Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe.

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Increased detection of enterovirus A71 infections, Germany, 2019.

We report on the increased circulation of enterovirus A71 in Germany in 2019. Strains were mainly identified in hospitalised patients with suspected aseptic meningitis/encephalitis. Molecular analysis showed co-circulation of EV-A71 sub-genogroups C1 and C4, a signal for physicians and public health authorities to include/intensify EV diagnostic in patients showing signs of aseptic meningitis, encephalitis or acute flaccid paralysis/myelitis.

[On-site detection of bioterrorism-relevant agents : Rapid detection methods for viruses, bacteria and toxins - capabilities and limitations]. / Vor-Ort-Nachweis bioterroristisch relevanter Agenzien : Schnelle Nachweismethoden für Viren, Bakterien und Toxine ­ Potenziale und Grenzen.

In Europe, besides the threat of terrorist attacks involving conventional methods such as explosive devices and automatic weapons, there is also a potential threat of terrorist groups using non-conventional material like biological agents in the scope of future attacks. Consequently, rapid and reliable detection systems for biological agents are being developed and tested continuously to inform crisis management. For environmental detection, a broad spectrum of different laboratory-based techniques has been developed for relevant biological agents. However for environmental samples, fast and reliable on-site detection methods are desired by first responders for rapid assessment.Based on different functional principles, generic, immunological and nucleic-acid-based on-site detection methods can be distinguished. Those should be facile, fast, sensitive, and specific. However, commercially available kits usually have limited sensitivity and often have not been validated independently. Furthermore in this context, the multitude of relevant biological agents that potentially have to be considered present in complex environmental matrices poses a serious challenge for reliable detection. Therefore, detailed knowledge of the specific scope of applications and the limitations of different analytical systems is necessary to evaluate the results obtained purposefully.The aim of this article is to provide an overview of the analytical principles, benefits and limitations of prevailing on-site environmental detection systems for bioterrorism-relevant viruses, bacteria and toxins. Despite promising developments the informative value of currently available on-site tests is still limited. Thus, expert laboratories have to conduct confirmatory testing.

Expression of tolerance associated gene-1, a mitochondrial protein inhibiting T cell activation, can be used to predict response to immune modulating therapies.

Immune modulating therapies gain increasing importance in treatment of patients with autoimmune diseases such as psoriasis. None of the currently applied biologics achieves significant clinical improvement in all treated patients. Because the therapy with biologics is cost intensive and sometimes associated with side effects, noninvasive diagnostic tools for early prediction of responders are of major interest. We studied the effects of Alefacept (LFA3Ig), an approved drug for treatment of psoriasis, on leukocytes in vitro and in vivo to identify gene markers predictive for treatment response and to further investigate its molecular mechanisms of action. In an open-label study, 20 psoriasis patients were treated weekly with 15 mg Alefacept over 12 wk. We demonstrate that transcription of the tolerance-associated gene (TOAG-1) is significantly up-regulated whereas receptor for hyaluronic acid mediated migration (RHAMM) transcription is down-regulated in PBMCs of responding patients before clinical improvement. TOAG-1 is exclusively localized within mitochondria. Overexpression of TOAG-1 in murine T cells leads to increased susceptibility to apoptosis. Addition of Alefacept to stimulated human T cells in vitro resulted in reduced frequencies of activated CD137(+) cells, increased TOAG-1 but reduced RHAMM expression. This was accompanied by reduced proliferation and enhanced apoptosis. Inhibition of proliferation was dependent on enhanced PDL1 expression of APCs. Thus, peripheral changes of TOAG-1 and RHAMM expression can be used to predict clinical response to Alefacept treatment in psoriasis patients. In the presence of APCs Alefacept can inhibit T cell activation and survival by increasing expression of TOAG-1 on T cells and PDL1 on APCs.

Enterovirus Surveillance (EVSurv) in Germany.

The major aim of the enterovirus surveillance (EVSurv) in Germany is to prove the absence of poliovirus circulation in the framework of the Global Polio Eradication Program (GPEI). Therefore, a free-of-charge enterovirus diagnostic is offered to all hospitals for patients with symptoms compatible with a polio infection. Within the quality proven laboratory network for enterovirus diagnostic (LaNED), stool and cerebrospinal fluid (CSF) samples from patients with suspected aseptic meningitis/encephalitis or acute flaccid paralysis (AFP) are screened for enterovirus (EV), typing is performed in all EV positive sample to exclude poliovirus infections. Since 2006, ≈200 hospitals from all 16 German federal states have participated annually. On average, 2500 samples (70% stool, 28% CSF) were tested every year. Overall, the majority of the patients studied are children <15 years. During the 15-year period, 53 different EV serotypes were detected. While EV-A71 was most frequently detected in infants, E30 dominated in older children and adults. Polioviruses were not detected. The German enterovirus surveillance allows monitoring of the circulation of clinically relevant serotypes resulting in continuous data about non-polio enterovirus epidemiology.

Peripheral Facial Nerve Palsy in Children With Enterovirus Infection.

Enteroviruses are one of the leading causes of central nervous system infections, but their causative role in peripheral facial nerve palsy is unresolved. We used data from a large national Enterovirus Surveillance Database to identify cases of facial nerve palsy, showing a rate of 3.8% of patients with facial nerve palsy to have enterovirus infection.

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.

External quality assessment study for ebolavirus PCR-diagnostic promotes international preparedness during the 2014 - 2016 Ebola outbreak in West Africa.

During the recent Ebola outbreak in West Africa several international mobile laboratories were deployed to the mainly affected countries Guinea, Sierra Leone and Liberia to provide ebolavirus diagnostic capacity. Additionally, imported cases and small outbreaks in other countries required global preparedness for Ebola diagnostics. Detection of viral RNA by reverse transcription polymerase chain reaction has proven effective for diagnosis of ebolavirus disease and several assays are available. However, reliability of these assays is largely unknown and requires serious evaluation. Therefore, a proficiency test panel of 11 samples was generated and distributed on a global scale. Panels were analyzed by 83 expert laboratories and 106 data sets were returned. From these 78 results were rated optimal and 3 acceptable, 25 indicated need for improvement. While performance of the laboratories deployed to West Africa was superior to the overall performance there was no significant difference between the different assays applied.

Effect of Different Omega-6/Omega-3 Polyunsaturated Fatty Acid Ratios on the Formation of Monohydroxylated Fatty Acids in THP-1 Derived Macrophages.

Omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) can modulate inflammatory processes. In western diets, the content of n-6 PUFA is much higher than that of n-3 PUFA, which has been suggested to promote a pro-inflammatory phenotype. The aim of this study was to analyze the effect of modulating the n-6/n-3 PUFA ratio on the formation of monohydroxylated fatty acid (HO-FAs) derived from the n-6 PUFA arachidonic acid (AA) and the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in THP-1 macrophages by means of LC-MS. Lipid metabolites were measured in THP-1 macrophage cell pellets. The concentration of AA-derived hydroxyeicosatetraenoic acids (HETEs) was not significantly changed when incubated THP-1 macrophages in a high AA/(EPA+DHA) ratio of 19/1 vs. a low ratio AA/(EPA+DHA) of 1/1 (950.6 ± 110 ng/mg vs. 648.2 ± 92.4 ng/mg, p = 0.103). Correspondingly, the concentration of EPA-derived hydroxyeicosapentaenoic acids (HEPEs) and DHA-derived hydroxydocosahexaenoic acids (HDHAs) were significantly increased (63.9 ± 7.8 ng/mg vs. 434.4 ± 84.3 ng/mg, p = 0.012 and 84.9 ± 18.3 ng/mg vs. 439.4 ± 82.7 ng/mg, p = 0.014, respectively). Most notable was the strong increase of 18-hydroxyeicosapentaenoic acid (18-HEPE) formation in THP-1 macrophages, with levels of 170.9 ± 40.2 ng/mg protein in the high n-3 PUFA treated cells. Thus our data indicate that THP-1 macrophages prominently utilize EPA and DHA for monohydroxylated metabolite formation, in particular 18-HEPE, which has been shown to be released by macrophages to prevent pressure overload-induced maladaptive cardiac remodeling.

Differential expression and function of α-mannosidase I in stimulated naive and memory CD4+ T cells.

N-linked protein glycosylation represents an important cellular process for modifying protein properties. It resembles a cascade of various enzymatic reactions, in which class I α-mannosidases play a central role. We and others have recently shown that N-glycosylation plays a major role for immune functions. We now analyzed the expression and function of α-mannosidase I in CD4(+) naive and memory T cells studying human and murine T cells. Alpha-mannosidase I function was altered by (i) treatment with Kifunensine, a specific inhibitor class I α-mannosidases, (ii) synthetic inhibitory RNA, and (iii) overexpression by retroviral gene transfer. T-cell activation was evaluated by CD69 expression, cytokine production and proliferation. Our results demonstrate (i) that α-mannosidase I transcription is transiently downregulated after T-cell activation with either polyclonal anti-CD3/CD28 antibodies or allogeneic CD19(+) B cells, and (ii) that α-mannosidase I exerts an inhibitory effect on T-cell activation. It is interesting to note that the inhibitory effect was restricted to naive CD4(+) T cells in both systems, human T cells and murine transgenic CD4(+)OT-II cells, whereas human memory T cells and primed CD4(+)OT-II cells remained unaffected. Alpha-mannosidase I inhibition reduced the activation threshold for naive but not already primed CD4(+)OT-II cells as the cells were able to respond to lower ovalbumin peptide concentrations and increased the rejection potential of alloreactive T cells in vivo. Thus, complex N-glycans generated by enzymes such as α-mannosidase I inhibit the activation of naive T cells. These findings could be used to improve the ex vivo priming of naive T cells for adaptive T-cell therapies.

Prevalence of transmitted drug resistance and impact of transmitted resistance on treatment success in the German HIV-1 Seroconverter Cohort.

BACKGROUND: The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort. METHODS: Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008). A genotypic resistance result was obtained for 1,276/1,312 (97.3%). The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml. RESULTS: Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (N = 158/1,276; 12.4%; CI(wilson) 10.7-14.3; p(for trend) = 0.25). NRTI resistance was predominant (7.5%) but decreased significantly over time (CI(Wilson): 6.2-9.1, p(for trend) = 0.02). NNRTI resistance tended to increase over time (NNRTI: 3.5%; CI(Wilson): 2.6-4.6; p(for trend)= 0.07), whereas PI resistance remained stable (PI: 3.0%; CI(Wilson): 2.1-4.0; p(for trend) = 0.24). Resistance to all drug classes was frequently caused by singleton resistance mutations (NRTI 55.6%, PI 68.4%, NNRTI 99.1%). The majority of NRTI-resistant strains (79.8%) carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains (susceptible 45.3%; 173/382 vs. resistant 65.5%; 40/61). The majority of patients in both groups were treated successfully within the first year after ART-initiation (susceptible: 89.9%; 62/69; resistant: 7/9; 77.8%). CONCLUSION: Overall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected with HIV might be related to the introduction of novel antiretroviral drugs and a wider use of genotypic resistance analysis prior to treatment initiation.

Generation of HCMV-specific T-cell lines from seropositive solid-organ-transplant recipients for adoptive T-cell therapy.

Chronically immunosuppressed patients, like solid-organ-transplant (SOT) recipients, are at increased risk for severe human cytomegalovirus (HCMV) infection. Despite the availability of effective antiviral drugs, lasting control of remaining viruses is dependent on an effective T-cell immunity. So in some patients conventional antiviral therapy cannot control the infection and prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Selective reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells derived from healthy donors is a safe and effective approach in hematopoietic stem cell transplant recipients. The aim of this study was to determine whether functional HCMV-specific T cells can also be generated from chronically immunosuppressed patients. Autologous CD4+/8+ T-cell lines directed against the HCMV protein IE-1 were generated from the peripheral blood of SOT patients using a recently developed modular protocol easily applicable to good-manufacturing-practice conditions. T-cell lines from SOT patients showed similar features as cells from healthy donors regarding phenotype, functionality (HCMV-specific killing, gene expression pattern, and cytokine secretion), IE-1 epitope recognition, and dominance of distinct T-cell receptor V beta families. Most importantly, this protocol also allowed the generation of T-cell lines from immunosuppressed patients with HCMV infection/chronic HCMV disease. Our data suggest the potential of this autologous approach for the treatment of SOT recipients suffering from HCMV infection/disease poorly responding to virostatic therapy.