A Monovalent and Trivalent MVA-Based Vaccine Completely Protects Mice Against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenge.

Venezuelan, eastern and western equine encephalitis viruses (EEV) can cause severe disease of the central nervous system in humans, potentially leading to permanent damage or death. Yet, no licensed vaccine for human use is available to protect against these mosquito-borne pathogens, which can be aerosolized and therefore pose a bioterror threat in addition to the risk of natural outbreaks. Using the mouse aerosol challenge model, we evaluated the immunogenicity and efficacy of EEV vaccines that are based on the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform: three monovalent vaccines expressing the envelope polyproteins E3-E2-6K-E1 of the respective EEV virus, a mixture of these three monovalent EEV vaccines (Triple-Mix) as a first approach to generate a multivalent vaccine, and a true multivalent alphavirus vaccine (MVA-WEV, Trivalent) encoding the polyproteins of all three EEVs in a single non-replicating MVA viral vector. BALB/c mice were vaccinated twice in a four-week interval and samples were assessed for humoral and cellular immunogenicity. Two weeks after the second immunization, animals were exposed to aerosolized EEV. The majority of vaccinated animals exhibited VEEV, WEEV, and EEEV neutralizing antibodies two weeks post-second administration, whereby the average VEEV neutralizing antibodies induced by the monovalent and Trivalent vaccine were significantly higher compared to the Triple-Mix vaccine. The same statistical difference was observed for VEEV E1 specific T cell responses. However, all vaccinated mice developed comparable interferon gamma T cell responses to the VEEV E2 peptide pools. Complete protective efficacy as evaluated by the prevention of mortality and morbidity, lack of clinical signs and viremia, was demonstrated for the respective monovalent MVA-EEV vaccines, the Triple-Mix and the Trivalent single vector vaccine not only in the homologous VEEV Trinidad Donkey challenge model, but also against heterologous VEEV INH-9813, WEEV Fleming, and EEEV V105-00210 inhalational exposures. These EEV vaccines, based on the safe MVA vector platform, therefore represent promising human vaccine candidates. The trivalent MVA-WEV construct, which encodes antigens of all three EEVs in a single vector and can potentially protect against all three encephalitic viruses, is currently being evaluated in a human Phase 1 trial.

Evaluation of modified Vaccinia Ankara-based vaccines against foot-and-mouth disease serotype A24 in cattle.

To prepare foot-and-mouth disease (FMD) recombinant vaccines in response to newly emerging FMD virus (FMDV) field strains, we evaluated Modified Vaccinia virus Ankara-Bavarian Nordic (MVA-BN®) as an FMD vaccine vector platform. The MVA-BN vector has the capacity to carry and express numerous foreign genes and thereby has the potential to encode antigens from multiple FMDV strains. Moreover, this vector has an extensive safety record in humans. All MVA-BN-FMD constructs expressed the FMDV A24 Cruzeiro P1 capsid polyprotein as antigen and the FMDV 3C protease required for processing of the polyprotein. Because the FMDV wild-type 3C protease is detrimental to mammalian cells, one of four FMDV 3C protease variants were utilized: wild-type, or one of three previously reported mutants intended to dampen protease activity (C142T, C142L) or to increase specificity and thereby reduce adverse effects (L127P). These 3C coding sequences were expressed under the control of different promoters selected to reduce 3C protease expression. Four MVA-BN-FMD constructs were evaluated in vitro for acceptable vector stability, FMDV P1 polyprotein expression, processing, and the potential for vaccine scale-up production. Two MVA-BN FMD constructs met the in vitro selection criteria to qualify for clinical studies: MVA-mBN360B (carrying a C142T mutant 3C protease and an HIV frameshift for reduced expression) and MVA-mBN386B (carrying a L127P mutant 3C protease). Both vaccines were safe in cattle and elicited low to moderate serum neutralization titers to FMDV following multiple dose administrations. Following FMDV homologous challenge, both vaccines conferred 100% protection against clinical FMD and viremia using single dose or prime-boost immunization regimens. The MVA-BN FMD vaccine platform was capable of differentiating infected from vaccinated animals (DIVA). The demonstration of the successful application of MVA-BN as an FMD vaccine vector provides a platform for further FMD vaccine development against more epidemiologically relevant FMDV strains.

Protective efficacy of monovalent and trivalent recombinant MVA-based vaccines against three encephalitic alphaviruses.

The three encephalitic alphaviruses, western, eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV) are potential biothreat agents due to high infectivity through aerosol exposure, ease of production in large amounts, and relative stability in the environment. Currently, there is no licensed vaccine for human use to these three encephalitic alphaviruses, and efforts to move vaccine candidates forward into clinical trials have not been successful. In this study, the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform was used to construct and produce three monovalent recombinant MVA-BN-based encephalitic alphavirus vaccines, MVA-BN-W, MVA-BN-E, and MVA-BN-V. Additionally, a MVA-BN-based construct was designed to produce antigens against all three alphaviruses, the trivalent vaccine MVA-BN-WEV. The protective efficacy of these vaccines was evaluated in vivo. Female BALB/c mice were immunized with two doses of each monovalent MVA-BN-based alphavirus vaccine, a mixture of the three monovalent vaccines, MVA-BN-W + E + V, or the trivalent vaccine MVA-BN-WEV at a four-week interval. Two weeks after the booster immunization, the mice were instilled intranasally with 5 × 103 to 1 × 104 plaque forming units of WEEV, EEEV, or VEEV. All mice immunized with monovalent vaccines survived the respective virus challenge without any signs of illness or weight loss, while all the control mice died. The triple mixture of vaccines or the trivalent vaccine also provided 90 to 100% protection to the mice against WEEV and VEEV challenges, and 60% to 90% protection against EEEV challenge. These data suggest that each monovalent MVA-BN-W, MVA-BN-E, and MVA-BN-V is a potential vaccine candidate against respective encephalitic alphavirus and the three monovalent vaccines can be given in a mixture (MVA-BN-W + E + V) or the trivalent vaccine MVA-BN-WEV can serve as a true multivalent vaccine without significantly reducing efficacy against WEEV and VEEV despite slightly reduced efficacy against EEEV challenge.

Correction: A prophylactic multivalent vaccine against different filovirus species is immunogenic and provides protection from lethal infections with Ebolavirus and Marburgvirus species in non-human primates.

[This corrects the article DOI: 10.1371/journal.pone.0192312.].

A prophylactic multivalent vaccine against different filovirus species is immunogenic and provides protection from lethal infections with Ebolavirus and Marburgvirus species in non-human primates.

The search for a universal filovirus vaccine that provides protection against multiple filovirus species has been prompted by sporadic but highly lethal outbreaks of Ebolavirus and Marburgvirus infections. A good prophylactic vaccine should be able to provide protection to all known filovirus species and as an upside potentially protect from newly emerging virus strains. We investigated the immunogenicity and protection elicited by multivalent vaccines expressing glycoproteins (GP) from Ebola virus (EBOV), Sudan virus (SUDV), Taï Forest virus (TAFV) and Marburg virus (MARV). Immune responses against filovirus GP have been associated with protection from disease. The GP antigens were expressed by adenovirus serotypes 26 and 35 (Ad26 and Ad35) and modified Vaccinia virus Ankara (MVA) vectors, all selected for their strong immunogenicity and good safety profile. Using fully lethal NHP intramuscular challenge models, we assessed different vaccination regimens for immunogenicity and protection from filovirus disease. Heterologous multivalent Ad26-Ad35 prime-boost vaccination regimens could give full protection against MARV (range 75%-100% protection) and EBOV (range 50% to 100%) challenge, and partial protection (75%) against SUDV challenge. Heterologous multivalent Ad26-MVA prime-boost immunization gave full protection against EBOV challenge in a small cohort study. The use of such multivalent vaccines did not show overt immune interference in comparison with monovalent vaccines. Multivalent vaccines induced GP-specific antibody responses and cellular IFNγ responses to each GP expressed by the vaccine, and cross-reactivity to TAFV GP was detected in a trivalent vaccine expressing GP from EBOV, SUDV and MARV. In the EBOV challenge studies, higher humoral EBOV GP-specific immune responses (p = 0.0004) were associated with survival from EBOV challenge and less so for cellular immune responses (p = 0.0320). These results demonstrate that it is feasible to generate a multivalent filovirus vaccine that can protect against lethal infection by multiple members of the filovirus family.

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant.IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.

IFN-γ Primes Keratinocytes for HSV-1-Induced Inflammasome Activation.

Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1ß and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1ß and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1ß and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1ß expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells.

A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines.

Immunotherapy with MVA-BN®-HER2 induces HER-2-specific Th1 immunity and alters the intratumoral balance of effector and regulatory T cells.

MVA-BN®-HER2 is a new candidate immunotherapy designed for the treatment of HER-2-positive breast cancer. Here, we demonstrate that a single treatment with MVA-BN®-HER2 exerts potent anti-tumor efficacy in a murine model of experimental pulmonary metastasis. This anti-tumor efficacy occurred despite a strong tumor-mediated immunosuppressive environment characterized by a high frequency of regulatory T cells (T(reg)) in the lungs of tumor-bearing mice. Immunogenicity studies showed that treatment with MVA-BN®-HER2 induced strongly Th1-dominated HER-2-specific antibody and T-cell responses. MVA-BN®-HER2-induced anti-tumor activity was characterized by an increased infiltration of lungs with highly activated, HER-2-specific, CD8+CD11c+ T cells accompanied by a decrease in the frequency of T(reg) cells in the lung, resulting in a significantly increased ratio of effector T cells to T(reg) cells. In contrast, administration of HER2 protein formulated in Complete Freund's Adjuvant (CFA) induced a strongly Th2-biased immune response to HER-2. However, this did not lead to significant infiltration of the tumor-bearing lungs by CD8+ T cells or the decrease in the frequency of T(reg) cells nor did it result in anti-tumor efficacy. In vivo depletion of CD8+ cells confirmed that CD8 T cells were required for the anti-tumor activity of MVA-BN®-HER2. Furthermore, depletion of CD4+ or CD25+ cells demonstrated that tumor-induced T(reg) cells promoted tumor growth and that CD4 effector cells also contribute to MVA-BN®-HER2-mediated anti-tumor efficacy. Taken together, our data demonstrate that treatment with MVA-BN®-HER2 controls tumor growth through mechanisms including the induction of Th1-biased HER-2-specific immune responses and the control of tumor-mediated immunosuppression.

Immediate-early expression of a recombinant antigen by modified vaccinia virus ankara breaks the immunodominance of strong vector-specific B8R antigen in acute and memory CD8 T-cell responses.

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses.

Hammerhead ribozymes with cleavage site specificity for NUH and NCH display significant anti-hepatitis C viral effect in vitro and in recombinant HepG2 and CCL13 cells.

BACKGROUND/AIMS: Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system. METHODS: For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively. RESULTS: A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct. CONCLUSIONS: This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.

Requirements for adeno-associated virus-derived non-viral vectors to achieve stable and site-specific integration of plasmid DNA in liver carcinoma cells.

BACKGROUND AND AIMS: Adeno-associated virus (AAV) is the only known virus capable of site-specific genomic integration in human cells. Thus, AAV-based vectors may be an attractive option to achieve prolonged transgene expression in human cells. We therefore studied the minimal elements of gene therapy vectors necessary for stable integration and tested the effectiveness of this approach in hepatoma cells. METHODS: Plasmids were constructed that contained a GFPneo fusion transgene with or without the AAV-inverted terminal repeats (ITRs). In addition, Rep protein was either encoded in CIS or supplied in TRANS by co-transfections. Stable clones were analyzed by Southern blotting for site-specific integration. RESULTS: The ITRs alone conferred neither stable nor site-specific transgene integration. Expression of Rep protein in CIS or TRANS resulted in an increased frequency of integration regardless of the presence of ITRs. It was shown that in the absence of the ITRs, other Rep-binding site (RBS) like sequences such as the ColE1 sequence present in plasmid backbones can function as RBS. Site-specific integration was achieved in up to 26% of clones derived from hepatoma cells. CONCLUSION: Both expression of Rep proteins and inclusion of a RBS are necessary for enhanced and stable integration of AAV-based non-viral vectors. A novel two-plasmid system capable of achieving stable and site-specific gene transfer in hepatoma cells is introduced.