Chimeric Yellow Fever/Dengue Virus as a Candidate Dengue Vaccine: Quantitation of the Dengue Virus-Specific CD8 T-Cell Response

We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against DEN-2 virus challenge, indicating that protection was mediated by the DEN-2 virus prM- and E-specific immune responses. YF/DEN vaccine-primed CD8 T cells expanded and were efficiently recruited into the central nervous systems of DEN-2 virus challenged mice. At 5 days after challenge, 3 to 4% of CD8 T cells in the spleen were specific for the prM and E proteins, and 34% of CD8 T cells in the central nervous system recognized these proteins. Depletion of either CD4 or CD8 T cells, or both, strongly reduced the protective efficacy of the YF/DEN virus, stressing the key role of the antiviral T-cell response

Cyclophosphamide Chemotherapy Sensitizes Tumor Cells to TRAIL-Dependent CD8 T Cell-Mediated Immune Attack Resulting in Suppression of Tumor Growth

Background: Anti-cancer chemotherapy can be simultaneously lymphodepleting and immunostimulatory. Pre-clinical models clearly demonstrate that chemotherapy can synergize with immunotherapy, raising the question how the immune system can be mobilized to generate anti-tumor immune responses in the context of chemotherapy. Methods and Findings: We used a mouse model of malignant mesothelioma, AB1-HA, to investigate T cell-dependent tumor resolution after chemotherapy. Established AB1-HA tumors were cured by a single dose of cyclophosphamide in a CD8 T cell- and NK cell-dependent manner. This treatment was associated with an IFN-α/β response and a profound negative impact on the anti-tumor and total CD8 T cell responses. Despite this negative effect, CD8 T cells were essential for curative responses. The important effector molecules used by the anti-tumor immune response included IFN-γ and TRAIL. The importance of TRAIL was supported by experiments in nude mice where the lack of functional T cells could be compensated by agonistic anti-TRAIL-receptor (DR5) antibodies. Conclusion: The data support a model in which chemotherapy sensitizes tumor cells for T cell-, and possibly NK cell-, mediated apoptosis. A key role of tumor cell sensitization to immune attack is supported by the role of TRAIL in tumor resolution and explains the paradox of successful CD8 T cell-dependent anti-tumor responses in the absence of CD8 T cell expansion

H5N1 Influenza Vaccine Formulated with AS03A Induces Strong Cross-Reactive and Polyfunctional CD4 T-Cell Responses

Objective Adjuvantation of an H5N1 split-virion influenza vaccine with AS03(A) substantially reduces the antigen dose required to produce a putatively protective humoral response and promotes cross-clade neutralizing responses. We determined the effect of adjuvantation on antibody persistence and B- and T-cell-mediated immune responses. Methods Two vaccinations with a split-virion A/Vietnam/1194/2004 (H5N1, clade 1) vaccine containing 3.75-30 mu g hemagglutinin and formulated with or without adjuvant were administered to groups of 50 volunteers aged 18-60 years. Results Adjuvantation of the vaccine led to better persistence of neutralizing and hemagglutination-inhibiting antibodies and higher frequencies of antigen-specific memory B cells. Cross-reactive and polyfunctional H5N1-specific CD4 T cells were detected at baseline and were amplified by vaccination. Expansion of CD4 T cells was enhanced by adjuvantation. Conclusion Formulation of the H5N1 vaccine with AS03(A) enhances antibody persistence and induces stronger T- and B-cell responses. The cross-clade T-cell immunity indicates that the adjuvanted vaccine primes individuals to respond to either infection and/or subsequent vaccination with strains drifted from the primary vaccine strain

Innate immunity defines the capacity of antiviral T cells to limit persistent infection

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence

Characterization of T-cell immune responses in clinical trials of the candidate RTS,S malaria vaccine

The candidate malaria vaccine RTS,S has demonstrated 45.7% efficacy over 18 months against all clinical disease in a phase-III field study of African children. RTS,S targets the circumsporozoite protein (CSP), which is expressed on the Plasmodium sporozoite during the pre-erythrocyte stage of its life-cycle; the stage between mosquito bite and liver infection. Early in the development of RTS,S, it was recognized that CSP-specific cell-mediated immunity (CMI) was required to complement CSP-specific antibody-mediated immunity. In reviewing RTS,S clinical studies, associations between protection and various types of CMI (CSP-specific CD4+ T cells and INF-γ ELISPOTs) have been identified, but not consistently. It is plausible that certain CD4+ T cells support antibody responses or co-operate with other immune-cell types to potentially elicit protection. However, the identities of vaccine correlates of protection, implicating either CSP-specific antibodies or T cells remain elusive, suggesting that RTS,S clinical trials may benefit from additional immunogenicity analyses that can be informed by the results of controlled human malaria infection studies

Mutagenesis of the RGD Motif in the Yellow Fever Virus 17D Envelope Protein

The envelope protein of yellow fever virus 17D (YFV-17D) contains a solvent-exposed RGD motif, which has led to the suggestion that integrins may function as cellular receptors for YFV-17D. We found that mutating the RGD motif to RGE had no effect on viral titers, whereas changing RGD to TGD, TGE, TAD, TAE, or RGS led to reduced titers. Substitution of RGD by RAD or RAE yielded RNA genomes that replicated in mammalian cells but could not spread to neighboring cells at 37°C. These mutants did spread through the cell monolayer at 30°C (both in mosquito cells and in SW13 cells) and viruses grown at this temperature were capable of infecting mammalian cells at 37°C. These results strongly suggest that RGD-mediated integrin binding does not play a major role in YFV-17D entry, since the RGD to RAD mutation, as well as many or all of the other mutations studied, should disrupt all RGD-dependent integrin binding. However, the RGD to RAD or RAE mutations (as well as TAD and TAE) severely destabilized the envelope protein at 37°C, providing an explanation for the observed phenotype. Implications of these findings are discussed in light of the fact that mutations that alter tropism or virulence in different flaviviruses are often found within the loop containing the RGD motif

Immunogenic anti-cancer chemotherapy as an emerging concept

Tumors can acquire mutations or hijack regulatory pathways of the host immune system to render them resistant to immune attack. Standard first line therapies such as chemotherapy and radiation were not thought to provoke natural immunity to cancer, but recent findings demonstrating that dying tumor cells present and release key signals to stimulate or evade neighboring leukocytes are challenging that view. Killing tumor cells in a manner that provides danger signals and tumor antigens in the right context promotes the engagement of innate and adaptive immunity; however, this response alone will not be effective against established cancer. Coincidently driving the immune response with specific monoclonal antibodies and other immunomodulators that activate and mature dendritic cells and co-stimulate T cells and other lymphocytes is one approach. Additionally releasing immune checkpoints and inhibiting tumor-derived molecules that prevent effective tumor immunity is another. Combined these approaches have enormous potential to improve the current outcomes from conventional cancer therapy

Impact of Epitope Escape on PD-1 Expression and CD8 T-Cell Exhaustion during Chronic Infection▿

During some persistent viral infections, virus-specific T-cell responses wane due to the antigen-specific deletion or functional inactivation (i.e., exhaustion) of responding CD8 T cells. T-cell exhaustion often correlates with high viral load and is associated with the expression of the inhibitory receptor PD-1. In other infections, functional T cells are observed despite high levels of pathogen persistence. The reasons for these different T-cell fates during chronic viral infections are not clear. Here, we tracked the fate of virus-specific CD8 T cells in lymphocytic choriomeningitis virus (LCMV)-infected mice during viral clearance, the persistence of wild-type virus, or the selection and persistence of a viral variant that abrogates the presentation of a single epitope. Viral clearance results in PD-1lo functional virus-specific CD8 T cells, while the persistence of wild-type LCMV results in high PD-1 levels and T-cell exhaustion. However, following the emergence of a GP35V→A variant virus that abrogates the presentation of the GP33 epitope, GP33-specific CD8 T cells remained functional, continued to show low levels of PD-1, and reexpressed CD127, a marker of memory T-cell differentiation. In the same animals and under identical environmental conditions, CD8 T cells recognizing nonmutated viral epitopes became physically deleted or were PD-1hi and nonfunctional. Thus, the upregulation of PD-1 and the functional inactivation of virus-specific T cells during chronic viral infection is dependent upon continued epitope recognition. These data suggest that optimal strategies for vaccination should induce high-magnitude broadly specific T-cell responses that prevent cytotoxic T-lymphocyte escape and highlight the need to evaluate the function of vaccine-induced T cells in the context of antigens presented during virus persistence

Role and plasticity of Th1 and Th17 responses in immunity to Staphylococcus aureus

The human commensal Staphylococcus aureus (SA) is a leading cause of skin/soft tissue and surgical-site infections, and bacteremia. Functional antibodies and T-cell-mediated immunity, particularly Th1/Th17 responses, are thought to mediate protection. Vaccine development may be hindered by modulation of vaccine-induced T cells by pathogen-activated immunoregulatory responses, e.g., via IL-10. We screened SA proteins for CD4+ T-cell-activating and IL-10/IL-17-inducing capacities using healthy donor-derived PBMCs. Responses were characterized (Th1/Th17/Th22/immunosuppressive IL-10-producing cells) using intracellular cytokine staining and flow cytometry. Phenotypic plasticity of Th1/Th17 cells was evaluated under pro- or anti-inflammatory conditions using modulatory cytokines. The impact of vaccination on SA-specific memory responses was assessed using samples from a clinical trial evaluating AS03-adjuvanted and non-adjuvanted multicomponent (CPS5/CPS8/α-toxin/ClfA) vaccines (NCT01160172). The donors exhibited SA-specific memory T-cell responses, indicative of pre-existing immunity to SA. We identified effective activators of Th1 responses (EbhA/IsaA/SdrE/MntC/Aaa/α-toxin), and Th17 and Th1/Th17 responses (EbhA/IsaA/SdrE and, to a lesser extent, α-toxin), but not of Th22 responses or IL-10 production. MRPII, IsdA, and ClfA were inefficient CD4+ T-cell activators in our assays. IL-10, likely produced by innate immune cells, influenced mainly Th1 cells by suppressing IFN-γ production. The memory CD4+ T-cells observed after long-term stimulation with α-toxin and ClfA indicated that vaccination with these proteins had induced expansion of pre-existing Th1 but not Th17 responses, without apparent adjuvant effect, confirming the trial data. The Th1/Th17-driving proteins (EbhA/IsaA/SdrE) shared low IL-10-promoting abilities and restricted phenotypic plasticity under pro- and anti-inflammatory conditions. Given the complex immunopathology and multiple virulence factors, identification of Th1/Th17-driving antigens, adjuvants and administration routes, and delineation of the role of memory responses, may advance vaccine development

Long-term persistence of cell-mediated and humoral responses to A(H1N1)pdm09 influenza virus vaccines and the role of the AS03 adjuvant system in adults during two randomized controlled trials

We investigated the role of AS03(A) (here AS03), an alpha-tocopherol oil- in-water emulsion-based adjuvant system, on the long-term persistence of humoral and cell-mediated immune responses to A(H1N1) pdm09 influenza vaccines. In two studies, a total of 261 healthy adults (<= 60 years old) were randomized to receive two doses of AS03-adjuvanted vaccine containing 3.75 mu g of hemagglutinin (HA) or nonadjuvanted vaccine containing 15 mu g of hemagglutinin (in study A) or 3.75 mu g of hemagglutinin (in study B) 21 days apart. Hemagglutination inhibition (HI) antibody, memory B-cell, and CD4(+)/CD8(+) T-cell responses were characterized up to 1 year following dose 1. We also assessed the effects of age and seasonal influenza vaccination history. AS03-adjuvanted (3.75 mu g HA) vaccine and nonadjuvanted vaccine at 15 mu g but not at 3.75 mu g HA elicited HI antibody responses persisting at levels that continued to meet European licensure criteria through month 12. At month 12, the geometric mean titer for AS03-adjuvanted vaccine was similar to that for nonadjuvanted (15-mu g) vaccine in study A (1: 86 and 1: 88, respectively) and higher than that for nonadjuvanted (3.75-mu g) vaccine in study B (1: 77 and 1: 35, respectively). A(H1N1) pdm09-specific CD4(+) T-cell and B-cell responses were stronger in AS03-adjuvanted groups and persisted only in these groups for 12 months at levels exceeding prevaccination frequencies. Advancing age and a seasonal vaccination history tended to reduce HI antibody and memory B-cell responses and, albeit less consistently, CD4(+) T-cell responses. Thus, AS03 seemed to enhance the persistence of humoral and cell-mediated responses to A(H1N1) pdm09 vaccine, allowing for antigen sparing and mitigating potential negative effects of age and previous seasonal vaccination