*2.6. VLP Vaccination Strategy, Regimen, and Dose*

Vaccination has the primary purpose of producing long-lasting protection against diseases. The choice of appropriate vaccine strategy, regimen, and dose is crucial for the success of vaccination. It becomes especially concerning when immaturity or senescence of the immune system can affect the efficacy of the immunization [77]. Different strategies of prime-boost vaccination against infectious diseases searching to improve humoral and cellular immunity have been studied [78,79]. These heterologous strategies induce efficient humoral and cellular responses to the same antigen presented by two different delivery systems. Priming with a DNA vaccine or viral vector followed by boosting with a protein-based vaccine usually induces a strong cellular immune response, with higher and more specific antibody production as compared to homologous delivery systems [80]. In the circumstances where

homologous protein-based vaccination induces strong humoral response but weak cellular immunity, the heterologous approach can be more productive. Below are examples of the prime-boost regimen:


They combine a better antibody and CD4<sup>+</sup> T cell response induced by protein antigen, efficient stimulation of T cell response by DNA vaccines, and improvement of the CD4+/CD8<sup>+</sup> T cells and antibody response by recombinant viral vectors. Similarly, different vectors have different immune characteristics and, therefore, induce a unique immune response to the immunodominant epitope, and reduce the immunity against the vector [79].

The choice of the optimal time and the frequency of repeated boosts can impact the quality of the immune response [81]. Understanding the process of establishing immune memory is valuable information for choosing the interval between the primary immunization and booster injections. Memory T cells with high proliferative potential are formed several weeks after the prime immunization, suggesting that the boosting should occur at least two or three months after the first immunization. A similar concept applies to antibody responses, as memory B cells need to undergo a germinal center response to develop [82]. Repeated boosting drives T cell towards terminal differentiation and recruits a subset of a previously generated memory cell; as a result, a heterologous population of memory T cells at several different stages can be found [78]. An appropriate immunization schedule can take advantage of this process.

The establishment of permanent protective T cell memory may depend on the antigen load and its kinetics. The possibility of mimicking pathogen replication enhances the T cell activity. A study using choriomeningitis virus (LCMV) in mice with different doses that prolong the antigen exposure showed a significant effect on the immune response, where high doses of the virus led T cell exhaustion, while low doses induced potent and durable T cell activation [55]. The ideal vaccination schedule starts with low doses, followed by a peak dose and finishes with low doses, instead of repeated doses of equivalent vaccine concentration [83]. This approach appears to be more suitable for therapeutic vaccines. In contrast, for prophylactic vaccines, when an effective B cell response is wanted, a simple vaccination schedule with an injection in a month or more apart may be preferable. The immune system can generate a reservoir of antigens in the germinal center, keeping B cells stimulated, which results in a stronger antibody response. Memory B cells and plasma cells favor this event that may contribute to the success of inactivated virus vaccines [76].
