A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes

Copyright @ 2012, American Society for Microbiology.Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E

Non-human primate to human immunobridging to infer the protective effect of an Ebola virus vaccine candidate

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Safety and long-term immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Sierra Leone: a combined open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2 trial

Background The Ebola epidemics in west Africa and the Democratic Republic of the Congo highlight an urgent need for safe and effective vaccines to prevent Ebola virus disease. We aimed to assess the safety and long-term immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vector-based vaccine, encoding glycoproteins from Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in Sierra Leone, a country previously affected by Ebola. Methods The trial comprised two stages: an open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2. The study was done at three clinics in Kambia district, Sierra Leone. In stage 1, healthy adults (aged ≥18 years) residing in or near Kambia district, received an intramuscular injection of Ad26.ZEBOV (5×1010 viral particles) on day 1 (first dose) followed by an intramuscular injection of MVA-BN-Filo (1×108 infectious units) on day 57 (second dose). An Ad26.ZEBOV booster vaccination was offered at 2 years after the first dose to stage 1 participants. The eligibility criteria for adult participants in stage 2 were consistent with stage 1 eligibility criteria. Stage 2 participants were randomly assigned (3:1), by computer-generated block randomisation (block size of eight) via an interactive web-response system, to receive either the Ebola vaccine regimen (Ad26.ZEBOV followed by MVA-BN-Filo) or an intramuscular injection of a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo on day 57 (second dose; control group). Study team personnel, except those with primary responsibility for study vaccine preparation, and participants were masked to study vaccine allocation. The primary outcome was the safety of the Ad26.ZEBOV and MVA-BN-Filo vaccine regimen, which was assessed in all participants who had received at least one dose of study vaccine. Safety was assessed as solicited local and systemic adverse events occurring in the first 7 days after each vaccination, unsolicited adverse events occurring in the first 28 days after each vaccination, and serious adverse events or immediate reportable events occurring up to each participant’s last study visit. Secondary outcomes were to assess Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second vaccine in a per-protocol set of participants (ie, those who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response) and to assess the safety and tolerability of the Ad26.ZEBOV booster vaccination in stage 1 participants who had received the booster dose. This study is registered at ClinicalTrials.gov, NCT02509494. Findings Between Sept 30, 2015, and Oct 19, 2016, 443 participants (43 in stage 1 and 400 in stage 2) were enrolled; 341 participants assigned to receive the Ad26.ZEBOV and MVA-BN-Filo regimen and 102 participants assigned to receive the MenACWY and placebo regimen received at least one dose of study vaccine. Both regimens were well tolerated with no safety concerns. In stage 1, solicited local adverse events (mostly mild or moderate injection-site pain) were reported in 12 (28%) of 43 participants after Ad26.ZEBOV vaccination and in six (14%) participants after MVA-BN-Filo vaccination. In stage 2, solicited local adverse events were reported in 51 (17%) of 298 participants after Ad26.ZEBOV vaccination, in 58 (24%) of 246 after MVA-BN-Filo vaccination, in 17 (17%) of 102 after MenACWY vaccination, and in eight (9%) of 86 after placebo injection. In stage 1, solicited systemic adverse events were reported in 18 (42%) of 43 participants after Ad26.ZEBOV vaccination and in 17 (40%) after MVA-BN-Filo vaccination. In stage 2, solicited systemic adverse events were reported in 161 (54%) of 298 participants after Ad26.ZEBOV vaccination, in 107 (43%) of 246 after MVA-BN-Filo vaccination, in 51 (50%) of 102 after MenACWY vaccination, and in 39 (45%) of 86 after placebo injection. Solicited systemic adverse events in both stage 1 and 2 participants included mostly mild or moderate headache, myalgia, fatigue, and arthralgia. The most frequent unsolicited adverse event after the first dose was headache in stage 1 and malaria in stage 2. Malaria was the most frequent unsolicited adverse event after the second dose in both stage 1 and 2. No serious adverse event was considered related to the study vaccine, and no immediate reportable events were observed. In stage 1, the safety profile after the booster vaccination was not notably different to that observed after the first dose. Vaccine-induced humoral immune responses were observed in 41 (98%) of 42 stage 1 participants (geometric mean binding antibody concentration 4784 ELISA units [EU]/mL [95% CI 3736–6125]) and in 176 (98%) of 179 stage 2 participants (3810 EU/mL [3312–4383]) at 21 days after the second vaccination. Interpretation The Ad26.ZEBOV and MVA-BN-Filo vaccine regimen was well tolerated and immunogenic, with persistent humoral immune responses. These data support the use of this vaccine regimen for Ebola virus disease prophylaxis in adults

Safety and immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in children in Sierra Leone: a randomised, double-blind, controlled trial

Background—Children account for a substantial proportion of cases and deaths from Ebola virus disease. We aimed to assess the safety and immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vectorbased vaccine, encoding glycoproteins from the Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in a paediatric population in Sierra Leone. Methods—This randomised, double-blind, controlled trial was done at three clinics in Kambia district, Sierra Leone. Healthy children and adolescents aged 1–17 years were enrolled in three age cohorts (12–17 years, 4–11 years, and 1–3 years) and randomly assigned (3:1), via computer-generated block randomisation (block size of eight), to receive an intramuscular injection of either Ad26.ZEBOV (5 × 1010 viral particles; first dose) followed by MVA-BN-Filo (1 × 108 infectious units; second dose) on day 57 (Ebola vaccine group), or a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo (second dose) on day 57 (control group). Study team personnel (except for those with primary responsibility for study vaccine preparation), participants, and their parents or guardians were masked to study vaccine allocation. The primary outcome was safety, measured as the occurrence of solicited local and systemic adverse symptoms during 7 days after each vaccination, unsolicited systemic adverse events during 28 days after each vaccination, abnormal laboratory results during the study period, and serious adverse events or immediate reportable events throughout the study period. The secondary outcome was immunogenicity (humoral immune response), measured as the concentration of Ebola virus glycoprotein-specific binding antibodies at 21 days after the second dose. The primary outcome was assessed in all participants who had received at least one dose of study vaccine and had available reactogenicity data, and immunogenicity was assessed in all participants who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response. This study is registered at ClinicalTrials.gov, NCT02509494. Findings—From April 4, 2017, to July 5, 2018, 576 eligible children or adolescents (192 in each of the three age cohorts) were enrolled and randomly assigned. The most common solicited local adverse event during the 7 days after the first and second dose was injection-site pain in all age groups, with frequencies ranging from 0% (none of 48) of children aged 1–3 years after placebo injection to 21% (30 of 144) of children aged 4–11 years after Ad26.ZEBOV vaccination. The most frequently observed solicited systemic adverse event during the 7 days was headache in the 12–17 years and 4–11 years age cohorts after the first and second dose, and pyrexia in the 1–3 years age cohort after the first and second dose. The most frequent unsolicited adverse event after the first and second dose vaccinations was malaria in all age cohorts, irrespective of the vaccine types. Following vaccination with MenACWY, severe thrombocytopaenia was observed in one participant aged 3 years. No other clinically significant laboratory abnormalities were observed in other study participants, and no serious adverse events related to the Ebola vaccine regimen were reported. There were no treatment-related deaths. Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second dose of the Ebola virus vaccine regimen were observed in 131 (98%) of 134 children aged 12–17 years (9929 ELISA units [EU]/mL [95% CI 8172–12 064]), in 119 (99%) of 120 aged 4–11 years (10 212 EU/mL [8419–12 388]), and in 118 (98%) of 121 aged 1–3 years (22 568 EU/mL [18 426–27 642]). Interpretation—The Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen was well tolerated with no safety concerns in children aged 1–17 years, and induced robust humoral immune responses, suggesting suitability of this regimen for Ebola virus disease prophylaxis in children

Persistent hepatitis C viral replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and a direct-acting antiviral

Abstract Exhaustion of antiviral CD8(+) T cells contributes to persistence of hepatitis C viral (HCV) infection. This immune response has proved difficult to restore by therapeutic vaccination, even when HCV replication is suppressed using antiviral regimens containing type I interferon. Because immunomodulatory effects of type I interferon may be a factor in poor T-cell priming, we undertook therapeutic vaccination in two chronically infected chimpanzees during treatment with a direct-acting antiviral (DAA) targeting the HCV NS5b polymerase protein. Immunization with genetic vaccines encoding the HCV NS3-NS5b nonstructural proteins during DAA treatment resulted in a multifunctional CD8(+) T-cell response. However, these antiviral CD8(+) T cells did not prevent persistent replication of DAA-resistant HCV variants that emerged during treatment. Most vaccine-induced CD8(+) T cells targeted class I epitopes that were not conserved in the circulating virus. Exhausted intrahepatic CD8(+) T-cell targeting-conserved epitopes did not expand after vaccination, with a notable exception. A sustained, multifunctional CD8(+) T-cell response against at least one intact class I epitope was detected in blood after vaccination. Persistence of HCV was not due to mutational escape of this epitope. Instead, failure to control HCV replication was likely caused by localized exhaustion in the liver, where CD8(+) T-cell expression of the inhibitory receptor programmed cell death 1 increased 25-fold compared with those in circulation. CONCLUSION: Treatment with a DAA during therapeutic vaccination provided transient control of HCV replication and a multifunctional T-cell response, primarily against nonconserved class I epitopes; exhaustion of liver-infiltrating CD8(+) T cells that target conserved epitopes may not be averted when DAA therapy fails prematurely due to emergence of resistant HCV variants

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited

T-cell immunity and hepatitis C virus reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen in a chimpanzee

Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T-cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)-free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct-acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T-cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T-cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape-prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies ▿ †

Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of ∼5.5 log10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes