key: cord-0877781-92rq5pvm authors: Garcia-Valtanen, Pablo; Hope, Christopher M.; Masavuli, Makutiro G.; Yeow, Arthur Eng Lip; Balachandran, Harikrishnan; Mekonnen, Zelalem A.; Al-Delfi, Zahraa; Abayasingam, Arunasingam; Agapiou, David; Stella, Alberto Ospina; Aggarwal, Anupriya; Bouras, George; Gummow, Jason; Ferguson, Catherine; O’Connor, Stephanie; McCartney, Erin M.; Lynn, David J.; Maddern, Guy; Gowans, Eric J.; Reddi, Benjamin AJ.; Shaw, David; Kok-Lim, Chuan; Beard, Michael R.; Weiskopf, Daniela; Sette, Alessandro; Turville, Stuart G.; Bull, Rowena A.; Barry, Simon C.; Grubor-Bauk., Branka title: SARS-CoV-2 Omicron variant escapes neutralising antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents. date: 2022-05-17 journal: Cell Rep Med DOI: 10.1016/j.xcrm.2022.100651 sha: 91baa5fde5ed176d36c4ce22ee60f478dd534c80 doc_id: 877781 cord_uid: 92rq5pvm COVID-19 convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with SARS-CoV-2. We evaluate humoral and T cell immunity against five Variants of Concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals and yet, serum neutralisation against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated Spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOC were impaired. These results indicate that increased antigen variability in VOCs impair humoral and Spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOC. Thus stressing the importance of vaccination programes. them the denomination variant of concern (VOC). 74 After primary infection and in parallel with the antibody response, symptomatic COVID-75 19 convalescents generate a robust CD4 + and CD8 + memory T cell response which targets a 76 wider range of antigens and epitopes than that covered by antibodies. 20-24 Importantly, the 77 breadth of SARS-CoV-2-specific T cell epitopes appears to be less sensitive to mutations 78 present in VOCs. 25, 26 It is unclear to what extent T cells can protect from re-infection and 79 progression to severe COVID-19. However, it is likely that T cell responses in convalescents, 80 which target most SARS-CoV-2 antigens, 20 could afford some of level of protection for many 81 J o u r n a l P r e -p r o o f months, even years. In fact, SARS-CoV-specific T cells can be detected in convalescents for 82 almost two decades. 27 83 While current vaccines are highly effective in preventing severe disease and death, and 84 booster vaccinations may temporarily circumvent dwindling efficacy over time, 28 next 85 generation vaccines, that can prevent virus transmission, are likely needed to end the 86 pandemic. 29, 30 Long-term studies of the evolution of immune correlates in convalescents, where the immune system has encountered an active live virus infection in 88 the presence of all its antigens, are necessary to elucidate the fine specificities and immune 89 functionality of antibody and T cell responses. In particular, the adaptability of pre-existing 90 immunity to mutated Spike antigens, present in VOC, is the key piece of information that is 91 still unanswered. 92 Compared to the majority of the world, South Australia is in an optimal position to 93 undertake studies on mid-to long-term immunity of COVID-19 due to 1) early and strict 94 border control measures with other countries, and other states within Australia, which were 95 enforced by health authorities in 2020-21, thus eliminating local transmission of the virus in 96 the community, and 2) South Australia has maintained a high testing rate with a total test 97 count of >2.2 M with only 899 positive cases of which only 9 were caused by unknown, locally-98 acquired contacts (accessed on 23/09/2021). 31 99 We present a COVID-19 immunity study at 12 months after PCR confirmed SARS-CoV-2 100 infection and in the complete absence of community transmission in a South Australian 101 cohort of 43 mild COVID-19 convalescents. An in-depth evaluation of multi-isotype antibody 102 responses, homologous pseudotyped virus, homologous and VOC live-virus serum 103 neutralisation activity, RBD-specific B cell populations and Spike and non-Spike SARS-CoV-2 104 J o u r n a l P r e -p r o o f specific CD8 + and CD4 + T cell immunity against ancestral and VOC antigenic epitopes, was 105 undertaken. Results were compared to age-and gender-matched COVID-19 naïve, healthy 106 individuals and to COVID-19 convalescent responses at 6 months after infection in the same 107 cohort. Longitudinal SARS-CoV-2 humoral responses in mild-COVID-19 convalescents. 110 The receptor binding domain (RBD) of SARS-CoV-2 Spike protein is the main target of 111 neutralising antibodies (nAb), and nAb titers decline in the months after COVID-19 infection. term SARS-CoV-2-specific humoral immunity 12 months post-infection. Spike-specific IgG + B 123 cells were also elevated in COVID-19 convalescents, but healthy control background 124 frequencies were also higher ( Figure S2 ), likely due to cross-reactivity. RBD-and Spike-specific 125 non-IgG + B cell frequencies were present a low rates (figure S2). 126 J o u r n a l P r e -p r o o f Obtaining long-term serum neutralisation data from communities free of circulating CoV-2, such as in this study, is difficult in other cohorts in the context of this pandemic. In our 128 cohort, sera in 64% of convalescents at 12 months yielded ID50 neutralisation titers 129 significantly above healthy background levels against the pseudotyped virus bearing a 130 Wuhan-like Spike protein, which is the same as the prevalent virus present in the community 131 when study participants were infected ( Figure 1D ). 132 Since early 2020, when the study participants were recruited, five VOCs, namely, Alpha Figure 1E ). Values were similar to those against pseudotyped 137 virus particles bearing the same Spike protein sequence in Figure 1D Spike-and non-Spike-specific T cell responses. 32 These T cells can be detected and quantified 152 by means of activation-induced marker (AIM) assays using SARS-CoV-2 antigen peptide pools 153 and flow cytometric analysis ( Figure S3 ). 20, 22, 23 For accurate interpretation in SARS-CoV-2 AIM 154 assays, naïve healthy controls must be included in the analysis to establish assay baseline 155 levels that arise from previous immunity to unrelated antigens/pathogens, and particularly to 156 seasonal human coronaviruses. 33 In our cohort, the relative frequency of CD4 + and CD8 + T 157 cells did not differ between the two time points (6 and 12 months) or between COVID-19 158 convalescents and healthy controls ( Figure S4A ). 159 AIM assays revealed that the frequency of circulating Spike-specific CD4 + T cells did not 160 significantly decrease between 6 and 12 moths post-infection (95% CI, from 0.35-0.91 at 5-6 161 months to 0.29-0.68) (Figure 2A ). The same trend was observed for non-Spike antigen CD4 + T 162 cells (from 0.22-0.43 to 0.18-0.37) and for the combined CD4 + T cell response, Spike + non-163 Spike (from 0.60-1.33 to 0.49-1.04) ( Figure 2A ). 164 Comparatively, the reduction of Spike-specific CD8 + T cell frequency over time was more 165 pronounced and statistically significant (95% CI, from 0.28-1.19 to 0.11-0.35, p<0.01) ( Figure 166 3B). No statistically significant differences were observed for the frequencies of CD8 + T cell 167 reacting to whole SARS-CoV-2 proteome pools A and B ( Figure 2B ). Convalescent AIM results 168 were compared to naïve healthy controls in all instances, corroborating significantly lower T 169 cell frequencies (baseline levels), consistent with previous AIM studies using the same peptide subset within CD8 + CD69 + CD137 + cells at 6 months (>65%) and at 12 months (>60%), 204 irrespective of antigen specificity ( Figure 4B ). 205 The presence of significant frequencies of SARS-CoV-2 antigen-specific CD4 + TEM and CD8 + 206 TEMRA at 12 months, has important implications, particularly in the absence of virus re-207 exposure, given that, in principle, these cells have a limited life-span. Importantly, a significant 208 decrease in these populations from 6 to 12 months suggests that in the absence of 209 restimulation by either infection or vaccination, these populations will eventually not be 210 detectable in circulation, or plateau at low frequencies for extended periods of time. In future, 211 the renewal mechanisms of both of these cell populations is an important question that needs 212 to be addressed. Functionality of Spike-specific T cells in 'high-responder' convalescents. For the context of this experiment and its results, it is important to note that our cohort was 257 infected in early 2020 in Australia, where SARS-CoV-2 with unmutated Spike amino acid 258 sequences was prevalent ( Figure 6A ). In order to clearly identify changes in functionality of T 259 cell immunity to Spike antigens present in VOCs, we calculated fold-change of cytokine Figure 6B ) and for IFNγ in CD8 + T cells compared to B.1.617.2 274 (p<0.001, Figure 6C ). Notably, GZMB and PRF1 expression in CD8 + T cells was completely 275 unaffected by changes in the Spike sequence ( Figure 6C ), suggesting that cellular cytotoxic 276 immune function may be most resistant to antigenic changes. 277 To further analyse our data, we used a cut-off fold-change value of -2, meaning a two-fold Figure S11C ). 290 Here, we report a loss of Spike-specific T cell functionality against VOCs, despite durable (12 291 months) maintainance in overall frequency, has been reported in COVID-19 convalescents. Circles represent AUC individual patient values (n=43 at 6 months, orange, and 12 months, yellow, 514 n=15 for healthy controls, blue), with mean value denoted by a horizontal black line. Seronegative 515 samples were assigned a value of 0.001 data visualisation purposes. 516 (C) SARS-CoV-2 RBD-specific (n=28) memory B cells (CD27 + ) were quantified 12 month post-infectoion 517 with corresponding specific tetramers and further characterised as IgG + . 518 Cell population-specific background ( Neutralisation activity was considered negative, value of zero, when neutralisation of initial serum 530 dilution was <50%. *, **, *** and **** denote P values < 0.05, 0.01, 0.001, 0.0001 respectively. ns = 531 not significant. The percentage of convalescents with neutralisation activity is indicated for each VOC 532 in the corresponding X-axis labels. 533 Spike-, non-Spike-and cumulative (Spike + non-Spike)-specific CD4 + T cells detected in Figure 2A . Cells 551 were classified as follows: naïve (TN, CCR7 + CD45RA + ), central memory (TCM, CCR7 + CD45RA -), effector 552 memory (TEM, CCR7 -CD45RA -) and terminally differentiated effector memory cells re-expressing 553 CD45RA (TEMRA, CCR7 -CD45RA + ). Frequencies are indicated as percentage of total SARS-CoV-2 antigen-554 specific CD4 + T cells within the total pool of immune cells with same phenotype in the patient's PBMCs. 555 (B) Doughnut charts indicating the proportion (%) of each immune phenotype in (A) of the total of 556 SARS-CoV-2-specific CD4 + T cells for each antigen. In (A) circles represent patient individual values. 557 Averages are denoted by a horizontal line and statistically significant differences between time points 558 indicated by asterisks. * and **denote P values < 0.05 and 0.01, respectively. ns = not significant. 559 Spike-, whole proteome pools A-and B-specific CD8+ T cells detected in Figure 2B . Immune 564 phenotypes were defined as in Figure 3 . Frequencies are indicated as percentage of total SARS-CoV-2 565 antigen-specific CD8+ T cells within the total pool of immune cells with same phenotype in the 566 patient's PBMCs. SARS-CoV-2 and CMV peptide megapools were kindly provided by Prof Alessandro Sette CD8-specific 733 peptide pools, 628 peptides restricted to the 12 most common HLA-A and HLA-B alleles and 734 partially covering the sequences of nsp1, nsp2, PLpro, nsp4, nsp6, nsp7, nucleocapsid 735 phosphoprotein, 3CL, nsp8, nsp9, nsp10, nsp14, RdRpol, Hel, nsp15, nsp16, surface 736 glycoprotein, ORF3a, ORF10, ORF6, ORF7a, ORF8, envelope protein, and membrane 737 glycoprotein were predicted in silico as previously described. 21 Peptides were divided into two 738 separate megapools, CD8_A and CD8_B. In Spike peptide pool, 15-mer peptides overlapping 739 by 10 amino acids and covering the entire Spike protein sequence were used (total of 253 740 peptides). For the non-Spike SARS-CoV-2 CD4 megapool 221 15-mer restricted to seven 741 common HLA-DR (Class II HLA) alleles and covering the entire SARS-CoV-2 proteome complete RPMI (cRPMI) medium 748 (40 U/mL penicillin, 40 ug/mL streptomycin, 2 mM L-Glutamine) with 5% (v/v) heat-749 inactivated human AB serum. Cells were then plated at 10 6 PBMC/well in u-bottom 96-well 750 plates and stimulated with 1 µg/mL of different SARS-CoV-2-megapools. Combined CD4 and 751 CD8 cytomegalovirus (CMV) megapool (1 µg/mL) and PHA 10µg/mL (Sigma Aldrich), were 752 included as positive controls. An equimolar amount of dimethyl sulfoxide (DMSO, vehicle) was 753 used as a negative control. PBMC were stimulated for 24 h at 37°C, 5% CO2 Cells were then washed and stained with (CD3 BUV737, CD4 BUV496, CD8 41-BB) BV421) for 20min, at room temperature in the dark. Fluorescence minus one (FMO) antigens: CD45RA, CCR7, CD134, CD69 and CD137 were added to PHA stimulated 759 cells. PBMC were washed and FACS Fix (0.4%PFA, 20g/L Glucose, Sodium Azide 0.02% in PBS) 760 was added for 20 min at room temperature, in the dark. Fixed cells were washed, 761 resuspended in FACS wash buffer and data was acquired on BD FACS Symphony. Data analysis 762 was performed using FCS Express TM (DeNovo Software Variant of concern Spike-specific peptide pools Gladbach, GER) were utilised to 767 test immune reactivity of COVID-19 convalescent T cells to mutated Spike epitopes present in 768 five VOCs. 26,75 All peptide pools consisted of 15-mer peptides with 11 aa overlap covering 769 Spike protein sequences affected by mutations in each VOC. Five VOC peptide pools 770 corresponding to mutated Spike sequences in SARS-CoV-2 variants B 34, 30, 41, 32 and 83 peptides, respectively) and five corresponding 772 control/reference pools with Wuhan aa sequences were compared in parallel along with 773 whole Spike peptide pool as positive control (pool described above). Mutations and deletions 774 represented in mutated pools are summarized in Table S1. For the assays, lyophilised peptides 775 were resuspended in sterile miliQ water as per the manufacturer's instructions at 30 nM (50 776 µg/mL Spike-specific T follicular helper cell quantification and intracellular cytokine staining in 778 Spike high responder convalescents Double responders (meeting criterion for both CD4 + and CD8 + T cells) and high CD4 + 782 with available PBMC samples were selected (n=15) for further analysis. Following methods 783 similar to other published prior to this study, 76 PBMCs were thawed and prepared for cell 784 culture as described for the AIM assay. Cells were pre-treated with 0.555 µg/mL of anti-CD40 785 blocking antibody (HB14, Miltenyi Biotec) for 15 min. Then, peptide pools were added to a 786 final concentration of 1 µg/mL (making anti-CD40 concentration 0.5µg/mL for the remainder 787 of the stimulation period) BD, 555029) were added to the 789 cells and incubated for an additional 4 hrs. Cells were then co-incubated with Fixable Viability 790 stain 780 (BD) and Fc Block (BD) for 20 min, RT, in the dark, washed with FACS buffer solution 791 and stained with surface stain mix CD14 APC-Cy7, CD20 APC-Cy7) for 20 min, RT, in the dark. Cells were washed with PBS and 793 subsequently fixed and permeabilized with Cytofix/Cytoperm TM (BD, 51-2090KZ) for 20 min the dark. Cells were then washed with Perm/Wash TM (BD, 51-2091KZ) and stained with 795 ICS stain Mix (CD154 PE, IFNγ PE-Cy7, TNFα APC, PRF1 FITC, IL-2 BV711, GZMB BV421 RT, in the dark. 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Both neutralising antibody and Spike-specific T cell responses were significantly affected by the Spike amino acid differences