Bimodal Effect of NKG2A Blockade on Intratumoral and Systemic CD8 T Cell Response Induced by Cancer Vaccine.

Immune check-point blockade (ICB) has revitalized cancer immunotherapy, showing unprecedented efficacy despite only a narrow number of indications and with limited long-term protection. Cancer vaccines are promising combination partners for ICB to widen the patient population profiting from these treatments. Therapeutic heterologous prime-boost vaccination with KISIMATM protein vaccine and VSV-GP-TAg oncolytic virus was shown to inflame the tumor microenvironment, promoting significant infiltration of antigen-specific CD8 T cells resulting in robust antitumoral efficacy in mouse tumor models, and clinical trials are currently ongoing. Here, we report the impact of NKG2A blockade on antitumoral CD8 T cell immune response elicited by KISIMA-VSV-GP-TAg vaccination in tumor mouse models. Combination therapy significantly reduced the amount of vaccine-induced exhausted CD8 T cells infiltrating the tumor, resulting in short-term improved tumor growth control and prolonged mouse survival, while it also influenced the establishment of systemic effector memory CD8 T cell response. Taken together, these data show a compartment-dependent effect of NKG2A blockade on cancer vaccine-induced T cell immunity, increasing intratumoral T cell efficacy and attenuating the development of peripheral effector memory CD8 T cell response.

Replication-Deficient Lymphocytic Choriomeningitis Virus-Vectored Vaccine Candidate for the Induction of T Cell Immunity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) represents a major burden to global health, and refined vaccines are needed. Replication-deficient lymphocytic choriomeningitis virus (rLCMV)-based vaccine vectors against cytomegalovirus have proven safe for human use and elicited robust T cell responses in a large proportion of vaccine recipients. Here, we developed an rLCMV vaccine expressing the Mtb antigens TB10.4 and Ag85B. In mice, rLCMV elicited high frequencies of polyfunctional Mtb-specific CD8 and CD4 T cell responses. CD8 but not CD4 T cells were efficiently boosted upon vector re-vaccination. High-frequency responses were also observed in neonatally vaccinated mice, and co-administration of rLCMV with Expanded Program of Immunization (EPI) vaccines did not result in substantial reciprocal interference. Importantly, rLCMV immunization significantly reduced the lung Mtb burden upon aerosol challenge, resulting in improved lung ventilation. Protection was associated with increased CD8 T cell recruitment but reduced CD4 T cell infiltration upon Mtb challenge. When combining rLCMV with BCG vaccination in a heterologous prime-boost regimen, responses to the rLCMV-encoded Mtb antigens were further augmented, but protection was not significantly different from rLCMV or BCG vaccination alone. This work suggests that rLCMV may show utility for neonatal and/or adult vaccination efforts against pulmonary tuberculosis.

A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.

STING Agonist Combined to a Protein-Based Cancer Vaccine Potentiates Peripheral and Intra-Tumoral T Cell Immunity.

Combining different immunotherapy approaches is currently building the future of immunotherapy, with the view to maximize anti-tumoral efficacy for larger patient population. The KISIMA™ platform allows the development of protein-based cancer vaccines able to induce tumor-specific T cell response resulting in anti-tumoral efficacy in various mouse models. Intra-tumoral administration of stimulator of interferon gene agonists (STINGa) was shown to induce a potent inflammatory response leading to the development of tumor-specific immunity. Here, we explored the efficacy and mechanisms of action of subcutaneous STINGa treatment combined with therapeutic vaccination in various mouse tumor models. This combinatory treatment highly enhanced frequency and effector function of both peripheral and intra-tumoral antigen-specific CD8 T cells, promoting potent IFNγ and TNFα production along with increased cytotoxicity. Moreover, combination therapy favorably modulated the tumor microenvironment by dampening immune-suppressive cells and increasing CD4 T cell infiltration together with their polarization toward Th1 phenotype. Combination with STINGa treatment improved the effect of therapeutic vaccination, resulting in a prolonged control and slower growth of B16-OVA and TC-1 tumors. Altogether, the results presented here highlight the potential of combining STINGa with a therapeutic protein vaccine for cancer treatment.

Novel Protein-Based Vaccine against Self-Antigen Reduces the Formation of Sporadic Colon Adenomas in Mice.

Novel immunopreventive strategies are emerging that show great promise for conferring long-term protection to individuals at high risk of developing colorectal cancer. The KISIMA vaccine platform utilizes a chimeric protein comprising: (1) a selected tumor antigen; (2) a cell-penetrating peptide to improve antigen delivery and epitope presentation, and (3) a TLR2/4 agonist to serve as a self-adjuvant. This study examines the ability of a KISIMA vaccine against achaete-scute family bHLH transcription factor 2 (Ascl2), an early colon cancer antigen, to reduce colon tumor formation by stimulating an anti-tumor immune response. Vaccine administrations were well-tolerated and led to circulating antibodies and antigen-specific T cells in a mouse model of colorectal cancer. To assess preventive efficacy, the vaccine was administered to mice either alone or in combination with the immune checkpoint inhibitor anti-PD-1. When delivered to animals prior to colon tumor formation, the combination strategy significantly reduced the development of colon microadenomas and adenomas, as compared to vehicle-treated controls. This response was accompanied by an increase in the intraepithelial density of CD3+ T lymphocytes. Together, these data indicate that the KISIMA-Ascl2 vaccine shows great potential to be a safe and potent immunopreventive intervention for individuals at high risk of developing colorectal cancer.

A TLR7/8 Agonist-Including DOEPC-Based Cationic Liposome Formulation Mediates Its Adjuvanticity Through the Sustained Recruitment of Highly Activated Monocytes in a Type I IFN-Independent but NF-κB-Dependent Manner.

Novel adjuvants, such as Toll-like receptors (TLRs) agonists, are needed for the development of new formulations able to circumvent limitations of current vaccines. Among TLRs, TLR7/8 agonists represent promising candidates, as they are well described to enhance antigen-specific antibody responses and skew immunity toward T helper (TH) 1 responses. We find here that the incorporation of the synthetic TLR7/8 ligand 3M-052 in a cationic DOEPC-based liposome formulation shifts immunity toward TH1 responses and elicits strong and long-lasting germinal center and follicular T helper cell responses in adult mice. This reflects the prolonged recruitment of innate cells toward the site of immunization and homing of activated antigen-loaded monocytes and monocyte-derived dendritic cells toward draining lymph nodes. We further show that this adjuvanticity is independent of type I IFN but NF-κB-dependent. Overall, our data identify TLR7/8 agonists incorporated in liposomes as promising and effective adjuvants to enhance TH1 and germinal center responses.

Targeting self and neo-epitopes with a modular self-adjuvanting cancer vaccine.

Induction of a potent CD4 and CD8 T-cell response against tumor-specific and tumor-associated antigen is critical for eliminating tumor cells. Recent vaccination strategies have been hampered by an inefficacious and low amplitude immune response. Here we describe a self-adjuvanted chimeric protein vaccine platform to address these challenges, characterized by a multidomain construction incorporating (i) a cell penetrating peptide (CPP) allowing internalization of several multiantigenic Major Histocompatibility Complex (MHC)-restricted peptides within (ii) the multiantigenic domain (Mad) and (iii) a TLR2/4 agonist domain (TLRag). Functionality of the resulting chimeric protein is based on the combined effect of the above-mentioned three different domains for simultaneous activation of antigen presenting cells and antigen cross-presentation, leading to an efficacious multiantigenic and multiallelic cellular immune response. Helper and cytotoxic T-cell responses were observed against model-, neo- and self-antigens, and were highly potent in several murine tumor models. The safety and the immunogenicity of a human vaccine candidate designed for colorectal cancer treatment was demonstrated in a non-human primate model. This newly engineered therapeutic vaccine approach is promising for the treatment of poorly infiltrated tumors that do not respond to currently marketed immunotherapies.

In-vivo gp100-specific Cytotoxic CD8+ T Cell Killing Assay.

Cytotoxic CD8+ T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans. Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8+ T cell melanoma epitope of the hgp10025-33 protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp10025-33 peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV. Measuring the ability of antigen-specific CD8+ T cells to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.

Identification of a novel population of highly cytotoxic c-Met-expressing CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met+ CTLs). Phenotypic and functional analysis of c-Met+ CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met- CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met+ CTLs in cell-mediated cytotoxicity reactions in vitro and in vivo and abrogates T-cell responses against metastatic melanoma in vivo Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met+ CTLs are a naturally occurring CD8+ T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8+ T-cell-mediated anti-tumor immunity.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

Cell-penetrating peptides-the Swiss Army knife of cancer vaccines.

Therapeutic cancer vaccination is an attractive treatment modality for cancer, but with limitations using existing whole-cell, peptide, or protein vaccines. We propose that a cell-penetrating peptide (CPP)-based vaccine delivering multi-epitopic antigens into antigen-presenting cells (APCs) offers great potential to induce an integrated antitumor immune response and robust, sustained therapeutic effect.

Novel Cell-Penetrating Peptide-Based Vaccine Induces Robust CD4+ and CD8+ T Cell-Mediated Antitumor Immunity.

Vaccines that can coordinately induce multi-epitope T cell-mediated immunity, T helper functions, and immunologic memory may offer effective tools for cancer immunotherapy. Here, we report the development of a new class of recombinant protein cancer vaccines that deliver different CD8(+) and CD4(+) T-cell epitopes presented by MHC class I and class II alleles, respectively. In these vaccines, the recombinant protein is fused with Z12, a novel cell-penetrating peptide that promotes efficient protein loading into the antigen-processing machinery of dendritic cells. Z12 elicited an integrated and multi-epitopic immune response with persistent effector T cells. Therapy with Z12-formulated vaccines prolonged survival in three robust tumor models, with the longest survival in an orthotopic model of aggressive brain cancer. Analysis of the tumor sites showed antigen-specific T-cell accumulation with favorable modulation of the balance of the immune infiltrate. Taken together, the results offered a preclinical proof of concept for the use of Z12-formulated vaccines as a versatile platform for the development of effective cancer vaccines.

Functional limitations of plasmacytoid dendritic cells limit type I interferon, T cell responses and virus control in early life.

Infant mortality from viral infection remains a major global health concern: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8⁺ T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC), which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8) is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.

Environmental and T cell-intrinsic factors limit the expansion of neonatal follicular T helper cells but may be circumvented by specific adjuvants.

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.

Homing and adhesion patterns determine the cellular composition of the bone marrow plasma cell niche.

According to commonly held concepts, plasma cell (PC) longevity in bone marrow (BM) depends upon their access to survival niches. These are thought to exist in nursery cell types, which support PCs by secreting PC survival factors. To better define PC survival niches and their functioning, we adoptively transferred traceable Blimp-1-(GFP) PCs into recipient mice lacking a proliferation-inducing ligand (APRIL), IL-6, or macrophage migration inhibitory factor. Transferred BMPCs were preferentially associated with Ly-6C(high) monocytes (normalized colocalization index: 9.84), eosinophils (4.29), and megakaryocytes (2.12). Although APRIL was essential for BMPC survival, PC recruitment into the proximity of nursery cells was unimpaired in APRIL-deficient mice, questioning the concept that the same factors account for attraction/retention of PCs as for their local survival. Rather, the order of colocalization with BMPCs (monocytes > eosinophils > megakaryocytes) reflected these cells' relative expression of CXCR4, VLA-4, and LFA-1, the homing and adhesion molecules that direct/retain PCs in the BM. This suggests a scenario wherein the cellular composition of the BMPC niche is defined by a common pattern of attraction/retention on CXCL12-abundant reticular docking cells. Thereby, PCs are directed to associate in a functional BM niche with hematopoietic CXCR4(+)VLA-4(+)LFA-1(+) nursery cells, which provide PC survival factors.

Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage.

Immunity to malaria has long been thought to be stage-specific. In this study we show that immunization of BALB/c mice with live erythrocytes infected with nonlethal strains of Plasmodium yoelii under curative chloroquine cover conferred protection not only against challenge by blood stage parasites but also against sporozoite challenge. This cross-stage protection was dose-dependent and long lasting. CD4(+) and CD8(+) T cells inhibited malaria liver but not blood stage. Their effect was mediated partially by IFN-gamma, and was completely dependent of NO. Abs against both pre-erythrocytic and blood parasites were elicited and were essential for protection against blood stage and liver stage parasites. Our results suggest that Ags shared by liver and blood stage parasites can be the foundation for a malaria vaccine that would provide effective protection against both pre-erythrocytic and erythrocytic asexual parasites found in the mammalian host.

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Protracted course of lymphocytic choriomeningitis virus WE infection in early life: induction but limited expansion of CD8+ effector T cells and absence of memory CD8+ T cells.

Viral infections in human infants frequently follow a protracted course, with higher viral loads and delayed viral clearance compared to viral infections in older children. To identify the mechanisms responsible for this protracted pattern of infection, we developed an infant infection murine model using the well-characterized lymphocytic choriomeningitis virus (LCMV) WE strain in 2-week-old BALB/c mice. In contrast to adult mice, in which viral clearance occurred as expected 8 days after infection, LCMV titers persisted for several weeks after infection of infant mice. LCMV-specific effector CD8(+) T cells were elicited in infant mice and fully functional on day 7 but rapidly waned and could not be recovered from day 12 onwards. We show here that this results from the failure of LCMV-specific CD8(+) T cells to expand and the absence of protective LCMV-specific memory CD8(+) T cells. Under these early life conditions, viral control and clearance are eventually achieved only through LCMV-specific B cells that contribute to protect infant mice from early death or chronic infection.