Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

Reduction of the risk of severe COVID-19 due to Omicron compared to Delta variant in Italy (November 2021 - February 2022).

OBJECTIVES: During 2022, Omicron became the dominant SARS-CoV-2 variant in Europe. This study aims to assess the impact of such variant on severe disease from SARS-CoV-2 compared with the Delta variant in Italy. METHODS: Using surveillance data, we assessed the risk of developing severe COVID-19 with Omicron infection compared with Delta in individuals aged ≥12 years using a multilevel negative binomial model adjusting for sex, age, vaccination status, occupation, previous infection, weekly incidence, and geographical area. We also analyzed the interaction between the sequenced variant, age, and vaccination status. RESULTS: We included 21,645 cases of SARS-CoV-2 infection where genome sequencing found Delta (10,728) or Omicron (10,917), diagnosed from November 15, 2021 to February 01, 2022. Overall, 3,021 cases developed severe COVID-19. We found that Omicron cases had a reduced risk of severe COVID-19 compared with Delta cases (incidence rate ratio [IRR] = 0.77; 95% confidence interval [CI]: 0.70-0.86). The largest difference was observed in cases aged 40-59 (IRR = 0.66; 95% CI: 0.55-0.79), while no protective effect was found in those aged 12-39 (IRR = 1.03; 95% CI: 0.79-1.33). Vaccination was associated with a lower risk of developing severe COVID-19 in both variants. CONCLUSION: The Omicron variant is associated with a lower risk of severe COVID-19 compared to infection with the Delta variant, but the degree of protection varies with age.

Evidence of immune response to BNT162b2 COVID-19 vaccine in systemic lupus erythematosus patients treated with Belimumab.

OBJECTIVES: To evaluate humoral and cell-mediated response after three doses of BNT162b2 SARS-CoV-2 vaccine in patients with systemic lupus erythematosus (SLE) treated with Belimumab (BLM). METHODS: SLE patients were vaccinated with three doses of BNT162b2-mRNA vaccine (two-dose primary vaccination, third booster dose after 6 months). The humoral immune response was assessed one and 6 months after the second dose (T1, T2), and 6 months after the booster dose (T3). Serological assay was performed (The Liaison® SARS-CoV-2 TrimericS IgG chemiluminescent). Spike-specific T-cell response was monitored 6 months after the second vaccine dose and the percentage of cytokines producing T cells was assessed by flow cytometry. RESULTS: Twelve patients [12F; median age 46 years (IQR 8.25); median disease duration 156 months (IQR 188)] were enrolled. At T1, all patients showed seroconversion (median anti-Spike IgG levels 1610 BAU/mL, IQR 1390). At T2--day of the third dose--a significant reduction of median anti-Spike IgG antibodies levels was observed [214 BAU/mL (IQR 94); p = 0.0009]. Anti-Spike IgG were significantly increased at T3, reaching a median value of 1440 BAU/mL (IQR 1316; p = 0.005). Despite declining humoral immunity, almost 60% of patients mounted a virus-specific CD4 + T-cell response 6 months after primary vaccination. CONCLUSIONS: BLM does not impair humoral response to primary BNT162b2 SARS-CoV-2 vaccination. During the follow-up, a decline in antibody levels is evident and the third dose is crucial to increase the specific immune response. Finally, we observed a recall T-cell response to the Spike antigen 6 months after the first vaccination cycle.

SARS-CoV-2 variants: what have we learnt so far? Commentary.

Besides the timely detection of different SARS-CoV-2 variants through surveillance systems, functional and modelling studies are essential to better inform public health response and preparedness. Here, the knowledge available so far on SARS-CoV-2 variants is discussed from different perspectives, in order to highlight the relevance of a multidisciplinary approach in countering the threat posed by this insidious virus.

First detection of SARS-CoV-2 lineage A.27 in Sardinia, Italy.

INTRODUCTION: Multiple variants of SARS-CoV-2, since the end of 2020 have emerged in many geographical areas and are currently under surveillance worldwide highlighting the continuing need for genomic monitoring to detect variants previously not yet identified. METHODS: In this study, we used whole-genome sequencing (WGS) and phylogenetic analysis to investigate A.27 lineage SARS-CoV-2 from Sardinia, Italy. RESULTS: The Italian A.27 lineage genomes from Sardinia appeared related in a clade with genomes from France. Among the key mutations identified in the spike protein, the N501Y and the L452R deserve attention as considered likely vaccine escape mutations. Additional mutations were also here reported. CONCLUSION: A combination of features could explain our data such as SARS-CoV-2 genetic variability, viral dynamics, the human genetic diversity of Sardinian populations, the island context probably subjected to different selective pressures. Molecular and genomic investigation is essential to promptly identify variants with specific mutations with potential impact on public health and vaccine formulation.

Analysis of Genomic Characteristics of SARS-CoV-2 in Italy, 29 January to 27 March 2020.

We performed next-generation sequencing (NGS), phylogenetic analysis, gene flows, and N- and O-glycosylation prediction on SARS-CoV-2 genomes collected from lab-confirmed cases from different Italian regions. To this end, a total of 111 SARS-CoV-2 genomes collected in Italy between 29 January and 27 March 2020 were investigated. The majority of the genomes belonged to lineage B.1, with some descendant lineages. The gene flow analysis showed that the spread occurred mainly from the north to the center and to the south of Italy, as confirmed by epidemiological data. The mean evolutionary rate estimated here was 8.731 × 10-4 (95% highest posterior density, HPD intervals 5.809 × 10-4 to 1.19 × 10-3), in line with values reported by other authors. The dated phylogeny suggested that SARS-CoV-2 lineage B.1 probably entered Italy between the end of January and early February 2020. Continuous molecular surveillance is needed to trace virus circulation and evolution.

Identification and characterization of SARS-CoV-2 clusters in the EU/EEA in the first pandemic wave: additional elements to trace the route of the virus.

A high-quality dataset of 3289 complete SARS-CoV-2 genomes collected in Europe and European Economic Area (EAA) in the early phase of the first wave of the pandemic was analyzed. Among all single nucleotide mutations, 41 had a frequency ≥ 1%, and the phylogenetic analysis showed at least 6 clusters with a specific mutational profile. These clusters were differentially distributed in the EU/EEA, showing a statistically significant association with the geographic origin. The analysis highlighted that the mutations C14408T and C14805T played an important role in clusters selection and further virus spread. Moreover, the molecular analysis suggests that the SARS-CoV-2 strain responsible for the first Italian confirmed COVID-19 case was already circulating outside the country.

Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe.

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

Genomic Analysis and Lineage Identification of SARS-CoV-2 Strains in Migrants Accessing Europe Through the Libyan Route.

Many African countries, representing the origin of the majority of refugees, asylum-seekers, and other migrants, toward regions bordering on the Mediterranean area, are experiencing sustained local transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sicily is one of the main entry gates of migrants crossing into Europe. We conducted a pilot study, based on the full-genome sequencing of SARS-CoV-2 strains isolated from migrants coming to Sicily by crossing the Mediterranean Sea, with the aim to investigate the viral genome polymorphism and to describe their genetic variations and the phylogenetic relationships. On June 21, a nongovernmental organization vessel rescued 210 migrants crossing the Mediterranean Sea from Libya to Sicily. Of them, 13.4% tested positive for SARS-CoV-2. Eighteen whole genome sequences were obtained to explore viral genetic variability. All but one of the sequences clustered with other viral African strains within the lineage A, whereas only one intermixed among B.1 lineage genomes. Our findings documented that most of the investigated migrants acquired SARS-CoV-2 infection before landing in Sicily. However, SARS-CoV-2 transmission during travel or in overcrowded Libyan immigrant camps and/or illegal transport boats could not be ruled out. SARS-CoV-2 molecular surveillance on migrants arriving in Europe through the Sicilian gate may improve the knowledge of global SARS-CoV-2 transmission dynamic also in light of the emergence of new variants.

Characteristics of SARS-CoV-2 variants of concern B.1.1.7, B.1.351 or P.1: data from seven EU/EEA countries, weeks 38/2020 to 10/2021.

We compared 19,207 cases of SARS-CoV-2 variant B.1.1.7/S gene target failure (SGTF), 436 B.1.351 and 352 P.1 to non-variant cases reported by seven European countries. COVID-19 cases with these variants had significantly higher adjusted odds ratios for hospitalisation (B.1.1.7/SGTF: 1.7, 95% confidence interval (CI): 1.0-2.9; B.1.351: 3.6, 95% CI: 2.1-6.2; P.1: 2.6, 95% CI: 1.4-4.8) and B.1.1.7/SGTF and P.1 cases also for intensive care admission (B.1.1.7/SGTF: 2.3, 95% CI: 1.4-3.5; P.1: 2.2, 95% CI: 1.7-2.8).

Selective pressure on SARS-CoV-2 protein coding genes and glycosylation site prediction.

BACKGROUND: An outbreak of a febrile respiratory illness due to the newly discovered Coronavirus, SARS-CoV-2, was initially detected in mid-December 2019 in the city of Wuhan, Hubei province (China). The virus then spread to most countries in the world. As an RNA virus, SARS-CoV-2 may acquire mutations that may be fixed. The aim of this study was to evaluate the selective pressure acting on SARS-CoV-2 protein coding genes. METHODS: Mutations and glycosylation site prediction were analyzed in SARS-CoV-2 genomes (from 464 to 477 sequences). RESULTS: Selective pressure on surface glycoprotein (S) revealed one positively selected site (AA 943), located outside the receptor binding domain (RBD). Mutation analysis identified five residues on the surface glycoprotein, with variations (AA positions 367, 458, 477, 483, 491) located inside the RDB. Positive selective pressure was identified in nsp2, nsp3, nsp4, nsp6, nsp12, helicase, ORF3a, ORF8, and N sub-sets. A total of 22 predicted N-glycosylation positions were found in the SARS-CoV-2 surface glycoprotein; one of them, 343N, was located within the RBD. One predicted N-glycosylation site was found in the M protein and 4 potential O-glycosylation sites in specific protein 3a sequences. CONCLUSION: Overall, the data showed positive pressure and mutations acting on specific protein coding genes. These findings may provide useful information on: i) markers for vaccine design, ii) new therapeutic approach, iii) information to implement mutagenesis experiments to inhibit SARS-CoV-2 cell entry. The negative selection identified in SARS-CoV-2 protein coding genes may help the identification of highly conserved regions useful to implement new future diagnostic protocols.