key: cord-0845203-akpskduy
authors: Low, Jia Ming; Low, Yue Wey; Zhong, Youjia; Lee, Cheuk Yiu Charlotte; Chan, Ming; Ng, Nicholas Beng Hui; Amin, Zubair; Ng, Yvonne Peng Mei
title: Titres and neutralising capacity of SARS-CoV-2-specific antibodies in human milk: a systematic review
date: 2021-07-12
journal: Arch Dis Child Fetal Neonatal Ed
DOI: 10.1136/archdischild-2021-322156
sha: 8a620c20f31d74d84109f41165cc24e17df498d6
doc_id: 845203
cord_uid: akpskduy

OBJECTIVE: Synthesise evidence on production of SARS-CoV-2 antibodies in human milk of individuals who had COVID-19, and antibodies’ ability to neutralise SARS-CoV-2 infectivity. DESIGN: A systematic review of studies published from 1 December 2019 to 16 February 2021 without study design restrictions. SETTING: Data were sourced from PubMed, MEDLINE, Embase, CNKI, CINAHL and WHO COVID-19 database. Search was also performed through reviewing references of selected articles, Google Scholar and preprint servers. Studies that tested human milk for antibodies to SARS-CoV-2 were included. PATIENTS: Individuals with COVID-19 infection and human milk tested for anti-SARS-CoV-2 neutralising antibodies. MAIN OUTCOME MEASURES: The presence of neutralising antibodies in milk samples provided by individuals with COVID-19 infection. RESULTS: Individual participant data from 161 persons (14 studies) were extracted and re-pooled. Milk from 133 (82.6%) individuals demonstrated the presence of anti-SARS-CoV-2 immunoglobulin A (IgA), IgM and/or IgG. Illness severity data were available in 146 individuals; 5 (3.4%) had severe disease, 128 (87.7%) had mild disease, while 13 (8.9%) were asymptomatic. Presence of neutralising antibodies in milk from 20 (41.7%) of 48 individuals neutralised SARS-CoV-2 infectivity in vitro. Neutralising capacity of antibodies was lost after Holder pasteurisation but preserved after high-pressure pasteurisation. CONCLUSION: Human milk of lactating individuals after COVID-19 infection contains anti-SARS-CoV-2-specific IgG, IgM and/or IgA, even after mild or asymptomatic infection. Current evidence demonstrates that these antibodies can neutralise SARS-CoV-2 virus in vitro. Holder pasteurisation deactivates SARS-CoV-2-specific IgA, while high-pressure pasteurisation preserves the SARS-CoV-2-specific IgA function.

Neonates can contract COVID-19 infection via vertical transmission or acquire it from the community. 1 While the risk of vertically transmitted COVID-19 infection to the neonate appears to be low, [2] [3] [4] neonates who get infected de novo were more likely to develop severe disease compared with older children. 5 6 Human milk offers protection against gastrointestinal and respiratory tract infections. 7 8 A metaanalysis 9 in October 2020 showed that SARS-CoV-2 genome is generally not found in human milk of COVID-19 infected individuals, yet SARS-CoV-2 antibodies are produced in human milk. 9 Since then, there have been further studies investigating the characteristics of the SARS-CoV-2-specific antibodies in human milk, providing valuable information on the functional and kinetic details of these antibodies. This information is critical to evaluate whether these antibodies protect at-risk neonates from COVID-19 infection.

A thorough understanding of SARS-CoV-2 antibodies in human milk would provide support to the recommendation for infected individuals to continue breast feeding. [10] [11] [12] An overview of the functional and kinetic features of COVID-19 infection-induced antibodies secreted will help investigators design studies to investigate antibody production induced by vaccination. This would guide recommendations on vaccination of lactating individuals and if human milk in vaccinated mothers may confer protection to their infants. Hence, we conducted this review to examine the presence, isotypes and binding characteristics of antibodies against SARS-CoV-2 in human milk, and to assess the neutralising capacity of these antibodies in vitro.

► Human milk of lactating individuals with COVID-19 infection contains anti-SARS-CoV-2-specific immunoglobulin G (IgG), IgM and/ or IgA.

► Evidence demonstrates that these antibodies from human milk of lactating individuals can neutralise SARS-CoV-2 virus in vitro. ► Widely used Holder pasteurisation deactivates SARS-CoV-2-specific IgA, while high-pressure pasteurisation preserves antibody activity.

A systematic review protocol was developed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) 13 

Two reviewers (JML and YWL) independently screened titles and abstracts, and full-text articles were assessed for inclusion. A third reviewer (NBHN) resolved any disagreements on study eligibility. Study authors were contacted for clarification if information on eligibility was unavailable/unclear. Inclusion criteria were lactating individuals with laboratory-confirmed COVID-19 infection using either quantitative real-time reverse transcription PCR (qRT-PCR) for SARS-CoV-2, or immunoassay such as ELISA for SARS-CoV-2 specific immunoglobulin G (IgG)/immunoglobulin M (IgM), who were infected during pregnancy or postpartum period. To ensure a comprehensive search on this topic, it was determined a priori that the review would include case reports, case series, cohort, case-control, cross-sectional studies and clinical trials. Review articles or articles written based on secondary data were excluded.

Citations of articles retrieved from database searches were exported into EndNote software V.X7 where duplicates were removed. Two reviewers (JML and YWL) independently extracted individual participant data from selected articles. Primary endpoint was presence of SARS-CoV-2-specific IgG and/or IgA in human milk of individuals with active COVID-19 or convalesced from COVID-19; where data regarding IgM were reported, these data were extracted.

The Murad reporting tool was used to assess quality of case series and case reports which include eight items under four domains (selection, ascertainment, causality, reporting). 14 Two reviewers (YWL and JML) completed the quality appraisal, with a third reviewer (NBHN) resolving inconsistencies.

One hundred and three articles were obtained from the systematic search and five articles from other sources (ie, reference lists of selected articles). After excluding duplicates and screening for titles and abstracts for articles that met inclusion criteria, 14 articles 15-28 were analysed; 6 were case reports, 3 were case-control studies, 5 were case series (table 1) . Flow diagram is presented in figure 1 .

Six studies fulfilled all domains, while 8 studies fulfilled three domains in quality assessment. Quality selection for the domain of subject selection was high in six studies (42.9%) with low risk of sampling bias where patients represented the whole experience of the investigator/centre. All studies diagnosed SARS-CoV-2 infection using RT-PCR and/or ELISA (ascertainment of exposure), and accurately ascertained outcome measures. All studies described cases with sufficient details for replication or allow practitioners to make inferences related to their own practice (high quality) (online supplemental material 2). 29 Illness severity data were available in 146 women from nine studies; 5 (3.4%) had severe disease, 128 (87.7%) had mild disease (ie, fever, loss of smell/taste, headache and fatigue), while 13 (8.9%) were asymptomatic. Samples were collected between 1 and 195 days post COVID-19 infection. Repeated samples were collected from most participants.

The type of infant feeding was stated in 110 individuals; 66 (60.0%) were breast feeding exclusively, 16 17 19 20 22 24 26 27 31 (28.2%) breastfed and supplemented their infants with formula milk, 21-23 and 13 (11.8%) fed only formula to their infants. 17 Four infants were infected with COVID-19; three were likely a result of vertical transmission. One infant was symptomatic at the same time as his mother via community transmission. 15 23 27 All four infants were breastfed and had mild disease.

One hundred and thirty-three of 161 (82.6%) individuals had either anti-SARS-CoV-2 IgA, IgM or IgG in human milk. 15 (table 2) . 16 Specific IgG antibodies against the nucleocapsid protein and spike protein regions of the virus (ie, anti-SARS-CoV-2 Anti-SARS-CoV-2 spike IgM was found in human milk of 12 of 23 (52%) individuals who were evaluated for this.

Included studies mostly used ELISA to determine presence of specific IgA, IgM and IgG. A study used microsphere immunoassay and two studies used an unspecific chemiluminescence assay (table 2) .

The neutralising capacity of human milk against SARS-CoV-2 was tested in three studies (table 3) . Samples from 20 of 48 individuals (41.7%) were found to neutralise SARS-CoV-2 infectivity in vitro. 16 22 25 In two studies, the neutralising capacity of these samples was also tested before and after pasteurisation using two methods, that is, Holder pasteurisation and high-pressure pasteurisation. 16 23 25 Holder pasteurisation uses heat treatment at 62.5°C for 30 min and is traditionally used in donor banks, whereas high-pressure pasteurisation is an alternative method which inactivates pathogens using cold water and hydrostatic pressure with no heat-induced damage to the milk. 30 Van Keulen et al 25 used a SARS-CoV-2 clinical isolate on modified Vero E6 cells to interrogate neutralising activity using 50% neutralising titres as the endpoint. Unsurprisingly, viral neutralisation was better in the presence of higher IgA levels in human milk. Non-pasteurised and high pressure pasteurised samples were effective at neutralising the virus. However, samples treated by Holder pasteurisation lost neutralising capacity. 25 Favara et al performed neutralisation assays on antibodies in human milk using a SARS-CoV-2 S-antigen-expressing pseudovirus. 16 IgA was the predominant antibody isotype found. The antibodies in the human milk sample were strongly neutralising but showed reduced neutralising capacity after Holder pasteurisation. 16 Pace et al showed that 21 of 34 (61.7%) samples possessed neutralising capacity 22 using microneutralisation assays (Vero E6/TMPRSS2 cells), with 50% neutralising titres as the readout. Through a multivariable regression model, it was found that neutralisation was mainly mediated by anti-RBD IgA antibodies. Samples in this study were unpasteurised. 22 In summary, the viral neutralising capacity of human milk was evaluated after Holder pasteurisation in the samples from 30 individuals 16 25 and high-pressure pasteurisation in samples from 29 individuals. 25 Although IgA levels as measured by ELISA remained unchanged, the neutralising function was significantly reduced or lost after Holder pasteurisation. High-pressure pasteurised-treated milk was effective at neutralising the virus.

This review summarises data of 161 lactating individuals with COVID-19 infection, extracted from 14 papers focusing on presence, isotype and binding characteristics of anti-SARS-CoV-2 antibodies in human milk, and neutralising capacity of human milk in vitro. Neutralisation is the ability of an antibody to block the infection process. In the context of COVID-19, this translates into the ability of an antibody to bind to the RBDof the surface spike proteins of SARS-CoV-2 and preventing its interaction with the ACE-2 receptor of target cells like respiratory epithelial cells. 28 By preventing infection of target cells, the neutralising activity of such antibodies is likely to correlate with immunity against future infection.

Despite majority (96.6%) of individuals only having mild or asymptomatic COVID-19 disease, most produced detectable SARS-CoV-2-specific antibodies (of either IgG, IgA or IgM subtype) in human milk. This is consistent with time to seroconversion in serum IgG responses after mild COVID-19 infections. 31 The potential protective capacity of human milk may also be long lasting, reported duration of milk antibody is currently 195 days after infection in one individual. While durations from first infective symptoms until testing of antibody are reported in table 2, median durability of human milk antibody production could not be reported due to heterogeneity of reporting.

Antigen-specific antibodies are enriched in human milk; of these isotypes, generally about 90% of the total antibodies is secretory IgA. IgA is the predominant antibody isotype conferring mucosal immunity and passive transfer during breast feeding would patrol mucosal surfaces of the breastfeeding infant for potential pathogens. 32 In our review, 82.6% of lactating individuals demonstrated presence of SARS-CoV-2 antibodies in human milk. In terms of isotype, milk of 62 of 79 (78.5%) tested individuals contained SARS-CoV-2-specific IgA. In fact, it has been demonstrated that for SARS-CoV-2, the majority of antibodies (60%) are IgA or secretory IgA. 33 As majority of studies in our review did not test for the presence of IgA, the true proportion of individuals who have SARS-COV-2-specific IgA is likely to be higher.

In terms of specificity, milk of 63 of 154 women were tested for RBD-specific antibodies, and the majority (87.3%) were found to be positive. Many studies found nucleocapsid and S2 subunit specific IgG or IgA, with a positivity ranging from 78.8% to 100%. Since these antibodies can be cross-reactive with non-SARS-CoV-2 coronaviridae, it is unclear whether these antibodies may confer protection against SARS-CoV-2. 28 34 Ultimately, neutralisation assays are the gold standard for estimation of functional capacity of antibodies. Although neutralisation tends to correlate with RBD-specific antibody levels in various serological studies, 35 our review found that only 20 of 

48 subjects (41.7%) with positive antibodies could neutralise SARS-CoV-2 infectivity in vitro. This underscores the importance of performing functional characterisation of antibodies and not just quantitative analysis. At present, there is a lack of standardisation for neutralisation assays. While the US Food and Drug Administration (FDA) has issued recommendations on titres of neutralising antibodies in convalescent plasma, it did not specify the level of virus neutralisation that should be achieved at these titres or how to measure it. 36 In addition, several strains of pseudovirus and live SARS-COV-2 variants, and modified target cells, are being used to measure neutralisation in laboratories. 25 Studies in our review also used multiple methods to determine neutralising activity of human milk antibodies, rendering it difficult to compare antibody function across studies. To be able to compare antibody function across centres, such standardisation is urgently needed. There is also uncertainty on whether in vitro neutralisation correlates with in vivo protection against infection.

The study of SARS-CoV-2-specific neutralisation capacity of human milk has presented a unique opportunity to revisit methods of pasteurisation that are used in donor human milk banks (HMB) worldwide. Holder pasteurisation is recommended in all international HMB guidelines. 37 It has been shown to effectively inactivate SARS-CoV-2 in human milk. 38 However, our review shows that Holder pasteurisation significantly reduces neutralisation capacity of SARS-CoV-2-specific IgA. The high temperature of 62.5°C has been shown to denature secretory IgA. 39 The issue of Holder pasteurisation being detrimental to the bioactivity of human milk deserves to be revisited, and alternative methods such as highpressure pasteurisation should be judiciously explored for HMBs. At present, data are scarce on the safety for microbiological control for alternative methods of pasteurisation. 40 As COVID-19 immunisations are rolled out, the risk-benefit ratio of vaccinating lactating mothers remains unclear owing to paucity of real-world data. However, in the context of a pandemic, the American College of Obstetricians and Gynaecologists 41 recommends that vaccines be offered to lactating individuals, based on the principle 42 that non-live vaccines are safe in lactation in general. Studies have shown that lactating individuals who received the mRNA vaccines have SARS-CoV-2-specific antibodies in human milk for up to 6 weeks after vaccination. 43 If human milk post vaccination is found to contain SARS-CoV-2-specific IgA with neutralizing function, this would lend further strengths to the recommendation to vaccinate lactating mothers. Since the kinetics of antibody production after natural infection and vaccination are closely related, our review informs future studies on this.

In summary, this review provides a snapshot of a dynamic milk immune response in lactating individuals with COVID-19. The majority of lactating individuals with COVID-19 produce human milk containing SARS-CoV-2-specific IgA, which in about half demonstrate in vitro neutralisation capacity. Larger studies on the quantity, function and durability of SARS-CoV-2-specific antibodies in human milk from COVID-19 convalescent and vaccinated individuals are warranted. More studies are required to determine if these antibodies confer passive immunity to breastfed infants.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement All data relevant to the study are included in the article or uploaded as supplemental information. The data were extracted from the published literature. The details of all such literature used are included in the manuscript.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

This article is made freely available for use in accordance with BMJ's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by BMJ. You may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. Higher neutralisation titre were correlated with higher concentration of RBDreactive IgA

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The authors wish to thank Dr Dimple Rajgor for helping with