key: cord-0872640-3msx73ty
authors: Goenka, Anu; Halliday, Alice; Gregorova, Michaela; Milodowski, Emily; Thomas, Amy; Williamson, Maia Kavanagh; Baum, Holly; Oliver, Elizabeth; Long, Anna E.; Knezevic, Lea; Williams, Alistair JK.; Lampasona, Vito; Piemonti, Lorenzo; Gupta, Kapil; Di Bartolo, Natalie; Berger, Imre; Toye, Ashley M.; Vipond, Barry; Muir, Peter; Bernatoniene, Jolanta; Bailey, Mick; Gillespie, Kathleen M.; Davidson, Andrew D.; Wooldridge, Linda; Rivino, Laura; Finn, Adam
title: Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2
date: 2021-06-09
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100327
sha: 0d38bc5a310dd4db47fdcbbf1eeed8a3be0d982c
doc_id: 872640
cord_uid: 3msx73ty

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in four infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein with corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ and/or TNF-ɑ producing T cells is comparable between infants and adults. On principal component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19.

The COVID-19 pandemic is responsible for unprecedented morbidity and mortality, particularly in the elderly, but significant disease appears rare in children. 1 Compared with older children, severe COVID-19 has been reported relatively more commonly in young infants. 1 Despite this, approximately one quarter of young infants infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are asymptomatic and there have been few reported deaths in this age group. 2 This is unexpected given that early life is a period of rapid transition for the immune system that renders infants vulnerable to severe respiratory viral infection such as those caused by respiratory syncytial virus and influenza. 3, 4 Few data are currently available describing SARS-CoV-2 immunity in infants less than 3 months old. We therefore evaluated antibody and cellular immune responses in a small cohort of young infants recovered from COVID-19.

Four infants aged less than 12 weeks old presented with fever without an obvious clinical focus to Bristol Royal Hospital for Children, UK over a four-week period in March 2020. Baseline characteristics of the infants (I1-I4), their mothers (M1-M4) and fathers (F1-F4) are shown in Table   1 . All parents experienced COVID-19 symptoms in the days preceding the development of symptoms in their infants, except two fathers (F3 and F4) who remained asymptomatic. The median age of the infants at presentation was 7 weeks (I1 -6 weeks; I2 -1 week; I3 -11 weeks corrected age; I4 -7 weeks). One infant was exclusively breastfed (I2), one was exclusively formula fed (I3), and two were mixed formula and breastfed (I1 and I4). There was no significant perinatal or past medical history, except in one infant (I3) who was born at 28 weeks' gestation and did not suffer significant complications of prematurity but had been recently admitted to hospital with rhinovirus bronchiolitis. Reduced peripheral lymphocyte counts of 1.2-2.1x10 9 /L cells/mm 3 (normal range 3.3-10.3x10 9 /L cells/mm 3 ) were observed in two infants (I1 and I2) but were normal in one infant (I4) and not measured in one infant (I3). C-reactive protein was measured in three infants (I1, I2 and I4) and was <1mg/L (normal range <5mg/L) in all. A transiently raised serum alanine aminotransferase with a peak of 207 units/L (normal range <33 units/L) was observed in one infant (I1). SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (qRT-J o u r n a l P r e -p r o o f PCR) was positive on nasopharyngeal swab in all four infants with a median (range) cycle threshold value of 24.4 (22.0-29.9 ). Empirical treatment with intravenous antibiotics was commenced in two infants and discontinued at 36 hours after negative blood and urine culture in one infant (I1), and after 14 days in the other (I2) from whom group B streptococcus was isolated from urine, but not blood culture. None of the infants required oxygen therapy or feeding support and all exhibited symptom resolution within two days. Following recovery, peripheral blood and saliva were obtained for immunological analyses at a similar median interval after the onset of COVID-19 symptoms from infants (78 days), parents (66 days) as well as matched adult controls (63 days) who had recovered from qRT-PCR-proven COVID-19 (Table 1) .

Human coronavirus infections typically result in the production of antibodies after [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] days that can persist for many months, some of which have neutralising activity and correlate with protection against re-infection. 5 As such, serological assays have played a pivotal role in developing our understanding of adaptive and potentially protective immune responses to SARS-CoV-2 infection. Infants have been shown to produce broadly neutralising antibodies rapidly to some viral infections including HIV, 6 but typically generate lower systemic and mucosal antibody titres to other respiratory infections compared with adults. 7, 8 In this study, we measured antibody responses to SARS-CoV-2 antigens using the luciferase immunoprecipitation system (LIPS) and an enzyme-linked immunosorbent assay (ELISA). All four infants exhibited robust serum IgG responses to the SARS-CoV-2 spike protein and its receptor-binding domain (RBD) (Figures 1A, 1B and S1). Although infants' serum concentrations of anti-spike/RBD IgG appeared higher than those of their parents and qRT-PCR confirmed adult controls, the difference was not statistically significant after adjustment for multiple comparisons. Concentrations of serum IgG directed against SARS-CoV-2 nucleoprotein were low but above pre-pandemic levels for infants and their parents ( Figures 1B and S1 ). Serum antibodies to SARS-CoV-2 antigens were not detected in the two asymptomatic parents. Relatively high serum IgA responses to spike and RBD were detected in 3 of the 4 infants ( Figure S1 ). None of the infants, and six of the parents/qRT-PCR confirmed adult controls, had SARS-CoV-2 specific IgM serum antibody titres clearly above those in pre-pandemic sera ( Figure S1 ). Infants also exhibited robust salivary anti-spike IgG and IgA responses ( Figures   1C and 1D) . The infant IgA response may reflect endogenous antibody production rather than J o u r n a l P r e -p r o o f acquisition from maternal breastmilk, because the exclusively formula fed infant (I3) exhibited relatively high IgA titres, compared with the more modest titres of the exclusively breastfed infant (I2) (Figures 1D and S1) . A virus neutralisation assay confirmed that the high anti-spike/RBD IgG titres in infant sera mirrored their functional ability to block SARS-CoV-2 entry into cells (Figures 1E, 1F and S2) . This is consistent with other reports demonstrating a direct relationship between high anti-spike/RBD titres and functional antibody responses in adults. 9 Thus, infants mounted robust and functional systemic and mucosal antibody responses to SARS-CoV-2 spike/RBD suggestive of clinically protective immunity. 10 Alongside antibodies, T-cells directed against SARS-CoV-2 have been observed in convalescent individuals. 11 Since IFN-γ has a key function in anti-viral cell-mediated immunity, 11 we measured its production by peripheral blood mononuclear cells (PBMCs) stimulated with peptide pools spanning SARS-CoV-2 proteins using an enzyme-linked immune absorbent spot (ELISpot) assay ( Figure S3A ). Like others, 11 we observed a significant correlation (r=0.82, p<0.001) between the concentration of serum anti-spike IgG and IFN-γ production by PBMCs in response to stimulation by spike peptide pools among the seropositive adults recovered from COVID-19 (Figure S3B ). Production of IFN-γ by PBMCs from infants and parents (alongside 4/5 PCR-proven adult COVID-19 controls) was detected following stimulation with spike peptide pools ( Figure 2A ).

The two asymptomatic parents exhibited IFN-γ production (Figure 2A ), which has been described in seronegative individuals and may represent SARS-CoV-2 exposure or cross-reactive T-cell immunity from seasonal coronaviruses. 12, 13 To further explore the antigen-specific cytokine production and its cellular source in infants, we measured IFN-γ and TNF-ɑ production by CD4+ and CD8+ T-cells using flow cytometric intracellular cytokine staining (ICS) following ex vivo stimulation of PBMCs with peptide pools spanning SARS-CoV-2 proteins ( Figure S2C ).

Comparable frequencies of cytokine positive CD4+ and CD8+ T-cells (defined as IFN-γ and/or TNF-ɑ positive) were detectable among infants' and parents' PBMCs following stimulation with spike and membrane/nucleocapsid peptide pools ( Figures 2B and 2C ). Given the low magnitude of infant cellular responses we observed ex vivo, compared with relatively high infant anti-SARS-CoV-2 antibody titres, we sought to determine their T-cell antigen-specificity by in vitro expansion with SARS-CoV-2 peptide pools. 14 Of the 3 infants from whom we had sufficient yield of PBMCs, J o u r n a l P r e -p r o o f all exhibited significant expansion of CD4+ T-cells reactive to peptide pools spanning spike as well as M/N protein pools, suggestive of antigen-specificity (Figures 2D, 2E and 2F) . Interestingly, infants' PBMCs appeared to exhibit a lower production of IFN-γ in response to spike protein compared with adults' by both ELISpot (Figure 2A ) and ICS ( Figures S3D and S3E ); although the difference was not statistically significant after adjustment for multiple comparisons. These apparent differences may be representative of the well-documented and generalised decreased type 1 cytokine producing-ability of infant T-cells, 15 which we also observed in response to mitogen stimulation ( Figure S3F ). Assessed by principal component analysis, the antibody and cellular immune response to SARS-CoV-2 in young infants collectively appeared distinct from those of their parents, despite the lack of statistical significance in individual assays after adjustment for multiple comparisons ( Figure 2G ).

These data suggest that the mild clinical course of COVID-19 reported in young infants may be associated with robust functional antibody responses and restrained IFN-γ production.

Describing the molecular mechanisms underlying the mild course of COVID-19 in infants during their period of vulnerability to other severe respiratory viral infections and contrasting them with those seen in severely affected adults might help explain the pathogenesis of severe COVID-19.

There are several limitations of our study. This is a small cohort and participants underwent sampling at a single time point only. As well as confirming our observations in a larger cohort of participants, it would be valuable in future studies to study both innate and adaptive responses in infants compared with adults, in the acute phase of COVID-19 and by longitudinal observations in convalescence. The current study is also restricted to individuals recovering from mildly symptomatic COVID-19, thereby potentially not representative of the significant proportion of young infants and adults with asymptomatic infection. In addition, we were unable to assess neutralising capacity of mucosal antibody due to low sample volume, and also had insufficient PBMCs to definitively demonstrate their antigen specificity by tetramer staining. Comparing infant and adult T cell responses to a broader range of epitopes such as non-structural SARS-CoV-2 peptides would be of interest in future studies. 11, 13, 14 J o u r n a l P r e -p r o o f ACKNOWLEDGMENTS We dedicate this manuscript to the memory of our colleague and co-author Alistair JK Williams whose enthusiasm and meticulous attention to detail played a key role in the development of the suite of COVID-19 serological assays developed at University of Bristol. We are very grateful to the families and control individuals who participated in the study. We thank the Bristol UNCOVER team for helpful discussions during the execution of this work and preparation of this manuscript.

We are also grateful to the Diabetes and Metabolism team for testing serum samples using the 

The authors declare that they have no competing interests. AlexaFluor 568 conjugated secondary antibody. Images were acquired and analysed using the ImageXpress Pico system. Scale bar represents 500µm.

Data points represent mean of technical duplicates for serum assays, or single observations for saliva ELISA. Individual families denoted by colour (1: red, 2: green, 3: blue; 4: orange); infants (coloured circles), fathers (coloured squares); mothers (coloured square with central marking); RT-PCR-confirmed adult COVID-19 controls (black squares); pre-pandemic sera (clear squares).

Significance determined by Kruskal-Wallis test with Bonferroni's correction for multiple comparisons. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anu Goenka (anu.goenka@bristol.ac.uk).

This study did not generate new unique reagents.

The datasets generated during this study have been uploaded to https://data.mendeley.com at http://dx.doi.org/10.17632/v78gcvxc2s.3

Clinical information and blood/saliva samples were obtained under research ethics approval of the 

Protein production for ELISA SARS-CoV-2 trimeric spike protein ectodomain and receptor binding domain (RBD) were produced in insect cells as previously described. 17 SARS-CoV-2 spike ectodomain was expressed in insect cells with pFastBac™ Dual (Thermo Fisher Scientific) plasmid as previously described, 18 and followed by a c-terminal octa-histidine tag for purification. For both spike and RBD, MultiBac baculovirus expression system was used to produce the proteins in Hi5 insect cells as previously described. 19 A similar purification protocol was used for both spike and RBD. Three days after infection, cell cultures expressing the spike or RBD protein were centrifuged at 1,000g for 10 min to collect the media with secreted protein as supernatant, which was again centrifuged at 5,000g

for 30 min. This media was then incubated with 7 mL (10 mL for RBD) HisPur Ni-NTA Superflow Agarose (Thermo Fisher Scientific) for each 3 L of expression for 1 hour at 4ºC. Next, Ni-NTA resin bound with spike or RBD was collected using a gravity flow column, followed by extensive wash with 15 column volume wash buffer (65 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.5).

Finally, a step gradient of elution buffer (65 mM NaH2PO4, 300 mM NaCl, 235 mM imidazole, pH readings at 492 nm for each well were subtracted from the OD at 620 nm then corrected for the average signal of blank wells from the same plate; ODs reported are an average of duplicate wells per sample.

Salivary antibodies specific for SARS-CoV-2 spike protein were detected with an ELISA based on the methodology described above with some modifications. Antigens were diluted in PBS and MICROLON ® plates (Griener Bio-One) were coated with 10 µg/mL spike protein overnight at 4 ºC.

Saliva was heat inactivated at 56 ºC for 30 minutes and centrifuged at 13,000g for 5 minutes to pellet debris. Saliva supernatants were assayed singly, diluted at either 1 in 10 (IgA) or 1 in 5 (IgG)

to a final volume of 100 µL per well. Secondary antibodies were as described for serum with concentrations optimised for saliva: IgA at 1:20,000 and IgG at 1:15,000. Plates were developed with 1-StepUltra TMB-ELISA Substrate Solution (Thermo Fisher) for 20 minutes and the reaction was quenched with 2M H2SO4 (Merck). All incubations were temperature controlled at 23 ºC. ODs were read at 450 nm and 570 nm using the same reader.

Detection and quantification of IgG specific to RBD was performed using an N-terminally nanoluciferase tagged monomeric RBD construct with competitive displacement based on previously described methodology. 20 To make the construct, modified coding sequences were designed and obtained as synthetic genes (Eurofins Genomics) allowing production of secretory (20 mM Tris Buffer, 150mM NaCl, pH 7.4 [TBST], and 0.05% casein in label incubation buffer only) with or without addition of unlabelled RBD (8x10 -8 mol/L). Immunocomplexes were precipitated using 2.5µl glycine-blocked Protein A Sepharose 4 fast flow (Cytiva) and 2.5µl ethanolamine-blocked Protein G sepharose (Cytiva) (washed 4 times in TBST) for 1hr with shaking (~700rpm) as previously described. 21 Precipitates were washed 5 times with TBST and then transferred to a 96-well OptiplateTM (Perkin-Elmer) and excess buffer removed by aspiration (end volume 30 uL). Nano-Glo® substrate (40 µL, Promega) was injected into each well immediately before counting in a Hidex Sense Beta (Hidex). Raw data were converted into units using a standard curve made by serially diluting a pool of positive samples in SARS-CoV-2 antibody negative human AB serum (Merck KGaA).

Heat inactivated serum samples ( were added to the plate with or without peptide pools (see below) in a total assay volume of 100 µL. PBMC incubated with R2 medium alone were used as negative (unstimulated) controls. PBMC stimulated with PMA at 1 µg/mL and ionomycin at 10 µg/mL (Sigma Aldrich), or anti-CD3 antibody (MABTECH, Mab CD3-2, 0.1% v/v) were used as positive controls (1-2x10 5 PBMC per well).

Antigen-specific cellular responses were measured following stimulation with an overlapping peptide library spanning the entire spike protein (divided across two pools: S1 and S2) (Mimotopes) at a final concentration of 2 µg/mL in R2. All assays were performed in duplicate.

Plates were incubated for 18 hours at 37°C/5% CO 2 in a humidified incubator. For development, plates were washed 5 times in PBS then incubated for 2 hours at room temperature with detection antibody (7-B6-1-biotin; 1µg/mL) in reagent diluent (PBS/0.5% FCS). Following incubation, plates were washed 5 times in PBS and incubated for 1 hour at room temperature with 0.1% v/v Streptavidin-ALP diluted in reagent diluent. Developed plates were protected from light and dried for 24-48 hours before image acquisition using C.T.L. ImmunoSpot S6 Ultra-V Analyzer. All plates J o u r n a l P r e -p r o o f were read using the same settings. Spot forming units (SFU) per million PBMC were calculated after subtraction of average background calculated from negative control wells.

Cryopreserved PBMC were thawed, washed and plated at 1x10 6 

PBMCs were thawed and washed with PBS 1% BSA. 20% of cells were pulsed in AIM-V medium with 2% human serum (Merck KGaA) with peptide pools from SARS-CoV-2 spike protein, membrane/nucleocapsid proteins at 5 µg/ml for 45 min at 37°C 5% CO 2 . After stimulation cells J o u r n a l P r e -p r o o f were washed in PBS 1% BSA and resuspended with remaining 80% of the PBMCs in AIM-V 2% human serum with 20 IU/ml of IL-2 (R&D Systems) and cultured for 10 days in 96-well U well plates at 0.6x10 6 cells /well, as previously described. 24 After 10-day expansion culture, cells were re-stimulated for 5 hours with SARS-CoV-2 peptide pools (as described above in ex vivo experiments) or unstimulated (media) control. The frequency of cytokine positive cells following restimulation was calculated by subtracting the frequency observed in a well containing cells without exogenous re-stimulation (i.e. media only) from a parallel well containing re-stimulated cells.

Statistical analysis and plots were produced using Prism (Version 9.0, GraphPad Software).

Comparisons of the antibody/cellular response between the infant and parent groups were made using the Mann Whitney U test. When the antibody/cellular responses of infants were compared with more than one adult group, the significance was determined by the Kruskal-Wallis test with

Bonferroni's correction for multiple comparisons. The Benjamini-Hochberg method was used to control for the False Discovery Rate (FDR) of multiple assays being performed on the same sample. The significance levels were set at p<0.05 and FDR<0.05. To investigate variation in immune responses of infants and parents, all features were reduced using principal component analysis (R statistical software version 4.0.2; procomp function). Data from all antibody and cellular assays were included and scaled. Missing values were imputed with group means for family 1 saliva IgG/IgA and IFNγ production by total PBMCs (ELISpot). Principal components were visualised and 95% confidence ellipses plotted using the factoextra package (Version 1.0.7).

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A) Serum neutralisation expressed as half maximal effective dose/dilution (ED50) of sera to achieve SARS-CoV-2 neutralisation. Significance determined by Kruskal-Wallis test with Bonferroni's correction for multiple comparisons; (B) Neutralisation of SARS-CoV-2 by serial dilutions of sera from pre-pandemic and RT-PCR confirmed adult controls; and (C) sera from each individual family. Neutralisation of SARS-CoV-2 measured by infection of Vero E6 cells with SARS-CoV-2 pre-incubated with decreasing concentrations of serum

All data points represent mean of technical duplicates

A) Representative image of an ELISpot plate following 18h stimulation with SARS-CoV-2 peptide pools (S1, S2 at 2µg/mL), unstimulated (media) or positive control stimulus (PMA 1µg/mL/Ionomycin 10µg/mL or anti-CD3 antibody 0.1% v/v) of PBMCs from adults recovered from RT-PCR confirmed COVID-19; (B) Correlation of IFN-γ production measured by ELISpot and anti-spike IgG measured by ELISA in seropositive adults; (C) flow cytometry gating strategy for intracellular cytokine staining (ICS); (D) and (E) IFN-γ production of CD4+ and CD8+ T cells (with and without naïve CD45RA + CCR7 + included respectively) measured by ICS of PBMCs following 5h ex vivo stimulation with SARS-CoV-2 peptide pools spanning spike (S1/S2) (1µg/mL)

fathers (coloured squares); mothers (coloured square with central marking); and RT-PCR confirmed adult COVID-19 controls