23rd September 2016
Animal and Plant Health Agency
Dr Sandrine Lesellier (APHA) and Dr Tharangani Herath (Harper Adams University) co-hosted a UK Veterinary Vaccinology Network Workshop on Mucosal Vaccinology at the Animal and Plant Health Agency on 23rd September 2016. The meeting was attended by over 37 researchers and industrial partners. The workshop focused on:
- Reviewing current platforms for delivery to mucosal surfaces
- To meet experts in the field
- Develop collaborations for the development of veterinary mucosal vaccines
- Assess gaps in Mucosal Vaccinology research
Five speakers were invited from varied fields of expertise, to highlight the general importance of mucosal vaccines for the veterinary industry and to support medical research, and describe some platforms already developed to support the mucosal delivery of vaccines (such as nano-particles and adjuvants) in a range of models (mice, fish, bovine, in-vitro models). Mucosal vaccines against Tuberculosis, Chronic Wasting Disease, Streptococcus equi, Enterotoxigenic, E. coli were used as examples.
The workshop opened with an Overview of Mucosal Vaccines and host-pathogen interactions in the intestine from Professor Neil Mabbott (The Roslin Institute). Although it is estimated that >90% of infections occur via mucosal surfaces, there is still a limited number of mucosal vaccines for the veterinary field, and even fewer for the medical field. The development of mucosal vaccines is challenging because of their generally low immunogenicity (hence the importance of adjuvants) and the need for innovative delivery systems. Uptake across the mucosal epithelium and antigen presentation must be targeted by vaccine formulations, considering host specificity (associated with species, sex, age, exposure to other pathogens and probiotics, etc). The epithelial M cells play an important role in this process and have generated a lot of scientific interest in the past 5 years. A technological novelty presented by Professor Mabbott was bovine enteroids, consisting of ex-vivo extracts of the enteric mucosa which circumvallate and develop similar cellular structures with physiological relevance to the gut (e.g. with the presence M cells and goblet cells) and could be used to study the presentation of bacteria/virus/proteins to the gut surface in vitro. Finally Professor Mabbott described the successful development of a mucosal Salmonella typhimurium based vaccine (strain expressing PrP) against Chronic Wasting Disease (CWD) which affects free ranging and farmed deer and has recently moved from the US to Europe (Norway).
Dr Phil Hogarth (APHA) discussed TB and BCG vaccination in his presentation on ‘Is mucosal vaccination a prerequisite for mucosal immunity against TB?’ His group showed that mice vaccinated with BCG by the intra-nasal route were better protected than after ID injection , which confirms that a mucosal vaccine, although not indispensable, can be considered for TB. They demonstrated a massive expansion of IL-2, TNF-a producing CD4 +T cells in the lung compared with the spleen and a better protection after intra-nasal delivery. The links between the persistence of the vaccine, immunological responses and protection are difficult to established, and it is particularly the case in the context of complex immunological profiles seen in tuberculosis infected models. TEM were shown to persist for longer than a year after vaccination, and their levels correlated with bacterial load. BCG was cleared from the lung rapidly, but persisted longer than a year in pooled draining lymph nodes. Intranasal vaccination induced an augmentation of CD4+ T cells in the parenchymal compartment of the lung and in the broncho-Alveolar Lavage (BAL), but not in the vascular compartment, whereas ID vaccination stimulated stronger relative responses in the spleen, so the route of vaccination clearly seems to influence the local immune responses.
In his talk ‘Alginate/chitosan microparticles for BCG encapsulation and mucosal delivery’ Professor Antonio Almeida (University of Lisbon) highlighted the need for mucosal adjuvants to help overcome the mucosal barriers, and the importance of the size, the geometry, the hydrophilic, and charge of vaccine particles to target mucosal surfaces and antigen presenting cells. Using this expertise, his group has developed and patented an oral inactivated vaccine against Streptococcus equi. He presented the nasal delivery of BCG with a Chitosan/Alginate encapsulation delivery system, aiming to increase the positive charge of vaccine particles and adhere better to negatively charged epithelial cells. Linoleic acid was also added to the formulation as a permeation enhancer. The formulation was optimised to allow a similar survival of BCG solution as in 0.9 % NaCl for 3 weeks +4°C and to reduce the natural tendency of bacteria to clump. In mice, enhanced IgA levels were measured in lung and intestinal secretions 26 weeks post-vaccination, and enhanced levels of IL-2, IL-4, IFN-g and TNF-a in the blood, with better results seen with the chitosan formulated vaccine.
Professor Ed Lavelle (Trinity College Dublin) presented ‘Adjuvants for mucosal vaccination’ presented first the importance of the first line of innate immune responses to stimulate strong responses against mucosal pathogens such as Enterotoxigenic E. coli. He then described the studies from his group on a potent mucosal adjuvant α-Galcer – an iNKT activating glycolipid presented by CD1d. An Enterotoxigenic E. coli antigen with alpha Galcer was delivered in Single multiple (Smpill®) capsules which protects the formulation against stomach acids and enzymes. The vaccine enhanced mucosal (gut) IgA and IgG responses following oral vaccination.
Professor Lavelle also discussed the mucosal adjuvant properties of chitosan, illustrated by an increase of the Th1 responses in the lung following nasal vaccination. His group demonstrated that chitosan enhances expression of the costimulatory molecules CD40 and CD86 on dendritic cells in the absence of inflammatory cytokines. Professor Lavelle’s group investigated the mechanism by which chitosan promoted dendritic cell activation and the induction of Th1 responses. The group demonstrated that chitosan drives the release of micochondrial DNA which is sensed by cGAS leading to STING dependent induction of type 1 interferons. In mice deficient in cGAS, STING or IFNAR, the chitosan induced Th1 response and IgG2c antibodies were absent.
The final presentation of the day was from Dr Tharangani Herath (Harper Adams University) who discussed ‘Fish mucosal immunity and vaccine development: known and future.’ Fish are the first vertebrate in evolution to share basic principles of human/mammalian immune systems. Mucosal barriers in fish include: intestine, skin, gills and nasopharynx. Dr Herath described the varying components of the immune system seen in different fish spp. as well as recent advances in fish mucosal immune system such as discovery of IgT, the mucosal specific immunoglobulin of fish. She also highlighted currently available mucosal vaccines in aquaculture and different delivery methods. Dr Herath also briefed ‘hygiene hypotheses’ phenomenon that may need more insight when trying to translate results generated from vaccine experiment from laboratory level with low microbial burden into field situation where fish experience high microbial burden. Dr Herath ended her presentation by addressing the limitations and challenges seen in mucosal vaccination within aquaculture. These included:
- Lack of optimized protective antigen doses for mucosal vaccines
- Lack of knowledge on oral tolerance
- Reduction of systemic antibodies due to prolonged exposure to oral vaccination
- Need for cellular markers
- Discovering immunostimulant able to enhance the performance of non-replicative mucosal vaccines
- Oral vaccines have to be tailor made according to the species
The workshop finally hosted a roundtable discussion where gaps in mucosal Vaccinology were highlighted:
Mucosal immunological correlates of protection require identifying, with specificity associated with pathogens. Can we measure markers of persistent memory response and link them with duration of immunity?
These correlates can be complex, depending on the host and the pathogen, and should include the early innate immune responses and secretory antibodies.
These markers may differ between hosts, hence the importance of conducting studies in the most appropriate animal model, ideally the final host. This is the strength of the veterinary field, compared with the medical one.
Trans-species and in-vitro assessments of vaccines are very valuable to reduce developmental costs. It was noted that conclusions drawn from mice sometimes fail in target species. A better understanding of the common mucosal cellular uptake mechanisms and antigen presentation is therefore essential, for which the use of mice is irreplaceable, as well as in other species.
Accessing negative results is important (ethical and cost justification). How could this been done collectively, in spite of lack of interest by journals for such results?
It would be useful to access catalogues of reagents/ tissues models/genome editing (gathered by scientists) and specific to mucosal responses in different animal species
The impact of microbiota, persistence of maternal immunity, age, sex, endocrinology on mucosal responses should not be underestimated and cross-disciplinary approach is always beneficial.
The successful development of mucosal vaccines has paved the way for further interesting research for academia and the industry, with main points of focus being the efficiency of vaccine delivery, uptake by the mucosal surface, immune responses stimulated in the lamina propria and in draining lymph nodes, and homing at mucosal surface exposed to pathogen, persistence of vaccine in the host and final protective efficacy, using final hosts and relevant animals and tissues models.