Observational study on antibody response to COVID-19 Vaccines in pAtients with gastro-entero-panCreatic Cancers and neuroendocrIne NeoplAsms on systemic TreatmEnts (VACCINATE). (preprint)

The Coronavirus disease-19 (COVID-19) pandemic dramatically impacted on oncologic patients' care. Since the introduction of vaccines and the demonstration of their benefit on frail patients, COVID-19 vaccinations were indicated also for oncologic population. However, data about the impact of anticancer-treatments and the timing between vaccinations and systemic therapy de-livery were not available. We aimed to evaluate potential factors influencing the outcome of COVID-19 vaccination in cancer patients. We prospectively collected data of patients undergoing COVID-19 vaccination with gas-tro-entero-pancreatic and neuroendocrine neoplasms, treated at our Institute, between 03/2021 and 12/2021. We enrolled 46 patients, 63.1% males; at the time of data collection 86.9% received two-doses of Pfizer-BioNTech and the remains Moderna vaccine. All patients obtained a subsequent im-mune-response. A significantly lower values of IgG for patients treated with chemotherapy versus other anti-cancer agents (p=0.004). No significant effect on immune-response was reported for both vaccinations performed ≤7 vs >7 days from the last systemic treatment (p=0.77) and lymphocytes count (p=0.11). The findings suggest that the optimal timing for COVID-19 vaccination and lymphocytes count are not the issue but rather the quality of the subset of lymphocytes before the vaccination to deter-mine the efficacy level of immune-response in this population.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape (preprint)

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy (preprint)

SARS-CoV-2 is constantly evolving leading to new variants. We analysed data from 4,400 SARS-CoV-2-positive samples in order to continue variant surveillance in Italy to evaluate their epidemiological and relative impact on public health in the period April-December 2021. The main circulating strain (76.2%) was Delta followed by Alpha (13.3%), Omicron (5.3%) and Gamma variants (2.9%). B.1.1 lineages, Eta, Beta, Iota, Mu and Kappa variants represented around 1% of cases. Overall, 48.2% of subjects were not vaccinated with a lower median age compared to vaccinated subjects (47 vs. 61 years). An increasing number of infections in vaccinated subjects was observed overtime, with the highest proportion in November (85.2%). Variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed among Delta variant, while subjects harboring Gamma variant showed the highest proportion of asymptomatics (21.6%), albeit also of deaths (5.4%). The Omicron variant was only found in vac-cinated subjects, of which 47% were hospitalized. Diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at national and international level. Our study pro-vides data on the rapid changes in the epidemiological landscape of SARS-CoV-2 variants in Italy.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift (preprint)

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab, S2X259 and S2H97, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

SARS-CoV-2 B.1.1.7 escape from mRNA vaccine-elicited neutralizing antibodies (preprint)

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (<1:4 to 3449) that were reduced against B.1.1.7 variant by 3.85 fold (IQR 2.68-5.28). This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralization was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 outof 29), but not in neutralizing mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background led to a further loss of neutralizing activity by vaccine-elicited antibodies over that conferred by the B.1.1.7 mutations alone. Further work is needed to establish the impact of these observations on protective vaccine efficacy in the context of the evolving B.1.1.7 lineage that will likely acquire E484K.

Impact of SARS-CoV-2 B.1.1.7 Spike variant on neutralisation potency of sera from individuals vaccinated with Pfizer vaccine BNT162b2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

The lethal triad: SARS-CoV-2 Spike, ACE2 and TMPRSS2. Mutations in host and pathogen may affect the course of pandemic. (preprint)

Variants of SARS-CoV-2 have been identified rapidly after the beginning of pandemic. One of them, involving the spike protein and called D614G, represents a substantial percentage of currently isolated strains. While research on this variant was ongoing worldwide, on December 20th 2020 the European Centre for Disease Prevention and Control reported a Threat Assessment Brief describing the emergence of a new variant of SARS-CoV-2, named B.1.1.7, harboring multiple mutations mostly affecting the Spike protein. This viral variant has been recently associated with a rapid increase in COVID-19 cases in South East England, with alarming implications for future virus transmission rates. Specifically, of the nine amino acid replacements that characterize the Spike in the emerging variant, four are found in the region between the Fusion Peptide and the RBD domain (namely the already known D614G, together with A570D, P681H, T716I), and one, N501Y, is found in the Spike Receptor Binding Domain - Receptor Binding Motif (RBD-RBM). In this study, by using in silico biology, we provide evidence that these amino acid replacements have dramatic effects on the interactions between SARS-CoV-2 Spike and the host ACE2 receptor or TMPRSS2, the protease that induces the fusogenic activity of Spike. Mostly, we show that these effects are strongly dependent on ACE2 and TMPRSS2 polymorphism, suggesting that dynamics of pandemics are strongly influenced not only by virus variation but also by host genetic background.

Surveillance of genetic diversity and evolution in locally transmitted SARS-CoV-2 in Pakistan during the first wave of the COVID-19 pandemic (preprint)

Surveillance of genetic diversity in the SARS-CoV-2 is extremely important to detect the emergence of more infectious and deadly strains of the virus. In this study, we monitored mutational events in the SARS-CoV-2 genome through whole genome sequencing. The samples (n=48) were collected from the hot spot regions of the metropolitan city Karachi, Pakistan during the four months (May 2020 to August 2020) of first wave of the COVID-19 pandemic. The data analysis highlighted 122 mutations, including 120 single nucleotide variations (SNV), and 2 deletions. Among the 122 mutations, there were 71 singletons, and 51 recurrent mutations. A total of 16 mutations, including 5 nonsynonymous mutations, were detected in spike protein. Notably, the spike protein missense mutation D614G was observed in 31 genomes. The phylogenetic analysis revealed majority of the genomes (36) classified as B lineage, where 2 genomes were from B.6 lineage, 5 genomes from B.1 ancestral lineage and remaining from B.1 sub-lineages. It was noteworthy that three clusters of B.1 sub-lineages were observed, including B.1.36 lineage (10 genomes), B.1.160 lineage (11 genomes), and B.1.255 lineage (5 genomes), which represent independent events of SARS-CoV-2 transmission within the city. The sub-lineage B.1.36 had higher representation from the Asian countries and the UK, B.1.160 correspond to the European countries with highest representation from the UK, Denmark, and lesser representation from India, Saudi Arabia, France and Switzerland, and the third sub-lineage (B.1.255) correspond to the USA. Collectively, our study provides meaningful insight into the evolution of SARS-CoV-2 lineages in spatio-temporal local transmission during the first wave of the pandemic.

Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation (preprint)

There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.

Impacts of 203/204: RG>KR mutation in the N protein of SARS-CoV-2 (preprint)

We present a structure-based model of phosphorylation-dependent binding and sequestration of SARS-CoV-2 nucleocapsid protein and the impact of two consecutive amino acid changes R203K and G204R. Additionally, we studied how mutant strains affect HLA-specific antigen presentation and correlated these findings with HLA allelic population frequencies. We discovered RG>KR mutated SARS-CoV-2 expands the ability for differential expression of the N protein epitope on Major Histocompatibility Complexes (MHC) of varying Human Leukocyte Antigen (HLA) origin. The N protein LKR region K203, R204 of wild type (SARS-CoVs) and (SARS-CoV-2) observed HLA-A*30:01 and HLA-A*30:21, but mutant SARS-CoV-2 observed HLA-A*31:01 and HLA-A*68:01. Expression of HLA-A genotypes associated with the mutant strain occurred more frequently in all populations studied. ImportanceThe novel coronavirus known as SARS-CoV-2 causes a disease renowned as 2019-nCoV (or COVID-19). HLA allele frequencies worldwide could positively correlate with the severity of coronavirus cases and a high number of deaths.

N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2 (preprint)

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2 (preprint)

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus. IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Human Identical Sequences of SARS-CoV-2 Promote Clinical Progression of COVID-19 by Upregulating Hyaluronan via NamiRNA-Enhancer Network (preprint)

The COVID-19 pandemic is a widespread and deadly public health crisis. The pathogen SARS-CoV-2 replicates in the lower respiratory tract and causes fatal pneumonia. Although tremendous efforts have been put into investigating the pathogeny of SARS-CoV-2, the underlying mechanism of how SARS-CoV-2 interacts with its host is largely unexplored. Here, by comparing the genomic sequences of SARS-CoV-2 and human, we identified five fully conserved elements in SARS-CoV-2 genome, which were termed as "human identical sequences (HIS)". HIS are also recognized in both SARS-CoV and MERS-CoV genome. Meanwhile, HIS-SARS-CoV-2 are highly conserved in the primate. Mechanically, HIS-SARS-CoV-2 RNA directly binds to the targeted loci in human genome and further interacts with host enhancers to activate the expression of adjacent and distant genes, including cytokines gene and angiotensin converting enzyme II (ACE2), a well-known cell entry receptor of SARS-CoV-2, and hyaluronan synthase 2 (HAS2), which further increases hyaluronan formation. Noteworthily, hyaluronan level in plasma of COVID-19 patients is tightly correlated with severity and high risk for acute respiratory distress syndrome (ARDS) and may act as a predictor for the progression of COVID-19. HIS antagomirs, which downregulate hyaluronan level effectively, and 4-Methylumbelliferone (MU), an inhibitor of hyaluronan synthesis, are potential drugs to relieve the ARDS related ground-glass pattern in lung for COVID-19 treatment. Our results revealed that unprecedented HIS elements of SARS-CoV-2 contribute to the cytokine storm and ARDS in COVID-19 patients. Thus, blocking HIS-involved activating processes or hyaluronan synthesis directly by 4-MU may be effective strategies to alleviate COVID-19 progression.

The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity (preprint)

SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

Development and Validation of a Laboratory Score Based on Blood Cell Parameters to Predict Survival in Hospitalized COVID-19 Patients (preprint)

Background: A relevant portion of patients with disease caused by the ‘severe acute respiratory syndrome coronavirus 2’ (COVID-19) experience poor outcome and several laboratory tests have been proposed to predict disease severity. Among others, dramatic changes in peripheral blood cells have been described. We developed and validated a laboratory score based on blood cell parameters to predict survival in hospitalized COVID-19 patients.Methods: We retrospectively analyzed 1619 blood cell counts from 226 COVID-19 patients, consecutively admitted to our institution, to select variables for inclusion in the severity score. The lymphocyte percentage, the immature granulocyte number, the highly fluorescent lymphocyte cell percentage, the neutrophils side fluorescent light, and the lymphocytes fluorescence light intensity were finally selected and the derived score then validated on a separate cohort of 140 COVID-19 patients.Findings: Using ROC curve analysis, a best cut-off for score of 30.6 was derived, which was associated to an overall 82.0% sensitivity (95% CI: 78-84) and 82.5% specificity (95% CI: 80-84), respectively, for detecting outcome. The score trend effectively separated the patients’ groups, starting on average two weeks before the end of the hospitalization period.Interpretation: The developed scoring system accurately predicted survival in hospitalized COVID-19 patients. As the laboratory parameters employed in the model are easily accessible on the modern haematology analyzers, the proposed score may offer a fast and reliable tool for supporting clinical decisions in COVID-19 patients.Funding Statement: None.Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The Institutional Review Board approved the study.

30-day mortality in patients hospitalized with COVID-19 during the first wave of the Italian epidemic: a prospective cohort study (preprint)

Background: Italy was the first European country hit by the COVID-19 pandemic and has the highest number of recorded COVID-19 deaths in Europe. Methods: This prospective cohort study of the correlates of the risk of death in COVID-19 patients was conducted at the Infectious Diseases and Intensive Care units of Luigi Sacco Hospital, Milan, Italy. The clinical characteristics of all the COVID-19 patients hospitalised in the early days of the epidemic (21 February -19 March 2020) were recorded upon admission, and the time-dependent probability of death was evaluated using the Kaplan-Meier method (censored as of 20 April 2020). Cox proportional hazard models were used to assess the factors independently associated with the risk of death. Results: Forty-eight (20.6%) of the 233 patients followed up for a median of 40 days (interquartile range 33-47) died during the follow-up. Most were males (69.1%) and their median age was 61 years (IQR 50-72). The time-dependent probability of death was 19.7% (95% CI 14.6-24.9%) 30 days after hospital admission. Age (adjusted hazard ratio [aHR] 2.08, 95% CI 1.48-2.92 per ten years more) and obesity (aHR 3.04, 95% CI 1.42-6.49) were independently associated with an increased risk of death, which was also associated with critical disease (aHR 8.26, 95% CI 1.41-48.29), C-reactive protein levels (aHR 1.17, 95% CI 1.02-1.35 per 50 mg/L more) and creatinine kinase levels above 185 U/L (aHR 2.58, 95% CI 1.37-4.87) upon admission. Conclusions: Case-fatality rate of patients hospitalized with COVID-19 in the early days of the Italian epidemic was about 20%. Our study adds evidence to the notion that older age, obesity and more advanced illness are factors associated to an increased risk of death among patients hospitalized with COVID-19.

GENOMIC CHARACTERISATION AND PHYLOGENETIC ANALYSIS OF SARS-COV-2 IN ITALY (preprint)

This report describes the isolation, the molecular characterization and the phylogenetic analysis of the first three complete genomes of SARS-CoV-2 isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.