key: cord-0985781-jgkd9jt9 authors: Gonçalves, Juliana; Juliano, A. Margarida; Charepe, Nádia; Alenquer, Marta; Athayde, Diogo; Ferreira, Filipe; Archer, Margarida; Amorim, Maria João; Serrano, Fátima; Soares, Helena title: Secretory IgA and T cells targeting SARS-CoV-2 spike protein are transferred to the breastmilk upon mRNA vaccination date: 2021-12-02 journal: Cell Rep Med DOI: 10.1016/j.xcrm.2021.100468 sha: fb36418612e41169d9ebb3318d1225e4eea2f84f doc_id: 985781 cord_uid: jgkd9jt9 In view of the data scarcity to guide decision-making, we evaluated how BNT162b2 and mRNA-1273 vaccines impact the immune response of lactating women and the protective profile of breastmilk. Compared to controls, lactating women had higher frequency of circulating RBD-memory B cells and higher anti-RBD antibody titers, but similar neutralizing capacity. We show that upon vaccination, immune transfer to breastmilk occurs through a combination of anti-spike secretory IgA (SIgA) antibodies and spike-reactive T cells. While we found that the concentration of anti-spike IgA in breastmilk might not be sufficient to directly neutralize SARS-CoV-2, our data suggest that cumulative transfer of IgA might provide the infant with effective neutralization capacity. Our findings put forward that breastmilk might convey both immediate, through anti-spike SIgA, as well as long-lived, via spike-reactive T cells, immune protection to the infant. Further studies are needed to address this possibility and to determine spike-T cells functional profile. In view of the data scarcity to guide decision-making, we evaluated how 24 BNT162b2 and mRNA-1273 vaccines impact the immune response of lactating 25 women and the protective profile of breastmilk. Compared to controls, lactating 26 women had higher frequency of circulating RBD-memory B cells and higher 27 anti-RBD antibody titers, but similar neutralizing capacity. We show that upon 28 vaccination, immune transfer to breastmilk occurs through a combination of 29 anti-spike secretory IgA (SIgA) antibodies and spike-reactive T cells. While we 30 found that the concentration of anti-spike IgA in breastmilk might not be 31 sufficient to directly neutralize SARS-CoV-2, our data suggest that cumulative 32 transfer of IgA might provide the infant with effective neutralization capacity. Our 33 findings put forward that breastmilk might convey both immediate, through anti-34 spike SIgA, as well as long-lived, via spike-reactive T cells, immune protection 35 Clinical trials of COVID-19 mRNA vaccines excluded lactating women, causing 45 a dearth of data to guide vaccine decision-making by health authorities 1 . This is 46 especially worrisome since infants are the children's group most affected by 47 COVID-19 2,3 . In view of the physiological alterations observed in lactating 48 women and of the crucial role of breastmilk in providing immunity to the suckling 49 infant, there is a pressing need to foresee how mRNA vaccines impact immune 50 responses in lactating mothers and to uncover the effector profile of breastmilk 51 transferred immune protection. 52 Infants have an immature immune system and rely on the transfer of maternal 53 immune cells and antibodies via breastmilk to provide them with immunity 4-8 . 54 Human breastmilk contains a wide variety of immunoglobulins, including IgA 55 (~90%), IgM (~8%) and IgG (~2%) 9 . While milk IgG mostly originates from 56 blood, IgA and IgM originate predominantly from mucosa-associated lymphatic 57 tissue (MALT) within the mammary gland 10,11 . At mucosal sites, IgA and IgM are 58 Table S3 ). Next, we purified 142 milk IgA through size exclusion chromatography (SEC) (Fig. S1 A, B) . Purified 143 IgA from pre-and post-vaccination samples eluted in a single peak 144 corresponding to polymeric SIgA, suggesting that vaccination did not result in 145 an influx of monomeric IgA from the blood (Fig. S1 C, D) . 146 Our data indicate that anti-spike-SIgA titers were unaffected by vaccine 147 boosting. While the concentration of spike-reactive IgA in breastmilk might not 148 be sufficient to directly neutralize viral infection, our data suggest that 149 cumulative transfer of IgA through feeding might provide the infant with effective 150 SARS-CoV-2 neutralization. 151 152 Lactating women have higher frequency of circulating RBD-reactive 153 While human IgA secreting B cells are preferentially retained in the mammary 155 gland, functional IgG secreting B cells can be found in higher frequencies in 156 breastmilk 11,22,41 . We sought to evaluate the presence of RBD-reactive B cells in 157 the milk following vaccination. We could only detect B cells in 5/23 milk 158 samples, which were overwhelming IgD -22 (Fig. 3A) . Due the limited number of 159 B cells detected we were not able to assess the presence of RBD-reactive milk 160 Mainly due to hormonal changes, lactating women display distinct immune 162 responses 42 . In the blood, a clear population of RBD-binding IgD -B cell 163 population could be detected post vaccine first dose, which remained unaltered 164 upon boost (Fig 3 B, C) . Both RBD-reactive plasmablasts and memory B cells 165 were detectable after vaccine prime, with only plasmablasts increasing in frequency upon boost (Fig. 3 B, C) . Compared to controls, breastfeeding 167 women have higher frequency of memory B cells and higher titers of circulating 168 anti-RBD IgA and IgG (Fig. 3 C, D) . Moreover, RBD-reactive memory B cells, 169 and overall RBD-reactive B cells, correlated with anti-spike IgG levels ( Fig. 3 E, 170 F), but not with neutralization titers (Fig. 3 E, F) . 171 Altogether, lactating women had higher frequencies of RBD-reactive circulating 172 memory B cells and higher RBD-IgG antibodies, when compared to controls. 173 174 Emerging evidence suggests the requirement of both antibody-mediated and T 176 cell-mediated immunity for effective protection against SARS-CoV-2 43,44 . To 177 detect spike specific CD4 + T cells, we used an Activation Induced Marker (AIM) 178 assay using OX40 and CD25 dual expression to detect spike reactivity 45-47 . We 179 could only robustly detect CD4 + T cells in the milk of 12 (52%) donors (Fig 4 A, 180 B). The absence of T cell detection in the other 11 samples was likely due to 181 insufficient milk volume available. After vaccine second dose, spike-reactive T 182 cells could be identified in all milk samples with detectable T cells, with 183 frequencies ranging from 0.7% to 9.1% ( Fig. 4 A, C) . Indicating that spike-T 184 cells are transferred to breastmilk, upon mRNA vaccination. 185 All lactating women possessed spike-T cells (median, 0.76%; IQR, 0.5-1.19) in 186 circulation after vaccine prime, and their frequency was not altered by 187 subsequent boost (Fig 4 D, E) , even though their activation state, measured by 188 CD69 expression, was decreased ( Fig. 4 D, E) . Curiously, it appears that after 189 vaccine boost, breastfeeding women have less spike-reactive CD4 + T cells 190 when compared to vaccinated controls (Fig. 4D, E) . In view of the role of CD4 + 191 T cells in B cell effector differentiation, we looked if there was an association 192 between circulating spike-reactive T cells and RBD-reactive B cells. There was 193 no correlation between spike-T cells and RBD-plasmablasts or memory B cells 194 (Fig. 4F) . 195 All together our data show that in addition to antibodies, milk also contains 196 spike-reactive T cells. These spike-reactive T cells might transfer long-lived 197 immunity to the suckling infant. Further information and requests for resources and reagents should be directed 396 to and will be fulfilled by the lead contact (helena.soares@nms.unl.pt). 397 This study did not generate new unique reagents. (Table S1 ). The second collection interval occurred upon opening of the 407 vaccination to the general population from June to September 2021 and 408 participants were recruited through social media platforms, pre-natal support 409 groups and/or word of mouth. In this second period, we collected 9 paired 410 samples of breastmilk and blood, ~11 days after the first (IQR, 10-15 days) and (Table S2) All data reported in this paper will be shared by the lead contact upon request. 544 This paper does not report original code. 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