OBJECTIVE:The aim of the study was to determine the prevalence of antibodies against hepatitis E virus (HEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Crimean-Congo haemorrhagic fever (CCHF) virus, rabies virus, Echinococcus spp., Brucella spp., as well as the colonisation with methicillin-resistant Staphylococcus aureus (MRSA) among Austrian veterinarians in dependence on their field of occupation, age, gender and, if applicable, the vaccination status. METHODS AND RESULTS:A total of 293 Austrian veterinary practitioners were included in the study. All study participants had to fill in a questionnaire regarding personal details, field of occupation and vaccination status against TBEV and rabies. Furthermore, a nasal swab for detection of colonisation with MRSA and a blood sample were taken. Antibodies against HEV, TBEV, WNV, CCHF virus and Echinococcus spp. were analysed by ELISA tests. Antibodies against Brucella spp. were measured by complement fixation test. Rabies antibodies were analysed by virus neutralisation test. Most study participants were vaccinated against rabies (93.2%) and TBEV (94.3%). Accordingly, a high prevalence of TBEV antibodies was found (95.9%). HEV antibodies were detected in 11.3% of the study participants, with a significantly higher seroprevalence in the oldest age group (23.3%). MRSA colonisation was age-depended and significantly higher in study participants with occupational livestock exposure (10.3% vs. 2.2%). Hygienic measures such as hand washing and disinfection led to less MRSA colonisation. Antibodies against WNV were found in 3.1% of the study participants. No antibodies against Brucella spp., Echinococcus spp. and CCHF were detected. CONCLUSIONS:Despite the limitations of the study design, the included veterinarians represent the Austrian average veterinarians regarding gender, age and field of occupation. The vaccination rate against TBEV and rabies was high. HEV seroprevalence was lower than in other studies, whereas MRSA colonisation rates and WNV seroprevalence were comparable to results of other European studies.

West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) are worldwide endemic zoonotic orthoflaviviruses, often co-circulating in the same areas. Serological studies in animals, mostly birds and horses, are important means to monitor the spread of these viruses and the infection risks for humans. However, cross-reactive antibodies to these structurally similar flaviviruses frequently impact serological differentiation in enzyme-linked immunosorbent assays (ELISAs), hence time-consuming virus neutralization tests (VNTs) have to be employed in laboratories with high biosafety level. This study presents ELISAs using recombinant flavivirus E proteins with point mutations in the conserved fusion loop domain (Equad proteins) for differentiating IgY or IgG antibodies against WNV, USUV or TBEV in ducks, geese, chickens and horses. Panels of 169 duck and goose sera, 101 chicken sera and 136 horse sera were tested in Equad ELISAs, which resulted in high sensitivity and specificity, further improved by a pre-absorption step for the differentiation of WNV and USUV antibodies. Equad ELISAs for poultry and horse sera enable the reliable differentiation of WNV, USUV and TBEV specific antibodies without the need for VNTs, which has important implications for conducting seroprevalence studies as well as for veterinary routine diagnosis.

Background: Serum and other blood-derived products are widely used in biomedical and biopharmaceutical processes, especially for the production of vaccines or cell therapeutic applications. To ensure quality and safety, each serum lot undergoes testing for sterility to minimize the risk of disease transmission. A currently performed standard procedure is gamma-irradiation of serum for effectively killing pathogens. However, gamma irradiation can only be performed in highly shielded, specialized facilities. Low-energy electron irradiation (LEEI) is an alternative, Good Manufacturing Practice (GMP) compatible method for sterilizing serum in a standard laboratory environment.Results: We directly compared the sensitivity of viruses belonging to the family of flaviviruses (Zika virus and bovine viral diarrhea virus) and parvoviruses (minute virus of mice) against LEEI in 90% serum or buffer. Although the reduction is more effective in buffer than in serum, the calculated decimal reduction dose values suggest that LEEI can efficiently sterilize serum by inactivating viruses, thereby increasing product safety and extending shelf life. Furthermore, LEEI-treated serum was tested as a supplement in cell culture and showed comparable performance to nonirradiated or gamma-irradiated serum when used for the cultivation of different cell lines.Conclusion: LEEI is able to inactivate viral contaminants in serum and offers significant advantages over gamma irradiation, as no large amounts of radioactivity are generated, on-site irradiation is possible, and space requirements are reduced due to fewer protective measures. These advantages allow the technology to be used in a GMP-compliant irradiation process.

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.

EDITORIAL article Front. Immunol., 09 January 2023Sec. Vaccines and Molecular Therapeutics Volume 13 - 2022 | https://doi.org/10.3389/fimmu.2022.1075335

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.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

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.

Introduction:West Nile Virus (WNV) is a zoonotic flavivirus transmitted by mosquitoes. Especially in the elderly or in immunocompromised individuals an infection with WNV can lead to severe neurological symptoms. To date, no human vaccine against WNV is available. The Envelope (E) protein, located at the surface of flaviviruses, is involved in the invasion into host cells and is the major target for neutralizing antibodies and therefore central to vaccine development. Due to their close genetic and structural relationship, flaviviruses share highly conserved epitopes, such as the fusion loop domain (FL) in the E protein, that are recognized by cross-reactive antibodies. These antibodies can lead to enhancement of infection with heterologous flaviviruses, which is a major concern for potential vaccines in areas with co-circulation of different flaviviruses, e.g. Dengue or Zika viruses.Material:To reduce the potential of inducing cross-reactive antibodies, we performed an immunization study in mice using WNV E proteins with either wild type sequence or a mutated FL, and WNV E domain III which does not contain the FL at all.Results and discussion:Our data show that all antigens induce high levels of WNV-binding antibodies. However, the level of protection against WNV varied, with the wildtype E protein inducing full, the other antigens only partial protection. On the other hand, serological cross-reactivity to heterologous flaviviruses was significantly reduced after immunization with the mutated E protein or domain III as compared to the wild type version. These results have indications for choosing antigens with the optimal specificity and efficacy in WNV vaccine development.

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.

Background The currently used SARS-CoV-2 mRNA vaccines have proven to induce a strong and protective immune response. However, functional relevance of vaccine-generated antibodies and their temporal progression are still poorly understood. Thus, the central aim of this study is to gain a better understanding of systemic and mucosal humoral immune response after mRNA vaccination with BNT162b2. Methods We compared antibody production against the S1 subunit and the RBD of the SARS-CoV-2 spike protein in sera of BNT162b2 vaccinees, heterologous ChAdOx1-S/BNT162b2 vaccinees and COVID-19 patients. We monitored the neutralizing humoral response against SARS-CoV-2 wildtype strain and three VOCs over a period of up to eight months after second and after a subsequent third vaccination. Results In comparison to COVID-19 patients, vaccinees showed higher or similar amounts of S1- and RBD-binding antibodies but similar or lower virus neutralizing titers. Antibodies peaked two weeks after the second dose, followed by a decrease three and eight months later. Neutralizing antibodies (nAbs) poorly correlated with S1-IgG levels but strongly with RBD-IgGAM titers. After second vaccination we observed a reduced vaccine-induced neutralizing capacity against VOCs, especially against the Omicron variant. Compared to the nAb levels after the second vaccination, the neutralizing capacity against wildtype strain and VOCs was significantly enhanced after third vaccination. In saliva samples, relevant levels of RBD antibodies were detected in convalescent samples but not in vaccinees. Conclusions Our data demonstrate that BNT162b2 vaccinated individuals generate relevant nAbs titers, which begin to decrease within three months after immunization and show lower neutralizing potential against VOCs as compared to the wildtype strain. Large proportion of vaccine-induced S1-IgG might be non-neutralizing whereas RBD-IgGAM appears to be a good surrogate marker to estimate nAb levels. A third vaccination increases the nAb response. Furthermore, the systemic vaccine does not seem to elicit readily detectable mucosal immunity.

Vaccines consisting of whole inactivated bacteria (bacterins) are generated by incubation of the pathogen with chemicals. This is a time-consuming procedure which may lead to less immunogenic material, as critical antigenic structures can be altered by chemical modification. A promising alternative approach is low-energy electron irradiation (LEEI). Like other types of ionizing radiation, it mainly acts by destroying nucleic acids but causes less damage to structural components like proteins. As the electrons have a limited penetration depth, LEEI is currently used for sterilization of surfaces. The inactivation of pathogens in liquids requires irradiation of the culture in a thin film to ensure complete penetration. Here, we describe two approaches for the irradiation of bacterial suspensions in a research scale. After confirmation of inactivation, the material can be directly used for vaccination, without any purification steps.

ABSTRACTBackgroundCurrently used vaccines to protect from COVID-19 mostly focus on the receptor-binding domain (RBD) of the viral spike protein, and induced neutralizing antibodies have shown to be protective. However, functional relevance of vaccine-generated antibodies are poorly understood on variants-of-concern (VOCs) and mucosal immunity.MethodsWe compared specific antibody production against the S1 subunit and the RBD of the spike protein, the whole virion of SARS-CoV-2, and monitored neutralizing antibodies in sera and saliva of 104 BNT162b2 vaccinees and 57 individuals with natural SARS-CoV-2 infection. Furthermore, we included a small cohort of 11 individuals which received a heterologous ChAdOx1-S/BNT162b2 prime-boost vaccination.ResultsVaccinated individuals showed higher S1-IgG antibodies in comparison to COVID-19 patients, followed by a significant decrease 3 months later. Neutralizing antibodies (nAbs) were poorly correlated with initial S1-IgG levels, indicating that these might largely be non-neutralizing. In contrast, RBD IgGAM was strongly correlated to nAbs, suggesting that RBD-IgGAM is a surrogate marker to estimate nAb concentrations after vaccination. The protective effect of vaccine- and infection-induced nAbs was found reduced towards B.1.617.2 and B.1.351 VOCs. NAb titers are significantly higher after third vaccination compared to second vaccination. In contrast to COVID-19 patients, no relevant levels of RBD specific antibodies were detected in saliva samples from vaccinees.ConclusionsOur data demonstrate that BNT162b2 vaccinated individuals generate relevant neutralizing antibodies, which begin to decrease within three months after immunization and show lower neutralizing potential to VOCs as compared to the original Wuhan virus strain. A third booster vaccination provides a stronger nAb antibody response than the second vaccination. The systemic vaccine does not seem to elicit readily detectable mucosal immunity.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that belong to the Japanese encephalitis virus serocomplex within the genus Flavivirus. Due to climate change and the expansion of mosquito vectors, flaviviruses are becoming endemic in increasing numbers of countries. WNV infections are reported with symptoms ranging from mild fever to severe neuro-invasive disease. Until now, only a few USUV infections have been reported in humans, mostly with mild symptoms. The serological diagnosis and differentiation between flavivirus infections, in general, and between WNV and USUV, in particular, are challenging due to the high degree of cross-reacting antibodies, especially of those directed against the conserved fusion loop (FL) domain of the envelope (E) protein. We have previously shown that E proteins containing four amino-acid mutations in and near the FL strongly reduce the binding of cross-reactive antibodies leading to diagnostic technologies with improved specificities. Here, we expanded the technology to USUV and analyzed the differentiation of USUV- and WNV-induced antibodies in humans. IgG ELISAs modified by an additional competition step with the heterologous antigen resulted in overall specificities of 93.94% for WNV Equad and 92.75% for USUV Equad. IgM antibodies against WNV could be differentiated from USUV IgM in a direct comparison using both antigens. The data indicate the potential of the system to diagnose antigenically closely related flavivirus infections.

Background: In 2015–2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. Methods: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. Results: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. Conclusions: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.

Among 713 equids sampled in northeastern Brazil during 2013–2018, West Nile virus seroprevalence was 4.5% (95% CI 3.1%–6.3%). Mathematical modeling substantiated higher seroprevalence adjacent to an avian migratory route and in areas characterized by forest loss, implying increased risk for zoonotic infections in disturbed areas.

Background: The diagnosis of SARS-CoV-2 infection relies on RT-PCR from nasopharyngeal swabs. The pre-analytical value of different methods of material harvesting for SARS-CoV-2 are unknown. Methods: We conducted a comprehensive investigation of the pre-analytical performance for different pharyngeal sampling procedures in hospitalized patients with confirmed SARS-CoV-2 infection. In addition to swabs taken simultaneously from different locations, saliva and pharyngeal lavages were also analyzed using RT-PCR. Results: In 10 COVID-19 patients, standard nasopharyngeal swabs detected 8 out of 10 positive patients, whereas swabs taken from the palatoglossal arch resulted in 9 correct-positive results. Brushing the posterior pharynx wall with swabs resulted in detection of 9 out of 10 positive patients with no difference using either dry swabs or liquid Amies medium. A strong correlation between Ct values of both swab materials was observed. Pharyngeal lavages yielded 6 out of 10 positive results in concordance with 85% of nasopharyngeal swabs in late-stage COVID-19 patients. Investigating 23 patients with early SARS-CoV-2 infection, pharyngeal lavages showed a concordance rate of 100% compared to nasopharyngeal swabs. Conclusions: The diagnostic performance of swabs taken from the palatoglossal arch in detecting SARS-CoV-2 infection is similar to that of specimens taken from the nasopharyngeal region. However, the former sampling method is associated with less discomfort and much easier to perform. Pharyngeal lavages may replace swabs for mass screening in early stages of SARS-CoV-2 infection. The predictive values are comparable, and the procedure is performed without exposing healthcare workers to transmission risks.

Background:With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these "off-the-shelf" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function.Methods:Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others.Results:Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30.Conclusions:The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.