*3.3. E*ff*ect of Platform Rigidity*

The influence of platform rigidity is also a topic of debate, and it is yet to be confirmed whether epitopes that are held as part of a rigid, semi-crystalline structure (e.g., HPV) possess the same immunological properties as epitopes that are part of a more flexible structure (e.g., modular CLPs). If this is the case, the use of long flexible linkers involved in some of the more complex conjugation strategies should be avoided. Likewise, the nature of the underlying VLP should be considered. There are several examples where enveloped VLPs and other lipid-based vaccine platforms have successfully been used for induction of protective immune responses; targeting either the native surface protein (e.g., the hepatitis B virus (HBV) surface antigen (HBsAg) [117]) or a displayed foreign antigen [118–120]. Recently, two studies have performed head-to-head comparisons between lipidand capsid-based VLP vaccines. Chen et al. showed that vaccination with a self-antigen peptide displayed on Qβ CLPs resulted in a 200-fold increase in antigen-specific IgG titers, compared to antigen presentation on liposome particles [113]. In contrast, Marini et al. compared Pfs25 antigen display via the Tag/Catcher conjugation system on either HBsAg lipid-based particles or AP205 CLPs, and showed no significant differences in antibody titers and functionality between the two vaccines [77]. The duration of the induced antibody responses was however not tested in either study. This could be an important factor to investigate, as the antibody longevity of licensed VLP-based vaccines differ substantially. When targeting many important pathogens, such as HIV, HPV and malaria, neutralizing antibody levels need to be maintained in order for the vaccine to be protective. This is due to the importance of preventing the initial infection of these pathogens. In such cases, memory B-cell dependent humoral responses would not be sufficient [121]. While the HBV vaccine has shown to be effective and has decreased HBV prevalence worldwide [122], the long-term efficacy of the HBV vaccine is memory B-cell dependent. In general, durable antibody responses are not induced, even after three doses [123]. The RTS,S vaccine, which is based on genetic incorporation of a malaria antigen into HBsAg VLPs, only showed short-term efficacy in clinical trials, due to a similar rapid decline in antibody levels [118,124,125]. In contrast, HPV CLPs are strong inducers of long-lived antibody-producing plasma cells [14,126]. These marked differences in the induced immune responses could reflect fundamental differences in the immunological properties of lipid- versus capsid-based backbones [127].
