There is increasing evidence from animal models that delivery of a vaccine direct to the respiratory mucosa may be a more protective route of vaccination. An understanding of the different T cell subsets within the lung would inform the design of vaccines targeting this route.
Lung T cell subsets
Thus far, three main subsets of lung resident memory cells have been defined: T effector memory cells (T
EM), T central memory cells (T
CM), and T resident memory cells (T
RM). Much of the available data on this has come from murine studies. It is not always clear how these findings relate to NHPs and humans. Most of the lung resident T memory cells are of the T
EM phenotype [
160], CD44
hi CD62L
lo CD127
hi [
141,
161]. The T
EM subset act in the first line of defence [
162], predominantly secreting Th1 cytokines [
160]. They are able to recirculate between blood, non-lymphoid tissues and lymph [
163]. Studies of individuals with LTBI have demonstrated an increased level of the exhaustion marker PD-1 on T cells, perhaps due to continuous antigenic stimulation [
164]. T cells from BCG vaccinated individuals were CD27+ but had low PD-1 expression, indicating an earlier stage of differentiation [
164]. Despite this, the antigen-specific CD4+ T cell response of BCG-vaccinated human new-borns wanes over the first year of life, suggesting that the T
EM population induced is unable to maintain persistent memory [
165]. In response to continuous antigen exposure, T
EM become terminally differentiated T effector (T
eff) cells, losing the ability to proliferate and migrate into the lung parenchyma, expressing the KLRG1 marker [
166,
167].
In contrast, IL-2-producing T
CM have a high proliferative capacity [
168], usually CD62
hi CD127
hi [
161], and derive from KLRG1
− precursors [
169]. This cell population is capable of rapid proliferation, evolving into large numbers of pro-inflammatory effectors upon antigen re-exposure [
168]. The lack of T
CM induction by intradermal BCG may underlie the loss of protective efficacy with time [
170], supported by findings that prevention of T
CM exiting the lymph nodes has no influence on the protection provided by BCG [
171]. This indicates that BCG promotes mainly T
EM- and T
eff-based responses [
171].
A recent study has challenged the conventional view that T
CM are necessary for vaccine-induced protection. A recombinant CMV-vectored TB vaccine achieved very high levels of protection against
M. tuberculosis challenge in NHPs which was associated with the induction of T
EM and transitional effector memory T cells (T
TrEM), not T
CM [
172]. The ability of T
CM to confer greater protection than T
EM is possibly best shown by adoptive transfer of the separate T subsets (Kaufmann et al.), in which T
CM markedly protected against TB in contrast with T
EM and T follicular helper (T
FH) cells [
126]. These T
CM cells had characteristic CXCR5
+ CCR7
+ expression and CXCR5 expression correlating with decreased lung pathology [
126].
In mice, VPM1002 delivered subcutaneously induced a significantly increased T
CM response compared with BCG, which was associated with improved protection after
M. tuberculosis challenge [
126]. Adoptive transfer of T
CM specific for
M. tuberculosis conferred protection, whereas adoptive transfer of T
FH alone did not [
126].
T
RM are CD44
hi CD62L
lo CD69
+ CD103
+ in phenotype [
141], like T
CM deriving from KLRG1
− precursors [
169]. T
RM permanently reside in non-lymphoid tissue, making them strategically placed for a rapid recall response [
138]. As a group, T
RM are highly heterogeneous, with some CD4+ T
RM displaying a regulatory profile (Foxp3
hi IL-10
hi) and others with a more effector profile (T-bet
+) [
141]. In contrast, airway resident CD8+ T
RM cells are more homogenous, expressing predominantly Th1 cytokines [
141]. In addition to their cytolytic role, CD8+ T
RM are also capable of activating bystander NK and B cells through IFN-ɣ, TNFα and IL-2 [
173]. Maintenance of T
RM may be reliant on the presence of live bacilli, as clearance of BCG in mice with chemotherapy abrogates the antigen-specific CD4+ T cell response [
166]. Of all the T memory cell subtypes, the mucosal transfer of CD8+ T
RM cells was associated with the most protection against
M. tuberculosis challenge on a per-cell basis [
141].
Despite promoting lung-localised T
RM, mucosal boosting with a protein/adjuvant candidate vaccine, H56:CAF01, did not enhance protection [
174]. H56 is a subunit vaccine, a fusion protein of the
M. tuberculosis antigens Ag85B, ESAT-6 and Rv2660c [
175], which has been combined with the liposome adjuvant CAF01. The parenteral priming followed by mucosal boosting did enhance early lung T cell response; however, mucosal boosting did not alter the cytokine profile nor conferred added protection [
174]. H56:IC31 administered systemically has been evaluated in a phase 2a trial (NCT01865487) [
176] and is currently recruiting for another larger scale phase 2 trial (NCT03512249).
In summary, less-differentiated CD4+ T cells seem to provide greater protection than more-differentiated effector T cells. Vaccine strategies should therefore attempt to induce these cell populations, which appear to be related to dose and persistence of the vaccine construct [
165,
177].
Mucosal TB vaccines
The concept of delivering a TB vaccine direct to the respiratory mucosa is nothing new. Nebulised BCG was demonstrated to be safe and immunogenic in terms of tuberculin skin test conversion in 1968 [
178]. There are concerns about intransal delivery after transient cases of facial nerve palsy following nasal subunit vaccination in two phase 1 clinical trials [
179,
180]. Furthermore, there were worries that a post-exposure vaccine could trigger Koch’s phenomenon, in which reinfection is marked by rapidly developing necrotic lesions caused by hypersensitivity to the mycobacteria [
181]. To date, this concerns appear unfounded, at least in BCG-primed individuals [
182].
Aerosolised MVA85A, a modified Vaccinia virus Ankara expressing Ag85A, was evaluated in a proof-of-concept phase 1 trial (NCT01497769) in BCG vaccinated healthy adults [
182]. In this trial, respiratory adverse events post-aerosol were rare, with no difference in occurrence compared with placebo [
182]. Aerosol delivery induced more potent brochoalveolar lavage Th1 responses compared with intradermal vaccination and comparable systemic responses [
182].
Adenoviruses are another promising candidate for use in a mucosal TB vaccine due to their natural tropism for respiratory epithelium [
183]. Two adenovirus-based TB vaccines are AdHu5Ag85A, which has demonstrated T cell responses despite pre-existing adenoviral immunity [
184], and ChAdOx1.85A [
185]. Both are currently being evaluated as a nebulised vaccine (NCT02337270 and NCT04121494). An adenovirus AdHu35 expressing the
M. tuberculosis antigens Ag85A, Ag5B and TB10.4, AERAS-402, had demonstrated robust cellular immune responses in the lungs of rhesus macaques, however this failed to confer added protection [
186]. Whilst in mice the accumulation and retention of memory CD4+ and CD8+ T cells within the airway lumen correlated with protection against TB, this was not observed in the macaques. This was potentially due to the very large doses of
M. tuberculosis used in the macaque trial [
186].
Mucosal BCG vaccination has been shown to confer superior protection in the lungs compared with intradermal BCG in mice [
139], and parenteral administration in guinea pigs [
187] and macaques [
188], associated with greater numbers of T
RM and an enhanced proliferative capacity of lung parenchymal CD4+ T cells [
139,
141]. The superior protection was specific to the lungs, with protection in the spleen equal to that conferred by the intradermal route [
139]. CXCR3 expression, key to the recruitment of CD8+ T cells [
189], was only found in lung parenchymal CD4+ T cells with mucosal BCG vaccination [
139]. A recent study in NHPs has demonstrated a superior protective effect of mucosal BCG immunisation compared with intradermal immunisation against low-dose repeated
M. tuberculosis infection [
190]. The mucosally immunised group showed higher local levels of polyfunctional Th17 cells, IL10 and mucosal IgA [
190]. Though this work is highly promising, novel methods for the immunomonitoring of aerosol vaccination are necessary, due to the invasive nature of bronchoscopy and bronchoalveolar lavage. Induced sputum is one possibility, having been used before as an immunoassay in TB patients [
191,
192].