Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals. (preprint)

Purpose Mass vaccination campaigns have reduced the incidence and severity of COVID-19. However, there is limited information about how patients with predominantly antibody-deficiencies (PAD) respond to COVID-19 vaccination. Here, we evaluated humoral and cellular responses developed in SARS-CoV-2-naïve PAD individuals after three mRNA-1273 vaccine doses.Methods Patients and healthy controls (HCs) were immunized at week 0 (w0) and w4. PAD individuals received an additional dose at w24. Blood samples were collected at w0, w4, w8, w24, and/or w28. We determined levels of anti-Spike and anti-RBD antibodies, Spike-specific IgG avidity, and neutralizing activity (Wuhan-Hu-1, Delta, and Omicron variants). Cellular responses were evaluated by IFN-γ ELISpot and flow cytometry.Results Unclassified primary antibody-deficiency patients (unPAD, n = 9) and HCs developed comparable vaccine-induced humoral responses. However, common variable immunodeficiency patients (CVID, n = 12) showed lower antibody responses than HCs. While the frequency of Spike-specific CD4 + T cells was similar between PAD patients and HCs, CD8 + T cells responses were reduced in CVID individuals. Both PAD groups showed lower levels of Spike-specific IFN-γ-producing T-cells. Combined immunodeficiency (CID, n = 1) and thymoma with immunodeficiency (TID, n = 1) patients developed cellular but not humoral responses after two immunizations. The third vaccine dose boosted humoral responses in most PAD patients, but had little effect on cellular immunity.Conclusion mRNA-1273 vaccine-induced immune responses in PAD individuals are heterogeneous, depend on the type and degree of antibody-deficiency, and should be immunomonitored to define a personalized vaccination strategy.

Demonstration of antibodies against SARS-CoV-2, neutralizing or binding, in seroconversion panels after mRNA-1273, BNT-162b2 and Ad26.COV2.S vaccine administration. (preprint)

Seroconversion panels were collected before and after vaccination with three COVID-19 vaccines: two mRNA vaccines (mRNA-1273 and BNT-162b2) and one adenovirus vector vaccine (Ad26.COV2.S). The panels were tested for antibody activity by chemiluminescent immunoassay, ELISA and one was tested in a pseudovirus neutralization assay. Participants positive for anti-SARS-CoV-2 antibodies before vaccination (18.6%) had a higher response to the first vaccine dose than participants who tested negative. For two-dose vaccines, older participants showed a lower response to the first dose than younger participants. All participants showed positive antibody responses after the second vaccine. For the adenovirus vector vaccine, two participants did not generate antibody responses two weeks and two months after vaccination. Three participants were negative at two weeks but positive at two months. Pseudovirus neutralization showed good correlation with antibody activity (correlation coefficient =0.78, p<0.0001). Antibody responses in participants over 45 years old tended to be less robust.

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

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.

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

Background SARS-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. Methods Plasma 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. Results Three 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. Conclusions While 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 summary While 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.

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

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. Findings 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 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. Conclusions 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, 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.

Inappropriate sinus tachycardia in post-covid-19 Syndrome

Background: Persistent symptoms after the acute phase of SARS-CoV-2 infection are referred to as 'post-COVID-19 syndrome' (PCS), with a reported incidence ranging between 35% and 87%. Fatigue, palpitations and exercise intolerance are common complains among PCS patients in whom unexplained sinus tachycardia, occasionally exacerbated by postural changes, is a frequent observation that remains poorly characterized. Purpose: We sought to characterize the prevalence of inappropriate sinus tachycardia (IST) and postural orthostatic tachycardia (POTS) in a consecutive and prospective population of patients with PCS. Methods: Consecutive patients with persistent symptoms 3 months after an acute SARS-CoV-2 infection were prospectively evaluated at a multi-disciplinary PCS unit. All patients were screened for IST or POTS and those with confirmed criteria underwent comprehensive cardiovascular examination including echocardiography, 24-hour Holter, Minnesota Living with Heart Failure Questionnaire (MLHFQ), six-minute walking test (6MWT) and inflammation and myocardial biomarkers. Two control patients, matched by age and gender, were assigned to each case: one with previous SARS-CoV-2 infection without PCS (group 2) and one without prior SARS-CoV-2 infection (group 3). Results: IST or POTS criteria were met in 34 out of the 200 PCS patients (17%). The mean age was 39 ± 10 years, with 29 women (91%). The interval from the index COVID-19 disease to the PCS diagnosis was 71 ± 17 days, with a majority of patients (n = 29,85%) not requiring hospital admission during the acute phase. At physical examination, the mean heart rate was 96 ± 3bpm at supine and 112 ± 17bpm at the upright position, with 8 patients fulfilling diagnostic criteria of POTS. No underlying structural heart disease, pro-inflammatory state, myocyte injury or hypoxia were identified among our patient population. The 6MWT showed a significantly diminished exercise capacity with a 59% of the estimated distance after adjustment by age, sex and body mass index;an impaired quality of life was also identified, as suggested by a median MLFHQ total score of 67 out of 105 points. The 24-hour Holter showed an increase in HR predominantly during daytime in group 1 (mean daytime HR of 94 ± 3bpm), an altered heart rate variability with a decrease in time domain parameters [PNN50 4 ± 4 in group 1 (vs. 11 ± 9 in group 2 and 18 ± 9 in group 3;p < 0.002)respectively;SD 100 ± 20 (vs. 127 ± 38 and 136 ± 13;p = 0.009) and a decrease in frequency domain parameters [LF 751 ± 450 (vs. 1721 ± 1009 and 2199 ± 920;p = 0.01), HF 336 ± 280 (vs. 823 ± 1200 and 1229 ± 630;p = 0.01)]. Conclusions: IST and its POTS variant are a prevalent condition among PCS patients and may at least partially explain the common symptoms of fatigue, impaired exercise and palpitations that characterize the PCS. Cardiac autonomic nervous system imbalance may account as a plausible pathophysiological mechanism of IST in PCS patients.

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

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.

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

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.

Publishing of COVID-19 Preprints in Peer-reviewed Journals, Preprinting Trends, Public Discussion and Quality Issues (preprint)

IntroductionCOVID-19-related (vs. non-related) articles appear to be more expeditiously processed and published in peer-reviewed journals. We aimed to evaluate: (i) whether COVID-19-related preprints were favoured for publication, (ii) preprinting trends and public discussion of the preprints and (iii) relationship between the publication topic (COVID-19-related or not) and quality issues. MethodsManuscripts deposited at bioRxiv and medRxiv between January 1 and October 21 were assessed for the probability of publishing in peer-reviewed journals, and those published were evaluated for submission-to-acceptance time. The extent of public discussion was assessed based on Altmetric and Disqus data. The Retraction Watch database and PubMed were used to explore the retraction of COVID-19 and non-COVID-19 articles and preprints. ResultsWith adjustment for the preprinting server and number of deposited versions, COVID-19-related preprints were more likely to be published within 120 days since the deposition of the first version (OR=1.99, 95%CI 1.76-2.25) as well as over the entire observed period (OR=1.49, 95%CI 1.36-1.62). Submission-to-acceptance was by 41.69 days (95%CI 46.56-36.80) shorter for COVID-19 articles. Public discussion of preprints was modest and COVID-19 articles were overrepresented in the pool of retracted articles in 2020. ConclusionCurrent data suggest a preference for publication of COVID-19-related preprints over the observed period.

Building a virtual summer research experience in cancer for high school and early undergraduate students: lessons from the COVID-19 pandemic (preprint)

BackgroundThe COVID-19 pandemic posed a unique challenge for summer research programs in 2020, particularly for programs aimed at hands-on experience for younger trainees. The Indiana University Melvin and Bren Simon Comprehensive Cancer Center supports two pipeline programs, which traditionally immerse high school juniors, seniors, and early undergraduate students from underrepresented populations in science in hands-on projects in cancer biology labs. However, due to social distancing policies during the pandemic and reduction of research operations, these students were not physically allowed on campus. Thus, the authors set out to strategically pivot to a wholly virtual curriculum and evaluate the Virtual Summer Research Experience in Cancer outcomes. MethodsThe virtual program included four components: 1. a core science and professional development curriculum led by high school teachers and senior undergraduates; 2. faculty-delivered didactic sessions on cancer science; 3. mentored, virtual research projects with research faculty; and 4. online networking events to encourage vertical mentoring. Outcomes data were measured using an 11-item Research Preparation scale, daily electronic feedback, and structured evaluation and feedback via Zoom weekly. ResultsOutcome data suggested high self-reported satisfaction with the virtual program. Outcome data also revealed the importance of coordination between multiple entities for seamless program implementation. This includes the active recruitment and participation of high school teachers and further investment in information technology capabilities of institutions. ConclusionsFindings reveal a path to educate and train high school and early undergraduate students in cancer research when hands-on, in-person training is not feasible. Virtual research experiences are not only useful to engage students during public health crises but can provide an avenue for cancer centers to expand their cancer education footprints to remotely located schools and universities with limited resources to provide such experiences to their students.

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

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.

In-Silico analysis reveals lower transcription efficiency of C241T variant of SARS-CoV-2 with host replication factors MADP1 and hnRNP-1 (preprint)

Novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has claimed more than 1.5 million lives worldwide and counting. As per the GISAID database, the genomics of SARS-CoV2 is extensively studied with more than 500 genome submissions per day. Out of several hotspot mutations within the SARS-CoV-2 genome, researchers have focused a lot on missense variants but the least work is done on the UTRs. One of the most frequent UTR variants in the SARS-CoV-2 genome is the C241T with a global frequency of more than 0.9. In the present study, the effect of the C241T mutation has been studied with respect to change in RNA structure and its interaction with the host replication factors MADP1 Zinc finger CCHC-type and RNA-binding motif 1 (hnRNP1). The results obtained from molecular docking and molecular dynamics simulation indicated weaker interaction of C241T mutant stem loops with host transcription factor MADP1 indicating reduced replication efficiency. The results are also correlated with increased recovery rates and decreased death rates of global SARS-CoV-2 cases.

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19 (preprint)

Two proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

SARS-CoV-2 infection suppresses ACE2 function and antiviral immune response in the upper respiratory tract of infected patients (preprint)

There is an urgent need to elucidate the molecular mechanisms underlying the transmissibility and pathogenesis of SARS-CoV-2. ACE2 is a host ectopeptidase with well-described anti-inflammatory and tissue protective functions and the receptor for the virus. Understanding SARS-CoV-2-ACE2 interaction and the expression of antiviral host genes in early infection phase is crucial for fighting the pandemic. We tested the significance of soluble ACE2 enzymatic activity longitudinally in positive nasopharyngeal swabs at two time points after symptom consultation, along with gene expression profiles of ACE2, its proteases, ADAM17 and TMPRRS2, and interferon-stimulated genes (ISGs), DDX58, CXCL10 and IL-6. Soluble ACE2 activity decreased during infection course, in parallel to ACE2 gene expression. On the contrary, SARS-CoV-2 infection induced expression of the ISG genes in positive SARS-CoV-2 samples at baseline compared to negative control subjects, although this increase wanes with time. These changes positively correlated with viral load. Our results demonstrate the existence of mechanisms by which SARS-CoV-2 suppress ACE2 expression and function casting doubt on the IFN-induced upregulation of the receptor. Moreover, we show that initial intracellular viral sensing and subsequent ISG induction is also rapidly downregulated. Overall, our results offer 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.