An Endogenous Retrovirus Vaccine Encoding an Envelope with a Mutated Immunosuppressive Domain in Combination with Anti-PD1 Treatment Eradicates Established Tumours in Mice.

Endogenous retroviruses (ERVs) account for 8% of our genome, and, although they are usually silent in healthy tissues, they become reactivated and expressed in pathological conditions such as cancer. Several studies support a functional role of ERVs in tumour development and progression, specifically through their envelope (Env) protein, which contains a region described as an immunosuppressive domain (ISD). We have previously shown that targeting of the murine ERV (MelARV) Env using virus-like vaccine (VLV) technology, consisting of an adenoviral vector encoding virus-like particles (VLPs), induces protection against small tumours in mice. Here, we investigate the potency and efficacy of a novel MelARV VLV with a mutated ISD (ISDmut) that can modify the properties of the adenoviral vaccine-encoded Env protein. We show that the modification of the vaccine's ISD significantly enhanced T-cell immunogenicity in both prime and prime-boost vaccination regimens. The modified VLV in combination with an α-PD1 checkpoint inhibitor (CPI) exhibited excellent curative efficacy against large established colorectal CT26 tumours in mice. Furthermore, only ISDmut-vaccinated mice that survived CT26 challenge were additionally protected against rechallenge with a triple-negative breast cancer cell line (4T1), showing that our modified VLV provides cross-protection against different tumour types expressing ERV-derived antigens. We envision that translating these findings and technology into human ERVs (HERVs) could provide new treatment opportunities for cancer patients with unmet medical needs.

Knockdown of Atg7 Induces Nuclear-LC3 Dependent Apoptosis and Augments Chemotherapy in Colorectal Cancer Cells.

Autophagy is a catabolic process that enables cells to degrade obsolete content and refuel energy depots. In colorectal cancer (CRC) autophagy has been shown to promote tumorigenesis through energy delivery in the condition of uncontrolled proliferation. With this study, we aimed at evaluating whether autophagy sustains CRC cell viability and if it impacts therapy resistance. Initially, a colorectal cancer tissue micro array, containing mucosa (n = 10), adenoma (n = 18) and adenocarcinoma (n = 49) spots, was stained for expression of essential autophagy proteins LC3b, Atg7, p62 and Beclin-1. Subsequently, central autophagy proteins were downregulated in CRC cells using siRNA technology. Viability assays, flow cytometry and immunoblotting were performed and three-dimensional cell culture was utilized to study autophagy in a tissue mimicking environment. In our study we found an upregulation of Atg7 in CRC. Furthermore, we identified Atg7 as crucial factor within the autophagy network for CRC cell viability. Its disruption induced cell death via triggering apoptosis and in combination with conventional chemotherapy it exerted synergistic effects in inducing CRC cell death. Cell death was strictly dependent on nuclear LC3b, since simultaneous knockdown of Atg7 and LC3b completely restored viability. This study unravels a novel cell death preventing function of Atg7 in interaction with LC3b, thereby unmasking a promising therapeutic target in CRC.

The potential of adenoviral vaccine vectors with altered antigen presentation capabilities.

Introduction: Despite their appeal as vaccine vectors, adenoviral vectors are yet unable to induce protective immune responses against some weakly immunogenic antigens. Additionally, the maximum doses of adenovirus-based vaccines are limited by vector-induced toxicity, causing vector elimination and diminished immune responses against the target antigen. In order to increase immune responses to the transgene, while maintaining a moderate vector dose, new technologies for improved transgene presentation have been developed for adenoviral vaccine vectors.Areas covered: This review provides an overview of different genetic-fusion adjuvants that aim to improve antigen presentation in the context of adenoviral vector-based vaccines. The influence on both T cell and B cell responses are discussed, with a main focus on two technologies: MHC class II-associated invariant chain and virus-like-vaccines.Expert opinion: Different strategies have been tested to improve adenovirus-based vaccinations with varying degrees of success. The reviewed genetic adjuvants were designed to increase antigen processing and MHC presentation, or promote humoral immune responses with an improved conformational antigen display. While none of the introduced technologies is universally applicable, this review shall give an overview to identify potential improvements for future vaccination approaches.

Virus-like vaccines against HIV/SIV synergize with a subdominant antigen T cell vaccine.

BACKGROUND: In non-human primates (NHPs) and humans, partial protection from HIV/SIV infection or suppression of replication is achievable by Env-binding antibodies and Gag-specific CD8+ T-cells targeting protective epitopes. Unfortunately, such T-cell responses are frequently dominated by responses to non-protective, variable epitopes. In this study we attempt to combine three independent approaches, each developed to prevent immunodominance of non-protective epitopes. These approaches were (1) vaccines consisting exclusively of putatively protective p24 Gag highly conserved elements (CEs), (2) vaccines using solely subdominant antigens which were acutely protective in a recent NHP trial, and (3) virus-encoded virus-like particle vaccines (virus-like vaccines/VLVs) using heterologous Env and Gag sequences to enable selection of broadly cross-reactive responses and to avoid immunodominance of non-conserved sequences in prime-boost regimens as previously observed. METHODS: We vaccinated outbred CD1 mice with HIV-1 clade B Gag/Env encoded in an adenoviral prime and SIVmac239 Gag/Env in an MVA boost. We combined this completely heterologous immunization regimen and the homologous SIVmac239 Gag/Env immunization regimen with an additional prime encoding SIV CEs and accessory antigens Rev, Vif and Vpr (Ad-Ii-SIVCErvv). T-cell responses were analyzed by intracellular cytokine staining of splenocytes and antibody responses by trimer-specific ELISA, avidity and isotype-specific ELISA. RESULTS: Env dominance could be avoided successfully in the completely heterologous prime-boost regimen, but Env immunodominance reappeared when Ad-Ii-SIVCErvv was added to the prime. This regimen did however still induce more cross-reactive Gag-specific CD8+ T-cells and Env-specific antibodies. Including Ad-Ii-SIVCErvv in the homologous prime-boost not only elicited accessory antigen-specific CD8+ memory T-cells, but also significantly increased the ratio of Gag- to Env-specific CD8+ T-cells. The CD4+ T-cell response shifted away from structural antigens previously associated with infection-enhancement. CONCLUSION: The homologous Gag/Env prime-boost with Ad-Ii-SIVCErvv prime combined acutely protective CD8+ T-cell responses to subdominant antigens and Env-binding antibodies with chronically protective Gag-specific CD8+ T-cells in outbred mice. This vaccine regimen should be tested in an NHP efficacy trial.

Adenovirus based virus-like-vaccines targeting endogenous retroviruses can eliminate growing colorectal cancers in mice.

Endogenous retroviruses (ERVs) that make up 8% of the human genome have been associated with the development and progression of cancer. The murine model system of the melanoma associated retrovirus (MelARV), which is expressed in different murine cancer cell lines, can be used to study mechanisms and therapeutic approaches against ERVs in cancer. We designed a vaccine strategy (Ad5-MelARV) of adenoviruses encoding the MelARV proteins Gag and Env that assemble in vivo into virus-like particles displaying the cancer-associated MelARV Env to the immune system. The novel vaccine was designed to induce both humoral as well as cellular immune responses in order to attack ERV expressing tumor cells. Despite a lack of antibody induction, we found that T cell responses were strong enough to prevent colorectal CT26 tumor growth and progression in BALB/c mice after a single vaccination before or after tumor challenge. A combination with the checkpoint inhibitor anti-PD-1 further increased the efficacy of the vaccination leading to complete tumor regression. Furthermore, immune responses in vaccinated mice were not restricted to only one cancer cell line but vaccinated animals were also protected from a rechallenge with the distinct breast cancer cell line 4T1. Thus, the developed vaccine strategy could represent a novel tool to successfully target diverse ERV-bearing tumors in cancer patients.

Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques.

The human adenovirus type 19a/64 (hAd19a) is a rare serotype in the human population that transduces human dendritic cells (DCs) and human muscle cells more efficiently than the well-characterized human adenovirus type 5 (hAd5). To further characterize the potential of this vector as a vaccine we designed replication deficient hAd19a, hAd5 and MVA vectors expressing a papillomavirus (PV) antigen fused to the human MHC class II associated invariant chain T cell adjuvant (hIi) and investigated their immunogenicity in vivo in mice and cynomolgus macaques. We initially showed that the hIi encoded in the hAd5 enhanced PV specific CD8+ T cell responses in mice. The T cell responses induced after hAd19a vaccination was similar to those induced by hAd5 vaccination. The hAd19a induced responses were not reduced in presence of preexisting Ad5 immunity in mice. In macaques both vaccines were equally potent at inducing CD8+ T cells after MVA boost, while the level of CD4+ T cell responses were found to be broader in hAd19a primed animals. These data demonstrate the potential of hAd19a as an alternative vector to hAd5 to elicit potent T cell responses to PV.