Optimizing the Illumina COVIDSeq laboratorial and bioinformatics pipeline on thousands of samples for SARS-CoV-2 Variants of Concern tracking.

The SARS-CoV-2 Variants of Concern tracking via Whole Genome Sequencing represents a pillar of public health measures for the containment of the pandemic. The ability to track down the lineage distribution on a local and global scale leads to a better understanding of immune escape and to adopting interventions to contain novel outbreaks. This scenario poses a challenge for NGS laboratories worldwide that are pressed to have both a faster turnaround time and a high-throughput processing of swabs for sequencing and analysis. In this study, we present an optimization of the Illumina COVID-seq protocol carried out on thousands of SARS-CoV-2 samples at the wet and dry level. We discuss the unique challenges related to processing hundreds of swabs per week such as the tradeoff between ultra-high sensitivity and negative contamination levels, cost efficiency and bioinformatics quality metrics.

Rapid decline of humoral response to two doses of BNT162b2 vaccine in patients with solid cancer after six months: The urgent need of the additional dose!

INTRODUCTION: We previously reported on the immunogenicity and safety of BNT162b2 in a large cohort of patients with cancer after the first and second doses (Di Noia et al., 2021) [1]. Herein, we present result after six months of follow-up. METHODS: This prospective study included patients affected by solid tumors and afferent to our institution who received two doses of BNT162b2 vaccine. A cohort of vaccinated healthcare workers (HCW) was used as control-group. Both cohorts were evaluated for the titer of anti-Spike (S) IgG at prefixed time-points (TPs). Time-point 4 was scheduled at 24-26 weeks after the second dose. RESULTS: In the current analysis, 400 patients and 232 healthcare workers were evaluated. Responders (IgG > 15 AU/mL) in patients group were 86.5% compared with 94.4% among healthcare workers. Also the IgG titer at TP4 was significantly inferior in patients than in healthcare workers (70.81 vs 134.64 AU/ml, p < 0.001). There was a more rapid decline of the antibody level from TP3 to TP4 in patients than in healthcare workers (1.78 vs 1.3 fold). The estimated IgG half-life was significantly shorter for patients (73 days) than in healthcare workers (118 days) as well as the time to reach negative serological status (340 vs 532 days). CONCLUSION: The decline of humoral response to the vaccine observed in patients with solid cancer after six months from the first dose support the urgent need of an early additional dose.

Antibody Persistence 6 Months Post-Vaccination with BNT162b2 among Health Care Workers.

BACKGROUND: We present immunogenicity data 6 months after the first dose of BNT162b2 in correlation with age, gender, BMI, comorbidities and previous SARS-CoV-2 infection. METHODS: An immunogenicity evaluation was carried out among health care workers (HCW) vaccinated at the Istituti Fisioterapici Ospitalieri (IFO). All HCW were asked to be vaccine by the national vaccine campaign at the beginning of 2021. Serum samples were collected on day 1 just prior to the first dose of the vaccine and on day 21 just prior to the second vaccination dose. Thereafter sera samples were collected 28, 49, 84 and 168 days after the first dose of BNT162b2. Quantitative measurement of IgG antibodies against S1/S2 antigens of SARS-CoV-2 was performed with a commercial chemiluminescent immunoassay. RESULTS: Two hundred seventy-four HWCs were analyzed, 175 women (63.9%) and 99 men (36.1%). The maximum antibody geometric mean concentration (AbGMC) was reached at T2 (299.89 AU/mL; 95% CI: 263.53-339.52) with a significant increase compared to baseline (p < 0.0001). Thereafter, a progressive decrease was observed. At T5, a median decrease of 59.6% in COVID-19 negative, and of 67.8% in COVID-19 positive individuals were identified with respect to the highest antibody response. At T1, age and previous COVID-19 were associated with differences in antibody response, while at T2 and T3 differences in immune response were associated with age, gender and previous COVID-19. At T4 and T5, only COVID-19 positive participants demonstrated a greater antibody response, whereas no other variables seemed to influence antibody levels. CONCLUSIONS: Overall our study clearly shows antibody persistence at 6 months, albeit with a certain decline. Thus, the use of this vaccine in addressing the COVID-19 pandemic is supported by our results that in turn open debate about the need for further boosts.

Design of a companion bioinformatic tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants.

BACKGROUND: Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. METHODS: Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. RESULTS: Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. CONCLUSIONS: This work provides a framework able to track down SARS-CoV-2 genomic variability.