Development of an objective gene expression panel as an alternative to self-reported symptom scores in human influenza challenge trials.

BACKGROUND: Influenza challenge trials are important for vaccine efficacy testing. Currently, disease severity is determined by self-reported scores to a list of symptoms which can be highly subjective. A more objective measure would allow for improved data analysis. METHODS: Twenty-one volunteers participated in an influenza challenge trial. We calculated the daily sum of scores (DSS) for a list of 16 influenza symptoms. Whole blood collected at baseline and 24, 48, 72 and 96 h post challenge was profiled on Illumina HT12v4 microarrays. Changes in gene expression most strongly correlated with DSS were selected to train a Random Forest model and tested on two independent test sets consisting of 41 individuals profiled on a different microarray platform and 33 volunteers assayed by qRT-PCR. RESULTS: 1456 probes are significantly associated with DSS at 1% false discovery rate. We selected 19 genes with the largest fold change to train a random forest model. We observed good concordance between predicted and actual scores in the first test set (r = 0.57; RMSE = -16.1%) with the greatest agreement achieved on samples collected approximately 72 h post challenge. Therefore, we assayed samples collected at baseline and 72 h post challenge in the second test set by qRT-PCR and observed good concordance (r = 0.81; RMSE = -36.1%). CONCLUSIONS: We developed a 19-gene qRT-PCR panel to predict DSS, validated on two independent datasets. A transcriptomics based panel could provide a more objective measure of symptom scoring in future influenza challenge studies. Trial registration Samples were obtained from a clinical trial with the ClinicalTrials.gov Identifier: NCT02014870, first registered on December 5, 2013.

Mycetoma Caused by Acremonium Species in a Patient with Chronic Granulomatous Disease.

Patients with chronic granulomatous disease are predisposed to fungal infections and are therefore routinely prescribed antifungal prophylaxis. We report a case where acremonium was responsible for causing a cutaneous infection (mycetoma) despite antifungal prophylaxis. Treatment with voriconazole was initiated and the infection gradually resolved. This case highlights the need for careful clinical follow-up and thorough investigation of patients who have a neutrophil immunodeficiency.

Transcriptomic profiling facilitates classification of response to influenza challenge.

UNLABELLED: Despite increases in vaccination coverage, reductions in influenza-related mortality have not been observed. Better vaccines are therefore required and influenza challenge studies can be used to test the efficacy of new vaccines. However, this requires the accurate post-challenge classification of subjects by outcome, which is limited in current methods that use artificial thresholds to assign 'symptomatic' and 'asymptomatic' phenotypes. We present data from an influenza challenge study in which 22 healthy adults (11 vaccinated) were inoculated with H3N2 influenza (A/Wisconsin/67/2005). We generated genome-wide gene expression data from peripheral blood taken immediately before the challenge and at 12, 24 and 48 h post-challenge. Variation in symptomatic scoring was found amongst those with laboratory confirmed influenza. By combining the dynamic transcriptomic data with the clinical parameters this variability can be reduced. We identified four subjects with severe laboratory confirmed influenza that show differential gene expression in 1103 probes 48 h post-challenge compared to the remaining subjects. We have further reduced this profile to six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3) that can be used to define these subjects. We have used this gene set to predict symptomatic infection from an independent study. This analysis gives further insight into host-pathogen interactions during influenza infection. However, the major potential value is in the clinical trial setting by providing a more quantitative method to better classify symptomatic individuals post influenza challenge. KEY MESSAGE: Differential gene expression signatures are seen following influenza challenge. Expression of six predictive genes can classify response to influenza challenge. The genomic influenza response classification replicates in an independent dataset.

A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory.

A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8(+) and CD4(+) HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.

Safety and immunogenicity of a candidate tuberculosis vaccine MVA85A delivered by aerosol in BCG-vaccinated healthy adults: a phase 1, double-blind, randomised controlled trial.

BACKGROUND: Intradermal MVA85A, a candidate vaccine against tuberculosis, induces high amounts of Ag85A-specific CD4 T cells in adults who have already received the BCG vaccine, but aerosol delivery of this vaccine might offer immunological and logistical advantages. We did a phase 1 double-blind trial to compare the safety and immunogenicity of aerosol-administered and intradermally administered MVA85A METHODS: In this phase 1, double-blind, proof-of-concept trial, 24 eligible BCG-vaccinated healthy UK adults were randomly allocated (1:1) by sequentially numbered, sealed, opaque envelopes into two groups: aerosol MVA85A and intradermal saline placebo or intradermal MVA85A and aerosol saline placebo. Participants, the bronchoscopist, and immunologists were masked to treatment assignment. The primary outcome was safety, assessed by the frequency and severity of vaccine-related local and systemic adverse events. The secondary outcome was immunogenicity assessed with laboratory markers of cell-mediated immunity in blood and bronchoalveolar lavage samples. Safety and immunogenicity were assessed for 24 weeks after vaccination. Immunogenicity to both insert Ag85A and vector modified vaccinia virus Ankara (MVA) was assessed by ex-vivo interferon-γ ELISpot and serum ELISAs. Since all participants were randomised and vaccinated according to protocol, our analyses were per protocol. This trial is registered with ClinicalTrials.gov, number NCT01497769. FINDINGS: Both administration routes were well tolerated and immunogenic. Respiratory adverse events were rare and mild. Intradermal MVA85A was associated with expected mild local injection-site reactions. Systemic adverse events did not differ significantly between the two groups. Three participants in each group had no vaccine-related systemic adverse events; fatigue (11/24 [46%]) and headache (10/24 [42%]) were the most frequently reported symptoms. Ag85A-specific systemic responses were similar across groups. Ag85A-specific CD4 T cells were detected in bronchoalveolar lavage cells from both groups and responses were higher in the aerosol group than in the intradermal group. MVA-specific cellular responses were detected in both groups, whereas serum antibodies to MVA were only detectable after intradermal administration of the vaccine. INTERPRETATION: Further clinical trials assessing the aerosol route of vaccine delivery are merited for tuberculosis and other respiratory pathogens. FUNDING: The Wellcome Trust and Oxford Radcliffe Hospitals Biomedical Research Centre.

Coadministration of seasonal influenza vaccine and MVA-NP+M1 simultaneously achieves potent humoral and cell-mediated responses.

Current seasonal influenza vaccines have reduced immunogenicity and are of suboptimal efficacy in older adults. We have previously shown that the novel candidate vaccine MVA-NP+M1 is able to boost memory T cell responses in adults aged 50-85 years. Preclinical studies have demonstrated that viral vectored vaccines can act as adjuvants when coadministered with protein-based vaccines. We have conducted a phase I clinical trial to compare the coadministration of seasonal influenza vaccine and MVA-NP+M1 with seasonal influenza vaccine alone in adults aged 50 years and above. This combination of vaccines was safe and well tolerated. T cell responses to internal influenza proteins were boosted to significantly higher levels in the group receiving MVA-NP+M1 compared with the group receiving seasonal influenza vaccine alone. Rates of seroprotection and seroconversion against the three vaccine strains were similar in both groups; however, there was a significant increase in the geometric mean titer ratio for the H3N2 component of seasonal influenza vaccine in the coadministration group. While some vaccine combinations result in immune interference, the coadministration of MVA-NP+M1 alongside seasonal influenza vaccine is shown here to increase some influenza strain-specific antibody responses and boost memory T cells capable of recognizing a range of influenza A subtypes.

Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved Influenza A antigens.

Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection.

Testing for hypersensitivity to seminal fluid-free spermatozoa.

Hypersensitivity to human semen (HHS) is an increasingly reported condition with symptoms manifested locally and systemically, which in some cases may result in anaphylaxis. This report describes four cases of HHS all with positive allergy skin prick tests to partner's whole semen. None of the patients elicited a response to seminal fluid-free washed spermatozoa. In cases of high risk of anaphylaxis, we recommend avoiding exposure to semen. By carrying out an allergy skin prick test on seminal fluid-free washed spermatozoa, the risk of immunogenic reaction to the spermatozoa could be eliminated.

A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years.

BACKGROUND: Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults. METHODS: Thirty volunteers (aged 50-85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8) plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+) T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach. RESULTS: Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) phenotype both before and after vaccination. CONCLUSIONS: MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT00942071.

ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: assessment of efficacy against mosquito bite challenge in humans.

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1-results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets.

T-cell responses in children to internal influenza antigens, 1 year after immunization with pandemic H1N1 influenza vaccine, and response to revaccination with seasonal trivalent-inactivated influenza vaccine.

BACKGROUND: During seasonal influenza epidemics, 5-15% of the population are affected with an illness having a nontrivial mortality, morbidity and economic burden. Inactivated influenza vaccines are routinely used to prevent influenza infection, primarily by inducing humoral immunity. In addition, trivalent-inactivated influenza vaccines have previously been shown to boost influenza-specific T-cell responses in a small percentage of adults. We investigate here the influenza-specific T-cell response, in children, 1 year after pandemic H1N1 vaccination and the ability to boost the T-cell response with trivalent-inactivated influenza immunization. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from children previously vaccinated with pandemic H1N1 vaccine, pre- and postseasonal 2010-2011 trivalent influenza vaccine (TIV) vaccination. Samples were analyzed by interferon-gamma enzyme-linked immunosorbent spot for reactogenicity toward internal influenza antigens (nucleoprotein, matrix protein 1 and nonstructural protein 1). RESULTS: Basal ex vivo T-cell responses to nucleoprotein, matrix protein 1 and nonstructural protein 1 measured by interferon-gamma enzyme-linked immunosorbent spot assay were significantly higher in those children who had previously received an AS03B-adjuvanted split virion pandemic vaccine 12 months earlier rather than a nonadjuvanted whole virion vaccine. Boosting of these responses, 21 days after 2010/2011 seasonal TIV vaccination was observed regardless of age or prior pandemic vaccination regime, although boosting was greater in those groups with the lowest initial response. CONCLUSIONS: We show here that children previously vaccinated with the 2009 pandemic H1N1 vaccine have measurable T-cell responses 1 year after vaccination. The magnitudes of these responses are dependent on both age of vaccine and type of pandemic H1N1 vaccine used. After 2010/2011 seasonal TIV vaccination, these T-cell responses undergo a small but significant boost.

Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man.

Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

Virus-specific cytotoxic T lymphocytes differentially express cell-surface leukocyte immunoglobulin-like receptor-1, an inhibitory receptor for class I major histocompatibility complex molecules.

Leukocyte immunoglobulin-like receptor-1 (LIR-1) is an inhibitory receptor that negatively regulates T cell effector functions after interaction with host class I major histocompatibility complex molecules and, additionally, binds to UL18, a human cytomegalovirus (HCMV)-encoded class I homologue. Here, we demonstrate that virus-specific cytotoxic T lymphocytes (CTLs) differentially express LIR-1, with high frequencies of expression on HCMV-specific CD8+ T cells and intermediate and low frequencies of expression on influenza virus-specific and Epstein-Barr virus (EBV)-specific CTLs, respectively. Expression of LIR-1 was dependent on CTL-antigen specificity and was associated with a differentiated effector memory phenotype, as demonstrated by decreased expression of CD28 and increased expression of CD57. During primary HCMV and EBV infections, expression of LIR-1 on virus-specific CTLs was low and increased slowly. These results indicate that expression of LIR-1 increases during differentiation of virus-specific CD8+ effector T cells. Furthermore, they suggest that a potential immunoregulatory function of UL18 may be to preferentially target highly differentiated HCMV-specific effector memory T cells during persistent infection.