Therapeutic Immunization Research Articles

Overcoming resistance to immune checkpoint blockade with RNA-loaded nanoparticles.

117 Background: While checkpoint blockade has shown promising survival benefits in patients with solid tumors, immune escape through loss of MHCI expression juxtaposed to an immunoregulatory milieu remain significant hurdles. To overcome these limitations, we developed a novel treatment platform, which leverages the use of commercially available and clinically translatable nanoparticles (NPs) that can be combined with tumor-derived RNA to activate systemic immunity and re-program the intratumoral microenvironment from a regulatory into an immune activated locale. Methods: Since local vaccination strategies are mired with poor immunogenicity, we assessed if i.v. delivery of tumor-derived RNA encapsulated in lipophilic NPs could activate peripheral and intratumoral antigen presenting cells (APCs) for induction of therapeutic anti-tumor immunity in pre-clinical murine melanoma models. Results: We identified a clinically translatable NP formulation that when administered intravenously, mediates release of inflammatory cytokines (i.e. CCL2, IFN-alpha) into serum, systemically activates host APCs in reticuloendothelial organs, and induces precipitous upregulation of MHCI and immune activation markers (i.e. CD86) within the tumor microenvironment. Both model-antigen encoding RNA and physiologically-relevant tumor-derived RNA, when encapsulated in NPs, could expand potent anti-tumor T-cell immunity. We demonstrated that RNA-NPs harness the anti-viral defense mechanism against tumor antigens in a type I interferon dependent manner, and can be further engineered to deliver combinatorial therapies by co-encapsulating mRNAs encoding for immunomodulatory molecules (i.e. HCV PAMPs, GM-CSF). In a pre-clinical melanoma model, RNA-NPs mediate anti-tumor efficacy and significantly enhance activity of immune checkpoint mAbs when used in combination. Conclusions: By employing a systemic RNA-NP formulation encoding for both tumor RNAs and immunomodulatory molecules, as an innovative and versatile platform for delivering combinatorial therapeutics via a single treatment modality, this platform can be harnessed to simultaneously target tumor antigens and re-program the intratumoral microenvironment

Abstract B28: Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective FcγR-engagement

Abstract The use of immunomodulatory mAbs to stimulate effective therapeutic anticancer immunity has shown clinical successes for the treatment of an increasing number of cancer types. An approach that is currently under clinical development uses agonistic anti-CD40 mAbs to induce APC activation, thereby harnessing the potency of the immune response to eradicate tumors. While engagement of the inhibitory Fcγ Receptor(FγγR)IIB is required for the in vivo agonistic activity of anti-mouse CD40 mAbs, a similar requirement for human mAbs has been disputed based on in vitro studies and in vivo studies performed either in wild-type or mouse FcγR knockout strains and thus fails to address the unique binding affinities and tissue distribution of human FcγRs. To overcome these limitations we generated and used unique mouse model fully humanized for its FcγRs and CD40, and revealed that FcγRIIB engagement is also essential for the activity of the human CD40 agonistic mAbs, while engagement of the activating FcγRIIA inhibits their activity. By engineering Fc variants of human mAbs with selective enhancement to FcγRIIB but not to FcγRIIA, significant improvement of antitumor immunity was observed in multiple tumor models. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using pre-clinical model that accurately reflects the unique affinities and cellular expression of human FcRs, thus providing a development approach for therapeutic human antibodies based on their requirement for selective FcγR interactions. Here we describe the selection of optimized next-generation Fc-engineered agonistic CD40 mAbs. Citation Format: Rony Dahan, Bryan C. Barnhart, Alan J. Korman, Jeffrey V. Ravetch. Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective FcγR-engagement. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B28.

Dendritic Cell Targeting Vaccine for HPV-Associated Cancer

Dendritic cells (DCs) are major antigen presenting cells that can efficiently prime and activate cellular immune responses. Delivering antigens to in vivo DCs has thus been considered as a promising strategy that could allow us to mount T cell-mediated therapeutic immunity against cancers in patients. Successful development of such types of cancer vaccines that can target in vivo DCs, however, requires a series of outstanding questions that need to be addressed. These include the proper selection of which DC surface receptors, specific DC subsets and DC activators that can further enhance the efficacy of vaccines by promoting effector T cell infiltration and retention in tumors and their actions against tumors. Supplementing these areas of research with additional strategies that can counteract tumor immune evasion mechanisms is also expected to enhance the efficacy of such therapeutic vaccines against cancers. After more than a decade of study, we have concluded that antigen targeting to DCs via CD40 to evoke cellular responses is more efficient than targeting antigens to the same types of DCs via eleven other DC surface receptors tested. In recent work, we have further demonstrated that a prototype vaccine (anti-CD40-HPV16.E6/7, a recombinant fusion protein of anti-human CD40 and HPV16.E6/7 protein) for HPV16-associated cancers can efficiently activate HPV16.E6/7-specific T cells, particularly CD8+ T cells, from the blood of HPV16+ head-and-neck cancer patients. Moreover, anti-CD40-HPV16.E6/7 plus poly(I:C) can mount potent therapeutic immunity against TC-1 tumor expressing HPV16.E6/7 protein in human CD40 transgenic mice. In this manuscript, we thus highlight our recent findings for the development of novel CD40 targeting immunotherapeutic vaccines for HPV16-associated malignancies. In addition, we further discuss several of key questions that still remain to be addressed for enhancing therapeutic immunity elicited by our prototype vaccine against HPV16-associated malignancies.

The Tat Protein Enhances CTL Responses and Therapeutic Immunity of Gag-Specific Exosome-Targeted T Cell-Based Gag/Tat-Texo Vaccine in Transgenic HLA-A2 Mice

Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8+T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tat- and Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naive mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also significantly lower than that (78/lung) observed in Gag-Texo-immunized mice. Taken together, this indicates that HIV-1 Gag/Tat-Texo capable of stimulating enhanced Gag-specific CTL responses and therapeutic immunity may become a new immunotherapeutic vaccine candidate for controlling virus in HIV-1 patients.

Cancer Vaccine Therapy Using Tumor Endothelial Cells as Antigens Suppresses Solid Tumor Growth and Metastasis.

Tumor angiogenesis plays an important role in tumor growth and metastasis, with tumor cells requiring nutrients and oxygen via blood flow for their proliferation. In comparison, angiogenesis also occurs under normal physiological conditions, such as wound healing and in the formation of the corpus luteum. Herein, we report on the development of a novel dendritic cell (DC) vaccine therapy using tumor endothelial cells (TECs) derived from tumor vessels as tumor antigens. After density gradient centrifugation and the detection of angiotensin-converting enzyme activities, a TEC-rich fraction was separated from solid tumor tissues. Prophylactic or therapeutic immunization using DCs pulsed with TECs as vaccine antigens significantly suppressed solid tumor growth in a Colon-26 colorectal adenocarcinoma tumor-bearing mouse model, compared with the use of tumor cells as DC vaccine antigens. Tumor tissues showed reduced angiogenesis. However, vaccination using DCs pulsed with TECs did not inhibit physiological angiogenesis as evidenced by a wound healing assay. Additionally, in a B16/BL6 mouse melanoma lung metastasis model, DC vaccination using TECs derived not only from the same tumor tissue but from a different type of tumor also suppressed metastasis. These results thus show that cancer vaccine therapy targeting TECs is an effective therapy against angiogenesis in several types of cancer, but does not affect normal blood vessel growth.

Improving the potency of DNA vaccine encoding HIV-1 Nef antigen using two endogenous adjuvants in mouse model.

DNA immunization can induce long-term immune responses, which are required to design an effective HIV vaccine. It was shown that antigen-expressing plasmids can increase the protective immunity against infectious diseases such as: influenza and malaria. However, DNA-based immunizations have poor immunogenicity, thus the use of potent immunoadjuvants can enhance their potency. In the current study, preparation of the recombinant HIV-1 Nef, Gp96 and HMGB1 DNA constructs was performed in bacterial system. Then, the immunogenicity of DNA construct harboring HIV-1 Nef gene (pcDNA-Nef) was studied using two endogenous adjuvants (pcDNA-HMGB1 and pcDNA-Gp96) in BALB/c mouse model. Our data showed that co-injection of pcDNA-Nef with pcDNA-HMGB1 effectively raised both humoral and cell-mediated immune responses in mice as compared to pcDNA-Nef adjuvanted with pcDNA-gp96. Indeed, co-immunization of HIV-1 Nef DNA with HMGB1 DNA significantly induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also cytotoxic T lymphocytes (CTLs) activity in comparison with other immunized groups. These findings suggest that the full length of HMGB1 gene could be a more efficient adjuvant for improvement of therapeutic HIV DNA-based immunization compared to the full length of gp96 gene (Tab. 1, Fig. 3, Ref. 58).

Selection of epitopes from self-antigens for eliciting Th2 or Th1 activity in the treatment of autoimmune disease or cancer.

Vaccines have been valuable tools in the prevention of infectious diseases, and the rapid development of new vectors against constantly mutating foreign antigens in viruses such as influenza has become a regular, seasonal exercise. Harnessing the immune response against self-antigens is not necessarily analogous or as achievable by iterative processes, and since the desired outcome includes leaving the targeted organism intact, requires some precision engineering. In vaccine-based treatment of autoimmunity and cancer, the proper selection of antigens and generation of the desired antigen-specific therapeutic immunity has been challenging. Both cases involve a threshold of existing, undesired immunity that must be overcome, and despite considerable academic and industry efforts, this challenge has proven to be largely refractory to vaccine approaches leveraging enhanced vectors, adjuvants, and administration strategies. There are in silico approaches in development for predicting the immunogenicity of self-antigen epitopes, which are being validated slowly. One simple approach showing promise is the functional screening of self-antigen epitopes for selective Th1 antitumor immunogenicity, or inversely, selective Th2 immunogenicity for treatment of autoimmune inflammation. The approach reveals the importance of confirming both Th1 and Th2 components of a vaccine immunogen; the two can confound one another if not parsed but may be used individually to modulate antigen-specific inflammation in autoimmune disease or cancer.

In Situ Vaccination with a TLR9 Agonist and Anti-OX40 Antibody Leads to Tumor Regression and Induces Abscopal Responses in Murine Lymphoma

Background:Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns on bacterial, fungal, or viral pathogens. Intratumoral (IT) injection of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 agonist, results in local tumor eradication but on its own is not able to induce a systemic anti-tumor immune response. OX40 is a potent costimulatory receptor that can potentiate the action of conventional T cells leading to their proliferation, effector function and survival, but can also inhibit or kill T regulatory cells by ADCC. In previous preclinical studies systemic injection of OX40 agonists increased antitumor immunity and improved survival.Scientific question:Does local injection of both CpG and low doses of an anti-OX40 agonistic antibody trigger a systemic anti-tumor immune response?Results:We implanted the A20 lymphoma tumor bilaterally on opposite sides of the abdomen. After tumors were established we administered microgram quantities of CpG and anti-OX40 antibody into the tumor on one side and monitored both the injected and the uninjected tumor sites. This treatment resulted in both a local and an abscopal effect on the contralateral, untreated tumor. In addition, the animals were protected from a second challenge with A20 cells. This anti-tumor effect was T cell dependent, since depletion of either CD4+ or CD8+ T cells abrogated the therapeutic effect. There was no evidence of toxicity or autoimmunity in the treated animals.To examine the potential of this maneuver to treat spontaneous, non-transplanted cancers we chose the mouse mammary tumor model- FVB/N-Tg(MMTV-PyVT)634Mul/J. These animals all develop invasive breast cancer tumors in all of their mammary glands by 12 weeks of age. Injections of CpG and anti-OX40 antibody into the first arising tumor not only prevented its growth but significantly reduced the incidence and outgrowth of subsequent tumors at un-injected susceptible mammary glands and reduced the number of lung metastases.Significance:TLR9 agonists and anti-OX40 antibodies are currently under clinical development for cancer treatment. We show here that combining anti-OX40 antibody with a TLR9 agonist at a single established tumor is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at distant sites in both transplantable and spontaneously occurring oncogene-driven murine tumors. This anti-tumor effect was long lasting, specific and required T cells.Impact:Our current results suggest that CpG and anti-OX40 are sufficient to induce fully protective and curative anti-tumor immune responses, even in spontaneously arising cancer. Anti-OX40 and CpG are both currently in phase-I clinical trials as single agents. Our results provide the rationale for testing these agents clinically in combination as described here, injected locally in low doses in order to induce therapeutic anti-lymphoma immunity. DisclosuresLevy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

Clinical and immunological assessment of therapeutic immunization with a subunit vaccine for recurrent ocular canine herpesvirus-1 infection in dogs

Latent canine herpesvirus-1 (CHV-1) infections are common in domestic dogs and reactivation of latent virus may be associated with recurrent ocular disease. The objectives of the present study were to evaluate the ability of a subunit CHV-1 vaccine to stimulate peripheral CHV-1 specific immunity and prevent recurrent CHV-1 ocular disease and viral shedding. Mature dogs with experimentally-induced latent CHV-1 infection received a 2-dose CHV-1 vaccine series. Recurrent ocular CHV-1 infection was induced by corticosteroid administration in the prevaccinal, short-term postvaccinal (2 weeks post-vaccination), and long-term postvacccinal (34 weeks post-vaccination) periods. Immunological, virological, and clinical parameters were evaluated during each study period. Quantitative assessment of peripheral immunity included lymphocyte immunophenotyping, proliferation response, and interferon-γ production; and CHV-1 virus neutralizing antibody production. In the present study, vaccination did not prevent development of ocular disease and viral shedding; however, there was a significant decrease in clinical ocular disease scores in the short-term postvaccinal period. Significant alterations in peripheral immunity detected in the dogs during the short-term and long-term postvaccinal periods included increased T and B lymphocyte subpopulation percentage distributions, increased lymphocyte expression of major histocompatibility complex class I and II, increased CHV-1 virus neutralizing antibody titers, decreased lymphocyte proliferation, and decreased interferon-γ production. Vaccination of latently infected mature dogs with the selected subunit CHV-1 vaccine was not effective in preventing recurrent ocular CHV-1 infection and viral shedding induced by corticosteroid administration. The vaccine did induce long-term CHV-1 specific immunity and may decrease the severity of clinical ocular disease in the immediate postvaccinal period.

Protection of mice against gastric colonization of Helicobacter pylori by therapeutic immunization with systemic whole cell inactivated vaccines

The protective effect of therapeutic immunization with heat inactivated Helicobacter pylori cells administered with aluminum phosphate as an adjuvant was evaluated with “Swiss albino mice” infected with H. pylori Sydney strain 1 (SS1). The presence of bacteria in histological sections of the stomach was evaluated to confirm the colonization of H. pylori. The infection dose was determined to be 1 × 108 cells which resulted to be the optimal concentration to sustain infection for required time. Systemic immunization of H. pylori 26695 and SS1 Whole cell heat inactivated vaccine were induced on mice. The IgG titer levels of high dose adjuvant vaccine of both strains were proportionate on the 7th and 14th day. Subsequently on the 21st and 28th day SS1 high dose adjuvant revealed a higher titer value. The Probability values were <0.0001 which is statistically significant. Moreover, therapeutic immunization with SS1 (WC) vaccine confers efficacious protection against H. pylori infection in mice. These results represent strong evidence for feasibility of therapeutic use of whole cell based vaccine formulations against H. pylori infection in animal model.

Abstract B014: Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective Fc receptor-engagement

Abstract The use of immunomodulatory mAbs to stimulate effective therapeutic anticancer immunity has shown clinical successes for the treatment of an increasing number of cancer types. An approach that is currently under clinical development uses agonistic anti-CD40 mAbs to induce APC activation, thereby harnessing the potency of the immune response to eradicate tumors. While engagement of the inhibitory Fcγ Receptor(FcγR)IIB is required for the in vivo agonistic activity of anti-mouse CD40 mAbs, a similar requirement for human mAbs has been disputed based on in vitro studies and in vivo studies performed either in wild-type or mouse FcγR knockout strains and thus fails to address the unique binding affinities and tissue distribution of human FcγRs. To overcome these limitations we generated and used unique mouse model fully humanized for its FcγRs and CD40, and revealed that FcγRIIB engagement is also essential for the activity of the human CD40 agonistic mAbs, including CP-870,893, while engagement of the activating FcγRIIA inhibits their activity. By engineering Fc variants of human mAbs with selective enhancement to FcγRIIB but not to FcγRIIA, significant improvement of antitumor immunity was observed in multiple tumor models. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using an appropriate pre-clinical model that accurately reflects the unique affinities and cellular expression of human FcRs, thus providing a development approach for therapeutic human antibodies based on their requirement for selective FcγR interactions. Here we describe the selection of optimized next-generation Fc-engineered agonistic CD40 mAbs. Citation Format: Rony Dahan, Jeffrey V. Ravetch. Improved therapeutic activity of agonistic, human anti-CD40 monoclonal Abs by selective Fc receptor-engagement [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B014.

Therapeutic HPV Cancer Vaccine Targeted to CD40 Elicits Effective CD8+ T-cell Immunity.

Human papillomavirus (HPV), particularly HPV16 and HPV18, can cause cancers in diverse anatomical sites, including the anogenital and oropharyngeal (throat) regions. Therefore, development of safe and clinically effective therapeutic vaccines is an important goal. Herein, we show that a recombinant fusion protein of a humanized antibody to CD40 fused to HPV16.E6/7 (αCD40-HPV16.E6/7) can evoke HPV16.E6/7-specific CD8+ and CD4+ T-cell responses in head-and-neck cancer patients in vitro and in human CD40 transgenic (hCD40Tg) mice in vivo The combination of αCD40-HPV16.E6/7 and poly(I:C) efficiently primed HPV16.E6/7-specific T cells, particularly CD8+ T cells, in hCD40Tg mice. Inclusion of montanide enhanced HPV16.E6/7-specific CD4+, but not CD8+, T-cell responses. Poly(I:C) plus αCD40-HPV16.E6/7 was sufficient to mount both preventative and therapeutic immunity against TC-1 tumors in hCD40Tg mice, significantly increasing the frequency of HPV16-specific CD8+ CTLs in the tumors, but not in peripheral blood. In line with this, tumor volume inversely correlated with the frequency of HPV16.E6/7-specific CD8+ T cells in tumors, but not in blood. These data suggest that CD40-targeting vaccines for HPV-associated malignancies can provide a highly immunogenic platform with a strong likelihood of clinical benefit. Data from this study offer strong support for the development of CD40-targeting vaccines for other cancers in the future. Cancer Immunol Res; 4(10); 823-34. ©2016 AACR.

Novel paradigm for immunotherapy of breast cancer by engaging prophylactic immunity against hepatitis B.

BackgroundImmunotherapy of patients suffering from the human epidermal growth factor receptor 2 overexpressing (HER-2+) breast cancers with the anti-HER-2 antibodies results in increase of the patients’ overall survival. However, no prophylactic vaccine is available against HER-2+ breast cancers. Although, prophylactic vaccine for human hepatitis B virus (HBV) is very effective.Specific aimThe specific aim of this work was to design, synthesize, and test bio-molecules which would engage prophylactic immunity against hepatitis B virus towards killing breast cancers cells.Methods and ResultsBy biomolecular engineering, we have created a novel family of biomolecules: antibody (anti-HER-2) × vaccine (HBsAg) engineered constructs (AVEC: anti-HER-2 × HBsAg). These biomolecules were utilized for redirecting, accelerating, and amplifying of the vaccination-induced, prophylactic immunity originally targeted against HBV as therapeutic immunity, newly targeted against HER-2+ breast cancers. Treatment of the HER-2+ breast cancer cells with AVEC: anti-HER-2 × HBsAg in blood of the patients, vaccinated with HBsAg, rapidly increased efficacy of killing of HER-2+ breast cancer cells over that attained with the naked anti-HER-2 antibodies.ConclusionNovel antibody-vaccine engineered constructs (AVEC) facilitate redirecting, accelerating, and amplifying of prophylactic, HBV vaccination-induced immunity as immunotherapy (RAAVIIT) of HER-2+ breast cancer. We currently streamline this novel therapeutic paradigm into clinical trials of breast and other cancers.

MPLA-coated hepatitis B virus surface antigen (HBsAg) nanocapsules induce vigorous T cell responses in cord blood derived human T cells

Chronic hepatitis B virus (HBV) infection is the most prevalent serious liver infection in the world. A frequent route of infection represents mother-to-child transmission. Efficient control of HBV replication depends on antigen-specific cellular immune response mediated by dendritic cells (DCs). Aim of the present study was to evaluate optimized adjuvant combinations, efficiently maturing monocyte-derived neonatal and adult dendritic cells (moDCs). In addition, the potential of polymeric HBsAg-nanocapsules (HBsAg-NCs) was investigated regarding up-take by moDCs and the subsequent induction of specific T cell responses in a human co-culture model. Simultaneous stimulation of moDCs with MPLA and IFNγ induced up-regulation of CD80 and HLA-DR along with vigorous secretion of IL-12p70. MPLA-coating of HBsAg-NCs promoted NCs-uptake by moDCs. Finally, MPLA-HBsAg-NCs-pulsed moDCs with IFNγ increased T cell proliferation and induced antigen-specific IFNγ release by T cells. The herein presented vaccine approach provides a rational for neonatal and therapeutic immunization strategies against HBV.

Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity

Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP) and dense granule (GRA) proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ) TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM) associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately control the development of host immune responses that provide effective antitumor immunity against established ovarian cancer.

Abstract 551: In situ vaccination with TLR9 agonist and anti-Ox40 antibody is sufficient to induce abscopal responses even in mice with spontaneous oncogene-driven tumors

Abstract Background: Toll-like receptors (TLRs) are components of the innate immune system that recognize pathogen-associated molecular patterns on bacterial, fungal, or viral pathogens. Intratumoral (IT) injection of unmethylated CG-enriched oligodeoxynucleotides (CpG), a TLR9 agonist, results in local tumor eradication but on its own is not able to induce a systemic anti-tumor immune response. OX40 is a potent costimulatory receptor that can potentiate the action of conventional T cells leading to their proliferation, effector function and survival, but can also inhibit or kill T regulatory cells by ADCC. In preclinical studies OX40 agonists increased antitumor immunity and improve tumor-free survival. Scientific question: Does local injection of a CpG with anti-OX40 agonistic antibody trigger a systemic anti-tumor immune response? Results: Using transplantable syngeneic tumor models, we implanted the tumor bilaterally in opposite sides of the abdomen. After tumors were established we administered CpG and anti-OX40 antibody into the tumor on one side and monitored both the injected and the uninjected sites. Mice bearing the A20 lymphoma tumors were cured and were protected from a second challenge with A20 cells. To examine the potential to treat spontaneous, non-transplanted cancers we chose the mouse mammary gland tumor model- FVB/N-Tg(MMTV-PyVT)634Mul/J. Injections of CpG and anti-OX40 antibody to the first arising tumor significantly reduced the incidence and outgrowth of subsequent tumors at un-injected susceptible mammary glands and the number of lung metastases. This anti tumor effect was T cell dependent, since depletion of either CD4+ or CD8+ T cells abrogated the therapeutic effect of in situ vaccination. Significance: TLR9 agonists and anti-OX40 antibodies are currently under clinical development for cancer treatment. We show here that combining anti-OX40 antibody with a TLR9 agonist at a single established tumor is sufficient to trigger a systemic anti-tumor response able to eradicate tumor at distant sites in both transplantable and spontaneously occurring oncogene-driven murine tumors. This anti-tumor effect was long lasting, specific and required T cells. Impact: We recently published positive results showing that the combination of CpG with antibodies against CTLA4 and OX40 induced a therapeutic immune response against transplantable syngeneic tumors (Marabelle, Levy, et al. JCI, 2013). Our current results suggest that CpG and anti-OX40 are sufficient to induce fully protective and curative anti tumor immune responses, even in spontaneously arising cancer. Anti-OX40 and CpG are both currently in phase-I clinical trials as single agents. Our results provide the rationale for testing these agents in combination in the form of locally injected inducers of therapeutic anti-cancer immunity. Citation Format: Idit Sagiv-Barfi, Debra K. Czerwinski, Ronald Levy. In situ vaccination with TLR9 agonist and anti-Ox40 antibody is sufficient to induce abscopal responses even in mice with spontaneous oncogene-driven tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 551.

Combined effect of Vacc-4x, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial

Immune priming before reversal of latency might be a component of a functional HIV cure. To assess this concept, we assessed if therapeutic HIV immunisation followed by latency reversal would affect measures of viral transcription, plasma viraemia, and reservoir size in patients with HIV on suppressive antiretroviral therapy. In this single-arm, phase 1B/2A trial, we recruited adults treated at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark (aged ≥18 years) with successfully treated HIV-1 with plasma RNA loads of less than 50 copies per mL for the previous year and CD4 counts of at least 500 cells per μL. Exclusion criteria included CD4 counts of less than 200 cells per μL within the past 2 years, active hepatitis B or C infections, and clinically significant cardiac disease, including QTc prolongation. Participants received six therapeutic intradermal HIV-1 immunisations with 12 mg/mL Vacc-4x and 0·6 mg/mL rhuGM-CSF over 12 weeks (at 0 weeks, 1 week, 2 weeks, 3 weeks, 11 weeks, and 12 weeks) before receiving 5 mg/m(2) intravenous romidepsin once a week for 3 weeks. This procedure was followed by analytical treatment interruption. Coprimary outcomes were changes in copies of HIV-1 DNA (total and integrated) per million CD4 T cells and infectious units per million (IUPM) resting memory CD4 T cells established by viral outgrowth, assessed in all patients receiving at least one dose of active treatment with assessable data. We assessed total HIV-1 DNA at screening, before romidepsin treatment, and 6 weeks after romidepsin treatment. We assessed integrated viral DNA at baseline, before romidepsin treatment, and 8 weeks after romidepsin treatment. We assessed IUPM at screening, 2 weeks before romidepsin treatment, and 6 weeks after romidepsin treatment. This trial is registered at ClinicalTrials.gov, number NCT02092116. Between May 19, 2014, and Oct 8, 2014, we enrolled 20 individuals, of whom 17 completed all Vacc-4x and rhuGM-CSF administrations and romidepsin infusions. 16 of 17 had assessable total HIV-1 DNA, 15 of 17 had assessable integrated HIV-1 DNA, and six of 17 had assessable IUPM at baseline and at one or more timepoints after study treatment. Total HIV-1 DNA declined from screening to 6 weeks after romidepsin treatment (mean reduction 39·7%, 95% CI -59·7 to -11·5; p=0·012). The decrease in integrated HIV-1 DNA from baseline to 8 weeks after romidepsin treatment was not significant (19·2%, -38·6 to 6·3; p=0·123). Among the six assessable participants, the mean reduction in IUPM from screening to 6 weeks after romidepsin treatment was 38·0% (95% CI -67·0 to -8·0; p=0·019). Of 141 adverse events, 134 (95%) were grade 1 and seven (5%) were grade 2-3. This in-vivo combinatorial approach provides the first evidence for the feasibility of a combined shock and kill strategy, but also emphasises that further optimisation of this strategy is needed to achieve a sizeable effect on the latent reservoir that will translate into clinically measurable benefits for people living with HIV-1. Bionor Pharma, the Research Council of Norway, and SkatteFUNN.

Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway.

CD8+ cytotoxic T lymphocyte (CTL) exhaustion is a chief issue for ineffective virus elimination in chronic infectious diseases. We generated novel ovalbumin (OVA)-specific OVA-Texo and HIV-specific Gag-Texo vaccines inducing therapeutic immunity. To assess their therapeutic effect in chronic infection, we developed a new chronic infection model by i.v. infecting C57BL/6 mice with the OVA-expressing adenovirus AdVova. During chronic AdVova infection, mouse CTLs were found to express the inhibitory molecules programmed cell-death protein-1 (PD-1) and lymphocyte-activation gene-3 (LAG-3) and to be functionally exhausted, showing a significant deficiency in T-cell proliferation, IFN-γ production and cytolytic effects. Naive CD8+ T cells upregulated inhibitory PD-ligand 1 (PD-L1), B- and T-lymphocyte attenuator and T-cell anergy-associated molecules (Grail and Itch) while down-regulating the proliferative response upon stimulation in mice with chronic infection. Remarkably, the OVA-Texo vaccine counteracted T-cell anergy and converted CTL exhaustion. The latter was associated with (i) the upregulation of a marker for CTL functionality, diacetylated histone-H3 (diAcH3), (ii) a fourfold increase in CTLs, occurring independent of host DCs or CD4+ T cells, and (iii) the restoration of CTL IFN-γ production and cytotoxicity. In vivo OVA-Texo-stimulated CTLs upregulated the activities of the mTORC1 pathway-related molecules Akt, S6, eIF4E and T-bet, and treatment of the CTLs with an mTORC1 inhibitor, rapamycin, significantly reduced the OVA-Texo-induced increase in CTLs. Interestingly, OVA-Texo-mediated CD40L signaling played a critical role in the observed immunological effects. Importantly, the Gag-Texo vaccine induced Gag-specific therapeutic immunity in chronic infection. Therefore, this study should have a serious impact on the development of new therapeutic vaccines for human immunodeficiency virus (HIV-1) infection.

Chimeric HBcAg virus-like particles presenting a HPV 16 E7 epitope significantly suppressed tumor progression through preventive or therapeutic immunization in a TC-1-grafted mouse model.

BackgroundTherapeutic human papillomavirus (HPV) vaccines are currently being developed. However, no therapeutic efficacy has been achieved in clinical trials for the treatment of cervical intraepithelial neoplasia or cancer. One of the important issues in increasing vaccine efficacy is determining the best way to enhance tumor antigen-specific cellular immune responses. This study aimed to explore the virus-like particles (VLPs) of hepatitis B core antigen (HBcAg) as potential therapeutic vaccine carriers and to assess its immunological characteristics.MethodsChimeric VLPs presenting a HPV 16 cytotoxic T lymphocytes epitope E749–57 (amino acid 49–57 of the E7 protein) were prepared using recombinant genes. C57BL/6 mice were immunized with VLPs and grafted with tumor cells TC-1 which is an E7-expressing tumorigenic cell line. The dynamic tumor growth was monitored and anti-tumor immune responses were investigated.ResultsUsing a preventive strategy, immunization with VLPs resulted in nearly complete suppression of tumor growth. In treatment studies, VLP immunization significantly suppressed the tumor progression in mice carrying 2–3 mm tumors and in those bearing even larger tumors with diameters up to 8–9 mm. The VLP structure was shown to be important to induce vigorous antitumor immunity and effects. In immunized mice, enhanced E749–57-specific cellular immune responses were evidenced by increased interferon (IFN)-γ expression and decreased interleukin (IL)-4 expression in splenic lymphocytes, as well as an elevated number of effector cells expressing IFN-γ in response to the in vitro stimulation of the specific peptide E749–57. In addition, effective immune memory after VLP immunization was maintained for at least 16 weeks, preventing significant tumor growth after subsequent TC-1 challenge.ConclusionWhile VLPs were highly immunogenic in stimulating humoral immunity, our results strongly indicated that VLPs, such as HBcAg particles, might also be potent therapeutic vaccine carriers to elicit robust cellular immune responses, even in the immunosuppressive microenvironment of a tumor.

Vitamin D increases the human endosomal TLR response against Mycobacterium tuberculosis nucleic acids

Abstract Mycobacterium tuberculosis (Mtb) infects macrophages where it avoids elimination by interfering with host defense mechanisms. Type I IFNs are produced by activation of endosomal TLRs after recognition of nucleic acids. TLR importance is underlined by the fact that the TLR-adaptor molecule MyD88 pathway in macrophages is important in confining Mtb within phagolysosomes, mycobacterial clearance, and an adequate innate and adaptive immune response against Mtb. Vitamin D (VD) has a positive effect in macrophage activation, phagocytosis, restriction of Mtb growth, and killing of the mycobacteria. We aimed to characterize the endosomal TLR-signaling of human macrophages, as they are appear crucial in determining the fate of the disease, and explore if variations in Type I IFN responses correlate with differences in Mtb nucleic acids derived from different Mtb genotypes. Stimulation with DNA from Mtb of different lineages elicited differential NF-κB and IRF inflammatory responses in THP-1 derived macrophages. We also studied the effect of VD on these responses, as it is known to activate macrophages and enhance their antimycobacterial activity. Pretreatment with VD increased the response to DNA from HN878 and virulent strain H37Rv. It also enhanced the response to RNA from M. smegmatis and attenuated M. bovis strain BCG. No previous study has evaluated Type I or Type II IFNs response differences between different Mtb strains. This is of great importance as type I IFNs appear to strongly inhibit macrophages responsiveness to IFN-γ, and are associated with active TB. Understanding VD-mediated activation occurring at the pulmonary alveoli will impact the design of novel therapeutic and successful immunization strategies.