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Recently, oncolytic vaccinia viruses (VACVs) have shown their potential to provide for clinically effective cancer treatments. The reason for this clinical usefulness is not only the direct destruction of infected cancer cells but also activation of immune responses directed against tumor antigens. For eliciting a robust antitumor immunity, a dominant T helper 1 (Th1) cell differentiation of the response is preferred, and such polarization can be achieved by activating the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. However, current VACVs used as oncolytic viruses to date still encode several immune evasion proteins involved in the inhibition of this signaling pathway. By inactivating genes of selected regulatory virus proteins, we aimed for a candidate virus with increased potency to activate cellular antitumor immunity but at the same time with a fully maintained replicative capacity in cancer cells. The removal of up to three key genes (C10L, N2L, and C6L) from VACV did not reduce the strength of viral replication, both in vitro and in vivo, but resulted in the rescue of IRF3 phosphorylation upon infection of cancer cells. In syngeneic mouse tumor models, this activation translated to enhanced cytotoxic T lymphocyte (CTL) responses directed against tumor-associated antigens and neo-epitopes and improved antitumor activity., Graphical abstract, The corresponding author and colleagues constructed and tested a replication-competent vaccinia virus with an improved capacity to activate the interferon regulatory factor 3 (IRF3) pathway as a candidate cancer therapeutic, showing increased antitumor immune responses and the ability to destroy tumors in mouse models.

Summary Background The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a respiratory disease with a case fatality rate of up to 35%. Given its potential to cause a public health emergency and the absence of efficacious drugs or vaccines, MERS is one of the WHO priority diseases warranting urgent research and development of countermeasures. We aimed to assess safety and tolerability of an anti-MERS-CoV modified vaccinia virus Ankara (MVA)-based vaccine candidate that expresses the MERS-CoV spike glycoprotein, MVA-MERS-S, in healthy adults. Methods This open-label, phase 1 trial was done at the University Medical Center Hamburg-Eppendorf (Hamburg, Germany). Participants were healthy men and women aged 18–55 years with no clinically significant health problems as determined during medical history and physical examination, a body-mass index of 18·5–30·0 kg/m2 and weight of more than 50 kg at screening, and a negative pregnancy test for women. A key exclusion criterion was a previous MVA vaccination. For the prime immunisation, participants received doses of 1 × 107 plaque-forming unit (PFU; low-dose group) or 1 × 108 PFU (high-dose group) MVA-MERS-S intramuscularly. A second identical dose was administered intramuscularly as a booster immunisation 28 days after first injection. As a control group for immunogenicity analyses, blood samples were drawn at identical study timepoints from six healthy adults, who did not receive any injections. The primary objectives of the study were safety and tolerability of the two dosage levels and reactogenicity after administration. Immunogenicity was assessed as a secondary endpoint by ELISA and neutralisation tests. T-cell immunity was evaluated by interferon-γ-linked enzyme-linked immune absorbent spot assay. All participants who were vaccinated at least once were included in the safety analysis. Immunogenicity was analysed in the participants who completed 6 months of follow-up. This trial is registered with ClinicalTrials.gov , NCT03615911 , and EudraCT, 2014-003195-23 Findings From Dec 17, 2017, to June 5, 2018, 26 participants (14 in the low-dose group and 12 in the high-dose group) were enrolled and received the first dose of the vaccine according to their group allocation. Of these, 23 participants (12 in the low-dose group and 11 in the high-dose group) received a second dose of MVA-MERS-S according to their group allocation after a 28-day interval and completed follow-up. Homologous prime–boost immunisation with MVA-MERS-S revealed a benign safety profile with only transient mild-to-moderate reactogenicity. Participants had no severe or serious adverse events. 67 vaccine-related adverse events were reported in ten (71%) of 14 participants in the low-dose group, and 111 were reported in ten (83%) of 12 participants in the high-dose group. Solicited local reactions were the most common adverse events: pain was observed in 17 (65%; seven in the low-dose group vs ten in the high-dose group) participants, swelling in ten (38%; two vs eight) participants, and induration in ten (38%; one vs nine) participants. Headaches (observed in seven participants in the low-dose group vs nine in the high-dose group) and fatigue or malaise (ten vs seven participants) were the most common solicited systemic adverse events. All adverse events resolved swiftly (within 1–3 days) and without sequelae. Following booster immunisation, nine (75%) of 12 participants in the low-dose group and 11 (100%) participants in the high-dose group showed seroconversion using a MERS-CoV S1 ELISA at any timepoint during the study. Binding antibody titres correlated with MERS-CoV-specific neutralising antibodies (Spearman's correlation r=0·86 [95% CI 0·6960–0·9427], p=0·0001). MERS-CoV spike-specific T-cell responses were detected in ten (83%) of 12 immunised participants in the low-dose group and ten (91%) of 11 immunised participants in the high-dose group. Interpretation Vaccination with MVA-MERS-S had a favourable safety profile without serious or severe adverse events. Homologous prime–boost immunisation induced humoral and cell-mediated responses against MERS-CoV. A dose–effect relationship was demonstrated for reactogenicity, but not for vaccine-induced immune responses. The data presented here support further clinical testing of MVA-MERS-S in larger cohorts to advance MERS vaccine development. Funding German Center for Infection Research.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

The severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on vaccinia virus MVA against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust synthesis of S antigen, make it a suitable candidate vaccine for industrial scale production. Vaccinated mice produced S antigen-specific CD8+ T cells and serum antibodies binding to S glycoprotein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.Significance StatementThe highly attenuated vaccinia virus MVA is licensed as smallpox vaccine, and as vector it is a component of the approved Adenovirus-MVA-based prime-boost vaccine against Ebola virus disease. Here we provide results from testing the COVID-19 candidate vaccine MVA-SARS-2-S, a poxvirus-based vector vaccine that proceeded to clinical evaluation. When administered by intramuscular inoculation, MVA-SARS-2-S expresses and safely delivers the full-length SARS-CoV-2 spike (S) protein, inducing balanced SARS-CoV-2-specific cellular and humoral immunity, and protective efficacy in vaccinated mice. Substantial clinical experience has already been gained with MVA vectors using homologous and heterologous prime-boost applications, including the immunization of children and immunocompromised individuals. Thus, MVA-SARS-2-S represents an important resource for developing further optimized COVID-19 vaccines.

In 2015 Zika virus (ZIKV) emerged for the first time in South America. The following ZIKV epidemic resulted in the appearance of a clinical phenotype with microcephaly and other severe malformations in newborns. So far, mechanisms of ZIKV induced damage to the fetus are not completely understood. Previous data suggest that ZIKV may bypass the placenta to reach the fetus. Thus, animal models for ZIKV infection are important to facilitate studies about ZIKV infection during pregnancy. Here, we used ultrasound based imaging (USI) to characterize ZIKV induced pathogenesis in the pregnant Type I interferon receptor-deficient (IFNAR-/-) mouse model. Based on USI we suggest the placenta to be a primary target organ of ZIKV infection enabling ZIKV spreading to the fetus. Moreover, in addition to direct infection of the fetus, the placental ZIKV infection may cause an indirect damage to the fetus through reduced uteroplacental perfusion leading to intrauterine growth retardation (IUGR) and fetal complications as early as embryonic day (ED) 12.5. Our data confirmed the capability of USI to characterize ZIKV induced modifications in mouse fetuses. Data from further studies using USI to monitor ZIKV infections will contribute to a better understanding of ZIKV infection in pregnant IFNAR-/- mice.

Nipah virus (NiV) is an emerging zoonotic virus that is transmitted by bats to humans and to pigs, causing severe respiratory disease and often fatal encephalitis. Antibodies directed against the NiV-glycoprotein (G) protein are known to play a major role in clearing NiV infection and in providing vaccine-induced protective immunity. More recently, T cells have been also shown to be involved in recovery from NiV infection. So far, relatively little is known about the role of T cell responses and the antigenic targets of NiV-G that are recognized by CD8 T cells. In this study, NiV-G protein served as the target immunogen to activate NiV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for the generation of MVA&ndash, NiV-G candidate vaccines expressing different versions of recombinant NiV-G. Overlapping peptides covering the entire NiV-G protein were used to identify major histocompatibility complex class I/II-restricted T cell responses in type I interferon receptor-deficient (IFNAR&minus, /&minus, ) mice after vaccination with the MVA&ndash, NiV-G candidate vaccines. We have identified an H2-b-restricted nonamer peptide epitope with CD8 T cell antigenicity and a H2-b 15mer with CD4 T cell antigenicity in the NiV-G protein. The identification of this epitope and the availability of the MVA&ndash, NiV-G candidate vaccines will help to evaluate NiV-G-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of NiV-G infection. Of note, a soluble version of NiV-G was advantageous in activating NiV-G-specific cellular immune responses using these peptides.

Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens.

Coronaviruses in the Middle East Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness and kills about a third of people infected. The virus is common in dromedary camels, which can be a source of human infections. In a survey for MERSCoV in over 1300 Saudi Arabian camels, Sabir et al. found that dromedaries share three coronavirus species with humans. Diverse MERS lineages in camels have caused human infections, which suggests that transfer among host species occurs quite easily. Haagmans et al. made a MERS-CoV vaccine for use in camels, using poxvirus as a vehicle. The vaccine significantly reduced virus excretion, which should help reduce the potential for transmission to humans, and conferred cross-immunity to camelpox infections. Science , this issue p. 81 , p. 77

The recent Ebola virus outbreak has highlighted the therapeutic potential of antisera and renewed interest in this treatment approach. While human convalescent sera may not be readily available in the early stages of an outbreak, antisera of animal origin can be produced in a short time frame. Here, we compared adjuvanted virus-like particles (VLP) with recombinant modified vaccinia virus Ankara and vesicular stomatitis virus (VSV), both expressing the Ebola virus antigens. The neutralizing antibody titers of rabbits immunized with adjuvanted VLPs were similar to those immunized with the replication-competent VSV, indicating that presentation of the antigen in its native conformation rather than de novo antigen expression is essential for production of functional antibodies. This approach also yielded high-titer antisera against Nipah virus glycoproteins, illustrating that it is transferable to other virus families. Multiple-step immunoglobulin G purification using a two-step 20–40% ammonium sulfate precipitation followed by protein A affinity chromatography resulted in 90% recovery of functionality and sustained in vivo stability. Adjuvanted VLP-based immunization strategies are thus a promising approach for the rapid generation of therapeutic antisera against emerging infections., Anti-serum: Rapid generation for viral outbreaks Passive immunity through the transfer of anti-serum represents the earliest clinical application of antibodies and is still widely used to this day in the form of anti-venoms. Veronika von Messling and colleagues at the Paul Ehrlich Institute investigate the potential of generating neutralizing anti-serum to the emerging viruses Ebola and Nipah. The authors compare different vaccination platforms in mice and rabbits and find that following multiple vaccine challenges, neutralizing antibody titers equivalent to that seen in convalescent patients could be obtained. Purification of the IgG fraction and processing into F(ab’)2 fragments has the potential to significantly reduce xeno-responses yet the authors find that neutralizing capacity is largely retained albeit at the cost of a shorter in vivo half-life. These findings offer the hope of rapidly generating large quantities of neutralizing anti-serum that could be used in a viral outbreak scenario.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8 + T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as vaccine against infectious diseases. Here, we assessed the in vivo distribution of a recombinant MVA candidate vaccine against the Middle Eastern Respiratory Syndrome (MVA-MERS-S) in mice. Intramuscularly inoculated mice were necropsied at different time points and examined by histology, immunohistochemistry and real-time PCR. We detected inflammation and myonecrosis at the parenteral site and hyperplasia of the draining lymph nodes. MVA-MERS-S did not result in detectable lesions in tissues peripheral to the parenteral site and draining lymph nodes. Real-time PCR analysis of >240 tissue samples detected MVA-DNA predominantly at the injection site and in the draining lymph nodes, and suggested continuous clearance of the candidate vaccine during the observation period. Levels of parenteral site inflammation and hyperplasia of draining lymph nodes were considered in line with immunological responses to vaccine inoculation.

Safety tested Modified Vaccinia virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. As a biologically well-characterized mutant virus, MVA facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza virus H5N1 or Ebola virus. This review summarizes our current knowledge about MVA as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology.

BACKGROUND: Modified vaccinia virus Ankara (MVA) is a promising viral vector platform for the development of an H5N1 influenza vaccine. Preclinical assessment of MVA-based H5N1 vaccines showed their immunogenicity and safety in different animal models. We aimed to assess the safety and immunogenicity of the MVA-haemagglutinin-based H5N1 vaccine MVA-H5-sfMR in healthy individuals.METHODS: In a single-centre, double-blind phase 1/2a study, young volunteers (aged 18-28 years) were randomly assigned with a computer-generated list in equal numbers to one of eight groups and were given one injection or two injections intramuscularly at an interval of 4 weeks of a standard dose (10(8) plaque forming units [pfu]) or a ten times lower dose (10(7) pfu) of the MVA-H5-sfMR (vector encoding the haemagglutinin gene of influenza A/Vietnam/1194/2004 virus [H5N1 subtype]) or MVA-F6-sfMR (empty vector) vaccine. Volunteers and physicians who examined and administered the vaccine were masked to vaccine assignment. Individuals who received the MVA-H5-sfMR vaccine were eligible for a booster immunisation 1 year after the first immunisation. Primary endpoint was safety. Secondary outcome was immunogenicity. The trial is registered with the Dutch Trial Register, number NTR3401.FINDINGS: 79 of 80 individuals who were enrolled completed the study. No serious adverse events were identified. 11 individuals reported severe headache and lightheadedness, erythema nodosum, respiratory illness (accompanied by influenza-like symptoms), sore throat, or injection-site reaction. Most of the volunteers had one or more local (itch, pain, redness, and swelling) and systemic reactions (rise in body temperature, headache, myalgia, arthralgia, chills, malaise, and fatigue) after the first, second, and booster immunisations. Individuals who received the 10(7) dose had fewer systemic reactions. The MVA-H5-sfMR vaccine at 10(8) pfu induced significantly higher antibody responses after one and two immunisations than did 10(7) pfu when assessed with haemagglutination inhibition geometric mean titre at 8 weeks against H5N1 A/Vietnam/1194/2004 (30·2 [SD 3·8] vs 9·2 [2·3] and 108·1 [2·4] vs 15·8 [3·2]). 27 of 39 eligible individuals were enrolled in the booster immunisation study. A single shot of MVA-H5-sfMR 10(8) pfu prime immunisation resulted in higher antibody responses after the booster immunisation than did two shots of MVA-H5-sfMR at the ten times lower dose.INTERPRETATION: The MVA-based H5N1 vaccine was well tolerated and immunogenic and therefore the vaccine candidates arising from the MVA platform hold great promise for rapid development in response to a future influenza pandemic threat. However, the immunogenicity of this vaccine needs to be compared with conventional H5N1 inactivated non-adjuvanted vaccine candidates in head-to-head clinical trials.FUNDING: European Research Council.

Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses.

Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic stability and growth characteristics of MVA-MERS-S make it a suitable candidate vaccine for clinical testing. Vaccinated mice produced high levels of serum antibodies neutralizing MERS-CoV. Thus, MVA-MERS-S may serve for further development of an emergency vaccine against MERS-CoV.

Infection with human cytomegalovirus (HCMV) can cause severe complications in newborns and immunocompromised patients, and a prophylactic or therapeutic vaccine against HCMV is not available. Here, we generated a HCMV vaccine candidate fulfilling the regulatory requirements for GMP-compliant production and clinical testing. A novel synthetic fusion gene consisting of the coding sequences of HCMV pp65 and IE1 having a deleted nuclear localization sequence and STAT2 binding domain was introduced into the genome of the attenuated vaccinia virus strain MVA. This recombinant MVA, MVA-syn65_IE1, allowed for the production of a stable ∼120kDa syn65_IE1 fusion protein upon tissue culture infection. MVA-syn65_IE1 infected CD40-activated B cells activated and expanded pp65- and IE1-specific T cells derived from HCMV-seropositive donors to at least equal levels as control recombinant MVA expressing single genes for pp65 or IE1. Additionally, we show that MVA-syn65_IE1 induced HCMV pp65- and IE1-epitope specific T cells in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Thus, MVA-syn65_IE1 represents a promising vaccine candidate against HCMV and constitutes a basis for the generation of a multivalent vaccine targeting relevant pathogens in immunocompromised patients.

Due to antigenic drift of influenza viruses, seasonal influenza vaccines need to be updated annually. These vaccines are based on predictions of strains likely to circulate in the next season. However, vaccine efficacy is greatly reduced in the case of a mismatch between circulating and vaccine strains. Furthermore, novel antigenically distinct influenza viruses are introduced into the human population from animal reservoirs occasionally and may cause pandemic outbreaks. To dampen the impact of seasonal and pandemic influenza, vaccines that induce broadly protective and long-lasting immunity are preferred. Because influenza virus-specific CD8 + T cells are directed mainly against relatively conserved internal proteins, like nucleoprotein (NP), they are highly cross-reactive and afford protection against infection with antigenically distinct influenza virus strains, so-called heterosubtypic immunity. Here, we used modified vaccinia virus Ankara (MVA) as a vaccine vector for the induction of influenza virus NP-specific CD8 + T cells. To optimize the induction of CD8 + T cell responses, we made several modifications to NP, aiming at retaining the protein in the cytosol or targeting it to the proteasome. We hypothesized that these strategies would increase antigen processing and presentation and thus improve the induction of CD8 + T cell responses. We showed that NP with increased degradation rates improved CD8 + T cell activation in vitro if the amount of antigen was limited or if CD8 + T cells were of low functional avidity. However, after immunization of C57BL/6 mice, no differences were detected between modified NP and wild-type NP (NPwt), since NPwt already induced optimal CD8 + T cell responses. IMPORTANCE Due to the continuous antigenic drift of seasonal influenza viruses and the threat of a novel pandemic, there is a great need for the development of novel influenza vaccines that offer broadly protective immunity against multiple subtypes. CD8 + T cells can provide immunity against multiple subtypes of influenza viruses by the recognition of relatively conserved internal antigens. In this study, we aimed at optimizing the CD8 + T cell response to influenza A virus by making modifications to influenza A virus nucleoprotein (NP) expressed from the modified vaccinia virus Ankara (MVA) vaccine vector. These modifications resulted in increased antigen degradation, thereby producing elevated levels of peptides that can be presented on major histocompatibility complex (MHC) class I molecules to CD8 + T cells. Although we were unable to increase the NP-specific immune response in the mouse strain used, this approach may have benefits for vaccine development using less-immunogenic proteins.

International audience; Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts. (C) 2016 Elsevier B.V. All rights reserved.

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo , we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1 −/− mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4 + and CD8 + T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and in mediating vaccine-induced protective immunity. In contrast, relatively little is known about the role of T cell responses and the antigenic targets of MERS-CoV that are recognized by CD8+ T cells. In this study, the highly conserved MERS-CoV nucleocapsid (N) protein served as a target immunogen to elicit MERS-CoV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for generating MVA-MERS-N expressing recombinant N protein. Overlapping peptides spanning the whole MERS-CoV N polypeptide were used to identify major histocompatibility complex class I/II-restricted T cell responses in BALB/c mice immunized with MVA-MERS-N. We have identified a H2-d restricted decamer peptide epitope in the MERS-N protein with CD8+ T cell antigenicity. The identification of this epitope, and the availability of the MVA-MERS-N candidate vaccine, will help to evaluate MERS-N-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of MERS-CoV infection.

Immunization with modified vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8 + T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR −/− ). We found a minimal dose of 10 5 PFU of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR −/− mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus-specific CD8 + T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen-specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naive CD8 + T cells by MVA immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. IMPORTANCE We demonstrate that single-shot low-dose immunizations with vaccinia virus MVA can rapidly induce T cell-mediated protective immunity against lethal orthopoxvirus infections. Our data provide new evidence for an efficient protective capacity of vaccination with replication-deficient MVA. These data are of important practical relevance for public health, as the effectiveness of a safety-tested, next-generation smallpox vaccine based on MVA is still debated. Furthermore, producing sufficient amounts of vaccine is expected to be a major challenge should an outbreak occur. Moreover, prevention of other infections may require rapidly protective immunization; hence, MVA could be an extremely useful vaccine for delivering heterologous T cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination.

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections., Author Summary The Chikungunya virus (CHIKV) represents a threat to Europe. This is supported by its recent introduction and autochthonous transmission in Italy and France in 2007 and 2010 respectively. CHIKV causes an acute febrile illness and severe joint pains that may last for several months in some patients. In absence of a proven effective treatment, safe and effective vaccines represent the best way to control outbreaks of CHIKV. This study describes the development of a vaccine candidate based on the Modified Vaccinia Ankara virus (MVA). MVA has been used to develop vaccines against several viruses and based on the safety and efficacy records of MVA vaccines, various candidate vaccines are currently in clinical trials. MVA expressing structural proteins of CHIKV were constructed and tested in AG129 mice for immunogenicity and protective ability. All animals that received MVA expressing E2 developed neutralizing antibodies and were protected against lethal challenge. Our study shows that recombinant MVA expressing the E2 protein of CHIKV represents a potential candidate vaccine for prevention of CHIKV infections in travellers or endemic areas.

Modified Vaccinia virus Ankara (MVA) is a tissue culture-derived, highly attenuated strain of vaccinia virus (VACV) exhibiting characteristic defective replication in cells from mammalian hosts. In the 1960s MVA was originally generated as a candidate virus for safer vaccination against smallpox. Now, MVA is widely used in experimental vaccine development targeting important infectious diseases and cancer. Versatile technologies for genetic engineering, large-scale production, and quality control facilitate R&D of recombinant and non-recombinant MVA vaccines matching today's requirements for new biomedical products. Such vaccines are attractive candidates for delivering antigens from pathogens against which no, or no effective vaccine is available, including emerging infections caused by highly pathogenic influenza viruses, chikungunya virus, West Nile virus or zoonotic orthopoxviruses. Other directions are seeking valuable vaccines against highly complex diseases such as AIDS, malaria, and tuberculosis. Here, we highlight examples of MVA candidate vaccines against infectious diseases, and review the efforts made to assess both the efficacy of vaccination and immune correlates of protection in preclinical studies.

Vaccination is highly effective in preventing various infectious diseases, whereas the constant threat of new emerging pathogens necessitates the development of innovative vaccination principles that also confer rapid protection in a case of emergency. Although increasing evidence points to T cell immunity playing a critical role in vaccination against viral diseases, vaccine efficacy is mostly associated with the induction of antibody responses. Here we analyze the immunological mechanism(s) of rapidly protective vaccinia virus immunization using mousepox as surrogate model for human smallpox. We found that fast protection against lethal systemic poxvirus disease solely depended on CD4 and CD8 T cell responses induced by vaccination with highly attenuated modified vaccinia virus Ankara (MVA) or conventional vaccinia virus. Of note, CD4 T cells were critically required to allow for MVA induced CD8 T cell expansion and perforin-mediated cytotoxicity was a key mechanism of MVA induced protection. In contrast, selected components of the innate immune system and B cell-mediated responses were fully dispensable for prevention of fatal disease by immunization given two days before challenge. In conclusion, our data clearly demonstrate that perforin-dependent CD8 T cell immunity plays a key role in MVA conferred short term protection against lethal mousepox. Rapid induction of T cell immunity might serve as a new paradigm for treatments that need to fit into a scenario of protective emergency vaccination., Author Summary Prophylactic use of vaccinia virus allowed eradication of human smallpox, one of the greatest successes in medicine. However there are concerns that variola virus, the infectious agent of smallpox, may be used as bioterroristic weapon and zoonotic monkeypox or cowpox remain threatening infections in humans. Thus, new developments of safe and rapidly protecting orthopoxvirus-specific vaccines have been initiated. The candidate vaccine modified vaccinia virus Ankara (MVA) was recently shown to protect against lethal systemic poxvirus disease even when applied shortly before or after infection of mice with ectromelia virus, the probably best animal model for human smallpox. Surprisingly, little is known about the protective mechanism of early immune responses elicited against orthopoxvirus infections. Here, we used the mousepox model to analyze the immunological basis of rapidly protective MVA vaccination. In contrast to common understanding of orthopoxvirus vaccine efficacy relying mainly on antibody mediated immunity, we observed unimpaired protection also in absence of B cells. Surprisingly, rapid protection by vaccination with MVA or conventional vaccinia virus was solely dependent on T cells, irrespective of the route of injection. Thus, our study suggests a key role for T cell immunity in rapidly protective immunization against orthopoxviruses and potentially other infectious agents.