ABSTRACT
BACKGROUND AND AIMS: Induction of functional helper CD4+ T cells is the hallmark of a protective immune response against hepatitis C virus (HCV), associated with spontaneous viral clearance. Heterologous prime/boost viral vectored vaccination has demonstrated induction of broad and polyfunctional HCV-specific CD8+ T cells in healthy volunteers; however, much less is known about CD4+ T-cell subsets following vaccination. APPROACH AND RESULTS: We analyzed HCV-specific CD4+ T-cell populations using major histocompatibility complex class II tetramers in volunteers undergoing HCV vaccination with recombinant HCV adenoviral/modified vaccinia Ankara viral vectors. Peptide-specific T-cell responses were tracked over time, and functional (proliferation and cytokine secretion) and phenotypic (cell surface and intranuclear) markers were assessed using flow cytometry. These were compared to CD4+ responses in 10 human leukocyte antigen-matched persons with HCV spontaneous resolution and 21 chronically infected patients treated with directly acting antiviral (DAA) therapy. Vaccination induced tetramer-positive CD4+ T cells that were highest 1-4 weeks after boosting (mean, 0.06%). Similar frequencies were obtained for those tracked following spontaneous resolution of disease (mean, 0.04%). In addition, the cell-surface phenotype (CD28, CD127) memory subset markers and intranuclear transcription factors, as well as functional capacity of peptide-specific CD4+ T-cell responses characterized after vaccination, are comparable to those following spontaneous viral resolution. In contrast, helper responses in chronic infection were infrequently detected and poorly functional and did not consistently recover following HCV cure. CONCLUSIONS: Helper CD4+ T-cell phenotype and function following HCV viral vectored vaccination resembles "protective memory" that is observed following spontaneous clearance of HCV. DAA cure does not promote resurrection of exhausted CD4+ T-cell memory in chronic infection.
Subject(s)
Antiviral Agents/therapeutic use , Hepacivirus/immunology , Hepatitis C, Chronic/therapy , T-Lymphocytes, Helper-Inducer/immunology , Viral Hepatitis Vaccines/administration & dosage , Adenoviridae/genetics , Cell Line , Female , Genetic Vectors/genetics , Healthy Volunteers , Hepacivirus/isolation & purification , Hepatitis C, Chronic/diagnosis , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/virology , Humans , Immunogenicity, Vaccine , Immunologic Memory , Male , Middle Aged , Remission, Spontaneous , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Hepatitis Vaccines/genetics , Viral Hepatitis Vaccines/immunology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunologyABSTRACT
BACKGROUND: Respiratory syncytial virus (RSV) disease is a major cause of infant morbidity and mortality. This Phase I, randomized, observer-blind, placebo- and active-controlled study evaluated an investigational vaccine against RSV (ChAd155-RSV) using the viral vector chimpanzee-adenovirus-155, encoding RSV fusion (F), nucleocapsid, and transcription antitermination proteins. METHODS: Healthy 18-45-year-old adults received ChAd155-RSV, a placebo, or an active control (Bexsero) at Days (D) 0 and 30. An escalation from a low dose (5 × 109 viral particles) to a high dose (5 × 1010 viral particles) occurred after the first 16 participants. Endpoints were solicited/unsolicited and serious adverse events (SAEs), biochemical/hematological parameters, cell-mediated immunogenicity by enzyme-linked immunospot, functional neutralizing antibodies, anti RSV-F immunoglobin (Ig) G, and ChAd155 neutralizing antibodies. RESULTS: There were 7 participants who received the ChAd155-RSV low dose, 31 who received the ChAd155-RSV high dose, 19 who received the placebo, and 15 who received the active control. No dose-related toxicity or attributable SAEs at the 1-year follow-up were observed. The RSV-A neutralizing antibodies geometric mean titer ratios (post/pre-immunization) following a high dose were 2.6 (D30) and 2.3 (D60). The ratio of the fold-rise (D0 to D30) in anti-F IgG over the fold-rise in RSV-A-neutralizing antibodies was 1.01. At D7 after the high dose of the study vaccine, the median frequencies of circulating B-cells secreting anti-F antibodies were 133.3/106 (IgG) and 16.7/106 (IgA) in peripheral blood mononuclear cells (PBMCs). The median frequency of RSV-F-specific interferon γ-secreting T-cells after a ChAd155-RSV high dose was 108.3/106 PBMCs at D30, with no increase after the second dose. CONCLUSIONS: In adults previously naturally exposed to RSV, ChAd155-RSV generated increases in specific humoral and cellular immune responses without raising significant safety concerns. CLINICAL TRIALS REGISTRATION: NCT02491463.
Subject(s)
Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus Vaccines , Adenoviridae , Adolescent , Adult , Animals , Antibodies, Neutralizing , Antibodies, Viral , Humans , Leukocytes, Mononuclear , Middle Aged , Nucleocapsid , Pan troglodytes , Respiratory Syncytial Virus Infections/prevention & control , Viral Proteins , Virion , Young AdultABSTRACT
The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host's memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words).
Subject(s)
Adenoviruses, Human/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory , Muromegalovirus/immunology , Viral Vaccines/immunology , Adenovirus Infections, Human/immunology , Adenovirus Infections, Human/prevention & control , Adenoviruses, Human/genetics , Adenoviruses, Human/pathogenicity , Animals , Coinfection/immunology , Coinfection/prevention & control , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Herpesviridae Infections/immunology , Herpesviridae Infections/prevention & control , Host-Pathogen Interactions/immunology , Humans , Lac Operon , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Models, Immunological , Muromegalovirus/genetics , Muromegalovirus/pathogenicity , Receptors, Interleukin-18/deficiency , Receptors, Interleukin-18/genetics , Receptors, Interleukin-18/immunology , Viral Vaccines/geneticsABSTRACT
CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-α chain 7.2 (Vα7.2)-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) Vα7.2(+) and CD161(++) Vα7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.
Subject(s)
Interleukin-12/immunology , Interleukin-18/immunology , Mucous Membrane/immunology , Natural Killer T-Cells/immunology , Receptors, Interleukin-18/metabolism , T-Lymphocyte Subsets/immunology , CD8 Antigens/metabolism , Cell Line , Cell Separation , Flow Cytometry , Humans , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Interleukin-18/pharmacology , Lymphocyte Activation , NK Cell Lectin-Like Receptor Subfamily B/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/drug effectsABSTRACT
CD4(+) T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+) cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+) compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+) T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul) participated. CCR5-expression defined a CD4(+) subpopulation of predominantly CD45R0(+) memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+) vs CCR5(-); healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4(+) T-cells (predominantly CD45RA(+) naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+)CD45R0(+)CD4(+) memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4(+) T-cell loss is primarily driven by non-specific immune activation.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1/immunology , Immunologic Memory , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Adult , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation , Female , HIV Infections/virology , Humans , Leukocyte Common Antigens/metabolism , Male , Viral Tropism , Young AdultABSTRACT
CD8(+) T cell memory inflation, first described in murine CMV (MCMV) infection, is characterized by the accumulation of high-frequency, functional Ag-specific CD8(+) T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of Ag is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus's low-level persistence and stochastic reactivation. We developed a new model of memory inflation based on a ß-galactosidase (ßgal)-recombinant adenovirus vector. After i.v. administration in C57BL/6 mice, we observed marked memory inflation in the ßgal96 epitope, whereas a second epitope, ßgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype, and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC class II. As in MCMV, only the inflating epitope showed immunoproteasome independence. These data define a new model for memory inflation, which is fully replication independent, internally controlled, and reproduces the key immunologic features of the CD8(+) T cell response. This model provides insight into the mechanisms responsible for memory inflation and, because it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Immunologic Memory , Memory Disorders/immunology , Memory Disorders/virology , Adenoviridae/genetics , Adenoviridae/immunology , Animals , CD8-Positive T-Lymphocytes/pathology , Epitopes, T-Lymphocyte/administration & dosage , Epitopes, T-Lymphocyte/immunology , Female , Genetic Vectors/administration & dosage , Immunologic Memory/genetics , Male , Memory Disorders/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Muromegalovirus/genetics , Muromegalovirus/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
Convincing correlates of protective immunity against tuberculosis have been elusive. In BALB/c mice, intranasal immunization with a replication-deficient recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (adenovirus-85A) induces protective lower respiratory tract immunity against pulmonary challenge with Mycobacterium tuberculosis, while intradermal immunization with adenovirus-85A does not. Here we report that intranasal immunization with adenovirus-85A induces expression of the chemokine receptor CXCR6 on lung CD8 T lymphocytes, which is maintained for at least 3 months. CXCR6-positive antigen-specific T cell numbers are increased among bronchoalveolar lavage-recoverable cells. Similarly, intranasal immunization with recombinant antigen 85A with adjuvant induces CXCR6 expression on lung CD4 cells in BALB/c and C57BL/6 mice, while a synthetic ESAT6(1-20) peptide with adjuvant induces CXCR6 expression in C57BL/6 mice. Parenteral immunization fails to do so. Upregulation of CXCR6 is accompanied by a transient elevation of serum CXCL16 after intranasal immunization, and lung cells cultured ex vivo from mice immunized intranasally show increased production of CXCL16. Administration of CXCL16 and cognate antigen intranasally to mice previously immunized parenterally increases the number of antigen-specific T lymphocytes in the bronchoalveolar lavage-recoverable population, which mediates inhibition of the early growth of Mycobacterium tuberculosis after challenge. We conclude that expression of CXCR6 on lung T lymphocytes is a correlate of local protective immunity against Mycobacterium tuberculosis after intranasal immunization and that CXCR6 and CXCL16 play an important role in the localization of T cells within lung tissue and the bronchoalveolar lavage-recoverable compartment.
Subject(s)
Biomarkers/analysis , CD8-Positive T-Lymphocytes/immunology , Lung/immunology , Mycobacterium tuberculosis/immunology , Receptors, CXCR/analysis , Tuberculosis Vaccines/immunology , Acyltransferases/genetics , Acyltransferases/immunology , Adenoviridae/genetics , Administration, Intranasal , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bronchoalveolar Lavage Fluid/cytology , CD4-Positive T-Lymphocytes/chemistry , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/chemistry , Female , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, CXCR6 , Tuberculosis Vaccines/administration & dosage , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunologyABSTRACT
The immune response to human cytomegalovirus (HCMV) infection is characterized by the accumulation of HCMV-specific CD8(+) T cells, particularly in the elderly; such expansions may impair immune responses to other pathogens. We investigated mechanisms underlying HCMV-specific expansions in 12 young and 21 old healthy subjects (although not all analyses were performed on all subjects). Phenotypically, HCMV-pentamer(+) CD8(+) T cells were characterized by marked Vß restriction, advanced differentiation (being predominantly CD27(-) CD28(-) ), and variable CD45RO/RA expression. Although more common and larger in older subjects, expansions had similar phenotypic characteristics in the young. In one old subject, repeated studies demonstrated stability in size and Vß distribution of pentamer(+) populations over 6 years. We tested whether HCMV-specific CD8(+) T-cell expansions arose from accelerated proliferation or extended lifespan by in vivo labelling with deuterated glucose and ex vivo Ki-67 expression. Uptake of deuterated glucose was lower in pentamer(+) cells than in pentamer(-) CD8(+) CD45RO(+) or CD8(+) CD45RA(+) cells in three old subjects, consistent with reduced proliferation and extended lifespan. Similarly Ki-67 labelling showed no evidence for increased proliferation in HCMV-specific CD8(+) expansions in older subjects, although pentamer(-) CD45RA(+) cells from young donors expressed very little Ki-67. We investigated Bcl-2 and CD95 as possible anti-apoptotic mediators, but neither was associated with pentamer-positivity. To investigate whether expansion represents a compensatory response to impaired functionality, we performed two tests of functionality, peptide-stimulated proliferation and CD107 expression; both were intact in pentamer(+) cells. Our data suggest that HCMV-specific CD8(+) expansions in older subjects accumulate by extended lifespan, rather than accelerated proliferation.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Adult , Aged , Aged, 80 and over , Apoptosis/immunology , Cell Proliferation , Cytomegalovirus Infections/virology , Histocompatibility Antigens Class I/immunology , Humans , Ki-67 Antigen/biosynthesis , Ki-67 Antigen/immunology , Phenotype , Proto-Oncogene Proteins c-bcl-2/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Young Adult , fas Receptor/immunologyABSTRACT
Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.
Subject(s)
Antiviral Agents/pharmacology , COVID-19/immunology , COVID-19/virology , Cross Reactions/immunology , Immunoassay/methods , SARS-CoV-2/physiology , T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/epidemiology , Cell Proliferation , Cytokines/metabolism , HEK293 Cells , Health Personnel , Humans , Immunoglobulin G/immunology , Immunologic Memory , Interferon-gamma/metabolism , Pandemics , Peptides/metabolism , SARS-CoV-2/drug effectsABSTRACT
Memory T cells can be divided into central-memory (T(CM)) and effector-memory (T(EM)) cells, which differ in their functional properties. Although both subpopulations can persist long term, it is not known whether they are maintained by similar mechanisms. We used in vivo labeling with deuterated glucose to measure the turnover of CD4(+) T cells in healthy humans. The CD45R0(+)CCR7(-) T(EM) subpopulation was shown to have a rapid proliferation rate of 4.7% per day compared with 1.5% per day for CD45R0(+)CCR7(+) T(CM) cells; these values are equivalent to average intermitotic (doubling) times of 15 and 48 d, respectively. In contrast, the CD45RA(+)CCR7(+) naive CD4(+) T cell population was found to be much longer lived, being labeled at a rate of only 0.2% per day (corresponding to an intermitotic time of approximately 1 yr). These data indicate that human CD4(+) T(EM) cells constitute a short-lived cell population that requires continuous replenishment in vivo.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Immunologic Memory , Adolescent , Adult , CD4 Antigens/biosynthesis , CD4-Positive T-Lymphocytes/metabolism , Cell Division , Deuterium/metabolism , Glucose/metabolism , Humans , Kinetics , Leukocyte Common Antigens/biosynthesis , Phenotype , Receptors, CCR7 , Receptors, Chemokine/biosynthesis , Receptors, Chemokine/metabolism , T-Lymphocyte Subsets/metabolism , T-Lymphocytes/metabolism , Time FactorsABSTRACT
Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM) in the gut. Conclusions: These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections.
ABSTRACT
Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.
Subject(s)
Hepatitis B virus/genetics , Hepatitis B/genetics , High-Throughput Nucleotide Sequencing/methods , Nanopore Sequencing/methods , Whole Genome Sequencing/methods , Adolescent , Adult , Cohort Studies , Data Accuracy , Female , Genome, Viral/genetics , Haplotypes , Hepatitis B/virology , Humans , Male , Plasmids/genetics , Polymorphism, Genetic , Viral Load/genetics , Young AdultABSTRACT
OBJECTIVES: Respiratory syncytial virus (RSV) causes respiratory infection across the world, with infants and the elderly at particular risk of developing severe disease and death. The replication-defective chimpanzee adenovirus (PanAd3-RSV) and modified vaccinia virus Ankara (MVA-RSV) vaccines were shown to be safe and immunogenic in young healthy adults. Here we report an extension to this first-in-man vaccine trial to include healthy older adults aged 60-75 years. METHODS: We evaluated the safety and immunogenicity of a single dose of MVA-RSV given by intra-muscular (IM) injection (nâ¯=â¯6), two doses of IM PanAd3-RSV given 4-weeks apart (nâ¯=â¯6), IM PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (nâ¯=â¯6), intra-nasal (IN) spray of PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (nâ¯=â¯6), or no vaccine (nâ¯=â¯6). Safety measures included all adverse events within one week of vaccination and blood monitoring. Immunogenicity measures included serum antibody responses (RSV- and PanAd3-neutralising antibody titres measured by plaque-reduction neutralisation and SEAP assays, respectively), peripheral B-cell immune responses (frequencies of F-specific IgG and IgA antibody secreting cells and memory B-cells by ex vivo and cultured dual-colour ELISpot assays respectively), and peripheral RSV-specific T-cell immune responses (frequencies of IFNγ-producing T-cells by ex vivo ELISpot and CD4+/CD8+/Tfh-like cell frequencies by ICS/FACS assay). RESULTS: The vaccines were safe and well tolerated. Compared with each individual baseline immunity the mean fold-changes in serum RSV-neutralising antibody, appearance and magnitude of F-specific IgG and IgA ASCs and expansion of CD4+/CD8+ IFNγ-producing T-cells in peripheral circulation were comparable to the results seen from younger healthy adults who received the same vaccine combination and dose. There were little/no IgA memory B-cell responses in younger and older adults. Expansion of IFNγ-producing T-cells was most marked in older adults following IM prime, with balanced CD4+ and CD8+ T cell responses. The RSV-specific immune responses to vaccination did not appear to be attenuated in the presence of PanAd3 (vector) neutralising antibody. CONCLUSIONS: PanAd3-RSV and MVA-RSV was safe and immunogenic in older adults and the parallel induction of RSV-specific humoral and cellular immunity merits further assessment in providing protection from severe disease.
Subject(s)
Drug Carriers , Immunity, Cellular , Immunity, Humoral , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/adverse effects , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Virus, Human/immunology , Administration, Intranasal , Adolescent , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibody-Producing Cells/immunology , B-Lymphocytes/immunology , Drug-Related Side Effects and Adverse Reactions/epidemiology , Female , Healthy Volunteers , Humans , Immunization Schedule , Injections, Intramuscular , Male , Mastadenovirus/genetics , Middle Aged , Respiratory Syncytial Virus Vaccines/administration & dosage , Respiratory Syncytial Virus Vaccines/genetics , Respiratory Syncytial Virus, Human/genetics , T-Lymphocytes/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/adverse effects , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Vaccinia virus/genetics , Young AdultABSTRACT
Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.
Subject(s)
DNA Damage , Radiation, Ionizing , Alpha Particles , Animals , CHO Cells , Cricetinae , DNA Repair , Gamma Rays , TemperatureABSTRACT
The signature DNA lesion induced by ionizing radiation is clustered DNA damage. Gamma radiation-induced clustered DNA damage containing base lesions was investigated in plasmid DNA under cell mimetic conditions and in two cell lines, V79-4 (hamster) and HF19 (human), using bacterial endonucleases Nth (endonuclease III) and Fpg (formamidopyrimidine DNA glycosylase). Following irradiation with 60Co gamma-rays, induction of double-strand breaks (DSB) and clustered DNA damage, revealed as DSB by the proteins, was determined in plasmid using the plasmid-nicking assay and in cells by either conventional pulsed field gel electrophoresis or a hybridization assay, in which a 3 Mb restriction fragment of the X chromosome is used as a radioactive labeled probe. Enzyme concentrations (30-60 ng/microg DNA) were optimized to minimize visualization of background levels of endogenous DNA damage and DSB produced by non-specific cutting by Fpg and Nth in cellular DNA. 60Co gamma-radiation produces a 1.8-fold increase in the yields of both types of enzyme sensitive sites, visualized as DSB compared with that of prompt DSB in plasmid DNA. In mammalian cells, the increase in yields of clustered DNA damage containing either Fpg or Nth sensitive sites compared with that of prompt DSB is 1.4-2.0- and 1.8-fold, respectively. Therefore, clustered DNA damage is induced in cells by sparsely ionizing radiation and their yield is significantly greater than that of prompt DSB.
Subject(s)
DNA Damage , DNA/radiation effects , Deoxyribonuclease (Pyrimidine Dimer) , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins , Gamma Rays , N-Glycosyl Hydrolases/metabolism , Animals , CHO Cells , Cell Line , Cricetinae , DNA-Formamidopyrimidine Glycosylase , Dose-Response Relationship, Radiation , Female , Fibroblasts/radiation effects , Fibroblasts/ultrastructure , Humans , Kinetics , Nucleic Acid Hybridization , Plasmids/radiation effectsABSTRACT
Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/ß. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.
Subject(s)
Lymphocyte Activation/physiology , Mucosal-Associated Invariant T Cells/physiology , Virus Diseases/immunology , Adult , Cells, Cultured , Coculture Techniques , Cytokines/metabolism , Female , Humans , Leukocytes, Mononuclear/physiology , MaleABSTRACT
INTRODUCTION: Respiratory syncytial virus (RSV) infection causes respiratory disease throughout life, with infants and the elderly at risk of severe disease and death. RSV001 is a phase 1 (first-in-man), open-label, dose-escalation, clinical trial of novel genetic viral-vectored vaccine candidates PanAd3-RSV and modified vaccinia virus Ankara (MVA)-RSV. The objective of RSV001 is to characterise the (primary objective) safety and (secondary objective) immunogenicity of these vaccines in healthy younger and older adults. METHODS AND ANALYSIS: Heterologous and homologous 'prime'/boost combinations of PanAd3-RSV and single-dose MVA-RSV are evaluated in healthy adults. 40 healthy adults aged 18-50â years test one of four combinations of intramuscular (IM) or intranasal (IN) PanAd3-RSV prime and IM PanAd3 or IM MVA-RSV boost vaccination, starting at a low dose for safety. The following year an additional 30 healthy adults aged 60-75â years test either a single dose of IM MVA-RSV, one of three combinations of IN or IM PanAd3-RSV prime and PanAd3-RSV or MVA-RSV boost vaccination used in younger volunteers, and a non-vaccinated control group. Study participants are self-selected volunteers who satisfy the eligibility criteria and are assigned to study groups by sequential allocation. Safety assessment includes the daily recording of solicited and unsolicited adverse events for 1â week after vaccination, as well as visit (nursing) observations and safety bloods obtained at all scheduled attendances. Laboratory measures of RSV-specific humoral and cellular immune responses after vaccination will address the secondary end points. All study procedures are performed at the Centre for Clinical Vaccinology and Tropical Medicine (CCVTM), Oxford, UK. ETHICS AND DISSEMINATION: RSV001 has clinical trial authorisation from the Medicines and Healthcare Products Regulatory Agency (MHRA) and ethics approval from NRES Berkshire (reference 13/SC/0023). All study procedures adhere to International Conference on Harmonisation (ICH) Good Clinical Practice guidelines. The results of the trial are to be published in peer-reviewed journals, conferences and academic forums. TRIAL REGISTRATION NUMBER: NCT01805921.
Subject(s)
Adenoviruses, Simian , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Viruses , Vaccination , Vaccinia virus , Viral Proteins , Adolescent , Adult , Aged , Clinical Protocols , Female , Genetic Vectors , Humans , Male , Middle Aged , Research Design , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Vaccines/adverse effects , Young AdultABSTRACT
Following exposure to vaccines, antigen-specific CD8(+) T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8(+) T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8(+) T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8(+) T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses.
Subject(s)
Adenoviridae/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Genetic Vectors/immunology , Immunologic Memory/immunology , Animals , Apoptosis/immunology , Cytomegalovirus/immunology , Humans , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Transcription Factors/immunology , Vaccination/methodsABSTRACT
Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease.
Subject(s)
Adenoviruses, Simian/genetics , Genetic Vectors/genetics , Pan troglodytes/virology , Respiratory Syncytial Virus Vaccines/adverse effects , Respiratory Syncytial Virus Vaccines/immunology , Respiratory Syncytial Viruses/immunology , Vaccinia virus/genetics , Adult , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Body Temperature , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dose-Response Relationship, Immunologic , Genetic Vectors/adverse effects , HEK293 Cells , Healthy Volunteers , Humans , Immunization, Secondary , Interferon-gamma/immunology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus Vaccines/genetics , VaccinationABSTRACT
BACKGROUND: BCG, the only licensed vaccine against tuberculosis, provides some protection against disseminated disease in infants but has little effect on prevention of adult pulmonary disease. Newer parenteral immunization prime boost regimes may provide improved protection in experimental animal models but are unproven in man so that there remains a need for new and improved immunization strategies. METHODS AND FINDINGS: Mice were immunized parenterally, intranasally or simultaneously by both routes with BCG or recombinant mycobacterial antigens plus appropriate adjuvants. They were challenged with Mycobacterium tuberculosis (Mtb) and the kinetics of Mtb growth in the lungs measured. We show that simultaneous immunization (SIM) of mice by the intranasal and parenteral routes is highly effective in increasing protection over parenteral BCG administration alone. Intranasal immunization induces local pulmonary immunity capable of inhibiting the growth of Mtb in the early phase (the first week) of infection, while parenteral immunization has a later effect on Mtb growth. Importantly, these two effects are additive and do not depend on priming and boosting the immune response. The best SIM regimes reduce lung Mtb load by up to 2 logs more than BCG given by either route alone. CONCLUSIONS: These data establish SIM as a novel and highly effective immunization strategy for Mtb that could be carried out at a single clinic visit. The efficacy of SIM does not depend on priming and boosting an immune response, but SIM is complementary to prime boost strategies and might be combined with them.