Modeling for influenza vaccines and adjuvants profile for safety prediction system using gene expression profiling and statistical tools

<div><p>Historically, vaccine safety assessments have been conducted by animal testing (<i>e</i>.<i>g</i>., quality control tests and adjuvant development). However, classical evaluation methods do not provide sufficient information to make treatment decisions. We previously identified biomarker genes as novel safety markers. Here, we developed a practical safety assessment system used to evaluate the intramuscular, intraperitoneal, and nasal inoculation routes to provide robust and comprehensive safety data. Influenza vaccines were used as model vaccines. A toxicity reference vaccine (RE) and poly I:C-adjuvanted hemagglutinin split vaccine were used as toxicity controls, while a non-adjuvanted hemagglutinin split vaccine and AddaVax (squalene-based oil-in-water nano-emulsion with a formulation similar to MF59)-adjuvanted hemagglutinin split vaccine were used as safety controls. Body weight changes, number of white blood cells, and lung biomarker gene expression profiles were determined in mice. In addition, vaccines were inoculated into mice by three different administration routes. Logistic regression analyses were carried out to determine the expression changes of each biomarker. The results showed that the regression equations clearly classified each vaccine according to its toxic potential and inoculation amount by biomarker expression levels. Interestingly, lung biomarker expression was nearly equivalent for the various inoculation routes. The results of the present safety evaluation were confirmed by the approximation rate for the toxicity control. This method may contribute to toxicity evaluation such as quality control tests and adjuvant development.</p></div

Evaluation of seasonal influenza vaccine with conventional animal safety test.

<p>A) The abnormal toxicity test was performed according to the Minimum Requirements of Biological Products. Each 5 ml vaccine was <i>i.p.</i> injected into rats, the body weight measured and lung tissues collected at day 1 after injection. B) Body weight change at day 1 after injection. NT: nontreated rat, SA: saline, PDv: pandemic H5N1 whole virion-derived vaccine with alum adjuvant, WPv: whole particle virion influenza vaccine, HAv: influenza HA vaccine, Man: manufacturer.</p

Evaluation of seasonal influenza vaccine with QGP.

<p>The relative gene expression levels of the <i>Gapdh</i> gene are indicated in each column (grades 1, 2 and 3, respectively). *Significant difference between B and C. **Significant difference between B, C and D, ***Significant difference between PD and WPv.</p

Comprehensive evaluation of influenza vaccine safety by zone classification.

<p>(A) The classification analysis using the probability value classified for RE. The probability indicated is the probability classified for RE by biomarker gene expression as served by the regression equation and the biomarker gene expression values. The probability is represented by 0 to 100%; thus, the threshold can be set to 50% probability. The results represent intranasal (<i>in</i>), intramuscular (<i>im</i>) and intraperitoneal (<i>ip</i>) vaccination routes. Each group constituted by 4 animals. Representative zone classification for RE-like toxicity risk assessment showing the intraperitoneal <i>vs</i> intramuscular vaccination route (B), and the intraperitoneal <i>vs</i> intranasal vaccination route (C). Each plot represents the mean value for each vaccine-treated group (4 animals/group). The broken lines separating the zones are drawn where the logit values were zero; the detailed method is described in Methods.</p

Validation of QGP with real-time PCR methods.

<p>A) GQP result was validated with real-time PCR methods. Bar graph indicates the real-time PCR results and dot blot indicates QGP results. B) Biomarkers were classified into three grades according to the relative expression level compared with WPv-treated rats.</p

Biomarkers to evaluate influenza vaccine safety.

<p>Biomarkers to evaluate influenza vaccine safety.</p

Changes in the expression levels of 18 lung biomarkers 16 hours after AddaVax^™-adjuvanted vaccination.

<p>At 16 h post-vaccination, mouse lungs were collected and total RNA was extracted from them. The total RNA was used for QGP analysis to assess the expression levels of the biomarkers. Their expression levels are shown as relative expression levels normalized against ß-actin. The data represents mean ±SD. In the figure, ip, im, and na indicate intraperitoneal, intramuscular and nasal vaccination routes, respectively. Each group constituted by 4 animals.</p

Changes in the expression levels of 18 lung biomarkers 16 hours after Poly I:C-adjuvanted vaccination.

<p>At 16 h post-vaccination, mouse lungs were collected and total RNA was extracted from them. The total RNA was used for QGP analysis to assess the expression levels of the biomarkers. Their expression levels are shown as relative expression levels normalized against ß-actin. The data represents mean ±SD. In the figure, ip, im, and na indicate intraperitoneal, intramuscular and nasal vaccination routes, respectively. Each group constituted by 4 animals.</p

Summary of biomarker studies.

<p>Biomarkers used in this study were strongly correlated with immune responses after influenza infection.</p

Influenza vaccine formulations used in study.

<p>Influenza vaccine formulations used in study.</p