Engineering PEG10 assembled endogenous virus-like particles with genetically encoded neoantigen peptides for cancer vaccination

Reviewer #1 (Public Review):

Tang et al present an important manuscript focused on endogenous virus-like particles (eVLP) for cancer vaccination with solid in vivo studies. The author designed eVLP with high protein loading and transfection efficiency by PEG10 self-assembling while packaging neoantigens inside for cancer immunotherapy. The eVLP was further modified with CpG-ODN for enhanced dendritic cell targeting. The final vaccine ePAC was proven to elicit strong immune stimulation with increased killing effect against tumor cells in 2 mouse models. Below are my specific comments:

(1) The figures were well prepared with minor flaws, such as missed scale bars in Figures 4B, 4K, 5B, and 5C. The author should also add labels representing statistical analysis for Figures 3C, 3D, and 3E. In Figure 6G, the authors should label which cell type is the data for.

(2) In Figure 3H, the antigen-presenting cells (APCs) increased significantly, but there was also a non-negligible 10% of APCs found in the control group, indicating some potential unwanted immune response; the authors need to explain this phenomenon or add a cytotoxic test on the normal liver or other cell lines for confirmation.

(3) In Figure 3I, the ePAC seems to have a very similar effect on cytotoxic T-cell tumor killing compared to the peptides + CpG group. If the concentrations were also the same, based on that, questions will arise as to what is the benefit of using the compact vector other than just free peptide and CpG? Please explain and elaborate.

(4) In the animal experiment in Figures 4F to L, the activation effect of APCs was similar between ePAC and CpG-only groups with no significance, but when it comes to the HCC mouse model in Figure 5, the anti-tumor effect was significantly increased between ePAC and CpG-only group. The authors should explain the difference between these two results.

Reviewer #2 (Public Review):

Summary:

The authors provided a novel antigen delivery system that showed remarkable efficacy in transporting antigens to develop cancer therapeutic vaccines.

Strengths:

This manuscript was innovative, meaningful, and had a rich amount of data.

Weaknesses:

There are still some issues that need to be addressed and clarified.

(1) The format of images and data should be unified. Specifically, as follows: a. The presentation of flow cytometry results; b, The color schemes for different groups of column diagrams.

(2) The P-value should be provided in Figures, including Figure 1F, 1H, 3C, 3D, and 3E.

(3) The quality of Figure 1C was too low to support the conclusion. The author should provide higher-quality images with no obvious background fluorescent signal. Meanwhile, the fluorescent image results of "Egfp+VSVg" group were inconsistent with the flow cytometry data. Additionally, the reviewer recommends that the authors use a confocal microscope to repeat this experiment to obtain a more convincing result.

(4) The survival situation of the mouse should be provided in Figure 5, Figure 6, and Figure 7 to support the superior tumor therapy effect of ePAC.

(5) To demonstrate that ePAC could trigger a strong immune response, the positive control group in Figure 4K should be added.

(6) In Figure 6G-I and other figures, the author should indicate the time point of detection. Meanwhile, there was no explanation for the different numbers of mice in Figure 6G-I. If the mouse was absent due to death, it may be necessary to advance the detection time to obtain a more convincing result.

(7) In Figure 6B, the rainbow color bar with an accurate number of maximum and minimum fluorescence intensity should be provided. In addition, the corresponding fluorescence intensity in Figure 6B should be noted.

(8) The quality of images in Figure 1D and Figure S1B could not support the author's conclusion; please provide higher-quality images.

(9) In Figure 2F, the bright field in the overlay photo may disturb the observation. Meanwhile, the scale bar should be provided in enlarged images.

Reviewer #3 (Public Review):

Summary:

The authors harnessed the potential of mammalian endogenous virus-like proteins to encapsulate virus-like particles (VLPs), enabling the precise delivery of tumor neoantigens. Through meticulous optimization of the VLP component ratios, they achieved remarkable stability and efficiency in delivering these crucial payloads. Moreover, the incorporation of CpG-ODN further heightened the targeted delivery efficiency and immunogenicity of the VLPs, solidifying their role as a potent tumor vaccine. In a diverse array of tumor mouse models, this novel tumor vaccine, termed ePAC, exhibited profound efficacy in activating the murine immune system. This activation manifested through the stimulation of dendritic cells in lymph nodes, the generation of effector memory T cells within the spleen, and the infiltration of neoantigen-specific T cells into tumors, resulting in robust anti-tumor responses.

Strengths:

This study delivered tumor neoantigens using VLPs, pioneering a new method for neoantigen delivery. Additionally, the gag protein of VLP is derived from mammalian endogenous virus-like protein, which offers greater safety compared to virus-derived gag proteins, thereby presenting a strong potential for clinical translation. The study also utilized a humanized mouse model to further validate the vaccine's efficacy and safety. Therefore, the anti-tumor vaccine designed in this study possesses both innovation and practicality.

Weaknesses:

(1) CpG-ODN is an FDA-approved adjuvant with various sequence structures. Why was CpG-ODN 1826 directly chosen in this study instead of other types of CpG-ODN? Additionally, how does DEC-205 recognize CpG-ODN 1826, and can DEC-205 recognize other types of CpG-ODN?

(2) Why was it necessary to treat DCs with virus-like particles three times during the in vitro activation of T cells? Can this in vitro activation method effectively obtain neoantigen-responsive T cells?

(3) In the humanized mouse model, the authors used Hepa1-6 cells to construct the tumor model. To achieve the vaccine's anti-tumor function, these Hepa1-6 cells were additionally engineered to express HLA-A0201. However, in the in vitro experiments, the authors used the HepG2 cell line, which naturally expresses HLA-A0201. Why did the authors not continue to use HepG2 cells to construct the tumor model, instead of Hepa1-6 cells?

(4) The advantages of low immunogenicity viruses as vaccines compared with conventional adenovirus and lentivirus, etc. should be discussed.

(5) In Figure 6B, the authors should provide statistical results.

(6.) The entire article demonstrates a clear logical structure and substantial content in its writing. However, there are still some minor errors, such as the misspelling of "Spleenic" in Figure 3B, and the sentence from line 234 should be revised.

(7) The authors demonstrated the efficiency of CpG-ODN membrane modification by varying the concentration of DBCO, ultimately determining the optimal modification scheme for eVLP as 3.5 nmol of DBCO. However, in Figure 2B, the author did not provide the modification efficiency when the DBCO concentration is lower than 3.5 nmol. These results should be provided.

(8) In Figure 3, the authors presented a series of data demonstrating that ePAC can activate mouse DC2.4 cells and BMDCs in vitro. However, in Figure 7, there is no evidence showing whether human DC cells can be activated by ePAC in vitro. This data should be provided.