Novel recombinant DNA vaccine candidates for human respiratory syncytial virus: Preclinical evaluation of immunogenicity and protection efficiency.

The development of safe and potent vaccines for human respiratory syncytial virus (HRSV) is still a challenge for researchers worldwide. DNA-based immunization is currently a promising approach that has been used to generate human vaccines for different age groups. In this study, novel HRSV DNA vaccine candidates were generated and preclinically tested in BALB/c mice. Three different versions of the codon-optimized HRSV fusion (F) gene were individually cloned into the pPOE vector. The new recombinant vectors either express full-length (pPOE-F), secretory (pPOE-TF), or M282-90 linked (pPOE-FM2) forms of the F protein. Distinctive expression of the F protein was identified in HEp-2 cells transfected with the different recombinant vectors using ELISA and immunofluorescence. Mice immunization verified the potential for recombinant vectors to elicit significant levels of neutralizing antibodies and CD8+ T-cell lymphocytes. pPOE-TF showed higher levels of gene expression in cell culture and better induction of the humoral and cellular immune responses. Following virus challenge, mice that had been immunized with the recombinant vectors were able to control virus replication and displayed lower inflammation compared with mice immunized with empty pPOE vector or formalin-inactivated HRSV vaccine. Moreover, pulmonary cytokine profiles of mice immunized with the 3 recombinant vectors were similar to those of the mock infected group. In conclusion, recombinant pPOE vectors are promising HRSV vaccine candidates in terms of their safety, immunogenicity and protective efficiency. These data encourage further evaluation in phase I clinical trials.

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.

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

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.

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.

GnRH-III based multifunctional drug delivery systems containing daunorubicin and methotrexate.

Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing two chemotherapeutic agents, daunorubicin and methotrexate, coupled to the GnRH-III decapeptide, which served as a targeting moiety. This represents a possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of anticancer drug-peptide bioconjugates. The multifunctional bioconjugates were prepared according to two drug design approaches recently developed by our group. Both bifunctional GnRH-III derivatives, [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) and [(8)Lys(Lys)]-GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Lys)-Pro-Gly-NH(2)), contain two free amino groups suitable for the attachment of two anticancer drugs, such as methotrexate and daunorubicin. The drugs were chosen with respect to their different mechanisms of action, with the goal of increasing the antitumor effect of the bioconjugates. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their in vitro stability/degradation in human serum and in the presence of rat liver lysosomal homogenate was investigated by liquid chromatography in combination with mass spectrometry. The influence of the multifunctional bioconjugates on the cell adhesion and cell proliferation was studied on Mono Mac 6 human leukemic monocytes. It was found that (1) all synthesized bioconjugates had in vitro cytostatic effect; (2) they were stable in human serum for at least 24 h; (3) they were hydrolyzed in the presence of lysosomal homogenate and (4) they exerted a moderate cell-cell adhesion inducing effect. These results demonstrate that multifunctional bioconjugates containing two different anticancer drugs attached to the same GnRH-III targeting moiety could be successfully prepared and resulted in higher in vitro cytostatic effect than the monofunctional bioconjugates containing either methotrexate or daunorubicin, in particular on HT-29 human colon cancer cells.

Design, synthesis, in vitro stability and cytostatic effect of multifunctional anticancer drug-bioconjugates containing GnRH-III as a targeting moiety.

Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) containing two lysine residues in positions 4 and 8, whose ε-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was used as a scaffold; an additional lysine residue was coupled to the ϵ-amino group of (8) Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.

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.

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

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.

Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7.

To allow vaccination irrespective of HLA type, DNA vaccines encoding full-length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full-length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic "gene-shuffled" variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full-length and gene-shuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full-length and the gene-shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein-70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T-cell responses. The E7-specific CD8(+) T cells induced by TTFC fusion vaccines were functional in terms of IFN-γ production, formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene-shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full-length and gene-shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV-positive carcinomas in future studies.

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.

Enhancement of immunogenicity of a therapeutic cervical cancer DNA-based vaccine by co-application of sequence-optimized genetic adjuvants.

Treatment of patients with cervical cancer by conventional methods (mainly surgery, but also radiotherapy and chemotherapy) results in a significant loss in quality of life. A therapeutic DNA vaccine directed to tumor-specific antigens of the human papilloma virus (HPV) could be an attractive treatment option. We have developed a nontransforming HPV-16 E7-based DNA vaccine containing all putative T cell epitopes (HPV-16 E7SH). DNA vaccines, however, are less immunogenic than protein- or peptide-based vaccines in larger animals and humans. In this study, we have investigated an adjuvant gene support of the HPV-16 E7SH therapeutic cervical cancer vaccine. DNA encoded cytokines (IL-2, IL-12, GM-CSF, IFN-gamma) and the chemokine MIP1-alpha were co-applied either simultaneously or at different time points pre- or post-E7SH vaccination. In addition, sequence-optimized adjuvant genes were compared to wild type genes. Three combinations investigated lead to an enhanced IFN-gamma response of the induced T cells in mice. Interestingly, IFN-gamma secretion of splenocytes did not strictly correlate with tumor response in tumor regression experiments. Gene-encoded MIP-1alpha applied 5 days prior to E7SH-immunization combined with IFN-gamma or IL-12 (3 days) or IL-2 (5 days) postimmunization lead to a significantly enhanced tumor response that was clearly associated with granzyme B secretion and target cells lysis. Our results suggest that a conditioning application and combination with adjuvant genes may be a promising strategy to enhance synergistically immune responses by DNA immunization for the treatment of cervical cancer.

An improved rearranged Human Papillomavirus Type 16 E7 DNA vaccine candidate (HPV-16 E7SH) induces an E7 wildtype-specific T cell response.

A new and very promising approach in vaccine development is the application of naked DNA. In comparison to conventional vaccines it offers several advantages, especially if there is a need for the development of low cost vaccines. Infection with high-risk human papillomaviruses (hr-HPVs) is the major risk factor for the development of cervical cancer (cc), the third most common cancer in women worldwide. The HPV E7 oncogene is constitutively expressed in HPV-infected cells and represents an excellent target for immune therapy of HPV-related disease. Therefore, we chose the HPV-16 E7 as model antigen in the development of a therapeutic DNA vaccine candidate. For safety reasons the use of a transforming gene like the HPV-16 E7 for DNA vaccination is not feasible in humans. In consequence we have generated an artificial ("shuffled") HPV-16 E7-gene (HPV-16 E7SH), containing all putative cytotoxic T-lymphocyte (CTLs) epitopes and exhibiting high safety features. Here, we show the induction of a strong E7-wildtype (E7WT) directed cellular and humoral immune response including tumor protection and regression after in vivo immunization in the murine system. Moreover, the vaccine candidate demonstrated immunogenicity in humans, demonstrated by priming of antigen-specific T cells in vitro. Importantly, the artificial HPV-gene has completely lost its transforming properties as measured in soft agar transformation assays. These results may be of importance for the development of vaccines based on oncogenes or oncoproteins.

Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth.

Colorectal carcinoma (CRC) is a major cause of morbidity and mortality in Western countries. It has so far been molecularly defined mainly by alterations of the Wnt pathway. We show here for the first time that aberrant activities of the signal transducer and activator of transcription STAT3 actively contribute to this malignancy and, thus, are a potential therapeutic target for CRC. Constitutive STAT3 activity was found to be abundant in dedifferentiated cancer cells and infiltrating lymphocytes of CRC samples, but not in non-neoplastic colon epithelium. Cell lines derived from malignant colorectal tumors lost persistent STAT3 activity in culture. However, implantation of colon carcinoma cells into nude mice resulted in restoration of STAT3 activity, suggesting a role of an extracellular stimulus within the tumor microenvironment as a trigger for STAT activation. STAT3 activity in CRC cells triggered through interleukin-6 or through a constitutively active STAT3 mutant promoted cancer cell multiplication, whereas STAT3 inhibition through a dominant-negative variant impaired IL-6-driven proliferation. Blockade of STAT3 activation in CRC-derived xenograft tumors slowed down their development, arguing for a contribution of STAT3 to colorectal tumor growth.

Modification of HPV 16 E7 genes: correlation between the level of protein expression and CTL response after immunization of C57BL/6 mice.

Immunization with a codon-optimized HPV 16 E7 gene was shown to yield higher levels of E7-specific cytotoxic T cells [Liu WJ, Gao F, Zhao KN, Zhao W, Fernando GJ, Thomas R, et al. Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 2002;301:43]. Here, we sought to verify the hypothesis that there is a direct correlation between the level of protein expression and immunogenicity in mice. We generated HPV 16 E7 expression plasmids where the genes were inserted either as authentic sequence (wt) or after optimizing the codons for use in mammalian cells (opt). For enhancement of translation of the E7 gene a 5' Kozak sequence (K) was added. Transfection experiments revealed the strength of expression in the order of E7opt+K, E7opt-K, E7wt+K and E7wt-K. After immunization of C57/B6 mice we observed an equally strong CD8+T-cell response with the E7opt plasmids (+ or -K), followed by the E7wt+K and E7wt-K DNAs. The same difference in efficiency was obtained in tumor protection experiments. Regression of pre-existing tumors and CTL activity was observed only with the E7opt+K plasmid. From these data, we conclude that the level of protein expression correlates with the efficiency of CTL response and hence testing by transfection of cells in culture may allow a pre-selection of expression plasmids prior to DNA immunization.

Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice.

We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1(165-173) peptide for ex vivo restimulation of splenocytes prior to analysis ((51)Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D(b)-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and (51)Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1 Delta N10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and (51)Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.

T cell response to human papillomavirus 16 E7 in mice: comparison of Cr release assay, intracellular IFN-gamma production, ELISPOT and tetramer staining.

Successful vaccination against infections by high-risk papillomaviruses aiming at the prevention of cervical cancer most likely requires the induction of neutralizing antibodies and human papillomavirus (HPV)-specific T cells directed against early viral proteins such as E7. Whereas the technology for detection of antibodies is well established, measurement of T cells is more cumbersome and standardization of assays is difficult. By using chromium release assay, ELISPOT, tetramer staining and intracellular IFN-gamma assay, we compared the levels of HPV 16 E7-specific T cells obtained after immunization of C57BL/6 mice with different DNA expression vectors. We found that all four assays gave highly comparable results. ELISPOT can be recommended for future studies as it indicates the presence of activated (i.e. IFN-gamma-secreting) T cells in a quantitative manner and combines high sensitivity with relatively low T cell demand.