Sustained Immune Response Research Articles

Protocols for Innate Lymphoid Cell Phenotypic and Functional Characterization: An Overview.

The innate immune system forms a first line of defense against infections and is an essential component in immune defense prior to the generation of sustained adaptive immune responses. Until recently this innate response was believed to be predominantly elicited by cells of myeloid origin. The last 10years have seen the discovery of an extensive family of innate lymphoid cells (ILCs) characterized by the ability to express cytokines, traditionally associated with activated T effector cells, and lack of expression of antigen receptors.ILCs are enriched at barrier surfaces and rapidly respond to alarmins within the tissue microenvironment. ILCs lack recombinant activating gene (RAG)-dependent rearranged receptors, lack myeloid and dendritic cell lineage defining factors, and possess lymphoid morphology. The first characterized innate lymphoid cells were natural killer cells (NK) cells and lymphoid tissue inducer (LTi) cells. NK cells, defined in 1975), mediate important early antigen independent immune responses. By contrast, LTi cells, defined in 1997, are vital for formation of lymph nodes during embryogenesis. In the past decade, research on ILCs has defined them as important regulators of barrier immunity. However, ILC phenotype and functional characterization is complex and requires highly specific protocols for delineating ILC specific function from other well-characterized immune cells. Within this book, each chapter provides an in-depth protocol that will expand on techniques used by laboratories to study ILC development, characterization and function in mice and humans.

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation.

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed “memory inflation”. Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation.

Sustained Cellular and Humoral Immune Responses From an Adenoviral Vector-based Respiratory Syncytial Virus Vaccine.

Sustained Cellular and Humoral Immune Responses From an Adenoviral Vector-based Respiratory Syncytial Virus Vaccine.

Oncolytic Viruses: Priming Time for Cancer Immunotherapy

New immuno-oncology therapies are improving cancer treatments beyond the former standard of care, as evidenced by the recent and continuing clinical approvals for immunotherapies in a broad range of indications. However, a majority of patients (particularly those with immunologically cold tumors) still do not benefit, highlighting the need for rational combination approaches. Oncolytic viruses (OV) both directly kill tumor cells and inflame the tumor microenvironment. While OV spread can be limited by the generation of antiviral immune responses, the initial local tumor cell killing can reverse the immunosuppressive tumor microenvironment, resulting in more effective release of tumor-associated antigens (TAAs), cross-presentation, and antitumoral effector T cell recruitment. Moreover, many OVs can be engineered to express immunomodulatory genes. Rational combination approaches to cancer immunotherapy include the use of OVs in combination with immune checkpoint inhibitors (ICIs) or adoptive T cell therapy (ACT) to promote sustained antitumoral immune responses. OV combinations have additive or synergistic efficacy in preclinical tumor models with ICIs or ACT. Several preclinical studies have confirmed systemic reactivation and proliferation of adoptively transferred antitumoral T cells in conjunction with oncolytic OVs (expressing cytokines or TAAs) resulting from the specific tumor cell killing and immunostimulation of the tumor microenvironment which leads to increased tumor trafficking, activity, and survival. Recent clinical trials combining OVs with ICIs have shown additive effects in melanoma. Additional clinical data in an expanded range of patient indications are eagerly awaited. The relative timings of OV and ICI combination remains under-studied and is an area for continued exploration. Studies systematically exploring the effects of systemic ICIs prior to, concomitantly with, or following OV therapy will aid in the future design of clinical trials to enhance efficacy and increase patient response rates.

Multiple viral proteins and immune response pathways act to generate robust long-term immunity in Sudan virus survivors

BackgroundProfiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. MethodsWe characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000−2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FindingsWe found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. InterpretationWe highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FundsThis research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Abstract 1494: Galectin 9 associates with PDL1 expression and promotes gastric cancer cell invasion

Abstract Background: The development of immune checkpoint inhibitors (immunotherapy, such as anti-PD1) that induce a sustained T-cell immune response with anti-tumor activity has been a breakthrough in oncology practice. However, in gastric cancer (GC), sustained responses to anti PD-1 therapy are only observed in approximately 20% of patients. The expression of alternative immune checkpoints such as Galectin 9 (Gal-9) and Tim3 has been associated to anti-PD1 resistance in other cancer types. In search of the factors that render gastric tumors resistant to immunotherapy, we hypothesized that Gal-9 expression in gastric cancer cells may enhance metastasis and immunosuppression. Methods: Bioinformatic analysis of gastric cancer data of patients with gastric adenocarcinoma from TCGA Database was performed. In vitro experiments with Gal-9 transfected gastric cancer cell line AGS were performed to elucidate Gal-9 gain of function effect on cell invasion and immune-suppressive proteins. Invasion assay using a matrigel coated transwell was performed within 24 hours of transfection. To determine changes in PDL1, E-Cadherin and N-Cadherin expression, cells were harvested 24h after transfection to analyze protein and mRNA levels through Western Blot and RT-PCR respectively. Results: TCGA database analysis demonstrate that Gal-9 expression is significantly correlated to PDL-1 expression in gastric cancer; furthermore, it was also positively correlated to immune cell markers such as FOXP3. In accordance, in vitro analysis demonstrates that exogenous expression of GAL9 results in an increase in PDL-1 protein and greater invasive potential of the AGS cell line. Conclusion: Taken together our results suggest that further studies should focus on the role played by Gal-9 is the establishment of an immune suppressive microenvironment in gastric tumors. FONDECYT 1180241, 1180173. CONICYT FONDAP 15130011, IMII P09/016F BMS CA209-8F3 Citation Format: Charlotte Hill, Camille Cabrolier, Elisa Cumsille, Maximiliano Arce, Mauricio P. Pinto, Maria Loreto Bravo, Matias Muñoz, Marcelo Garrido, Gareth I. Owen. Galectin 9 associates with PDL1 expression and promotes gastric cancer cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1494.

Life-long control of cytomegalovirus (CMV) by T resident memory cells in the adipose tissue results in inflammation and hyperglycemia.

Cytomegalovirus (CMV) is a ubiquitous herpesvirus infecting most of the world’s population. CMV has been rigorously investigated for its impact on lifelong immunity and potential complications arising from lifelong infection. A rigorous adaptive immune response mounts during progression of CMV infection from acute to latent states. CD8 T cells, in large part, drive this response and have very clearly been demonstrated to take up residence in the salivary gland and lungs of infected mice during latency. However, the role of tissue resident CD8 T cells as an ongoing defense mechanism against CMV has not been studied in other anatomical locations. Therefore, we sought to identify additional locations of anti-CMV T cell residency and the physiological consequences of such a response. Through RT-qPCR we found that mouse CMV (mCMV) infected the visceral adipose tissue and that this resulted in an expansion of leukocytes in situ. We further found, through flow cytometry, that adipose tissue became enriched in cytotoxic CD8 T cells that are specific for mCMV antigens from day 7 post infection through the lifespan of an infected animal (> 450 days post infection) and that carry markers of tissue residence. Furthermore, we found that inflammatory cytokines are elevated alongside the expansion of CD8 T cells. Finally, we show a correlation between the inflammatory state of adipose tissue in response to mCMV infection and the development of hyperglycemia in mice. Overall, this study identifies adipose tissue as a location of viral infection leading to a sustained and lifelong adaptive immune response mediated by CD8 T cells that correlates with hyperglycemia. These data potentially provide a mechanistic link between metabolic syndrome and chronic infection.

A late B lymphocyte action in dysfunctional tissue repair following kidney injury and transplantation

The mechanisms initiating late immune responses to an allograft are poorly understood. Here we show, via transcriptome analysis of serial protocol biopsies from kidney transplants, that the initial responses to kidney injury correlate with a late B lymphocyte signature relating to renal dysfunction and fibrosis. With a potential link between dysfunctional repair and immunoreactivity, we investigate the immunological consequences of dysfunctional repair examining chronic disease in mouse kidneys 18 months after a bilateral ischemia/reperfusion injury event. In the absence of foreign antigens, a sustained immune response involving both innate and adaptive immune systems accompanies a transition to chronic kidney damage. At late stages, B lymphocytes exhibite an antigen-driven proliferation, selection and maturation into broadly-reacting antibody-secreting cells. These findings reveal a previously unappreciated role for dysfunctional tissue repair in local immunomodulation that may have particular relevance to transplant-associated immunobiology.

Drug Delivery for Cancer Immunotherapy and Vaccines.

Cancer cells are able to avoid immune surveillance and exploit the immune system to grow and metastasize. With the development of nano- and micro-particles, there has been a growing number of immunotherapy delivery systems developed to elicit innate and adaptive immune responses to eradicate cancer cells. This can be accomplished by training resident immune cells to recognize and eliminate cells with tumor-associated antigens or by providing external stimuli to enhance tumor cell apoptosis in the immunosuppressive tumor microenvironment (TME). In this review we will focus on nano- and micro-particle (NP and MP) based immunotherapies and vaccines used to elicit a potent and sustained antitumor immune response.

An Antibody Designed to Improve Adoptive NK-Cell Therapy Inhibits Pancreatic Cancer Progression in a Murine Model.

Natural killer (NK) cells are primary immune cells that target cancer cells and can be used as a therapeutic agent against pancreatic cancer. Despite the usefulness of NK cells, NK-cell therapy is limited by tumor cell inhibition of NK-cell homing to tumor sites, thereby preventing a sustained antitumor immune response. One approach to successful cancer immunotherapy is to increase trafficking of NK cells to tumor tissues. Here, we developed an antibody-based NK-cell-homing protein, named NK-cell-recruiting protein-conjugated antibody (NRP-body). The effect of NRP-body on infiltration of NK cells into primary and metastatic pancreatic cancer was evaluated in vitro and in murine pancreatic ductal adenocarcinoma models. The NRP-body increased NK-cell infiltration of tumors along a CXCL16 gradient (CXCL16 is cleaved from the NRP-body by furin expressed on the surface of pancreatic cancer cells). CXCL16 induced NK-cell infiltration by activating RhoA via the ERK signaling cascade. Administration of the NRP-body to pancreatic cancer model mice increased tumor tissue infiltration of transferred NK cells and reduced the tumor burden compared with that in controls. Overall survival of NRP-body-treated mice (even the metastasis models) was higher than that of mice receiving NK cells alone. In conclusion, increasing NK-cell infiltration into tumor tissues improved response to this cancer immunotherapy. The combination of an NRP-body with NK-cell therapy might be useful for treating pancreatic cancer.

A Viral Nanoparticle Cancer Vaccine Delays Tumor Progression and Prolongs Survival in a HER2+ Tumor Mouse Model.

Human epidermal growth factor receptor 2 (HER2) overexpression is associated with aggressive tumors with increased incidence of metastasis and recurrence. Therapeutic antibodies such as Trastuzumab inhibit tumor growth through blockade of HER2 receptors. However, the short lifespan of such therapeutic antibodies necessitates repeat administrations with ensuing cardiac toxicity and development of resistance, while offering no protection against relapse. Cancer vaccines targeting HER2 can overcome these shortcomings of passive immunotherapy by instigating an endogenous and sustained immune response and memory against the cancer antigen. The efficacy of a viral nanoparticle (VNP)-based cancer vaccine is demonstrated here in activating a potent anti-HER2 immune response that delays progression of primary tumors as well as metastases and prolongs survival in mice. The results illustrate that the VNP-based vaccine instigates HER2-specific antibodies as well as effector and memory T cells, which contributes to the effectiveness of the vaccine. Given the highly aggressive course of HER2+ cancers, inhibition of disease progression by such cancer vaccines could provide a critical window for interventions with other adjuvant therapies. Moreover, the immune memory generated by this viral nanoparticle-based cancer vaccine could mitigate relapse of the disease.

Luciferase-assisted detection of extracellular ATP and ATP metabolites during immunogenic death of cancer cells.

The efficacy of cancer chemotherapy is enhanced by induction of sustainable anti-tumor immune responses. Such responses involve accumulation of immunogenic mediators, such as extracellular ATP and ATP metabolites, within the tumor microenvironment. Recent studies have identified nucleotide-permeable plasma membrane channels or pores that are activated as early downstream consequences of different regulated cell death pathways: pannexin-1 channels in apoptosis, MLKL pores in necroptosis, and gasdermin-family pores in pyroptosis. This chapter describes the use of highly quantitative and semi-high-throughput methods based on the ATP sensor luciferase to measure dynamic changes in extracellular ATP, ADP, and AMP in tissue/cell culture models of cancer cells during various modes of regulated cell death in response to chemotherapeutic drugs, death receptors, or metabolic perturbation.

Engineering Nanoparticles for Targeted Remodeling of the Tumor Microenvironment to Improve Cancer Immunotherapy.

Owing to the fast-paced growth and cross-infiltration of oncology, immunology and molecular biology, tumor immunotherapy technology represented by immune checkpoint blockade and chimeric antigen receptor (CAR) T cell therapy has lately made remarkable advancements. In comparison with traditional chemotherapy, immunotherapy has the potential to elicit a stronger sustained antitumor immune response in those patients who have advanced malignant malignancies. In spite of the advancements made, a significant number of clinical research works have validated that an extensive proportion of cancer patients still manifest insensitivity to immunotherapy, primarily because of the immunomodulatory interactions between tumor cells and the immunosuppressive tumor microenvironment (TME), together mediating the immune tolerance of tumors and accordingly impacting the positive response to immunotherapy. The intricate immunosuppressive networks formed by stromal cells, inflammatory cells, vasculature, extracellular matrix (ECM), and their secreted cytokines in the TME, play a pivotal role in tumor immune escape. Specific blocking of inhibition pathways in the TME is expected to effectively prevent immune escape and tolerance of tumor cells in addition to their metastasis, accordingly improving the antitumor immune response at various phases of tumor growth. Emerging nanoscale targeted drug carriers truly suit this specific requirement due to their specificity, biocompatibility, and convenience of production. This review emphasizes recent attempts to remodel the tumor immune microenvironment using novel nanoparticles, which include specifically eliminating immunosuppressive cells, reprogramming immune regulatory cells, promoting inflammatory cytokines and blocking immune checkpoints. Targeted remodeling of the immunosuppressive TME using well-designed and fabricated nanoparticles provides a promising strategy for improving the effectiveness of current immunotherapy and is greatly significant.

Effective effectors: How T cells access and infiltrate the central nervous system.

Several Phase II and III clinical trials have demonstrated that immunotherapy can induce objective responses in otherwise refractory malignancies in tumors outside the central nervous system. In large part, effector T cells mediate much of the antitumor efficacy in these trials, and potent antitumor T cells can be generated through vaccination, immune checkpoint blockade, adoptive transfer, and genetic manipulation. However, activated T cells must still traffic to, infiltrate, and persist within tumor in order to mediate tumor lysis. These requirements for efficacy pose unique challenges for brain tumor immunotherapy, due to specific anatomical barriers and populations of specialized immune cells within the central nervous system that function to constrain immunity. Both autoimmune and infectious diseases of the central nervous system provide a wealth of information on how T cells can successfully migrate to the central nervous system and then engender sustained immune responses. In this review, we will examine the commonalities in the efferent arm of immunity to the brain for autoimmunity, infection, and tumor immunotherapy to identify key factors underlying potent immune responses.

Single Dose of a Polyanhydride Particle-Based Vaccine Generates Potent Antigen-Specific Antitumor Immune Responses.

Many factors affect vaccine efficacy. One of the most salient is the frequency and intervals of vaccine administration. In this study, we assessed the vaccine administration modality for a recently reported polyanhydride-based vaccine formulation, shown to generate antitumor activity. Polyanhydride particles encapsulating ovalbumin (OVA) were prepared using a double-emulsion technique and subcutaneously delivered to mice either as a single-dose or as prime-boost vaccine regimens in which two different time intervals between prime and boost were assessed (7 or 21 days). This was followed by measurement of cellular and humoral immune responses, and subsequent challenge of the mice with a lethal dose of E.G7-OVA cells to evaluate tumor protection. Interestingly, a single dose of the polyanhydride particle-based formulation induced sustained OVA-specific cellular immune responses just as effectively as the prime-boost regimens. In addition, mice receiving single-dose vaccine had similar levels of protection against tumor challenge compared with mice administered prime-boosts. In contrast, measurements of OVA-specific IgG antibody titers indicated that a booster dose was required to stimulate strong humoral immune responses, since it was observed that mice administered a prime-boost vaccine had significantly higher OVA-specific IgG1 serum titers than mice administered a single dose. These findings indicate that the requirement for a booster dose using these particles appears unnecessary for the generation of effective cellular immunity.

Acetalated Dextran Microparticles for Codelivery of STING and TLR7/8 Agonists.

Vaccines are the most effective tool for preventing infectious diseases; however, subunit vaccines, considered the safest type, suffer from poor immunogenicity and require adjuvants to create a strong and sustained immune response. As adjuvants, pathogen-associated molecular patterns (PAMPs) offer potent immunostimulatory properties and defined mechanisms of action through their cognate pattern recognition receptors (PRRs). Their activity can be further enhanced through combining two or more PAMPs, particularly those that activate multiple immune signaling pathways. However, the cytosolic localization of many PRRs requires intracellular delivery of PAMPs for optimal biological activity, which is particularly true of the stimulator of interferon genes (STING) PRR. Using acetalated dextran (Ace-DEX) microparticles (MPs) encapsulating STING agonist 3'3'-cyclic GMP-AMP (cGAMP) combined with soluble PAMPS, we screened the effect of codelivery of adjuvants using primary mouse bone marrow derived dendritic cells (BMDCs). We identified that codelivery of cGAMP MPs and soluble Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) elicited the broadest cytokine response. cGAMP and R848 were then coencapsulated within Ace-DEX MPs via electrospray. Using the model antigen ovalbumin, we observed that Ace-DEX MPs coencapsulating cGAMP and R848 (cGAMP/R848 Ace-DEX MPs) induced antigen-specific cellular immunity, and a balanced Th1/Th2 humoral response that was greater than cGAMP Ace-DEX MPs alone and PAMPs delivered in separate MPs. These data indicate that polymeric Ace-DEX MPs loaded with STING and TLR7/8 agonists represent a potent cellular and humoral vaccine adjuvant.

PIN105 - THE PUBLIC HEALTH IMPACT OF TICK-BORNE ENCEPHALITIS VACCINATION ON THE SLOVENIAN ADULT POPULATION

Despite Slovenia having one of the highest Tick-Borne Encephalitis (TBE) incidence rates in Europe, TBE vaccination coverage in Slovenia remains low in the general population. This study evaluates the potential public health impact of implementing an additional TBE universal mass vaccination (UMV) campaign for Slovenian adults of 45-50 years of age (YOA). A lifetime Markov cohort model was developed to reproduce the natural history of TBE infection in Europe. The study population of Slovenian adults, 45-50 YOA (total cohort size: 177,353), was followed using a 1-year cycle length, during which the probability of infection and moving to a specific health state was determined by age- and gender-specific incidence rates (corrected for underreporting) and specific state-to-state transition probabilities. It was assumed that the vaccination coverage was 50% and that the vaccinated population would be fully compliant with the conventional TBE vaccination schedule. Following the outcomes of recently published literature on the sustained immune response after TBE vaccination, an extension of the booster time-interval was explored from 3 or 5 years to 10 years irrespective of age. Implementing a UMV campaign for Slovenian adults of 45-50 YOA would reduce the total number of TBE cases by 761 cases (23.4%), of primary phase symptoms by 282 cases (23.4%) as well as of secondary phase symptoms by 19 cases (22.5%). Consequently, 332 to 498 days of hospitalization could be averted. An extension of the booster time-interval would prevent 52 cases (6.8%) less while the amount of secondary phase symptoms would increase by 1 case (5.2%). Nevertheless, the total amount of required booster doses would be reduced by 230,867 doses (39.5%). Implementing a TBE UMV campaign in the 45-50 YOA adult Slovenian population would result in a significant public health impact irrespective of an extended booster time-interval for the TBE-vaccine schedule.

Hepatitis B Vaccination in Patients Receiving Oral Antiviral Therapy Without Hepatitis B Immunoglobulin After Liver Transplant

Our study aimed to determine if a double-dose pre-S containing hepatitis B virus (HBV) vaccination (Sci-B-Vac) could elicit an adequate and sustainable immune response in HBV patients who developed spontaneous hepatitis B surface antibody (anti-HBs) response after liver transplant. Patients and MethodsAll patients who received transplants for HBV-related disease for >1 year with normal graft function and hepatitis B surface antigen seronegativity were evaluated. They received a 40-μg HBV vaccine if they were responders in our previous vaccine trial, if anti-HBs was positive for >1 year after liver transplant (LT), or if a peak anti-HBs at any time point after LT was >100 mIU/mL. Primary endpoint was the development of anti-HBs ≥ 10 mIU/mL from previous negative value or a 1-log increase from baseline. ResultsA total of 86 patients were recruited; 5 were responders from a previous trial; 45 patients had detectable anti-HBs >1 year after LT, and 36 patients had an anti-HBs >100 mIU/mL. All (5/5, 100%) previous responders responded to booster vaccination. For the remaining 81 patients, 10 of 81 (12.3%) responded. ConclusionAll previous responders responded to booster vaccination, implying durability and memory of HBV immune response, which is an important prerequisite for definitive host immunity for HBV. In patients who had spontaneous anti-HBs production after LT, a single vaccination can induce response in 12.3% of patients.

Abstract 1726: A novel delivery platform containing up to 14 neoantigens can induce robust immune responses in a single formulation

Abstract Neoantigens are emerging targets for personalized cancer vaccines that provide patient specific cancer immunotherapies. Many algorithms have been developed to select the most immunogenic neoantigens to include, however not all neoantigens chosen will generate equivalent immune responses, nor may they induce effective anti-tumour activity. To maximize immunological activity, selected peptides should be delivered simultaneously in a formulation that can stimulate potent, sustained immune responses to many different peptides. The DepoVaxTM platform is an oil-based system that uses lipids to incorporate many different types of antigens and adjuvants into a single formulation. Using a set of neoantigens identified from murine B16-F10 melanoma, we optimized a DepoVax formulation method that allows us to incorporate up to 14 neoantigens with a polynucleotide based adjuvant in a single formulation. These selected neoantigens irrespective of their solubility and hydrophobicity were formulated in DepoVax with the contents completely soluble in oil. C57BL/6 mice were vaccinated with 14 synthetic long peptide neoantigens (each 27 amino acids in length) prepared in DepoVax or in an aqueous formulation containing poly ICLC adjuvant. The immune responses were assessed eight days later by IFN-γ ELISPOT using splenocytes. Several of the peptides generated strong immune responses that were significantly higher in mice vaccinated with the DepoVax formulation compared to the aqueous formulation. To assess the contribution of CD8+ and CD4+ T cell responses, splenocytes from vaccinated mice were stimulated with an immunogenic peptide and intracellular IFN-γ/TNF-α producing CD8+ or CD4+ T cells were detected by flow cytometry. The highest production of TNF-α was detected by CD8+ T cells. Biological activity of the vaccines was assessed after one month storage at -20, 5 and 25 °C by IFN-γ ELISPOT assay; no significant difference was detected compared to the initial results. Analytical characterization of 14 peptides in DepoVax carried out using high-performance liquid chromatography (RP-HPLC), detected no significant chemical modifications or degradation of peptides after storage at -20 °C for up to 3 months compared to the initial results. These results demonstrate that the DepoVax platform can incorporate at least 14 neoantigens in a single formulation. Neoantigens formulated in DepoVax are stable for at least 3 months and our manufacturing method can incorporate peptides with a wide range of physical and chemical characteristics. This formulation generates strong CD8+ T cell responses, in excess of those induced by an aqueous formulation. The DepoVax platform is a promising solution to inducing robust immune responses to multiple neoantigens in a single formulation. Citation Format: Valarmathy Kaliaperumal, Genevieve Weir, Rajkannan Rajagopalan, Arthvan Sharma, Heather Torrey, Alecia MacKay, Ava Vila-Leahey, Cynthia Tram, Andrea Penwell, Leeladhar Sammatur, Marianne Stanford. A novel delivery platform containing up to 14 neoantigens can induce robust immune responses in a single formulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1726.

Immunization With a DNA Vaccine Cocktail Encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 Genes Protects Mice Against Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic disease caused by the intracellular protozoan Toxoplasma gondii; and a major source of infection in humans is via ingestion of T. gondii tissue cysts. Ultimately, the goal of anti-toxoplasmosis vaccines is to elicit a sustainable immune response, capable of preventing formation of the parasite tissue cysts—or, at least, to restrain its growth. In this study, we formulated a cocktail DNA vaccine and investigated its immunologic efficacy as a protection against the establishment of T. gondii cysts in the mouse brain. This multicomponent DNA vaccine, encoded the TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 genes, which play key roles in the pathogenesis of T. gondii infection. Results showed that mice immunized via intramuscular injection three times, at 2-week intervals with this multicomponent DNA vaccine, mounted a strong humoral and cellular immune response, indicated by significantly high levels of total IgG, CD4+ and CD8+ T lymphocytes, and antigen-specific lymphocyte proliferation when compared with non-immunized mice. Immunization also induced a mixed Th1/Th2 response, with a slightly elevated IgG2a to IgG1 ratio. The increased production of proinflammatory cytokines gamma-interferon, interleukin-2, and interleukin-12 (p < 0.0001) correlated with increased expression of p65/RelA and T-bet genes of the NF-κB pathway. However, no significant difference was detected in level of interleukin-4 (p > 0.05). The number of brain cysts in immunized mice was significantly less than those in non-immunized mice (643.33 ± 89.63 versus 3,244.33 ± 96.42, p < 0.0001), resulting in an 80.22% reduction in the parasite cyst burden. These findings indicate that a multicomponent DNA vaccine, encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 genes, shows promise as an immunization strategy against chronic toxoplasmosis in mice, and calls for a further evaluation in food-producing animals.