Broad and potent neutralizing antibodies are elicited in vaccinated individuals following Delta/BA.1 breakthrough infection

Despite the success of COVID-19 vaccines in preventing infection and/or severe disease, there has been an increase in SARS-CoV-2 infections in vaccinated individuals owing to the waning vaccine-derived immunity, and the emergence of new variants which encode escape mutations in Spike. Following breakthrough infection in vaccinated individuals, an increase in neutralization breadth has been observed in sera/plasma. However, how exposure to a heterologous Spike broadens the neutralizing response at the monoclonal antibody (mAb) level is not fully understood. Through isolation of 119 mAbs from three individuals receiving two doses of BNT162b2 vaccine before becoming Delta or Omicron/BA.1 infected, we show that serum breadth occurs due to the presence of somatically mutated mAbs with broad neutralization activity indicative of re-activation and maturation of B cells generated through previous COVID-19 vaccination. Isolated mAbs frequently show reduced neutralization of current circulating variants including BA.2.75.2, XBB, XBB.1.5, and BQ.1.1 confirming continuous selective pressure on Spike to evolve and evade neutralization. However, isolation of mAbs that display effective cross-neutralization against all variants indicates the presence of conserved epitopes on the receptor binding domain and a lesser extent the N-terminal domain. These findings have implications for the selection of Spike antigens for next-generation COVID-19 vaccines. IMPORTANCE With the emergence of SARS-CoV-2 viral variants, there has been an increase in infections in vaccinated individuals. Here, we isolated monoclonal antibodies (mAbs) from individuals experiencing a breakthrough infection (Delta or BA.1) to determine how exposure to a heterologous Spike broadens the neutralizing antibody response at the monoclonal level. All mAbs isolated had reactivity to the Spike of the vaccine and infection variant. While many mAbs showed reduced neutralization of current circulating variants, we identified mAbs with broad and potent neutralization of BA.2.75.2, XBB, XBB.1.5, and BQ.1.1 indicating the presence of conserved epitopes on Spike. These results indicate that variant-based vaccine boosters have the potential to broaden the vaccine response.</p

Higher percentage of SARS-CoV-2 Spike-reactive memory B cells are detected in the extended booster group.

Percentage of Spike-reactive memory B cells were determined by flow cytometry. A) Example pre-bleed (baseline) PBMC staining for SARS-CoV-2 naïve individual. B) Example post 2nd vaccine PBMC staining for SARS-CoV-2 naïve individual. Example of full flow analysis shown if S1A Fig. C) Percentage Spike-reactive memory B cells at baseline and post 2nd vaccine were determined by flow cytometry. Previously infected individuals shown in red/grey. Extended and short interval groups shown with triangle and circle symbols, respectively. D’Agostino and Pearson tests were performed to determine normality. Based on this result, differences between groups were assessed using a Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons post hoc. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001. D) Percentage of IgG, IgM or other Spike-reactive memory B cells for each vaccine group. E) Correlation between Spike IgG ED50 and percentage of Spike-reactive memory B cells. F) Correlation between ID50 against Wuhan-1 pseudotyped virus and percentage of Spike-reactive memory B cells. (Spearman’s correlation, r; a linear regression was used to calculate the goodness of fit, r2).</p

SARS-CoV-2 Spike-reactive memory B cells measured before and after vaccination.

A) Example FACS gating for pre-vaccination sample (baseline) and from post-vaccination sample (post 2nd) from SARS-CoV-2 naïve individual. B) Frequency of memory B cells in the total B cell population at baseline and post 2nd vaccine for matched donors. (TIF)</p

Neutralization against omicron sub-lineages following 3 vaccine doses and/or BA.1/BA.2 breakthrough infection.

Neutralization breadth and potency was measured against D614G, BA.1, BA.2 and BA.4/BA.5. Analysis of sera collected A) 3-weeks post 3rd BNT162b2 vaccine dose (post 3rd), B) 6-months post 3rd vaccine (6m-post 3rd) and C) following breakthrough infection (post-BTI) (presumed to be BA.1 or BA.2). D) Comparison of titres 3-weeks (post 3rd) and 6-months post 3rd boost (6m-post 3rd). E) Comparison of titres 3-weeks post 3rd boost (post 3rd) and following BTI (post-BTI). Samples from a single individual are joined. Short and long booster groups shown with circle and triangle symbols, respectively. Individuals infected with SARS-CoV-2 prior to vaccination are shaded grey. Based on this result, multiple Mann-Whitney tests or unpaired t tests using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli were employed to determine significance between groups. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001.</p