*2.7. Usage of Adjuvants*

Adjuvants may be needed to increase the antigen reservoir and enhance vaccine efficacy. For almost a century, Aluminum and Freund's adjuvants were used to improve vaccine immunogenicity. Nowadays, many others have been developed [84]. Preclinical studies show that some VLP vaccines do not need additional adjuvants to produce a robust immune response since they have intrinsic adjuvant properties to activate TLR. In mice, the ssRNA into the VLP [85] induces higher antibody titers of IgG2a, IgG2b and IgA isotypes [86], while VLP with the ssRNA removed, elicit IgG1 antibodies, indicative of a Th2 response [18,87]. Overall, VLP are very immunogenic, even in the absence of TLR-mediated stimuli. However, TLR ligands increase the immune response of a vaccine, as observed in Cervarix, an HPV-derived VLP used in combination with MPL, which is a ligand for TLR4 [40]. The TLR9 ligands CpG-containing oligodeoxynucleotides (CpG-ODNs), are also used in vaccines to enhance B and T cell responses to a given recombinant epitope. CpG package into VLP is resistant to DNase I digestion and does not induce the undesirable side effect (splenomegaly and lethal toxic shock) caused by the free form of CpG. Vaccination with CpG-loaded VLP protected from infection with vaccinia virus and eradicated established solid fibrosarcoma tumors [84]. Therefore, VLP presenting the CpG

might represent a valuable approach to induce CTL responses [76]. Another way to enhance the VLP cell-mediated immune response is to present the universal T-assisted pan-HLA-DR-binding epitope (PADRE) on the nanoparticles. The RHDV VLP carrying PADRE and displaying HPV16 E6 peptides successfully prolonged survival of mice with pre-existing HPV tumors [25].

## **3. VLP as Cancer Vaccines**

In the past decade, cancer immunotherapy with checkpoint inhibitors has achieved a remarkable clinical outcome in patients with advanced stages of cancer [88]. However, the high cost for such therapies and the low percentage of patient responders highlight the need to find new immune-based strategies to fight cancer, which are both cost-effective and efficacious for a broad patient base [89,90]. Human cancer emerges through a combination of genetic and epigenetic changes that favor its survival. The tumor-associated antigen and tumor-specific antigens (neo-antigens) also create an opportunity for tumor detection and destruction by the immune system. However, cancer cells have adopted several strategies to evade immune recognition, leading to a state of tumor tolerance [91]. Biological response modifiers (such as vaccines) have the potential to fight cancer and can be used to break tumor tolerance. The vaccine must deliver a large number of selected cancer antigens to promote B cell and DC activation and antigen presentation to T cells. As mentioned above, with the VLP platform, even short peptides identified in tumor cells (that otherwise would be quickly cleared) can be presented to DCs to induce cellular or humoral response against cancer. Favorable results obtained by preventive VLP-based vaccines against cancer caused by infectious diseases—for instance, HBV and HPV—reinforce this concept, and many attempts are now ongoing for the development of VLP-based vaccines against many non-infection-related cancers. In the following sections, we review some relevant examples.

## *3.1. Cervical Cancer*

The current FDA-approved HPV vaccines are derived from HPV capsid protein L1 formulated with aluminum-containing adjuvants—aluminum hydroxyphosphate sulfate for Gardasil and the AS04 adjuvant system, composed of aluminum hydroxide and the TLR ligand 3-*O*-desacyl-4.-monophosphoryl lipid A (MPL) for Cervarix [92]. These vaccines are highly immunogenic and induce long-acting neutralizing antibodies. Their formulation is complex and expensive, which prohibits affordability in developing countries, where 85% of cervical cancer cases occur.

A preclinical study showed that another VLP-HPV vaccine using a neutralizing epitope from the HPV minor protein L2 could offer a robust immune response using a lower-cost production methodology and improved clinical applicability [23]. For this new approach, HPV16L2 was displayed at the N-terminus of MS2 VLP and at the AB loop of PP7 VLP. The L2-MS2 and L2-PP7 VLP were produced in a dry powder version using Spray Dryer (SD) and had their thermostability storage testing at 4 ◦C, 37 ◦C, and room temperature for one month. Spray Transmission Electron Microscopy (TEM) showed that SD and storage at the tested temperatures do not affect the nanoparticles' integrity or stability, preserving their ability to generate HPV-neutralizing antibodies. Immunization with reconstituted L2-MS2 VLP elicited high titers of anti-L2 IgG. The immunized mice were challenged with HPV Pseudo Virus (PsV), and their protection was compared with that induced by Gardasil immunization. When tested with two homologous PsV types (PsV31 and PsV45), L2-MS2-immunized mice were efficiently protected from both infections, while Gardasil failed to protect against the PsVs45 challenge. This work showed that L2-MS2 vaccines could be produced in a dry powder version with stability up to seven months at room temperature [23]. For other VLP-based vaccines against HPV cervical cancer, see [62].
