Design and Immunological Validation of Macaca fascicularis Papillomavirus Type 3 Based Vaccine Candidates in Outbred Mice: Basis for Future Testing of a Therapeutic Papillomavirus Vaccine in NHPs.

Patrick Neckermann,Cordula Pertl,Silke Schrödel,Benedikt Asbach,Peter Johannes Holst,Christian Thirion,Torsten Willert,Ralf Wagner,Ditte Rahbaek Boilesen

doi:10.3389/fimmu.2021.761214

Abstract

Persistent human papillomavirus (HPV) infections are causative for cervical neoplasia and carcinomas. Despite the availability of prophylactic vaccines, morbidity and mortality induced by HPV are still too high. Thus, an efficient therapy, such as a therapeutic vaccine, is urgently required. Herein, we describe the development and validation of Macaca fascicularis papillomavirus type 3 (MfPV3) antigens delivered via nucleic-acid and adenoviral vectors in outbred mouse models. Ten artificially fused polypeptides comprising early viral regulatory proteins were designed and optionally linked to the T cell adjuvant MHC-II-associated invariant chain. Transfected HEK293 cells and A549 cells transduced with recombinant adenoviruses expressing the same panel of artificial antigens proved proper and comparable expression, respectively. Immunization of outbred CD1 and OF1 mice led to CD8+ and CD4+ T cell responses against MfPV3 antigens after DNA- and adenoviral vector delivery. Moreover, in vivo cytotoxicity of vaccine-induced CD8+ T cells was demonstrated in BALB/c mice by quantifying specific killing of transferred peptide-pulsed syngeneic target cells. The use of the invariant chain as T cell adjuvant enhanced the T cell responses regarding cytotoxicity and in vitro analysis suggested an accelerated turnover of the antigens as causative. Notably, the fusion-polypeptide elicited the same level of T-cell responses as administration of the antigens individually, suggesting no loss of immunogenicity by fusing multiple proteins in one vaccine construct. These data support further development of the vaccine candidates in a follow up efficacy study in persistently infected Macaca fascicularis monkeys to assess their potential to eliminate pre-malignant papillomavirus infections, eventually instructing the design of an analogous therapeutic HPV vaccine.

Highlights

Despite efficient prophylactic vaccines and the possibility to screen for cervical lesions, infection with human papillomavirus (HPV) was still responsible for more than 340.000 cervical cancer-related deaths worldwide in 2020 [1]
In order to enable future validation of a therapeutic concept in non-human primates, the above antigens were derived from Macaca fascicularis papillomavirus type 3 (MfPV3), which was shown to persist and induce low-grade squamous intraepithelial lesions (LSIL)-like lesions in the cervix of breeding female cynomolgus macaques (Macaca fascicularis) [20, 21]
Antigens were conceived as (i) read-through polypeptides with all four MfPV3 early antigens encoded in one open reading frame, (ii) alternatively E1-E2 and E6-E7 fusions were linked via a p2A site [38] supporting translational coupling and co-expression of the two fusion proteins or (iii) as single expression units

Summary

Introduction

Despite efficient prophylactic vaccines and the possibility to screen for cervical lesions, infection with human papillomavirus (HPV) was still responsible for more than 340.000 cervical cancer-related deaths worldwide in 2020 [1]. There are three approved prophylactic vaccines providing near complete protection against vaccine-targeted HPV types, yet vaccine uptake is incomplete [3]. These vaccines do not lead to the eradication of pre-infected cells, since they target the major capsid protein L1, which is not expressed in infected basal layer- and cervical cancer cells [4,5,6,7]. Whereas most HPV infections are spontaneously cleared within months, some persist for years [8] These can progress towards low-grade squamous intraepithelial lesions (LSIL), which can further progress to high-grade squamous intraepithelial lesions (HSIL) and cervical cancer. The expression pattern of viral proteins changes during progression of SILs: in LSIL, mainly the early proteins E1/E2 are expressed, whereas in HSIL and transformed cells, E6/E7 are highly expressed and E2 expression is low or absent [9, 10]

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