Mucosal immunization with a low-energy electron inactivated respiratory syncytial virus vaccine protects mice without Th2 immune bias.

The respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections associated with numerous hospitalizations. Recently, intramuscular (i.m.) vaccines against RSV have been approved for elderly and pregnant women. Noninvasive mucosal vaccination, e.g., by inhalation, offers an alternative against respiratory pathogens like RSV. Effective mucosal vaccines induce local immune responses, potentially resulting in the efficient and fast elimination of respiratory viruses after natural infection. To investigate this immune response to an RSV challenge, low-energy electron inactivated RSV (LEEI-RSV) was formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) or 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DD-LEEI-RSV) for vaccination of mice intranasally. As controls, LEEI-RSV and formalin-inactivated-RSV (FI-RSV) were used via i.m. vaccination. The RSV-specific immunogenicity of the different vaccines and their protective efficacy were analyzed. RSV-specific IgA antibodies and a statistically significant reduction in viral load upon challenge were detected in mucosal DD-LEEI-RSV-vaccinated animals. Alhydrogel-adjuvanted LEEI-RSV i.m. showed a Th2-bias with enhanced IgE, eosinophils, and lung histopathology comparable to FI-RSV. These effects were absent when applying the mucosal vaccines highlighting the potential of DD-LEEI-RSV as an RSV vaccine candidate and the improved performance of this mucosal vaccine candidate.

Mucosal Application of a Low-Energy Electron Inactivated Respiratory Syncytial Virus Vaccine Shows Protective Efficacy in an Animal Model.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections in the elderly and in children, associated with pediatric hospitalizations. Recently, first vaccines have been approved for people over 60 years of age applied by intramuscular injection. However, a vaccination route via mucosal application holds great potential in the protection against respiratory pathogens like RSV. Mucosal vaccines induce local immune responses, resulting in a fast and efficient elimination of respiratory viruses after natural infection. Therefore, a low-energy electron irradiated RSV (LEEI-RSV) formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) was tested ex vivo in precision cut lung slices (PCLSs) for adverse effects. The immunogenicity and protective efficacy in vivo were analyzed in an RSV challenge model after intranasal vaccination using a homologous prime-boost immunization regimen. No side effects of PC-LEEI-RSV in PCLS and an efficient antibody induction in vivo could be observed. In contrast to unformulated LEEI-RSV, the mucosal vaccination of mice with PC formulated LEEI-RSV showed a statistically significant reduction in viral load after challenge. These results are a proof-of-principle for the use of LEEI-inactivated viruses formulated with liposomes to be administered intranasally to induce a mucosal immunity that could also be adapted for other respiratory viruses.

Apicomplexan parasites are attenuated by low-energy electron irradiation in an automated microfluidic system and protect against infection with Toxoplasma gondii.

Radiation-attenuated intracellular parasites are promising immunization strategies. The irradiated parasites are able to invade host cells but fail to fully replicate, which allows for the generation of an efficient immune response. Available radiation technologies such as gamma rays require complex shielding constructions and are difficult to be integrated into pharmaceutical production processes. In this study, we evaluated for the first time low-energy electron irradiation (LEEI) as a method to generate replication-deficient Toxoplasma gondii and Cryptosporidium parvum. Similar to other radiation technologies, LEEI mainly damages nucleic acids; however, it is applicable in standard laboratories. By using a novel, continuous, and microfluidic-based LEEI process, tachyzoites of T. gondii and oocysts of C. parvum were irradiated and subsequently analyzed in vitro. The LEEI-treated parasites invaded host cells but were arrested in intracellular replication. Antibody-based analysis of surface proteins revealed no significant structural damage due to LEEI. Similarly, excystation rates of sporozoites from irradiated C. parvum oocysts were similar to those from untreated controls. Upon immunization of mice, LEEI-attenuated T. gondii tachyzoites induced high levels of antibodies and protected the animals from acute infection. These results suggest that LEEI is a useful technology for the generation of attenuated Apicomplexan parasites and has potential for the development of anti-parasitic vaccines.

Low-Energy Electron Irradiation of Tick-Borne Encephalitis Virus Provides a Protective Inactivated Vaccine.

Tick-borne encephalitis virus (TBEV) is a zoonotic flavivirus which is endemic in many European and Asian countries. Humans can get infected with TBEV usually via ticks, and possible symptoms of the infection range from fever to severe neurological complications such as encephalitis. Vaccines to protect against TBEV-induced disease are widely used and most of them consist of whole viruses, which are inactivated by formaldehyde. Although this production process is well established, it has several drawbacks, including the usage of hazardous chemicals, the long inactivation times required and the potential modification of antigens by formaldehyde. As an alternative to chemical treatment, low-energy electron irradiation (LEEI) is known to efficiently inactivate pathogens by predominantly damaging nucleic acids. In contrast to other methods of ionizing radiation, LEEI does not require substantial shielding constructions and can be used in standard laboratories. Here, we have analyzed the potential of LEEI to generate a TBEV vaccine and immunized mice with three doses of irradiated or chemically inactivated TBEV. LEEI-inactivated TBEV induced binding antibodies of higher titer compared to the formaldehyde-inactivated virus. This was also observed for the avidity of the antibodies measured after the second dose. After viral challenge, the mice immunized with LEEI- or formaldehyde-inactivated TBEV were completely protected from disease and had no detectable virus in the central nervous system. Taken together, the results indicate that LEEI could be an alternative to chemical inactivation for the production of a TBEV vaccine.

Publisher Correction: Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Automated application of low energy electron irradiation enables inactivation of pathogen- and cell-containing liquids in biomedical research and production facilities.

Ionizing radiation is widely used to inactivate pathogens. It mainly acts by destroying nucleic acids but causes less damage to structural components like proteins. It is therefore highly suited for the sterilization of biological samples or the generation of inactivated vaccines. However, inactivation of viruses or bacteria requires relatively high doses and substantial amounts of radiation energy. Consequently, irradiation is restricted to shielded facilities-protecting personnel and the environment. We have previously shown that low energy electron irradiation (LEEI) has the same capacity to inactivate pathogens in liquids as current irradiation methods, but generates much less secondary X-ray radiation, which enables the use in normal laboratories by self-shielded irradiation equipment. Here, we present concepts for automated LEEI of liquids, in disposable bags or as a continuous process. As the electrons have a limited penetration depth, the liquid is transformed into a thin film. High concentrations of viruses (Influenza, Zika virus and Respiratory Syncytial Virus), bacteria (E. coli, B. cereus) and eukaryotic cells (NK-92 cell line) are efficiently inactivated by LEEI in a throughput suitable for various applications such as sterilization, vaccine manufacturing or cell therapy. Our results validate the premise that for pathogen and cell inactivation in liquids, LEEI represents a suitable and versatile irradiation method for standard biological research and production laboratories.

Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor.

Many Bacteria and Archaea employ the heterodisulfide reductase (Hdr)-like sulfur oxidation pathway. The relevant genes are inevitably associated with genes encoding lipoate-binding proteins (LbpA). Here, deletion of the gene identified LbpA as an essential component of the Hdr-like sulfur-oxidizing system in the Alphaproteobacterium Hyphomicrobium denitrificans. Thus, a biological function was established for the universally conserved cofactor lipoate that is markedly different from its canonical roles in central metabolism. LbpAs likely function as sulfur-binding entities presenting substrate to different catalytic sites of the Hdr-like complex, similar to the substrate-channeling function of lipoate in carbon-metabolizing multienzyme complexes, for example pyruvate dehydrogenase. LbpAs serve a specific function in sulfur oxidation, cannot functionally replace the related GcvH protein in Bacillus subtilis and are not modified by the canonical E. coli and B. subtilis lipoyl attachment machineries. Instead, LplA-like lipoate-protein ligases encoded in or in immediate vicinity of hdr-lpbA gene clusters act specifically on these proteins.