Single MVA-SARS-2-ST/N Vaccination Rapidly Protects K18-hACE2 Mice against a Lethal SARS-CoV-2 Challenge Infection.

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.

Protective MVA-ST Vaccination Robustly Activates T Cells and Antibodies in an Aged-Hamster Model for COVID-19.

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.

MVA-based vaccine candidates encoding the native or prefusion-stabilized SARS-CoV-2 spike reveal differential immunogenicity in humans.

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.

SARS-CoV-2 inactivation in laboratory animal tissues with 4% formaldehyde or 5% glutaraldehyde for transfer to biosafety level 1 laboratories.

The SARS-CoV-2 pandemic required the immediate need to transfer inactivated tissue from biosafety level (BSL)-3 to BSL-1 areas to enable downstream analytical methods. No validated SARS-CoV-2 inactivation protocols were available for either formaldehyde (FA)-fixed or glutaraldehyde (GA)-fixed tissues. Therefore, representative tissue from ferrets and hamsters was spiked with 2.2 × 106 tissue culture infectious dose 50% per ml (TCID50/ml) SARS-CoV-2 or were obtained from mice experimentally infected with SARS-CoV-2. SARS-CoV-2 inactivation was demonstrated with 4% FA or 5% GA at room temperature for 72 hours by a titer reduction of up to 103.8 TCID50/ml in different animal tissues with a maximum protein content of 100 µg/mg and a thickness of up to 10 mm for FA and 8 mm for GA. Our protocols can be easily adapted for validating the inactivation of other pathogens to allow for the transfer of biological samples from BSL-3 areas to BSL-1 laboratories.

The effect of Toll-like receptor agonists on the immunogenicity of MVA-SARS-2-S vaccine after intranasal administration in mice.

Background and aims: Modified Vaccinia virus Ankara (MVA) represents a promising vaccine vector for respiratory administration to induce protective lung immunity including tertiary lymphoid structure, the bronchus-associated lymphoid tissue (BALT). However, MVA expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein (MVA-SARS-2-S) required prime-boost administration to induce high titers of anti-Spike antibodies in serum and bronchoalveolar lavage (BAL). As the addition of adjuvants enables efficient tailoring of the immune responses even to live vaccines, we tested whether Toll-like receptor (TLR)-agonists affect immune responses induced by a single dose of intranasally applied MVA-SARS-2-S. Methods: We intranasally immunized C57BL/6 mice with MVA-SARS-2-S vaccine in the presence of either TLR3 agonist polyinosinic polycytidylic acid [poly(I:C)], TLR4 agonist bacterial lipopolysaccharide (LPS) from Escherichia coli, or TLR9 agonist CpG oligodeoxynucleotide (CpG ODN) 1826. At different time-points after immunization, we analyzed induced immune responses using flow cytometry, immunofluorescent microscopy, and ELISA. Results: TLR agonists had profound effects on MVA-SARS-2-S-induced immune responses. At day 1 post intranasal application, the TLR4 agonist significantly affected MVA-induced activation of dendritic cells (DCs) within the draining bronchial lymph nodes, increasing the ratio of CD11b+CD86+ to CD103+CD86+ DCs. Nevertheless, the number of Spike-specific CD8+ T cells within the lungs at day 12 after vaccination was increased in mice that received MVA-SARS-2-S co-administered with TLR3 but not TLR4 agonists. TLR9 agonist did neither significantly affect MVA-induced DC activation nor the induction of Spike-specific CD8+ T cells but reduced both number and size of bronchus-associated lymphoid tissue. Surprisingly, the addition of all TLR agonists failed to boost the levels of Spike-specific antibodies in serum and bronchoalveolar lavage. Conclusions: Our study indicates a potential role of TLR-agonists as a tool to modulate immune responses to live vector vaccines. Particularly TLR3 agonists hold a promise to potentiate MVA-induced cellular immune responses. On the other hand, additional research is necessary to identify optimal combinations of agonists that could enhance MVA-induced humoral responses.

Newly Designed Poxviral Promoters to Improve Immunogenicity and Efficacy of MVA-NP Candidate Vaccines against Lethal Influenza Virus Infection in Mice.

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail.

Short- and Long-Interval Prime-Boost Vaccination with the Candidate Vaccines MVA-SARS-2-ST and MVA-SARS-2-S Induces Comparable Humoral and Cell-Mediated Immunity in Mice.

The COVID-19 pandemic caused significant human health and economic consequences. Due to the ability of SARS-CoV-2 to spread rapidly and to cause severe disease and mortality in certain population groups, vaccines are essential for controlling the pandemic in the future. Several licensed vaccines have shown improved protection against SARS-CoV-2 after extended-interval prime-boost immunizations in humans. Therefore, in this study, we aimed to compare the immunogenicity of our two Modified Vaccinia virus Ankara (MVA) based COVID-19 candidate vaccines MVA-SARS-2-S and MVA-SARS-2-ST after short- and long-interval prime-boost immunization schedules in mice. We immunized BALB/c mice using 21-day (short-interval) or 56-day (long-interval) prime-boost vaccination protocols and analyzed spike (S)-specific CD8 T cell immunity and humoral immunity. The two schedules induced robust CD8 T cell responses with no significant differences in their magnitude. Furthermore, both candidate vaccines induced comparable levels of total S, and S2-specific IgG binding antibodies. However, MVA-SARS-2-ST consistently elicited higher amounts of S1-, S receptor binding domain (RBD), and SARS-CoV-2 neutralizing antibodies in both vaccination protocols. Overall, we found very comparable immune responses following short- or long-interval immunization. Thus, our results suggest that the chosen time intervals may not be suitable to observe potential differences in antigen-specific immunity when testing different prime-boost intervals with our candidate vaccines in the mouse model. Despite this, our data clearly showed that MVA-SARS-2-ST induced superior humoral immune responses relative to MVA-SARS-2-S after both immunization schedules.

MERS-CoV‒Specific T-Cell Responses in Camels after Single MVA-MERS-S Vaccination.

We developed an ELISPOT assay for evaluating Middle East respiratory syndrome coronavirus (MERS-CoV)‒specific T-cell responses in dromedary camels. After single modified vaccinia virus Ankara-MERS-S vaccination, seropositive camels showed increased levels of MERS-CoV‒specific T cells and antibodies, indicating suitability of camel vaccinations in disease-endemic areas as a promising approach to control infection.

Direct intranodal tonsil vaccination with modified vaccinia Ankara vaccine protects macaques from highly pathogenic SIVmac251.

Human immunodeficiency virus (HIV) is a mucosally transmitted virus that causes immunodeficiency and AIDS. Developing efficacious vaccines to prevent infection is essential to control the epidemic. Protecting the vaginal and rectal mucosa, the primary routes of HIV entry has been a challenge given the significant compartmentalization between the mucosal and peripheral immune systems. We hypothesized that direct intranodal vaccination of mucosa associated lymphoid tissue (MALT) such as the readily accessible palatine tonsils could overcome this compartmentalization. Here we show that rhesus macaques primed with plasmid DNA encoding SIVmac251-env and gag genes followed by an intranodal tonsil MALT boost with MVA encoding the same genes protects from a repeated low dose intrarectal challenge with highly pathogenic SIVmac251; 43% (3/7) of vaccinated macaques remained uninfected after 9 challenges as compared to the unvaccinated control (0/6) animals. One vaccinated animal remained free of infection even after 22 challenges. Vaccination was associated with a ~2 log decrease in acute viremia that inversely correlated with anamnestic immune responses. Our results suggest that a combination of systemic and intranodal tonsil MALT vaccination could induce robust adaptive and innate immune responses leading to protection from mucosal infection with highly pathogenic HIV and rapidly control viral breakthroughs.

Mouse models in COVID-19 research: analyzing the adaptive immune response.

The emergence of SARS-CoV-2, the severe acute respiratory syndrome coronavirus type 2 causing the COVID-19 pandemic, resulted in a major necessity for scientific countermeasures. Investigations revealing the exact mechanisms of the SARS-CoV-2 pathogenesis provide the basis for the development of therapeutic measures and protective vaccines against COVID-19. Animal models are inevitable for infection and pre-clinical vaccination studies as well as therapeutic testing. A well-suited animal model, mimicking the pathology seen in human COVID-19 patients, is an important basis for these investigations. Several animal models were already used during SARS-CoV-2 studies with different clinical outcomes after SARS-CoV-2 infection. Here, we give an overview of different animal models used in SARS-CoV-2 infection studies with a focus on the mouse model. Mice provide a well-established animal model for laboratory use and several different mouse models have been generated and are being used in SARS-CoV-2 studies. Furthermore, the analysis of SARS-CoV-2-specific T cells during infection and in vaccination studies in mice is highlighted.

Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity.

The SARS-CoV-2 spike (S) glycoprotein is synthesized as a large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as a candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S-infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/S2 cleavage site and K986P and V987P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of broadly reactive anti-S antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome.

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

An Equine Model for Vaccination against a Hepacivirus: Insights into Host Responses to E2 Recombinant Protein Vaccination and Subsequent Equine Hepacivirus Inoculation.

Equine hepacivirus (EqHV) is the closest known genetic homologue of hepatitis C virus. An effective prophylactic vaccine is currently not available for either of these hepaciviruses. The equine as potential surrogate model for hepacivirus vaccine studies was investigated, while equine host responses following vaccination with EqHV E2 recombinant protein and subsequent EqHV inoculation were elucidated. Four ponies received prime and booster vaccinations (recombinant protein, adjuvant) four weeks apart (day -55 and -27). Two control ponies received adjuvant only. Ponies were inoculated with EqHV RNA-positive plasma on day 0. Blood samples and liver biopsies were collected over 26 weeks (day -70 to +112). Serum analyses included detection of EqHV RNA, isotypes of E2-specific immunoglobulin G (IgG), nonstructural protein 3-specific IgG, haematology, serum biochemistry, and metabolomics. Liver tissue analyses included EqHV RNA detection, RNA sequencing, histopathology, immunohistochemistry, and fluorescent in situ hybridization. Al-though vaccination did not result in complete protective immunity against experimental EqHV inoculation, the majority of vaccinated ponies cleared the serum EqHV RNA earlier than the control ponies. The majority of vaccinated ponies appeared to recover from the EqHV-associated liver insult earlier than the control ponies. The equine model shows promise as a surrogate model for future hepacivirus vaccine research.

Protective CD8+ T Cell Response Induced by Modified Vaccinia Virus Ankara Delivering Ebola Virus Nucleoprotein.

The urgent need for vaccines against Ebola virus (EBOV) was underscored by the large outbreak in West Africa (2014-2016). Since then, several promising vaccine candidates have been tested in pre-clinical and clinical studies. As a result, two vaccines were approved for human use in 2019/2020, of which one includes a heterologous adenovirus/Modified Vaccinia virus Ankara (MVA) prime-boost regimen. Here, we tested new vaccine candidates based on the recombinant MVA vector, encoding the EBOV nucleoprotein (MVA-EBOV-NP) or glycoprotein (MVA-EBOV-GP) for their efficacy after homologous prime-boost immunization in mice. Our aim was to investigate the role of each antigen in terms of efficacy and correlates of protection. Sera of mice vaccinated with MVA-EBOV-GP were virus-neutralizing and MVA-EBOV-NP immunization readily elicited interferon-γ-producing NP-specific CD8+ T cells. While mock-vaccinated mice succumbed to EBOV infection, all vaccinated mice survived and showed drastically decreased viral loads in sera and organs. In addition, MVA-EBOV-NP vaccinated mice became susceptible to lethal EBOV infection after depletion of CD8+ T cells prior to challenge. This study highlights the potential of MVA-based vaccines to elicit humoral immune responses as well as a strong and protective CD8+ T cell response and contributes to understanding the possible underlying mechanisms.

Intranasal Delivery of MVA Vector Vaccine Induces Effective Pulmonary Immunity Against SARS-CoV-2 in Rodents.

Antigen-specific tissue-resident memory T cells (Trms) and neutralizing IgA antibodies provide the most effective protection of the lungs from viral infections. To induce those essential components of lung immunity against SARS-CoV-2, we tested various immunization protocols involving intranasal delivery of a novel Modified Vaccinia virus Ankara (MVA)-SARS-2-spike vaccine candidate. We show that a single intranasal MVA-SARS-CoV-2-S application in mice strongly induced pulmonary spike-specific CD8+ T cells, albeit restricted production of neutralizing antibodies. In prime-boost protocols, intranasal booster vaccine delivery proved to be crucial for a massive expansion of systemic and lung tissue-resident spike-specific CD8+ T cells and the development of Th1 - but not Th2 - CD4+ T cells. Likewise, very high titers of IgG and IgA anti-spike antibodies were present in serum and broncho-alveolar lavages that possessed high virus neutralization capacities to all current SARS-CoV-2 variants of concern. Importantly, the MVA-SARS-2-spike vaccine applied in intramuscular priming and intranasal boosting treatment regimen completely protected hamsters from developing SARS-CoV-2 lung infection and pathology. Together, these results identify intramuscular priming followed by respiratory tract boosting with MVA-SARS-2-S as a promising approach for the induction of local, respiratory as well as systemic immune responses suited to protect from SARS-CoV-2 infections.

Activation of interferon regulatory factor 3 by replication-competent vaccinia viruses improves antitumor efficacy mediated by T cell responses.

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.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

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.

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination.

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 modified vaccinia virus Ankara (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 expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8+ T cells and serum antibodies binding to S protein 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.

Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are evaluated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The newly established ELISA was evaluated using 59 sera of infected or vaccinated animals, including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% were achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.