*3.2. Hepatocellular Carcinoma*

The acute and chronic hepatitis caused by HBV can progress to cirrhosis and eventually to hepatocellular carcinoma (HCC). The current HBV vaccines are based on VLP of the S antigen; the most recently approved, HEPLISAV-B, has CpG as adjuvant [92]. These vaccines reduce the occurrence of HBV infection but are ineffective in treating existing HCC. The use of multiple epitopes' VLP was tested as a vaccine for HCC treatment in a preclinical study. The HBV X protein is highly expressed in HCC. High frequency of epitopes of HBV X displayed at HBV core protein elicited epitope-specific CD8+ T cells and a more forceful response than a single peptide showing that multiepitope-loaded VLP augment immunogenicity for each epitope by increasing the number of CTLs resulting in a significant anti-tumor response [28]. It is a proof of concept of the potential efficacy of VLP-based vaccines for HCC therapy.

#### *3.3. Skin Cancer*

Melanoma is an antigenic and immunogenic cancer with well-characterized tumor-associated antigens. Ideally, a melanoma-preventing vaccine should expand the number of spontaneous CTLs produced against the tumor at the tumor site and throughout the body as long-term immune surveillance [93].

A notable example of the potentiality of VLP against melanoma comes from a preclinical study using VLP not decorated with tumor antigens but with inherent immunogenic properties.

In situ vaccination, using the VLP generated by the plant virus Cowpea Mosaic Virus (CPMV), was shown to reduce B16F10 melanoma and generate systemic anti-tumor immunity [29]. The approach of in situ vaccination using VLP is limited, but local immunization can strongly modulate the local microenvironment and the anti-tumor immunity. Empty CPMV (eCPMV) VLP lacking any nucleic acid were tested in in vitro cultures of bone marrow-derived DCs (BMDCs) and primary macrophages from C57BL/6 mice. Twenty-four hours later, an increase in pro-inflammatory cytokines (IL-6, IL-1β, IL-12p40, IFN-γ and TNK-α) was observed, providing evidence that the empty VLP is intrinsically immunostimulatory. Notably, the VLP were produced in plants and, therefore, were endotoxin free. When tested in non-tumor-bearing mice, inhalation of eCPMV activated Ly6G+ neutrophils that up-regulated the CD11b activation marker and the CD86 co-stimulatory molecule. Inhalation of eCMVP by mice bearing lung melanoma metastases, induced by an intravenous (i.v.) injection of B16F10 cells, alters the lung microenvironment, with a significant increase in tumor-infiltrating neutrophils (TINs) and CD11b+Ly6G+-activated neutrophils as well as a reduction in CD11b−Ly6G<sup>+</sup> quiescent neutrophils. In addition, weekly intratracheal administration of 100 μg of eCPMV reduced the number of metastatic foci both in mice i.v. injected with B16F10 cells and in mice developing spontaneous lung metastases after a subcutaneous challenge with 4T1 mammary cancer cells [29]. Mice depleted of neutrophils and those lacking IL-12, IFN-γ or T, B, and NK cells failed to respond to the eCPMV inhalation therapy, confirming that neutrophils can disturb the tolerogenic nature of the tumor microenvironment and orchestrate an immune response that leads to tumor elimination. It probably occurs due to activation of pre-existing or de novo-induced anti-tumor responses, or both [29]. Administration of eCPMV in situ was also effective for treating dermal tumors. When the VLP were injected intra-tumorally in mice bearing cutaneous tumors induced by B16F10 melanoma cells or CT26 colon cancer cells, tumor growth was delayed. In the case of B16F10 melanomas, 50% of the treated mice rejected the tumor. These surviving mice were also protected from a re-injection of B16F10 cells, indicating the induction of a protective systemic immune response.

Another important preclinical study was based on the use of VLP decorated with the H-2D<sup>b</sup> immunodominant epitope of the human glycoprotein 100 (gp10025-33) [26], a melanoma-associated antigen [94]. The epitope was incorporated into rabbit hemorrhagic disease virus (RHDV) VLP in one, two, or three copies, resulting in gp100.1L, gp100.2L, and gp100.3L VLP, respectively. All three VLP induced a robust CD8<sup>+</sup> T cell proliferation, and gp100.2L and gp100.3L also induced a significant enhancement of IFN-γ production [26]. The activation of T cell proliferation was confirmed in in vivo experiments for gp100.2L and gp100.3L. These two versions of VLP also showed a therapeutic effect against murine melanomas. Mice vaccinated with the VLP remained tumor free over 60 days, which is an indication of the induction of a specific anti-tumor immunity. The mono or dimannosylation of gp100-VLP enhanced VLP uptake into APCs, but the anti-tumor T cell response was only enhanced by dimannosylation [26].

Another proof of the ability of VLP-based vaccines to prevent B16F10 melanoma comes from a study in which a recombinant cucumber-mosaic VLP (CuMV) (~ 30 nm) was used. [24]. The universal tetanus toxoid (TT) epitope TT830-843 has been genetically fused to CuMV (CuMVTT) and is displayed in the interior surface to avoid interference with TT-specific antibodies. The TT epitope serves as an additional T helper cell epitope, especially in elderly patients. CuMVTT was labeled with AF488 and was used in the study of drainage dynamics. The study showed that fast and effective drainage of VLP, that reached the regional LN in just one minute. A version of these VLP expressing the H-2Db restricted p33 peptide from LCMV antigen (CuMVTTp33-VLP) was formulated with the micron-sized microcrystalline tyrosine (MCT) adjuvant. MCT facilitated depot formation and prolonged the release of the VLP up to 9 days compared to 4 days of the free VLP form. With only one vaccination, CuMVTTp33 VLP formulated with MCT can induce high levels of p33-specific T cells and release of the cytokines IFN-γ and TNF-α. When this formulation was tested in mice bearing tumors from B16F10 melanoma cells expressing the p33 antigen, tumor progression was inhibited with a significant increase in total CD8<sup>+</sup> T cells and p33-specific T cell infiltration, showing proper anti-tumor protection [24].

A recent study proposed a therapeutic vaccine for melanoma, in which the loaded CpG Qβ-VLP and the desired epitope are combined with bio-orthogonal copper-free click chemistry; Dibenzocyclooctyne NHS ester (DBCO) is used as a cross-linking agent. This new method for peptide conjugation was shown to be superior to SMPH not only for coupling efficiency but also for inciting CTL and IFN-γ response in vivo. The developed vaccine was created by the conjugation of peptides from tumor-specific germline (immunopeptidomics) and mutated (whole-exome sequencing) CTL epitopes from B16F10 melanoma cells. Three different multitarget (MTV) vaccines were originated: 1) germline-multitarget vaccine (GL-MTV), 2) mutated epitope vaccine (mutated-MTV), and 3) a combination of both (Mix-MTV). Subsequently, they were tested in mice with transplanted B16F10 melanoma tumors in combination with anti-CD25. It was observed that the three vaccines inhibited the tumor progression, the Mix-MTV vaccination being more effective for anti-tumor protection. It was also the only one that showed significant tumor growth reduction. Compared with the control group and mutant MTV, the Mix-MTV vaccine was more effective in increasing CD8<sup>+</sup> T cell density. For Mix-MTV vaccination, higher IFN-γ production was also observed. Data show that the combination of germline and mutant epitopes in a vaccine is more effective against growing B16F10 melanoma tumors [21].
