Beta-Cyclodextrins as affordable antivirals to treat coronavirus infection

The SARS-CoV-2 pandemic made evident that we count with few coronavirus-fighting drugs. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety and tolerability profiles. We began elaborating a list of 116 drugs previously used to treat other pathologies or characterized in pre-clinical studies with potential to treat coronavirus infections. We next employed molecular modelling tools to rank the 44 most promising inhibitors and tested their efficacy as antivirals against a panel of and {beta} coronavirus, e.g., the HCoV-229E and SARS-CoV-2 viruses. Four drugs, OSW-1, U18666A, hydroxypropyl-{beta}-cyclodextrin (H{beta}CD) and phytol, showed antiviral activity against both HCoV-229E (in MRC5 cells) and SARS-CoV-2 (in Vero E6 cells). The mechanism of action of these compounds was studied by transmission electron microscopy (TEM) and by testing their capacity to inhibit the entry of SARS-CoV-2 pseudoviruses in ACE2-expressing HEK-293T cells. The entry was inhibited by H{beta}CD and U18666A, yet only H{beta}CD could inhibit SARS-CoV-2 replication in the pulmonary cells Calu-3. With these results and given that cyclodextrins are widely used for drug encapsulation and can be safely administered to humans, we further tested 6 native and modified cyclodextrins, which confirmed {beta}-cyclodextrins as the most potent inhibitors of SARS-CoV-2 replication in Calu-3 cells. All accumulated data points to {beta}-cyclodextrins as promising candidates to be used in the therapeutic treatments for SARS-CoV-2 and possibly other respiratory viruses.

Safety and Immunogenicity of a Recombinant Protein RBD Fusion Heterodimer Vaccine against SARS-CoV-2: preliminary results of a phase 1-2a dose-escalating, randomized, double-blind clinical trial.

BackgroundWe report safety, tolerability, and immunogenicity of a recombinant protein RBD-fusion heterodimeric vaccine against SARS-CoV-2 (PHH-1V). MethodsA dose-escalation, phase 1-2a, randomized clinical trial was performed in Catalonia, Spain. Each cohort had one safety sentinel that received PHH-1V vaccine of the corresponding dose, and remaining participants were randomly assigned to receive PHH-1V formulations [10{micro}g (n=5), 20{micro}g (n=10), 40{micro}g (n=10)] or control BNT162b2 (n=5). Two intramuscular doses (0-21 days) were administered. Primary endpoint was solicited events 7 days after each vaccination and secondary-exploratory endpoints were humoral and cellular immunogenicity. Findings30 young healthy adults were enrolled, thirteen were female. Vaccines were safe, well tolerated. The most common solicited events for all groups were tenderness and pain at the site of injection. The proportion of subjects with at least one reported local and/or systemic solicited adverse events (AE) after first or second vaccine dose were lowest in PHH-1V (n=21, 84%) than control group (n=5, 100%). AE were mild to moderate, and no severe AE nor AE of special interest were reported. All participants had a >4-fold change at day 35 in total binding antibodies from baseline. Variants of concern (VOC) alpha, beta, delta and gamma were evaluated using a SARS-CoV-2 pseudovirus-based neutralization assay. All groups had a significant geometric mean fold rise (p<.0001) at day 35 against all studied VOC. Similar results were obtained when a full replicative virus neutralization assay was carried out. InterpretationPHH-1V was safe, well tolerated, and induced robust humoral responses. These data support further exploration of PHH-1V in larger studies. FundingHIPRA ClinicalTrials.gov IdentifierNCT05007509 Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed up until August 1, 2021, with the terms "SARS-CoV-2", "COVID-19" and "vaccine". We initially identified 12,952 results but when added the terms "clinical trial" and "variants" this number decreased to 50. Of these references twelve were clinical trials, and although several vaccines were under development, and the ones that were already approved for administration in the general population described the neutralization effect to the different circulating variants of concern, we could not find any reference to a vaccine developed using variants of concern instead of ancestral Wuhan strain. Added value of this studyTo the best of our knowledge, our study is the first clinical trial to assess the effect as a primary series of a recombinant protein receptor-binding domain fusion heterodimer PHH-1V vaccine against SARS-CoV-2 not including the ancestral strain in its composition. This vaccine contains RBD from B{middle dot}1{middle dot}351 (beta) and B{middle dot}1{middle dot}1{middle dot}7 (alpha) variants and is co-formulated with an oil-in-water adjuvant emulsion. In this first-in-human randomized clinical trial, two doses of the SARS-CoV-2 PHH-1V vaccine in a range of 10 to 40 {micro}g/dose were safe and well-tolerated and induced robust humoral immune responses to different circulating variants of concern, including alpha (B1{middle dot}1{middle dot}7), beta (B{middle dot}1{middle dot}351), delta (B{middle dot}1{middle dot}617{middle dot}2) and gamma (P{middle dot}1). Additionally, the PHH-1V 40{micro}g dose vaccine elicited moderated cellular immune responses, particularly to variants of concern alpha and delta. Implications of all the available evidenceThese findings indicate that the recombinant protein receptor-binding domain fusion heterodimer vaccine PHH-1V is safe and immunogenic. Phase 2b and Phase 3 clinical trials are ongoing to further investigate its safety and protective efficacy as heterologous booster.

Safety and immunogenicity of the protein-based PHH-IV compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated against COVID-19: a multicentre, randomised, double-blind, non-inferiority phase IIb trial.

Background: A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14, 28 and 98 days after vaccine administration. Methods: The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine -either heterologous (PHH-1V group) or homologous (BNT162b2 group)- in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus [≥]65 years) with approximately 10% of the sample enrolled in the older age group. The primary endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies (PBNA) against the ancestral Wuhan-Hu-1 strain after the PHH-1V or the BNT162b2 boost, and the safety and tolerability of PHH-1V as a boost. The secondary endpoints were to compare changes in levels of neutralizing antibodies against different variants of SARS-CoV-2 and the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides. The exploratory endpoint was to assess the number of subjects with SARS-CoV-2 infections [≥]14 days after PHH-1V booster. This study is ongoing and is registered with ClinicalTrials.gov, NCT05142553. Findings: From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n=522) or BNT162b2 (n=260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14, 28 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1.68 (p<0.0001), 1.31 (p=0.0007) and 0.86 (p=0.40) for the ancestral Wuhan-Hu-1 strain; 0.62 (p<0.0001), 0.65 (p<0.0001) and 0.56 (p=0.003) for the Beta variant; 1.01 (p=0.92), 0.88 (p=0.11) and 0.52 (p=0.0003) for the Delta variant; and 0.59 (p=<0.0001), 0.66 (p<0.0001) and 0.57 (p=0.0028) for the Omicron BA.1 variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4+ and CD8+ T-cells expressing IFN-{gamma} on day 14. There were 458 participants who experienced at least one adverse event (89.3%) in the PHH-1V and 238 (94.4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79.7% and 89.3%), fatigue (27.5% and 42.1%) and headache (31.2 and 40.1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10.14%) for the PHH-1V group and 30 (11.90%) for the BNT162b2 group (p=0.45), and none of the subjects developed severe COVID-19. Interpretation: Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, although it does not reach a non-inferior neutralizing antibody response against the Wuhan-Hu-1 strain at days 14 and 28 after vaccination, it does so at day 98. PHH-1V as a heterologous booster elicits a superior neutralizing antibody response against the previous circulating Beta and Delta SARS-CoV-2 variants, as well as the currently circulating Omicron BA.1. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. Funding: HIPRA SCIENTIFIC, S.L.U.

Comparison between mid-nasal swabs and buccal swabs for SARS-CoV-2 detection in mild COVID-19 patients

BackgroundThe use of rapid antigen diagnostics tests (Ag-RDT) has gained widespread acceptance as an alternative method for diagnosis of COVID-19 outside of health care settings. Various authors have reported that saliva is a reliable specimen, alternative to nasopharyngeal and mid-nasal swabs, to detect SARS-CoV-2 infections by RT-PCR. We assessed the performance of buccal swabs containing saliva for SARS-CoV-2 detection by Ag-RDT, using mid-nasal specimens as a reference in the northern area of Barcelona (Catalonia, Spain) MethodsIn the context of routine clinical diagnosis of mild COVID-19 patients, we enrolled 300 adults in a study to directly compare mid-nasal swabs and saliva specimens for SARS-CoV-2 detection by Ag-RDT.. When mid-nasal and buccal Ag-RDTs showed discordant results, a third mid-nasal swab was collected and analysed by RT-PCR. ResultsPaired samples were successfully obtained in 300 suspected cases of SARS-CoV-2 infection. Of the 300 paired samples, Ag-RDT with the mid-nasal swab detected 139 (46.3%) positive COVID-19 cases. In comparison, buccal swabs showed a sensitivity and specificity of 31.7% (44/139) and 98.8% (159/161), respectively. 65 discordant results with positive mid-nasal swabs and negative buccal swabs were tested by RT-qPCR. All samples tested by Rt-PCR resulted positive, with a mean cycle threshold (Ct) of 28.3 (SD 7.3). ConclusionOur findings show that mid-nasal swabs have better performance than buccal swabs for detecting SARS-CoV-2 with Ag-RDT tests. Of note, the sensitivity of buccal samples was affected in samples with high viral loads (Ct<33), suggesting that buccal swabs might not be sensitive enough to detect individuals at risk of transmission. Taken together, the existing literature and the results provided in our analysis we advise against the use of buccal specimens for SARS-CoV-2 diagnostics with Ag-RDT.

Cross-sectional study to assess the efficacy of SARS-CoV-2 vaccination in patients with monoclonal gammopathies

SARS-CoV-2 vaccination is the most effective strategy to protect patients with haematologic malignancies against severe COVID-19, but primary vaccine responses are less effective in this population. Here, we characterized the humoral responses following 3 months after mRNA-based vaccines in patients at different stages of the same plasma cell diseases, including monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and multiple myeloma on first line therapy (MM), compared to a healthy control population matched by sex and age. We observed that plasmas from uninfected MM patients after 3 months post-vaccine have lower SARS-CoV-2 specific IgG and IgA antibodies and decreased neutralization capacity compared with MGUS and SMM individuals, and a group of healthy controls. Importantly, we detected significantly higher plasma neutralization capacity in MM individuals who recovered from COVID-19 compared to their uninfected counterparts, highlighting that hybrid immunity elicit stronger immune responses even in this immunocompromised population. In contrast to MM group, no differences in the vaccine-induced humoral response were observed between uninfected MGUS, SMM and healthy individuals. In conclusion, a booster vaccine dose is recommended in uninfected MM patients to develop an adequate and effective humoral response to SARS-CoV-2 vaccine.

Convalescent plasma for outpatients with early COVID-19

BackgroundConvalescent plasma (CP) for hospitalized patients with COVID-19 has not demonstrated clear benefits. However, data on outpatients with early symptoms are scarce. We aimed to assess whether treatment with CP administered during the first 7 days of symptoms reduced the disease progression or risk of hospitalization of outpatients. MethodsTwo double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when <20% of their predefined sample size had been recruited. A Bayesian adaptive individual patient data meta-analysis was implemented. Analyses were done with Bayesian proportional odds and logistic models, where odds ratios (OR)<1.0 indicate a favorable outcome for CP. Fourteen study sites across the Netherlands and Catalonia in Spain participated in the trial. The two studies included outpatients aged [≥]50 years and diagnosed with COVID-19 and symptomatic for [≤]7days. The intervention consisted of one unit (200-300mL) of CP with a predefined minimum level of antibodies. The two primary endpoints were (a) a 5-point disease severity scale (fully recovered by day 7 or not, hospital or ICU admission and death) and (b) a composite of hospitalization or death. ResultsOf 797 patients included, 390 received CP and 392 placebo. At baseline, they had a median age of 58 years, 1 comorbidity, symptoms for 5 days and 93% tested negative for SARS-CoV-2 S-protein IgG antibodies. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The OR of CP for an improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311). The OR for hospitalization or death was 0.919 (CI 0.592-1.416). The effect of CP on hospital admission or death was largest in patients with [≤]5 days of symptoms (OR 0.658, 95% CI 0.394-1.085). CP did not decrease the time to full symptom resolution (p=0.62). ConclusionTreatment with CP of outpatients in the first 7 days of symptoms did not improve the outcome of COVID-19. The possible beneficial effect in patients with [≤]5 days of symptoms requires further study. RegistrationNCT04621123 and NCT04589949 on https://www.clinicaltrials.gov Funding sourceZONMW, the Netherlands, grant number 10430062010001. SUPPORT-E, grant number 101015756 YoMeCorono, www.tomecorono.com The Fight AIDS and Infectious Diseases Foundation with funding from the pharmaceutical company Grifols S.A

Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

Limited immune responses after three months of BNT162b2 vaccine in SARS-CoV-2 uninfected elders living in long-term care facilities

BackgroundSARS-CoV-2 vaccination is the most effective strategy to protect elders living in long-term care facilities (LTCF) against severe COVID-19, but primary vaccine responses are less effective in older adults. Here, we characterized the humoral responses following 3 months after mRNA/BNT162b2 vaccine in institutionalized elders. MethodsPlasma levels of specific SARS-CoV-2 total IgG, IgM and IgA antibodies were measured before and 3 months after vaccination in elders living in LTCF. Neutralization capacity was assessed in a pseudovirus neutralization assay against WH1 (original) and B.1.617.2/Delta variants. A group of younger adults was used as reference group. ResultsThree months after vaccination, uninfected-elders presented reduced specific SARS-CoV-2 IgG levels and significantly lower neutralization capacity against the WH1 and Delta virus compared to vaccinated uninfected younger individuals. In contrast, COVID-19 recovered elders showed significantly higher specific SARS-CoV-2 IgG levels after vaccination than younger counterparts, while showing similar neutralization activity against WH1 virus and increased neutralization capacity against Delta variant. Despite previously infected elders elicit potent cross-reactive immune responses similarly to younger individuals, higher quantities of specific SARS-CoV-2 IgG antibodies are required to reach the same neutralization levels. ConclusionsWhile hybrid immunity seems to be active in previously infected elders after three months from mRNA/BNT162b2 vaccination, humoral immune responses are diminished in COVID-19 uninfected vaccinated residents living in LTCF. These results suggests that a vaccine booster dose should be prioritized for this particularly vulnerable population. Word summaryWhile previously infected and vaccinated elders living in LTCF had comparable neutralizing antibody levels to younger individuals, vaccinated uninfected-residents showed limited neutralization capacity against both original and delta variants. Hybrid immunity seems to be active in elders and can be relevant to design vaccine boosting campaigns.

Preclinical efficacy, safety, and immunogenicity of PHH-1V, a second-generation COVID-19 vaccine candidate based on a novel recombinant RBD fusion heterodimer of SARS-CoV-2.

The current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease and decrease in mortality rates. However, new variants of concern (VoCs) are continuously evolving, making the development of new accessible COVID-19 vaccines essential in order to mitigate the pandemic. Here we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine candidate (PHH-1V) consisting of an RBD fusion heterodimer containing the B.1.351 and B.1.1.7 SARS-CoV-2 VoCs and formulated with the SQBA adjuvant, an oil-in-water emulsion produced by HIPRA. BALB/c and K18-hACE2 mice were immunized with different recombinant RBD fusion heterodimer doses, following a two-dose prime-and-boost schedule. Vaccination induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralising activity against various VoCs with a good tolerability profile. Significantly, a 10-{micro}g or 20-{micro}g RBD fusion heterodimer/dose vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice. These findings demonstrate the feasibility of this recombinant vaccine strategy.

Antibody kinetics to SARS-CoV-2 at 13.5 months, by disease severity

BackgroundUnderstanding humoral responses and seroprevalence in SARS-CoV-2 infection is essential for guiding vaccination strategies in both infected and uninfected individuals. MethodsWe determine the kinetics of IgM against the nucleocapsid (N) and IgG against the spike (S) and N proteins of SARS-CoV-2 in a cohort of 860 health professionals (healthy and infected) in northern Barcelona. We model the kinetics of IgG and IgM at nine time points over 13.5 months from infection, using non-linear mixed models by sex and clinical disease severity. ResultsOf the 781 participants who were followed up, 478 (61.2%) became infected with SARS-CoV-2. Significant differences were found for the three antibodies by disease severity and sex. At day 270 after diagnosis, median IgM(N) levels were already below the positivity threshold in patients with asymptomatic and mild-moderate disease, while IgG(N, S) levels remained positive to days 360 and 270, respectively. Kinetic modelling showed a general rise in both IgM(N) and IgG(N) levels up to day 30, followed by a decay whose rate depended on disease severity. IgG(S) levels increased at day 15 and remained relatively constant over time. ConclusionsWe describe kinetic models of IgM(N) and IgG(N, S) SARS-CoV-2 antibodies at 13.5 months from infection and disease spectrum. Our analyses delineate differences in the kinetics of IgM and IgG over a year and differences in the levels of IgM and IgG as early as 15 days from symptoms onset in severe cases. These results can inform public health policies around vaccination criteria. Funded by the regional Ministry of Health of the Generalitat de Catalunya (Call COVID19-PoC SLT16_04; NCT04885478)

Clinical course impacts early kinetics and long-term magnitude and amplitude of SARS-CoV-2 neutralizing antibodies beyond one year after infection

BackgroundUnderstanding the determinants of long-term immune responses to SARS-CoV-2 and the concurrent impact of vaccination and emerging variants of concern will guide optimal strategies to achieve global protection against the COVID-19 pandemic. MethodsA prospective cohort of 332 COVID-19 patients was followed beyond one year. Plasma neutralizing activity was evaluated using HIV-based reporter pseudoviruses expressing different SARS-CoV-2 spikes and was longitudinally analyzed using mixed-effects models. FindingsLong-term neutralizing activity was stable beyond one year after infection in mild/asymptomatic and hospitalized participants. However, longitudinal models suggest that hospitalized individuals generate both short- and long-lived memory B cells, while outpatient responses were dominated by long-lived B cells. In both groups, vaccination boosted responses to natural infection, although viral variants, mainly B.1.351, reduced the efficacy of neutralization. Importantly, despite showing higher neutralization titers, hospitalized patients showed lower cross-neutralization of B.1.351 variant compared to outpatients. Multivariate analysis identified severity of primary infection as the factor that independently determines both the magnitude and the inferior cross-neutralization activity of long-term neutralizing responses. ConclusionsNeutralizing response induced by SARS-CoV-2 is heterogeneous in magnitude but stable beyond one year after infection. Vaccination boosts these long-lasting natural neutralizing responses, counteracting the significant resistance to neutralization of new viral variants. Severity of primary infection determines higher magnitude but poorer quality of long-term neutralizing responses.

SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant

SARS-CoV-2 variants display enhanced transmissibility and/or immune evasion and can be generated in humans or animals, like minks, thus generating new reservoirs. The continuous surveillance of animal susceptibility to new variants is necessary to predict pandemic evolution. In this study we demonstrate that, compared to the B.1 SARS-CoV-2 variant, K18-hACE2 transgenic mice challenged with the B.1.351 variant displayed a faster progression of infection. Furthermore, we also report that B.1.351 can establish infection in wildtype mice, while B.1 cannot. B.1.351-challenged wildtype mice showed a milder infection than transgenic mice, confirmed by detectable viral loads in oropharyngeal swabs and tissues, lung pathology, immunohistochemistry and serology. In silico models supported these findings by demonstrating that the Spike mutations in B.1.351 resulted in increased affinity for both human and murine ACE2 receptors. Overall, this study highlights the plasticity of SARS-CoV-2 animal susceptibility landscape, which may contribute to viral persistence and expansion.

Anti-SARS-CoV-2 hyperimmune immunoglobulin provides potent and robust neutralization capacity and antibody-dependent cellular cytotoxicity and phagocytosis induction through N and S proteins

BackgroundAlthough progressive COVID-19 vaccinations provide a significant reduction of infection rate in the short-to mid-term, effective COVID-19 treatments will continue to be an urgent need. MethodsWe have functionally characterized the anti-SARS-CoV-2 hyperimmune immunoglobulin (hIG) prepared from human COVID-19 convalescent plasma. SARS-CoV-2 virus neutralization was evaluated by four different methodologies (plaque reduction, virus induced cytotoxicity, TCID50 reduction and immunofluorimetry-based methodology) performed at four different laboratories and using four geographically different SARS-CoV-2 isolates (one each from USA and Italy; two from Spain). Two of the isolates contained the D614G mutation. Neutralization capacity against the original Wuhan SARS-CoV-2 straom and variants (D614G mutant, B.1.1.7, P.1 and B.1.351 variants) was evaluated using a pseudovirus platform expressing the corresponding spike (S) protein. The capacity to induce antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) was also evaluated. ResultsAll the SARS-CoV-2 isolates tested were effectively neutralized by hIG solutions. This was confirmed by all four methodologies showing potent neutralization capacity. Wild-type SARS-CoV-2 and variants were effectively neutralized as demonstrated using the pseudovirus platform. The hIG solutions had the capacity to induce ADCC and ADCP against SARS-CoV-2 N and S proteins but not the E protein. Under our experimental conditions, very low concentrations (25-100 {micro}g IgG/mL) were required to induce both effects. Besides the S protein, we observed a clear and potent effect triggered by antibodies in the hIG solutions against the SARS-CoV-2 N protein. ConclusionsThese results show that, beyond neutralization, other IgG Fc-dependent pathways may play a role in the protection from and/or resolution of SARS-CoV-2 infection when using hIG COVID-19 products. This could be especially relevant for the treatment of more neutralization resistant SARS-CoV-2 variants of concern.

Plitidepsin has a positive therapeutic index in adult patients with COVID-19 requiring hospitalization.

Plitidepsin is a marine-derived cyclic-peptide that inhibits SARS-CoV-2 replication at low nanomolar concentrations by the targeting of host protein eEF1A (eukaryotic translation-elongation-factor-1A). We evaluated a model of intervention with plitidepsin in hospitalized COVID-19 adult patients where three doses were assessed (1.5, 2 and 2.5 mg/day for 3 days, as a 90-minute intravenous infusion) in 45 patients (15 per dose-cohort). Treatment was well tolerated, with only two Grade 3 treatment-related adverse events observed (hypersensitivity and diarrhea). The discharge rates by Days 8 and 15 were 56.8% and 81.8%, respectively, with data sustaining dose-effect. A mean 4.2 log10 viral load reduction was attained by Day 15. Improvement in inflammation markers was also noted in a seemingly dose-dependent manner. These results suggest that plitidepsin impacts the outcome of patients with COVID-19. One-Sentence SummaryPlitidepsin, an inhibitor of SARS-Cov-2 in vitro, is safe and positively influences the outcome of patients hospitalized with COVID-19.

Siglec-1 on dendritic cells mediates SARS-CoV-2 trans-infection of target cells while on macrophages triggers proinflammatory responses

COVID-19 pandemic is not yet under control by vaccination, and effective antivirals are critical for preparedness. Here we report that macrophages and dendritic cells, key antigen presenting myeloid cells (APCs), are largely resistant to SARS-CoV-2 infection. APCs effectively captured viruses within cellular compartments that lead to antigen degradation. Macrophages sense SARS-CoV-2 and released higher levels of cytokines, including those related to cytokine storm in severe COVID-19. The sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) present on APCs, which interacts with sialylated gangliosides on membranes of retroviruses or filoviruses, also binds SARS-CoV-2 via GM1. Blockage of Siglec-1 receptors by monoclonal antibodies reduces SARS-CoV-2 uptake and transfer to susceptible target cells. APCs expressing Siglec-1 and carrying SARS-CoV-2 are found in pulmonary tissues of non-human primates. Single cell analysis reveals the in vivo induction of cytokines in those macrophages. Targeting Siglec-1 could offer cross-protection against SARS-CoV-2 and other enveloped viruses that exploit APCs for viral dissemination, including those yet to come in future outbreaks.

Highly functional Cellular Immunity in SARS-CoV-2 Non-Seroconvertors is associated with immune protection

The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naive and low effector CD8+ T cells in NSC. Moreover, polyfunctional Nucleocapsid (NP)-specific CD8+ T-cell responses, as well as reduced levels of T-cell activation monitored by PD-1 and activation-induced markers, were distinctive immunological traits in NSC. Longitudinal data support the stability of the NSC phenotype over three months. Our results implicate highly functional SARS-CoV-2 Spike and NP T-cell responses with low immune activation in protection from disease severity in the absence of seroconversion. SUMMARYTo understand SARS-CoV-2 specific T-cell immunity in the absence of seroconversion, we characterized immunological features of Non-Seroconvertors recovered from infection. Highly functional specific T-cell responses and low immune activation were determinants of immune protection from severe disease.

Previous SARS-CoV-2 infection increases B.1.1.7 cross-neutralization by vaccinated individuals.

To assess the potential impact of predominant circulating SARS-CoV-2 variants on neutralizing activity of infected and/or vaccinated individuals, we analyzed neutralization of pseudoviruses expressing the spike of the original Wuhan strain, the D614G and B.1.1.7 variants. Our data show that parameters of natural infection (time from infection and infecting variant) determined cross-neutralization. Importantly, upon vaccination, previously infected individuals developed equivalent B.1.1.7 and Wuhan neutralizing responses. In contrast, uninfected vaccinees showed reduced neutralization against B.1.1.7. FundingThis study was funded by Grifols, the Departament de Salut of the Generalitat de Catalunya, the Spanish Health Institute Carlos III, CERCA Programme/Generalitat de Catalunya, and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos.

Performance characteristics of five antigen-detecting rapid diagnostic test (Ag-RDT) for SARS-CoV-2 asymptomatic infection: a head-to-head benchmark comparison

Background Mass testing for early identification and isolation of infectious COVID-19 individuals, irrespective of concurrent symptoms, is an efficacious strategy to reduce disease transmission. Antigen-detecting rapid diagnostic tests (Ag-RDT) appear as a potentially suitable tool for mass testing on account of their ease-of-use, fast turnaround time, and low cost. However, benchmark comparisons are scarce, particularly in the context of unexposed asymptomatic individuals. Methods We used nasopharyngeal specimens from unexposed asymptomatic individuals to assess five Ag-RDTs: PanBio™ COVID-19 Ag Rapid test (Abbott), CLINITEST® Rapid COVID-19 Antigen Test (Siemens), SARS-CoV-2 Rapid Antigen Test (Roche Diagnostics), SARS-CoV-2 Antigen Rapid Test Kit (Lepu Medical), and COVID-19 Coronavirus Rapid Antigen Test Cassette (Surescreen). Samples were collected between December 2020-January 2021 during the third wave of the epidemic in Spain. Findings The analysis included 101 specimens with confirmed positive PCR results and 185 with negative PCR. For the overall sample, the performance parameters of Ag-RDTs were as follows: Abbott assay, sensitivity 38·6% (95% CI 29·1–48·8) and specificity 99·5% (97–100%); Siemens, sensitivity 51·5% (41·3–61·6) and specificity 98·4% (95·3–99·6); Roche, sensitivity 43·6% (33·7–53·8) and specificity 96·2% (92·4–98·5); Lepu, sensitivity 45·5% (35·6–55·8) and specificity 89·2% (83·8–93·3%); Surescreen, sensitivity 28·8% (20·2–38·6) and specificity 97·8% (94·5–99·4%). For specimens with cycle threshold (Ct) <30 in RT-qPCR, all Ag-RDT achieved a sensitivity of at least 70%, with Siemens, Roche, and Lepu assays showing sensitivities higher than 80%. In models according to population prevalence, all Ag-RDTs will have a NPV >99% and a PPV<50% at 1% prevalence. Interpretation Two commercial, widely available assays can be used for SARS-CoV-2 antigen testing to achieve sensitivity in specimens with a Ct<30 and specificity of at least 80% and 96%, respectively. Estimated negative and positive predictive values suggests the suitability of Ag-RDTs for mass screenings of SARS-CoV-2 infection in the general population. Funding Blueberry diagnostics, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, and #YoMeCorono.org crowdfunding campaign. Evidence before this study In December 2020, we searched on PubMed for articles containing the terms “antigen”, “test” (or Ag-RDT), and “SARS-CoV-2” or “COVID-19” either in the title or the abstract. Our search yielded 79 entries corresponding to articles written in English. Of them, 33 were articles presenting the diagnostic performance of qualitative lateral-flow antigen-detecting rapid diagnostic tests (Ag-RDT). Four of these articles reported the results of head-to-head comparisons of various Ag-RDTs; in all cases, the number of tests was lower than the recommended for retrospective assessments of diagnostic performance (i.e., minimum of 100 PCR positive and 100 PCR negative). Furthermore, all head-to-head comparisons found in the literature included specimens obtained among individuals with varying disease status (none of which asymptomatic), thus limiting the adequacy of the estimates for an asymptomatic screening strategy. Added value of this study We compared for the first time head-to-head five Ag-RDT using a powered set of fresh respiratory specimens PCR-confirmed positive or negative, collected from unexposed asymptomatic individuals during screening campaigns for early detection of SARS-CoV-2 infection. The sample size was large enough to draw robust conclusions. Our analysis identified four Ag-RDTs (i.e., assays marketed by Abbott, Siemens, Roche, and Surescreen) with specificity higher than 96%. Despite the low sensitivity for the overall sample (range 29% to 51%), the corresponding values for the subset of samples with Ct <30 were higher than 80% for Siemens, Roche, and Lepu assays. The estimated NPV for a screening performed in an area with 1% prevalence would be >99% for all tests, while the PPV would be <50%. Implications of all the available evidence Current data on the diagnostic performance of Ag-RDTs is heterogeneous and precludes benchmark assessments. Furthermore, the screening of asymptomatic populations is currently not considered among the intended uses of Ag-RDT, mostly because of lack of evidence on test performance in samples from unexposed asymptomatic individuals. Our findings add to the current evidence in two ways: first, we provide benchmarking data on Ag-RDTs, assessed head-to-head in a single set of respiratory specimens; second, we provide data on the diagnostic performance of Ag-RDTs in unexposed asymptomatic individuals. Our findings support the idea that Ag-RDTs can be used for mass screening in low prevalence settings and accurately rule out a highly infectious case in such setting.

Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in hamsters

Reinfections with SARS-CoV-2 have already been documented in humans, although its real incidence is currently unknown. Besides having great impact on public health, this phenomenon raises the question if immunity generated by a single infection is sufficient to provide sterilizing/protective immunity to a subsequent SARS-CoV-2 re-exposure. The Golden Syrian hamster is a manageable animal model to explore immunological mechanisms able to counteract COVID-19, as it recapitulates pathological aspects of mild to moderately affected patients. Here, we report that SARS-CoV-2-inoculated hamsters resolve infection in the upper and lower respiratory tracts within seven days upon inoculation with the Cat01 (G614) SARS-CoV-2 isolate. Three weeks after primary challenge, and despite high titers of neutralizing antibodies, half of the animals were susceptible to reinfection by both identical (Cat01, G614) and variant (WA/1, D614) SARS-CoV-2 isolates. However, upon re-inoculation, only nasal tissues were transiently infected with much lower viral replication than those observed after the first inoculation. These data indicate that a primary SARS-CoV-2 infection is not sufficient to elicit a sterilizing immunity in hamster models but protects against lung disease.

Stable neutralizing antibody levels six months after mild and severe COVID-19 episode

Understanding mid-term kinetics of immunity to SARS-CoV-2 is the cornerstone for public health control of the pandemic and vaccine development. However, current evidence is rather based on limited measurements, thus losing sight of the temporal pattern of these changes1-6. In this longitudinal analysis, conducted on a prospective cohort of COVID-19 patients followed up to 242 days, we found that individuals with mild or asymptomatic infection experienced an insignificant decay in neutralizing activity that persisted six months after symptom onset or diagnosis. Hospitalized individuals showed higher neutralizing titers, which decreased following a two-phase pattern, with an initial rapid decline that significantly slowed after day 80. Despite this initial decay, neutralizing activity at six months remained higher among hospitalized individuals. The slow decline in neutralizing activity at mid-term contrasted with the steep slope of antibody titers change, reinforcing the hypothesis that the quality of immune response evolves over the post-convalescent stage4,5.