Plitidepsin Is a Broad-Spectrum Antiviral Shaping the Cell Proteostatic Balance

Background: Different viruses employ similar pathways for replication, revealing key intracellular hotspots to target with host-directed therapies and achieve a broad-spectrum antiviral activity. Plitidepsin is a clinically approved antitumoral agent that blocks the elongation factor eEF1A required for protein translation. This drug counteracts SARS-CoV-2 replication and shows a favorable safety profile in COVID-19 patients. Yet, the precise antiviral mechanism of action of plitidepsin remains unknown. Method(s): Here we used a deep quantitative proteomic analysis to measure the impact of plitidepsin on the proteome of SARS-CoV-2-infected Vero E6 cells. This was complemented with transmission electron microscopy assays, which unraveled the subcellular and morphological changes associated to plitidepsin treatment. In addition, we performed functional in vitro assays to dissect the antiviral activity of plitidepsin against SARS-CoV-2 and other viruses. Result(s): We found that this drug inhibited the synthesis of all SARS-CoV-2 proteins in a dose-dependent manner. These included the R1AB polyproteins, which facilitate the synthesis of non-structural proteins involved in the formation of double membrane vesicles (DMV) required for viral replication. Plitidepsin reduced DMV formation and the morphogenesis of new viruses, having a greater impact on viral than on host proteins. Less than 14% of the cellular proteome was significantly affected by plitidepsin, inducing the up-regulation of key molecules associated with protein biosynthesis, such as the translation initiation factors eIF4A2 and eIF2S3. Therefore, plitidepsin induced a compensatory state that rescued protein translation. This proteostatic response explains how cells preserve the cellular proteome after treatment with a translation inhibitor such as plitidepsin. In addition, it suggests that plitidepsin could inhibit other RNA-dependent and non-integrated DNA viruses, as we confirmed in vitro using Zika virus, Hepatitis C virus replicon and Herpes simplex virus. However, the compensatory proteostasis induced by plitidespin also explains why this drug failed to inhibit the replication of integrated DNA proviruses such as HIV-1. Conclusion(s): Unraveling the mechanism of action of host-directed therapies like plitidepsin is imperative to define the indications and antiviral profile of these compounds. This knowledge will be key to develop broad-spectrum treatments and have them ready to deploy when future pandemic viruses break through.

HIGH-RESOLUTION MAPPING OF T-CELL HYBRID IMMUNITY TO THE ENTIRE SARS-CoV-2 PROTEOME

Background: To understand T-cell responses to SARS-CoV-2, it is essential to define the contribution of infection versus immunization to virus-specific hybrid immunity. Here, we characterized the breadth and magnitude of T-cell responses to the entire SARS-CoV2 proteome over a 2-year follow-up period in infected and vaccinated (CoV2+Vac+) and vaccinated and infected (Vac+CoV2+) individuals. Method(s): We selected samples from 38 (19 CoV2+ and 19 CoV2-, time1, T1) ProHEpiC-19 cohort participants, a prospective, longitudinal study starting in March 2020 involving 7,776 healthcare workers in Spain. Longitudinal samples were available from 10 of them after a 3-dose mRNA vaccination, including 5 CoV2+Vac+ and 5 Vac+CoV2+, at 824.5 and 250.5 days from symptoms onset (DfSO, time 2, T2). We measured the breadth and magnitude of IFN-y T-cell responses by ELISpot assay in cryopreserved PBMCs, using a 15-mer overlapping peptide (OLP) library of 2,790 SARS-CoV-2 peptides in 100 pools. Result(s): We identified immunodominant T-cell responses in S1, S2, nsp3, Env, NC, and M proteins across the SARS-CoV2 proteome. We observed an increased breadth of T-cell responses (responding pools over the entire region) to S1 (44 - 30%) and S2 (31 - 40%) in CoV2+Vac+ and Vac+CoV2+, respectively. In addition, CoV2+Vac+ had an exclusive and sustained response to M. We found significantly stronger responses in CoV2+Vac+ (P=0.0313). Particularly the total magnitude was greater in CoV2+Vac+ vs. Vac+CoV2+ in S1 (4476.88 vs. 1498.53), Env (457.34 vs. 250.50), and M (455.13 vs. 0.00) but not in S2 and nsp3. The total number of peptides for deconvolution was higher in CoV2+Vac+ (32 peptides) than in Vac+CoV2+ (3 peptides) during the follow-up. Seventy-five percent of the responses targeted S, and 25% M, ORF1a, and Env. Conclusion(s): These results profile immunodominant T-cell responses in S1, S2, nsp3, Env, NC, and M proteins across the entire SARS-CoV2 proteome. The data delineate differences in the number of T-cell responses primed hybrid immunity by infection previous to vaccination (CoV2+Vac+), being broader and of higher magnitude and underlining an exclusive T-cell response to the M region. Overall, these findings identify differences in long-term T-cell hybrid immunity primed by infection or vaccination, which may have implications in protection from re-infection and vaccine design.

Cytokine Profile in Different Post-Covid-19 Condition Phenotypes

Background: At least 10% of SARS-CoV-2 infected patients suffer from persistent symptoms for >12 weeks, known as post-COVID-19 condition (PCC) or Long Covid. Reported symptomatology is diverse with >200 physical and neurological debilitating symptoms. Here, we analyzed pro-inflammatory cytokine levels as a potential mechanism underlying persistent symptomatology. Method(s): Clinical data and samples used belong to the KING cohort extension, which includes clinically well characterized PCC (N=358, 59 persistent symptoms evaluated), COVID-19 recovered and uninfected subjects. We used Gower distances to calculate symptom's similarity between PCC and Ward's hierarchical clustering method to identify different symptom patterns among PCC patients. Cytokine levels of randomly selected PCC, recovered and uninfected subjects (N=193) were measured on plasma samples collected >6 months after acute infection using the 30-Plex Panel for Luminex. Mann- Whitney t-test was used to compare PCC vs recovered groups and Kruskal-Wallis t-test for >2 groups comparisons (PCC vs recovered vs Uninfected and within PCC clusters). FDR correction was applied for statistical significance (p-adj). Result(s): Hierarchical clustering identified 5 different PCC clusters according to their symptomatology, where PCC3 and PCC5 clusters showed higher prevalence of women ( >80%) and more persistent symptoms, while acute COVID-19 was mild in >80% of the patients. We selected 91 PCC (belonging to each cluster), 57 recovered and 45 uninfected subjects for cytokine profiling (Table 1). 13 soluble markers were significantly elevated (IL-1beta, Eotaxin, MIP-1beta, MCP-1, IL-15, IL-5, HGF, IFN-alpha, IL-1RA, IL-7, MIG, IL-4 and IL-8) in PCC and recovered groups compared to uninfected subjects (all p-adj< 0.04). In addition, PCC subjects tended towards higher levels of IL-1RA compared to recovered group (padj= 0.071). Within PCC clusters, FGF-basic and RANTES were elevated while IL-2 and MIG were decreased in PCC3 and PCC5 compared to the other PCC clusters (all p-adj< 0.04). TNF-alpha, IP-10, G-CSF and MIP-1alpha were decreased in PCC3 and PCC5 not reaching statistical significance (all p-adj=0.07). Conclusion(s): Some cytokines remained altered in all SARS-CoV-2 infected subjects independently of persistent symptoms after 6 months from acute infection. Differences between PCC and recovered individuals are limited after correction. Importantly, PCC cytokine profiles showed differences between clusters, which suggests different PCC subsyndromes with distinct etiology. Subjects Characteristics (Table Presented).

IDENTIFICATION OF CLINICAL FEATURES ASSOCIATED WITH SARS-CoV-2 REINFECTIONS

Background: Over 600 million of COVID-19 cases have been reported. A remarkable fragment of these cases are reinfections, which are mostly explained by the genomic variability of the SARS-CoV-2 variants. However, little is known about other factors fostering these reinfections. Method(s): We recorded clinical and demographic data from subjects (N=3303, March 2020 - March 2022) with at least 2 PCR+ events separated by >=90 days, analyzed by the Microbiology Department, Northern Metropolitan Clinical Laboratory from Germans Trias i Pujol Hospital (Spain). Data collected included: age, sex, comorbidities, adjusted morbidity group (GMA), hospitalization, symptomatology, NAAT (PCR, TMA) tests, antigen tests, serology, and vaccination. Temporal data was encoded using Python, and demographic characterization was performed under R. Result(s): We identified 2344 cases of confirmed reinfections, where the 2 PCR+ events were separated by >=90 days and a negative test was obtained between episodes. 72.2% of reinfected subjects were females with a median age of 45 IQR [28-63] years. Age density analysis showed three peaks at 24, 45, and 85 years, probably mostly composed of young people, who usually are less cautious, healthcare workers, and people living in nursing homes, respectively, being all of them groups prone to be tested. Regarding health status, 86.2% of participants had at least one chronic condition, with 40.5% of patients having chronic conditions in >=4 systems based on GMA assessment. Interestingly, 75.2% of reinfected subjects < 26 years had at least one chronic condition. 121 (4.2%) participants were hospitalized during a COVID-19 episode, highlighting 8.3% (N=10) of them hospitalized during the reinfection (half of them vaccinated before hospitalization), and 5% (N=6) of them during both infections. The severity of the second infection may be caused by a diminished acquired immunity after the first infection. Time between reinfections density analysis provided three peaks at ~200, ~400, and ~600 days, corresponding with time between waves. A decrease of reinfections was observed between 40 and 100 days after vaccination, which would be the period of highest protection against reinfection. Conclusion(s): SARS-CoV-2 reinfections are more prevalent among women. Importantly, people with an undermined health status, independently of age, are more sensitive to reinfections, but in most of the cases no hospitalization was required. Finally, vaccination seems to have a short protective effect on reinfection.

Severity Clusters and Likelihood of Recovery from Post-Covid-19 Condition

Background: The Post-COVID-19 Condition (PCC) is a novel, long-lasting, poorly understood and highly disabling post-viral syndrome, which poses enormous healthcare, economic and socio-political challenges. Lack of validated biomarkers forces clinical management to be based on clinical definitions, which are imprecise. In the clinic, symptoms tend to present in clusters, which have yet to be properly defined. Also, it is unclear how often PCC resolves, and which factors influence PCC resolution. Method(s): To delineate PCC presentation clusters and explore factors related with PCC resolution, we performed a 2-year prospective cohort study in individuals who recovered from acute COVID-19 regardless of its acute and post-acute severity. All subjects were systematically followed in the outpatient post-COVID-19 clinic of a tertiary care hospital in Spain. PCC was defined as per the WHO 2021 definition. Persistent symptoms were those present >3 months after acute COVID-19, and lasting for >2 consecutive months. PCC recovery was the absence of PCC symptoms during >3 consecutive months. Symptom clusters were identified using Gower's distance matrices, dendograms, PCA and Silhouette techniques. Factors associated with PCC recovery were identified using a directed acyclic graph approach. Result(s): 548 subjects were included;341 (62%) had PCC. The latter were mostly females (69.8%) with mean age of 47.9 (SD 12.2) years. Only 38.1% required hospitalization and 9% required high-flow oxygen during acute COVID-19. Their most frequent comorbidities were allergy (31.4%), obesity (24.8%), dyslipidemia (24.0%) and hypertension (19.6%). At least 3 symptom clusters with additive symptoms were identified: considering only symptoms present in >35% of subjects, Cluster A was enriched in fatigue and dyspnea;Cluster B had Cluster A symptoms plus headache, arthralgia and neurocognitive complains;Cluster C had Cluster B symptoms plus chest pain and tachycardia. PCC recovery was achieved by 26 (7.6%) individuals over 2 years. Male sex (RR 3.01;CI 1.4-6.3), ICU admission (RR 7.85;CI 2.6-23.2), metabolic comorbidity (RR 2.07;CI 1.1-4.1), and mild acute COVID-19 (RR 2.70;CI 1.1-4.6) increased the likelihood of PCC recovery. Conversely, subjects with muscle pain, impaired attention, dyspnea, and tachycardia were less likely to recover from PCC (RR 0.26;CI 0.13-0.52). Conclusion(s): At least 3 severity clusters can be identified in the PCC. Over the first 2 years, only a minority of subjects fully recover from PCC.

Cetylpyridinium Chloride Mouthwash to Reduce Shedding of Infectious SARS-CoV-2: A Double-Blind Randomized Clinical Trial.

The airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via respiratory fluids and droplets suggests that mouthwashes containing substances with virucidal activity can help reduce viral spread. We conducted a multicenter, double-blind, placebo-controlled, randomized trial to assess the virucidal activity of cetylpyridinium chloride (CPC) mouthwashes. Outpatients who tested positive for SARS-CoV-2 infection with or without symptoms were randomized to perform washes and gargles for 1 min with 15 mL of either colored distilled water or 0.07% CPC (Vitis CPC Protect) mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load and the nucleocapsid protein levels, both in saliva at 1 and 3 h after the intervention. In total, 118 patients were enrolled and randomized (mean [SD], age 46 [14] y). Thirteen of 118 participants (11%) did not complete follow-up or had insufficient sample volume for testing and were excluded from the analysis. The assessment of the viral load showed no significant differences between groups at any of the investigated points. However, the levels of SARS-CoV-2 nucleocapsid protein of lysed viruses were significantly higher in the CPC group compared with the control group at 1 h (adjusted difference 269.3 pg/mL; 95% confidence interval [CI], 97.1-441.5) and at 3 h postintervention (561.1 pg/mL; 95% CI, 380.0-742.2). In nonhospitalized patients with asymptomatic or mild symptomatic SARS-CoV-2 infection, a 0.07% CPC mouthwash, compared to placebo, was associated with a significant increase of nucleocapsid protein levels in saliva, indicating enhanced disruption of viral particles.

SARS-CoV-2 NEUTRALIZING RESPONSE beyond 1 YEAR after INFECTION

Background: Understanding 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. Methods: A 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. Results: Long-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 responses of non-hospitalized were dominated by long-lived B cells. In both groups, vaccination boosted responses to natural infection. In unvaccinated participants, viral variants, mainly beta, reduced the efficacy of long-term (>300 days from infection) neutralization. Importantly, despite showing higher neutralization titers, hospitalized patients showed lower cross-neutralization of beta variant compared to non-hospitalized. 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. Conclusion: Neutralizing 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 and should help counteract the resistance to neutralization of variants of concern such as the beta variant. Severity of primary infection determines higher magnitude but poorer quality of long-term neutralizing responses.

UNRAVELING the ANTIVIRAL ACTIVITY of PLITIDEPSIN by ULTRASTRUCTURAL ANALYSIS

Background: The use of compounds against highly conserved cellular host factors required to complete the replication cycle of distinct viruses such as SARS-CoV-2 offers a common solution to diverse viral threats. This approach is especially relevant for pan-antiviral effects given that viruses converge at intracellular steps such as viral genome replication and protein production. Currently, there are only a limited number of approved drugs involved in targeting intracellular host factors. One of these compounds is plitidiepsin, which has shown a potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Plitidepsin inhibits nucleocapsid viral protein expression and viral induced cytopathic effect in vitro. In addition, it also reduces genomic and subgenomic RNA expression. However, how plitidepsin exerts its antiviral activity remains unknown. Methods: Current models of SARS-CoV-2 replication propose that upon viral fusion, non-structural viral proteins form a replication-transcription complex that associates to compartments with a double membrane vesicle (DMV) morphology that shelters the viral genome replication. Here we have used an electron microcopy analysis to explore the antiviral effect of plitidepsin and its impact on SARS-CoV-2 replication and DMV formation on target Vero E6 cells. Results: This ultrastructural analysis allowed to recapitulate the SARS-CoV-2 infectious life cycle, where evident viral DMV formation was observed as well as viral budding events along with cell-associated viruses. However, in cells treated with plitidepsin at different non-toxic concentrations (0.2 and 0.05 μ M) there was a lack of viral DMV formation and a complete absence of viral particles. Complementary SARS-CoV-2 nucleocapsid and dsRNA immunogold labelling unambiguously confirmed the lack of viral replication in plitidepsin-treated cells. Overall, these data indicate that plitidepsin treatment abrogated the formation of DMVs, and the detection of nucleocapsid or dsRNA viral products. Conclusion: Electron microscopy ultrastructural analysis coupled to immunogold labelling of SARS-CoV-2 products offer a unique approach to understand how antivirals work. This knowledge is key to identify the mechanism of action of promising compounds interfering with host factors whose implication in strategic biological processes can be applied as pan-antiviral strategies.

CETYLPYRIDINIUM CHLORIDE MOUTHWASHES to REDUCE the SHEDDING of VIABLE SARS-CoV-2

Background: SARS-CoV-2 is spread via airborne transmission. Mouthwashes containing virucidal compounds can help reduce viral spread. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by disrupting viral membranes both in vitro and in vivo. Methods: We tested the capacity of CPC-containing mouthwashes to inhibit SARS-CoV-2 entry into target cells by using a luciferase-based assay with a reporter lentivirus pseudotyped with the SARS-CoV-2 spike protein. The replication-competent SARS-CoV-2 B.1.1.7 and D614G variants were also assayed. Viral envelope disruption by CPC's virucidal effect was measured by dynamic light-scattering analyses (DSL). We confirmed these results by modifying an ELISA that detects the SARS-CoV-2 nucleocapsid (NC), which was used in the absence of its own lysis buffer. The effect of CPC in the saliva of individuals with CoVID-19 was assessed in a double-blind, placebo-controlled, randomized clinical trial. SARS-CoV-2 positive patients were randomized to gargle either water or 0.07% CPC mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load by RT-PCR and the NC protein levels by ELISA, both in saliva at 1h and 3h post-intervention. Results: CPC-containing mouthwashes inhibited SARS-CoV-2 viral fusion in vitro in a dose-dependent manner and decreased more than a 1000 times the viral TCID50 in target cells, regardless of the variant tested. The ELISA and the DSL analyses pointed to the effective disruption of the integrity of viral membranes after treatment with CPC. The clinical study performed with 105 patients showed no significant differences in viral RNA load at 1h and 3h post-treatment in saliva between placebo and CPC-treated groups. However, the levels of SARS-CoV-2 NC protein of lysed viruses were significantly higher in the CPC group at 1h and 3h post-intervention. Conclusion: CPC decreased more than a 1000 times the infectivity of SARS-CoV-2 in vitro and was effective against different SARS-CoV-2 variants. In CoVID-19 patients, the use of a 0.07% CPC mouthwash correlated with a statistically significant increase of NC protein levels in saliva, indicating enhanced disruption of viral particles. CPC-containing mouth rinses can represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

CONVALESCENT PLASMA for OUTPATIENTS with EARLY COVID-19: A RANDOMIZED TRIAL

Background: Trials on convalescent plasma (CP) for hospitalized patients with COVID-19 have not demonstrated clear benefits. However, data on outpatients with early symptoms are limited. We studied if treatment with CP reduces disease burden of outpatients treated in the first 7 days of symptoms. Methods: Two double blind randomized trials (NCT04621123, NCT04589949) were merged. Pooling of data started 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. A DSMB monitored the accumulating data for efficacy. Patients aged ≥50, diagnosed with COVID-19 and symptomatic for ≤7days were eligible for participation. The intervention was 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. Secondary endpoints were efficacy in patients with ≤5days of symptoms and time to full symptom resolution. Results: Of 797 patients included, 390 received CP and 392 placebo. They had a median age of 58, 1 comorbidity, symptoms for 5 days and 93% tested negative for SARS-CoV-2 S-protein IgG antibodies. 74 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 ≤5days of symptoms (OR 0.658, 95% CI 0.394-1.085). CP did not decrease the time to full symptom resolution (p=0.62). Conclusion: Treatment with CP of outpatients in the first 7 days of symptoms did not improve outcome of COVID-19. The possible beneficial effect in patients with ≤5days of symptoms requires further study.

Mouthwashes with CPC Reduce the Infectivity of SARS-CoV-2 Variants In Vitro.

Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity in these oral rinses and their exact mechanism of action remain unknown. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium compound in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting the viral fusion step with target cells after disrupting the integrity of the viral envelope. We also found that CPC-containing mouth rinses decreased more than a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. This activity was effective for different SARS-CoV-2 variants, including the B.1.1.7 or Alpha variant originally identified in United Kingdom, and in the presence of sterilized saliva. CPC-containing mouth rinses could therefore represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

Stable neutralizing-antibody levels 6 months after mild and severe COVID-19 episode

Background: One of the fundamental pillars of SARS-CoV-2 pandemic control and vaccine development is understanding mid-and long-term immunity. Early humoral response has been extensively studied, however data on what recovered individuals are still scarce and the most recent studies are based on few time points over time, which limits the comprehension of the longitudinal pattern of the potential changes. In this study we have evaluated the neutralizing activity and IgG antibody titer against SARS-CoV-2 in mild/ asymptomatic and hospitalized COVID-19 individuals, over a 6-month period. Methods: We have evaluated the kinetics of the humoral immune response in 210 individuals infected by SARS-CoV-2 covering the first and second waves of COVID-19 outbreak in Catalonia (Spain). IgG antibody titer was evaluated with an in-house sandwich ELISA against the S2 subunit, the binding domain receptor (RBD) and the nucleoprotein (NP) and the neutralizing activity was evaluated by a neutralization assay with HIV reporter pseudoviruses expressing SARS-CoV-2 S protein. Statistical analyses were carried out using mixed-effects non-linear and linear models. Results: Most study participants developed a neutralizing humoral response against SARS-CoV-2, however the maximum neutralization titer was 10-fold lower in mild/asymptomatic individuals compared to those with a more severe illness. We observed a slow and progressive decay of neutralizing activity in individuals with mild or asymptomatic disease throughout the 6-month period. In hospitalized individuals, half maximal neutralization activity was achieved on day 10 and showed an initial rapid decline that significantly slowed and remained nearly flat after day 80. Despite this, activity at six months remained higher in hospitalized individuals compared to mild symptomatic participants. On the other hand, we observed that IgG antibody titers against S2, RBD and NP had a more marked fall without showing differences in the decay pattern between individuals with different degree of severity of the disease. Conclusion: Our data suggest that the neutralizing activity remains relatively stable for more than 6 months despite the decline in IgG antibodies, suggesting that the quality of immune response evolves and allows maintaining the neutralizing activity despite the decay in antibody titers. Our results provide a more detailed picture of the behavior of the natural humoral immune response over time that complements the current evidence on mid-term immunity.

Early core signature of soluble factors in mild to moderate SARSCOV-2 infection

Background: Understanding the kinetics of early immune responses to SARSCoV-2 infection is critical to identify potentials biomarkers of disease outcome. A myriad of soluble mediators including, pro-inflammatory, immune-suppressors and growth factors, play a relevant role in the disease progression. However, to date, limited data is available about the role of soluble factors and most studies focus only in severe cases with limited follow-up. Here, we studied with high resolution the kinetics of soluble mediators in mild to moderate cases of SARSCoV-2 infection 1-90 days from symptom onset (DfSO). Methods: We selected individuals from the ProHEpiC-19 cohort study that included mainly healthcare workers with a PCR+ and mild or moderate disease within 1-14 DfSO. IgG and IgM levels were determined by ELISA. We selected plasma samples (n=30) in the range of 1-90 DfSO, and performed a Luminex multiplex assay including 45 soluble human factors. Results: We identified a core signature including 19 highly correlated soluble factors at 1-14 DfSO, based on clustering analysis. The core signature contained three sub-clusters: #1 (RANTES, IL13, TGFa, PDGF-AB, PDGF-AA, EGF, MIP1b, CD40L and GROb), #2 (G-CSF, PDL1-B7, Fractalkine, IL8, IFNg, Granzyme B and IL10) and #3 (IL7, IL6, and VEGF). We found major changes in #2 and #3 cluster composition between 1-14 and 30-45 DfSO, due to the loss of PDL1-B7, Fractalkine, IL8, IL7, IL6, and VEGF association. Moreover, by 60-75 DfSO, the soluble factor association in #2 and #3 disappeared from the core signature. In addition, we observed a negative correlation between IgG and IgM levels with IL4 production at 1-14 DfSO (IgG: ρ =-0.82, p=0.012;IgM ρ=-0.83, p=0.011). Similarly, a negative correlation was observed between Igs and Mip3a at 30-45 DfSO (IgG: ρ=-0.78, p=0.023;IgM: ρ =-0.81, p=0.022). Conclusion: We delineated a core signature of soluble factors in mild to moderate SARS-CoV-2 infection, including growth factors, chemokines and pro-inflammatory cytokines. The longitudinal follow-up of this signature revealed significant changes during the 1-90 DfSO. This information can provide new insights for the definition of biomarkers for patient stratification in mild or moderate SARS-Cov-2 infection. Further data is needed to understand the association between IL4 and Mip3a with low Igs levels.

High-resolution mapping of T-cell immunity to the entire sarscov-2 proteome

Background: Since the discovery of SARS-CoV-2, researchers have put major efforts towards the understanding of virus-specific cellular immunity. However, the identification of epitope-and protein specific T-cell responses is limited to bioinformatic approaches, use of total viral proteins or peptide mega pools. To overcome these current limitations, we performed a high-resolution mapping using IFN-y ELISpot and peptide sets covering the entire CoV-2 proteome. Methods: We synthetized a 15-mer peptide library of 2790 peptides (11 amino acid overlap) covering a CoV-2cons proteome sequence based on 1700 sequences. We designed a mega matrix of consecutive and non-consecutive peptide pools with 20 to 35 peptides per pool. We assessed T-cell responses in cryopreserved PBMCs from IgG+ SARS-CoV-2 infected individuals (N=13), who recovered from mild/moderate infection, 90-190 Days from off-set symptoms. Also, we expanded PBMCs in the presence of anti-CD3 and IL-2 during 3 weeks and performed a comparative ELISpot using total and expanded PBMCs. Results: Frequencies of T-cell responses from positive peptide pools revealed 40% of responses targeting S2, 20% against S1, 10% against M, and 6% against nsp3 and NP, respectively. The strongest responses were targeting S2 and S1 (median values of 540 and 240 IFN-y SFC/106, respectively), followed by nsp3, NP and M. We observed a median of 13 deconvoluted reactive peptides across the entire proteome per tested individual. The breadth of responses ranged from 1-8 targeted proteins with a median of 2. In addition, we mapped responses in subproteins 3C-LP, nsp6, nsp10 (Orf1ab), and alternative reading frames. We also identified responses to peptide sequences conserved across pan-coronavirus strains Orf1b (n=2), S (n=1) and M (n=1). Following expansion, we observed a loss of CD4+ T-cells in cultured cells and altered peptide-recognition profiles characterized by a loss of S2 and an increase of nsp3 responses. Conclusion: We characterize protein hierarchy in terms of breadth and magnitude by high-resolution mapping of T-cell responses against the entire CoV-2 proteome. The most frequently targeted and immunogenic regions were S2 and S1. We identify responses to small proteins, alternative reading frames and conserved regions across coronaviruses. This data brings new insight into the complexity of CoV-2 T-cell responses and crucial information for vaccine design.

SARS-CoV-2 non-seroconvertors present t-cell responses with decreased activation

Background: Many immune studies of SARS-CoV-2 (CoV-2) infection have focused on the generation of virus-specific as a means of protection. However, a small group of CoV-2 infected individuals called Non-seroconverters (NSC), do not generate antibodies but experience a mild or moderate disease course. Identifying mechanism of CoV-2 control in NSC may inform the development of novel therapeutics and vaccines approaches. Methods: We identified eleven CoV-2 NSC (3.6%) from the King-cohort study (PI-20-217). NSC were defined by a positive CoV-2 PCR at the time of diagnosis in the absence of IgG, IgA and IgM in serum and plasma measured by two independent ELISA techniques. For comparison, we identify groups of CoV-2 convalescent (n=15) and low-neutralizers (n=15). We measured T-cell responses to the CoV-2 Spike (S) and Nucleocapsid (NP) recombinant proteins in PBMCs by ELISPOT and flow cytometry. We combined T-cell surface and lineage markers together with PD-1, functional (TNF, IFN-y, and IL-2) and activation induced markers (AIM: CD25, CD137 and OX40). Results: We identified CoV-2 specific CD4+ and CD8+ T-cells against the S and the NP in NSC individuals. All NSC responded to S by production of one or more cytokine in either CD4+ or CD8+ T-cells, and 57% responded to NP. Specific-CD8+ T cells against S in NSC were characterized by IFN-y, and TNF production, and we observed higher levels of TNF production as compared to low neutralizers (p=0.02). No differences were found in IFN-y, IL-2 and TNF production in S-specific CD4+ T cells between groups, nor in NP CD8+ or CD4+ T-cell responses. The levels of CD137/OX40 in CD8+ and CD4+ T cells were significantly lower in NSC in response to S (p=0.006, and p=0.012). Also, lower levels of PD-1 were observed in CD8+ T cells in response to NP in NSC (p=0.017). Conclusion: We provide evidence of SARS-CoV2 cellular immunity in NSC individuals despite the absence of humoral neutralizing responses. CD8+ and CD4+ T cells against the S and NP were present in NSC and characterized by TNF production in CD8+ T-cells in responses to S when compared to low neutralizers. Decreased levels of activation markers were observed in NSCs following S and NP stimulation. We propose a protective role of cellular immunity in NSC potentially driven by preexisting cellular responses.

Supression of ACE2 function and antiviral immune response by SARS-CoV-2 infection

Background: SARS-CoV-2 receptor angiontensin-conveting-enzyme 2 (ACE2) is also a protective factor that contributes to reduce inflammation and fibrosis in tissues. An active form of ACE2 can be released from the cell surface by host proteases ADAM17 and TMPRSS2, being the latter also necessary for viral entry. Due to its properties, the administration of soluble recombinant ACE2 has been proposed as a SARS-CoV-2 treatment. Here, we assess the role of ACE2 activity and antiviral immune response at the site of infection in nasopharyngeal swabs of SARS-CoV-2 patients, to unravel its effect on inflammation cascade and infection outcome. Methods: Soluble enzymatic activity of ACE2 was measured in nasopharyngeal swabs at the time of PCR positivity (mean time from symptom=4d) and 3 days after in a cohort of mild SARS-CoV-2 patients (n=40, mean age=42y) and in uninfected controls. Gene expression profiles of ACE2, its proteases, ADAM17 and TMPRRS2, and interferon-stimulated genes (ISGs), DDX58, CXCL10 and IL-6 were also evaluated by RT-qPCR. Results: Both ACE2 activity and mRNA expression decreased significantly during infection course in paired samples of SARS-CoV-2 infected subjects (p=0.048 and p<0.001, respectively), although differences between infected and uninfected subjects were only seen at mRNA level (p<0.001) Importantly, both ACE2 activity and mRNA expression showed a positive correlation with viral load (rho=0.352, p-value=0.0259), suggesting that viral infection is influencing ACE2 function. Similarly, infection downregulates TMPRSS2 expression (pvalue< 0.01), but not ADAM17, further indicating the viral-induced regulation of host receptors. In contrast to ACE2 data, a clear induction of IFN stimulated genes, CXCL10, IL-6 and DDX58 (RIG-I), is observed upon infection (p-value<0.05 in all cases), demonstrating that SARS-CoV-2 induces an antiviral response and suggesting that ACE2 is not an ISG. This increased expression of ISGs is directly linked to viral load (rho=0.6177, p-value<0.0001;rho=0.4026, p-value=0.0110;rho=0.3024, p-value=0.0613, respectively) but it is rapidly reversed during infection course. Conclusion: Overall, our results demonstrate the existence of mechanisms by which SARS-CoV-2 suppress ACE2 expression and function, intracellular viral detection and subsequent ISG induction, offering new insights into ACE2 dynamics and inflammatory response in the human upper respiratory tract that may contribute to understand the early antiviral host response to SARS-CoV-2 infection.

A placebo-controlled ati trial of HTI vaccines in early treated HIV infection

Background: HTI is a novel HIV vaccine immunogen designed at redirecting cellular immune responses to HIV targets associated with viral control. Methods: The AELIX-002 trial (NCT03204617) was a randomized, singlecenter, placebo-controlled trial to evaluate the safety, immunogenicity and antiviral effect of DNA.HTI (D), MVA.HTI (M) and ChAdOx1.HTI (C) vaccines after discontinuation of ART in early-treated people living with HIV (PLWH). 45 participants were randomized (2:1) to receive heterologous prime-boost vaccination regimens consisting of DDDMM followed by CCM, or matched placebo (P). During a 24-week analytical treatment interruption (ATI), plasma viral load (pVL) was monitored weekly and ART was resumed if pVL >100,000 copies/mL, or >10,000 copies/mL over 8 weeks, and/or CD4<350. Results: A total of 45 participants received DDDMM (n=30) or PPPPP (n=15). Of the 45 participants, 41 further completed the CCM (n=26) or PPP (n=15) regimen and entered the ATI. Immunizations were well tolerated, with no SAEs, and were immunogenic in 97% of vaccine recipients (defined by a >2-fold increase in HTI-specific T cell responses compared to baseline). Median (range) increase in total frequencies of HTI-specific T cells from baseline was 1,499 (120 to 3,150) SFC/million PBMC. At time of ATI start, 71% (0 to 100) of the total anti- HIV-1 T-cell response was HTI-specific. For participants without any potentially beneficial HLA class I alleles (32 of the 41), 8 (40%) of the vaccinees and 1 (8%) of the placebo recipients were able to remain off ART for 22 weeks (Δ 32%, 80%CI [7.6;55.7]);with pVL <2,000 copies/mL being observed in 5 and 1 vaccine and placebo recipients, respectively. Magnitude of HTI-specific responses at the time of ATI start positively correlated with time off ART in vaccinees (Rho 0.65, p < 0.01). Decay in total or intact HIV proviral DNA from baseline to ATI was similar between vaccine and placebo arms. Conclusion: HTI vaccines were safe and highly immunogenic in early-treated PLWH with a prolonged time off ART seen in vaccinees with non-beneficial HLA class I alleles. Time off ART positively correlated with vaccine-induced HTIspecific T cell responses at ATI start. Multivariate analysis for other correlates of response is ongoing. These encouraging data strongly support the use of HTIbased vaccines as the backbone of combination cure regimens such as with the TLR7 agonist vesatolimod, which is currently being evaluated in the AELIX-003 study (NCT04364035).