Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice

Abstract Background Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. Methods In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize γ-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. Results High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. Conclusions The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs

The fundamental left-right asymmetry in the Germanic verb cluster

Cinque (2005, 2009, 2014a) observes that there is an asymmetry in the possible ordering of dependents of a lexical head before versus after the head. A reflection on some of the concepts needed to develop Cinque’s ideas into a theory of neutral word order reveals that dependents need to be treated separately by class. The resulting system is applied to the problem of word order in the Germanic verb cluster. It is shown that there is an extremely close match between theoretically derived expectations for clusters made up of auxiliaries, modals, causative ‘let’, a main verb, and verbal particles. The facts point to the action of Cinque’s fundamental left-right asymmetry in language in the realm of the verb cluster. At the same time, not all verb clusters fall under Cinque’s generalization, which, therefore, argues against treating all cases of restructuring uniformly

Titanium Salan Complexes Displays Strong Antitumor Properties In Vitro and In Vivo in Mice

The anticancer activity of titanium complexes has been known since the groundbreaking studies of Köpf and Köpf-Maier on titanocen dichloride. Unfortunately, possibly due to their fast hydrolysis, derivatives of titanocen dichloride failed in clinical studies. Recently, the new family of titanium salan complexes containing tetradentate ONNO ligands with anti-cancer properties has been discovered. These salan complexes are much more stabile in aqueous media. In this study we describe the biological activity of two titanium salan complexes in a mouse model of cervical cancer. High efficiency of this promising complex family was demonstrated for the first time in vivo. From these data we conclude that titanium salan complexes display very strong antitumor properties exhibiting only minor side effects. Our results may influence the chemotherapy with metallo therapeutics in the future

The combination of TLR-9 adjuvantation and electroporation-mediated delivery enhances in vivo antitumor responses after vaccination with HPV-16 E7 encoding DNA

Therapeutic DNA vaccination is an attractive adjuvant option to conventional methods in the fight against cancer, like surgery radiotherapy and chemotherapy. Despite strong antitumor effects that were observed in small animals with different antigens, DNA-based vaccines remain weakly immunogenic in large animals and primates compared to protein-based vaccines. Here, we sought to enhance the immunogenicity of a therapeutic nontransforming cervical cancer DNA vaccine (HPV-16 E7SH) by introduction of a highly optimized CpG cassette into the plasmid backbone as well as by an optimized DNA delivery using an advanced electroporation (EP) technology. By integrating the means for agent administration and EP into a single device, this technology enables a simple, one-step procedure that facilitates reproducibility. We found that highly optimized CpG motifs alone triggers an enhanced IFN-γ and granzyme B response in Elispot assays as well as stronger tumor regression. Furthermore, these effects could be dramatically enhanced when the CpG cassette containing plasmid was administered via the newly developed EP technology. These data suggest that an optimized application of CpG-enriched DNA vaccines may be an attractive strategy for the treatment of cancer. Collectively, these results provide a basis for the transfer of preclinical therapeutic DNA-based immunization studies into successful clinical cancer trials

An artificial PAP gene breaks self-tolerance and promotes tumor regression in the TRAMP model for prostate carcinoma

Prostate cancer (PCa) is the most commonly diagnosed type of cancer in men in western industrialized countries. As a public health burden, the need for the invention of new cost-saving PCa immunotherapies is apparent. In this study, we present a DNA vaccine encoding for the prostate-specific antigen prostatic acid phosphatase (PAP) linked to the J-domain and the SV40 enhancer sequence. The PAP DNA vaccine induced a strong PAP-specific cellular immune response after electroporation (EP)-based delivery in C57BL/6 mice. Splenocytes from mice immunized with PAP recognized the naturally processed PAP epitopes, indicating that vaccination with the PAP-J gene broke its self-tolerance against PAP. Remarkably, DNA vaccination with PAP-J inhibited tumor growth in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model that closely resembled human PCa. Therefore, this study highlights a novel cancer immunotherapy approach with the potential to control PCa in clinical settings

Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling).

Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism

Synthesis and X-ray structure analysis of a heptacoordinate titanium(IV)-bis-chelate with enhanced in vivo antitumor efficacy

Chelate stabilization of a titanium(IV)–salan alkoxide by ligand exchange with 2,6-pyridinedicarboxylic acid (dipic) resulted in heptacoordinate complex 3 which is not redox-active, stable on silica gel and has increased aqueous stability. 3 is highly toxic in HeLa S3 and Hep G2 and has enhanced antitumor efficacy in a mouse cervical-cancer model