Critical role of perforin-dependent CD8+ T cell immunity for rapid protective vaccination in a murine model for human smallpox.

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.

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection.

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.

Uptake of antigens from modified vaccinia Ankara virus-infected leukocytes enhances the immunostimulatory capacity of dendritic cells.

BACKGROUND AIMS: Modified vaccinia Ankara (MVA) is a promising vaccine vector for infectious diseases and malignancies. It is fundamental to ascertain its tropism in human leukocyte populations and immunostimulatory mechanisms for application in immunotherapy. METHODS: Human peripheral blood mononuclear cells (PBMC) and leukocyte subpopulations [monocyte-derived dendritic cells (DC), monocytes and B cells] were infected with MVA in order to evaluate their infection rate, changes in surface markers, cytokine expression and apoptosis. RESULTS: Monocytes, DC and B cells were most susceptible to MVA infection, followed by natural killer (NK) cells. Monocytes were activated strongly, with upregulation of co-stimulatory molecules, major histocompatibility complex (MHC) molecules and chemokine (C-C motif) receptor (CCR7), while immature DC showed partial activation and B cells were inhibited. Furthermore, expression of chemokine (C-X-C motif) ligand (CXCL10), tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-12p70 was enhanced but IL-1ß and IL-10 were stable or even downregulated. MVA induced a high apoptosis rate of antigen-presenting cells (APC). Nevertheless, incubation of MVA-infected leukocytes with uninfected immature DC (iDC) led to complete maturation of the DC. Subsequently, the matured DC were able to stimulate cytomegalovirus (CMV)-immediate early protein (IE1)-specific T cells. CONCLUSIONS: MVA induces a T-helper (Th)-1-polarizing cytokine expression in APC. Furthermore, incubation of MVA-infected leukocytes with uninfected iDC leads to complete maturation of the DC and may be the basis for cross-presentation of MVA-encoded antigens. Thus this approach seems to be an ideal model for further studies with MVA-encoded viral antigens regarding immunotherapy and vaccination strategies.

Modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of CCL2 expression.

Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4(+) lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.

Vaccination with a Modified Vaccinia Virus Ankara-based vaccine protects mice from allergic sensitization.

BACKGROUND: Currently, no treatment is available for food allergy and strict avoidance of the allergenic food remains the only way to manage the allergy. New strategies leading to a safe and efficacious food allergy treatment are required. Modified vaccinia virus Ankara (MVA), which allows high levels of expression of recombinant protein in vivo and gives rise to a Th1-biased specific immune response, was used as a prophylactic vaccine in a murine model of ovalbumin (OVA) allergy. METHODS: An MVA-OVA vector vaccine was prepared. Female BALB/c mice were vaccinated twice with a MVA-OVA vector vaccine, followed by sensitization with OVA plus alum. OVA-specific immunoglobulin E(IgE) activity was measured by mediator release from rat basophilic leukaemia cells, whereas specific IgG subclass titers were determined by enzyme-linked immunosorbent assay. RESULTS: Expression of immunological active OVA in mammalian cells was demonstrated. OVA-specific IgE levels in sera from MVA-OVA-vaccinated mice were reduced and appeared delayed. The vaccine-mediated immune modulation was dose-dependent; the highest vaccine dose protected 50% of the animals from allergic sensitization. Upon sensitization, similar OVA-specific IgG1 titers were found in all mice, but the OVA-specific IgG2a antibody levels were strongly increased in MVA-OVA-vaccinated mice, signifying a Th1-biased and, non-allergic immune response. CONCLUSIONS: Prophylactic vaccination with MVA-OVA delays and in part even prevents the onset of a successful allergen-specific sensitization. Recombinant MVA, which fulfills the requirements for clinical application, is a promising candidate vector for the development of novel approaches to allergen-specific prophylactic vaccination and specific immunotherapy.

A synthetic human cytomegalovirus pp65-IE1 fusion antigen efficiently induces and expands virus specific T cells.

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.

Vaccinia virus replication is not affected by APOBEC3 family members.

BACKGROUND: The APOBEC3G protein represents a novel innate defense mechanism against retroviral infection. It facilitates the deamination of the cytosine residues in the single stranded cDNA intermediate during early steps of retroviral infection. Most poxvirus genomes are relatively A/T-rich, which may indicate APOBEC3G-induced mutational pressure. In addition, poxviruses replicate exclusively in the cytoplasm where APOBEC3G is located. It was therefore tempting to analyze whether vaccinia virus replication is affected by APOBEC3G. RESULTS: The replication of vaccinia virus, a prototype poxvirus, was not, however, inhibited in APOBEC3G-expressing cells, nor did other members of the APOBEC3 family alter vaccinia virus replication. HIV counteracts APOBEC3G by inducing its degradation. However, Western blot analysis showed that the levels of APOBEC3G protein were not affected by vaccinia virus infection. CONCLUSION: The data indicate that APOBEC3G is not a restriction factor for vaccinia virus replication nor is vaccinia virus able to degrade APOBEC3G.

CD40 ligand-triggered human dendritic cells mount interleukin-23 responses that are further enhanced by danger signals.

Interleukin (IL)-23 is a heterodimeric cytokine composed of the IL-23-specific subunit p19 and the p40 subunit which also constitutes part of IL-12. IL-23 propagates development of Th17 cells, a novel T cell subset which produces IL-17 but no interferon-gamma or IL-4. For both, IL-23 and IL-23-driven IL-17, a crucial role in autoimmune diseases such as experimental autoimmune encephalomyelitis, collagen-induced arthritis, and colitis is well accepted. Recent studies indicate that there is also a role for IL-23 and IL-17 in tumorigenesis, promoting tumor growth and vascularization, and affecting tumor incidence. We show that human CD14(+) peripheral blood monocyte-derived dendritic cells (DC), as used for clinical applications in anti-tumor immunization strategies, produce high amounts of IL-23. CD40-triggering of immature and mature DC but not of primary monocytes induced a rapid expression of high levels of IL-23, free p40, and minor levels of IL-12. Upon stimulation of DC subsets with a variety of different danger signals such as single stranded and double stranded RNA, bacterial components or viral infections, IL-23 expression pattern was analyzed. Interestingly, co-stimulation with CD40L enabled IL-23 expression by DC subsets towards danger signals to which they have been unresponsive upon single stimulation. Furthermore, we detected two novel splice variants of the IL-23-specific subunit p19 that could be associated with the regulation of IL-23 expression. Data presented here might have an impact on DC-based cancer vaccination strategies and contribute to a better understanding of the complex regulation of the heterodimeric cytokine IL-23.

Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells.

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro-differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

Postexposure immunization with modified vaccinia virus Ankara or conventional Lister vaccine provides solid protection in a murine model of human smallpox.

BACKGROUND: Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain. METHODS: We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV). RESULTS: In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses. CONCLUSIONS: ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.

MVA-based H5N1 vaccine affords cross-clade protection in mice against influenza A/H5N1 viruses at low doses and after single immunization.

Human infections with highly pathogenic avian influenza viruses of the H5N1 subtype, frequently reported since 2003, result in high morbidity and mortality. It is feared that these viruses become pandemic, therefore the development of safe and effective vaccines is desirable. MVA-based H5N1 vaccines already proved to be effective when two immunizations with high doses were used. Dose-sparing strategies would increase the number of people that can be vaccinated when the amount of vaccine preparations that can be produced is limited. Furthermore, protective immunity is induced ideally after a single immunization. Therefore the minimal requirements for induction of protective immunity with a MVA-based H5N1 vaccine were assessed in mice. To this end, mice were vaccinated once or twice with descending doses of a recombinant MVA expressing the HA gene of influenza virus A/Vietnam/1194/04. The protective efficacy was determined after challenge infection with the homologous clade 1 virus and a heterologous virus derived from clade 2.1, A/Indonesia/5/05 by assessing weight loss, virus replication and histopathological changes. It was concluded that MVA-based vaccines allowed significant dose-sparing and afford cross-clade protection, also after a single immunization, which are favorable properties for an H5N1 vaccine candidate.

Protective efficacy of several vaccines against highly pathogenic H5N1 avian influenza virus under experimental conditions.

Although several vaccines have been developed to protect against highly pathogenic avian influenza of subtype H5N1 'Asia' their efficiency has primarily been assessed individually. Thus, a direct comparison of their performance is still lacking. The following study was conducted to compare the protective efficacy of three commercially available inactivated vaccines based on influenza virus strains of subtypes H5N2 (vaccine A), H5N9 (vaccine B), and H5N3 (vaccine C), as well as two hemagglutinin expressing experimental vector vaccines (modified vaccinia virus Ankara-H5 and Newcastle disease virus-H5) against a lethal dose of highly pathogenic H5N1 avian influenza virus in chickens. To assess their potential as emergency vaccines, a single immunisation was performed for all vaccines, despite the recommendation of a double-vaccination schedule for commercial vaccines B and C. Overall, all vaccines induced clinical protection against challenge infection 3 weeks after immunisation. No mortality was observed in chickens immunised with vaccine A and viral shedding could not be detected. Immunisation with NDV-H5, vaccine C and MVA-H5 conferred also protection against lethal challenge. However, viral RNA was detected by real-time RT-PCR in swabs of 10%, 20% and 50% of animals, and 0%, 10% and 30% of animals, respectively, shed infectious virus. Immunisation with vaccine B was less protective since 50% of the vaccinated animals shed infectious virus after challenge and 20% of the chickens succumbed to disease. These results indicate that the NDV-H5 vectored vaccine is similarly effective as the best inactivated vaccine. Considering the advantage of live NDV which can be administered via spray or drinking water as well as the potential use of this H5 expressing vector vaccine for an easy DIVA (differentiating infected from vaccinated animals) strategy, NDV-H5 could represent an alternative for extensive vaccination against avian influenza in chickens.

Vaccination with recombinant modified vaccinia virus Ankara expressing bovine respiratory syncytial virus (bRSV) proteins protects calves against RSV challenge.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and calves. Bovine RSV (bRSV) is a natural pathogen for cattle, and bRSV infection in calves shares many features with the human infection. Thus, bRSV infection in cattle provides the ideal setting to evaluate the safety and efficacy of novel RSV vaccine strategies. Here, we have evaluated the efficacy and safety of modified vaccinia virus Ankara (rMVA)-based vaccine candidates, expressing the bovine RSV-F protein, either or not in combination with the G protein, in colostrums-deprived SPF calves born by caesarean section. Vaccination induced bRSV-specific IgG and CD8 T cell responses. Importantly, no IgE responses were detected. After bRSV challenge, rMVA vaccinated calves experienced less severe symptoms of lower respiratory tract disease compared to the mock-immunized control group. Immunized animals showed reduced pulmonary virus loads, and no eosinophilic infiltration or enhanced respiratory distress. In conclusion, candidate rMVA/bRSV vaccines induced protective and safe immune responses in calves.

Recombinant modified vaccinia virus Ankara-based vaccine induces protective immunity in mice against infection with influenza virus H5N1.

Since 2003, the number of human cases of infections with highly pathogenic avian influenza viruses of the H5N1 subtype is still increasing, and, therefore, the development of safe and effective vaccines is considered a priority. However, the global production capacity of conventional vaccines is limited and insufficient for a worldwide vaccination campaign. In the present study, an alternative H5N1 vaccine candidate based on the replication-deficient modified vaccinia virus Ankara (MVA) was evaluated. C57BL/6J mice were immunized twice with MVA expressing the hemagglutinin (HA) gene from influenza virus A/Hongkong/156/97 (MVA-HA-HK/97) or A/Vietnam/1194/04 (MVA-HA-VN/04). Subsequently, recombinant MVA-induced protective immunity was assessed after challenge infection with 3 antigenically distinct strains of H5N1 influenza viruses: A/Hongkong/156/97, A/Vietnam/1194/04, and A/Indonesia/5/05. Our data suggest that recombinant MVA expressing the HA of influenza virus A/Vietnam/1194/04 is a promising alternative vaccine candidate that could be used for the induction of protective immunity against various H5N1 influenza strains.

Short-term, but not post-exposure, protection against lethal orthopoxvirus challenge after immunization with modified vaccinia virus Ankara.

Safety-tested vaccinia virus (VACV) MVA serves as a candidate third-generation vaccine against smallpox. Here, MVA immunization of mice shortly before or after lethal respiratory challenge with VACV Western Reserve was investigated. Whilst post-exposure treatment failed to protect animals, immunizations on day 2 prior to challenge were fully protective. On the day of challenge, MVA inoculation may prevent death, but not onset of severe respiratory disease. After intranasal MVA application, massive influx of leukocytes (such as neutrophils, macrophages, natural killer cells and T cells) was found in the lungs of the animals, indicating the contribution of innate responses to protection. Correspondingly, in RAG-1-/- mice, MVA inoculation delayed onset of disease significantly, but did not prevent fatal infection. Thus, short-term protection required a tight interplay of both innate and adaptive antiviral immunity. These data suggest that, in addition to conventional vaccination, MVA may serve for potent emergency prophylaxis against orthopoxvirus infection.

Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara.

Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-DeltaE3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2'-5'OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2alpha (eIF2alpha) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-DeltaE3L-infected mouse embryonic fibroblasts from Pkr(0/0) mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.

Protective and disease-enhancing immune responses induced by recombinant modified vaccinia Ankara (MVA) expressing respiratory syncytial virus proteins.

Modified vaccinia Ankara (MVA) recombinants expressing single or multiple RSV surface proteins (F or G) are promising potential vaccines. We studied humoral and cellular responses induced by MVA-F and MVA-G in mice, comparing them to a formalin inactivated RSV preparation (FI-RSV) known to increase disease severity. MVA-F or MVA-G vaccination enhanced weight loss during RSV challenge, but did not show the lung eosinophilia seen after FI-RSV vaccination. FI-RSV induced a stronger total RSV IgG response than the MVA recombinants, but very little IgG2a. MVA recombinants induced cytokine responses biased towards IFNgamma and IL-12, while FI-RSV induced strong IL-4/5 responses in the lungs during RSV challenge. Thus, MVA vaccines induce a favourable immune profile in RSV disease but retain the potential to enhance disease.