rAcMNPV–Sf9 and (B) wild-type HaSNPV–HzAM1 systems. The solid line represents the empirical correlation, while the dashed line represents the Power–Nielsen model-derived correlation. The error bars represent the 95% confidence interval.

5-fold and 2-fold higher than that of the MTT and AlamarBlue assays respectively. A one-way ANOVA (using Scheffe’s procedure) showed that titers obtained from the SCT assay were significantly different from those of the ACT assays (P < 0.05), for most of the virus stocks tested (Table 1). Furthermore, the SCT assay exhibited much better reproducibilities than the ACT assays, with a 95% confidence interval of 13.7% (Table 1).

3.3. Suspension culture titration assay for HaSNPV and rAcMNPV (model-independent) Fig. 4A and B shows the empirical correlations between the SCT assay-derived PCD/ICD ratio and the ACT assay-derived MOI (after log-transformation), for the rAcMNPV–Sf9 and HaSNPV–HzAM1 systems respectively. The rAcMNPV standard curve exhibited an excellent linear fit (R2 = 0.9976) with the experimental data (with endpoint titration based MOIs), and is described in Eq. (8). The empirical standard curve (solid line) was close to that predicted by the rAcMNPV model (dashed line), and can be used to titer infections across a wide range of PCD/ICD ratios (1.5–7) or MOIs (0.00002

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Table 1 Comparison of the efficacy and reproducibility of adherent culture titration (ACT) and model-dependent suspension culture titration (SCT) assays for HaSNPV. Virus titration assay

MTT (ACT) AlamarBlue (ACT) SCT Multiple comparisons

MTT–AlamarBlue MTT–SCT AlamarBlue–SCT a

Budded virus titer (× 107 PFU/mL)

Average 95% CI of assay (%)

Stock virus 1

Stock virus 2

Stock virus 3

0.78 (±0.28)a 1.79 (±0.21) 4.13 (±0.58)

0.55 (±0.36) 1.01 (±0.31) 2.37 (±0.33)

0.23 (±0.15) 0.89 (±0.50) 1.39 (±0.18)

Significance of mean differences (˛ = 0.05) using Scheffe’s procedure Stock virus 1

Stock virus 2

Stock virus 3

0.369 0.000 0.000

0.846 0.001 0.002

0.451 0.027 0.241

The brackets enclose the 95% confidence interval (CI) of the mean virus titer from triplicate titration assays.

to 0.2 PFU/cell) (Fig. 4A). log

 PCD  ICD

= −0.1602(±0.0090) × log(MOI) + 0.0968(±0.0265){AcMNPV}

(8)

The HaSNPV standard curve also exhibited an excellent linear fit (R2 = 0.9981) with the experimental data (with plaque assay based MOIs), and is described in Eq. (9). The empirical standard curve (solid line) was close to that predicted by the HaSNPV model (dashed line) (Fig. 4B), but it did not have a wide range of PCD/ICD ratios (2–5) or MOIs (0.35–1.7 PFU/cell) when compared to the rAcMNPV standard curve, which is a reflection on the much poorer kinetics of HaSNPV infections (Pedrini et al., 2011), and the lower cell yields of the HzAM1/VPM3 culture system. log

±55.2 ±32.8 ±13.7

 PCD  ICD

= −0.5522(±0.0194) × log(MOI) + 0.4778(±0.0047){HaSNPV}

(9)

The empirical standard curves described above allow future virus titrations to be carried out entirely using a suspension culturebased system, as follows. Triplicated shaker infections are first set up at a defined ICD and virus volumetric fraction FV , from which the PCD/ICD ratio is obtained after 3–4 d.p.i. for HaSNPV or 1–2 d.p.i. for rAcMNPV (the PCD timing is dependent on the infection kinetics of each virus). The MOI is then estimated from the standard curve based on the PCD/ICD ratio, after which the virus inoculum BV titer is calculated using Eq. (5). The estimated variability (95% confidence interval) of BV titers obtained from the triplicated model-independent SCT assay (based on cell count variability) is 1.6% for coulter counter-measured cell densities, and 12.2% for hemocytometer-measured cell densities. 4. Discussion The first part of this study compared two ACT assays (MTT and AlamarBlue) with a new model-dependent SCT assay, in terms of ease of use and reproducibility for wild-type HaSNPV titration. Significant challenges were encountered for both ACT assays, which contributed toward poor titer reproducibilities (Table 1). A previous study comparing different titration methods for rAcMNPV also reported similarly poor reproducibilities for these assays (Roldao et al., 2009). For the MTT assay, it was difficult to prevent some loss of cells during the supernatant removal step (despite a prior microplate centrifugation step), leading to variability in estimating the formazan content. It was also difficult to solubilize the formazan salts completely in each well using DMSO, which further contributed

to assay variability. The AlamarBlue assay had some advantages over the MTT assay, as it was faster to complete (3 instead of 5 days) and lacked supernatant removal and dye solubilization steps. However, the time advantage of the AlamarBlue assay was more impressive in rAcMNPV titrations (Pouliquen et al., 2006), whereby only 1 day was required for completion. HaSNPV exhibited much poorer infection kinetics than rAcMNPV (Pedrini et al., 2011), hence cell growth inhibition was slow for wells that received diluted (low MOI) virus inocula, and a longer infection period was required to acquire the full growth inhibition curve. Furthermore, the variability of the AlamarBlue assay in this study was approximately 50% higher than that in the original protocol (Pouliquen et al., 2006). The earlier study may have had better assay reproducibilities due to the use of an automated liquid handling workstation, which greatly simplified the task of precision-addition of assay reagents (cells, medium, virus and dye) to the numerous wells of a microplate. However, such automated systems are expensive, and are unlikely to be widely available in research laboratories, especially those that are capable of preserving culture sterility for prolonged incubation periods (necessary for HaSNPV titration). The plaque assay, used as a means of converting MTT or AlamarBlue assay outcomes from TCLD50 to PFU basis (Fig. 3), was also challenging to get right, being highly dependent on operator technique and good cell condition (optimum growth rate and adherence ability) as described earlier. Furthermore, the poor infection kinetics of HaSNPV and the predominantly Many-Polyhedra (MP) phenotype of the ODV-derived virus stock (Pedrini et al., 2011; Reid and Lua, 2005), resulted in the formation of very small plaques that can be difficult to identify, which contributed to some subjectivity in this assay. To improve the performance of the plaque assay, the HzAM1 stock cells used were grown in serum-containing medium and adapted to adherent cultures, in contrast to the suspension-adapted cells used in the MTT and AlamarBlue assays. This ensured that a uniformly distributed and strongly adhered cell monolayer was formed on the microplate surface, that was not easily dislodged during pipetting steps and when the agarose overlay was poured, which is important for optimum plaque formation. Furthermore, a centrifugation step was carried out after virus inoculation and prior to agarose overlaying, to maximize the efficiency of virus adsorption onto the cell monolayer (Dee and Shuler, 1997b). This study has shown that the Power–Nielsen model of baculovirus infection (Power et al., 1994) can be used as an effective basis for BV titration, by predicting the MOI of infected suspension cell cultures via their initial and final cell densities. This model-dependent SCT assay was more promising than the MTT and AlamarBlue assays for HaSNPV titration, being supportive of consistently higher titer estimations and much better reproducibilities (Table 1). The better performance of the SCT assay may be attributed to the simplicity of the protocol, which did not feature

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manually repetitive (and potentially error-prone) steps such as those required for the MTT and AlamarBlue assays. Furthermore, suspension cultures may support more reproducible infections, due to their well-mixed environment, leading to efficient mass transfer of nutrients, oxygen and virus inoculum. For example, suspension cultures have been reported to support faster virus binding kinetics in the rAcMNPV–Sf21 system (Dee and Shuler, 1997a), higher cell densities and BV yields in the HzSNPV/HzAM1 system (McIntosh et al., 2001), and superior recombinant protein production in the rAcMNPV/Sf9 and rBmNPV/Bm5 systems (Kioukia et al., 1995; Zhang et al., 1994), when compared to adherent cultures. The adherent culture environment may support less reproducible virus infections because diffusion is the main mass transfer mechanism, which is not as efficient in delivering nutrients, oxygen and virus to cells (Guarino et al., 2005). While a model is an invaluable tool for both virus titration and process optimization in the baculovirus expression system, and should be a prerequisite of any process scale-up initiative, it may not be employed easily by groups that do not have a computational background, despite the wide availability of in vitro baculovirus expression models in the literature (De Gooijer et al., 1992; Dee and Shuler, 1997a; Enden et al., 2005; Hu and Bentley, 2000; Power et al., 1994; Roldao et al., 2007; Sanderson et al., 1999). The Power–Nielsen model predicted that a linear correlation exists between log(PCD/ICD) and log(MOI), which suggested that a standard curve can be developed for model-independent virus titration. This study has confirmed that such an approach is feasible, by first preparing a dilution series from a virus standard, which are then titered using conventional ACT assays (to obtain the MOI data) and used to set up shaker infections (to obtain the PCD/ICD data). Using this method, standard curves of excellent linearity were constructed for two viruses with very different infection kinetics, HaSNPV and rAcMNPV (Fig. 4). It may be argued that potentially highly variable ACT assays are incompatible with a new SCT assay aimed at better objectivity and reproducibility. However, the PCD/ICD ratio is best correlated to an actual unit of infection (PFU) to maximize the assay’s accessibility, and there are no better established and recognised means of measuring infectious BV titers than plaque assays (for wild-type viruses like HaSNPV) or endpoint assays (for reporter gene-enabled viruses like rAcMNPV). Provided that the ACT assays are well-replicated to reduce their variability, as performed in this study, high quality standard curves would be obtained. In principle, each standard curve need only be generated once, although re-validation of key sections (e.g. beginning, middle and end of the correlation) is prudent if there are significant changes to the production system, since infection kinetics can be profoundly affected by the cell line, growth medium or virus isolate used (Lynn, 2000; Pedrini et al., 2006; Pedrini et al., 2011). Once the standard curve is generated, the model-independent SCT assay is very simple and resource-efficient to implement, requiring only a small number of suspension culture infections to be set up per virus sample, needing only cell counts to obtain the PCD/ICD ratio, and using equipment that are widely available in cell culture laboratories. Since cell counts are very reproducible, the SCT assay’s variability (95% confidence interval) from replicate measurements is consequently very moderate, being around 2–12% depending on whether automated or manual cell counts are employed. This contrasts favourably with the 95% confidence intervals of the MTT and AlamarBlue assays tested in this study for HaSNPV (Table 1), and those of a variety of ACT and SCT asssays tested previously for rAcMNPV, including the MTT (39%), AlamarBlue (55%), cell size (26%), growth cessation (71%) and flow cytometry (26%) assays (Roldao et al., 2009). Furthemore, the SCT assay would be as accurate as the plaque or endpoint assay used to generate the MOI component of its standard curve.

Another key advantage of the model-independent SCT assay is that it is applicable to a wide range of virus infection kinetics, as demonstrated by the derivation of standard curves for ‘fast’ (rAcMNPV) and ‘slow’ (HaSNPV) viruses (Fig. 4). In contrast, most baculovirus titration assays are developed and optimized for rAcMNPV (in particular the rAcMNPV–Sf9 system), due to its status as the dominant baculovirus expression vector (Jarvis, 2009; Mena and Kamen, 2011). The superior infection kinetics of rAcMNPV, in terms of fast binding rate, short latency period prior to progeny BV production, rapid BV production rate and high volumetric BV titers (Pedrini et al., 2011; Power et al., 1994), are ‘titration friendly’ characteristics which promote the formation of large easier-to-score plaques in plaque assays, and allow low MOI infections to proceed efficiently in endpoint dilution-type (e.g. endpoint, MTT, AlamarBlue) assays. However, when infection kinetics are poor (such as those of HaSNPV), these ACT assays become less effective and reproducible, due to cell overgrowth and subsequent nutrient limitation at high virus dilution factors. Consideration for poor infection kinetics also appears to be absent from alternative SCT assays based on cell size change (Janakiraman et al., 2006), cell growth cessation (Roldao et al., 2009) and flow cytometry (Roldao et al., 2009). While the model-independent SCT assay appears to be very competitive against existing titration assays for ‘slow’ viruses like HaSNPV, it is also a promising alternative means of titering rAcMNPV due to its simplicity and resource-efficiency (once the one-off standard curve has been generated). In comparison, plaque assays are very dependent on operator expertise, while most of the other existing ACT or SCT assays require a dose response curve to be generated for each virus sample being titrated, which is a laborious exercise involving the setup of a large number of replicated microplate or shaker flask infections to accommodate a wide range of virus dilutions (Janakiraman et al., 2006; Kitts and Green, 1999; Mena et al., 2003; Pouliquen et al., 2006; Roldao et al., 2009). However, the new SCT assay has a drawback for rAcMNPV titration, being relatively longer in timeframe (24–48 h) when compared to faster methods (18–24 h) such as the AlamarBlue, cell size, growth cessation and flow cytometry assays (Roldao et al., 2009). The rAcMNPV standard curve derived from this study (Fig. 4A) may be a useful means of titering other types of rAcMNPV constructs, if an approximate titer estimation is sufficient, and provided that the cell-line and medium used are the same as those in this study. In conclusion, a new suspension culture titration (SCT) assay has been developed to measure infectious baculovirus titers, by correlating the PCD/ICD ratio of infections (suspension culture-derived) with the corresponding MOIs applied (adherent culture-derived). Standard curves of log(PCD/ICD) vs log(MOI) with excellent linearity have been generated for two representative baculoviruses (HaSNPV and rAcMNPV). Virus titrations are then carried out by setting up suspension culture infections from the desired virus stock, measuring the PCD/ICD ratio, and estimating the applied MOI via the standard curve. The SCT titration assay has the advantages of protocol simplicity, relatively low assay variability, time and resource efficiencies, and applicability to a wide range of virus infection kinetics. Acknowledgements The authors wish to acknowledge financial support from the Australian Research Council (Linkage Grant LP0989824) and Agrichem Pty Ltd, and the Iranian Government Ministry of Education for provision of a PhD scholarship. References Baxter, R., Patriarca, P.A., Ensor, K., Izikson, R., Goldenthal, K.L., Cox, M.M., 2011. Evaluation of the safety, reactogenicity and immunogenicity of FluBlok (R)

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