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. 2021 Feb 12;9(2):149.
doi: 10.3390/vaccines9020149.

An Oncolytic Adenovirus Encoding SA-4-1BBL Adjuvant Fused to HPV-16 E7 Antigen Produces a Specific Antitumor Effect in a Cancer Mouse Model

Alejandra G Martinez-Perez  1 , Jose J Perez-Trujillo  1 , Rodolfo Garza-Morales  2 , Norma E Ramirez-Avila  1 , Maria J Loera-Arias  1 , Jorge G Gomez-Gutierrez  2 , Odila Saucedo-Cardenas  1   3 , Aracely Garcia-Garcia  1 , Humberto Rodriguez-Rocha  1 , Roberto Montes-de-Oca-Luna  1
Affiliations

An Oncolytic Adenovirus Encoding SA-4-1BBL Adjuvant Fused to HPV-16 E7 Antigen Produces a Specific Antitumor Effect in a Cancer Mouse Model

Alejandra G Martinez-Perez et al. Vaccines (Basel). .

Abstract

Human papillomaviruses (HPVs) are responsible for about 25% of cancer cases worldwide. HPV-16 E7 antigen is a tumor-associated antigen (TAA) commonly expressed in HPV-induced tumors; however, it has low immunogenicity. The interaction of 4-1BBL with its receptor induces pleiotropic effects on innate, adaptive, and regulatory immunity and, if fused to TAAs in DNA vaccines, can improve the antitumor response; however, there is low transfection and antitumor efficiency. Oncolytic virotherapy is promising for antitumor gene therapy as it can be selectively replicated in tumor cells, inducing cell lysis, and furthermore, tumor cell debris can be taken in by immune cells to potentiate antitumor responses. In this study, we expressed the immunomodulatory molecule SA-4-1BBL fused to E7 on an oncolytic adenovirus (OAd) system. In vitro infection of TC-1 tumor cells and NIH-3T3 non-tumor cells with SA/E7/4-1BBL OAd demonstrated that only tumor cells are selectively destroyed. Moreover, protein expression is targeted to the endoplasmic reticulum in both cell lines when a signal peptide (SP) is added. Finally, in an HPV-induced cancer murine model, the therapeutic oncolytic activity of OAd can be detected, and this can be improved when fused to E7 and SP.

Keywords: 4-1BBL; HPV-16 antigen; cancer vaccines; oncolytic virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of SP/SA/E7/4-1BBL protein expression in HEK-293 cells. (a) Fifty-five kDa bands corresponding to the protein of interest at MOI concentrations of 0, 5, 10, 20, 30, and 40; normalized results with actin as an endogenous control. (b) Densitometry graph where greater expression of SP/SA/E7/4-1BBL protein is observed in a dose-dependent manner, relative to the expression of actin. MOI, multiplicity of infection.
Figure 2
Figure 2
Subcellular localization of SP/SA/E7/4-1BBL recombinant protein in (a) HEK-293, (b) TC-1, and (c) NIH/3T3 cell lines. Blue channel DAPI signal for the nucleus, red channel signal for E7 antigen, green channel signal for calnexin; merge (yellow stain) shows co-localization of signals for E7 and calnexin. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 3
Figure 3
In vitro antitumor effect of oncolytic adenovirus expressing SP/SA/E7/4-1BBL. Cell viability percentage of TC-1 cells was measured by crystal violet assay after infection with adenoviruses at different MOIs, which decreases while MOI increases, compared to infection-free control. *** (p < 0.001).
Figure 4
Figure 4
Specific oncolytic effect against tumoral cells. Evaluation of cellular density by light-field microscopy of TC-1 (a) and NIH/3T3 (b) cells after infection with oncolytic adenoviruses. Both cell lines were infected with different MOIs and incubated for 72 h. The TC-1 tumor cell line displays detachment and a decrease in cell density as MOI increases; nevertheless, the NIH/3T3 non-tumor cell line was unaltered. 1 = 0 MOI, 2 = 250 MOI, 3 = 2500 MOI, 4 = 5000 MOI. Scale bars 100 µm.
Figure 5
Figure 5
Cell viability percentage graph after 72 h post-infection with recombinant oncolytic adenoviruses. The TC-1 cell line displayed 43% mortality at an MOI of 2500 * (p < 0.05) and 67% at an MOI of 5000 ** (p < 0.01), while the non-tumor NIH/3T3 cell line exhibited no significant changes.
Figure 6
Figure 6
In vivo therapeutic antitumor effect of oncolytic adenovirus. (a) Tumor volume was measured after the first administration of oncolytic adenovirus DNA or PBS at 14 days after tumor challenge with 5 × 104 TC-1 cells in the right flank by subcutaneous injection. Groups (n = 7) of 6- to 8-week-old C57BL/6 mice were injected intratumorally with 2.5 × 108 UI of oncolytic adenovirus two times (one per week). The DNA construct was administrated on shaved abdominal skin with 1 µg of DNA using a gene gun system. PBS was used as a negative control. Tumor volumes are represented by mean ± SEM. Mice were euthanized when the tumor volume was higher than 1800 mm3. (b) Survival graph after TC-1 tumor challenge. Two-way ANOVA and post-hoc Tukey’s tests were performed.
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