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Puumala orthohantavirus (PUUV) is an emerging zoonotic virus that was first discovered in the Puumala region of Finland in the early 1980s and is the primary etiological agent of nephropathia epidemica (NE), a milder form of a life-threatening disease known as hemorrhagic fever with renal syndrome (HFRS). PUUV and other members of the Old World hantaviruses (OWHVs) predominantly circulate in rodents or insectivores across Eurasia, accounting for several thousand of reported HFRS cases every year (with many more unreported/misdiagnosed cases suspected). The rodent reservoir of PUUV is the common bank vole ( Myodes (M.) glareolus ), and transmission of the virus to humans occurs via inhalation of contagious aerosols and through contact with contaminated droppings or urine. Although PUUV is the subject of extensive research, due to its potential to cause severe disease outcomes in humans and its considerable economic and social impact, neither licensed vaccines nor specific antiviral treatments are available against PUUV. However, many important advancements have been made in terms of PUUV research over the last years. This included the elucidation of its glycoproteins, the discovery of broadly neutralizing hantavirus antibodies as therapeutic candidates and expanded research on the mRNA vaccine technology which will likely enable the development of strong PUUV vaccine candidates in the near future. Currently, there is still a lack of suitable animal models for the preclinical evaluation of experimental vaccines and antivirals, which hampers vaccine and antiviral development. Current attempts to decrease hantavirus-associated human infections rely primarily on prevention and countermeasures for rodent control, including reduced contact to droppings, saliva and urine, and disinfection of areas that are contaminated with rodent excreta. Here, we review these recent advances and other aspects including PUUV prevalence, virus biology, diagnosis and clinical features, and current animal models for vaccine and treatment development.

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.

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.

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.

The rapid development and deployment of injectable coronavirus disease 2019 (COVID-19) vaccines - in combination with non-pharmaceutical interventions and development of treatment options - significantly contributed to a decrease in both infection and mortality rates during the pandemic and saved millions of lives. However, injectable vaccines do not robustly and consistently induce a mucosal immune response, which is considered a key factor to prevent infection with and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, a tremendous effort is being made globally to develop next generation COVID-19 vaccines, which are capable of inducing a robust mucosal immune response in addition to a strong systemic cellular and humoral immune response. Mucosal COVID-19 vaccines have been evaluated successfully in preclinical and clinical trials, in which protection against SARS-CoV-2 infection has been demonstrated. This protective efficacy was associated with the upregulation of secretory IgA antibodies and the maturation of tissue-resident memory cells in the respiratory tract, which, together with an induced systemic immune response, significantly reduced viral replication and transmission in animal models. However, only five active mucosal vaccines (plus one ‘passive’ vaccine) have received approval for human use and robust data on their efficacy in inducing mucosal immune responses in humans and in blocking infection and transmission are missing. This highlights the importance of expanded research in this field. In this review, we aim to summarize what is known about these currently licensed vaccines, with an emphasis on the key findings obtained in both preclinical and clinical studies.

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. In a clinical trial, Fathi et al. show that a booster vaccination with a vector vaccine candidate against the highly pathogenic Middle East Respiratory Syndrome coronavirus is safe and strongly improves the immunity generated by primary immunization.

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.

Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause a serious disease involving the central nervous system (tick-borne encephalitis, TBE). Although approved inactivated vaccines are available, the number of TBE cases is rising, and breakthrough infections in fully vaccinated subjects have been reported in recent years. In the present study, we generated and characterized a recombinant Modified Vaccinia virus Ankara (MVA) for the delivery of the pre-membrane (prM) and envelope (E) proteins of TBEV (MVA-prME). MVA-prME was tested in mice in comparison with a licensed vaccine FSME-IMMUN® and proved to be highly immunogenic and afforded full protection against challenge infection with TBEV. Our data indicate that MVA-prME holds promise as an improved next-generation vaccine for the prevention of TBE.

The rapid and reliable diagnostics of highly pathogenic bacteria under restricted field conditions poses one of the major challenges to medical biodefense, especially since false positive or false negative reports might have far-reaching consequences. Fluorescence in situ hybridization (FISH) has the potential to represent a powerful microscopy-based addition to the existing molecular-based diagnostic toolbox. In this study, we developed a set of FISH-probes for the fast, matrix independent and simultaneous detection of thirteen highly pathogenic bacteria in different environmental and clinical sample matrices. Furthermore, we substituted formamide, a routinely used chemical that is toxic and volatile, by non-toxic urea. This will facilitate the application of FISH under resource limited field laboratory conditions. We demonstrate that hybridizations performed with urea show the same specificity and comparable signal intensities for the FISH-probes used in this study. To further simplify the use of FISH in the field, we lyophilized the reagents needed for FISH. The signal intensities obtained with these lyophilized reagents are comparable to freshly prepared reagents even after storage for a month at room temperature. Finally, we show that by the use of non-toxic lyophilized field (NOTIFy)-FISH, specific detection of microorganisms with simple and easily transportable equipment is possible in the field.

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.