Neoantigen-based personalized cancer vaccines: the emergence of precision cancer immunotherapy.

INTRODUCTION: The field of cancer therapy has undergone a major transformation in less than a decade due to the introduction of checkpoint inhibitors, the advent of next generation sequencing and the discovery of neoantigens. The key observation that the breadth of each patient's immune response to the unique mutations or neoantigens present in their tumor is directly related to their survival has led oncologists to focus on driving immune responses to neoantigens through vaccination. Oncology has entered the era of precision immunotherapy, and cancer vaccine development is undergoing a paradigm shift. AREAS COVERED: Neoantigens are short peptide sequences found in tumors, but not noncancerous tissues, the vast majority of which are unique to each patient. In addition to providing a description of the distinguishing features of neoantigen discovery platforms, this review will address cross-cutting personalized cancer vaccine design themes and developmental stumbling blocks. EXPERT OPINION: Immunoinformatic pipelines that can rapidly scan cancer genomes and identify 'the best' neoantigens are in high demand. Despite the need for such tools, immunoinformatic methods for identifying neoepitopes in cancer genomes are diverse and have not been well-validated. Validation of 'personalized vaccine design pipelines' will bring about a revolution in neoantigen-based vaccine design and delivery.

Multi-step screening of neoantigens' HLA- and TCR-interfaces improves prediction of survival.

Improvement of risk stratification through prognostic biomarkers may enhance the personalization of cancer patient monitoring and treatment. We used Ancer, an immunoinformatic CD8, CD4, and regulatory T cell neoepitope screening system, to perform an advanced neoantigen analysis of genomic data derived from the urothelial cancer cohort of The Cancer Genome Atlas. Ancer demonstrated improved prognostic stratification and five-year survival prediction compared to standard analyses using tumor mutational burden or neoepitope identification using NetMHCpan and NetMHCIIpan. The superiority of Ancer, shown in both univariate and multivariate survival analyses, is attributed to the removal of neoepitopes that do not contribute to tumor immunogenicity based on their homology with self-epitopes. This analysis suggests that the presence of a higher number of unique, non-self CD8- and CD4-neoepitopes contributes to cancer survival, and that prospectively defining these neoepitopes using Ancer is a novel prognostic or predictive biomarker.

Highly conserved, non-human-like, and cross-reactive SARS-CoV-2 T cell epitopes for COVID-19 vaccine design and validation.

Natural and vaccine-induced SARS-CoV-2 immunity in humans has been described but correlates of protection are not yet defined. T cells support the SARS-CoV-2 antibody response, clear virus-infected cells, and may be required to block transmission. In this study, we identified peptide epitopes associated with SARS-CoV-2 T-cell immunity. Using immunoinformatic methods, T-cell epitopes from spike, membrane, and envelope were selected for maximal HLA-binding potential, coverage of HLA diversity, coverage of circulating virus, and minimal potential cross-reactivity with self. Direct restimulation of PBMCs collected from SARS-CoV-2 convalescents confirmed 66% of predicted epitopes, whereas only 9% were confirmed in naive individuals. However, following a brief period of epitope-specific T-cell expansion, both cohorts demonstrated robust T-cell responses to 97% of epitopes. HLA-DR3 transgenic mouse immunization with peptides co-formulated with poly-ICLC generated a potent Th1-skewed, epitope-specific memory response, alleviating safety concerns of enhanced respiratory disease associated with Th2 induction. Taken together, these epitopes may be used to improve our understanding of natural and vaccine-induced immunity, and to facilitate the development of T-cell-targeted vaccines that harness pre-existing SARS-CoV-2 immunity.

The Effects of Japanese Encephalitis Vaccine and Accelerated Dosing Scheduling on the Immunogenicity of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine: A Phase II, Randomized, Open-Label, Single-Center Trial in Adults Aged 18 to 45 Years in the United States.

BACKGROUND: Dengue is a global health problem requiring an effective, safe dengue vaccine. METHODS: We report the results of a phase II, randomized, open-label, single-center trial in adults aged 18 to 45 years in the United States designed to explore the effects of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine (CYD-TDV, Dengvaxia) when administered on its designated schedule (months 0, 6, and 12) or on an accelerated dosing schedule (months 0, 2, and 6) and/or given before, or concomitantly with, a vaccine against Japanese encephalitis (JE). RESULTS: Based on dengue virus serotype-specific neutralizing antibody (NAb), the accelerated dosing schedule was comparable to the 0, 6, and 12-month schedule. Giving JE vaccine concurrently with CYD-TDV did not result in an increase in overall NAb titers. Immunophenotyping of peripheral blood mononuclear cells revealed an increase in activated CD8+ T cells after CYD-TDV vaccination, a phenomenon that was greatest for the JE vaccine primed. CONCLUSIONS: We conclude that an accelerated dosing schedule of CYD-TDV results in essentially equivalent dengue serotype-specific NAb titers as the currently used schedule, and there may be an early benefit in antibody titers and activated CD8+ T cells by the administration of the JE vaccine before CYD-TDV vaccination.

Analysis of MHC Class I Processing Pathways That Generate a Response to Vaccinia Virus Late Proteins.

Use of recombinant viral vectors encoding nonnative Ags is an attractive mechanism for the generation of protective Ab, CD4+ T cell (TCD4+), and CD8+ T cell (TCD8+) responses in vivo following immunization. However, the life cycle and tropism of the viral vector, and its interactions with various components of the immune system, must be fully understood to maximize the efficacy of any vaccination strategies. Ab and TCD4+ responses typically target native Ags driven by late promoters in vaccinia virus (VACV)-based vectors. However, it has been demonstrated that model Ags driven by late promoters in recombinant VACV vectors do not stimulate TCD8+ responses, whereas identical Ags driven by early promoters stimulate strong responses. Conversely, TCD8+ can be generated against some natural late VACV Ags. We explored this dichotomy by investigating the Ag presentation pathways responsible for presentation of natural late VACV Ags in mice. We found that all of the late VACV Ags we examined could be cross-primed (i.e., presented by uninfected professional APC), as well as directly presented by infected dendritic cell populations. However, one Ag was only presented by professional APC populations and was not the target of a protective TCD8+ response. Therefore, there is no generalized blockade in Ag presentation of late VACV Ags, and expression of nonnative Ags driven by a late promoter allows production of large quantities of Ag that may allow simultaneous targeting of both TCD4+ and Ab responses, as well as TCD8+ responses, in the future.

Targeting CCR8 Induces Protective Antitumor Immunity and Enhances Vaccine-Induced Responses in Colon Cancer.

CCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) and is known to be critical for CCR8+ Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8+ tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that mAb therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of tumor-resident CD4+CCR8+ Tregs. Tumor-specific CD8+ T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented de novo induction and suppressive function of Tregs without affecting CD8+ T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a Listeria monocytogenes-based immunotherapy. Anti-CCR8 mAb therapy synergized with L. monocytogenes-based immunotherapy to significantly delay growth of established tumors and to prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies.Significance: Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity and prolong patient survival. Cancer Res; 78(18); 5340-8. ©2018 AACR.

Reduced B lymphoid kinase (Blk) expression enhances proinflammatory cytokine production and induces nephrosis in C57BL/6-lpr/lpr mice.

BLK, which encodes B lymphoid kinase, was recently identified in genome wide association studies as a susceptibility gene for systemic lupus erythematosus (SLE), and risk alleles mapping to the BLK locus result in reduced gene expression. To determine whether BLK is indeed a bona fide susceptibility gene, we developed an experimental mouse model, namely the Blk+/-.lpr/lpr (Blk+/-.lpr) mouse, in which Blk expression levels are reduced to levels comparable to those in individuals carrying a risk allele. Here, we report that Blk is expressed not only in B cells, but also in IL-17-producing γδ and DN αß T cells and in plasmacytoid dendritic cells (pDCs). Moreover, we found that solely reducing Blk expression in C57BL/6-lpr/lpr mice enhanced proinflammatory cytokine production and accelerated the onset of lymphoproliferation, proteinuria, and kidney disease. Together, these findings suggest that BLK risk alleles confer susceptibility to SLE through the dysregulation of a proinflammatory cytokine network.

γδ T cells acquire effector fates in the thymus and differentiate into cytokine-producing effectors in a Listeria model of infection independently of CD28 costimulation.

Both antigen recognition and CD28 costimulation are required for the activation of naïve αß T cells and their subsequent differentiation into cytokine-producing or cytotoxic effectors. Notably, this two-signal paradigm holds true for all αß T cell subsets, regardless of whether they acquire their effector function in the periphery or the thymus. Because of contradictory results, however, it remains unresolved as to whether CD28 costimulation is necessary for γδ T cell activation and differentiation. Given that γδ T cells have been recently shown to acquire their effector fates in the thymus, it is conceivable that the contradictory results may be explained, in part, by a differential requirement for CD28 costimulation in the development or differentiation of each γδ T cell effector subset. To test this, we examined the role of CD28 in γδ T cell effector fate determination and function. We report that, although IFNγ-producing γδ T (γδ-IFNγ) cells express higher levels of CD28 than IL-17-producing γδ T (γδ-17) cells, CD28-deficiency had no effect on the thymic development of either subset. Also, following Listeria infection, we found that the expansion and differentiation of γδ-17 and γδ-IFNγ effectors were comparable between CD28(+/+) and CD28(-/-) mice. To understand why CD28 costimulation is dispensable for γδ T cell activation and differentiation, we assessed glucose uptake and utilization by γδ T cells, as CD28 costimulation is known to promote glycolysis in αß T cells. Importantly, we found that γδ T cells express higher surface levels of glucose transporters than αß T cells and, when activated, exhibit effector functions over a broader range of glucose concentrations than activated αß T cells. Together, these data not only demonstrate an enhanced glucose metabolism in γδ T cells but also provide an explanation for why γδ T cells are less dependent on CD28 costimulation than αß T cells.

Connexin43 is dispensable for phagocytosis.

Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

Processing of recombinant Listeria monocytogenes proteins for MHC class I presentation follows a dedicated, high-efficiency pathway.

CD8(+) T lymphocytes recognize short peptides of ∼8-10 aa bound to MHC class I molecules (pMHC) on the surface of APCs. These peptides can be generated from either endogenous proteins synthesized by the biosynthetic machinery of the presenting cell or from exogenously sourced proteins. Because much of the research characterizing the MHC class I processing pathway has focused on endogenously synthesized proteins, it is not known whether differences exist in the processing pathway followed by endogenously synthesized versus exogenously sourced proteins. To highlight potential differences in the processing of endogenous versus exogenous proteins, we developed a model system to measure the efficiency of pMHC generation from nearly identical recombinant proteins expressed from vaccinia virus and Listeria monocytogenes. In these experiments, we uncovered a striking difference in the way recombinant Listeria Ags are processed and presented when compared with endogenously synthesized viral proteins. Specifically, we find that pMHC production from secreted Listeria proteins occurs at the same rate, independent of the cellular half-life of the protein from which it is derived, whereas the rate of pMHC production from endogenously synthesized viral proteins is absolutely dependent on its protein half-life. Accordingly, our data demonstrate the existence of a distinct and highly efficient MHC class I presentation pathway used for the processing of at least some exogenously synthesized proteins.

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells.

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded ß(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.

Viral and bacterial minigene products are presented by MHC class I molecules with similar efficiencies.

MHC class I molecules present short peptides, usually 8-10 amino acids in length, to CD8(+) T cells. These peptides are typically generated from full-length endogenously synthesized proteins degraded by the antigen processing machinery of the target cell. However, exogenous proteins, whether originating from intracellular bacteria or parasites or via phagocytosis during cross-presentation, can also be processed for presentation by MHC class I molecules. It is currently not known whether endogenously synthesized proteins and proteins acquired from exogenous sources follow the same presentation pathway. One clue that the processing pathways followed by endogenous and exogenous proteins may not be identical is the vastly different presentation efficiencies reported for viral versus bacterial antigens. Because class I antigen processing involves multiple steps, we sought to determine where in the processing pathway these differences in efficiency occur. To accomplish this, we expressed identical minimal peptide determinants from viral and bacterial vectors using a minigene expression system and determined the rate of peptide-MHC generation per molecule of minigene product synthesized. We found that peptides expressed from either the viral or bacterial vector were presented with virtually identical efficiencies. These results suggest that differences in the processing pathways followed by endogenous versus exogenous proteins most likely occur at a point prior to where free peptide is liberated from full-length protein.

Compartmentalized MHC class I antigen processing enhances immunosurveillance by circumventing the law of mass action.

MHC class I molecules function to display peptides generated from cellular and pathogen gene products for immune surveillance by CD8(+) T cells. Cells typically express approximately 100,000 class I molecules, or approximately 1 per 30,000 cellular proteins. Given "one protein, one peptide" representation, immunosurveillance would be heavily biased toward the most abundant cell proteins. Cells use several mechanisms to prevent this, including the predominant use of defective ribosomal products (DRiPs) to generate peptides from nascent proteins and, as we show here, compartmentalization of DRiP peptide generation to prevent competition from abundant cytosolic peptides. This provides an explanation for the exquisite ability of T cells to recognize peptides generated from otherwise undetected gene products.

The exception that reinforces the rule: crosspriming by cytosolic peptides that escape degradation.

The nature of crosspriming immunogens for CD8(+) T cell responses is highly controversial. By using a panel of T cell receptor-like antibodies specific for viral peptides bound to mouse D(b) major histocompatibility complex class I molecules, we show that an exceptional peptide (PA(224-233)) expressed as a viral minigene product formed a sizeable cytosolic pool continuously presented for hours after protein synthesis was inhibited. PA(224-233) pool formation required active cytosolic heat-shock protein 90 but not ER g96 and uniquely enabled crosspriming by this peptide. These findings demonstrate that exceptional class I binding oligopeptides that escape proteolytic degradation are potent crosspriming agents. Thus, the feeble immunogenicity of natural proteasome products in crosspriming can be attributed to their evanescence in donor cells and not an absolute inability of cytosolic oligopeptides to be transferred to and presented by professional antigen-presenting cells.

Characterization of rapidly degraded polypeptides in mammalian cells reveals a novel layer of nascent protein quality control.

Approximately 30% of polypeptides synthesized by mammalian cells are degraded with a half-life of <10 min by proteasomes. These rapidly degraded polypeptides (RDPs) constitute the bulk of proteasome substrates and are the principal source of viral and self-peptide ligands for major histocompatibility complex class I molecules. Here we provide evidence that approximately 75% of RDPs are degraded by the standard ubiquitin 26 S proteasome system and that their degradation is regulated by modulating Hsc70 activity in cells. Surprisingly, the remaining approximately 25% of RDPs are degraded without ubiquitylation by 20 S proteasomes independently of 19 S regulators and in a manner that is largely unaffected by modulating Hsc70 activity. This latter pathway is utilized for generating an antigenic peptide from viral-defective ribosomal products. The dichotomy in the behavior of RDPs points to a novel quality control level for nascent proteins that is independent of the well established Hsc70-ubiquitin 26 S proteasome pathway.

Corticosterone impairs MHC class I antigen presentation by dendritic cells via reduction of peptide generation.

The presentation of viral peptide-MHC class I complexes by antigen presenting cells, such as dendritic cells (DCs), is obligatory for the generation of antiviral effector and memory CD8(+) cytotoxic T lymphocyte (CTL) responses. Prolonged psychological stress is immunosuppressive and undermines primary and memory CTL-mediated antiviral immunity; however, the mechanisms involved are unknown. Using a panel of novel reagents and techniques, we quantitatively measured the effect of the stress-induced hormone corticosterone (CORT) on the efficiency of DCs to process and present virally expressed antigen, characterized the conditions for this CORT-mediated effect, and delineated the components of the MHC class I pathway that were affected. We found that physiologically relevant levels of CORT, prior to infection and acting via the glucocorticoid receptor, suppressed the formation of peptide-MHC class I complexes on the surface of infected DCs. We further showed that this suppression of peptide-MHC class I complexes is via the action of CORT on elements of the class I pathway upstream from TAP that are involved in the generation of antigenic peptides. This CORT-mediated suppression of peptide-class I complexes on DCs also resulted in a marked reduction of their ability to activate a specific T cell hybridoma. These findings offer a mechanism contributing to the stress-induced suppression of host defenses against viral diseases and have implications for the efficacy of antiviral vaccines. At the most fundamental cellular level, this impairment of antigen processing has implications for the regulation of protein degradation in all cells, which is critical to many aspects of immune function.

CD8+ T cell cross-priming via transfer of proteasome substrates.

"Cross-priming" describes the activation of naïve CD8+ T cells by professional antigen-presenting cells that have acquired viral or tumor antigens from "donor" cells. Antigen transfer is believed to be mediated by donor cell-derived molecular chaperones bearing short peptide ligands generated by proteasome degradation of protein antigens. We show here that cross-priming is based on the transfer of proteasome substrates rather than peptides. These findings are potentially important for the rational design of vaccines that elicit CD8+ T cell responses.

Proteasomes get by with lots of help from their friends.

Proteasomes can't do it all. It was previously known that aminopeptidases frequently degrade proteasome-generated peptides. Now it appears that another protease, tripeptidyl peptidase II (TPP II), plays a critical role in cleaving proteasomal produced peptides into shorter peptides that can then be degraded by aminopeptidases.

Phagosomes are competent organelles for antigen cross-presentation.

The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.