Viral Replication In Patients Research Articles

Real-world safety and effectiveness of tenofovir alafenamide for 144 weeks in Japanese patients with chronic hepatitis B.

Tenofovir alafenamide (TAF), a prodrug of tenofovir, delivers high levels of active drug to hepatocytes and is given in a lower dose than tenofovir disoproxil fumarate (TDF). TAF reduces viral replication in patients with chronic hepatitis B (CHB) similar to TDF and has shown a lower risk of the renal and bone toxicities associated with TDF use. This post-marketing surveillance study examined the safety and effectiveness of TAF in treatment-naïve and -experienced CHB patients who received TAF for 144 weeks at real-world clinical sites in Japan. Safety assessments included the incidence of adverse drug reactions (ADRs), renal and bone events, and changes in selected laboratory parameters. Effectiveness was based on the proportion of patients with HBV DNA levels below the lower limit of quantitation or <29 IU/mL. This analysis included 580 patients; 18.4% of whom were treatment-naïve. The cumulative incidence of ADRs was 0.21 per 100 person-months, and the incidence of serious ADRs was 0.01 (95% CI, 0.00-0.04) per 100 person-months. There were no ADRs of declines in estimated glomerular filtration rates, renal failure or proximal tubulopathy. The most common ADR was hypophosphataemia in seven (1.2%) patients. Two (0.4%) patients each had decreased blood phosphorus, bone mineral density decreased, dizziness and alopecia. Overall, the proportion of virologically suppressed patients increased from 68.8% at baseline to 97.5% at Week 144. These results confirm the real-world safety and effectiveness of TAF in Japanese patients with CHB and are consistent with the findings of other evaluations of the safety and efficacy of TAF in CHB.

Interferon system in typical chronic and atypically occurring chronic active herpes virus infections

Objective: to study the characteristics of the functioning of the IFN system, the presence of autoantibodies to INFα in patients suffering from atypical chronic active herpesvirus infections (ACA-HVI) in comparison with patients with a typical course of chronic herpesvirus infections (CHVI).Materials and methods: under our supervision were 485 patients of both sexes aged 23 to 70 years, suffering from chronic herpes virus infections, of which 335 patients suffered from AHA-HVI and 150 people suffered from CHVI. The comparison group was 250 conditionally healthy individuals (CG). The complex of the study included methods for detecting herpesviruses: serodiagnostics, PCR-RT. The IFN system (spontaneous and induced production, serum concentration) was tested by ELISA. The study was approved by the ethics board and informed consent was obtained from all patients.Results: the incidence of various mono-mixed herpesvirus infections in patients with ACA-HVI (mono – 26,6% and mixed – 73.4%) and CHVI (mono – 23.1% and mixed – 76.9%) was determined, with EBV dominance in patients of both groups. Serum IFNα deficiency was detected in 100% of cases in both groups, and IFNα in 67% in ACA-HVI and 57% in CHVI. At the same time, significant differences were found between the ACA-HVI and HGVI groups in the level of IFNα reduction: 10 and 5 times, respectively, and for IFNγ – 2.0 and 2.6 times, respectively. The induced IFNα production decreased by 89.1% in ACA-HVI and 47.2% in CHVI. A decrease in induced IFNγ production is characteristic of 50% of patients in both groups. At the same time, the level of induced production of IFN α in patients with ACA-HVI was 9 times lower than in the control group and 4.75 times lower than in the group of patients with CHVI. And the level of induced IFNγ production was 2 times lower compared to CHVI and the control group.Conclusions: when assessing the state of the IFN system in patients with various chronic herpes virus infections, significant differences were revealed. Thus, the most pronounced manifestations of interferonopathy, consisting in a significant decrease in serum IFNα and IFNγ and defects in induced IFN production of both types, are observed statistically significantly more often in the group of patients with an atypical course of the disease than in the group of patients with a typical course of CHVI. The most pronounced disorders in the IFN system and the lack of recovery of IFNα and IFNγ levels in the interrelational period cause atypicity of the course and active viral replication in patients with ACA-HVI.

Efficacy of antiviral therapy and host–virus interactions visualised using serial liver sampling with fine-needle aspirates

Novel therapies for chronic hepatitis B (CHB), such as RNA interference, targetall viral RNAs for degradation, whereas nucleoside analogues are thought to block reverse transcription with minimal impact on viral transcripts. However, limitations in technology and sampling frequency have been obstacles to measuring actual changes in HBV transcription in the liver of patients starting therapy. We used elective liver sampling with fine-needle aspirates (FNAs) to investigate the impact of treatment on viral replication in patients with CHB. Liver FNAs were collected from patients with CHB at baseline and 12 and 24 weeks after starting tenofovir alafenamide treatment. Liver FNAs were subjected to single-cell RNA sequencing and analysed using the Viral-Track method. HBV was the only viral genome detected and was enriched within hepatocytes. The 5' sequencing technology identified protein-specific HBV transcripts and showed that tenofovir alafenamide therapy specifically reduced pre-genomic RNA transcripts with little impact on HBsAg or HBx transcripts. Infected hepatocytes displayed unique gene signatures associated with an immunological response to viral infection. Longitudinal liver sampling, combined with single-cell RNA sequencing, captured the dynamic impact of antiviral therapy on the replication status of HBV and revealed host-pathogen interactions at the transcriptional level in infected hepatocytes. This sequencing-based approach is applicable to early-stage clinical studies, enabling mechanistic studies of immunopathology and the effect of novel therapeutic interventions. Infection-dependent transcriptional changes and the impact of antiviral therapy on viral replication can be measured in longitudinal human liver biopsies using single-cell RNA sequencing data.

FRI-148 - Retrospective study of hepatitis C virus antibodies and active viral replication in patients with severe mental illness in a Spanish university hospital

FRI-148 - Retrospective study of hepatitis C virus antibodies and active viral replication in patients with severe mental illness in a Spanish university hospital

HBV Core-specific CD4+ T cells correlate with sustained viral control upon off-treatment in HBeAg-positive chronic hepatitis B patients

Background & aimsTreatment with nucleos(t)ide analogue (NA) efficiently suppresses viral replication in patients with chronic HBV infection, yet HBV relapses frequently upon NA withdrawal; the detailed immunomodulatory compounds for sustained viral control of HBV upon NA interruption have yet to be fully clarified. This study aimed to elucidate the role of T cells specific for distinct HBV peptides in sustained response upon discontinuation of antiviral treatment. MethodsA total of 48 patients with HBeAg-positive chronic hepatitis B receiving NA treatment and withdrawal were included longitudinally in a retrospective and prospective cohort. Enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) assays were performed to detect IFN-γ producing HBV-specific T cells following stimulation with overlapping peptides covering the whole HBV genome after 10 days of in vitro expansion. ResultsICS assays revealed that T cells specific for HBV Core and Polymerase induced more robust IFN-γ responses compared to Envelope and HBx. Notably, at the time of NA discontinuation, the intensity and breadth of HBV Core peptides-induced responses, predominately targeted by CD4+ T cells but not CD8+ T cells, were associated with sustained viral control upon off-treatment. Further exploration of longitudinal features in patients with sustained viral control revealed that the breadth of HBV-specific T cell responses does not increase following treatment cessation. ConclusionThis report emphasizes the essential role of HBV Core-specific CD4+ T cells in sustained response after therapy withdrawal, indicating it is a potential candidate for immunotherapeutic approaches in chronic HBV patients.

Controversies in the Design of Strategies for the Cure of HIV Infection.

The cure for chronic human immunodeficiency virus (HIV) infections has been a goal pursued since the antiretroviral therapy that improved the clinical conditions of patients became available. However, the exclusive use of these drugs is not enough to achieve a cure, since the viral load rebounds when the treatment is discontinued, leading to disease progression. There are several theories and hypotheses about the biological foundations that prevent a cure. The main obstacle appears to be the existence of a latent viral reservoir that cannot be eliminated pharmacologically. This concept is the basis of the new strategies that seek a cure, known as kick and kill. However, there are other lines of study that recognize mechanisms of persistent viral replication in patients under effective treatment, and that would modify the current lines of research on the cure of HIV. Given the importance of these concepts, in this work, we propose to review the most recent evidence on these hypotheses, covering both the evidence that is positioned in favor and against, trying to expose what are some of the challenges that remain to be resolved in this field of research.

Upper respiratory tract viral load quantification in COVID-19 patients at hospital admission and its association with disease severity.

There are aspects of COVID-19 pathogenesis that are still unknown. To determine the relationship between severity, mortality and viral replication in patients with COVID-19. Clinical characteristics, severity and mortality of 203 patients hospitalized for COVID-19 were analyzed and correlated with viral load (VL) and threshold cycle (TC) at admission; nasopharyngeal swab was obtained. Mean VLs in surviving patients with mild to moderate, moderate to severe and severe disease were the following: 6.8 x 106, 7.6 x 107 and 1.0 x 109, respectively; and in patients with critical disease who died, VL was 1.70 x 109. TCs were 26.06, 24.07, 22.66 and 21.78 for the same groups. In those who died, a higher mean VL was observed at admission in comparison with those who survived (1.7 x 109 vs. 9.84 x 106; p < 0.001). A significant correlation was observed between VL, severity and death (r = 0.254, p < 0.045 and r = 0.21, p < 0.015). High VL was associated with increased in-hospital mortality in comparison with low VL (OR = 2.926, p < 0.017). SARS-CoV-2 VL determined at hospital admission might classify risk simultaneously with other factors described in COVID-19.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFβ) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFβ to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFβ affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFβ target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFβ to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFβ interaction may be promising in the development of a new AIDS therapeutic strategy.

Qualitative plasma viral load determination as a tool for screening of viral reservoir size in PWH.

Suppression of viral replication in patients on antiretroviral therapy (ART) is determined by plasma viral load (pVL) measurement. Whenever pVL reaches values below the limit of quantification, the qualitative parameter 'target detected' or 'target not detected' is available but often not reported to the clinician. We investigated whether qualitative pVL measurements can be used to estimate the viral reservoir size. The study recruited 114 people with HIV (PWH) who are stable on ART between 2016 and 2018. The percentage of pVL measurements qualitatively reported as 'target detected' (PTD) within a 2-year period was calculated. t-DNA and US-RNA were used to estimate viral reservoir size and were quantified on peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. A median of 6.5 pVL measurements over a 2-year period was evaluated for each participant to calculate PTD. A positive correlation was found between t-DNA and PTD (r = 0.24; P = 0.011) but not between US-RNA and PTD (r = 0.1; P = 0.3). A significantly lower PTD was observed in PWH with a small viral reservoir, as estimated by t-DNA less than 66 copies/106 PBMCs and US-RNA less than 10 copies/106 PBMCs, compared with PWH with a larger viral reservoir (P = 0.001). We also show that t-DNA is detectable whenever PTD is higher than 56% and that ART regimen does not affect PTD. Our study shows that PTD provides an efficient parameter to preselect participants with a small viral reservoir based on already available pVL data for future HIV cure trials.

Inhibiting Keratinocyte-Derived Signal Transducer and Activator of Transcription 6 Improved Atopic Dermatitis in Mice

Pruritus is one of the most common symptoms in dermatology and is associated with significant patient morbidity, including atopic dermatitis (AD) (Meng et al., 2018). Signal transducer and activator of transcription 6 (STAT6) is implicated in AD (Howell et al., 2011) by acting as the upstream mediator of T helper 2 responses and promotes T helper 2 cell differentiation and phenotype (Elo et al., 2010). Increased IL-4 and IL-13 exacerbate STAT6 transcription (Howell et al., 2011; Kubo et al., 1997), resulting in enhanced viral replication in patients with AD.

Prolonged viral replication in patients with hematologic malignancies hospitalized with COVID-19.

Not available.

RNA SARS-CoV-2 Persistence in the Lung of Severe COVID-19 Patients: A Case Series of Autopsies.

The exact role of viral replication in patients with severe COVID-19 has not been extensively studied, and it has only been possible to demonstrate the presence of replicative virus for more than 3 months in a few cases using different techniques. Our objective was to study the presence of RNA SARS-CoV-2 in autopsy samples of patients who died from COVID-19 long after the onset of symptoms. Secondary superimposed pulmonary infections present in these patients were also studied. We present an autopsy series of 27 COVID-19 patients with long disease duration, where pulmonary and extrapulmonary samples were obtained. In addition to histopathological analysis, viral genomic RNA (gRNA) and viral subgenomic RNA (sgRNA) were detected using RT-PCR and in situ hybridization, and viral protein was detected using immunohistochemistry. This series includes 26 adults with a median duration of 39 days from onset of symptoms to death (ranging 9–108 days), 92% of them subjected to immunomodulatory therapy, and an infant patient. We detected gRNA in the lung of all but one patient, including those with longer disease duration. SgRNA was detected in 11 out of 17 patients (64.7%) with illness duration up to 6 weeks and in 3 out of 9 patients (33.3%) with more than 6 weeks of disease progression. Viral protein was detected using immunohistochemistry and viral mRNA was detected using in situ hybridization in 3 out of 4 adult patients with illness duration of <2 weeks, but in none of the 23 adult patients with an illness duration of >2 weeks. A remarkable result was the detection of viral protein, gRNA and sgRNA in the lung cells of the pediatric patient after 95 days of illness. Additional pulmonary infections included: 9 acute bronchopneumonia, 2 aspergillosis, 2 cytomegalovirus, and 1 BK virus infection. These results suggest that in severe COVID-19, SARS-CoV-2 could persist for longer periods than expected, especially in immunocompromised populations, contributing to the persistence of chronic lung lesions. Additional infections contribute to the fatal course of the disease.

Are Humanized Mouse Models Useful for Basic Research of Hepatocarcinogenesis through Chronic Hepatitis B Virus Infection?

Chronic hepatitis B virus (HBV) infection is a global health problem that can lead to liver dysfunction, including liver cirrhosis and hepatocellular carcinoma (HCC). Current antiviral therapies can control viral replication in patients with chronic HBV infection; however, there is a risk of HCC development. HBV-related proteins may be produced in hepatocytes regardless of antiviral therapies and influence intracellular metabolism and signaling pathways, resulting in liver carcinogenesis. To understand the mechanisms of liver carcinogenesis, the effect of HBV infection in human hepatocytes should be analyzed. HBV infects human hepatocytes through transfer to the sodium taurocholate co-transporting polypeptide (NTCP). Although the NTCP is expressed on the hepatocyte surface in several animals, including mice, HBV infection is limited to human primates. Due to this species-specific liver tropism, suitable animal models for analyzing HBV replication and developing antivirals have been lacking since the discovery of the virus. Recently, a humanized mouse model carrying human hepatocytes in the liver was developed based on several immunodeficient mice; this is useful for analyzing the HBV life cycle, antiviral effects of existing/novel antivirals, and intracellular signaling pathways under HBV infection. Herein, the usefulness of human hepatocyte chimeric mouse models in the analysis of HBV-associated hepatocarcinogenesis is discussed.

SARS-CoV-2 Disrupts Proximal Elements in the JAK-STAT Pathway.

ABSTRACTSARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Association of Physical Activity with the Risk of Hepatocellular Carcinoma in Patients with Chronic Hepatitis B.

Simple SummaryAlthough viral replication in patients with a chronic hepatitis B (CHB) infection is effectively suppressed by potent antiviral therapy such as entecavir or tenofovir, the risk of hepatocellular carcinoma (HCC) development in CHB patients cannot be totally eliminated. Thus, control of modifiable risk factors for HCC development such as lifestyle modification is important to minimize the HCC risk. In this study, we analyzed a nationwide population-based cohort to evaluate whether there is a significant association between physical activity and development of HCC in CHB patients treated with entecavir or tenofovir. Results in this study suggest that physical activity was significantly associated with a lower risk of HCC development in CHB patients treated with potent antiviral therapy. Increasing physical activity can have beneficial outcomes on HCC development in CHB patients treated with entecavir or tenofovir.Background and Aims: In the general population, previous studies reported that physical activity was associated with risk of hepatocellular carcinoma (HCC) development. However, it is unclear whether physical activity is associated with risk of HCC development in patients with chronic hepatitis B (CHB). We aimed to elucidate the association between physical activity and risk of HCC development in CHB patients. Methods: This nationwide cohort study involved treatment-naive patients with CHB (n = 9727) who started treatment with entecavir or tenofovir and answered self-reported questionnaires between January 2012 and December 2017, using data from the Korean National Health Insurance Service database. The primary endpoint was development of HCC. Multivariable Cox regression and competing risk analyses were used. Results: During a median follow-up of 3.1 years, cumulative HCC incidence rates were 8.3%. There was an inverse association between physical activity and the risk of HCC (p < 0.001). Patients with 1000–1500 metabolic equivalent task (MET)-min/week, compared to those without physical activity, showed a significantly lower risk of HCC in both patients without cirrhosis (adjusted hazard ratio [aHR] 0.66, p = 0.02) and patients with cirrhosis (aHR 0.61, p = 0.02). In patients who were younger (<60), male, without diabetes, and with high BMI, amounts of physical activity of 1000–1500 MET-min/week showed an inverse association with the risk of HCC (aHR 0.65, 0.63, 0.65, and 0.64, respectively, all p < 0.05). Conclusion: Physical activity was significantly associated with a low risk of HCC in CHB patients treated with entecavir or tenofovir.

COVID-19, diabetes y el sistema inmunológico

En el año de 2019 en la provincia de Wuhan, China, se reportó un brote de neumonías atípicas, con síntomas similares a los del Síndrome Respiratorio Agudo Severo (SARS), brote reportado en el 2003, y al Síndrome Respiratorio del Oriente Medio (MERS), brote reportado en 2012 en Arabia Saudita. Esta enfermedad emergente fue causada por el nuevo coronavirus “Síndrome Respiratorio Agudo Severo Coronavirus 2” (SARS-CoV-2) o también denominada COVID-19 del inglés Coronavirus desease 2019, caracterizado por presentar severos problemas respiratorios y diversas manifestaciones extrapulmonares. Al momento se ha reportado una elevada tasa de mortalidad en individuos del sexo masculino, en adultos mayores de 60 años, y en personas con comorbilidades como diabetes obesidad, hipertensión y problemas cardiacos. La diabetes es una enfermedad caracterizada por resistencia a insulina, pérdida de función de células β en el páncreas, y desbalance del sistema inmune por el estado de inflamación crónica. En pacientes graves con COVID-19, se ha observado una secreción alterada de citocinas pro inflamatorias, si esto ocurre en un paciente diabético, que ya tiene un estado inflamatorio per se, se induce inflamación exacerbada y una falla multiorgánica que puede desencadenar en la muerte de los pacientes. Diversos autores señalan como hipótesis que la hiperglicemia favorece la replicación viral, por lo que en un paciente “diabético con infección COVID-19”, es primordial mantener un control glicémico adecuado. Los tratamientos para COVID-19 se han enfocado en disminuir los síntomas, el uso de antivirales, antiinflamatorios, anticuerpos monoclonales y plasma convaleciente COVID-19 ha mostrado resultados variables. Para el control de esta infección viral es necesaria una vacuna, al momento existen más de 150 candidatos, con una eficiencia que va del rango de 70 al 95%, pero aún falta determinar la inmunidad a largo plazo en las personas vacunadas. Esta revisión aborda las características de la infección por SARS-CoV2 y la respuesta inmunitaria en pacientes diabéticos frente a COVID-19.

SARS-CoV-2 engages inflammasome and pyroptosis in human primary monocytes

Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with leukopenia and uncontrolled inflammatory response in critically ill patients. A better comprehension of SARS-CoV-2-induced monocyte death is essential for the identification of therapies capable to control the hyper-inflammation and reduce viral replication in patients with 2019 coronavirus disease (COVID-19). Here, we show that SARS-CoV-2 engages inflammasome and triggers pyroptosis in human monocytes, experimentally infected, and from patients under intensive care. Pyroptosis associated with caspase-1 activation, IL-1ß production, gasdermin D cleavage, and enhanced pro-inflammatory cytokine levels in human primary monocytes. At least in part, our results originally describe mechanisms by which monocytes, a central cellular component recruited from peripheral blood to respiratory tract, succumb to control severe COVID-19.

High levels of SARS-CoV-2-specific T cells with restricted functionality in severe courses of COVID-19.

BACKGROUNDPatients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) differ in the severity of disease. We hypothesized that characteristics of SARS-CoV-2–specific immunity correlate with disease severity.METHODSIn this study, SARS-CoV-2–specific T cells and antibodies were characterized in uninfected controls and patients with different coronavirus disease 2019 (COVID-19) disease severity. SARS-CoV-2–specific T cells were flow cytometrically quantified after stimulation with SARS-CoV-2 peptide pools and analyzed for expression of cytokines (IFN-γ, IL-2, and TNF-α) and markers for activation, proliferation, and functional anergy. SARS-CoV-2–specific IgG and IgA antibodies were quantified using ELISA. Moreover, global characteristics of lymphocyte subpopulations were compared between patient groups and uninfected controls.RESULTSDespite severe lymphopenia affecting all major lymphocyte subpopulations, patients with severe disease mounted significantly higher levels of SARS-CoV-2–specific T cells as compared with convalescent individuals. SARS-CoV-2–specific CD4+ T cells dominated over CD8+ T cells and closely correlated with the number of plasmablasts and SARS-CoV-2–specific IgA and IgG levels. Unlike in convalescent patients, SARS-CoV-2–specific T cells in patients with severe disease showed marked alterations in phenotypical and functional properties, which also extended to CD4+ and CD8+ T cells in general.CONCLUSIONGiven the strong induction of specific immunity to control viral replication in patients with severe disease, the functionally altered characteristics may result from the need for contraction of specific and general immunity to counteract excessive immunopathology in the lung.FUNDINGThe study was supported by institutional funds to MS and in part by grants of Saarland University, the State of Saarland, and the Rolf M. Schwiete Stiftung.

A multi centre randomized open label trial of chloroquine for the treatment of adults with SARS-CoV-2 infection in Vietnam

Background: COVID-19 is a respiratory disease caused by a novel coronavirus (SARS-CoV-2) and causes substantial morbidity and mortality. There is currently no vaccine to prevent COVID-19 or therapeutic agent to treat COVID-19. This clinical trial is designed to evaluate chloroquine as a potential therapeutic for the treatment of hospitalised people with COVID-19. We hypothesise that chloroquine slows viral replication in patients with COVID-19, attenuating the infection, and resulting in more rapid decline of viral load in throat/nose swabs. This viral attenuation should be associated with improved patient outcomes. Method: The study will start with a 10-patient prospective observational pilot study following the same entry and exclusion criteria as for the randomized trial and undergoing the same procedures. The main study is an open label, randomised, controlled trial with two parallel arms of standard of care (control arm) versus standard of care with 10 days of chloroquine (intervention arm) with a loading dose over the first 24 hours, followed by 300mg base orally once daily for nine days. The study will recruit patients in three sites in Ho Chi Minh City, Vietnam: the Hospital for Tropical Diseases, the Cu Chi Field Hospital, and the Can Gio COVID hospital. The primary endpoint is the time to viral clearance from throat/nose swab, defined as the time following randomization until the midpoint between the last positive and the first of the negative throat/nose swabs. Viral presence will be determined using RT-PCR to detect SARS-CoV-2 RNA. Discussion: The results of the study will add to the evidence-based guidelines for management of COVID-19. Given the enormous experience of its use in malaria chemoprophylaxis, excellent safety and tolerability profile, and its very low cost, if proved effective then chloroquine would be a readily deployable and affordable treatment for patients with COVID-19. Trial registration: Clinicaltrials.gov NCT04328493 31/03/2020

Combined Prophylactic and Therapeutic Use Maximizes Hydroxychloroquine Anti-SARS-CoV-2 Effects in vitro.

While the SARS-CoV-2 pandemic is heavily hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 and Caco-2 cells. This suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.