Evaluating Novel Quantification Methods for Infectious Baculoviruses

Abstract

Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research.

Keywords: flow cytometry; gp64 protein; infectivity titer; native baculovirus; process monitoring; quantitative polymerase chain reaction; viral RNA; virus quantification.

Figure 1

Amplification plots for the qPCR evaluation of different baculovirus concentrations in the range of 0 (i.e., blank)−1.1 × 10⁷ IU/mL (A) as well as the linear regression over the concentration range (B). The number of replicate measurements was n = 39 for 1.1 × 10² IU/mL and 1.1 × 10⁷ IU/mL and n = 18 for all other concentrations and the blank.

Figure 2

Amplification plots of the baculovirus DNA by qPCR measurements. The DNA was extracted from samples taken either from the supernatant or from the cell pellet of infected cells after 5 min (i.e., 0 h) of incubation and compared to a negative control (A). The amount of viral DNA detectable in the cell pellet was monitored over the course of 7 h (B) and compared to values after 18 h of infection (C). All samples were prepared in triplicates, with error bars only being displayed in (A) to allow a clearer view of the data.

Figure 3

Development of the flow cytometric quantification protocol for baculoviruses (either wild-type or virus expressing the green fluorescent protein, BV-wt and BV-GFP, respectively). In a first approach, (A) the detection capability for both viruses was determined without further method optimization after 18 h of cell infection and subsequent flow cytometric detection of fluorescent cells. Depending on the staining procedure, green (no staining, left column), yellow (PE staining, middle column), or red fluorescence (APC staining, right column) was evaluated. The infection kinetics of the three approaches (B) indicate the earliest possible time of quantification at which the linear calibration range was subsequently determined for each individually optimized strategy (C). Error bars depict standard deviation of technical triplicates (A,B) and 18 replicates with n = 6 on three different days for (C).

Figure 4

Quantification of the percentage of fluorescing cells after BV infection and fluorescent labeling of the mRNA of the viral gp64 protein within the first four hours of infection. A multiplicity of infection (MOI) of 1 was used and compared to a negative control without virus infection (error bars indicate the standard deviation of technical triplicates).