Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known as the etiological agent of coronavirus disease 2019 (COVID-19). Extrapulmonary manifestations of COVID-19 have gained increasing recognition as significant contributors to disease severity and long-term complications. The aim of this study is to investigate the neuroprotective properties of vaccines based on modified Vaccinia Virus Ankara (MVA) against SARS-CoV-2 infection in K18-hACE2 mice using different immunization protocols. Animals received PBS, vector, recombinant MVA expressing native (S) or stabilized (ST) SARS-CoV-2 spike protein, nucleocapsid protein (N) or both ST and N protein twice, followed by infection with SARS-CoV-2 four weeks later. In further experiments, mice were immunized only once and infected two days (Emergency experiment) or four weeks (Prime experiment) later. Both the control groups and the animals immunized with vaccines expressing only N-protein showed mild to moderate, lymphohistiocytic meningoencephalitis, microgliosis and numerous virus antigen-positive neurons in the brains and to a lesser extent in the retinas. Groups immunized four weeks prior to infection with vaccines containing viral spike protein showed no or minimal inflammatory changes and no neuroinvasion. Animals infected two days after immunization showed milder lesions than unvaccinated control groups.

To date, no vaccine has been approved against Zika virus (ZIKV). Any such vaccine must be safe to use during pregnancy and needs to consider immune interactions from prior flavivirus exposure given these viruses co-circulate and share the same mosquito vector. To address these issues, we have developed a vaccine using a modified vaccinia Ankara (MVA) virus. Here, we describe the development of this MVA vectored Zika vaccine that encodes structural proteins, premembrane and envelope, and non-structural (NS) proteins, NS3 and NS5 (MVA-ZIKA-B). A two-dose vaccination regimen with MVA-ZIKA-B resulted in significant serum neutralizing activity and induced polyfunctional T cell responses that protected in a mouse model of Zika, rendering virus undetectable across multiple tissues after virus challenge. Vaccine-associated protection was also characterised by high IgG antibodies against the ZIKV envelope and potent interferon-gamma (IFNγ)-positive T cell responses when stimulated ex vivo with ZIKV peptides. Further stability studies showed that the vaccine antigens were stably expressed, and the vaccine progressed to GMP manufacturing. A phase I human trial with MVA-ZIKA-B is ongoing.

Zika virus (ZIKV) outbreak of 2015 was associated with microcephaly and congenital birth defects in children born to pregnant women infected with ZIKV. Using the highly susceptible Type I Interferon Receptor-deficient mouse-model, we demonstrate that a single emergency vaccination with a non-replicating MVA-ZIKV vaccine, when administered as early as 2-days before challenge fully protected non-pregnant and pregnant mice and fetuses against lethal ZIKV-infection. Early protection was associated with the rapid emergence of ZIKV-specific CD8+ T cell responses; depletion of CD8+ T cells resulted in the loss of protection supporting a critical role for CD8+ T cells in the early protective efficacy of MVA-ZIKV. Neutralizing antibody responses were induced later than the CD8+ T cell responses, suggesting that it may play a role in later stages of infection. Our results suggest that MVA-ZIKV induces potent anamnestic cellular immunity early after infection, contributing to its protective efficacy against rapid ZIKV challenge.

Lassa virus (LASV) is circulating in rodents in several countries in West Africa and is the causative agent of the zoonotic disease Lassa fever. Several vaccine candidates have been successfully tested in preclinical and clinical research, while no LASV-specific vaccines or antiviral treatments have been licensed to date. Approximately 500,000 human cases of Lassa fever are estimated to occur every year. However, the high percentage (~80%) of asymptomatic cases and the low frequency of reporting systems in endemic regions demonstrate that Lassa fever cases are highly underreported. Given the frequent spread of the virus by travellers to non-endemic regions, the need for effective vaccines and treatments becomes clear. Here, we describe the generation and preclinical evaluation of two recombinant Lassa virus candidate vaccines, MVA-GP and MVA-NP, which are based on the highly attenuated modified vaccinia virus Ankara (MVA) strain. Constructed in the MVA vector, the MVA-GP vaccine delivers the glycoprotein (GP) of the prototype LASV Josiah strain (lineage IV), whereas the MVA-NP vaccine expresses the nucleoprotein (NP) from the Lassa virus Togo strain (lineage VII). Two immunizations of either MVA-GP or MVA-NP induced substantial polyfunctional Lassa virus-specific CD8+ and CD4+ T cell responses, respectively, in humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice (HLA-A*0201/DR1 transgenic mice). The identified human Lassa virus-specific T cell epitopes were in agreement with recently discovered T cell epitopes found in Lassa fever survivors. Further studies are warranted to characterize these recombinant MVA-Lassa virus vaccine candidates in other preclinical models and investigate their potential to be characterized in clinical studies in humans.

Background MERS-CoV is a respiratory pathogen with a case-fatality rate of 36%, and for which no vaccines currently licensed. MVA-MERS-S is a candidate vaccine based on recombinant modified vaccinia virus Ankara (MVA). In this study, the safety, immunogenicity, and optimal dose schedule of MVA-MERS-S was assessed in individuals with previous exposure to SARS-CoV-2 infections and vaccines. Methods We conducted a multicentre, double-blind, randomised controlled phase 1b clinical trial at two university medical centres in Germany and the Netherlands. Healthy volunteers aged 18-55 years were assigned by computer randomisation to receive three intramuscular injections of 107 or 10$ plaque-forming units (PFU) of MVA-MERS-S, with two treatment groups each of either 28-day or 56-day intervals between the initial two doses, and one control arm that received only placebo, at a ratio of 2:2:2:2:1. The third dose was given after 224 days. The sponsor, clinical laboratory staff, and participants were masked to both vaccine dose and dosing interval. The primary outcome safety, assessed in the all participants who had received at least one injection; daily solicited vaccine reactions were recorded after each dose for 7 days, unsolicited adverse events for 28 days, and serious adverse events throughout study. The secondary outcome was humoral immunogenicity, measured with vaccine-induced geometric mean antibody concentrations and seroconversion rates, analysed in all participants who received at least three allocated treatments. This study is registered at ClinicalTrials.gov (NCT04119440) and is completed. Findings Between 26 July, 2021, and 3 March, 2022, 244 volunteers were screened, 177 of whom were eligible 140 were randomly assigned either to the 28-day 107 PFU group (n=32), 56-day 107 PFU group (n=31), 28-day 10$ PFU group (n=31), 56-day 10$ PFU group (n=30), or placebo group (n=16). In total, 178 doses were administered of 107 PFU of MVA-MERS-S, 174 of 10$ PFU, and 164 doses of placebo, and 139 participants received least one injection. 73 (53%) were female and 66 (48%) were male. No serious vaccine-related adverse events occurred. Solicited local reactions were mild in 288 (93%, 95% CI 90-96) of 309 reports and consisted primarily of pain tenderness. Pain or tenderness (of any severity) occurred after 69 (39%, 32-46) of 178 107 PFU injections, 138 (79%; 73-85) of 174 10$ PFU injections, and 18 (11%; 7-11) of 164 placebo injections. Of 595 reported solicited systemic reactions, 479 (81%, 77-83) were graded as mild. Systemic reactions of any grade occurred after 77 (43%; 36-51) 107 PFU injections, 102 (59%; 51-66) 10$ PFU injections, and 67 (41%; 34-49) of 164 placebo injections. At 28 days after the second dose, MERS-CoV neutralising antibodies were highest for participants assigned to 56-day 10$ PFU, with geometric mean ratios of 7.2 (95% CI 3.9-13.3) for the 56-day 10$ PFU group versus the 28-day 10$ PFU group (p<00001), 3.9 (2.1-7.2) for the 56-day 10$ PFU group versus the 56-day 107 PFU group (p=0.0031), and 5.4 (2.9-10.0) for the 56-day 10$ PFU group versus the 28-day 107 PFU group (p=0.0003). Interpretation MVA-MERS-S was safe and immunogenic in individuals with previous and concurrent SARS-CoV-2 exposure. The second vaccination with the 10$ PFU dose of MVA-MERS-S elicited a stronger humoral immune response when administered 56 days after the first dose than a 28-day interval. Further studies are needed to verify these findings in groups at risk for MERS-CoV exposure, and at risk of severe disease, including older individuals and those with relevant comorbidities. Funding Coalition for Epidemic Preparedness Innovations, the German Centre for Infection Research, and the German Research Foundation.Copyright (c) 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.

Mucosal vaccines may reduce both infection and transmission by engaging local immunity, yet the immunological pathways they activate in humans remain poorly defined. Here, we present a comprehensive systems vaccinology analysis of a Modified Vaccinia virus Ankara (MVA)-based SARS-CoV-2 vaccine candidate tested in two distinct phase 1 clinical trials as a booster vaccination with either inhaled or intramuscular delivery, and benchmarked findings against published mRNA vaccine data. Longitudinal multi-omics profiling of peripheral blood and bronchoalveolar lavage revealed that inhaled vaccination induces a distinct immunological signature, characterized by effector CD8+ T cell enrichment in the respiratory tract with minimal systemic perturbation. Inhaled delivery elicited limited systemic antibody responses, yet cellular immunity was comparable to that induced by intramuscular vaccination. Transcriptional profiling uncovered shared innate and proliferative programs across both delivery routes, with temporal decoupling of gene programs associated with humoral versus cellular immunity. Machine learning identified a robust early blood transcriptional signature predictive of antibody production. These findings offer a compelling molecular rationale for mucosal vaccine strategies and highlight actionable targets for optimizing mucosal vaccines. ClinicalTrials.gov identifier: NCT05226390 , NCT04895449 .

The Brighton Collaboration Benefit-Risk Assessment of VAccines by TechnolOgy (BRAVATO) was formed to evaluate the safety and other key features of new platform technology vaccines. This manuscript provides an overview of Modified Vaccinia virus Ankara (MVA)-vectored vaccines and reviews molecular and biological key features of this platform. In particular, this review aims to provide fundamental information about the promising candidate vaccine MVA-MERS-S which has been evaluated successfully in different preclinical animal models and has undergone clinical testing including a phase Ib study involving more than 170 participants. Infectious diseases continue to be a major cause of human death worldwide. In this context, emerging zoonotic infectious diseases pose a particular challenge for public health systems. In the last two decades, three different respiratory coronaviruses, including the Middle East respiratory syndrome Coronavirus (MERS-CoV) have emerged. For many years, safe and efficacious vaccines have been a major tool to combat infectious diseases. Here, we report on a promising candidate vaccine (MVA-MERS-S) against MERS-CoV based on MVA. Upon application, MVA-MERS-S has been well tolerated and immunogenic, inducing both, cellular and humoral immune responses in different animal models and humans. We demonstrate that the MVA vector platform, with the example of MVA-MERS-S, is a viable and effective tool for producing safe, immunogenic, and efficient vaccines against emerging infectious diseases.

Over the last decade, heterologous prime–boost vaccination regimens have been established as a promising strategy to enhance immune responses and make optimal use of the advantages of different vaccine platforms. Modified vaccinia virus Ankara (MVA), a replication-deficient poxviral vector with an established safety profile, is under clinical investigation as a versatile recombinant vaccine platform against various infectious diseases. In the context of coronavirus disease 2019 (COVID-19), a recombinant MVA-based vaccine candidate expressing the prefusion-stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (MVA-ST) has demonstrated safety, immunogenicity and protection in preclinical studies using different animal models. Furthermore, a phase Ib clinical trial in healthy adults showed that MVA-ST is safe, well-tolerated and immunogenic when used as a booster following mRNA priming. In this study, we evaluated heterologous prime–boost vaccination regimens using MVA-ST as a booster in Syrian hamsters. Hamsters were primed with an mRNA vaccine (BNT162b2, BioNTech/Pfizer) or the adenoviral vector vaccine Ad26.COV2.S (Janssen) and subsequently boosted with MVA-ST at a dose of 10⁸ p.f.u. These heterologous vaccination regimens induced robust protection against severe SARS-CoV-2 disease, with superior immunogenicity compared to homologous MVA-ST vaccination. Notably, even a lower booster dose (10⁷ p.f.u.) of MVA-ST following mRNA priming conferred strong protection against SARS-CoV-2 challenge infection, while still associated with limited viral shedding from the upper respiratory tract. These findings highlight the potential of MVA-ST as a heterologous booster to enhance the immunogenicity and protective efficacy of existing COVID-19 vaccines and also to improve vaccination strategies against other emerging pathogens.

Background: Parenteral COVID-19 vaccines induce strong systemic immunity, but they do not typically trigger pronounced respiratory immunity. In this context, mucosally applied vaccines might help to induce local immune responses for early viral clearance and reduced viral transmission. Methods: In this investigator-initiated, open-label single-dose phase I trial, we analyzed the immunogenicity and safety of the vaccine candidate MVA-SARS-2-ST administered as an inhalation boost in COVID-19-immunized adults (n=23). MVA-SARS-2-ST represents a replication-deficient vector vaccine candidate built on the recombinant Modified Vaccinia virus Ankara (MVA) platform and expresses a prefusion-stabilized version of the full-length spike glycoprotein of SARS-CoV-2. Results: While there was no increase in spike-specific antibodies in the blood, the inhalation of 107 infectious units (IU) MVA-SARS-2-ST led to an increase in IFN-γ release after re-stimulation of whole blood with spike peptides. This enhanced IFN-γ release peaked at day 7 and remained detectable for at least 140 days after vaccination. Notably, selectively individuals with a history of COVID-19 (nucleocapsid protein (NCP)-seropositive study participants), but not individuals without a history of COVID-19 (NCP-seronegative study participants), showed a trend towards increased spike-specific IgA in the lung after inhalation of 107 IU MVA-SARS-2-ST. In contrast, inhaled application of MVA-SARS-2-ST robustly induced spike-specific CD4+ and CD8+ T cell responses in the lung in both NCP-seronegative and NCP-seropositive individuals. Conclusions: Collectively, our study demonstrated that a single booster inhalation of 107 IU MVA-SARS-2-ST did not have a relevant impact on the humoral immune response, but induced specific T cell responses in blood and lung. ### Competing Interest Statement Jens M. Hohlfeld reports grants for clinical trial conduct to his institution from Astellas Pharma GmbH, AstraZeneca, Bayer AG, Boehringer Ingelheim Pharma GmbH & Co. KG, Calibr at Scripps Research, Chiesi, CSL Behring, Desitin Arzneimittel GmbH, EpiEndo, F. Hoffmann-La Roche AG, Genentech, Inc., OM Pharma SA, ReAlta Life Sciences, Sanofi-Aventis Deutschland GmbH, and personal fees from Boehringer Ingelheim Pharma GmbH & Co. KG, Celerion, and Cureteq, all of which are outside the submitted work. ### Clinical Trial NCT05226390 ### Funding Statement This study was funded by MWK Niedersachsen (14-76103-184 CORONA-11/20); DZIF (TTU 01.934 and FF 01.941); DFG (EXC 2155 RESIST); DZL (grant 82DZL002B1). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of Hannover Medical School gave ethical approval for this work (no. 10012\_AMG\_mono_2021). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the corresponding author.

Existing parenteral SARS-CoV-2 vaccines protect against severe disease but do not reliably prevent infection or reinfection. Inhaled vaccines may elicit localized immunity in the respiratory tract, a principal entry site for SARS-CoV-2. In this investigator-initiated, single-center, open-label phase 1 trial ( NCT05226390 , ClinicalTrials.gov ), 23 healthy adults previously immunized with EU-approved SARS-CoV-2 vaccines received a single inhaled dose of Modified Vaccinia virus Ankara-(MVA)-SARS-2-ST (1×10 7 IU), engineered to express a prefusion-stabilized SARS-CoV-2 spike. Participants were followed for 140 days. Primary endpoints included solicited local and systemic reactogenicity through day 7, unsolicited adverse events through day 28, serious adverse events throughout, and changes in spirometry and laboratory parameters. Secondary endpoints were changes in SARS-CoV-2 S1-specific IgG in serum and bronchoalveolar lavage; exploratory endpoints included changes in S1-specific IgA in serum and bronchoalveolar lavage, and methacholine responsiveness. No serious adverse events were observed. Over 28 days, mild or moderate adverse events occurred in 87% of participants, predominantly cough, headache, and fatigue, all resolved. Pulmonary function and methacholine responsiveness were stable, except for one transient 20% decrease in FEV1 on Day 14 that normalized subsequently. Serum IgG responses remained minimal, whereas a subset displayed increased bronchoalveolar IgA. A single inhaled booster dose of MVA-SARS-2-ST was safe and generally well tolerated. While systemic antibody levels did not rise substantially, the observed mucosal IgA response in some participants points to a localized mucosal effect. Further studies are warranted to clarify underlying mechanisms and the significance of this response in diverse populations.

The availability of influenza vaccines that can induce broadly protective immune responses is highly desirable and could also mitigate the impact of future influenza pandemics. Ideally, these vaccines also induce virus-specific CD8 + T cells, which have been identified as an independent correlate of protection. In the present study, we explored the use of an artificial immunogen that comprises of twenty highly conserved influenza virus CD8 + T cell epitopes with an HLA coverage of 99.5% of the world population. The highly attenuated viral vector Modified Vaccinia virus Ankara (MVA) was used to deliver the artificial poly-epitope sequence (rMVA-PE) and by using T cell lines raised against individual epitopes, we confirmed that the epitopes are liberated from the artificial immunogen. For efficient antigen processing and presentation, the epitopes were separated by spacer sequences. Stimulation of peripheral blood mononuclear cells of HLA-typed blood donors with rMVA-PE resulted in the activation of influenza virus-specific T cell responses. Furthermore, immunization of humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice (HLA-A*02:01) with rMVA-PE induced influenza virus-specific CD8 + T cell responses. Thus, rMVA-PE proved to be immunogenic both in vitro and in vivo and constitutes a promising vaccine candidate for the induction of cross-reactive CD8 + T cell responses that could afford protection against antigenically distinct influenza A viruses (IAV) of various subtypes and species, and is currently considered for further clinical testing.

Vaccinia viruses (VACV) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified Vaccinia virus Ankara (MVA) is a particularly altered VACV strain that is highly immunogenic, incapable of replicating in mammalian hosts, and broadly used as a safe vector for vaccination. Alternatively, Western Reserve (WR) or Copenhagen (Cop) are VACV strains that efficiently replicate in cancer cells and therefore are used to develop oncolytic viruses. However, the immune evasion capacity of WR or Cop hinders their ability to elicit antitumor immune responses, which is crucial for efficacy in the clinic. Here, we describe a new VACV strain named Immune-Oncolytic Vaccinia virus Ankara (IOVA), which combines efficient replication in cancer cells with induction of immunogenic tumor cell death (ICD). IOVA was engineered from an MVA ancestor and shows superior cytotoxicity in tumor cells. In addition, the IOVA genome incorporates mutations that lead to massive fusogenesis of tumor cells, which contributes to improved antitumor effects. In syngeneic mouse tumor models, induction of ICD results in robust antitumor immunity directed against tumor neo-epitopes and eradication of large established tumors. These data present IOVA as an improved immunotherapeutic oncolytic vector.

In response to the COVID-19 pandemic, multiple vaccines were developed using platforms such as viral vectors and mRNA technology. Here, we report humoral and cellular immunogenicity data from human phase 1 clinical trials investigating two recombinant Modified Vaccinia virus Ankara vaccine candidates, MVA-SARS-2-S and MVA-SARS-2-ST, encoding the native and the prefusion-stabilized SARS-CoV-2 spike protein, respectively. MVA-SARS-2-ST was more immunogenic than MVA-SARS-2-S, but both were less immunogenic compared to licensed mRNA- and ChAd-based vaccines in SARS-CoV-2 naïve individuals. In heterologous vaccination, previous MVA-SARS-2-S vaccination enhanced T cell functionality and MVA-SARS-2-ST boosted the frequency of T cells and S1-specific IgG levels when used as a third vaccination. While the vaccine candidate containing the prefusion-stabilized spike elicited predominantly S1-specific responses, immunity to the candidate with the native spike was skewed towards S2-specific responses. These data demonstrate how the spike antigen conformation, using the same viral vector, directly affects vaccine immunogenicity in humans.

Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-β pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.

Background/Objectives: Marburg virus (MARV) is the etiological agent of Marburg Virus Disease (MVD), a rare but severe hemorrhagic fever disease with high case fatality rates in humans. Smaller outbreaks have frequently been reported in countries in Africa over the last few years, and confirmed human cases outside Africa are, so far, exclusively imported by returning travelers. Over the previous years, MARV has also spread to non-endemic African countries, demonstrating its potential to cause epidemics. Although MARV-specific vaccines are evaluated in preclinical and clinical research, none have been approved for human use. Modified Vaccinia virus Ankara (MVA), a well-established viral vector used to generate vaccines against emerging pathogens, can deliver multiple antigens and has a remarkable clinical safety and immunogenicity record, further supporting its evaluation as a vaccine against MARV. The rapid availability of safe and effective MVA-MARV vaccine candidates would expand the possibilities of multi-factored intervention strategies in endemic countries. Methods: We have used an optimized methodology to rapidly generate and characterize recombinant MVA candidate vaccines that meet the quality requirements to proceed to human clinical trials. As a proof-of-concept for the optimized methodology, we generated two recombinant MVAs that deliver either the MARV glycoprotein (MVA-MARV-GP) or the MARV nucleoprotein (MVA-MARV-NP). Results: Infections of human cell cultures with recombinant MVA-MARV-GP and MVA-MARV-NP confirmed the efficient synthesis of MARV-GP and MARV-NP proteins in mammalian cells, which are non-permissive for MVA replication. Prime-boost immunizations in C57BL/6J mice readily induced circulating serum antibodies binding to recombinant MARV-GP and MARV-NP proteins. Moreover, the MVA-MARV-candidate vaccines elicited MARV-specific T-cell responses in C57BL/6J mice. Conclusions: We confirmed the suitability of our two backbone viruses MVA-mCherry and MVA-GFP in a proof-of-concept study to rapidly generate candidate vaccines against MARV. However, further studies are warranted to characterize the protective efficacy of these recombinant MVA-MARV vaccines in other preclinical models and to evaluate them as vaccine candidates in humans.

The sudden emergence of SARS-CoV-2 demonstrates the need for new vaccines that rapidly protect in the case of an emergency. In this study, we developed a recombinant MVA vaccine co-expressing SARS-CoV-2 prefusion-stabilized spike protein (ST) and SARS-CoV-2 nucleoprotein (N, MVA-SARS-2-ST/N) as an approach to further improve vaccine-induced immunogenicity and efficacy. Single MVA-SARS-2-ST/N vaccination in K18-hACE2 mice induced robust protection against lethal respiratory SARS-CoV-2 challenge infection 28 days later. The protective outcome of MVA-SARS-2-ST/N vaccination correlated with the activation of SARS-CoV-2-neutralizing antibodies (nABs) and substantial amounts of SARS-CoV-2-specific T cells especially in the lung of MVA-SARS-2-ST/N-vaccinated mice. Emergency vaccination with MVA-SARS-2-ST/N just 2 days before lethal SARS-CoV-2 challenge infection resulted in a delayed onset of clinical disease outcome in these mice and increased titers of nAB or SARS-CoV-2-specific T cells in the spleen and lung. These data highlight the potential of a multivalent COVID-19 vaccine co-expressing S- and N-protein, which further contributes to the development of rapidly protective vaccination strategies against emerging pathogens.

Aging is associated with a decline in immune system functionality. So-called immunosenescence may impair the successful vaccination of elderly people. Thus, improved vaccination strategies also suitable for an aged immune system are required. Modified Vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus that has been established as a multipurpose viral vector for vaccine development against various infections. We characterized a recombinant MVA expressing a prefusion-stabilized version of SARS-CoV-2 S protein (MVA-ST) in an aged-hamster model for COVID-19. Intramuscular MVA-ST immunization resulted in protection from disease and severe lung pathology. Importantly, this protection was correlated with a potent activation of SARS-CoV-2 specific T-cells and neutralizing antibodies. Our results suggest that MVA vector vaccines merit further evaluation in preclinical models to contribute to future clinical development as candidate vaccines in elderly people to overcome the limitations of age-dependent immunosenescence.

Licensed vaccines against the Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging pathogen of concern, are lacking. The modified vaccinia virus Ankara vector-based vaccine MVA-MERS-S, expressing the MERS-CoV-spike glycoprotein (MERS-S), is one of 3 candidate vaccines in clinical development and elicits robust humoral and cellular immunity. Here, we identified for the first time a MERS-S-specific CD8(+) T-cell epitope in an HLA-A*03:01/HLA-B*35:01-positive vaccinee using a screening assay, intracellular cytokine staining, and in silico epitope prediction. As evidence from MERS-CoV infection suggests a protective role of long-lasting CD8(+) T-cell responses, the identification of epitopes will facilitate longitudinal analyses of vaccine-induced T-cell immunity.

Tick-borne encephalitis (TBE) is a serious neurological disease caused by TBE virus (TBEV). Because antiviral treatment options are not available, vaccination is the key prophylactic measure against TBEV infections. Despite the availability of effective vaccines, cases of vaccination breakthrough infections have been reported. The multienzymatic non-structural protein 3 (NS3) of orthoflaviviruses plays an important role in polyprotein processing and virus replication. In the present study, we evaluated NS3 of TBEV as a potential vaccine target for the induction of protective immunity. To this end, a recombinant modified vaccinia virus Ankara that drives the expression of the TBEV NS3 gene (MVA-NS3) was constructed. MVA-NS3 was used to immunize C57BL/6 mice. It induced NS3-specific immune responses, in particular T cell responses, especially against the helicase domain of NS3. However, MVA-NS3-immunized mice were not protected from subsequent challenge infection with a lethal dose of the TBEV strain Neudoerfl, indicating that in contrast to immunity to prME and NS1, NS3-specific immunity is not an independent correlate of protection against TBEV in this mouse model.

The emergence of hitherto unknown viral pathogens presents a great challenge for researchers to develop effective therapeutics and vaccines within a short time to avoid an uncontrolled global spread, as seen during the coronavirus disease 2019 (COVID-19) pandemic. Therefore, rapid and simple methods to identify immunogenic antigens as potential therapeutical targets are urgently needed for a better pandemic preparedness. To address this problem, we chose the well-characterized Modified Vaccinia virus Ankara (MVA)-T7pol expression system to establish a workflow to identify immunogens when a new pathogen emerges, generate candidate vaccines, and test their immunogenicity in an animal model. By using this system, we detected severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) nucleoprotein (N)-, and spike (S)-specific antibodies in COVID-19 patient sera, which is in line with the current literature and our observations from previous immunogenicity studies. Furthermore, we detected antibodies directed against the SARS-CoV-2-membrane (M) and -ORF3a proteins in COVID-19 patient sera and aimed to generate recombinant MVA candidate vaccines expressing either the M or ORF3a protein. When testing our candidate vaccines in a prime-boost immunization regimen in humanized HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice, we were able to demonstrate M- and ORF3a-specific cellular and humoral immune responses. Hence, the established workflow using the MVA-T7pol expression system represents a rapid and efficient tool to identify potential immunogenic antigens and provides a basis for future development of candidate vaccines.