Sequence of androgen receptor-targeted vaccination with androgen deprivation therapy affects anti-prostate tumor efficacy.

RATIONALE: Androgen deprivation therapy (ADT) is the primary treatment for recurrent and metastatic prostate cancer. In addition to direct antitumor effects, ADT has immunomodulatory effects such as promoting T-cell infiltration and enhancing antigen processing/presentation. Previous studies in our laboratory have demonstrated that ADT also leads to increased expression of the androgen receptor (AR) and increased recognition of prostate tumor cells by AR-specific CD8+T cells. We have also demonstrated that ADT combined with a DNA vaccine encoding the AR significantly slowed tumor growth and improved the survival of prostate tumor-bearing mice. The current study aimed to investigate the impact of the timing and sequencing of ADT with vaccination on the tumor immune microenvironment in murine prostate cancer models to further increase the antitumor efficacy of vaccines. METHODS: Male FVB mice implanted with Myc-CaP tumor cells, or male C57BL/6 mice implanted with TRAMP-C1 prostate tumor cells, were treated with a DNA vaccine encoding AR (pTVG-AR) and ADT. The sequence of administration was evaluated for its effect on tumor growth, and tumor-infiltrating immune populations were characterized. RESULTS: Vaccination prior to ADT (pTVG-AR → ADT) significantly enhanced antitumor responses and survival. This was associated with increased tumor infiltration by CD4+ and CD8+ T cells, including AR-specific CD8+T cells. Depletion of CD8+T cells prior to ADT significantly worsened overall survival. Following ADT treatment, however, Gr1+ myeloid-derived suppressor cells (MDSCs) increased, and this was associated with fewer infiltrating T cells and reduced tumor growth. Inhibiting Gr1+MDSCs recruitment, either by using a CXCR2 antagonist or by cycling androgen deprivation with testosterone replacement, improved antitumor responses and overall survival. CONCLUSION: Vaccination prior to ADT significantly improved antitumor responses, mediated in part by increased infiltration of CD8+T cells following ADT. Targeting MDSC recruitment following ADT further enhanced antitumor responses. These findings suggest logical directions for future clinical trials to improve the efficacy of prostate cancer vaccines.

Investigation of Immunostimulatory Effects of IFN-γ Cytokine and CD40 Ligand Costimulatory Molecule for Development of HIV-1 Therapeutic Vaccine Candidate.

The most crucial disadvantage of DNA-based vaccines is their low immunogenicity; therefore, finding an effectual adjuvant is essential for their development. Herein, immunostimulatory effects of IFNγ cytokine and a CD40 ligand (CD40L) costimulatory molecule are evaluated as combined with an antigen, and also linked to an antigen in mice. For this purpose, after preparation of the HIV-1 Nef, IFNγ, and CD40L DNA constructs, and also their recombinant protein in an Escherichia coli expression system, nine groups of female BALB/c mice are immunized with different regimens of DNA constructs. About 3 weeks and also 3 months after the last injection, humoral and cellular immune responses are assessed in mice sera and splenocytes. Additionally, mice splenocytes are exposed to single-cycle replicable (SCR) HIV-1 virions for evaluating their potency in the secretion of cytokines in vitro. The data indicate that the linkage of IFNγ and CD40L to Nef antigen can significantly induce the Th-1 pathway and activate cytotoxic T lymphocytes compared to other regimens. Moreover, groups receiving the IFNγ-Nef and CD40L-Nef fusion DNA constructs show higher secretion of IFNγ and TNF-α from virion-infected lymphocytes than other groups. Therefore, the IFNγ-Nef and CD40L-Nef fusion DNA constructs are suggested to be a potential option for development of an efficient HIV-1 vaccine.

The adjuvant effect of polymuramil, a NOD1 and NOD2 agonist, differs when immunizing mice of different inbred lines with nonstructural hepatitis C virus (Flaviviridae: Hepacivirus)proteins and is synergistically enhanced in combination with pyrogenalum, a TLR4 agonist.

INTRODUCTION: Hepatitis C is a liver disease with high chronicity, the cause of cirrhosis and hepatocarcinoma. The main obstacle to controlling hepatitis C is the lack of vaccines. The aim of the work was to compare the immunogenic activity of nonstructural recombinant proteins NS3, NS4 and NS5B of hepatitis C virus (HCV) as components of a subunit candidate vaccine and to analyze the adjuvant properties of two available commercial drugs, polymuramil and pyrogenalum. MATERIALS AND METHODS: BALB/c, DBA/2J and C57BL/6 mice were immunized with nonstructural proteins without adjuvants or with polymuramyl (NOD1 and NOD2 agonist) and pyrogenalum (TLR-4 agonist). The activity of antibodies was determined in ELISA, the cellular response - by antigen-specific lymphocyte proliferation and by production of IFN-γ in vitro. RESULTS: Recombinant proteins showed different immunogenicity. NS4 induced antibodies more efficiently than NS3 and NS5B. Significant differences were found in the immune response of three inbred lines mice: the level of IFN-γ in BALB/c and DBA/2J mice induced by NS5B protein was 30 times higher than in C57Bl/6 mice. In contrast, the induction of antibodies in BALB/c mice was lower than in C57Bl/6 and DBA/2J. Polymuramil did not increase the humoral response to NS5B and enhanced the cellular response only in C57BL/6 mice. The combined use of polymuramil with pyrogenalum significantly increased both the humoral and cellular response of mice to all recombinant HCV proteins. CONCLUSION: Different immunogenic properties and different functions of recombinant non-structural HCV proteins indicate the feasibility of their combined inclusion in subunit vaccines. It was established for the first time that immunization with HCV proteins with a complex adjuvant (polymuramyl + pyrogenalum) has a synergistic effect, significantly exceeding the effect of each of them separately.

Immunization with a multi-antigen targeted DNA vaccine eliminates chemoresistant pancreatic cancer by disrupting tumor-stromal cell crosstalk.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance. METHODS: We used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth. RESULTS: We establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors. CONCLUSIONS: These findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

Recombinant HBsAg-S and RFRP-3 DNA vaccine promotes reproduction hormone secretion in sheep.

RF-amide related peptides (RFRP) have been proposed as critical regulators of gonadotropin secretion in mammals. This study was designed to construct a DNA vaccine and investigate the effect of vaccine encoding RFRP-3 on reproduction physiology in ewe. A recombinant vaccine was constructed using two copies of the RFRP-3 gene and HBsAg-S that generate a fusion protein to induce an immunology response. Results showed this recombinant vaccine could produce a significant antibody titer in the treated animals (P < 0.05). The specific RFRP-3 antibody response induced by the vaccine was detected at week 2 with a peak at week 6 after the initial immunization. Furthermore, we found that ewes inoculated with pVAX-tPA-HBsAg-S-2RFRP-asd vaccine significantly raised the concentration of GnRH, LH and E2 in serum compared to the control group. LH and E2 concentration in the treated ewes (Group T) was significantly higher than that in control ewes (Group C) at weeks 10, 12 and 14 after the initial immunization, respectively (P < 0.05). Therefore, RFRP-3 can be used as a target for DNA immunization to promote reproductive hormone secretion in ewes and RFRP-3 gene immunization might be a candidate tool to regulate mammal reproduction.

DNA vaccine dual-expressing viral hemorrhagic septicemia virus glycoprotein and C-C motif chemokine ligand 19 induces the expression of immune-related genes in zebrafish (Danio rerio).

Glycoprotein (G protein)-based DNA vaccines are effective in protecting aquaculture fish from rhabdoviruses but the degree of immune response they elicit depends on plasmid concentration and antigen cassette. Here, we developed a DNA vaccine using the viral hemorrhagic septicemia virus G (VG) gene and chemokine (C-C motif) ligand 19 (CCL19)a.2 regulated by the CMV promoter as the molecular adjuvant. After transfection of the prepared plasmid (pVG + CCL19) into epithelioma papulosum cyprini cells, mRNA expression was confirmed through quantitative real-time polymerase chain reaction. The vaccine was intramuscularly injected into zebrafish (Danio rerio), and 28 days after immunization, viral hemorrhagic septicemia virus (105 TCID50/10 µl/fish) was intraperitoneally injected. A survival rate of 68% was observed in the pVG + CCL19 group but this was not significantly different from the survival rate of fish treated with pVG alone, that is, without the adjuvant. However, the expression of interferon- and cytokine-related genes in the spleen and kidney tissues of zebrafish was significantly increased (p < 0.05) on days 1, 3, 7, and 14 after immunization. Thus, CCL19a.2 induced an initial immune response as a molecular adjuvant, which may provide initial protection against virus infection before vaccination-induced antibody formation. This study provides insights on the functions of CCL19a.2 adjuvant in DNA vaccines.

ATP stabilised and sensitised calcium phosphate nanoparticles as effective adjuvants for a DNA vaccine against cancer.

Cancer vaccines based on DNA encoding oncogenes have shown great potential in preclinical studies. However, the efficacy of DNA vaccines is limited by their weak immunogenicity because of low cellular internalisation and insufficient activation of dendritic cells (DCs). Calcium phosphate (CP) nanoparticles (NPs) are biodegradable vehicles with low toxicity and high loading capacity of DNA but suffer from stability issues. Here we employed adenosine triphosphate (ATP) as a dual functional agent, i.e. stabiliser for CP and immunological adjuvant, and applied the ATP-modified CP (ACP) NPs to the DNA vaccine. ACP NP-enhanced cellular uptake and improved transfection efficiency of DNA vaccine, and further showed the ability to activate DCs that are critical for them to prime T cells in cancer immunotherapy. As a result, a higher level of antigen-specific antibody with stronger tumour growth inhibition was achieved in mice immunised with the ACP-DNA vaccine. Overall, this one-step synthesised ACP NPs are an efficient nano-delivery system and nano-adjuvant for cancer DNA vaccines.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

HPV-16 E6/E7 DNA tattoo vaccination using genetically optimized vaccines elicit clinical and immunological responses in patients with usual vulvar intraepithelial neoplasia (uVIN): a phase I/II clinical trial.

BACKGROUND: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis. METHOD: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays. RESULTS: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response. CONCLUSIONS: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit. TRIAL REGISTRATION NUMBER: NTR4607.

Synthetic multiepitope neoantigen DNA vaccine for personalized cancer immunotherapy.

Neoantigen-based personalized vaccination has emerged as a viable method for tumor immunotherapy. Here we set up a DNA-based neoantigen vaccine platform with comprehensive identification of individual somatic mutations using whole-exome sequencing (WES) and RNA-seq, bioinformatic prediction of neo-epitopes, dendritic cell (DC)-based efficacy prevalidation of vaccine candidates, optimization of the DNA vaccine and its nanocarrier and adjuvant, and preparation of a liposome-encapsulated multiepitope DNA vaccine. The DNA vaccine was efficiently uptaken by DCs and induced effective immune response against mouse melanoma cells, leading to significant inhibition of melanoma tumor growth and reduction of lung metastasis in a mouse model. Numerous intratumoral infiltrated CD8+ T-cells with specific in vitro killing ability towards melanoma cells were identified. Our study offers evidence that a multiepitope neoantigen DNA vaccine in a nanocarrier can be exploited for personalized tumor immunotherapy and as a reliable prevalidation approach for rapid enrichment of effective neoantigens.

A minigene DNA vaccine encoding peptide epitopes derived from Galectin-1 has protective antitumoral effects in a model of neuroblastoma.

We recently identified Galectin-1 (Gal-1), a ß-galactoside-binding lectin, as a novel immune regulator in neuroblastoma (NB). Here, we characterized the tolerogenic function of Gal-1 within the CD8+ T cell compartment and further evaluated its relevance as an antigen for effective DNA vaccination against NB in a mouse model. NB cells with Gal-1 knockdown (NXS-2L) exhibited significantly reduced tumor growth compared to NXS-2 NB cells. Administration of anti-CD8 antibodies prevented this antitumor effect, with primary tumor growth comparable to that from Gal-1 (G1)-sufficient NB cells. Peptide epitope screening with online databases and in silico docking experiments predicted the sequences "FDQADLTI" (#1), "GDFKIKCV" (#2), and "AHGDANTI" (#3) to have superior H2-KK binding affinities and "KFPNRLNM" (#4), "DGDFKIKCV" (#5), and "LGKDSNNL" (#6) to have superior H2-DD binding affinities. Minigenes encoding G1-KK (#1-#2-#3), G1-DD (#4-#5-#6) and the triplet with the highest affinity, G1-H (#1-#2-#4), were generated and cloned into a ubiquitin-containing plasmid (pU). Mice receiving pU-G1-KK or pU-G-1H presented a reduction in the s.c. tumor volume and weight of up to 80% compared to control mice; this reduction was associated with increased cytotoxicity of isolated splenocytes from vaccinated animals. Vaccination with pUG1-DD showed a lower capability to suppress primary tumor progression. In conclusion, Gal-1 expression by NB negatively regulates CD8+ T cells. Vaccination with DNA plasmids encoding Gal-1 epitopes overcomes immune escape, enhances CD8+ T cell-dependent immunity and displays effective antitumor activity against NB.

Current Update on Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine Development with a Special Emphasis on Gene Therapy Viral Vector Design and Construction for Vaccination.

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease (COVID-19) caused by the novel coronavirus SARS-coronavirus 2 (CoV-2). To combat the devastating spread of SARS-CoV-2, extraordinary efforts from numerous laboratories have focused on the development of effective and safe vaccines. Traditional live-attenuated or inactivated viral vaccines are not recommended for immunocompromised patients as the attenuated virus can still cause disease via phenotypic or genotypic reversion. Subunit vaccines require repeated dosing and adjuvant use to be effective, and DNA vaccines exhibit lower immune responses. mRNA vaccines can be highly unstable under physiological conditions. On the contrary, naturally antigenic viral vectors with well-characterized structure and safety profile serve as among the most effective gene carriers to provoke immune response via heterologous gene transfer. Viral vector-based vaccines induce both an effective cellular immune response and a humoral immune response owing to their natural adjuvant properties via transduction of immune cells. Consequently, viral vectored vaccines carrying the SARS-CoV-2 spike protein have recently been generated and successfully used to activate cytotoxic T cells and develop a neutralizing antibody response. Recent progress in SARS-CoV-2 vaccines, with an emphasis on gene therapy viral vector-based vaccine development, is discussed in this review.

New generation of DNA-based immunotherapy induces a potent immune response and increases the survival in different tumor models.

BACKGROUND: Strategies to increase nucleic acid vaccine immunogenicity are needed to move towards clinical applications in oncology. In this study, we designed a new generation of DNA vaccines, encoding an engineered vesicular stomatitis virus glycoprotein as a carrier of foreign T cell tumor epitopes (plasmid to deliver T cell epitopes, pTOP). We hypothesized that pTOP could activate a more potent response compared with the traditional DNA-based immunotherapies, due to both the innate immune properties of the viral protein and the specific induction of CD4 and CD8 T cells targeting tumor antigens. This could improve the outcome in different tumor models, especially when the DNA-based immunotherapy is combined with a rational therapeutic strategy. METHODS: The ability of pTOP DNA vaccine to activate a specific CD4 and CD8 response and the antitumor efficacy were tested in a B16F10-OVA melanoma (subcutaneous model) and GL261 glioblastoma (subcutaneous and orthotopic models). RESULTS: In B16F10-OVA melanoma, pTOP promoted immune recognition by adequate processing of both MHC-I and MHC-II epitopes and had a higher antigen-specific cytotoxic T cell (CTL) killing activity. In a GL261 orthotopic glioblastoma, pTOP immunization prior to tumor debulking resulted in 78% durable remission and long-term survival and induced a decrease of the number of immunosuppressive cells and an increase of immunologically active CTLs in the brain. The combination of pTOP with immune checkpoint blockade or with tumor resection improved the survival of mice bearing, a subcutaneous melanoma or an orthotopic glioblastoma, respectively. CONCLUSIONS: In this work, we showed that pTOP plasmids encoding an engineered vesicular stomatitis virus glycoprotein, and containing various foreign T cell tumor epitopes, successfully triggered innate immunity and effectively promoted immune recognition by adequate processing of both MHC-I and MHC-II epitopes. These results highlight the potential of DNA-based immunotherapies coding for viral proteins to induce potent and specific antitumor responses.

Combination of vasculature targeting, hypofractionated radiotherapy, and immune checkpoint inhibitor elicits potent antitumor immune response and blocks tumor progression.

BACKGROUND: Tumor endothelial marker 1 (TEM1) is a protein expressed in the tumor-associated endothelium and/or stroma of various types of cancer. We previously demonstrated that immunization with a plasmid-DNA vaccine targeting TEM1 reduced tumor progression in three murine cancer models. Radiation therapy (RT) is an established cancer modality used in more than 50% of patients with solid tumors. RT can induce tumor-associated vasculature injury, triggering immunogenic cell death and inhibition of the irradiated tumor and distant non-irradiated tumor growth (abscopal effect). Combination treatment of RT with TEM1 immunotherapy may complement and augment established immune checkpoint blockade. METHODS: Mice bearing bilateral subcutaneous CT26 colorectal or TC1 lung tumors were treated with a novel heterologous TEM1-based vaccine, in combination with RT, and anti-programmed death-ligand 1 (PD-L1) antibody or combinations of these therapies, tumor growth of irradiated and abscopal tumors was subsequently assessed. Analysis of tumor blood perfusion was evaluated by CD31 staining and Doppler ultrasound imaging. Immunophenotyping of peripheral and tumor-infiltrating immune cells as well as functional analysis was analyzed by flow cytometry, ELISpot assay and adoptive cell transfer (ACT) experiments. RESULTS: We demonstrate that addition of RT to heterologous TEM1 vaccination reduces progression of CT26 and TC1 irradiated and abscopal distant tumors as compared with either single treatment. Mechanistically, RT increased major histocompatibility complex class I molecule (MHCI) expression on endothelial cells and improved immune recognition of the endothelium by anti-TEM1 T cells with subsequent severe vascular damage as measured by reduced microvascular density and tumor blood perfusion. Heterologous TEM1 vaccine and RT combination therapy boosted tumor-associated antigen (TAA) cross-priming (ie, anti-gp70) and augmented programmed cell death protein 1 (PD-1)/PD-L1 signaling within CT26 tumor. Blocking the PD-1/PD-L1 axis in combination with dual therapy further increased the antitumor effect and gp70-specific immune responses. ACT experiments show that anti-gp70 T cells are required for the antitumor effects of the combination therapy. CONCLUSION: Our findings describe novel cooperative mechanisms between heterologous TEM1 vaccination and RT, highlighting the pivotal role that TAA cross-priming plays for an effective antitumor strategy. Furthermore, we provide rationale for using heterologous TEM1 vaccination and RT as an add-on to immune checkpoint blockade as triple combination therapy into early-phase clinical trials.

Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve.

BACKGROUND: Different types of tumors have varying susceptibility to immunotherapy and hence require different treatment strategies; these cover a spectrum ranging from 'hot' tumors or those with high mutational burden and immune infiltrates that are more amenable to targeting to 'cold' tumors that are more difficult to treat due to the fewer targetable mutations and checkpoint markers. We hypothesized that an effective anti-tumor response requires multiple agents that would (1) engage the immune response and generate tumor-specific effector cells; (2) expand the number and breadth of the immune effector cells; (3) enable the anti-tumor activity of these immune cells in the tumor microenvironment; and (4) evolve the tumor response to widen immune effector repertoire. METHODS: A hexatherapy combination was designed and administered to MC38-CEA (warm) and 4T1 (cool) murine tumor models. The hexatherapy regimen was composed of adenovirus-based vaccine and IL-15 (interleukin-15) superagonist (N-803) to engage the immune response; anti-OX40 and anti-4-1BB to expand effector cells; anti-PD-L1 (anti-programmed death-ligand 1) to enable anti-tumor activity; and docetaxel to promote antigen spread. Primary and metastatic tumor growth inhibition were measured. The generation of anti-tumor immune effector cells was analyzed using flow cytometry, ELISpot (enzyme-linked immunospot), and RNA analysis. RESULTS: The MC38-CEA and 4T1 tumor models have differential sensitivities to the combination treatments. In the 'warm' MC38-CEA, combinations with two to five agents resulted in moderate therapeutic benefit while the hexatherapy regimen outperformed all these combinations. On the other hand, the hexatherapy regimen was required in order to decrease the primary and metastatic tumor burden in the 'cool' 4T1 model. In both models, the hexatherapy regimen promoted CD4+ and CD8+ T cell proliferation and activity. Furthermore, the hexatherapy regimen induced vaccine-specific T cells and stimulated antigen cascade. The hexatherapy regimen also limited the immunosuppressive T cell and myeloid derived suppressor cell populations, and also decreased the expression of exhaustion markers in T cells in the 4T1 model. CONCLUSION: The hexatherapy regimen is a strategic combination of immuno-oncology agents that can engage, expand, enable, and evolve the immune response and can provide therapeutic benefits in both MC38-CEA (warm) and 4T1 (cool) tumor models.

Broad Neutralization Responses Against Oncogenic Human Papillomaviruses Induced by a Minor Capsid L2 Polytope Genetically Incorporated Into Bacterial Ferritin Nanoparticles.

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.

In pancreatic cancer, chemotherapy increases antitumor responses to tumor-associated antigens and potentiates DNA vaccination.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is an almost incurable tumor that is mostly resistant to chemotherapy (CT). Adaptive immune responses to tumor-associated antigens (TAA) have been reported, but immunotherapy (IT) clinical trials have not yet achieved any significant increase in survival, confirming the suppressive environment of PDA. As CT has immune-modulating properties, we investigated the effect of gemcitabine (GEM) in antitumor effector responses to TAA in patients with PDA. METHODS: The IgG antibody repertoire in patients with PDA before and after CT was profiled by serological proteome analysis and ELISA and their ability to activate complement-dependent cytotoxicity (CDC) was measured. Peripheral T cells were stimulated in vitro with recombinant TAA, and specific proliferation, IFN-γ/IL-10 and CD8+/Treg ratios were measured. Mice that spontaneously developed PDA were treated with GEM and inoculated with an ENO1 (α-Enolase) DNA vaccine. In some experimental groups, the effect of depleting CD4, CD8 and B cells by specific antibodies was also evaluated. RESULTS: CT increased the number of TAA recognized by IgG and their ability to activate CDC. Evaluation of the IFN-γ/IL-10 ratio and CD8+/Treg ratios revealed that CT treatment shifted T cell responses to ENO1, G3P (glyceraldheyde-3-phosphate dehydrogenase), K2C8 (keratin, type II cytoskeletal 8) and FUBP1 (far upstream binding protein 1), four of the most recognized TAA, from regulatory to effector. In PDA mice models, treatment with GEM prior to ENO1 DNA vaccination unleashed CD4 antitumor activity and strongly impaired tumor progression compared with mice that were vaccinated or GEM-treated alone. CONCLUSIONS: Overall, these data indicate that, in PDA, CT enhances immune responses to TAA and renders them suitable targets for IT.

A DNA-Launched Nanoparticle Vaccine Elicits CD8+ T-cell Immunity to Promote In Vivo Tumor Control.

Cytolytic T cells (CTL) play a pivotal role in surveillance against tumors. Induction of CTL responses by vaccination may be challenging, as it requires direct transduction of target cells or special adjuvants to promote cross-presentation. Here, we observed induction of robust CTL responses through electroporation-facilitated, DNA-launched nanoparticle vaccination (DLnano-vaccines). Electroporation was observed to mediate transient tissue apoptosis and macrophage infiltration, which were deemed essential to the induction of CTLs by DLnano-vaccines through a systemic macrophage depletion study. Bolus delivery of protein nano-vaccines followed by electroporation, however, failed to induce CTLs, suggesting direct in vivo production of nano-vaccines may be required. Following these observations, new DLnano-vaccines scaffolding immunodominant melanoma Gp100 and Trp2 epitopes were designed and shown to induce more potent and consistent epitope-specific CTL responses than the corresponding DNA monomeric vaccines or CpG-adjuvanted peptide vaccines. DNA, but not recombinant protein, nano-vaccinations induced CTL responses to these epitopes and suppressed melanoma tumor growth in mouse models in a CD8+ T-cell-dependent fashion. Further studies to explore the use of DLnano-vaccines against other cancer targets and the biology with which they induce CTLs are important.

A multiple T cell epitope comprising DNA vaccine boosts the protective efficacy of Bacillus Calmette-Guérin (BCG) against Mycobacterium tuberculosis.

BACKGROUND: Approximately 80% - 90% of individuals infected with latent Mycobacterium tuberculosis (Mtb) remain protected throughout their life-span. The release of unique, latent-phase antigens are known to have a protective role in the immune response against Mtb. Although the BCG vaccine has been administered for nine decades to provide immunity against Mtb, the number of TB cases continues to rise, thereby raising doubts on BCG vaccine efficacy. The shortcomings of BCG have been associated with inadequate processing and presentation of its antigens, an inability to optimally activate T cells against Mtb, and generation of regulatory T cells. Furthermore, BCG vaccination lacks the ability to eliminate latent Mtb infection. With these facts in mind, we selected six immunodominant CD4 and CD8 T cell epitopes of Mtb expressed during latent, acute, and chronic stages of infection and engineered a multi-epitope-based DNA vaccine (C6). RESULT: BALB/c mice vaccinated with the C6 construct along with a BCG vaccine exhibited an expansion of both CD4 and CD8 T cell memory populations and augmented IFN-γ and TNF-α cytokine release. Furthermore, enhancement of dendritic cell and macrophage activation was noted. Consequently, illustrating the elicitation of immunity that helps in the protection against Mtb infection; which was evident by a significant reduction in the Mtb burden in the lungs and spleen of C6 + BCG administered animals. CONCLUSION: Overall, the results suggest that a C6 + BCG vaccination approach may serve as an effective vaccination strategy in future attempts to control TB.

Ovarian granulosa cell tumor characterization identifies FOXL2 as an immunotherapeutic target.

Granulosa cell tumors (GCT) are rare ovarian malignancies. Due to the lack of effective treatment in late relapse, there is a clear unmet need for novel therapies. Forkhead Box L2 (FOXL2) is a protein mainly expressed in granulosa cells (GC) and therefore is a rational therapeutic target. Since we identified tumor infiltrating lymphocytes (TILs) as the main immune population within GCT, TILs from 11 GCT patients were expanded, and their phenotypes were interrogated to determine that T cells acquired late antigen-experienced phenotypes and lower levels of PD1 expression. Importantly, TILs maintained their functionality after ex vivo expansion as they vigorously reacted against autologous tumors (100% of patients) and against FOXL2 peptides (57.1% of patients). To validate the relevance of FOXL2 as a target for immune therapy, we developed a plasmid DNA vaccine (FoxL2-tetanus toxin; FoxL2-TT) by fusing Foxl2 cDNA with the immune-enhancing domain of TT. Mice immunization with FoxL2-TT controlled growth of FOXL2-expressing ovarian (BR5) and breast (4T1) cancers in a T cell-mediated manner. Combination of anti-PD-L1 with FoxL2-TT vaccination further reduced tumor progression and improved mouse survival without affecting the female reproductive system and pregnancy. Together, our results suggest that FOXL2 immune targeting can produce substantial long-term clinical benefits. Our study can serve as a foundation for trials testing immunotherapeutic approaches in patients with ovarian GCT.