5. Influence of multiplicity of infection (MOI) on synchronous baculovirus infections 5.1. Introduction

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1 5. Influence of multiplicity of infection (MOI) on synchronous baculovirus infections 5.1. Introduction The symptoms of baculovirus infection vary greatly between cells in the same cell culture, even if all cells are infected within a very short time period. These differences manifest themselves in (1) a large spread in time until cell lysis occurs from 45hpi to 90hpi as shown in chapter 3, (2) an up to 20-fold difference in the specific viral recombinant protein yield as shown in section 4.4, and (3) a cell volume increase that is inconsistent with simple uniform theories as discussed in appendix 9.4. These differences (or asynchronies) between infected cells could in theory be attributed to the number of infectious viruses an individual cell receives (η).the role of η, however, remains controversial. In synchronous recombinant AcMNPV infections of Sf9 cells, viral recombinant protein production and/or the viability have been observed to depend on the MOI by Naggie & Bentely (1998), Dalal & Bentley (1999), and Satio et al. (2002), whereas Radford et al. (1997, figure 6), Kioukia et al. (1995), and Schopf et al. (1990) observed no such behaviour. The different observations have led to the two- and three dimensional models, where the former assume no η dependency (section 1.3.1) while the latter assume a η dependency (section 1.3.2). Most of the studies were not specifically designed to investigate the influence of η and data from synchronous infections are often compared with data from asynchronous infections. If a culture is infected for example with an MOI of 1PFU/cell, 36% of all cells do not become infected during the primary infection (Equation 19). By the time the infected cells start to release BV at ~17hpi, the uninfected cell density has almost doubled whereas the infected cells had ceased cell division. Therefore, the observed behaviour is the average from two equally sized populations with one lagging 17 hours behind in the infection cycle. The timing of the onset of secondary infections is almost impossible to determine accurately as no adequate analytical method exists and one has to relay on modelling solely. Further, it can be speculated that secondary infection does not occur through the adsorption of released BV but rather through collisions with infected cells that have BV attached to their cell wall possibly a long time prior to release. Due to these reasons, asynchronous infections cannot be used to compare the effect of η on the timing and intensities of infection cycle markers. In this chapter, experimental data are presented that specifically demonstrate that η does not influence the responses of BEV infected Sf9 cells. 47

2 5.2. Experimental setup The concept The multiplicity of infection (MOI) is a measure of the average number of infectious virus particles a cell receives. Assuming that all cells are equally likely to become infected (A 7), η can be estimated by the Poisson distribution (Equation 19); in contrast to the Equation 4 (p.9) the restriction that only adsorptions which occur within the first hpi are relevant (A 8) is now removed as this assumption was introduced purely for the sake of an easier construction of the mathematical model and has no biologically supported basis. 21 p ( η ) η MOI e = η! MOI Equation 19: Probability that a cell adsorbs η infectious particles determined by Poisson distribution. A 7: A 17: All cells are equally likely to become infected. All added viruses adsorb. A culture that is infected with an MOI of 15 PFU/cell has <1% of all cells infected with an η < 6 PFU/cell, and <1% of all cells infected with an η > 25 PFU/cell (Figure 22). If η causes the observed asynchronies within a synchronously infected culture, it must be expected that cells infected with 6 PFU/cell behave differently than cells infected with 25 PFU/cell. A culture that is infected with an MOI of 5 PFU/cell has 76% of all cells infected with an η < 6 PFU/cell and 0.7% of non-infected cells; a culture infected with an MOI of 45 PFU/cell has 99.9% of all cells infected with an η > 25 PFU/cell (Figure 22). Figure 22: A 7: A 17: Probability distribution of η for cultures infected at different MOIs. All cells are equally likely to become infected. All added viruses adsorb. 21 According to Nielsen (2000), α can be estimated as 0.66 for an infection in serum free medium with an MOI of 15PFU/cell and a TOI of 10 6 cells/ml 48

3 Hence, if η causes the observed asynchronies within a synchronously infected culture, these asynchronies must be observed in deviating behaviours of cultures infected at MOIs of 5, 15, and 45 PFU/cell The setup MOI experiment I. Four 1050ml shaker flask cultures of Sf9 cells in HyQ SFX medium (Hy Clone) were seeded at a cell density of ~0.8*10 6 cells/ml, 14 hours prior to infection to allow some time for adaptation to the new environment; the inocula used for the four cultures were derived from the same parent culture. Three of these cultures were infected with MOIs of 4.7, 14.1, or 38.0 PFU/cell respectively. The βgal-bev stock contained 1.3*10 8 PFU/ml with a 95% confidence interval of 0.98*10 8 to 1.76*10 8 PFU/ml, the vdna copy number per PFU ratio was 450 genomes/pfu; the endpoint titration assay was performed immediately prior to the experiment to ensure accurate readings. Cell densities, viabilities, cell volume distributions, internal & total β-galactosidase concentrations, and budded virus concentrations were monitored throughout the complete infection cycle. The reliability of this data set might have been impaired due to the poor quality of the virus stock used (virus titres >10 9 PFU/ml, and genomes/pfu ratios << 100 genomes/pfu can be achieved in good virus stocks). Around 35hpi, the control culture was lost due to bacterial contamination. For these reasons, the experiment was repeated. MOI experiment II. Four 900ml shaker flask cultures of Sf9 cells in HyQ SFX medium (Hy Clone) were seeded at a cell density of ~0.6*10 6 cell/ml, 13 hours prior to infection; the same inoculum was used for the four cultures. Three of these cultures were infected with MOIs of 4.9, 15.2, or 46.1 PFU/cell respectively. The βgal-bev stock contained 2.6*10 9 PFU/ml with a 95% confidence interval of 2.1*10 9 to 3.2*10 9 PFU/ml, the vdna copy number per PFU ratio was 27 genomes/pfu; the endpoint titration assay was performed immediately prior to the experiment to ensure accurate readings. Additionally to the state variables monitored in MOI Experiment I the specific vdna copy numbers (n vdna ) was also monitored Results of the MOI experiments Total cell density. The total cell densities (Figure 23A and G) remained constant at the cell density at time of infection (TOI) confirming that virtually all cells were infected shortly after the addition of the virus stock. Smaller shaker flasks often show an increase in the cell density post infection by up to 49

4 20%, I speculate that this effect is due to cells that detach from the flask wall; this effect is thus less pronounced in larger volumes. The control cultures continued to grow exponentially. The control culture of MOI Experiment I was lost at 35hpi due to a contamination. The control culture of MOI Experiment II achieved a final cell density of 1.9*10 7 cells/ml. The 95% confidence interval was calculated according to section and lies in the range of 11 to 13% of the determined value. Median cell volume. The median cell volume (Figure 23B and H) was computed by fitting a lognormal function to the cell volume distribution as determined by Elzone readings. The cell volume increased from ~5hpi rapidly until ~20hpi, when the volume increase slowed slightly down. Around 65hpi, the peaks of viable and dead cells became difficult to separate. By then, the median cell volumes of MOI Experiment I and II had increased from 1680µm 3 to 2820µm 3 and from 1490µm 3 to 2670µm 3 respectively. The volume increased by 1.68±0.02 fold and 1.79±0.1 fold respectively. The MOI 46 culture in Experiment II started with a slightly higher cell volume and maintained this difference during the whole infection cycle. No influence from the different MOIs was observed in the timing and intensity of the cell swelling. The cell volume of the control culture decreased by 20% during the observed 65 hours. σ g of cell volume. The geometric standard deviation (σ g ) of the cell volume distribution (Figure 23C and I) was estimated by fitting a lognormal distribution to the Elzone data peak with the higher cell volume. σ g is a measure of the spread of the lognormal transformed cell volume in the culture, a small σ g indicates a narrow distribution. All infected cultures showed the same timing of the drop in σ g from 4hpi to 13hpi. Within the same experiments, all infected cultures showed the same intensity of σ g drop, expect the MOI 4.9 culture in Experiment II. It is not entirely clear why this difference has occurred; however, one might speculate that a sluggish infection at a lower MOI does result in a less pronounced decrease in σ g rather than a difference in the timing of the event. The control culture showed a slow decrease in σ g as it approaches the stationary phase. Specific Budded Virus titer (BV). The specific budded virus titers (Figure 23D and J) in the supernatant started to increase around 17hpi in an approximately linear fashion for all MOIs up to ~40hpi in MOI Experiment I and to the end of the culture in the MOI Experiment II. The differences in timing and intensity of the budding rate between the different MOIs lay well within the error of the analytical method (95% confidence interval 40%). The specific BV yield was about 2fold higher in MOI Experiment I than what is usually observed (Power, Reid et al. 1994). This is interesting for two reasons: firstly, the virus with the worse quality 50

5 produced a higher virus titer; and secondly, the difference occurred in spite of a seemingly good repetition of the first experiment. Specific β-galactosidase. The specific total β-galactosidase concentration (Figure 23E and K) starts to increase at around 23hpi and continues until ~70hpi the end of the culture. The production phases of the two different experiments and their different MOIs were virtually identical. The MOI 4.9 culture in Experiment II might have a delay of up to 2 hours but reached the same yield (0.15 Units / cell). The release of β-galactosidase into the supernatant commenced at ~40hpi. The timing and rate of β- galactosidase release was independent of the MOI. Again, a slight delay in β-galactosidase release could be observed in the MOI 4.9 culture. The 95% confidence interval was estimated to lie around 5% and was almost exclusively introduced through pipetting errors. Viability. The viability of the infected cell cultures (Figure 23F and L) decreased slowly from 97% to ~94% (MOI Experiment I) and to ~90% (MOI Experiment II) at ~40hpi. Thereafter, cell lysis increased rapidly and the viability dropped at a constant rate. Again, there were virtually no differences in the timing and rate of the decreasing viability observed between the different MOIs. The viability of the control culture remained at a high viability (>97%) throughout the duration of the infection. The 95% confidence interval was calculated as described in section (p.16) and lies within 3abs%. Specific viral DNA copy numbers (n vdna ). The specific viral DNA copy number (Figure 24) of the MOI Experiment II shows four distinct phases: 1.) Up to ~2hpi, the n vdna increased slowly due to the adsorption of viruses. 2.) Up to ~5hpi the adsorption of virus particles stagnated at an MOI proportional level; however, it cannot be excluded that some infectious vdna were already replicating during that time camouflaged by the adsorbed non-infectious particles. 3.) Up to ~15hpi the n vdna increased at an almost exponential rate. 4.) Subsequently, the replication rate slowed down and levelled off at a similar specific viral copy number for all MOIs. The final specific viral DNA copy number was independent of the MOI. The exponential increase in the viral copy number was delayed by less than one hour for the lower MOIs. This delay is expected, as the MOI 5 needs 3 replication cycles more than the MOI 45 to achieve the same n vdna. In fact, a delay of up to 4 hours could have been expected between the MOI 5 and MOI 45 as a minimum doubling time of ~1.25 (standard deviation = 0.06) hours was estimated from Figure 24. The coefficient of variation of the measurements is 17%. The scale of the specific viral genome copy numbers shown in Figure 24 is subjected to a considerable error due to the inaccuracy of the estimation of the standard concentration. However, the estimated 200,000 genome copies per cell lie 51

6 in the same range as previously observed (Rosinski, Reid et al. 2002). In contrast to this previous work, I observed a slightly longer viral replication phase up to 25hpi, but the replication rate slowed down from 15hpi. The replication rate can be estimated more accurately than the absolute copy number as it is independent of the scaling of the ordinate. By the comparison of the specific budded virus concentration (Figure 23D and J) and specific viral copy number (Figure 24), it can be seen that only a small fraction (2-5%) of the produced viral genomes eventually bud out. 52

7 4 x 106 A ) MOI Experiment I G ) MOI Experiment II x tot / (cells / ml) med. cell volume / µm B ) H ) σ g cell volume / log e ( µm) C ) τ / hpi MOI 4.7 MOI 14 MOI 38 control I ) τ / hpi MOI 4.9 MOI 15 MOI 46 control 53

8 MOI Experiment I MOI Experiment II BV / (PFU / cell) D ) J ) 0 tot.& sup. β-gal / (U / cell) E ) K ) 80 viability / % F ) τ / hpi MOI 4.7 MOI 14 MOI 38 control L ) τ / hpi MOI 4.9 MOI 15 MOI 46 control Figure 23: β-gal BEV infection of 3 shaker flask Sf9 cell cultures at ~1*10 6 cells/ml at an MOI of approximately 5, 15, and 45 PFU/cell respectively in 900ml HyQ SFX medium. All recorded state variables of the infected cultures are virtually identical between the different MOIs. The experiment has been repeated; Experiment I: Figure A to F, Experiment II: Figure G to L and Figure MOIexp2.m 54

9 10 6 Specific Viral Genome Density Figure 24: n vdna / (genomes / cell) MOI 4.9 MOI 15 MOI τ / hpi Specific viral genome copy number was determined by real time PCR for MOI Experiment II. The minimal genome doubling time lies around 1.25h Discussion No significant differences could be observed between MOI infections of 5 to 45PFU/cell in all monitored state variables. Further, no significant differences between MOI infections of 20 and 100PFU/cell in SF900II medium were observed for cell densities, cell volumes, cell volume distributions (σ g ), total and external β-gal concentrations, and viabilities as shown in appendix 9.5. Therefore, it can be concluded that η has no or a negligible effect on the responses of Sf9 cells in HyQ SFX medium at a cell density of ~1*10 6 cells/ml. This statement can most likely be extended to a PCD range up to the OPCD, because the cell specific β-gal yield below the OPCD is equal for MOIs of 5 and 10PFU/cell (Figure 37A, p.88). One group observed a positive dependency of the β-gal yield on MOI at a high PCD and a weak negative dependency at a low PCD (Licari and Bailey 1991). A possible reason for the observed dependency of β-gal yields between MOIs of 1, 5, and 20PFU/cell by Naggie & Bentley (1998) might therefore be that their PCD 24 of 1.3*10 6, 1.5*10 6 and 1.1*10 6 cells/ml respectively in Ex-Cell 401 medium was above the OPCD. Indeed, the data by Hensler et al. (1994, Table 2) suggest that the OPCD of Sf9 cells in Ex-Cell 401 lies around 0.9*10 6 to 1*10 6 cells/ml. When scrutinising Naggie & Bentley s data, it is found that the infection at the lowest PCD results the highest specific β-gal yield, 23 MOIexp2.m 24 Note that the values for the PCD were extracted from Naggie & Bentley s figure plotted on a logarithmic scale and might therefore be subjected to small errors. 55

10 and the infection at the highest PCD results in the lowest β-gal yield reflecting the expected behaviour for infections above the OPCD. The viability of the MOI 20 PFU/cell (PCD 1.1*10 6 cells/ml) infection resembles strongly the data presented in Figure 23F & L, whereas the MOI 5 and 1 PFU/cell infection both showed sluggish death kinetics; my interpretation of this effect is that in the culture with MOI 1 PFU/cell (PCD 1.3*10 6 cells/ml) this effect is caused by an asynchronous infection and in the MOI 5 PFU/cell (PCD 1.5*10 6 cells/ml) by substrate limitation. The data presented by Naggie & Bentley (1998) were the strongest published data in support of an η dependent infection cycle at low cell densities that could be found by an intensive literature search. In light of these data and the analysis presented in this chapter, it can be concluded that η does not explain the observed discrepancies in the responses of individual insect cells to BEV infections as described in section 5.1 at least not for PCD below the OPCD. Infections above the OPCD are not interesting from an economic stand point, because the overall yield of the recombinant protein, occlusion bodies, or BV decreases dramatically due to substrate limitation and possibly accumulation of inhibitory factors (see also appendix 9.1). Consequently, there is very little information available about factors affecting the infection cycle above the OPCD. Licari & Bailey (1991) observed a logarithmically increasing dependency of the β-gal yield on the MOI when infecting Sf9 cells during the late exponential phase (presumably above the OPCD) in stationary culture, suggesting a dependency on η at PCD > OPCD. If the infection cycle is dependent on η, it seems compelling to assume that the influence on the infection cycle is caused by viral template limitation. Under non substrate limiting condition, the specific viral DNA template concentration reaches copies per cell during the production phase (Figure 24); but even if under substrate limiting condition only a fraction of these vdna templates are produced, it seems implausible that the number of vdna copies could be the limiting factor for the progression of the cell cycle. Personally, I can think of no mechanism that suddenly would make the infection cycle dependent on η above a certain cell density. The work by Licari & Bailey (1991) remain the best published study of BEV infection at high cell densities. However, a more stringent investigation according to today s standards would certainly be helpful to diffuse doubts about the η dependency at high cell densities. Such a study would need to take into account that (1) virus addition at high cell densities inevitably means supplementing substrate, (2) the binding rate is reduced (Wong 1997, Figure 6.13), and (3) medium replacement increases the OPCD by 1.5 to 2fold (Wong, Peter et al. 1996; Radford, Cavegn et al ). 56