Differential Effects of the Second SARS-CoV-2 mRNA Vaccine Dose on T Cell Immunity in Naïve and COVID-19 Recovered Individuals (preprint)

The rapid development and deployment of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naïve individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naïve individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. Here we characterized, SARS-CoV-2 spike-specific humoral and cellular immunity in naïve and previously infected individuals during and after two-doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naïve individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.Funding: Research reported in this publication was supported in part by National Cancer Institute of the NIH (5R01HD102614-02; R01CA249204 and R01CA248984) and ISMMS seed fund to EG. The authors gratefully acknowledge use of the services and facilities of the Tisch Cancer Institute supported by the NCI Cancer Center Support Grant (P30 CA196521). MS was supported by a NCI training grant (T32CA078207). This work was supported by ISMMS seed fund to JO; Instituto de Salud Carlos III, COV20-00668 to RCR; Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181) co-financed by European Development Regional Fund “A way to achieve Europe” to EP; Instituto de Salud Carlos III, Spain (COV20/00170); Government of Cantabria, Spain (2020UIC22-PUB 0019) to MLH; Instituto de Salud Carlos III (PI16CIII/00012) to PP; Fondo Social Europeo e Iniciativa de Empleo Juvenil YEI (Grant PEJ2018-004557-A) to MPE; REDInREN 016/009/009 ISCIII; This project has received funding from the European Union’s Horizon 2020 research and innovation programme VACCELERATE under grant agreement No [101037867] to JO.Conflict of Interest: AB declares the filling of a patent application relating to the use of peptide pools in whole blood for detection of SARS-CoV-2 T cells (pending). The other authors declare no competing interests.Ethical Approval: The study protocols for the collection of clinical specimens from individuals with and without SARS-CoV-2 infection were reviewed and approved by Hospital La Paz, Hospital 12 de Octubre, Hospital Gregorio Marañón, IIS-Fundación Jimenez Díaz, Hospital Universitario Marqués de Valdecilla-IDIVAL and Hospital Puerta de Hierro Clinical Research Ethics Committee (CEIm), and Mount Sinai Hospital Institutional Review Board (IRB).

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals (preprint)

The rapid development and deployment of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has been questioned. Here we characterized SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during full BNT162b2 vaccination. Our results demonstrate that the second dose increases both the humoral and cellular immunity in naive individuals. On the contrary, the second BNT162b2 vaccine dose results in a reduction of cellular immunity in COVID-19 recovered individuals, which suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.

Evolution of antibodies against SARS-CoV-2 over seven months: experience of the Nationwide Seroprevalence ENE-COVID Study in Spain (preprint)

Abstract Objectives: To analyse temporal trends in SARS-CoV-2 anti-nucleocapsid IgG throughout the four rounds of the nationwide seroepidemiologic study ENE-COVID (April-November 2020), and to compare the fourth-round results of two immunoassays detecting antibodies against nucleocapsid and to S protein receptor-binding domain (RBD). Methods: A chemiluminescent microparticle immunoassay (CMIA) was offered to all participants in the first three rounds (Abbott; anti-nucleocapsid IgG). In the fourth round we offered this test and a chemiluminescence immunoassay (CLIA) (Beckman; anti-RBD IgG) to i) a randomly selected sub-cohort, ii) participants who were IgG-positive in any of the three first rounds; and iii) participants who were IgG-positive in the fourth round by point-of-care immunochromatography. Results: Immunoassays involving 10,153 participants (82.2% of people invited to donate samples) were performed in the fourth round. A total of 2595 participants (35.1% of participants with immunoassay results in the four rounds) were positive for anti-nucleocapsid IgG in at least one round. Anti-nucleocapsid IgG became undetectable in 43.3% of participants with positive first-round results. Pneumonia was more frequent in participants with anti-nucleocapsid IgG in all four rounds (11.2%) than those in which IgG became undetectable (2.4%). In fourth round, anti-nucleocapsid and anti-RBD IgG were detected in 5.5% and 5.4% participants of the randomly selected sub-cohort, and in 26.6% and 25.9% participants with at least one previous positive result, respectively. Agreement between techniques was 90.3% (kappa: 0.72). Conclusions: The response of IgG to SARS-CoV-2 is heterogeneous and conditioned by infection severity. A substantial proportion of the SARS-CoV-2 infected population may have negative serologic results in the post-infection months.

Convalescent Plasma for COVID-19: A multicenter, randomized clinical trial (preprint)

Background: Passive immunotherapy with convalescent plasma (CP) is a potential treatment for COVID-19 for which evidence from controlled clinical trials is lacking. Methods: We conducted a multi-center, randomized clinical trial in patients hospitalized for COVID-19. All patients received standard of care treatment, including off-label use of marketed medicines, and were randomized 1:1 to receive one dose (250-300 mL) of CP from donors with IgG anti-SARS-CoV-2. The primary endpoint was the proportion of patients in categories 5, 6 or 7 of the COVID-19 ordinal scale at day 15. Results: The trial was stopped after first interim analysis due to the fall in recruitment related to pandemic control. With 81 patients randomized, there were no patients progressing to mechanical ventilation or death among the 38 patients assigned to receive plasma (0%) versus 6 out of 43 patients (14%) progressing in control arm. Mortality rates were 0% vs 9.3% at days 15 and 29 for the active and control groups, respectively (log-rank p=0.056). No significant differences were found in secondary endpoints. At inclusion, patients had a median time of 8 days (IQR, 6-9) of symptoms and 49,4% of them were positive for anti-SARS-CoV-2 IgG antibodies. Conclusions: Convalescent plasma could be superior to standard of care in avoiding progression to mechanical ventilation or death in hospitalized patients with COVID-19. The strong dependence of results on a limited number of events in the control group prevents drawing firm conclusions about CP efficacy from this trial. (Funded by Instituto de Salud Carlos III; NCT04345523).

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro (preprint)

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

Engineered Trimeric ACE2 Binds and Locks "Three-up" Spike Protein to Potently Inhibit SARS-CoVs and Mutants (preprint)

SARS-CoV-2 enters cells via ACE-2, which binds the spike protein with moderate affinity. Despite a constant background mutational rate, the virus must retain binding with ACE2 for infectivity, providing a conserved constraint for SARS-CoV-2 inhibitors. To prevent mutational escape of SARS-CoV-2 and to prepare for future related coronavirus outbreaks, we engineered a de novo trimeric ACE2 (T-ACE2) protein scaffold that binds the trimeric spike protein with extremely high affinity (KD < 1 pM), while retaining ACE2 native sequence. T-ACE2 potently inhibits all tested pseudotyped viruses including SARS-CoV-2, SARS-CoV, eight naturally occurring SARS-CoV-2 mutants, two SARSr-CoVs as well as authentic SARS-CoV-2. The cryo-EM structure reveals that T-ACE2 can induce the transit of spike protein to "three-up" RBD conformation upon binding. T-ACE2 thus represents a promising class of broadly neutralizing proteins against SARS-CoVs and mutants.

From Face-to-Face to Online Modality: Implications for Undergraduate Learning While the World is Temporarily Closed in the Age of COVID-19 (preprint)

The second half of the Spring 2020 semester has been an unprecedented time globally due to the ongoing coronavirus disease 2019 (COVID-19). COVID-19 pandemic has forced more than one billion students out of school, has disrupted the world and led all university courses switched to online instruction social distancing actions taken to limit the spread of the virus. The aim of the present study is to evaluate the pandemic related changes for undergraduate students, to assess their perspectives related to their learning, experiences in two courses, and to discuss the far-reaching potential implications for the upcoming summer and fall semesters. An electronic survey was conducted to gather data on the student perceptions and learning characteristics of this transition from face-to-face (F2F) to online at a medium-sized university in the Southeast in the Spring 2020 semester. Nearly 88% of the participants indicated that the COVID-19 pandemic impacted their education, while 19% indicated that they prefer online over F2F learning. Furthermore, the online modality significantly increased attendance in General Biology I. Our study also showed that the usage of live conferencing and digital applications increased due to the pandemic. The current research fills the gap in the existing literature by providing the first study on the effects of the ongoing COVID-19 pandemic on undergraduate learning and experiences in the most unique dual modality of the Spring 2020 semester.

SARS-CoV-2 infection fatality risk in a nationwide seroepidemiological study (preprint)

ObjectiveTo estimate the range of the age- and sex-specific infection fatality risk (IFR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on confirmed coronavirus disease 2019 (COVID-19) deaths and excess all-cause deaths. DesignNationwide population-based seroepidemiological study combined with two national surveillance systems. Setting and participantsNon-institutionalized Spanish population of all ages. Main outcome measuresThe range of IFR was calculated as the observed number of COVID-19 deaths and excess deaths divided by the estimated number of SARS-CoV-2 infections in the non-institutionalized Spanish population. Laboratory-confirmed COVID-19 deaths were obtained from the National Epidemiological Surveillance Network (RENAVE) and excess all-cause deaths from the Monitoring Mortality System (MoMo) up to July 15, 2020. SARS-CoV-2 infections were derived from the estimated seroprevalence by a chemiluminiscent microparticle immunoassay for IgG antibodies in 61,092 participants in the ENE-COVID nationwide serosurvey between April 27 and June 22, 2020. ResultsThe overall IFR (95% confidence interval) was 0.8% (0.8% to 0.9%) for confirmed COVID-19 deaths and 1.1% (1.0% to 1.2%) for excess deaths. The IFR ranged between 1.1% (1.0% to 1.2%) and 1.4% (1.3% to 1.5%) in men and between 0.6% (0.5% to 0.6%) and 0.8% (0.7% to 0.8%) in women. The IFR increased sharply after age 50, ranging between 11.6% (8.1% to 16.5%) and 16.4% (11.4% to 23.2%) in men [≥]80 years and between 4.6% (3.4% to 6.3%) and 6.5% (4.7% to 8.8%) in women [≥]80 years. ConclusionThe sharp increase in SARS-CoV-2 IFR after age 50 was more marked in men than in women. Fatality from COVID-19 is substantially greater than that reported for other common respiratory diseases such as seasonal influenza. WHAT IS ALREADY KNOWN ON THIS TOPICInfection fatality risk (IFR) for SARS-CoV-2 is a key indicator for policy decision making, but its magnitude remains under debate. Case fatality risk, which accounts for deaths among confirmed COVID-19 cases, overestimates SARS-CoV-2 fatality as it excludes a large proportion of asymptomatic and mild-symptomatic infections. Population-based seroepidemiological studies are a valuable tool to properly estimate the number of infected individuals, regardless of symptoms. Also, because ascertainment of deaths due to COVID-19 is often incomplete, the calculation of the IFR should be complemented with data on excess all-cause mortality. In addition, data on age- and sex-specific IFR are scarce, even though age and sex are well known modifiers of the clinical evolution of COVID-19. WHAT THIS STUDY ADDSUsing the ENE-COVID nationwide serosurvey and two national surveillance systems in Spain, this study provides a range of age- and sex-specific IFR estimates for SARS-CoV-2 based on laboratory-confirmed COVID-19 deaths and excess all-cause deaths. The risk of death was very low among infected individuals younger than 50 years, but it increased sharply with age, particularly among men. In the oldest age group ([≥]80 years), it was estimated that 12% to 16% of infected men and 5% to 6% of infected women died during the first epidemic wave.

Phylodynamics of SARS-CoV-2 transmission in Spain (preprint)

ObjectivesSARS-CoV-2 whole-genome analysis has identified three large clades spreading worldwide, designated G, V and S. This study aims to analyze the diffusion of SARS-CoV-2 in Spain/Europe. MethodsMaximum likelihood phylogenetic and Bayesian phylodynamic analyses have been performed to estimate the most probable temporal and geographic origin of different phylogenetic clusters and the diffusion pathways of SARS-CoV-2. ResultsPhylogenetic analyses of the first 28 SARS-CoV-2 whole genome sequences obtained from patients in Spain revealed that most of them are distributed in G and S clades (13 sequences in each) with the remaining two sequences branching in the V clade. Eleven of the Spanish viruses of the S clade and six of the G clade grouped in two different monophyletic clusters (S-Spain and G-Spain, respectively), with the S-Spain cluster also comprising 8 sequences from 6 other countries from Europe and the Americas. The most recent common ancestor (MRCA) of the SARS-CoV-2 pandemic was estimated in the city of Wuhan, China, around November 24, 2019, with a 95% highest posterior density (HPD) interval from October 30-December 17, 2019. The origin of S-Spain and G-Spain clusters were estimated in Spain around February 14 and 18, 2020, respectively, with a possible ancestry of S-Spain in Shanghai. ConclusionsMultiple SARS-CoV-2 introductions have been detected in Spain and at least two resulted in the emergence of locally transmitted clusters, with further dissemination of one of them to at least 6 other countries. These results highlight the extraordinary potential of SARS-CoV-2 for rapid and widespread geographic dissemination.