Low-Energy Electron Irradiation Efficiently Inactivates the Gram-Negative Pathogen Rodentibacter pneumotropicus—A New Method for the Generation of Bacterial Vaccines with Increased Efficacy

Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy

Cellular co-repressors as functional interactors of the papillomavirus E8^E2C protein

Das E2 Gen von Papillomviren kodiert für mehrere Regulatorproteine, welche die Transkription und Replikation des viralen Genoms kontrollieren und somit die Expression der viralen Onkoproteine (E6/E7) modulieren. Da das E2 Gen häufig durch Integration in das Genom der Wirtszelle in Zervixkarzinomen deletiert ist, wurde postuliert, dass Störungen in der Onkogenexpression zur malignen Progression beitragen könnten. Neben dem vollständigen E2 Protein kodieren mehrere humane Papillomviren (HPV) für ein E8^E2C Protein, das ein potenter Repressor der viralen Transkription und Genomreplikation ist. Das HPV31 E8^E2C Protein besteht aus der E8 Repressordomäne und der E2C Domäne, welche sequenzspezifische Bindung an DNA und Dimerisierung zwischen E2 Proteinen vermittelt. Vorbefunde legten nahe, dass E8^E2C zelluläre Proteine wie Histondeacetylasen rekrutiert und so als Repressor fungiert. Um den Mechanismus der E8^E2C-vermittelten Repression genauer aufzuklären, wurden einerseits ein genetischer Screen in Hefe durchgeführt und andererseits 31E8^E2C und assoziierte Proteine aus humanen Zellen quantitativ immunopräzipitiert. Dadurch konnten das „Chromodomain Helicase DNA binding protein 6“ (CHD6) sowie Komponenten des „nuclear receptor corepressor complex“ (N-CoR) als neue Interaktionspartner identifiziert werden. CHD6 ist Teil eines Multiproteinkomplexes, der vermutlich Transkription reguliert. Die Bindung von CHD6 erfolgte über die E2C Domäne und die des N-CoR Komplex über die E8 Repressordomäne. Beide Interaktionen sind hochkonserviert, da sie auch für die E8^E2C Proteine von HPV16 und 18 nachweisbar waren. Mutationsanalysen der 31E2C Domäne und RNA Interferenz-Experimente belegten, dass die E8^E2C/CHD6 Interaktion zur transkriptionellen Repression des E6/E7 Promoters beiträgt. CHD6 interagiert auch mit 31E2 und dies trägt zur transkriptionellen Repression aber nicht zur Aktivierung durch E2 bei. Mutationsanalysen von 16, 18 und 31E8^E2C belegten, dass die Rekrutierung des N-CoR Komplexes für deren Repressionsaktivität wichtig ist. Ein artifizieller Repressor bestehend aus der KRAB-Domäne des zellulären KOX1 Proteins und der 31E2C Domäne verhielt sich funktionell wie E8^E2C, was nahelegt, dass die Repression des E6/E7 Promotors keine spezifische Eigenschaft viraler Proteine ist. Zusammenfassend lässt sich sagen, dass die transkriptionelle Repression durch E8^E2C durch die Rekrutierung unterschiedlicher Multiproteinkomplexe sowohl über die E8 Repressordomäne als auch über die E2C-Domäne vermittelt wird.The papillomavirus E2 gene encodes for several regulatory proteins, which control the transcription and replication of the viral genome and thereby modulate the viral oncogene (E6/E7) expression. Since the E2 open reading frame is often disrupted after integration in the host cell genome in cervical carcinomas it has been postulated that dysfunctions in the oncogene expression contribute to malignant progression. Beside the full length E2 protein several human papillomaviruses encode for the E8^E2C protein which is a potent repressor of the viral transcription and genome replication. The HPV31 E8^E2C protein consists of the E8 repression domain and the E2C domain which mediates sequence specific DNA binding and dimerisation between E2 proteins. Recent results indicate that E8^E2C recruits cellular proteins like e.g. Histone deacetylases, thereby acting as a repressor. To further elucidate the mechanism of the E8^E2C mediated repression, on the one hand a genetic screen in yeast was performed and on the other hand 31E8^E2C associated proteins were quantitavely co-immunoprecipitated out of human cells. Thus the „Chromodomain Helicase DNA binding protein 6“ (CHD6) as well as components of the “nuclear receptor corepressor complex“ (N-CoR could be identified as novel interaction partners. CHD6 is a part of a multi protein complex that regulates transcription. The binding of CHD6 occurred in the E2C domain and the N-Cor binding in the E8 repression domain. Both interactions are highly conserved, since they could be shown with the E8^E2C proteins of HPV16 and 18. Mutational analyses of the E2C domain and RNA interference experiments validated that the E8^E2C/CHD6 interaction contributes to the transcriptional repression of the E6/E7 promotor. CHD6 also interacts with 31E2 and this contributes to transcriptional repression but not to activation by E2. Mutational analyses of HPV16, 18 and 31 E8^E2C confirmed that the recruitment of the N-Cor complex is necessary for their repression activity. An artificial repressor consisting from the KRAB-domain of the cellular KOX1 protein and the 31E2C domain functionally acted like E8^E2C. This suggests that the repression of the E6/E7 promotor is not a unique property of viral proteins. In summary the transcriptional repression by E8^E2C is mediated by the recruitment of different multi protein complexes by the E8 domain as well as by the E2C domain

Immunization with an adjuvanted low-energy electron irradiation inactivated respiratory syncytial virus vaccine shows immunoprotective activity in mice

Respiratory syncytial virus (RSV) is a pathogen causing severe lower respiratory tract disease in infants and the elderly. In spite of the great need for a vaccine against RSV, currently there is no licensed product on the market. A very early vaccine candidate developed in the 1960s based on formaldehyde inactivation (FI) turned out to instead enhance the disease. Our novel inactivation method applied low-energy electron irradiation (LEEI) to produce a killed RSV vaccine. LEEI yielded inactivated virus particles with a reproducible virus antigen conservation above 70%, while FI resulted in highly variable antigen conservation. Immunization of mice with LEEI-RSV elicited a strong immune response, resulting in a drastic reduction in viral load upon challenge in two independent studies. These results have implications for the development of an RSV vaccine and should be validated in further preclinical and clinical studies

A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles

Growth Inhibition of HeLa Cells Is a Conserved Feature of High-Risk Human Papillomavirus E8^E2C Proteins and Can Also Be Achieved by an Artificial Repressor Protein▿ †

Infections with certain human papillomaviruses (HPV), such as type 16 (HPV16), 18, or 31, are a necessary risk factor for the development of cervical cancer. Transcript analyses of several HPV revealed that the viral E2 gene encodes both the E2 regulator protein and the E8∧E2C protein, which differ in their amino termini. Up to now, functional studies have focused on HPV31 E8∧E2C and demonstrated that it is a potent repressor of viral transcription and replication. However, recent analyses of HPV16 genomes have suggested that E8∧E2C proteins may differ in their activities. Therefore, we performed a comparative analysis of E8∧E2C proteins of HPV16, -18, and -31. All E8∧E2C proteins potently inhibited HPV E6/E7 oncogene promoters, and also displayed long-distance transcriptional-repression activities. Furthermore, the expression of all E8∧E2C proteins inhibited the growth of HeLa cells. Expression of E8∧E2C proteins rapidly increased the protein levels of the E6 and E7 targets p53 and p21, consistent with the repression of the endogenous HPV18 E6/E7 promoter. All E8∧E2C proteins induced G1 arrest more efficiently than E2 proteins and activated senescence markers. Furthermore, we demonstrate that the 31E8 domain can be functionally replaced by the KRAB repression domain derived from KOX1. The KRAB-E2C fusion protein possesses long-distance transcriptional-repression activity and inhibits the growth of HeLa cells comparably to E8∧E2C. Taken together, our results suggest that the E8∧E2C proteins of HPV16, -18, and -31 are highly conserved transcriptional repressors that inhibit the growth of HeLa cells by repression of E6/E7 transcription but do not have proapoptotic activities

Eimeria tenella oocysts attenuated by low energy electron irradiation (LEEI) induce protection against challenge infection in chickens

In vitro and in vivo studies were performed to assess whether Eimeria tenella (E. tenella) oocysts, exposed to low energy electron irradiation (LEEI), might be considered potential vaccine candidates against cecal coccidiosis. Sporulated oocysts were exposed to LEEI of 0.1 kGy to 10.0 kGy. Reproduction inhibition assays (RIA) were performed in MDBK cells to assess infectivity of sporozoites excysted from irradiated and non-irradiated oocysts. LEEI of 0.1 kGy or 0.5 kGy resulted in 73.2% and 86.5% inhibition of in vitro reproduction (%I RIA ), respectively. Groups of 12 one day old (D1) chicken were orally inoculated with Paracox®-8 (G1), 2.0 × 10 3 non-irradiated oocysts (G2) or 1.0 × 10 4 irradiated oocysts exposed to LEEI of 0.1 kGy (G3, G4) or 0.5 kGy (G5). Chicken of groups G1, G2, G4 and G5 were challenged 3 weeks later (D21) by a single inoculation of 7.5 × 10 4 non-attenuated oocysts of the same strain while G3 remained unchallenged. All chickens were subject to necropsy 7 days after challenge (D28) to estimate lesion scores (LS) and oocyst index (OI). A positive control (PC, non-vaccinated, challenged) and a negative control (NC, non-vaccinated, non-challenged) were kept in parallel. Chicken of group G5 had similar weight gain as the Paracox®-8 group (G1) after challenge and higher weight gains as compared to the other vaccinated groups. Feed conversion ratio (FCR) did not differ between chickens inoculated with oocysts irradiated with 0.5 kGy (G5) and negative control (NC) before challenge (1.25–1.52). After challenge FCR was 1.99 (G5) to 2.23 (G4) in the vaccinated chicken compared to 1.76 in group NC. LS and OI were significantly lower in all vaccinated groups as compared to group PC. Progeny oocysts collected from the feces of chickens following vaccination with irradiated oocysts exhibited lower in vitro infectivity/reproduction in MDBK cells with %I RIA of 89.7% and 82.4% for progeny of oocysts irradiated with 0.5 kGy and 0.1 kGy, respectively, suggesting hereditary attenuation by LEEI treatment. Seroconversion was demonstrated by ELISA before challenge (D21) in all vaccinated groups, however, chicken inoculated with irradiated oocysts displayed higher antibody levels than those inoculated with precocious oocysts (G1). In Western blot analysis chicken vaccinated with virulent (G2) or 0.1 kGy-irradiated E. tenella oocysts (G3, G4) showed more protein bands compared to G5 (0.5 kGy). We conclude that LEEI could be a promising technology for production of attenuated oocyst vaccines

Correlation of humoral immune responses to different SARS-CoV-2 antigens with virus neutralizing antibodies and symptomatic severity in a German COVID-19 cohort

Monitoring the humoral protective immune response and its durability after SARS-CoV-2 infections is essential for risk assessment of reinfections, the improvement of diagnostic methods and the evaluation of vaccine trials. We have analyzed neutralizing antibodies and IgG responses specific to different antigens, including the inactivated whole-virion of SARS-CoV-2, the spike subunit 1 protein and its receptor binding domain, as well as the nucleocapsid protein. We show the dynamic developments of the responses from the early convalescent stages up to 9 months post symptoms onset in follow-up samples from 57 COVID-19 patients with varying clinical severity. By correlating antibody signals to neutralizing titres, valid diagnostic markers for the estimation of neutralizing protection could be identified

Interaction of NCOR/SMRT repressor complexes with papillomavirus E8^E2C proteins inhibits viral replication

Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins