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    Issue Date: November 2023

    Human Milk Antibodies Against Covid: Potent and Persistent

    • Mammal mothers pass on antibodies in milk that are specific to pathogens they encounter during their lifetime.  
    • COVID-19 infection and mRNA vaccination for SARS-CoV-2 in pregnant or lactating human mothers both result in the production of milk antibodies specific to and able to neutralize the SARS-CoV-2 virus.
    • The response to vaccine is dominated by IgG antibodies and is short-lived, whereas the response to infection is dominated by sIgA and can last up to a year, if not longer.

    As the COVID-19 pandemic enters its fourth year and Americans line up to receive the third formulation of the SARS-CoV-2 mRNA vaccine [1], there is one group that should be right at the front. “The lactating population should be first in line to get the vaccine,” explains milk immunologist Dr. Rebecca Powell, Assistant Professor in the Icahn School of Medicine at Mount Sinai. “Even if the mothers don’t think they need it, they should get vaccinated for the protective effect it has on their baby.”

    The protective effect Powell refers to is passive immunity—the maternal transfer of pathogen-specific antibodies to the fetus via the placenta and to infants in human milk. Human infants are born with immature and naïve immune systems and first encounters with pathogens can be challenging for even fully-developed immune systems—something many people experienced with the novel SARS-CoV-2 virus. These encounters would be even more difficult for an infant with a reduced capacity to make antibodies and other important immune cells. The transfer of maternal antibodies specific to pathogens the infant is most likely to encounter is an evolved mechanism that protects infants from infection while their own immune systems are still developing. 

    Powell has been studying the maternal transfer of antibodies directed at SARS-CoV-2 in human milk since the start of the pandemic and was one of the first to report the presence of SARS-CoV-2-reactive milk antibodies from mothers who had previously tested positive for COVID-19 [2, 3]. Powell’s advice for lactating people to get vaccinated is based on a growing body of research [4-10], including data from her own lab, that indicates SARS-CoV-2 mRNA vaccination while pregnant or nursing results in a robust milk antibody response. Passive immunity may have evolved to protect infants from pathogens that had previously infected their mother, but because vaccines were designed to mimic infection, they can also increase pathogen-specific milk antibodies (an immunological two for one!). 

    SARS-CoV-2 Milk Antibodies are Potent

    Maternal COVID mRNA vaccination produces a strong response from immunoglobulin G (IgG) and, to a lesser extent, IgA in the milk [4-11]. Both types of milk antibodies have demonstrated neutralization capabilities against SARS-CoV-2, suggesting protective effects for infants who would otherwise have no level of defense [4-11]. IgG and IgA are common antibodies in the bloodstream and their increase in milk after vaccination can be explained by the intramuscular delivery of the COVID mRNA vaccines. Muscle tissue is highly vascular, which means that the antigen contained in the vaccine—in this case, an mRNA sequence that codes for part of the spike protein that surrounds the virus—is quickly moved into circulation and detected by the immune system. Levels of milk IgG and IgA specific to the spike protein mirror serum levels (or systemic immunity), peaking in the weeks following vaccination and then waning over time [4-11]. This is why lactating individuals that have previously received a vaccine are urged to get their booster—keeping milk antibody levels high provides better protection for infants. 

    Unlike vaccines, SARS-CoV-2 does not take an intramuscular route and instead infects mucosal surfaces such as the nose, throat, and lungs. As a result, the milk antibody response to infection looks very different than the vaccine-specific antibody response. “COVID-19 affects the immune system like other respiratory viruses that promote a classic milk response to mucosal infections,” explains Powell.

    This “classic” response includes the production of secretory IgA (sIgA) as the predominant antibody in milk, with little IgG. Milk IgA that originates from serum is monomeric (i.e., made up of just one IgA molecule). In contrast, milk sIgA is manufactured in the mammary gland; two IgA molecules are attached by a joining chain and then combined with a secretory component. Rather than pulling IgA molecules from the circulation, the IgA that eventually become milk sIgA are derived from special antibody-producing cells in the mother’s gut called GALT (gut associated lymphoid tissue). Human milk sIgA is the main responding factor in the nursing infant’s mucosal immune system; the secretory component protects the antibody from enzymatic degradation, allowing sIgA to thrive in areas where other antibodies might not survive, including the gastrointestinal tract [3, 11-13].  

    Over the last three years, Powell and colleagues [2, 3, 13] have demonstrated that COVID-19 infection among lactating individuals almost universally results in the production of potent antibodies that target the very areas that SARS-CoV-2 is known to infect. COVID-19-positive mothers were found to produce milk with high levels of sIgA specific to the SARS-CoV-2 spike protein and the levels of these antibodies in their milk were correlated with effective neutralization [2, 3, 13]. Even more impressive, the mammary gland continued to produce these virus-neutralizing, spike-specific sIgA well after mothers had recovered from their COVID-19 infection.

    SARS-CoV-2 Milk Antibodies are Persistent

    Powell’s lab started collecting milk samples from mothers that recovered from COVID-19 at the start of the pandemic (spring 2020) and continued collection as mothers continued to produce milk. They found that despite decreases in serum levels of sIgA and other antibodies after infection, milk sIgA levels were durable across lactation [2, 3, 13]. 

    “My suspicion, based on the literature, is that there are long-lived B cells in the gut and these cells continue to produce antibodies even after they start waning in the serum,” says Powell. “I don’t think COVID-19 is unique in eliciting this long-lived antibody response, this is just part of our evolved response to mucosal infections.”

    Just how long lived might these antibodies be? In a new paper [13], Powell’s lab reports that milk sIgA specific to SARS-CoV-2 spike protein were present at significant levels in 94% of samples for 9–12 months after infection. Because they had been following the same group of mothers, they knew none of the mothers included in the study were reinfected with COVID-19 and none of the mothers had been vaccinated for COVID-19. Moreover, the novelty of SARS-CoV-2 to humans meant that no one in the study group had previous exposure. And, just to be sure these weren’t antibodies that were commonly found in human milk, Powell’s team used milk samples that were collected for other studies prior to the pandemic as a control [2, 3, 13]. “We are able to show that these [sIgA] antibodies are definitely from the initial COVID infection and not just there by chance,” says Powell. “I didn’t expect it to be so long lasting. This is the most important thing we’ve shown in our work.”

    Despite finding that natural immunity from COVID-19 infection has the potential to produce a more robust and durable milk immune response than COVID-19 vaccination, Powell still recommends that pregnant or lactating mothers get vaccinated. The risks of COVID-19 infection, particularly in pregnant women who have reduced lung capacity and are immuno-compromised, are just too great and vaccination does provide a robust antibody response, just not the same type of antibodies for the same duration. The mRNA vaccines were not designed with the milk immune response in mind; indeed, lactating people were excluded from any clinical trials. Powell hopes that this will change. 

    “The goal of my lab’s work is to design vaccines targeted to the lactating population that will also produce an ideal response to protect infants,” explains Powell. “The ideal response would be long-lived, neutralizing sIgA because we have evolved to produce this antibody class to protect nursing infants.”

    To get such a response, however, might require a different mode of delivery from the usual intramuscular route or alterations to the current formulation. But a new study [7] looked at how the current vaccine could be optimized for lactating people by changing the timing of inoculation. Sixty-two Israeli women were given their second dose of the Pfizer mRNA vaccine (BNT162b2) during either their second or third trimester of pregnancy. Vaccination during the third trimester resulted in the expected milk vaccine-specific antibody response, high IgG and lower IgA [7]. However, vaccination during the second trimester had the opposite result, higher IgA and lower IgG. There was also greater persistence of IgA antibodies with second trimester vaccination than is usually observed after mRNA vaccination [7]. 

    Although these results seem to suggest that it might be possible to mimic the milk immune response to COVID-19 infection by targeting vaccination to a certain time frame during pregnancy, there are two important caveats. First, the study measured total IgA and the assays did not distinguish between IgA and sIgA. Second, the investigators did not control for asymptomatic COVID-19 infection across their cohort meaning they cannot say with certainty that the effect they measured is solely due to the timing of the vaccine. Hopefully, future studies will overcome these limitations and provide guidance to help pregnant mothers optimize protection for their infants through vaccination. “Vaccine design should absolutely consider the lactating population,” says Powell. 

    References

    1. U.S. Food and Drug Administration. (2023, September 11). FDA takes action on updated mRNA covid-19 vaccines to better protect against currently circulating variants [Press release]. https://www.fda.gov/news-events/press-announcements/fda-takes-action-updated-mrna-covid-19-vaccines-better-protect-against-currently-circulating variants
    2. Fox A, Marino J, Amanat F, Krammer F, Hahn-Holbrook J, Zolla-Pazner S, Powell RL. Evidence of a significant secretory-IgA-dominant SARS-CoV-2 immune response in human milk following recovery from COVID-19. MedRxiv. 2020 May 8: 2020-05.
    3. Fox A, Marino J, Amanat F, Krammer F, Hahn-Holbrook J, Zolla-Pazner S, Powell RL. Robust and specific secretory IgA against SARS-CoV-2 detected in human milk. Iscience. 2020 Nov 20; 23(11).
    4. Gray KJ, Bordt EA, Atyeo C, Deriso E, Akinwunmi B, Young N, Baez AM, Shook LL, Cvrk D, James K, De Guzman R. 2021. COVID-19 vaccine response in pregnant and lactating women: a cohort study.
    5. Golan Y, Prahl M, Cassidy A, Wu AH, Jigmeddagva U, Lin CY, Gonzalez VJ, Basilio E, Warrier L, Buarpung S, Asiodu IV. Immune response during lactation after anti-SARS-CoV2 mRNA vaccine. medRxiv. 2021 Mar 12: 2021-03.
    6. Young BE, Seppo AE, Diaz N, Rosen-Carole C, Nowak-Wegrzyn A, Vasquez JM, Ferri-Huerta R, Nguyen-Contant P, Fitzgerald T, Sangster MY, Topham DJ. Association of human milk antibody induction, persistence, and neutralizing capacity with SARS-CoV-2 infection vs mRNA vaccination. JAMA Pediatrics. 2022 Feb 1;176(2): 159-68.
    7. Kigel A, Vanetik S, Mangel L, Friedman G, Nozik C, Terracina C, Taussig D, Dror Y, Samra H, Mandel D, Lubetzky R. Maternal immunization during the second trimester with BNT162b2 mRNA vaccine induces a tobust IgA tesponse in human milk: a prospective cohort study. The American Journal of Clinical Nutrition. 2023 Sep 1;118(3): 572-8.
    8. Dimitroglou M, Sokou R, Iacovidou N, Pouliakis A, Kafalidis G, Boutsikou T, Iliodromiti Z. Anti-SARS-CoV-2 immunoglobulins in human milk after coronavirus disease or vaccination—time frame and duration of detection in human milk and factors that affect their titers: a systematic review. Nutrients. 2023 Apr 14;15(8): 1905.
    9. Baird JK, Jensen SM, Urba WJ, Fox BA, Baird JR. SARS-CoV-2 antibodies detected in human breast milk post-vaccination. MedRxiv. 2021 Mar 2: 2021-02.
    10. Esteve-Palau E, Gonzalez-Cuevas A, Guerrero ME, Garcia-Terol C, Alvarez MC, Casadevall D, Diaz-Brito V. Quantification of specific antibodies against SARS-CoV-2 in breast milk of lactating women vaccinated with an mRNA vaccine. JAMA Network Open. 2021 Aug 2;4(8): e2120575.
    11. Pieri M, Maniori MA, Shahabian L, Kanaan E, Paphiti-Demetriou I, Pipis S, Felekkis K, Nicolaidou V, Papaneophytou C. Survival of vaccine-induced human milk SARS-CoV-2 IgG, IgA and SIgA immunoglobulins across simulated human infant gastrointestinal digestion. Nutrients. 2022 Aug 17;14(16): 3368.
    12. Donald K, Petersen C, Turvey SE, Finlay BB, Azad MB. Secretory IgA: Linking microbes, maternal health, and infant health through human milk. Cell Host & Microbe. 2022 May 11;30(5): 650-9.
    13. Yang X, Fox A, DeCarlo C, Pineda N, Powell RL. The secretory IgA response in human milk against the SARS-CoV-2 spike is highly durable and neutralizing for at least 1 year of lactation postinfection. Breastfeeding Medicine. 2023 Aug 1;18(8): 602-11.

    Whey Protein Powder Lessens Alzheimer’s Disease Symptoms in Mice

    • Milk fat globule membrane, a component of whey protein, is rich in phospholipids, which play an important role in brain health and development. 
    • Mice carrying three genes that cause Alzheimer’s disease did poorly on tests of learning and memory, but their performance improved after three months of consuming whey protein powder.
    • Whey protein powder may be exerting its brain-bolstering effect via a signaling pathway called PPAR, which regulates inflammation in the brain.

    New therapies for Alzheimer’s disease are sorely needed, and some studies have hinted that molecules called phospholipids may bolster cognition and brain health. Whey protein powder, which is enriched in phospholipids, consist of a complex mixture of milk fat globule membrane (MFGM), whey protein, and casein derived from bovine milk. According to a recent study, MFGM may be the component of whey protein powder that helps alleviate cognitive and physiological pathology in a mouse model of the Alzheimer’s disease (1). 

    Phospholipids are present in high concentrations in the brain. Researchers have found a decrease of certain phospholipids in older adults experiencing cognitive decline (2). Enriching phospholipids in the diet of mice improves short-term learning and memory (3). What’s more, MFGM in particular has been shown to increase the expression of genes involved in brain function in rats (4) and possibly to contribute to cognitive performance in human infants (5).

    To determine whether MFGM may ameliorate aspects of Alzheimer’s disease, researchers in China tested its effect on a mouse model in which the animals were engineered to have three mutations associated with genetic forms of the disease. These mice develop the hallmark physiological symptoms of Alzheimer’s, including amyloid beta plaque deposits, neurofibrillary tangles caused by the accumulation of a protein called tau, and brain inflammation. They also have learning and memory deficits as measured by their performance on maze tests. 

    Through pathological and multiomics analysis, the researchers elucidated the effect of whey protein powder with MFGM in ameliorating inflammation and improving cognitive function of AD mice. They examined the effects of whey protein powder by studying the brains and behavior of control mice (without the mutations) that ate regular chow, mice with the mutations that ate regular chow, and mice with the mutation that were fed the protein powder in addition to chow. After three months of ingesting whey protein powder, the Alzheimer’s disease model mice showed stronger performance in water maze tests—and thus improved cognitive performance—as well as on a test for anxiety, compared with Alzheimer’s disease mice that did not consume whey protein powder. Mice that consumed whey protein powder also had lower levels of amyloid beta molecules their brains and lower levels of tau protein.

    Additionally, the researchers pinpointed how the dietary whey protein powder may have exerted its brain-protecting effect. In studies on homogenized cortical tissue, they identified dozens of genes  that were differentially expressed in the control mice, the Alzheimer’s disease mice, and the Alzheimer’s disease mice that consumed whey protein powder. These differences in expression were especially strong in genes associated with inflammatory signaling pathways. They also identified several metabolites in cortical tissue that were present in different amounts among the three groups. Most of these belonged to a group of lipid or lipid-like molecules and many enriched the fatty acid metabolism pathway. 

    One metabolic pathway in particular, called the PPAR signaling pathway, which acts as a lipid sensor and regulates lipid metabolism, was implicated in these differences. In further studies the researchers found a regulatory effect of whey protein powder on the PPAR pathway, which in turn affected a related signaling pathway called mTOR. mTor has been linked to inflammatory factors in the brains of people with Alzheimer’s disease (6).

    Overall, the study suggests that dietary whey protein powder merits further inquiry for treating at least some aspects of neurodegenerative disease. “Our results provide new insights into the underlying mechanism of [whey protein powder] and more evidence for its efficacy in alleviating” Alzheimer’s disease, the authors write in the study (1). 

    References

    1. Li Y, Zhang ZH, Huang SL, Yue ZB, Yin XS, Feng ZQ, Zhang XG, Song GL. Whey Protein Powder with Milk Fat Globule Membrane Attenuates Alzheimer’s Disease Pathology in 3×Tg-AD Mice by Modulating Neuroinflammation Through the Peroxisome Proliferator-activated Receptor γ Signaling Pathway. J Dairy Sci. 2023: 106(8):5253-5265. 
    2. Liu TT, Pang SJ, Jia SS, Man QQ, Li YQ, Song S, Zhang J. Association of Plasma Phospholipids with Age-Related Cognitive Impairment: Results from a Cross-Sectional Study. Nutrients. 2021:13(7):2185. 
    3. Schipper L, van Dijk G, Broersen LM, Loos M, Bartke N, Scheurink AJ, van der Beek EM. A Postnatal Diet Containing Phospholipids, Processed to Yield Large, Phospholipid-Coated Lipid Droplets, Affects Specific Cognitive Behaviors in Healthy Male Mice. J Nutr. 2016:146(6):1155-1161. 
    4. Brink LR, Lönnerdal B. The Role of Milk Fat Globule Membranes in Behavior and Cognitive Function Using a Suckling Rat Pup Supplementation Model. J Nutr Biochem. 2018:58:131-137. (Preprint)
    5. Hernell O, Timby N, Domellöf M, Lönnerdal B. Clinical Benefits of Milk Fat Globule Membranes for Infants and Children. J Pediatr. 2016:173 Suppl:S60-5.
    6. Thakur S, Dhapola R, Sarma P, Medhi B, Reddy DH. Neuroinflammation in Alzheimer’s Disease: Current Progress in Molecular Signaling and Therapeutics. Inflammation. 2023:46(1):1-17. 

    Preserving Milk Proteins while Inactivating Pathogens

    • Milk banks use Holder pasteurization to inactivate pathogens in human milk.
    • Holder pasteurization also degrades many bioactive milk proteins important for infant health.
    • High-pressure processing preserves human milk proteins while inactivating pathogens.

    Human milk provides important health and growth benefits for infants, particularly preterm infants. But parents of preterm infants are not always able to provide them with sufficient milk, in which case preterm infants are often fed donor human milk from nonprofit milk banks [1-3]. 

    To reduce the risk of transmitting pathogens to infants through donor milk, milk banks apply a heat treatment known as Holder pasteurization (HoP) to donor human milk. HoP involves heating the milk to 62.5 ºC for 30 minutes, and results in a more than 5-log—or 100,000-fold—reduction in the major bacterial and viral pathogens in milk, inactivating 99.999% of them [4]. 

    HoP is thus excellent at making donor milk safe from most pathogens. But at the same time, it damages some bioactive milk proteins, including enzymes such as bile salt-stimulated lipase (BSSL), lactoferrin, and immune proteins such as immunoglobulin (Ig) A and IgG [5-9]. Unfortunately, this degradation of human milk proteins by HoP means that preterm infants may not be getting all the health benefits of consuming human milk.

    “I’ve been really interested in all the bioactives of human milk for a long time, particularly the proteins, and I’m interested in what all those things can do but also how to best preserve them so that all those proteins stay in the intact forms and have the best opportunity to improve the health of infants,” says David Dallas, Associate Professor in the nutrition program of the College of Health at Oregon State University. “Holder pasteurization is actually quite a good method, but there are some bioactive proteins that are still lost by Holder pasteurization, so the question was, can we improve upon that?”

    Preserving some of the bioactive compounds in human milk could help improve the growth of preterm infants. “One of the key proteins that we were interested in is called bile salt-stimulated lipase, and it contributes to a large degree to fat digestion in preterm infants,” says Dallas. BSSL degradation by HoP could affect infant fat absorption and growth. “There have been some studies that show that if you compare very premature infants fed their own parents’ milk versus donor pasteurized milk, the ones fed parents’ own milk grow much better and they also have shown they have a 30–40% higher lipid absorption compared with Holder-pasteurized milk,” he says. “I thought that was really cool because growth is a hugely important thing for preterm infants and is still somewhat of an unsolved problem in preterm infant nutrition, and lipids are the highest calorie source of milk, so it’s really important that lipids are fully absorbed,” says Dallas.

    Preserving milk proteins could also benefit infant immunity. Holder pasteurization partially degrades immune proteins called immunoglobulins that are very important for protecting infants from pathogens, says Dallas. “So there’s a lot of key proteins and the idea is let’s preserve them as well as we can,” he says. “As I learned about how heat processing can denature and cause other changes to proteins, I’ve been interested in trying to find better ways to process milk, and we heard about high-pressure processing as another method that has some promise for human milk and decided to examine that,” he says.

    High-pressure processing (HPP) uses water to transmit pressure without applying heat [10]. It is currently used in the food industry to inactivate microorganisms in products such as juice and some meats and seafoods. HPP is thought to inactivate microbes by disrupting their cell membranes and some microbial proteins [11].

    In a new study, Dallas investigated the minimum HPP conditions required to inactivate pathogenic bacteria and examined how this treatment affected bioactive milk proteins [12]. He and his colleagues spiked donor milk with bacteria and bacterial spores and examined what HPP conditions would deliver a 5-log reduction in pathogenic bacteria. “I think this is the first paper that did the actual spiking experiments with human milk as opposed to just using the natural abundances of bacteria that might be present in the milk, and we did that to ensure that we could achieve the same kind of standards as Holder pasteurization,” says Dallas. The researchers pooled raw human donor milk and inoculated it with several relevant pathogens such as Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii, as well as spores of Bacillus subtilis and Paenibacillus spp.

    Dallas discovered HPP conditions that inactivated these microbes just as well as HoP. “We used 500 megapascals of pressure for nine minutes and we kept the temperature as close to zero as possible, and that was sufficient to create a greater than five-log reduction of the bacteria that we tested,” he says.

    Dallas then examined how HPP affected the bioactive proteins in human milk, in collaboration with Oregon State food microbiologist Joy Waite-Cusic, a co-author on the study. “We found better preservation of certain proteins that we tested including better preservation of IgA, IgG, IgM, lactoferrin, elastase, polymeric immunoglobin receptor, and BSSL activity,” he says. “So we were pretty pleased with those results,” he says. In contrast to HPP, HoP eliminates lactoferrin, elastase, and BSSL in human milk and causes a significant decrease in IgA, IgG, and IgM antibodies and polymeric immunoglobin receptor.

    These findings suggest that high-pressure processing of human donor milk could preserve more bioactive proteins than HoP and thus improve infant health outcomes. Improved immunoglobulin and lactoferrin levels could provide better protection against pathogenic bacteria and viruses, better elastase retention could improve protein digestion, and improved BSSL activity could improve lipid digestion and infant growth [13-15].

    Dallas says more studies need to be done before milk banks start using HPP on milk. “I think there’s still more work to do to verify that it’s the right technique, that it will be beneficial,” he says. “I think that for milk banks, probably the  number one concern is safety, so you have to have a really good safety profile,” says Dallas. Among other things, he plans to look at how HPP affects viruses. “In order to be as good as Holder pasteurization, it doesn’t necessarily have to be able to get rid of all viruses, but it should probably at least be able to do the equivalent of what Holder pasteurization does to convince milk banks, so that’s key,” says Dallas. 

    Milk banks also have practical and logistical considerations to consider, given that HPP equipment is large and expensive. “What we do is we pick up milk from a milk bank and then we take it to a high-pressure processing company, they charge for processing, and you tell them what settings you want to do and they’ll do that for you,” says Dallas. “That’s what we’re doing for our feeding studies, and I think that could be a feasible solution for milk banks,” he says.

    In follow-up experiments, Dallas plans to test the effects of HPP on other nutrients and milk components, including milk fat globules and exosomes. “It’s possible that by preserving those things better, we could have even better health outcomes,” he says. “Then we need to have testing in infants to show some sort of improvement, whether that’s growth or gut health outcomes,” says Dallas. 

    Dallas is currently doing a study where preterm infants in the neonatal intensive care unit are fed HPP and HoP milk. “We’re feeding the same baby one day with high-pressure processed milk, and the next day with Holder-pasteurized milk, and then we are looking at their stool samples and we are going to be comparing which one has better lipid absorption, so which one has the least amount of fat still in their stool,” he says. “The hope is that because we preserve the bile salt-stimulated lipase better, that we should have better lipid absorption in high-pressure processed milk-fed infants, so that’s what we’re working on now,” says Dallas. If he’s able to show better lipid absorption, he plans to then move on to a longer-term feeding study to look for changes in growth patterns between babies fed HoP milk and HPP milk.

    In the future, Dallas plans to continue refining milk treatment methods to preserve even more milk proteins and components while inactivating more pathogens. For example, neither HoP nor HPP can inactivate spore-forming bacteria, and the search is on for methods that can do that without degrading milk proteins. “If you’re doing some sort of mechanical process that degrades bacteria, chances are it can also degrade other things in milk, so it’s a really hard challenge,” says Dallas. “Ideally we could create a process that gets rid of bacteria and spores and also better preserves proteins,” he says.

    But HPP already improves upon HoP by providing an equivalent inactivation of pathogens while doing a better job of retaining key bioactive proteins. “It’s definitely  moving towards something that’s a better preservation technique,” says Dallas. “It shows that there is more flexibility in the human milk space to try some innovative techniques, so we need creativity in this space and it would be great to have more engineers and more food scientists entering this space,” he says.

    References

    1. Hill PD, Aldag JC, Chatterton RT, Zinaman M. Comparison of milk output between mothers of preterm and term infants: the first 6 weeks after birth. J Hum Lact. 2005 Feb;21(1):22-30.
    2. Unger S, Gibbins S, Zupancic J, O’Connor DL. DoMINO: Donor milk for improved neurodevelopmental outcomes. BMC Pediatr. 2014 May 13;14:123.
    3. Wight NE. Donor human milk for preterm infants. J Perinatol. 2001 Jun;21(4):249-54.
    4. Kontopodi E, Hettinga K, Stahl B, van Goudoever JB, M van Elburg R. Testing the effects of processing on donor human Milk: Analytical methods. Food Chem. 2022 Mar 30;373(Pt A):131413.
    5. Koh J, Victor AF, Howell ML, Yeo JG, Qu Y, Selover B, Waite-Cusic J, Dallas DC. Bile salt-stimulated lipase activity in donor breast milk influenced by pasteurization techniques. Front Nutr. 2020 Nov 12;7:552362.
    6. Silvestre D, Miranda M, Muriach M, Almansa I, Jareño E, Romero FJ. Antioxidant capacity of human milk: effect of thermal conditions for the pasteurization. Acta Paediatr. 2008 Aug;97(8):1070-4.
    7. Paulaviciene IJ, Liubsys A, Eidukaite A, Molyte A, Tamuliene L, Usonis V. The effect of prolonged freezing and Holder pasteurization on the macronutrient and bioactive protein compositions of human milk. Breastfeed Med. 2020 Sep;15(9):583-8.
    8. Demazeau G, Plumecocq A, Lehours P, Martin P, Couëdelo L, Billeaud C. A new high hydrostatic pressure process to assure the microbial safety of human milk while preserving the biological activity of its main components. Front Public Health. 2018 Nov 6;6:306.
    9. Espinosa-Martos I, Montilla A, de Segura AG, Escuder D, Bustos G, Pallás C, Rodríguez JM, Corzo N, Fernández L. Bacteriological, biochemical, and immunological modifications in human colostrum after Holder pasteurisation. J Pediatr Gastroenterol Nutr. 2013 May;56(5):560-8.
    10. Viazis S, Farkas BE, Jaykus LA. Inactivation of bacterial pathogens in human milk by high-pressure processing. J Food Prot. 2008 Jan;71(1):109-18.
    11. Peila C, Emmerik NE, Giribaldi M, Stahl B, Ruitenberg JE, van Elburg RM, Moro GE, Bertino E, Coscia A, Cavallarin L. Human milk processing: A systematic review of innovative techniques to ensure the safety and quality of donor milk. J Pediatr Gastroenterol Nutr. 2017 Mar;64(3):353-61.
    12. Liang N, Mohamed HM, Kim BJ, Burroughs S, Lowder A, Waite-Cusic J, Dallas DC. High-pressure processing of human milk: A balance between microbial inactivation and bioactive protein preservation. J Nutr. 2023 Sep;153(9):2598-611.
    13. Lönnerdal B. Bioactive proteins in human milk: health, nutrition, and implications for infant formulas. J Pediatr. 2016 Jun;173 Suppl:S4-9.
    14. Kell DB, Heyden EL, Pretorius E. The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria. Front Immunol. 2020 May 28;11:1221.
    15. Koh J, Victor AF, Howell ML, Yeo JG, Qu Y, Selover B, Waite-Cusic J, Dallas DC. Bile salt-stimulated lipase activity in donor breast milk influenced by pasteurization techniques. Front Nutr. 2020 Nov 12;7:552362.

    Contributed by

    Dr. Sandeep Ravindran

    Freelance Science Writer

    Sandeepr.com

    Immune Cells Produce a Distinct Subset of Antibodies in Human Milk

    • Human milk and serum from an individual share a cocktail of IgA antibodies that occur in distinct forms in the two fluids.
    • Repertoires of IgA antibodies are unique and have almost no overlap between individuals. 
    • IgA1 in serum occurs mostly in the monomer form. 
    • Human milk IgA is entirely in the secretory form and found largely as polymers with 24 antibodies linked to each other.

    In response to antigens, immune cells known as B cells produce large amounts of antibodies that bind to foreign molecules either to neutralize them or to mark them for destruction by other cells. Circulating antibodies comprise 15 to 25 percent of all proteins in human serum and are also present in maternal milk where they confer immune protection early in life [1]. But precisely which antibodies enter human milk, and how they do so, has been a mystery. 

    Different B cells synthesize a wide range of immunoglobulin, and—in theory—cells throughout the human body could produce 1016 to 1018 distinct kinds of antibodies. In reality, however, only a few hundred kinds of antibodies dominate the population, forming about 50 to 90 percent of circulating immunoglobulins both in serum and in human milk, according to a study by researchers at Utrecht University in the Netherlands [2].  

    Antibodies that enter human milk must undergo additional processing so they can cross epithelial cell layers. To accomplish this, two or more immunoglobulin molecules are linked to each other via a joining (J-) chain. This linked up polymer binds to a receptor to be transported across layers of epithelial cells and is then cleaved to form the secretory form of the immunoglobulin. The researchers, led by analytic chemist Albert Heck, set out to learn whether one kind of immunoglobulin in serum and milk, known as IgA1, originated from the same cells. “IgAs play a very critical role in the early immune development of the infant,” said Kelly Dingess, a senior scientist at Danone Nutritional Research who co-authored the study [2]. 

    In 2021, the team characterized the IgA1 antibodies present in the serum and milk of two healthy human donors at multiple time points over the first four months of lactation [2]. There was little variation in the immunoglobulins present in human milk over that time period. About 80 percent of secretory IgA1 molecules (sIgA1) present at 16 weeks of lactation were the same as those present in the first week. Although many of these immunoglobulins were similar across the serum and milk of a single donor, there were almost no antibodies common between the two donors. “Nobody produces the exact same antibodies as another person. This is really highly individual, specific, and unique,” Dingess said in an interview. “Also, all of the antibodies that we find in our blood are not necessarily the antibodies we find in milk either.”

    In a subsequent study, the authors examined serum and human milk IgA1 to understand the similarities and differences between antibodies in these two fluids [3]. The team compared paired milk and serum samples from three healthy donors between the ages of 27 and 35 years. The samples were collected approximately once a month over a period of three months.  As before, the researchers found that there was little variation in the antibody profile of samples from a single donor over time, and almost no commonality amongst the IgA1 clones present in samples from different donors. 

    The team identified approximately 370400 IgA1 clones in serum, and approximately 600 sIgA1 clones in human milk. Overall, about 45 percent of the IgA1 molecules in serum were also detected in milk. 

    Because the classical theory is that an antibody-producing B cell produces only one antibody, molecules common in milk and serum should both have the J-chain, which is necessary for antibodies to be transported into milk. The authors looked for dimers and other polymers of IgA antibodies both in serum and milk. They found that 83 percent of IgA in serum is present as monomers, and only 17 percent occurs in the dimeric J-chain linked form. But nearly half of IgA in milk was present in the dimer form. The team also reported that 33 percent was present as trimers and 17 percent in a tetramer form, with four IgA molecules linked to each other. Although these same IgA1 molecules bearing the J-chain were present in serum, they often existed in the monomeric or dimeric form, not the polymers detected in milk. “Typically, when people think of antibodies found in blood, they think of this very simple Y-like structure, whereas the antibodies found in milk can be highly complex,” Dingess said in the interview. 

    Secretory IgAs in milk are important for training the infant immune system to protect against infections and in helping to establish the infant gut microbiome, Dingess added, and the secretory J-chain protects the antibody from being digested in the infant gut. “This becomes really important because we need those antibodies to survive the stomach and the gastrointestinal tract,” she said.

    Identifying differences in the structures of immunoglobulins present in milk and serum is a crucial step toward understanding whether they also play different roles in protection against infections and in the development of a healthy gut microbiome. Although other components of milk, such as human milk oligosaccharides (HMOs), are typically thought to regulate the gut microbiome, IgA plays an important role as well, Dingess said. “Milk is a complex system,” she said. “It’s not any one thing that does something. All of these things that are really working together.” 

    References

    1. Gonzalez-Quintela A, Alende R, Gude FA, Campos J, Rey J, Meijide LM, Fernandez-Merino C, Vidal C. Serum levels of immunoglobulins (IgG, IgA, IgM) in a general adult population and their relationship with alcohol consumption, smoking and common metabolic abnormalities. Clinical & Experimental Immunology. 2008 Jan;151(1):42-50.
    2. Bondt A, Dingess KA, Hoek M, van Rijswijck DM, Heck AJ. A direct MS-Based approach to profile human milk secretory immunoglobulin A (IgA1) reveals donor-specific clonal repertoires with high longitudinal stability. Frontiers in Immunology. 2021 Dec 6;12:789748.
    3. Dingess KA, Hoek M, van Rijswijk DM, Tamara S, den Boer MA, Veth T, Damen MJ, Barendregt A, Romijn M, Juncker HG, van Keulen BJ. Identification of common and distinct origins of human serum and breastmilk IgA1 by mass spectrometry-based clonal profiling. Cellular & Molecular Immunology. 2023 Jan;20(1):26-37.

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