Fluoxetine and Sertraline Potently Neutralize the Replication of Distinct SARS-CoV-2 Variants.

The pandemic caused by SARS-CoV-2 is still a major health problem. Newly emerging variants and long-COVID-19 represent a challenge for the global health system. In particular, individuals in developing countries with insufficient health care need easily accessible, affordable and effective treatments of COVID-19. Previous studies have demonstrated the efficacy of functional inhibitors of acid sphingomyelinase against infections with various viruses, including early variants of SARS-CoV-2. This work investigated whether the acid sphingomyelinase inhibitors fluoxetine and sertraline, usually used as antidepressant molecules in clinical practice, can inhibit the replication of the former and recently emerged SARS-CoV-2 variants in vitro. Fluoxetine and sertraline potently inhibited the infection with pseudotyped virus-like particles and SARS-CoV-2 variants D614G, alpha, delta, omicron BA.1 and omicron BA.5. These results highlight fluoxetine and sertraline as priority candidates for large-scale phase 3 clinical trials at different stages of SARS-CoV-2 infections, either alone or in combination with other medications.

Immune responses in COVID-19 patients during breakthrough infection with SARS-CoV-2 variants Delta, Omicron-BA.1 and Omicron-BA.5.

Background: Breakthrough infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are increasingly observed in vaccinated individuals. Immune responses towards SARS-CoV-2 variants, particularly Omicron-BA.5, are poorly understood. We investigated the humoral and cellular immune responses of hospitalized COVID-19 patients during Delta and Omicron infection waves. Methods: The corresponding SARS-CoV-2 variant of the respective patients were identified by whole genome sequencing. Humoral immune responses were analyzed by ELISA and a cell culture-based neutralization assay against SARS-CoV-2 D614G isolate (wildtype), Alpha, Delta (AY.43) and Omicron (BA.1 and BA.5). Cellular immunity was evaluated with an IFN-γ ELISpot assay. Results: On a cellular level, patients showed a minor IFN-γ response after stimulating PBMCs with mutated regions of SARS-CoV-2 variants. Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced. Double-vaccinated patients with Delta breakthrough infection showed a significantly increased neutralizing antibody response against Delta compared to double-vaccinated uninfected controls (median complete neutralization titer (NT100) 640 versus 80, p<0.05). Omicron-BA.1 infection increased neutralization titers against BA.1 in double-vaccinated patients (median NT100 of 160 in patients versus 20 in controls, p=0.07) and patients that received booster vaccination (median NT100 of 50 in patients versus 20 in controls, p=0.68). For boosted patients with BA.5 breakthrough infection, we found no enhancing effect on humoral immunity against SARS-CoV-2 variants. Conclusion: Neutralizing antibody titers against Omicron-BA.1 and especially BA.5 were strongly reduced in SARS-CoV-2 breakthrough infections. Delta and Omicron-BA.1 but not Omicron-BA.5 infections boosted the humoral immunity in double-vaccinated patients and patients with booster vaccination. Despite BA.5 breakthrough infection, those patients may still be vulnerable for reinfections with BA.5 or other newly emerging variants of concern.

Cell-to-cell spread inhibiting antibodies constitute a correlate of protection against herpes simplex virus type 1 reactivations: A retrospective study.

Background: Herpes simplex viruses (HSV) cause ubiquitous human infections. For vaccine development, knowledge concerning correlates of protection is essential. Therefore, we investigated (I) if humans are in principle capable producing cell-to-cell spread inhibiting antibodies against HSV and (II) whether this capacity is associated with a reduced HSV-1 reactivation risk. Methods: We established a high-throughput HSV-1-ΔgE-GFP reporter virus-based assay and evaluated 2,496 human plasma samples for HSV-1 glycoprotein E (gE) independent cell-to-cell spread inhibiting antibodies. Subsequently, we conducted a retrospective survey among the blood donors to analyze the correlation between the presence of cell-to-cell spread inhibiting antibodies in plasma and the frequency of HSV reactivations. Results: In total, 128 of the 2,496 blood donors (5.1%) exhibited high levels of HSV-1 gE independent cell-to-cell spread inhibiting antibodies in the plasma. None of the 147 HSV-1 seronegative plasmas exhibited partial or complete cell-to-cell spread inhibition, demonstrating the specificity of our assay. Individuals with cell-to-cell spread inhibiting antibodies showed a significantly lower frequency of HSV reactivations compared to subjects without sufficient levels of such antibodies. Conclusion: This study contains two important findings: (I) upon natural HSV infection, some humans produce cell-to-cell spread inhibiting antibodies and (II) such antibodies correlate with protection against recurrent HSV-1. Moreover, these elite neutralizers may provide promising material for immunoglobulin therapy and information for the design of a protective vaccine against HSV-1.

Long-term cellular immune response in immunocompromised unvaccinated COVID-19 patients undergoing monoclonal antibody treatment.

Immunocompromised patients are at increased risk for a severe course of COVID-19. Treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection with anti-SARS-CoV-2 monoclonal antibodies (mAbs) has become widely accepted. However, the effects of mAb treatment on the long-term primary cellular response to SARS-CoV-2 are unknown. In the following study, we investigated the long-term cellular immune responses to SARS-CoV-2 Spike S1, Membrane (M) and Nucleocapsid (N) antigens using the ELISpot assay in unvaccinated, mAb-treated immunocompromised high-risk patients. Anti-SARS-CoV-2 mAb untreated though vaccinated COVID-19 immunocompromised patients, vaccinated SARS-CoV-2 immunocompromised patients without COVID-19 and vaccinated healthy control subjects served as control groups. The cellular immune response was determined at a median of 5 months after SARS-CoV-2 infection. Our data suggest that immunocompromised patients develop an endogenous long-term cellular immune response after COVID-19, although at low levels. A better understanding of the cellular immune response will help guide clinical decision making for these vulnerable patient cohorts.

GNB3 c.825c>T polymorphism influences T-cell but not antibody response following vaccination with the mRNA-1273 vaccine.

Background: Immune responses following vaccination against COVID-19 with different vaccines and the waning of immunity vary within the population. Genetic host factors are likely to contribute to this variability. However, to the best of our knowledge, no study on G protein polymorphisms and vaccination responses against COVID-19 has been published so far. Methods: Antibodies against the SARS-CoV-2 spike protein and T-cell responses against a peptide pool of SARS-CoV-2 S1 proteins were measured 1 and 6 months after the second vaccination with mRNA-1273 in the main study group of 204 participants. Additionally, antibodies against the SARS-CoV-2 spike protein were measured in a group of 597 participants 1 month after the second vaccination with mRNA-1273. Genotypes of GNB3 c.825C>T were determined in all participants. Results: The median antibody titer against the SARS-CoV-2 spike protein and median values of spots increment in the SARS-CoV-2 IFN-γ ELISpot assay against the S1-peptide pool were significantly decreased from months 1 to 6 (p < 0.0001). Genotypes of GNB3 c.825C>T had no influence on the humoral immune response. At month 1, CC genotype carriers had significantly increased T-cell responses compared to CT (p = 0.005) or TT (p = 0.02) genotypes. CC genotype carriers had an almost 6-fold increased probability compared to TT genotype carriers and an almost 3-fold increased probability compared to T-allele carriers to mount a SARS-CoV-2-specific T-cell response above the median value. Conclusion: CC genotype carriers of the GNB3 c.825C>T polymorphism have an increased T-cell immune response to SARS-CoV-2, which may indicate better T-cell-mediated protection against COVID-19 after vaccination with mRNA-1273.

Cellular and Humoral Immunity against Different SARS-CoV-2 Variants Is Detectable but Reduced in Vaccinated Kidney Transplant Patients.

In kidney transplant (KTX) patients, immune responses after booster vaccination against SARS-CoV-2 are inadequately examined. We analyzed these patients a median of four months after a third/fourth vaccination and compared them to healthy controls. Cellular responses were analyzed by interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpot assays. Neutralizing antibody titers were assessed against SARS-CoV-2 D614G (wild type) and the variants alpha, delta, and omicron by a cell culture-based neutralization assay. Humoral immunity was also determined by a competitive fluorescence assay, using 11 different variants of SARS-CoV-2. Antibody ratios were measured by ELISA. KTX patients showed significantly lower SARS-CoV-2-specific IFN-γ responses after booster vaccination than healthy controls. However, SARS-CoV-2-specific IL-2 responses were comparable to the T cell responses of healthy controls. Cell culture-based neutralizing antibody titers were 1.3-fold higher in healthy controls for D614G, alpha, and delta, and 7.8-fold higher for omicron (p < 0.01). Healthy controls had approximately 2-fold higher concentrations of potential neutralizing antibodies against all 11 variants than KTX patients. However, more than 60% of the KTX patients displayed antibodies to variants of SARS-CoV-2. Thus, KTX patients should be partly protected, due to neutralizing antibodies to variants of SARS-CoV-2 or by cross-reactive T cells, especially those producing IL-2.

The GNB3 c.825C>T (rs5443) polymorphism and protection against fatal outcome of corona virus disease 2019 (COVID-19).

Background and aims: Albeit several factors which influence the outcome of corona virus disease (COVID-19) are already known, genetic markers which may predict the outcome of the disease in hospitalized patients are still very sparse. Thus, in this study, we aimed to analyze whether the single-nucleotide polymorphism (SNP) rs5443 in the gene GNB3, which was associated with higher T cell responses in previous studies, might be a suitable biomarker to predict T cell responses and the outcome of COVID-19 in a comprehensive German cohort. Methods: We analyzed the influence of demographics, pre-existing disorders, laboratory parameters at the time of hospitalization, and GNB3 rs5443 genotype in a comprehensive cohort (N = 1570) on the outcome of COVID-19. In a sub cohort, we analyzed SARS-CoV-2-specific T cell responses and associated GNB3 rs5443 genotypes. We investigated the influence of all factors on COVID-19 fatality in multivariable analysis. Results: We found a younger patient age, normotension or absence of diabetes mellitus or cardiovascular diseases, normal blood cell counts, and low inflammatory markers at hospital admission were protective factors against fatal course of disease. In addition, the rs5443 TT genotype was significantly associated with protection against COVID-19 fatality (OR: 0.60, 95% CI: 0.40-0.92, p = 0.02). We also observed significantly increased SARS-CoV-2-specific T cell responses in rs5443 TT genotype carriers (p = 0.01). Although we observed a significant association of the factors described previously in univariate analysis, only a younger age of the patients, normal blood cell counts, and the GNB3 rs5443 TT genotype remained independent predictors against COVID-19 fatality in multivariable analysis. Conclusion: Immutable predictors for COVID-19 fatality are relatively rare. In this study we could show that the TT genotype of the SNP rs5443 in the gene GNB3 is associated with protection against COVID-19 fatality. It was as well correlated to higher SARS-CoV-2-specific T cell responses, which could result in a milder course of disease in those patients. Based on those observations we hereby provide a further prognostic biomarker, which might be used in routine diagnostics as a predictive factor for COVID-19 mortality already upon hospitalization.

Cellular and Humoral Immunity after the Third Vaccination against SARS-CoV-2 in Hematopoietic Stem-Cell Transplant Recipients.

Protecting vulnerable groups from severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is mandatory. Immune responses after a third vaccination against SARS-CoV-2 are insufficiently studied in patients after hematopoietic stem-cell transplantation (HSCT). We analyzed immune responses before and after a third vaccination in HSCT patients and healthy controls. Cellular immunity was assessed using interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpots. Furthermore, this is the first report on neutralizing antibodies against 11 variants of SARS-CoV-2, analyzed by competitive fluorescence assay. Humoral immunity was also measured by neutralization tests assessing cytopathic effects and by ELISA. Neither HSCT patients nor healthy controls displayed significantly higher SARS-CoV-2-specific IFN-γ or IL-2 responses after the third vaccination. However, after the third vaccination, cellular responses were 2.6-fold higher for IFN-γ and 3.2-fold higher for IL-2 in healthy subjects compared with HSCT patients. After the third vaccination, neutralizing antibodies were significantly higher (p < 0.01) in healthy controls, but not in HSCT patients. Healthy controls vs. HSCT patients had 1.5-fold higher concentrations of neutralizing antibodies against variants and 1.2-fold higher antibody concentrations against wildtype. However, half of the HSCT patients exhibited neutralizing antibodies to variants of SARS-CoV-2, which increased only slightly after a third vaccination.

COVID-19 in Elderly, Immunocompromised or Diabetic Patients-From Immune Monitoring to Clinical Management in the Hospital.

The novel, highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic of acute respiratory illness worldwide and remains a huge threat to the healthcare system's capacity to respond to COVID-19. Elderly and immunocompromised patients are at increased risk for a severe course of COVID-19. These high-risk groups have been identified as developing diminished humoral and cellular immune responses. Notably, SARS-CoV-2 RNA remains detectable in nasopharyngeal swabs of these patients for a prolonged period of time. These factors complicate the clinical management of these vulnerable patient groups. To date, there are no well-defined guidelines for an appropriate duration of isolation for elderly and immunocompromised patients, especially in hospitals or nursing homes. The aim of the present study was to characterize at-risk patient cohorts capable of producing a replication-competent virus over an extended period after symptomatic COVID-19, and to investigate the humoral and cellular immune responses and infectivity to provide a better basis for future clinical management. In our cohort, the rate of positive viral cultures and the sensitivity of SARS-CoV-2 antigen tests correlated with higher viral loads. Elderly patients and patients with diabetes mellitus had adequate cellular and humoral immune responses to SARS-CoV-2 infection, while immunocompromised patients had reduced humoral and cellular immune responses. Our patient cohort was hospitalized for longer compared with previously published cohorts. Longer hospitalization was associated with a high number of nosocomial infections, representing a potential hazard for additional complications to patients. Most importantly, regardless of positive SARS-CoV-2 RNA detection, no virus was culturable beyond a cycle threshold (ct) value of 33 in the majority of samples. Our data clearly indicate that elderly and diabetic patients develop a robust immune response to SARS-CoV-2 and may be safely de-isolated at a ct value of more than 35.

Early Treatment with Monoclonal Antibodies or Convalescent Plasma Reduces Mortality in Non-Vaccinated COVID-19 High-Risk Patients.

Vulnerable patients such as immunosuppressed or elderly patients are at high risk for a severe course of COVID-19 upon SARS-CoV-2 infection. Immunotherapy with SARS-CoV-2 specific monoclonal antibodies (mAb) or convalescent plasma represents a considerable treatment option to protect these patients from a severe or lethal course of infection. However, monoclonal antibodies are not always available or less effective against emerging SARS-CoV-2 variants. Convalescent plasma is more commonly available and may represent a good treatment alternative in low-income countries. We retrospectively evaluated outcomes in individuals treated with mAbs or convalescent plasma and compared the 30-day overall survival with a patient cohort that received supportive care due to a lack of SARS-CoV-2 specific therapies between March 2020 and April 2021. Our data demonstrate that mAb treatment is highly effective in preventing severe courses of SARS-CoV-2 infection. All patients treated with mAb survived. Treatment with convalescent plasma improved overall survival to 82% compared with 61% in patients without SARS-CoV-2 targeted therapy. Our data indicate that early convalescent plasma treatment may be an option to improve the overall survival of high-risk COVID-19 patients. This is especially true when other antiviral drugs are not available or their efficacy is significantly reduced, which may be the case with emerging SARS-CoV-2 variants.

Long-Term SARS-CoV-2 Specific Immunity Is Affected by the Severity of Initial COVID-19 and Patient Age.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the greatest medical challenge. Although crucial to the future management of the pandemic, the factors affecting the persistence of long-term SARS-CoV-2 immunity are not well understood. Therefore, we determined the extent of important correlates of SARS-CoV-2 specific protection in 200 unvaccinated convalescents after COVID-19. To investigate the effective memory response against the virus, SARS-CoV-2 specific T cell and humoral immunity (including virus-neutralizing antibodies) was determined over a period of one to eleven months. SARS-CoV-2 specific immune responses were present in 90% of individual patients. Notably, immunosuppressed patients did not have long-term SARS-CoV-2 specific T cell immunity. In our cohort, the severity of the initial illness influenced SARS-CoV-2 specific T cell immune responses and patients' humoral immune responses to Spike (S) protein over the long-term, whereas the patients' age influenced Membrane (M) protein-specific T cell responses. Thus, our study not only demonstrated the long-term persistence of SARS-CoV-2 specific immunity, it also determined COVID-19 severity and patient age as significant factors affecting long-term immunity.

Humoral and Cellular Vaccination Responses against SARS-CoV-2 in Hematopoietic Stem Cell Transplant Recipients.

The cellular response to SARS-CoV-2 vaccination and infection in allogeneic hematopoietic stem cell transplant (HSCT) recipients is not yet clear. In the current study, HSCT recipients prior to and post vaccination were tested for SARS-CoV-2-specific humoral and cellular immunity. Antibodies against spike (S) 1 were assessed by Anti-SARS-CoV-2 IgG ELISA (Euroimmun). Cellular immunity was analyzed by an in house interferon-gamma ELISpot and T-SPOT.COVID (Oxford Immunotec), using altogether seven SARS-CoV-2-specific antigens. In 117 HSCT patients vaccinated twice, SARS-CoV-2 IgG antibodies were significantly higher than in HSCT controls pre vaccination (p < 0.0001). After the second vaccination, we observed a median antibody ratio of 4.7 and 68% positive results, whereas 35 healthy controls reached a median ratio of 9.0 and 100% positivity. ELISpot responses in patients were significantly (p < 0.001) reduced to ≤33% of the controls. After the second vaccination, female HSCT patients and female healthy controls showed significantly higher antibody responses than males (6.0 vs. 2.1 and 9.2 vs. 8.2, respectively; p < 0.05). Cellular immunity was diminished in patients irrespective of sex. In conclusion, especially male HSCT recipients showed impaired antibody responses after SARS-CoV-2 vaccination. Changing the vaccine schedule or composition could help increase vaccine responses.

Comparison of SARS-CoV-2- and HCoV-Specific T Cell Response Using IFN-γ ELISpot.

Herd immunity is essential to control severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), especially in immunocompromised patients. Convalescent individuals should be vaccinated later due to vaccine shortage, as studies show that neutralizing antibodies generated during infection are stable for at least 6 months. Cellular immunity is also detectable for months. However, there is evidence of cross-reactivity of T cells with human endemic coronaviruses (HCoVs). Here, we show that cross-reactivity-which may prevent the specific detection of SARS-CoV-2-specific T cell responses-can be avoided if cells are stimulated with the N-terminus of the spike protein in IFN-γ ELISpot. In contrast to previous studies, we examined T-cell responses against all four known HCoVs using IFN-γ ELISpot in 19 convalescent volunteers and 10 fully vaccinated volunteers. In addition, we performed Spearman analyses to detect cross-reactivity of T cells. We observed no correlation between T-cell responses against SARS-CoV-2 and human endemic coronaviruses, either in the whole cohort or in the individual groups. The use of the respective stimuli could lead to a more accurate assessment of cellular immunity in recovered individuals. This testing procedure could help to define the best time point at which convalescents should receive SARS-CoV-2 vaccination.

SARS-CoV-2-specific humoral and cellular immunity in two renal transplants and two hemodialysis patients treated with convalescent plasma.

When patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten recovery, is contraindicated. Antiviral treatment with convalescent plasma (CP) could be an alternative treatment option. In this case report, we present two kidney transplant recipients and two hemodialysis patients who were infected with SARS-CoV-2 and received CP. Antibodies against the receptor-binding domain in the S1 subunit of the SARS-CoV-2 spike protein were determined sequentially by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and neutralization assay and specific cellular responses by interferon-gamma ELISpot. Before treatment, in both kidney transplant recipients and one hemodialysis patient antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers (≤1:40). ELISpot responses in the four patients were either weak or absent. After CP treatment, we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific cellular responses. After intermittent clinical improvement, one kidney transplant recipient again developed typical symptoms on Day 12 after treatment and received a second cycle of CP treatment. Altogether, three patients clinically improved and could be discharged from the hospital. However, one 83-year-old multimorbid patient deceased. Our data suggest that the success of CP therapy may only be temporary in patients with chronic kidney disease; which requires close monitoring of viral load and antiviral immunity and possibly an adaptation of the treatment regimen.

Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2-Specific IgG.

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells.