Tecovirimat for the treatment of severe Mpox in Germany.

BACKGROUND: In May 2022, a multi-national mpox outbreak was reported in several non-endemic countries. The only licensed treatment for mpox in the European Union is the orally available small molecule tecovirimat, which in Orthopox viruses inhibits the function of a major envelope protein required for the production of extracellular virus. METHODS: We identified presumably all patients with mpox that were treated with tecovirimat in Germany between the onset of the outbreak in May 2022 and March 2023 and obtained demographic and clinical characteristics by standardized case report forms. RESULTS: A total of twelve patients with mpox were treated with tecovirimat in Germany in the study period. All but one patient identified as men who have sex with men (MSM) who were most likely infected with mpox virus (MPXV) through sexual contact. Eight of them were people living with HIV (PLWH), one of whom was newly diagnosed with HIV at the time of mpox, and four had CD4+ counts below 200/µl. Criteria for treatment with tecovirimat included severe immunosuppression, severe generalized and/or protracted symptoms, a high or increasing number of lesions, and the type and location of lesions (e.g., facial or oral soft tissue involvement, imminent epiglottitis, or tonsillar swelling). Patients were treated with tecovirimat for between six and 28 days. Therapy was generally well-tolerated, and all patients showed clinical resolution. CONCLUSIONS: In this cohort of twelve patients with severe mpox, treatment with tecovirimat was well tolerated and all individuals showed clinical improvement.

Murine hepatocytes do not support persistence of Hepatitis D virus mono-infection in vivo.

BACKGROUNDS & AIMS: As a result of the limited availability of in vivo models for hepatitis D virus (HDV), treatment options for HDV chronically infected patients are still scant. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as HDV entry receptor has enabled the development of new infection models. AIM: To comparatively assess the efficacy and persistence of HDV mono-infection in murine and human hepatocytes in vivo. METHODS: Mice with humanized NTCP (hNTCPed84-87 mice) were generated by editing amino acid residues 84-87 of murine NTCP in C57BL/6J mice. HDV infection was assessed in hNTCPed84-87 mice and in immune deficient uPA/SCID/beige (USB) mice, whose livers were reconstituted with human or murine (hNTCPed84-87 ) hepatocytes. Livers were analysed between 5 and 42 days post-HDV inoculation by qRT-PCR, immunofluorescence and RNA in situ hybridization (ISH). RESULTS: hNTCPed84-87 mice could be infected with HDV genotype 1 or 3. ISH analysis demonstrated the presence of antigenomic HDV RNA positive murine hepatocytes with both genotypes, proving initiation of HDV replication. Strikingly, murine hepatocytes cleared HDV within 21 days both in immunocompetent hNTCPed84-87 mice and in immunodeficient USB mice xenografted with murine hepatocytes. In contrast, HDV infection remained stable for at least 42 days in human hepatocytes. Intrinsic innate responses were not enhanced in any of the HDV mono-infected cells and livers. CONCLUSION: These findings suggest that in addition to NTCP, further species-specific factors limit HDV infection efficacy and persistence in murine hepatocytes. Identifying such species barriers may be crucial to develop novel potential therapeutic targets of HDV.

Non-organ-specific autoantibodies with unspecific patterns are a frequent para-infectious feature of chronic hepatitis D.

Infections with hepatotropic viruses are associated with various immune phenomena. Hepatitis D virus (HDV) causes the most severe form of viral hepatitis. However, few recent data are available on non-disease-specific and non-organ-specific antibody (NOSA) titers and immunoglobulin G (IgG) levels in chronic hepatitis D (CHD) patients. Here, we examined the NOSA titers and IgG levels of 40 patients with CHD and different disease courses and compared them to 70 patients with chronic hepatitis B (CHB) infection. 43% of CHD patients had previously undergone treatment with pegylated interferon-α (IFN-α). The antibody display of 46 untreated patients diagnosed with autoimmune hepatitis (AIH) was used as a reference. The frequency of elevated NOSA titers (CHD 69% vs. CHB 43%, p < 0.01), and the median IgG levels (CHD 16.9 g/L vs. CHB 12.7 g/L, p < 0.01) were significantly higher in CHD patients than in patients with CHB, and highest in patients with AIH (96%, 19.5 g/L). Also, the antinuclear antibody pattern was homogeneous in many patients with AIH and unspecific in patients with viral hepatitis. Additionally, f-actin autoantibodies were only detectable in patients with AIH (39% of SMA). In CHD patients, IgG levels correlated with higher HDV viral loads, transaminases, and liver stiffness values. IgG levels and NOSA were similar in CHD patients irrespective of a previous IFN-α treatment. In summary, autoantibodies with an unspecific pattern are frequently detected in CHD patients with unclear clinical relevance.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

High-resolution analysis of individual spike peptide-specific CD4+ T-cell responses in vaccine recipients and COVID-19 patients.

Objectives: Potential differences in the breadth, distribution and magnitude of CD4+ T-cell responses directed against the SARS-CoV-2 spike glycoprotein between vaccinees, COVID-19 patients and subjects who experienced both ways of immunisation have not been comprehensively compared on a peptide level. Methods: Following virus-specific in vitro cultivation, we determined the T-cell responses directed against 253 individual overlapping 15-mer peptides covering the entire SARS-CoV-2 spike glycoprotein using IFN-γ ELISpot and intracellular cytokine staining. In vitro HLA binding was determined for selected peptides. Results: We mapped 955 single peptide-specific CD4+ T-cell responses in a cohort of COVID-19 patients (n = 8), uninfected vaccinees (n = 16) and individuals who experienced both infection and vaccination (n = 11). Patients and vaccinees (two-time and three-time vaccinees alike) had a comparable number of CD4+ T-cell responses (median 26 vs. 29, P = 0.7289). Most of these specificities were conserved in B.1.1.529 and the BA.4 and BA.5 sublineages. The highest magnitude of these in vitro IFN-γ CD4+ T-cell responses was observed in COVID-19 patients (median 0.35%), and three-time vaccinees showed a higher magnitude than two-time vaccinees (median 0.091% vs. 0.175%, P < 0.0001). Twelve peptide specificities were each detected in at least 40% of subjects. In vitro HLA binding showed promiscuous presentation by DRB1 molecules for several peptides. Conclusion: Both SARS-CoV-2 infection and vaccination prime broadly directed T-cell responses directed against the SARS-CoV-2 spike glycoprotein. This comprehensive high-resolution analysis of spike peptide specificities will be a useful resource for further investigation of spike-specific T-cell responses.

Clinical characteristics and comparison of longitudinal qPCR results from different specimen types in a cohort of ambulatory and hospitalized patients infected with monkeypox virus.

BACKGROUND: The ongoing monkeypox virus outbreak includes at least 7553 confirmed cases in previously non-endemic countries worldwide as of July 2022. Clinical presentation has been reported as highly variable, sometimes lacking classically described systemic symptoms, and only small numbers of cutaneous lesions in most patients. The aim of this study was to compare clinical data with longitudinal qPCR results from lesion swabs, oropharyngeal swabs and blood in a well characterized patient cohort. METHODS: 16 male patients (5 hospitalized, 11 outpatients) were included in the study cohort and serial testing for monkeypox virus-DNA carried out in various materials throughout the course of disease. Laboratory analysis included quantitative PCR, next-generation sequencing, immunofluorescence tests and virus isolation in cell culture. RESULTS: All patients were male, between age 20 and 60, and self-identified as men having sex with men. Two had a known HIV infection, coinciding with an increased number of lesions and viral DNA detectable in blood. In initial- and serial testing, lesion swabs yielded viral DNA-loads at, or above 106 cp/ml and only declined during the third week. Oropharyngeal swabs featured lower viral loads and returned repeatedly negative in some cases. Viral culture was successful only from lesion swabs but not from oropharyngeal swabs or plasma. DISCUSSION: The data presented underscore the reliability of lesion swabs for monkeypox virus-detection, even in later stages of the disease. Oropharyngeal swabs and blood samples alone carry the risk of false negative results, but may hold value in pre-/asymptomatic cases or viral load monitoring, respectively.

Strong Replication Interference Between Hepatitis Delta Viruses in Human Liver Chimeric Mice.

BACKGROUND: Hepatitis D Virus (HDV) is classified into eight genotypes with distinct clinical outcomes. Despite the maintenance of highly conserved functional motifs, it is unknown whether sequence divergence between genotypes, such as HDV-1 and HDV-3, or viral interference mechanisms may affect co-infection in the same host and cell, thus hindering the development of HDV inter-genotypic recombinants. We aimed to investigate virological differences of HDV-1 and HDV-3 and assessed their capacity to infect and replicate within the same liver and human hepatocyte in vivo. METHODS: Human liver chimeric mice were infected with hepatitis B virus (HBV) and with one of the two HDV genotypes or with HDV-1 and HDV-3 simultaneously. In a second set of experiments, HBV-infected mice were first infected with HDV-1 and after 9 weeks with HDV-3, or vice versa. Also two distinct HDV-1 strains were used to infect mice simultaneously and sequentially. Virological parameters were determined by strain-specific qRT-PCR, RNA in situ hybridization and immunofluorescence staining. RESULTS: HBV/HDV co-infection studies indicated faster spreading kinetics and higher intrahepatic levels of HDV-3 compared to HDV-1. In mice that simultaneously received both HDV strains, HDV-3 became the dominant genotype. Interestingly, antigenomic HDV-1 and HDV-3 RNA were detected within the same liver but hardly within the same cell. Surprisingly, sequential super-infection experiments revealed a clear dominance of the HDV strain that was inoculated first, indicating that HDV-infected cells may acquire resistance to super-infection. CONCLUSION: Infection with two largely divergent HDV genotypes could be established in the same liver, but rarely within the same hepatocyte. Sequential super-infection with distinct HDV genotypes and even with two HDV-1 isolates was strongly impaired, suggesting that virus interference mechanisms hamper productive replication in the same cell and hence recombination events even in a system lacking adaptive immune responses.