Verification of pigs as an appropriate model for the development of human vaccines targeting the entero-mammary pathway to elicit a robust secretory IgA response in human milk

Secretory Immunoglobulin A (sIgA) is the dominant immunoglobulin (Ig) in human mucosae and their fluids and is critical in protecting these surfaces from infection [1, 2]. sIgA is most often comprised of dimeric IgA linked by joining (j) chain. This dimer is secreted by the B cell, with j-chain marking it for transport by polymeric Ig receptor (pIgR) on epithelial cells into mucosal fluids [3]. pIgR is cleaved as it transports, leaving a domain known as secretory component (SC) attached, creating sIgA [4, 5]. SC renders it highly resistant to degradation in harsh mucosal environments [6, 7]. Human milk Ig is ~90% sIgA [8-10]. B cells that produce human milk sIgA originate from the gut associated lymphoid tissue (GALT), via the entero-mammary pathway [5, 11-13]. This pathway is evident in humans in that mammary B cells display adhesion receptors similar to GALT B cells, and milk sIgA exhibits strong reactivity for enteric microbes [14-20]. Our work analyzing the milk antibody response to SARS-CoV-2 infection found robust sIgA is elicited that is exceptionally long-lasting, a critical finding that is likely representative of any mucosal infection where immunogens ultimately present in the GALT [21-23]. In contrast, after COVID-19 vaccination, the response is IgG-dominant with low or undetectable sIgA, all waning within 3-6 months. We have also shown that influenza-specific milk sIgA is poorly boosted after seasonal vaccination [24]. Certainly, intramuscular (IM) vaccines do not elicit significant milk sIgA [24, 25]. Intranasal live-attenuated influenza vaccine is not better than IM vaccine for eliciting milk sIgA or IgG [26]. There is a true need to design vaccines aimed to elicit potent, specific sIgA responses in milk to protect breastfed infants. We expect such vaccines would be best designed to target the GALT of a lactating woman. However, animal models for such targeted vaccine design do not exist. The entero-mammary pathway is not universal; for example, little evidence has been found for this pathway in sheep or cows, who's milk contains mainly serum IgG [27]. Early studies in mice and pigs suggested IgA-secreting GALT B cells traffic to the mammary gland, and in these species, sIgA predominates in milk [27]. Mice are not a good model animal for our work, given the paucity of milk produced and, unlike humans, their milk IgG is actively transported from the infant gut into the systemic circulation during the neonatal period [6, 28]. Pigs are likely more ideal. As such, this exploratory project aims to verify that pigs are an appropriate pre-clinical model for the human entero- mammary pathway and therefore, our future vaccine work. Using state-of-the-art single cell RNA sequencing, we will determine if, compared to other immune compartments, the cellular adhesion marker profiles of GALT and mammary gland (and/or milk) B cells destined to produce/producing sIgA are most similar; and the expressed Ig of these B cells are also highly related. The overall goal of this foundational work is to use pigs as a model for the development of targeted vaccines for lactating women that elicit robust, long-lasting milk sIgA to protect the breastfed infant. This work is focused on the unique needs of lactating women.