On the lookout for influenza viruses in Italy during the 2021-2022 season: Along came A(H3N2) viruses with a new phylogenetic makeup of their hemagglutinin.

AIMS: To assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy. MATERIALS AND METHODS: 12,393 respiratory specimens (nasopharyngeal swabs or broncho-alveolar lavages) collected from in/outpatients with influenza illness in the period spanning from January 1, 2022 (week 2022-01) to May 31, 2022 (week 2022-22) were analysed to identify IV genome and were molecularly characterized by 12 laboratories throughout Italy. A(H3N2) evolution was studied by conducting an in-depth phylogenetic analysis of the hemagglutinin (HA) gene sequences. The predicted vaccine efficacy (pVE) of vaccine strain against circulating A(H3N2) viruses was estimated using the sequence-based Pepitope model. RESULTS: The overall IV-positive rate was 7.2% (894/12,393), all were type A IVs. Almost all influenza A viruses (846/894; 94.6%) were H3N2 that circulated in Italy with a clear epidemic trend, with 10% positivity rate threshold crossed for six consecutive weeks from week 2022-11 to week 2022-16. According to the phylogenetic analysis of a subset of A(H3N2) strains (n=161), the study HA sequences were distributed into five different genetic clusters, all of them belonging to the clade 3C.2a, sub-clade 3C.2a1 and the genetic subgroup 3C.2a1b.2a.2. The selective pressure analysis of A(H3N2) sequences showed evidence of diversifying selection particularly in the amino acid position 156. The comparison between the predicted amino acid sequence of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and the study strains revealed 65 mutations in 59 HA amino acid positions, including the substitution H156S and Y159N in antigenic site B, within major antigenic sites adjacent to the receptor-binding site, suggesting the presence of drifted strains. According to the sequence-based Pepitope model, antigenic site B was the dominant antigenic site and the p(VE) against circulating A(H3N2) viruses was estimated to be -28.9%. DISCUSSION AND CONCLUSION: After a long period of very low IV activity since public health control measures have been introduced to face COVID-19 pandemic, along came A(H3N2) with a new phylogenetic makeup. Although the delayed 2021-2022 influenza season in Italy was characterized by a significant reduction of the width of the epidemic curve and in the intensity of the influenza activity compared to historical data, a marked genetic diversity of the HA of circulating A(H3N2) strains was observed. The identification of the H156S and Y159N substitutions within the main antigenic sites of most HA sequences also suggested the circulation of drifted variants with respect to the 2021-2022 vaccine strain. Molecular surveillance plays a critical role in the influenza surveillance architecture and it has to be strengthened also at local level to timely assess vaccine effectiveness and detect novel strains with potential impact on public health.

Six months SARS-CoV-2 serology in a cohort of mRNA vaccinated subjects over 90 years old.

Ageing is associated with a progressive decline and remodelling of the immune system. Also, the efficacy of COVID-19 vaccines has been observed to depend on subjects' age. The post-vaccination data about patients aged > 90 years old is scarcely represented in the literature. The antibody titre profiles of elderly vaccinated subjects (age > 90 years old) were evaluated and compared with profiles obtained in a younger population (age 23-69 years old). To the best of our knowledge, this is the first report providing post-vaccination serological data in subjects aged 90 + years old. This study suggests that distinct SARS-CoV-2 viral-specific antibody response profiles vary based on anti-N serostatus, age, and sex in the very elderly adults. The data obtained could impact the organisation of the vaccination campaign (i.e., prioritisation strategies, administration of additional doses) and the factors that facilitate intentions to receive the vaccination among elderly adults (i.e., vaccine effectiveness).

Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA.

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA.

Harmonization of six quantitative SARS-CoV-2 serological assays using sera of vaccinated subjects.

BACKGROUND AND AIMS: Vaccines, to limit SARS-CoV-2 infection, were produced and reliable assays are needed for their evaluation. The WHO produced an International-Standard (WHO-IS) to facilitate the standardization/comparison of serological methods. The WHO-IS, produced in limited amount, was never tested for reproducibility. This study aims at developing a reproducible and accessible working standard (WS) to complement the WHO-IS. MATERIALS AND METHODS: Sera from vaccinated individuals were used to produce the WSs. The WHO-IS, the WSs and single serum samples (n = 48) were tested on 6 quantitative serological devices. Neutralization assays were performed for the 48 samples and compared with their antibody titers. RESULTS: The WS carry an antibody titer 20-fold higher than the WHO-IS. It was reproducible, showed both good linearity and insignificant intra- and inter-laboratory variability. However, the WSs behave differently from the WHO-IS. Analysis of the 48 samples showed that single correlation factors are not sufficient to harmonize results from different assays. Yet, all the devices predict neutralization activity based on the antibody titer. CONCLUSIONS: A reproducible and highly concentrated WS, specific for IgG against SARS-CoV-2 Spike-glycoprotein was produced. Such characteristics make it particularly suited for the harmonization of commercially available assays and the consequent evaluation of post-vaccinated individuals.

Diagnosis of COVID-19 in children guided by lack of fever and exposure to SARS-CoV-2.

BACKGROUND: The objective of this study is to test how certain signs and symptoms related to COVID-19 in children predict the positivity or negativity of the SARS-CoV-2 nasopharyngeal swab in children. METHODS: We review the data of children who were tested for SARS-CoV-2 for a suspected infection. We compared the clinical characteristics of the subjects who tested positive and negative, including the sensibility, positive and negative predictive value of different combination of signs and symptoms. RESULTS: Of all the suspected infected, 2596 tested negative (96.2%) and 103 tested positive (3.8%). The median age was 7.0 and 5.3 years for the positive and negative ones, respectively. The female to male ratio was ~1:1.3. Fever and respiratory symptoms were mostly reported. Most positive children had a prior exposure to SARS-CoV-2-infected subjects (59.2%). A total of 99.3% of patients without fever nor exposure to the virus proved negative to the SARS-CoV-2 test. CONCLUSIONS: Our study suggests that a child without fever or contact with infected subjects is SARS-CoV-2 negative. If this were to be confirmed, many resources would be spared, with improved care of both COVID-19 and not COVID-19-affected children. IMPACT: Key message: lack of fever and exposure to SARS-CoV-2-infected people highly predicts a negative results of the SARS-CoV-2 nasopharyngeal swab in the paediatric population. Added value to the current literature: this is the first article to prove this point. IMPACT: reduction of emergency department accesses of children with suspected SARS-CoV-2 infection; increased outpatient management of children with cough or other common respiratory symptoms of infancy; sparing of many human and material health resources.

School in Italy: a safe place for children and adolescents.

BACKGROUND: During the first SARS-CoV-2 pandemic phase, the sudden closure of schools was one of the main measures to minimize the spread of the virus. In the second phase, several safety procedures were implemented to avoid school closure. To evaluate if the school is a safe place, students and staff of two school complexes of Rome were monitored to evaluate the efficacy of prevention measures inside the school buildings. METHODS: Oral secretions specimens were collected from 1262 subjects for a total of 3431 samples, collected over a 3 months period. Detection of Coronavirus SARS-CoV-2 was performed by real-time PCR. Target genes were represented by E gene, RdRP/S gene and N gene. RESULTS: Among the 3431 samples analyzed, just 16 sample resulted as positive or low positive: 1 sample in the first month, 12 samples in the second month and 3 in the third month. In each period of evaluation, all positive children attended different classes. CONCLUSIONS: Even if the school has the potential for spreading viruses, our preliminary results show the efficacy of the implementations undertaken in this setting to minimize virus diffusion. Our evidence suggests that school does not act as an amplifier for transmission of SARS-CoV-2 and can be really considered a safe place for students.