**6. Importance** *in vitro* **and** *in vivo* **experiments using peptide-based vaccine prototypes**

After forming a synthetic vaccine prototype, the cytotoxicity of the bioconjugate of the peptides and biopolymers is first determined (generally we use MTT analysis). After the apoptotic effect of the prototype on living cells is measured by flow cytometric detection, the vaccine prototype with the most viable cell number should be selected for further study [62]. After all these methods, immunization is the next step. We immunize BALB/c mice with each one of the peptides biopolymer conjugates or peptides loaded nanoparticles following conventional immunization protocol. The goal is to identify the most antigenic vaccine prototype. The

antibodies are measured in blood (for humeral response such as; T and B lymphocytes, IgGs) or splenic (cellular response like ILs and IFNs) samples from the immunized BALB/c mice via the indirect enzyme-linked immunosorbent assay (ELISA) to determinate the highest antibody level. Thus, the most suitable peptidebased vaccine prototypes will be identified for future clinical phase studies. In brief, cell culture and toxicity studies are important before the analyses the effect of vaccine prototype in vivo [62].
