A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal ...domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate-based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies.
The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the ...effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody
. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab
, S2X259
and S2H97
. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.
Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. ...In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.
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•SARS-CoV-2 RBD is immunodominant and accounts for 90% of serum neutralizing activity•RBD antibodies decline with a half-life of ∼50 days, but their avidity increases•Structural definition of a SARS-CoV-2 RBD antigenic map using monoclonal antibodies•ACE2-binding site dominates SARS-CoV-2 polyclonal neutralizing antibody responses
Serological analyses of ∼650 SARS-CoV-2-exposed individuals show that 90% of the serum or plasma neutralizing activity targets the virus receptor-binding domain, with structural insights revealing how distinct types of neutralizing antibodies targeting the ACE2-binding site dominate the immune response against SARS-CoV-2 spike.
Advanced breast cancer (BC) impact immune cells in the blood but whether such effects may reflect the presence of early BC and its therapeutic management remains elusive.
To address this question, we ...used multiparametric flow cytometry to analyze circulating leukocytes in patients with early BC (n = 13) at the time of diagnosis, after surgery, and after adjuvant radiotherapy, compared to healthy individuals. Data were analyzed using a minimally supervised approach based on FlowSOM algorithm and validated manually.
At the time of diagnosis, BC patients have an increased frequency of CD117
CD11b
granulocytes, which was significantly reduced after tumor removal. Adjuvant radiotherapy increased the frequency of CD45RO
memory CD4
T cells and CD4
regulatory T cells. FlowSOM algorithm analysis revealed several unanticipated populations, including cells negative for all markers tested, CD11b
CD15
, CD3
CD4
CD8
, CD3
CD4
CD8
, and CD3
CD8
CD127
CD45RO
cells, associated with BC or radiotherapy.
This study revealed changes in blood leukocytes associated with primary BC, surgical removal, and adjuvant radiotherapy. Specifically, it identified increased levels of CD117
granulocytes, memory, and regulatory CD4
T cells as potential biomarkers of BC and radiotherapy, respectively. Importantly, the study demonstrates the value of unsupervised analysis of complex flow cytometry data to unravel new cell populations of potential clinical relevance.
Introduction
Circulating tumor DNA (ctDNA) correlates with the response to therapy in different types of cancer. However, in patients with locally advanced rectal cancer (LARC), little is known about ...how ctDNA levels change with neoadjuvant chemoradiation (Na-ChRT) and how they correlate with treatment response. This work aimed to explore the value of serial liquid biopsies in monitoring response after Na-ChRT with the hypothesis that this could become a reliable biomarker to identify patients with a complete response, candidates for non-operative management.
Materials and Methods
Twenty-five consecutive LARC patients undergoing long-term Na-ChRT therapy were included. Applying next-generation sequencing (NGS), we characterized DNA extracted from formalin-fixed paraffin embedded diagnostic biopsy and resection tissue and plasma ctDNA collected at the following time points: the first and last days of radiotherapy (T
0
, T
end
), at 4 (T
4
), 7 (T
7
) weeks after radiotherapy, on the day of surgery (T
op
), and 3–7 days after surgery (T
post-op
). On the day of surgery, a mesenteric vein sample was also collected (T
IMV
). The relationship between the ctDNA at those time-points and the tumor regression grade (TRG) of the surgical specimen was statistically explored.
Results
We found no association between the disappearance of ctDNA mutations in plasma samples and pathological complete response (TRG1) as ctDNA was undetectable in the majority of patients from Tend on. However, we observed that the poor (TRG 4) response to Na-ChRT was significantly associated with a positive liquid biopsy at the T
op
.
Conclusions
ctDNA evaluation by NGS technology may identify LARC patients with poor response to Na-ChRT. In contrast, this technique does not seem useful for identifying patients prone to developing a complete response.
Objective: Primary uterine leiomyosarcomas(ULMS) are rare,and the optimal treatment is controversial.We aimed to assess the outcome and prognostic factors in a multicenter population of women treated ...for primary ULMS.Methods: We retrospectively collected data of 110 women treated in 19 institutions of the Rare Cancer Network(RCN).Inclusion criteria consisted of a pathology report confirming the diagnosis of ULMS,aged 18–80 years,complete International Federation of Gynecology and Obstetrics(FIGO) stage information,complete information on treatment,and a minimum follow-up of 6 months.Local control(LC) and locoregional control(LRC),overall survival(OS) and disease-free survival(DFS) rates were computed using the Kaplan-Meier method.Univariate analysis was implemented using the log rank test,and multivariate analysis using the Cox model.Results: All patients underwent surgery.Seventy-five patients(68%) received adjuvant radiotherapy(RT),including brachytherapy in 18(16%).Seventeen patients(15%) received adjuvant chemotherapy.Median follow-up was 58(range,6–240) months.Five-year OS and DFS rates were 50% and 34%,and LC and LRC rates were 88%and 72%,respectively.On multivariate analysis,independent favorable prognostic factors were younger age,FIGO stage I,small tumor size,previous uterine disease,and no vascular invasion for OS and DFS.FIGO stage was the only favorable factor influencing LRC.Adjuvant local or systemic treatments did not improve the outcomes.Eight patients treated with RT presented a grade 3 acute toxicity,and only one patient with grade 3 late toxicity.Conclusions: In this large population of primary ULMS patients,we found good results in terms of LC and LRC.Nevertheless,OS remains poor,mainly due to the occurrence of distant metastases.An early diagnosis seemed to improve the prognosis of the patients.Adjuvant local or systemic treatments,or more aggressive surgicalprocedures such as the Wertheim procedure,did not seem to impact the outcome.
The identification of CD4
T cell epitopes is instrumental for the design of subunit vaccines for broad protection against coronaviruses. Here we demonstrate in COVID-19-recovered individuals a robust ...CD4
T cell response to naturally processed SARS-CoV-2 spike (S) and nucleoprotein (N), including effector, helper, and memory T cells. By characterizing 2943 S-reactive T cell clones from 34 individuals, we found that 34% of clones and 93% of individuals recognized a conserved immunodominant S346-365 region within the RBD comprising nested HLA-DR- and HLA-DP-restricted epitopes. Using pre- and post-COVID-19 samples and S proteins from endemic coronaviruses, we identify cross-reactive T cells targeting multiple S protein sites. The immunodominant and cross-reactive epitopes identified can inform vaccination strategies to counteract emerging SARS-CoV-2 variants.
To assess feasibility and safety of conventionally fractionated radiotherapy (cfRT) in patients with hepatocellular carcinoma (HCC).
Patients with histologically confirmed stage cT1-4, cN0-1 HCC and ...Child-Pugh Score (CPS) A or B disease were included in a phase I multicenter trial. Metastatic HCC were allowed if ≥90% of total tumor volume was located within the liver. Patients were enrolled onto five dose-escalation levels (54-70Gy in 2Gy fractions) based on a modified 3 + 3 design, with cohorts of five patients instead of three patients in dose levels 4 and 5. Primary trial endpoint was dose-limiting toxicity (DLT), as specifically defined for 17 clinical and nine laboratory parameters as grade ≥3 or ≥4 toxicity (CTCAE vs. 3). The threshold to declare a dose level as maximum tolerated dose (MTD) was defined as a DLT rate of ≤16.7% in dose levels 1-3, and ≤10% in dose levels 4-5. Best objective response of target liver lesions and adverse events (AE's) were assessed as secondary endpoints.
The trial was terminated early in DL 3 due to low accrual. Nineteen patients were recruited. Fifteen patients were evaluable for the primary and 18 for the secondary endpoints. Maximum tolerated dose was not reached. One patient in dose level 1, and one patient in dose level 2 experienced DLT (lipase > 5xULN, and neutrophils <500/μL respectively). However, dose level 3 (62Gy) was completed, with no DLTs in 3 patients. Overall, 56% of patients had a partial response and 28% showed stable disease according to RECIST. No signs of radiation induced liver disease (RILD). Two patients in dose level 3 experienced lymphocytopenia grade 4, with no clinical impact.
Conventionally fractionated radiotherapy of 58Gy to even large HCC was safe for patients with CPS A and B. 62Gy was delivered to three patients without any sign of clinically relevant increased toxicity. The maximum tolerated dose could not be determined.
ClinicalTrials.gov identifier NCT00777894 , registered October 21st, 2008.
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 time frame. 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 prefusion 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 sublineages, 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.
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•SARS-CoV-2 infection and vaccines induce specific memory B cells that reach steady-state levels•mRNA COVID-19 vaccines elicit prefusion Spike-specific memory B cell responses•Postfusion Spike-specific memory B cells cross-react with human betacoronaviruses•Antibody avidity rises over time raising resilience to escape by variants of concern
Immunology; Virology
Introduction:
Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the androgen receptor (AR), through ACE2 receptor and TMPRSS2, to enter nasal and upper airways epithelial cells. Genetic ...analyses revealed that
HSD3B1
P1245C polymorphic variant increases dihydrotestosterone production and upregulation of TMPRSS2 with respect to P1245A variant, thus possibly influencing SARS-CoV-2 infection. Our aim was to characterize the
HSD3B1
polymorphism status and its potential association with clinical outcomes in hospitalized patients with COVID-19 in Southern Switzerland.
Materials and Methods:
The cohort included 400 patients hospitalized for COVID-19 during the first wave between February and May 2020 in two different hospitals of Canton Ticino. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue blocks, and
HSD3B1
gene polymorphism was evaluated by Sanger sequencing. Statistical associations were verified using different test.
Results:
HSD3B1
polymorphic variants were not associated with a single classical factor related to worse clinical prognosis in hospitalized patients with SARS-CoV-2. However, in specific subgroups,
HSD3B1
variants played a clinical role: intensive care unit admission was more probable in patients with P1245C diabetes compared with P1245A individuals without this comorbidity and death was more associated with hypertensive P1245A>C cases than patients with P1245A diabetes without hypertension.
Discussion:
This is the first study showing that
HSD3B1
gene status may influence the severity of SARS-CoV-2 infection. If confirmed, our results could lead to the introduction of
HSD3B1
gene status analysis in patients infected with SARS-CoV-2 to predict clinical outcome.
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