Vaccination and Immune Checkpoint Inhibitors: Does Vaccination Increase the Risk of Immune-related Adverse Events? A Systematic Review of Literature.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have become part of cancer treatments. Their main side effects are immune-related adverse events (irAEs). So far, there has been no recommendation regarding routine vaccinations during ICIs treatment. Clinicians are aware of the risk of irAEs increases in this specific situation. The aim of this review of literature is to summarize the main studies about vaccination and ICIs interactions. METHODS: A systematic assessment of literature articles was performed by searching in PubMed (MEDLINE), and major oncology meeting following PRISMA guidelines. RESULTS: This review highlights the lack of literature. Indeed, most of the studies published were about influenza vaccination. Vaccination for patients under ICIs causes a humoral response and seems to be associated with an increase rate of seroconversion. Interestingly vaccination may provoke irAEs in ICIs-treated patients. So far, inactivated vaccines have not been contraindicated during ICI treatment. CONCLUSION: Larger prospective studies are needed in order to define a consensus on the use of vaccines under immunotherapy.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

Novel Sperm and Gonadotropin-releasing Hormone-based Recombinant Fusion Protein: Achievement of 100% Contraceptive Efficacy by Co-immunization of Male and Female Mice.

Improvements in long-term female contraception can be achieved by vaccinating with sperm-derived proteins. Here, recombinant proteins comprising either (i) N- (amino acid residues 1-80) or C- (amino acid residues 76-126) terminal fragments of mouse sperm protein 17 (Sp17) fused to the promiscuous T non-B cell epitope of tetanus toxoid (TT), amino acid residues 830-844 followed by di-lysine linker (KK) (TT-KK-Sp17N or TT-KK-Sp17C , respectively) or (ii) mouse equatorin (amino acid residues 21-185) fused to the T non-B cell epitope of bovine RNase (amino acid residues 94-104) were expressed in Escherichia coli. Immunization of female FVB/J mice, using alum as an adjuvant, led to the generation of high antibody titers against the above proteins. Antibodies against both N- and C-terminal fragments of Sp17 reacted with the entire capacitated mouse spermatozoa, whereas those against equatorin reacted exclusively with the equatorial region. Despite the reactivity of all immune sera, only sera from mice immunized with TT-KK-Sp17N and TT-KK-Sp17C significantly reduced mouse in vitro fertilization. Mating studies of the immunized females with un-immunized male mice revealed the highest infertility in the TT-KK-Sp17C -immunized group. In an attempt to further boost the immune response, the C-terminal fragment of Sp17 was expressed as fusion protein with a tandem repeat of gonadotropin-releasing hormone (GnRH) (Sp17C -GnRH2 ). Immunization of both male and female mice with Sp17C -GnRH2 led to higher contraceptive efficacy compared to mice immunized with TT-KK-Sp17C . Interestingly, mating studies wherein partners were both immunized with Sp17C -GnRH2 showed a complete failure of female mice to conceive. Thus, immunization of both males and females with Sp17C -GnRH2 has the potential to increase contraceptive efficacy. Mol. Reprod. Dev. 83: 1048-1059, 2016. © 2016 Wiley Periodicals, Inc.

Poor functional immune recovery in aged HIV-1-infected patients following successfully treatment with antiretroviral therapy.

Aging is now a well-recognized characteristic of the HIV-infected population and both AIDS and aging are characterized by a deficiency of the T-cell compartment. The objective of the present study was to evaluate the impact of antiretroviral (ARV) therapy in recovering functional response of T cells to both HIV-1-specific ENV peptides (ENV) and tetanus toxoid (TT), in young and aged AIDS patients who responded to ARV therapy by controlling virus replication and elevating CD4(+) T cell counts. Here, we observed that proliferative response of T-cells to either HIV-1-specific Env peptides or tetanus toxoid (TT) was significantly lower in older antiretroviral (ARV)-treated patients. With regard to cytokine profile, lower levels of IFN-γ, IL-17 and IL-21, associated with elevated IL-10 release, were produced by Env- or TT-stimulated T-cells from older patients. The IL-10 neutralization by anti-IL-10 mAb did not elevate IFN-γ and IL-21 release in older patients. Finally, even after a booster dose of TT, reduced anti-TT IgG titers were quantified in older AIDS patients and it was related to both lower IL-21 and IFN-γ production and reduced frequency of central memory T-cells. Our results reveal that ARV therapy, despite the adequate recovery of CD4(+) T cell counts and suppression of viremia, was less efficient in recovering adequate immune response in older AIDS patients.

Tetanus shot may improve glioblastoma treatment.

Preconditioning the immune system with a tetanus/diphtheria toxoid significantly improved the effectiveness of dendritic cell immunotherapy and extended overall survival in a small, randomized study of patients with glioblastoma.

Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

IL-10-overexpressing B cells regulate innate and adaptive immune responses.

BACKGROUND: Distinct human IL-10-producing B-cell subsets with immunoregulatory properties have been described. However, the broader spectrum of their direct cellular targets and suppressive mechanisms has not been extensively studied, particularly in relation to direct and indirect IL-10-mediated functions. OBJECTIVE: The aim of the study was to investigate the effects of IL-10 overexpression on the phenotype and immunoregulatory capacity of B cells. METHODS: Primary human B cells were transfected with hIL-10, and IL-10-overexpressing B cells were characterized for cytokine and immunoglobulin production by means of specific ELISA and bead-based assays. Antigen presentation, costimulation capacity, and transcription factor signatures were analyzed by means of flow cytometry and quantitative RT-PCR. Effects of IL-10-overexpresing B cells on Toll-like receptor-triggered cytokine release from PBMCs, LPS-triggered maturation of monocyte-derived dendritic cells, and tetanus toxoid-induced PBMC proliferation were assessed in autologous cocultures. RESULTS: IL-10-overexpressing B cells acquired a prominent immunoregulatory profile comprising upregulation of suppressor of cytokine signaling 3 (SOCS3), glycoprotein A repetitions predominant (GARP), the IL-2 receptor α chain (CD25), and programmed cell death 1 ligand 1 (PD-L1). Concurrently, their secretion profile was characterized by a significant reduction in levels of proinflammatory cytokines (TNF-α, IL-8, and macrophage inflammatory protein 1α) and augmented production of anti-inflammatory IL-1 receptor antagonist and vascular endothelial growth factor. Furthermore, IL-10 overexpression was associated with a decrease in costimulatory potential. IL-10-overexpressing B cells secreted less IgE and potently suppressed proinflammatory cytokines in PBMCs, maturation of monocyte-derived dendritic cells (rendering their profile to regulatory phenotype), and antigen-specific proliferation in vitro. CONCLUSION: Our data demonstrate an essential role for IL-10 in inducing an immunoregulatory phenotype in B cells that exerts substantial anti-inflammatory and immunosuppressive functions.

Increased frequencies of CD11b(+) CD33(+) CD14(+) HLA-DR(low) myeloid-derived suppressor cells are an early event in melanoma patients.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population characterized by immunosuppressive activity. Elevated levels of MDSC in peripheral blood are found in inflammatory diseases as well as in malignant tumors where they are supposed to be major contributors to mechanisms of tumor-associated tolerance. We investigated the frequency and function of MDSC in peripheral blood of melanoma patients and observed an accumulation of CD11b(+) CD33(+) CD14(+) HLA-DR(low) MDSC in all stages of disease (I-IV), including early stage I patients. Disease progression and enhanced tumor burden did not result in a further increase in frequencies or change in phenotype of MDSC. By investigation of specific MDSC-associated cytokines in patients' sera, we found an accumulation of IL-8 in all stages of disease. T-cell proliferation assays revealed that MDSC critically contribute to suppressed antigen-specific T-cell reactivity and thus might explain the frequently observed transient effects of immunotherapeutic strategies in melanoma patients.

Cytokine responses in relation to age, gender, body mass index, Mycobacterium tuberculosis infection, and otitis media among Inuit in Greenland.

OBJECTIVES: To evaluate the cytokine response pattern in Inuit in Greenland in relation to age, gender, body mass index (BMI), Mycobacterium tuberculosis infection (MTI), and otitis media (OM) to assess whether Inuit may have signs of impaired immune responsiveness to infection. METHODS: A cross-sectional health assessment was conducted among inhabitants of Maniitsoq, West Greenland, in 2009, and several health outcomes were measured. The prevalence of MTI, overweight, and obesity was assessed among 263 school children and 137 adults, and OM was assessed among the children. Cytokine responses were measured in whole blood cultures after stimulation with phytohemagglutinin or purified protein derivative (PPD). Associations between cytokine concentrations, age, gender, BMI, MTI, and OM were estimated by linear regression. RESULTS: Adults had generally higher cytokine concentrations than children. Children with MTI had 2.7 times higher interleukin (IL)-10 concentrations than those without (P = 0.01), and girls had 80% higher IL-10 than boys (P < 0.01) after phytohemagglutinin stimulation. Interferon (IFN)γ and tumor necrosis factor (TNF) concentrations were strongly elevated among children (P(IFNγ) < 0.001 and P(TNF) < 0.001) and adults (P(IFNγ) < 0.001 and P(TNF) <0.01) with MTI compared to those without after PPD stimulation. Adult women had significantly lower IFNγ (P = 0.03) and TNF (P = 0.04) concentrations than men. TNF was positively correlated with BMI in children (P = 0.01), and IL-10 was positively correlated with BMI in adults (P = 0.0004) after PPD stimulation. CONCLUSION: We found cytokine patterns similar to those reported from other immune competent study populations. Therefore, the study does not support the suggestion that Inuit may have impaired immune reactivity to infection.

B-cell exposure to self-antigen induces IL-10 producing B cells as well as IL-6- and TNF-α-producing B-cell subsets in healthy humans.

Human B cells are able to secrete IL-10 after stimulation with mitogens, but their ability to produce IL-10 and regulate T-cell responses after stimulation with self-antigens is unclear. We co-cultured thyroglobulin-pulsed B cells from healthy donors with autologous T cells and observed production of IL-10 and TGF-ß, in addition to TNF-α and IL-6. Pulsing with foreign antigen, tetanus toxoid (TT), induced a Th1-response with minimal IL-10 production. After thyroglobulin-pulsing, 1.10±0.50% of B cells and 1.00±0.20% of CD4(+) T cells produced IL-10, compared to 0.29±0.19% of B cells (P=0.01) and 0.13±0.15% of CD4(+) T cells (P=0.006) following TT-pulsing. Thyroglobulin-stimulated, IL-10-secreting B cells were enriched within CD5(+) and CD24(high) cells. While thyroglobulin-pulsed B cells induced only modest proliferation of CD4(+) T cells, B cells pulsed with TT induced vigorous proliferation. Thus, B cells mediate self-antigen-specific IL-10, TNF-α and IL-6 production in co-cultures with T cells and contribute actively to these cytokine secretions.

Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation.

Antigen-specific proliferation is a critical function of memory T cells that is often utilised to measure vaccine immunogenicity and T cell function. We proposed that measurement of intracellular expression of the nuclear protein, Ki67, could reliably assess specific T cell proliferation in vitro. Ki67 was expressed in CD4+ and CD8+ T cells that had undergone in vitro proliferation after 6-day culture of human whole blood or PBMC with antigens. T cells cultured with no antigen did not express Ki67. When compared to current flow cytometry based proliferation assays, Ki67 detected proliferating cells with greater sensitivity than BrdU incorporation, whereas its sensitivity was similar to dye dilution of Oregon Green (OG), a CFSE derivative. Overall, the magnitude and cytokine expression profile of proliferating T cells detected by Ki67 expression correlated strongly with T cells detected with BrdU or OG. The intra-assay variability of Ki67 proliferation was 2-3% for CD4+ T cells, and 10-16% for CD8+ T cells. Finally, we demonstrate that the Ki67 assay detects tetanus toxoid-specific CD4+ T cell proliferation after infant vaccination with tetanus toxoid (TT). Overall our data suggest that intracellular Ki67 expression provides a specific, quantitative and reproducible measure of antigen-specific T cell proliferation in vitro.

Altered T cell costimulation during chronic hepatitis B infection.

T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

Monitoring the immune response using real-time PCR.

Induction of an immune response to a particular antigen is the basis of vaccination. This has been done for years to prevent infectious diseases, and has the potential for the treatment of cancer. The immune response is nowadays more precisely modulated rather than simply induced, like in case of immunotherapy of allergic diseases. Likewise, autoimmune diseases are associated with an inappropriate immune response, and many efforts are made for specifically inhibiting this unwanted response. A possible line of attack is the induction of an antigen-specific immune tolerance, which also has a use in the field of transplantation, where allogeneic responses are deleterious for the graft. In all of these fields of fundamental and clinical medicine, the modulation of immune response requires the assistance of laboratory tests, among which real-time PCR appears more and more helpful. This chapter describes a protocol to quantify immune-related mRNAs using reverse transcription-real-time PCR. The transcripts can be quantified in cultured cells or in cultured whole blood, after an incubation period in the presence of the antigen to which the immune response is analyzed. This is the typical approach to evaluate the efficacy of a vaccine. The transcripts can also be quantified directly in the biological sample, giving information about the in vivo immune status of the individual. The techniques to achieve these different methods are described, and are illustrated by the analysis of the response against the toxoid tetanus antigen.

Broad influenza-specific CD8+ T-cell responses in humanized mice vaccinated with influenza virus vaccines.

The development of novel human vaccines would be greatly facilitated by the development of in vivo models that permit preclinical analysis of human immune responses. Here, we show that nonobese diabetic severe combined immunodeficiency (NOD/SCID) beta(2) microglobulin(-/-) mice, engrafted with human CD34+ hematopoietic progenitors and further reconstituted with T cells, can mount specific immune responses against influenza virus vaccines. Live attenuated trivalent influenza virus vaccine induces expansion of CD8+ T cells specific to influenza matrix protein (FluM1) and nonstructural protein 1 in blood, spleen, and lungs. On ex vivo exposure to influenza antigens, antigen-specific CD8+ T cells produce IFN-gamma and express cell-surface CD107a. FluM1-specific CD8+ T cells can be also expanded in mice vaccinated with inactivated trivalent influenza virus vaccine. Expansion of antigen-specific CD8+ T cells is dependent on reconstitution of the human myeloid compartment. Thus, this humanized mouse model permits preclinical testing of vaccines designed to induce cellular immunity, including those against influenza virus. Furthermore, this work sets the stage for systematic analysis of the in vivo functions of human DCs. This, in turn, will allow a new approach to the rational design and preclinical testing of vaccines that cannot be tested in human volunteers.

Development of T cell-mediated immunity after autologous stem cell transplantation: prolonged impairment of antigen-stimulated production of gamma-interferon.

The conditioning regimens for autologous SCT (auto-SCT) lead to impairment of the immune system and concomitant increase in susceptibility to infections. We studied the recovery of cellular immunity by in vitro analysis of T-cell proliferation and cytokine production profiles during the first 15 months after auto-SCT in patients with multiple myeloma and non-Hodgkin's lymphoma. PBMC were collected at 6, 9 and 15 months after transplantation and stimulated with a combination of CD2 and CD28 monoclonal antibodies, with PHA or with tetanus toxoid as recall antigen. A multiplex enzyme linked immunoassay was used to determine levels of Th1 cytokines IL-2, IFN-gamma and tumour-necrosis factor-alpha (TNF-alpha), Th2 cytokines IL-4, IL-5 and IL-13, the regulatory cytokine IL-10 and the proinflammatory cytokines IL-1alpha, IL-1beta, IL-6 and the chemokine IL-8. T-cell proliferation progressively increased from 6 to 15 months after auto-SCT. Overall, cytokine production increased after auto-SCT. Production of Th2 cytokines IL-5 and IL-13 was superior to production of Th1 cytokines IFN-gamma and TNF-alpha. We hypothesize that prolonged impairment of IFN-gamma production might contribute to the relatively high incidence of viral infections after auto-SCT.

Phenotypic and functional effects of heat shock protein 90 inhibition on dendritic cell.

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-gamma secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.

Frequency analysis of HLA-B7-restricted Epstein-Barr virus-specific cytotoxic T lymphocytes in patients with multiple sclerosis and healthy controls.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of multiple sclerosis (MS), however, the mechanisms by which EBV may be involved in MS are unknown. We here have investigated the frequency of EBV-specific cytotoxic T lymphocytes (CTL) in human leukocyte antigen (HLA)-B7(+) patients with MS and healthy controls using enzyme-linked immunospot assays and seven previously characterized HLA-B7-restricted immunogenic EBV peptides. Overall, there were no significant differences in the frequency of EBV-specific CTL between both groups. These data do not support the hypothesis that EBV could play a role in MS by inducing quantitatively altered EBV-specific CTL responses. Other pathogenic mechanisms for EBV in MS remain to be elucidated.