Cameron Sargent and Dr. Eric Wilson, Microbiology and Molecular Biology
Introduction
Because skin provides an effective physical barrier, most pathogens that invade the body do so at mucosal sites like the nose and mouth. As such, finding methods of improving mucosal immunity is key to providing greater defense against communicable diseases, the cause of approximately one-quarter of all deaths worldwide [1]. Improving vaccination, the technique in which the body is exposed to a weakened or nonvirulent dosage of a pathogen or toxin to promote an immune response specific to that foreign material, is a potential way of enhancing mucosal immunity. Vaccination has been used for several decades in medicine, nearly eradicating diseases like polio and reducing the impact of several others. Despite much success, however, the development of more effective vaccination strategies is necessary to protect against the pathogens that still cause much sickness and death. In particular, vaccines that specifically enhance the immune response in mucosal tissues, where nearly all germs enter the body, are in high demand. For example, vaccines capable of directing immunity to the lungs to resist influenza or to the small intestine to resist rotavirus would be powerfully effective in protecting against these diseases.
Understanding the factors directing lymphocyte accumulation to mucosal tissues and identifying ways of stimulating that accumulation are critical to improving mucosal immunity through vaccination. In previous studies, Dr. Wilson has identified that the chemokine CCL28 and its receptor CCR10 are essential to directing lymphocytes to the mucosal tissue of the mammary glands [2,3]. Among these lymphocytes, antibody-secreting cells (ASCs) migrate to the mammaries and produce an array of antibodies that are passed on to progeny through breastfeeding to create passive immunity. In this study, we investigated ways of using vaccination to increase the secretion of antibodies specific to a particular antigen in mucosal tissues like the mammary glands. Other studies suggested that vitamin D3 could be used as an effective adjuvant, a harmless material included in a vaccine to boost the response to the antigen, in improving mucosal immunity. Vitamin D3 was found to increase ASC and antibody levels in various mucosal tissues [4] as well as CCR10 expression in skin-homing T cells, a different type of lymphocyte [5]. Together with Dr. Wilson’s previous research, we used these studies to deduce the hypothesis that the use of vitamin D3 as a vaccine adjuvant increases the expression of CCR10 in ASCs and facilitates ASC accumulation to the mammary glands. We tested this hypothesis by using mouse models immunized with an ovalbumin antigen and with or without the vitamin D3 adjuvant. Many of these mice were mated to allow the collection of milk samples in addition to feces and serum. We analyzed the antibody concentrations of these samples using ELISAs and assessed the chemokines involved in ASC homing using ELISPOT. We also tested the efficacy of a variety of immunization strategies as well as cholera toxin as an adjuvant.
Methodology
In the first experiment testing ASC accumulation to the mammary glands, mice were bred according to standard breeding protocols. After confirming mating, mice were immunized subcutaneously in the footpad with 30 μl immunizations containing 200 μg ovalbumin and with or without 0.1 μg vitamin D3. Mice were immunized one day after breeding and again 14 days later. Fecal samples were collected from mice one week after the initial immunization, at the time of the booster immunization, and nine days following the birth of mouse pups; samples were dissolved in PBS and centrifuged to pellet solid waste. Milk and serum samples were collected from mice nine days following the birth of mouse pups; fatty cream was removed from milk via centrifugation. The concentrations of all IgA and ovalbumin-specific IgA antibodies in fecal, milk, and serum samples were analyzed using an ELISA protocol from the lab. Over the course of the year, a total of 11 mice were immunized with vitamin D3 and a total of 11 mice were immunized without vitamin D3; fecal samples from all 11 of each type were assessed by ELISA, and milk and serum samples were collected and assessed from 3 of each type.
In addition to the ELISA analysis of antibody secretion in mice immunized with or without the vitamin D3 adjuvant, ELISPOT was used to partially determine the role of CCR10 in lymphocyte homing to tissues in the immune system. Mice were immunized and bred as before. Cells were harvested from the spleen and mesenteric lymph nodes nine days after the birth of pups. Cells were exposed to a CCL28 gradient across a semi-permeable migration membrane and then analyzed using ELISPOT.
Several immunization techniques were also assessed to determine the most effective method for eliciting immunity using an adjuvant in immunization. Using vaccine solutions adapted from those described above, immunizations were administered by injection in the mouse scruff, intraperitoneal cavity, and hind leg hock; by pipetting into the nasal cavity and beneath the tongue; and by oral gavage. Fecal samples were collected from all mice, and serum and milk samples were collected from mice immunized intranasally, sublingually, and orally. ELISAs were used to measure antibody concentrations in the different samples.
Lastly, cholera toxin (CT) was investigated as a potential adjuvant for vaccination, again using ovalbumin as an antigen. Vaccines were administered intranasally, sublingually, and orally, delivering 200 μg ovalbumin with or without 4 μg CT. Fecal, milk, and serum samples were collected as before and analyzed using ELISA.
Results
ELISAs of samples collected from mice, half immunized with the vitamin D3 adjuvant and half without, revealed that the average concentration of ovalbumin-specific antibodies in milk from mice immunized with vitamin D3 was 1.02 times higher than mice immunized without vitamin D3; that the average concentration of ovalbumin-specific antibodies in feces from mice immunized with vitamin D3 was 1.89 times higher than mice immunized without vitamin D3; and that the average concentration of ovalbumin-specific antibodies in serum from mice immunized with vitamin D3 was 3.56 times lower than mice immunized without vitamin D3. Because of high variability in the results from individual experiments and a low sample size, however, none of these ratios were statistically significant. ELISPOT revealed that mice immunized without vitamin D3 had a higher percentage of ASCs that were CCL28-homing (49.7%) than mice immunized with vitamin D3 (21.1%), but these results were likewise not statistically significant. ELISA analysis of samples testing different immunization strategies was unable to identify a robust method for producing consistent and increased antibody levels in mice immunized with the adjuvant. Immunization with CT suggested that it might be a more potent adjuvant than vitamin D3 but that it also could not induce a significant improvement in mucosal immunity.
Discussion
Altogether, the results gathered from several experiments did not support our hypothesis that the use of vitamin D3 as a vaccine adjuvant increases the expression of CCR10 in ASCs and facilitates their accumulation to the mammary glands. One possible reason for this is that vitamin D3 simply does not have the effect on mucosal immunity that we supposed. Another possible reason is that we were unable to test our hypothesis appropriately. Because the mechanism by which vitamin D3 might elicit an improved response is unknown, we were unable to simplify our experiments beyond immunizing and breeding mice as described. This approach included many unavoidable variables. Despite synchronizing menstrual cycles, the timing of mice mating and gestation were not uniform. Collection of feces, milk, and blood from mice was often impacted by mouse behavior as well. Furthermore, successfully implementing the designed experiments demanded much more deliberation and attention than anticipated and errors were introduced by human factors such as being unable to eliminate leakages during immunizations or being unable to maintain strict timing on all steps of the experiments due to class schedules and other conflicts.
Conclusion
Despite literature and preliminary investigation both suggesting that vitamin D3 would improve tissue-specific mucosal immunity, this study was unable to gather evidence in favor of our hypothesis that vitamin D3 could be used in directing lymphocyte accumulation specifically to mucosal tissues. This study also investigated another adjuvant and different immunization strategies, but was unable to identify a method of producing targeted immunity. Using mouse models to assess vaccination strategies proved more difficult than initially anticipated due to significant time constraints and the inability to control mouse behavior. Future experiments could be improved by eliminating variables through more deliberate control of all possible factors and by using more robust assays.
Sources
- World Health Statistics 2013, World Health Organization: Italy.
- Wilson, E. and E.C. Butcher, CCL28 controls immunoglobulin (Ig)A plasma cell accumulation in the lactating mammary gland and IgA antibody transfer to the neonate. J Exp Med, 2004. 200(6): p. 805-9.
- Morteau, O., et al., An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation. J Immunol, 2008. 181(9): p. 6309-15.
- Enioutina, E.Y., et al., The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant. Vaccine, 1999. 17(23- 24): p. 3050-64.
- Sigmundsdottir, H., et al., DCs metabolize sunlight-induced vitamin D3 to ‘program’ T cell attraction to the epidermal chemokine CCL27. Nat Immunol, 2007. 8(3): p. 285-93.