Alpha (B.1.1.7) and Delta (B.1.617.2 - AY.40) SARS-CoV-2 variants present strong neutralization decay at M4 post-vaccination and a faster replication rates than D614G (B.1) lineage.

Dear Editor,

We read with interest the article by Dimeglio et al., showing the importance of the SARS-CoV-2 anti-S antibody response. Along with previous results showing a strong decrease of antibody and neutralization titers after vaccination, , especially for Gamma variant, their results highlight the usefulness of assessing antibody levels among HealthCare Workers (HCWs) and to monitor the effects of emerging SARS-CoV-2 variants. Likewise, the article by Douxfils et al., demonstrated post-vaccination decreasing neutralizing antibodies titers and highlighted the difficulty to obtain a definite protection threshold. They also could not verified the effects of Alpha and Delta variants. Recently, Goldberg et al., assessed Delta breakthrough infections among a large vaccinated population and demonstrated a decreasing protection in all age groups a few months after receiving a full vaccination scheme. To date, little data are still available regarding comparative neutralizing capability of antibodies against Alpha and Delta variants several months after vaccination schemes. Moreover, the infectious capacity of the Delta variant, that could explain in part its epidemiological success, is not characterized. In this study, we investigated the replicative cycle of Delta variant compared to the Alpha and B.1 variants. In addition, we also provided new elements and live virus neutralisation data that strengthen the analysis of vaccinated HCWs antibody responses up to 4 months after the second vaccine dose.

Replication kinetic was performed in triplicates on Vero E6 (ATCC, R CRL-1586) and A549 expressing ACE2 and TMPRSS2 receptors (InvivoGen, a549-hace2tpsa) by analyzing SARS-CoV-2 infectious titers, RNA and N antigen levels. The neutralizing titers of 45 sera from 9 BNT162b2 vaccinated HCWs, up to 5 months after the first vaccine dose, were analyzed by live virus neutralization assays with B.1 (EPI_ISL_4537783), Alpha (EPI_ISL_4536454) and Delta (EPI_ISL_4536228) strains. Decomplemented sera were subjected to serial two-fold dilutions (1:25 to 1:12800), incubated with 50μL of diluted virus at 2 × 103 PFU/mL in a 96-well plate at 37 °C, 5% CO2 for 60 min. Then 3 × 104 cells Vero E6 cells were added to each well before being incubated at 37 °C, 5% CO2.

Lower titers of infectious particles production were observed after 24 h for strain B.1 (titers comprised on VeroE6 from 2 to 220 PFU/mL for each replicate) than both Alpha and Delta strains (from 4000 to 40000 PFU/mL on VeroE6, p = 0.04) (Fig. 1 ). The viral RNA production in culture supernatants were also similar at 24h post-infection for Alpha and Delta variants but approximately 10 times higher than strain B.1 with both cell models (Vero E6: p = 0.02; A549: p = 0.03). Similar results were obtained with N antigen quantification but as soon as 15 h after cell infection with similar titers for Alpha and Delta variants and higher than with B.1 (Vero E6: p = 0.02; A549: p = 0.02). This difference last up to 48 h (Vero E6: p = 0.02; A549: p = 0.02) (Fig. 1). For all our measurements, a plateau with similar production levels was obtained for all variants since 72 hours.

Fig. 1

Evaluation of viral production kinetics for Delta (B.1.617.2 – AY.40) strain, in blue, Alpha (B.1.1.7), in green and B.1, in red. Viral production has been assessed on two cell lines, Vero E6 and A549, and by RT-qPCR for RNA viral load, plaque assay for infectious particles and ELISA for N antigen production assessment. Results were obtained in triplicates (indicated by dot shapes).

We also evaluated the level of antibodies neutralization for the Delta strain after full BNT162b2 vaccination. Live virus neutralization showed a maximum neutralization titer one month after the second dose of vaccine for all 9 HCWs. The median dilution of neutralizing sera were similar or slightly lower with Delta and Alpha, at 1:6400 [InterQuartile Range (IQR): 1:3200–1:6400] and 1:6400 [1:3200–1:12800], respectively, than with the B.1 strain, at 1:12800 [1:6400–1:12800]. Four months after the second dose of vaccine, all tested sera showed a decreasing neutralization activity, still slightly lower with Delta, at a median of 1:400 [IQR: 1:200–1:800], 1:100 [1:50–1:200] and 1:25 [1:25–1:50] for B.1, Alpha and Delta, respectively (Fig. 2 ).

Fig. 2

Seroneutralization titers obtained among vaccinated healthcare workers for B.1, Alpha (B.1.1.7) and Delta (B.1.617.2 – AY.40) strains up to 4 months after the second vaccine dose. The A panel present the seroneutralization titers kinetics for each vaccinated HCWs. The administration of the second dose, 30 days after the first dose, is indicated by the vertical dashed line. The B panel represent median and interquartiles at each evaluated time point (i.e. day 0, week 4, week 8 and week 20 after the first vaccine dose).

Many efforts are focusing on vaccines efficacy against various variants, , a cornerstone for public health policies. However, live-virus neutralization follow-up data are still scarce today. A recent work demonstrated a 3–5-fold decreased Delta susceptibility 5 weeks after the second BNT162b2 vaccine dose compared to Alpha variant. In the present study, we confirm and strengthen those results by observing strongly decreased neutralizing titers 4 months after the second BNT162b2 vaccine injection with Delta, but also Alpha and B.1 variants.

Other important concerns about Delta variant, especially regarding its epidemiological success, are the potential differences on the global viral fitness that has not been characterized to date. Here, we investigated the viral replication kinetics of B.1, Alpha and Delta variants. We evidenced a shorter replication cycle and a quicker production rate of infectious viral particles with both Alpha and Delta strains than with B.1. The 10-times higher in vitro replication levels from 24 to 48 h post-infection, observed with Alpha and Delta strains, are in line with its higher viral loads and 24 h earlier consultation observed among infected patients.8, 9, 10 The Delta variant, which quickly replaced the Alpha and other variants, presents the same in vitro replication profile than the Alpha strain (Fig. 1). Thus, if a shorter replication rate could have helped the Alpha strain to successfully emerge over historical variants, and could be a pre-requisite for future variant expansion, other factors must have participated to the emergence of Delta over Alpha. The lower sensitivity to neutralizing antibody is probably a part of this equation.

In conclusion, we highlight a shorter replication cycle and a quicker production of viral materials and infectious particles with Alpha and Delta lineages compared to B.1 lineage. This is expected to play a role and explain in part the higher viral loads, higher transmissibility, and the large epidemiological success of Alpha and Delta variants. We also report a decreased neutralizing titers of BNT162b2 vaccine elicited antibodies against Delta correlated with a decreased sera neutralizing activity four months after complete vaccine scheme in HCWs for all tested strains. These observations highlight the question of vaccine humoral protection lasting and enhance the need for close cellular immunity evaluations against new variants.

Funding

None declared.

CRediT authorship contribution statement

Samuel Lebourgeois: Conceptualization, Visualization, Investigation, Funding acquisition, Formal analysis, Data curation, Writing – original draft. Reyene Menidjel: Investigation, Formal analysis, Data curation, Visualization. Houssem Redha Chenane: Investigation, Formal analysis, Data curation, Visualization. Valentine Marie Ferré: Investigation, Formal analysis, Data curation, Visualization. Gilles Collin: Investigation, Formal analysis, Data curation, Visualization. Florence Damond: Visualization, Investigation, Writing – review & editing. Romain Coppée: Visualization, Investigation, Writing – review & editing. Yazdan Yazdanpanah: Visualization, Investigation, Writing – review & editing. Jean-François Timsit: Visualization, Investigation, Writing – review & editing. Nadhira Houhou-Fidouh: Conceptualization, Visualization, Funding acquisition, Supervision. Diane Descamps: Conceptualization, Visualization, Funding acquisition, Supervision. Charlotte Charpentier: Conceptualization, Visualization, Investigation, Funding acquisition, Data curation, Supervision, Writing – original draft, Writing – review & editing. Benoit Visseaux: Conceptualization, Visualization, Investigation, Funding acquisition, Data curation, Supervision, Writing – original draft, Writing – review & editing.

Declaration of Competing Interest

The authors have no relevant competing interest to disclose in relation to this work.