key: cord-0767582-fwry91bz authors: Gidari, Anna; Sabbatini, Samuele; Bastianelli, Sabrina; Pierucci, Sara; Busti, Chiara; Monari, Claudia; Pasqua, Barbara Luciani; Dragoni, Filippo; Schiaroli, Elisabetta; Zazzi, Maurizio; Francisci, Daniela title: Cross-neutralization of SARS-CoV-2 B.1.1.7 and P.1 variants in vaccinated, convalescent and P.1 infected date: 2021-07-25 journal: J Infect DOI: 10.1016/j.jinf.2021.07.019 sha: 1f99672d6492e267738691f2b339e3ee75867afb doc_id: 767582 cord_uid: fwry91bz OBJECTIVES: : The emergence of new variants of concern (VOCs) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) around the world significantly complicated the exit from Coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to evaluate the serum neutralizing activity of three cohorts. METHODS: : BNT162b2-elicited serum (N=103), candidates as hyper-immune plasma donors (N=90) and patients infected with the SARS-CoV-2 P.1 variant (N=22) were enrolled. Three strains of SARS-CoV-2 have been tested: 20A.EU1, B.1.1.7 (alpha) and P.1 (gamma). Neutralizing antibodies (NT-Abs) titers against SARS-CoV-2 were evaluated. RESULTS: : B.1.1.7 and P.1 are less efficiently neutralized by convalescent wild-type infected serums if compared to 20A.EU1 strain (mean titer 1.6 and 6.7-fold lower respectively). BNT162b2 vaccine-elicited human sera show an equivalent neutralization potency on the B.1.1.7 but it is significantly lower for the P.1 variant (mean titer 3.3-fold lower). Convalescent P.1 patients are less protected from other SARS-CoV-2 strains with an important reduction of neutralizing antibodies against 20A.EU1 and B.1.1.7, about 12.2 and 10.9-fold, respectively. CONCLUSIONS: : BNT162b2 vaccine confers immunity against all the tested VOCs, while previous SARS-CoV-2 infection may be less protective. More than one year after the declaration of Coronavirus disease 2019 (COVID-19) as a pandemic 1 , severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still spreading around the world causing serious public health concerns. A number of effective vaccines are being administered at an unprecedented pace, decreasing the incidence and severe consequences of SARS-CoV-2 infection. However, massive and prolonged worldwide replication let SARS-CoV-2 rapidly explore its genetic space and new variants of concern (VOCs) eventually emerged by the end of 2020. Briefly, VOC The vaccination campaign started by the end of December 2020 with the administration of BNT162b2 (Comirnaty® -BioNTech / Pfizer) COVID-19 mRNA vaccine, firstly to healthcare workers. The original aim of this study was to evaluate the dynamics and duration of SARS-CoV-2 neutralizing activity of BNT162b2-elicited antibody. The subsequent emergence of the different VOCs allowed us to expand the study design and analyze the cross-neutralization pattern with the different variants by using sera from subjects vaccinated with BNT162b2 or infected with different virus lineages. In particular, samples from patients infected with the SARS-CoV-2 P.1 variant have been included in the study together with the ones having a history of SARS-CoV-2 infection from June to October 2020 and candidates as hyper-immune plasma donors. Design, setting and participants. All experiments were performed using three SARS-CoV-2 strains isolated in our Biosafety Level 3 (BSL3) virology laboratory at Santa Maria della Misericordia Hospital, Perugia, Italy, as previously described 6 . Briefly, the transport medium (UTM) of a nasopharyngeal swab was incubated with a 1:1 nystatin (10,000 U/mL) and penicillin-streptomycin (10,000 U/mL) mixture for 1 h at 4°C to remove bacterial/fungal contamination. The suspension was centrifuged at 400 × g for 10 min, and the supernatant was inoculated on an African green monkey kidney clone E6 (Vero E6) cells monolayer maintained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C with 5% CO 2 . The viral titer in the supernatant was determined by Half-maximal Tissue Culture Infectious Dose (TCID50) endpoint dilution assay 7 and stock aliquots were stored at -80°C. Whole-genome sequencing of multiple isolates was used to identify a SARS-CoV-2 genome belonging to clade 20A.EU1 (lineage B.1) and clustered with viruses circulating in Italy in spring 2020, a SARS-CoV-2 genome belonging to clade B.1.1.7 (better known as alpha variant), and a SARS-CoV-2 genome belonging to clade P.1 (also known as gamma variant) 8 . The SARS-CoV-2 clade 20A.EU1 (lineage B.1) strain was isolated in May 2020 from a symptomatic patient during the first wave of infections. SARS-CoV-2 clade B.1.1.7 and P.1 were isolated on January 2021 during the third wave. Single virus stock aliquots were thawed immediately before each experiment and discarded after use. Neutralizing antibodies (NT-Abs) titers against SARS-CoV-2 were evaluated using flat-bottom tissue culture 96-well microtiter plates as previously published 9 . One day prior to the experiment, Vero E6 cells (2.5 × 10 4 /well) were cultivated in MEM + 10% FBS in a 96-well plate incubated at 37°C + 5% CO 2 . Serum samples of each study cohort were heat-inactivated for 30 min at 56°C before to be two-fold serially diluted from 1:10 to 1:640 with MEM + 2% FBS and seeded into 96well microtiter plates. Each sample was tested in duplicate and mixed with an equal volume of medium containing 50 TCID50 of SARS-CoV-2 selected strains and the plates were incubated for 30 min at 37°C. Sera with known neutralization titer and medium were used as positive and negative control, respectively. Subsequently, the mixtures were transferred to Vero E6 cell containing plates and incubated for 72 h at 37°C + 5% CO 2 . Then, the medium was removed and the cells were fixed and stained with 0.25% crystal violet and 10% formalin for 30 min at room temperature. The plates were washed to remove excess staining and the absorbance at 595nm was measured using a microtiter plate reader (Multiskan Fc Photometer, Thermo Fisher Scientific). The neutralizing titer was determined as the maximum dilution showing reduction ≥ 90% of CPE respect to the virus control. As shown in Figure 1 A-C, three cohorts of subjects were enrolled in this study. This cohort was composed of 103 healthcare workers vaccinated with the BNT162b2 COVID-19 mRNA vaccine (Figure 1A) . We postulated that patients with a history of SARS-CoV-2 infection acquired from June to October that time 4 . We enrolled 90 convalescent patients as candidates as hyper-immune plasma donors ( Figure 1B) . CoV-2 infection. The distribution of NT-Ab titers is shown in Figure 2B , F. In detail, the median NT-Ab titer was (Figure 2C, G) . The median NT-Ab titer was significantly higher for 20A than for B.1.1.7 (mean titer 1.8-fold higher) and P.1 (mean titer 8.5-fold higher) (p<0.0001) and it was higher for B.1.1.7 than for P.1 (mean titer 4.3-fold mean titer) (p<0.0001). Twenty-two patients with ascertained SARS-CoV-2 P.1 infection acquired from 21st November 2020 to 8th February 2021 in the Umbria region were enrolled, including 5 health care workers who already had a dose of BNT162b2 vaccine (Figure 1C ). Two patients were tested at multiple time points, one 2 and the other 3 times. Each measure was considered separately, therefore, a total of 25 samples were considered for the analysis. The mean age was 61.1 ± 19. The distribution of the 25 sera according to NT-Ab titers is shown in Figure 2D , H. No correlation between NT-Ab titre and time passed since diagnosis was found for any of the three lineages. The 23 . Indeed, in our study, vaccine sera neutralized both 20A and B.1.1.7 strains with the same efficacy. We separately analyzed a subgroup of high NT-Abs titer convalescent patients (NT-Ab≥1:160) and we found that these sera could not be considered as hyperimmune to B.1.1.7 and P.1 VOCs because they were significantly less effective in neutralizing these variants. Thus, the emergence of VOCs can abrogate this therapeutic option when the SARS-CoV-2 lineage of donors and recipient do not match. To the best of our knowledge, this is the first study that evaluated neutralizing activity of sera from P.1 variant infected patients to other SARS-CoV-2 lineages. As expected, we found an important reduction of NT-Abs against 20A and B.1.1.7 strains (12.2 and 10.9-fold, respectively). Considering that patients with a previous wild-type infection had a titer reduction of 6.7 and 1.58-fold on P.1 and B.1.1.7, it appears that patients with a previous P.1 infection are less protected from further SARS-CoV-2 reinfections from other variants. The impact of VOCs on serum neutralization activity and protection from SARS-CoV-2 Nothing to declare. World Health Organization. General's opening remarks at the media briefing on COVID-19 SARS-CoV-2 Variant Classifications and Definitions Is recurrence possible in coronavirus disease 2019 (COVID-19)? 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Patients selection Flow chart. Coronavirus disease Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2; Neutralizing antibodies NT-Abs titers against SARS-CoV-2 were evaluated using flat-bottom tissue culture 96-well microtiter plate serum dilution assay. Serums have been tested against 20A.EU1, B.1.1.7 and P.1 strains isolated from symptomatic patients with Coronavirus disease 2019 (COVID-19). Panels A, B, C and D show NT-Abs titers distribution for the three strains. Panels E, F, G and H show how NT-Abs serum titers of each patient change for the three variants Thanks to the healthcare workers of Santa Maria della Misericordia Hospital that spontaneously participated to this study, once again their contribute was crucial.