Role of E5 from HPV16 in the Evasion of the Immune Response
, Minerva Maldonado-Gama 1, Marisela Méndez-Armenta 3, Fernando Esquivel-Guadarrama 4,* and Lourdes Gutierrez-Xicotencatl 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn Abstract author should focus what are in this review they are going do
Role of E5 in cell transformation look like authors discussing regulation not transformation et al, please correct it.
In innate immune they should have mentioned some immune cells like they have been mentioned for adaptive immune response.
Author Response
Reviewer 1
- In Abstract author should focus what are in this review they are going do.
Answer: We rewrote the abstract for a better integration of the information and strengthened the aim of the review, as the reviewer suggested (lines 16 to 30):
“Human papillomavirus type 16 (HPV16) persistence relies on early viral mechanisms that synchronize oncogenic signaling with immune evasion, with the E5 oncoprotein serving as a central regulator of the viral cycle and the initiation of cell transformation. This review integrates current evidence on how E5 reconfigures host cell dynamics: first, by hijacking signaling pathways such as EGFR, MAPK/ERK, and PI3K/AKT to drive keratinocyte proliferation and survival; and second, by organizing a multi-step immune evasion strategy. We detail how E5 suppresses innate antiviral responses, specifically by repressing IFN-κ and IFN-β via interference with IRF1, TGF-β/SMAD, STING, and MAVS signaling. Simultaneously, E5 interferes with the adaptive immunity by disrupting MHC-I trafficking and impairing MHC-II maturation. Furthermore, preclinical studies utilizing various vaccine platforms targeting HPV16 E5 have demonstrated the capacity to reduce tumor burden and significantly increase survival rates. By integrating these molecular and immunological checkpoints, we highlight the role of E5 in sustaining viral persistence and underscore its potential as a high-value target for next-generation immunotherapeutic and vaccine-based strategies.”
- Role of E5 in cell transformation look like authors discussing regulation not transformation et al, please correct it.
Answer: We have rewritten several sections of the now Section 3 to emphasize the role of E5 in the transformation process (lines 77-137). All this new information has been highlighted in the manuscript in yellow.
- In innate immune they should have mentioned some immune cells like they have been mentioned for adaptive immune response.
Answer: The literature regarding E5-mediated modulation of innate immunity is narrow in scope. Since HPV infects only keratinocyte precursors, studies aimed at elucidating the role of E5 in modulating the innate immune response have relied solely on epithelial-derived cell lines. Thus, the only cells in the innate immune response affected by E5 in keratinocytes are Natural killer T (NKT) cells, since E5 sequesters the CD1 protein within endosomes, preventing their activation. This point is described in lines 134-137.
Reviewer 2 Report
Comments and Suggestions for AuthorsPerez-Morales et al review the role of E5 protein of HPV16 in the evasion of immune responses. Overall, this is a well-written and interesting topic, some issues need to be clarified and improved.
Major comments
It is not made clear whether these events are specific to HPV16 or also occur with other high-risk HPV. How different are the E5, E6, E7 proteins in the 14 different high-risk HPV variants? A discussion is necessary.
The authors focus in the introduction of infection of Keratinocytes. A paragraph should be added on infection of other cell types, e.g. relevant to other types of cancers such as cervical cancer or nasopharyngeal cancers.
Figure 1. Some writings are in Spanish. Font size is too small. The numbers 1 and 2 from the legend should be pointed out also in the figure. Not clear what the red cross is. E5 protein should be clearly shown and labelled.
3.1 is too long and should be separated with subtitles, e.g. referring to the pathways shown in Figure 2. Possibly the parts addressing regulatory effects of E6 and E7 should be shown as a separate paragraph.
Figure 2: E5, E6 and E7 should be clearly shown and labelled. Not clear what the red star is, same symbols for E5 should be used as in Figure 1. Font size is too small.
A Figure depicting the events occurring for MHC and E5 in Section 4 should be added.
Additon of the Scheme/Figure depicting the infection cycle of HPV and the expression changes of E5, E6, and E7 during the life cycle of HPV would be useful.
Abbreviations should be in alphabetic order
Minor comments
Line 26: antigen presentation by which cells?
Line 32: These epithelial cells are the primary target of HPV infection. The E5 oncoprotein ….
Line 63: resulting in formation of three… forming three
Line 64: is E5 also in the plasma membranes?
Line 79: …degradation of anti-apoptotic Bax
Liens 94-108: It should be specified in which cells this occurs
Line 126: upregulation of ISC… what is ISC? Not listed in the abbreviations
Line 163: These finding suggest that E5 may interfere with IRF1 access to DNA…. Not clear whether E5 is in the nucleus or whether this is an indirect effect
Line 227: …NF-kB in other places it is NFkB
Lines 265-277: In which cells this occurs, cell type should be clearly mentioned.
Line 351: the inclusion of E5…not clear, is it the inhibition of E5? And how is it inhibited?
Abbreviations: MAVS … remove bracket at the end
Author Response
Reviewer 2
Perez-Morales et al review the role of E5 protein of HPV16 in the evasion of immune responses. Overall, this is a well-written and interesting topic, some issues need to be clarified and improved.
Major comments
- It is not made clear whether these events are specific to HPV16 or also occur with other high-risk HPV. How different are the E5, E6, E7 proteins in the 14 different high-risk HPV variants? A discussion is necessary.
Answer: The reviewer is right that the HPV type is not clearly specified in the document. The main HPV type studied is type 16, which is around 70% associated with cancer, and that was the reason for omitting the HPV type. Besides, while HPV16 E5 is a potent collaborator in the transformation process, in other HR-HPV types, E5´s role is considered dispensable, as E5 gene expression is lost when the cell is fully transformed, although E6 and E7 expressions remain during this process. To clarify this information in this review, we added information in the conclusion section (lines 422-436):
“All 14 HR-HPV types share the same basic genomics blueprints. E6 and E7 are highly conserved across HR-HPV types and maintain their core functions: binding and degrading tumor suppressor proteins (p53 and pRb, respectively). Meanwhile, E5 is the most variable among the HR-HPV types (Jones, et al., 2024). For instance, while HPV16 E5 is highly hydrophobic with three transmembrane domains, other types can vary in length and membrane orientation, and expression can be lost during viral genome integration into host DNA once the cell is fully transformed (DiMaio & Petti, 2013). However, persistent expression of E6 and E7 is required to maintain the malignant phenotype of cancer cells. In contrast, in LR-HPVs, the viral DNA typically remains episomal, maintaining E5 within its natural regulatory framework and contributing to benign proliferation without inducing the genomic instability characteristic of HR-HPV integration (Venuti, et al., 2011). Therefore, the oncogenic potential of the viral type results from synergy between the combined efficacy of E6/E7 in dismantling p53/pRb, which is essential for cancer maintenance, and the supportive activity of their specific E5, which enhances the early stages of transformation by promoting cell proliferation and immune evasion (Busukala & Banks, 2021; Zhang, et al., 2025).”
References:
Jones RN, Miyauchi S, Roy S, Boutros N, Mayadev JS, Mell LK, Califano JA, Venuti A, Sharabi AB. Computational and AI-driven 3D structural analysis of human papillomavirus (HPV) oncoproteins E5, E6, and E7 reveal significant divergence of HPV E5 between low-risk and high-risk genotypes. Virology. 2024; 590:109946. doi: 10.1016/j.virol.2023.109946
DiMaio D, Petti LM. The E5 proteins. Virology. 2013 Oct;445(1-2):99-114. doi: 10.1016/j.virol.2013.05.006.
Basukala O, Banks L. The Not-So-Good, the Bad and the Ugly: HPV E5, E6 and E7 Oncoproteins in the Orchestration of Carcinogenesis. Viruses. 2021 Sep 22;13(10):1892. doi: 10.3390/v13101892
Zhang, Y., Qiu, K., Ren, J., Zhao, Y., & Cheng, P. (2025). Roles of human papillomavirus in cancers: oncogenic mechanisms and clinical use. Signal Transduction and Targeted Therapy, 10. doi.org/10.1038/s41392-024-02083-w
- The authors focus in the introduction of infection of Keratinocytes. A paragraph should be added on infection of other cell types, e.g. relevant to other types of cancers such as cervical cancer or nasopharyngeal cancers.
Answer: We agree with the reviewer that the focus remains exclusively on keratinocytes. This emphasis is intentional, as the HPV life cycle occurs in stratified mucosal epithelia. The virus exhibits a specific tropism for precursor epithelial cells that will differentiate into keratinocytes; consequently, in vitro models using these cell types are the gold standard for mimicking the physiological environment of infection. Thus, keratinocyte-based models are used to study the molecular mechanisms of viral entry, persistence, immune evasion, and transformation.
- Figure 1. Some writings are in Spanish. Font size is too small. The numbers 1 and 2 from the legend should be pointed out also in the figure. Not clear what the red cross is. E5 protein should be clearly shown and labelled.
Answer: We have reviewed Figure 1 and made all the changes the reviewer pointed out: labels are in English, the font size has been increased for better visibility, and the numbers 1 and 2 have been added to the figure. The red cross has been modified, moved over an arrow signal, and defined in the figure legend as “blocked pathway”.
- 1 is too long and should be separated with subtitles, e.g. referring to the pathways shown in Figure 2. Possibly the parts addressing regulatory effects of E6 and E7 should be shown as a separate paragraph. (Lourdes)
Answer: We addressed this part and have separated this section into two, although now it is Section 4: 4.1 Inhibition of IFN-k production and 4.2 Inhibition of IFNs type I expression. We decided to remove the section on the regulatory effects of E6 and E7 from the text, as these oncogenes are not the primary focus of the review, and the information was not necessary to explain the inhibitory effect of E5 on the immune response.
- Figure 2: E5, E6 and E7 should be clearly shown and labelled. Not clear what the red star is, same symbols for E5 should be used as in Figure 1. Font size is too small
Answer: The correction for the E5 symbol in figures 1 and 2 has been addressed, as the reviewer suggested, and the font size has also been increased. Symbols for E6 and E7 were not added to the figure because information about these oncogenes is not the main point of the figure, and the text related to them was removed from the manuscript.
- A Figure depicting the events occurring for MHC and E5 in Section 4 should be added.
Answer: As the reviewer suggested, a new figure for E5 and MHC in section 4 has been added to clarify this part of the review.
- Addition of the Scheme/Figure depicting the infection cycle of HPV and the expression changes of E5, E6, and E7 during the life cycle of HPV would be useful.
Answer: We thank the reviewer for the suggestion. However, since this review focuses on the role of E5 as a modulator of the innate immune response, we do not consider that an additional figure on the HPV life cycle would enrich the manuscript. On the other hand, several reviews that focus on the HPV life cycle and address the topic in greater depth have been published, for example:
- Abbreviations should be in alphabetic order
Answer: We have corrected the abbreviations in alphabetical order as the reviewer pointed out.
Minor comments
Line 26: antigen presentation by which cells?
Answer: The abstract was rewritten, and the information that the reviewer pointed out has been modified.
Line 52: These epithelial cells are the primary target of HPV infection. The E5 oncoprotein ….
Answer: This part has been modified as the reviewer suggested, and it is highlighted in yellow.
Line 63: resulting in formation of three… forming three
Answer: We had corrected this as follows (line 79):
“characterized by three transmembrane α-helices (residues 8-30, 37-52, and 58-76).”
Line 64: is E5 also in the plasma membranes?
Answer: Yes, the E5 is also present in the plasma membrane. We inadvertently omitted this information from the manuscript; it has been corrected on line 81. The reference for this information is the same.
Line 79: …degradation of anti-apoptotic Bax
Answer: We had corrected this information in line 96-97 as the reviewer suggested.
Liens 94-108: It should be specified in which cells this occurs
Answer: We have modified this paragraph and incorporated the information the reviewer requested and (lines 118- 137).
Line 126: upregulation of ISC… what is ISC? Not listed in the abbreviations
Answer: The reviewer refers to the abbreviation ISGs, which stands for Interferon-stimulated genes. This acronym is listed in the abbreviation list and defined on line 53.
Line 163: These finding suggest that E5 may interfere with IRF1 access to DNA…. Not clear whether E5 is in the nucleus or whether this is an indirect effect
Answer: The reviewer is right that the information in this line is ambiguous. We have modified this information as follows (lines 192-194):
“These findings suggest that E5 indirectly interferes with IRF´s transcriptional activity by blocking its interaction with the ISRE region in the ISG promoter, which reduces IFN-k expression”.
Line 227: …NF-kB in other places it is NFkB
Answer: The reviewer is right, and we have corrected this as NF-kB
Lines 265-277: In which cells this occurs, cell type should be clearly mentioned.
Answer: We agree with the reviewer that the cell type needs to be defined in this paragraph. We added the proper information as follows (lines 293-294):
“Antigen-presenting cells use complex machinery to process and display peptide-MHC complexes on their cell surface for recognition by T cells. ”
Line 351: the inclusion of E5…not clear, is it the inhibition of E5? And how is it inhibited?
Answer: We have clarified this part for the reviewer in lines 463 to 467 as required.
“Consequently, the same mechanisms that allow E5 to orchestrate immune subversion also render it a promising target for immunotherapy. Thus, incorporating E5 into peptide, DNA, or recombinant viral vector platforms, researchers have elicited robust CD4+/CD8+ cellular immunity and a Th1-biased response, leading to potent anti-tumor activity [69-72, 81].”
Abbreviations: MAVS … remove bracket at the end
Answer: We have corrected this error in the final version of the manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
your manuscript titled: " Role of E5 from HPV16 in the evasion of the immune response" summarized 91 articles and present molecular mechanism and patchways of E5 oncoprotein roles. Manuscript is written in a clear and interesting way and structured of four main parts. Core of the review is interactions of E5 oncoprotein with innate and adaptive immune response. Some references represents the newest literature position in this field.
For increased value of this manuscript, I listed some suggestions below:
- figure 1: all descriptions on figure should be in english
- in the manuscript there is no information of methodology of this review; how criteria of the manuscript chosen were applied? please add methodological section
- did you find any in vivo, clinical or in vitro studies that present the E5 protein potential for clinics? in the conclusion section you suggest this possibility, but I didn't find this information suppported by studies in the manuscript
- you write about vaccines based on E5, with antitumour activity. Please provide some information about anti-HPV vaccines that are available, mechanism of its actions. Why vaccines based on E5 may be better? could you find some articles about E5 based vaccines or clinicall studies?
Author Response
Reviewer 3
Your manuscript titled: " Role of E5 from HPV16 in the evasion of the immune response" summarized 91 articles and present molecular mechanism and pathways of E5 oncoprotein roles. Manuscript is written in a clear and interesting way and structured of four main parts. Core of the review is interactions of E5 oncoprotein with innate and adaptive immune response. Some references represents the newest literature position in this field.
For increased value of this manuscript, I listed some suggestions below:
- Figure 1: all descriptions on figure should be in English.
Answer: We have reviewed Figure 1 and made all the necessary changes
- In the manuscript there is no information of methodology of this review; how criteria of the manuscript chosen were applied? please add methodological section (Lourdes)
Answer: We agree with the reviewer that a methodology section is required to strengthen the selection of the information utilized for this review. We added a methodology section as follows: (lines 62-76)
“2. Methodology
This article employs a narrative review methodology to provide a comprehensive qualitative synthesis of current knowledge on the role of HPV16 E5 oncoprotein in modulating the host immune response. A comprehensive literature search was conducted across the PubMed, Scopus, and Web of Science databases using keywords including “HPV E5”, “immune evasion”, “innate immune response”, “adaptive immune response”, “interferon signaling”, “TGF-β pathway”, “MHC-I and MHC-II”, and “antigen presentation”.
The selection of sources prioritized seminal mechanistic studies and recent peer-reviewed research, elucidating the molecular interactions of E5 in human epithelial models. Data were synthesized using a thematic analysis approach, categorizing E5´s activities into four different axes: (1) E5 in cell transformation, (2) innate immune response, (3) adaptive immune response, and (4) E5 as a therapeutic target. This review focuses on the synergy between E5-mediated signaling disruption and the evasion of innate and adaptive immune surveillance.”
- Did you find any in vivo, clinical or in vitro studies that present the E5 protein potential for clinics? in the conclusion section you suggest this possibility, but I didn't find this information suppported by studies in the manuscript.
Answer: There are no clinical protocols reported in which E5 is used as a vaccine against HPV-induced lesions or tumors. On the other hand, there are a few preclinical studies in mice using E5 as a vaccine against HPV-induced tumors. We included these studies in the review in Section 6 “Prophylactic and therapeutic E5-based vaccine against HPV-induced tumors” (lines 371-420), to clarify the information in the conclusions section.
- You write about vaccines based on E5, with antitumour activity. Please provide some information about anti-HPV vaccines that are available, mechanism of its actions. Why vaccines based on E5 may be better? could you find some articles about E5 based vaccines or clinicall studies?
Answer: We have added in the manuscript Section 6 that includes this new information (lines 371-420).
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe revised manuscript is much improved, most of the suggestions and concerns of the reviewers are sufficiently addressed.
Minor corrections
Line 21: ...organizing...orchestrating or promoting
Font of the text is not homogenous throughout the text of the manuscript
