Monte Carlo simulation of the Spearman-Kaerber TCID50.

BACKGROUND: In the biological sciences the TCID50 (median tissue culture infective dose) assay is often used to determine the strength of a virus. METHODS: When the so-called Spearman-Kaerber calculation is used, the ratio between the pfu (the number of plaque forming units, the effective number of virus particles) and the TCID50, theoretically approaches a simple function of Eulers constant. Further, the standard deviation of the logarithm of the TCID50 approaches a simple function of the dilution factor and the number of wells used for determining the ratios in the assay. However, these theoretical calculations assume that the dilutions of the assay are independent, and in practice this is not completely correct. The assay was simulated using Monte Carlo techniques. RESULTS: Our simulation studies show that the theoretical results actually hold true for practical implementations of the assay. Furthermore, the simulation studies show that the distribution of the (the log of) TCID50, although discrete in nature, has a close relationship to the normal distribution. CONCLUSION: The pfu is proportional to the TCID50 titre with a factor of about 0.56 when using the Spearman-Kaerber calculation method. The normal distribution can be used for statistical inferences and ANOVA on the (the log of) TCID50 values is meaningful with group sizes of 5 and above.

A randomized, double-blind, dose-finding Phase II study to evaluate immunogenicity and safety of the third generation smallpox vaccine candidate IMVAMUNE.

IMVAMUNE is a Modified Vaccinia Ankara (MVA)-based virus that is being developed as a safer 3rd generation smallpox vaccine. In order to determine the optimal dose for further development, a double-blind, randomized Phase II trial was performed testing three different doses of IMVAMUNE in 164 healthy volunteers. All three IMVAMUNE doses displayed a favourable safety profile, with local reactions as the most frequent observation. The 1 x 10(8)TCID(50) IMVAMUNE dose induced a total antibody response in 94% of the subjects following the first vaccination and the highest peak seroconversion rates by ELISA (100%) and PRNT (71%). This IMVAMUNE dose was considered to be optimal for the further clinical development of this highly attenuated poxvirus as a safer smallpox vaccine.

Modified vaccinia Ankara strains with identical coding sequences actually represent complex mixtures of viruses that determine the biological properties of each strain.

Modified vaccinia Ankara (MVA) was developed by serial passages on chicken embryo fibroblast cells. After passage 570, the virus was considered homogenous and genetically stable. Three MVA strains (MVA-572, MVA-I721 and MVA-BN) have been analyzed and shown to be 100% genetically identical; although significant differences in their phenotypes were illustrated. All MVA strains except MVA-BN replicated in human cells, or killed immune suppressed mice. Viruses isolated from dead animals were shown to represent variants present within MVA-572 or MVA-I721 used to inoculate the mice. These subpopulations were shown to encode mutations, or contain less than the six deletions associated with MVA and had significantly altered phenotypes compared to the parental MVA strains. MVA is a complex polyclonal mixture of viruses, the composition of which governs the phenotype.

Safety and immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine.

A Phase I trial was performed to investigate the safety and immunogenicity of the third generation smallpox vaccine MVA-BN (IMVAMUNE), a highly attenuated clone derived from the Modified Vaccinia Virus Ankara strain 571, in naive and pre-immunized subjects. A total of 86 healthy subjects received the vaccine in five groups using different doses and routes of administration. All 38 subjects seroconverted in the groups receiving the highest dose (10(8) TCID50). All vaccinations were well tolerated with mainly mild or moderate pain at the injection site being the most frequent symptom. The results indicate that MVA-BN has the potential to be developed as an efficient and safe alternative to the conventional smallpox vaccines such as Lister-Elstree or Dryvax. Unique attributes render it a promising candidate for prophylactic mass immunization, even in subjects for whom conventional smallpox vaccines are contraindicated.