LTA1 is a safe, intranasal enterotoxin-based adjuvant that improves vaccine protection against influenza in young, old and B-cell-depleted (μMT) mice

Enterotoxin-based adjuvants including cholera toxin and heat-labile toxin (LT) are powerful manipulators of mucosal immunity; however, past clinical trials identified unacceptable neurological toxicity when LT or mutant AB5 adjuvant proteins were added to intranasal vaccines. Here, we examined the isolated enzymatic A1 domain of LT (LTA1) for intranasal safety and efficacy in combination with influenza (flu) vaccination. LTA1-treated mice exhibited no neurotoxicity, as measured by olfactory system testing and H&E staining of nasal tissue in contrast with cholera toxin. In vaccination studies, intranasal LTA1 enhanced immune responses to inactivated virus antigen and subsequent protection against H1N1 flu challenge in mice (8-week or 24-months). In addition, lung H1N1 viral titers post-challenge correlated to serum antibody responses; however, enhanced protection was also observed in μMT mice lacking B-cells while activation and recruitment of CD4 T-cells into the lung was apparent. Thus, we report that LTA1 protein is a novel, safe and effective enterotoxin adjuvant that improves protection of an intranasal flu vaccination by a mechanism that does not appear to require B-cells.


Results
LTA1 does not cause neurological toxicity as observed with nasal administration of AB 5 enterotoxins. Previous nasal vaccines with LT adjuvants were found to be unacceptable due to neurological toxicity that was not apparent in pre-clinical studies 14 . A newer methodology to evaluate IN toxicity of enterotoxins is olfactory behavioral testing which identifies effects on cranial nerve 1 23 . To verify our hypothesis that the lack of a B-subunit and GM1-binding precludes development of neurological toxicity after LTA1 IN administration, we performed a habituation-dishabituation test for olfactory system function (Fig. 1A). In this test, mice are first habituated to filter paper placement in their cage (containing mineral oil) then evaluated for subsequent investigation of new filter papers containing novel odors. In pilot analyses, we determined that 5-10 μg of CT IN was optimal for detection of olfactory system decline 24 h later (as higher doses limited mouse movement due to respiratory distress and lower doses were comparable to naïve mice, see Supplementary Fig. 1). We then treated mice with CT or 30 μg LTA1 IN and 24 h later performed the olfactory habituation-dishabituation test. Unlike CT-treated mice, LTA1-treated and untreated mice investigated cheese or shrimp odors significantly more times than their habituation control (i.e., in comparison with their third trial with mineral oil; Supplementary Video 1 and Fig. 1B). Compilation of all times investigating specific odors (from three trials) also indicated non-significant difference from LTA1-treated mice and untreated, whereas significantly lower investigations were observed with CT-treated mice in cheese and shrimp trials (Fig. 1C, respectively P = 0.07 or P < 0.01).
Although major histological changes in nasal tissue were not previously been reported with LT 26 , we also further examined mice post-behavioral testing for histopathologic changes in three H&E-or toluidine blue-stained transverse sections through the nasal cavity according to established guidelines 27 and depicted in Fig. 2A (top). CT-treated mice exhibited neutrophil infiltration (arrows) and mild, acute inflammation in subepithelial tissue of nasal passages immediately posterior to the incisors and at the level of the incisive papilla but not by the second molar tooth and also a significantly higher level of mast cells ( Fig. 2A,B and Supplementary Fig. 2). No significant inflammatory changes were detected in nasal passages of LTA1-treated or untreated mice. Thus, LTA1 exhibits improved neurological safety compared with an AB 5 enterotoxin after IN delivery.

Immune responses to IN influenza immunization are enhanced with LTA1 adjuvant and exhibit dose-dependent responses for antibody generation.
To investigate whether LTA1 can boost immunity to an IN vaccine, we used the inactivated antigen from the injectable split-virus commercial flu vaccine Fluzone ® (FZ). FZ formulations at 3.6 μg total hemagglutinin (HA) content were admixed with 1, 5 or 10 μg LTA1 and delivered by prime/boost IN immunization to mice (days 0, 21) with sample analyses performed two weeks after either immunization (day 14 or 35) (Fig. 3A). For all analyses, two doses yielded higher levels of antibodies and recall cytokine responses than one and the addition of LTA1 adjuvant increased detectable FZ-specific responses ( Fig. 3 and Supplementary Fig. 3). Significant induction of serum IgG1 in the FZ group was observed compared to naïve animals on day 35, but only FZ + LTA1 groups developed antigen-specific serum IgG2a or bronchoalveolar lavage fluid (BAL) IgA. In general, development of antibody responses was adjuvant dose-dependent and maximal levels were observed with the highest dose of adjuvant (dark green bars, FZ + LTA1 10 μg, Fig. 3B).
We also evaluated memory cytokines responses to recall antigen using ex vivo cultured splenocytes (Fig. 3C,D). Robust cytokine secretion was observed in the FZ group compared to the naïve group including Th1 cytokines (INFγ, IL-2, TNFα) and other cytokines or proteases linked to protection from flu (GM-CSF 28 , MIP-1β). Inclusion of LTA1 augmented these responses and also promoted generation secreted factors IL-6 and Granzyme B (a serine protease that contributes to apoptosis of infected cells) 29,30 . However, with a few exceptions (e.g., Granzyme B, GM-CSF), the level of cytokine secretion was not adjuvant dose-dependent. Thus, pursuant to these results, we chose the 10 μg LTA1 dose for subsequent animal studies. LTA1 adjuvant enhances protection from H1N1 flu challenge in FZ-vaccinated mice. We next examined if mice immunized with FZ and LTA1 adjuvant would improve protection from H1N1 challenge. First, we immunized mice with IN delivery of FZ alone or with 10 μg LTA1 (FZ + LTA1), then challenged them 2 weeks later with lethal pandemic Influenza strain A/California H1N1 (Fig. 4A). All mice were monitored for mortality and morbidity (e.g. weight loss). We found this single FZ + LTA1 immunization resulted in 100% protection from H1N1 mortality compared with only ~60% survival after FZ immunization (P = 0.06, Fig. 4B). FZ + LTA1 animals initially exhibited weight loss on days 2-4 post-challenge but recovered more rapidly than did FZ-immunized animals (*P ≤ 0.05 for weight loss on study days 6-11 post-challenge, Fig. 4C). We also observed improved protection from H1N1 survival and weight loss in a prime/boost model with 24-month old mice immunized with FZ + LTA1 compared to FZ ( Supplementary Fig. 4). In conclusion, LTA1 adjuvant improves vaccine-mediated protection from H1N1 challenge in mice. LTA1-mediated protection from H1N1 flu challenge is associated with lower lung viral titers, acute-phase response IL-6 cytokine, and higher serum IgM, IgG, and IgA antibodies. We next During each trial, mice were exposed to filter paper laced with mineral oil, cheese odor or shrimp odor as indicated. (B) Mean + SEM times investigating filter paper during each trial from 3 experiments totaling 5 mice/group. Significance is indicated (*untx, # LTA1, ** ,## P ≤ 0.01, *** ,### P ≤ 0.001) for trials with increased investigations compared to the habituation control (3 rd trial) using two-way repeated measures ANOVA with Dunnett's post-test. No CT trials were significantly different than habituation control. (C) Mean + SEM times investigating filter paper compiled by odor for all three trials. Significance is indicated (**P ≤ 0.01) using ANOVA with Dunnett's post-test compared to untreated.
wanted to confirm our survival and weight loss results by evaluating if viremia or acute-phase response cytokine IL-6 during H1N1 challenge would be similarly improved by the addition of LTA1 adjuvant to vaccination. To do so we tested level of H1N1 viral RNA or IL-6 RNA in the lungs of mice day 3 or day 6 post-challenge comparing unvaccinated, FZ-vaccinated, or FZ + LTA1 vaccinated groups after 1 or 2 immunizations (Fig. 4D-I). In all instances, we observed significantly lower levels of lung H1N1 viral RNA in the FZ + LTA1 mice compared the FZ only group (P < 0.05 for day 6 post-H1N1 in primed animals; P < 0.001 for day 3 or 6 post-H1N1 in prime/boosted animals; Fig. 4D,G). Similarly, we observed significantly lower levels of lung IL-6 RNA in the FZ + LTA1 mice compared the FZ only group (P < 0.05 for day 6 post-H1N1 in primed animals; P < 0.001 for day 3 or P < 0.05 day 6 post-H1N1 in prime/boosted animals; Fig. 4E,H). Using data collected from all animals, we also observed that the level of viral RNA significantly correlated to lung IL-6 for each experimental conditions (Spearman's P < 0.001 and r > 0.7; Fig. 4F,I).
We also evaluated serum FZ-specific IgM, IgG, and IgA antibody responses and number of total CD19+ B-cells in the lungs from these animals post-challenge ( Fig. 5A,B). We observed significant changes in post-challenge levels of serum IgM, IgG, and lung B-cells post-challenge in mice immunized with FZ + LTA compared to those immunized just with FZ after 1 immunization or 2 immunizations (P < 0.05 or greater). Serum IgA responses were also elevated in the FZ + LTA1 group, but only significantly different from FZ at 6-days post-H1N1 challenge after prime immunization (P < 0.05). Using data collected from all animals all timepoints, we also observed that the level of serum IgM, IgG, or IgA significantly and inversely correlated to levels of lung H1N1 viral RNA (Spearman's P < 0.001 and r < −0.65; Fig. 5C). Thus, LTA1 immunized animals were better protected from high levels of H1N1 viral replication as well as viral associated acute phase responses IL-6 cytokine secretion while exhibiting higher levels of humoral immune responses post-challenge (anti-FZ serum antibodies).

LTA1-mediated protection from H1N1 flu challenge does not require B-cells. B-cells are crit-
ical to protection from influenza, as mice lacking mature B-cells (μMT mice) have worse survival outcomes post-challenge 31 . In addition, changes in serological responses (e.g., HA-neutralizing antibody titers) are the primary correlate of vaccine efficacy for existing or new clinical influenza vaccines 32 . We also observed higher levels of B-cells and serum antibodies in FZ + LTA1 vaccinated animals than in other tested groups (Fig. 5). To investigate if the protective effects of LTA1 adjuvant flu vaccine require intact humoral responses, we immunized μMT mice with FZ + LTA1 twice on days 0, 21 and then compared their responses to lethal H1N1 infection on day 35 with that of naïve μMT mice. Immunized mice were completely protected from mortality after flu challenge (P < 0.001) and also exhibited minimal weight loss compared with naïve μMT mice (significant beginning day 6 post-infection; Fig. 6A).
To confirm these results, we next evaluated animals from μMT or C57Bl/6 (wild-type or WT) mice 6 days after H1N1-challenge who had been immunized prime/boost with either FZ or FZ + LTA1. Similar to previous results www.nature.com/scientificreports www.nature.com/scientificreports/ ( Fig. 4), we observed lower lung H1N1 viral levels in FZ + LTA1 mice compared with FZ in either μMT or WT strains (P < 0.01 in μMT mice or P < 0.001 in WT mice, Fig. 6B). In addition, all μMT mice irrespective of immunization history had significantly reduced levels of % or total lung CD19+ B-cells ( Fig. 6C-E) and no detectable anti-FZ serum IgM, IgG, or IgA 6-days post-H1N1 challenge (Fig. 6F). No differences in B-cells or antibody levels were observed between FZ or FZ + LTA1 groups in μMT mice. Thus, despite previously observed humoral effects with FZ + LTA1 immunization, the protective effects of LTA1-adjuvanted flu vaccine is not dependent upon B-cells.

LTA1-mediated protection from H1N1 flu challenge is associated with lung CD4 T-cells. Since
B-cells were not required for LTA1 mediated protection from flu challenge, we next evaluated changes to other immune cell populations in the lungs of μMT or WT mice 6 days after H1N1-challenge who had been immunized prime/boost with either FZ or FZ + LTA1. We observed higher levels of % CD3 T-cells and lower % of levels of NK cells recruited into the lungs of FZ + LTA1 μMT or WT groups post-challenge (P < 0.05 or greater, Fig. 7A,B). Within this CD3 T-cell population in both mouse strains, the proportion of CD4 T-cells was greater in FZ + LTA1 groups (P < 0.001, Fig. 7C), while % of CD8 T-cells or NKT cells were smaller or not-significantly different. Further evaluation in WT mice, indicated that CD4 T-cells in the FZ + LTA1 animals were also expressing significantly high levels of early activation marker CD69 than unvaccinated or FZ mice on days 3 and 6 post-H1N1 challenge (P < 0.001, Fig. 7D,E). Evaluation of INFγ RNA levels in the lungs of these mice was not significantly different between vaccinated mice groups (Fig. 7F), indicating no evidence for stronger Th1 responses. In contrast, slightly higher levels of IL-22 RNA were observed in FZ + LTA1 mice compared with FZ (P < 0.01 for day 6, Fig. 7G). IL-22 secretion by T-cells has previously been shown to be critical for epithelial repair during influenza infection 33,34 , but has not been explored as a mechanism in enhanced protection from enterotoxin based adjuvants. Thus, the protective effects of LTA1-adjuvanted flu vaccine are associated with CD4 T-cell activation and possibly IL-22 secretion.  (F) Correlation of lung RNA data from D&E with Spearman correlation coefficient (r) and P-value indicated. (G) Lung H1N1 viral RNA and (E) lung IL-6 RNA levels by qRT-PCR and quantified as fold change to unvaccinated/H1N1 infected mice were analyzed from day 3 or day 6 post-H1N1 challenged mice (n-4) previously vaccinated prime/boost (day 0, 21) and challenged on day 35. (I) Correlation of lung RNA data from G&H with Spearman correlation coefficient (r) and P-value indicated. Data reported as mean + SEM with significance indicated by log-rank (survival), t-test with Bonferroni-Dunn correction (weight), or by oneway ANOVA with Dunnett's post-test compared to FZ group (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ns = not significant).

Discussion
In this study, we identified LTA1 as a safe, effective adjuvant for IN delivery with the potential to overcome past clinical safety issues observed with the LT-adjuvant flu vaccine. This is the first report of LTA1 enhancing vaccine protection against flu challenge or any infectious challenge. This clearly refutes the long-held belief that the A1 domain of LT is not stable enough to bind to or be taken up at mucosal surfaces and mediate immunologic stimulation. Additionally, this is the first study to demonstrate safety of an LT-based IN adjuvant using behavioral testing as evidence for lack of neurological toxicity in comparison with holotoxin. Lastly, we provide insight into the protective mechanisms of LTA1, identifying that B-cells are not required for adjuvanted vaccine protection against flu challenge but CD4 T-cells are highly activated. These results have direct implications for vaccine development and use of LTA1 as a novel IN adjuvant.   www.nature.com/scientificreports www.nature.com/scientificreports/ Enterotoxin-based adjuvants are well known inducers of mucosal immunity, but their use in IN vaccination was hindered by increased risk of Bell's palsy (estimated at 13 cases per 10,000 vaccine recipients) compared with non-vaccinated individuals 14 . One benefit of this past clinical observation is that other severe adverse events (e.g., local site reactogenicity, increased risk for cancer, birth defects, or pulmonary complications etc.) were never observed in pre-clinical and clinical studies 14,26 . Thus, the specific safety risks that must be mitigated for a safe LT-based IN adjuvant are clearer than with an untried adjuvant. There is no pre-clinical model of Bell's palsy or other assessment of cranial nerve 1 reported with enterotoxin use. Therefore, we utilized the olfactory habituation-dishabituation test that assesses neurotoxicity through changes to cranial nerve 7 and olfactory system function (Fig. 1) 23 . While we previously speculated that removal of the B-subunit would limit neurological toxicity 24 , this is the first demonstration of nontoxicity after nasal delivery of a LT-based adjuvant (at a 3-30x expected dose) using this methodology. Combined with our previous data demonstrating poor antigenic potential 25 , this indicates LTA1 is an attractive option to overcome past clinical trial safety concerns. It is also worth mentioning, that LTA1 is also distinct from any other proposed IN enterotoxin adjuvant because it is based on LT protein and has no specific binding or cell-targeting component. For example, CTA1-DD has also been presented as an IN adjuvant but it is composed of a B-cell targeting synthetic component based on protein A and the A1 domain of CT (which is differs from LT by level of enzymatic activity and quality of immune responses) 35,36 . However, continued investigations with LTA1 both for neurological safety and in comparison with related adjuvants are warranted.
In our experiments, we utilized a flu vaccination model to determine the ability of LTA1 to improve vaccine efficacy. However, our results in this model are relevant since optimal protection against influenza has yet to be achieved 6 . Seasonal and pandemic influenza (flu) are considered major public health threats despite annual vaccine campaigns 37,38 . Addition of an adjuvant can overcome limited vaccine efficacy in high-risk populations (i.e., young, elderly) and also allow dose-sparing in case of vaccine shortages due to the 6-9 month production time for new vaccines 37,39,40 . Lastly, while traditionally IN vaccines may not perform as well as injectables, they often are a popular needle-free option, as observed with FluMist ® vaccination campaigns in US schools 41 . Thus, an adjuvanted IN vaccine may improve vaccine efficacy particularly for high-risk groups and improve logistics of mass delivery during seasonal disease or pandemic outbreaks. In this study, we were able to show IN vaccination with an optimized dose of LTA1 admixed with an inactivated virus antigen improved survival, weight loss, and viremia from lethal H1N1 challenge (Fig. 4, Supplementary Fig. 4). Thus, LTA1, like its parent molecule LT, can boost immunity to flu antigens and has potential as a novel adjuvant for IN vaccine strategies.
We observed increases in humoral immunity in LTA1 adjuvanted vaccination that correlated with reduced lung viremia (Fig. 5). Despite this observation, μMT mice immunized twice with LTA1 + FZ were protected from severe weight loss and death from lethal H1N1 challenge and still exhibited reduced lung viremia (Fig. 6). Immunized mice did exhibit a slight decrease in weight post-challenge (5-10%) that differed from that observed in non-transgenic adult mice (seen in Supplementary Fig. 4). μMT mice have a disruption in the IgM heavy chain and therefore don't develop mature B-cells and are estimated to have at least 50-fold more sensitivity to influenza infection 31,42 . This indicates that classic B-cell functions, including antigen presentation and neutralizing antibodies are not required for LTA1 adjuvant effects, though B-cell responses may be necessary for maximal adjuvant-mediated protection. In both μMT and WT mice, we observed high levels of CD4 T-cells in the lungs during infection (Fig. 7). This suggests that LTA1-mediated protective responses may be dependent upon CD4 T-cells or both B-cells and CD4 T-cells may play redundant roles in protection that should be explored in future studies.
In conclusion, these results support LTA1 as a unique mucosal adjuvant that has broad potential for flu and other vaccines. The impetus for this adjuvant development comes as successful control of infectious diseases through vaccination and other public health measures has reduced levels of circulating infections, thereby widening the gap between acceptable risks of side effects versus benefits from disease protection afforded by vaccination. This ever-evolving dialog alters how vaccines are formulated and scheduled, even precluding the use of older, effective live-attenuated vaccines or resulting in the phasing out of whole-killed vaccines due to reactogenicity and/or risk of disease, such as with pertussis and polio vaccines [2][3][4][5] . However with altered vaccine formulations, tradeoffs in immunity are being created, including gaps in protection observed currently with both polio and pertussis vaccines that protect against clinical disease but not transmission. Adjuvants such as LTA1 that allow needle-free delivery and can promote mucosal immunity to subunit vaccination 24 or inactivated vaccines (as reported here) can play the critical role of filling these key gaps in protection in the continuously changing landscape of vaccination and disease prevention policies.

Methods
Vaccines and adjuvant proteins. LTA1 and CT were produced from E. coli clones expressing recombinant protein as previously described 25,43 . Proteins were stored lyophilized and freshly resuspended prior to use (CT) or kept frozen at −20 °C until use (LTA1). The endotoxin content of all proteins was < 1 EU/mg. The commercial Mice. Seven-to-9-week old C57BL/6 female mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
For aged mice experiments, Balb/c mice were purchased at 6-to-8-weeks of age from NCI Charles River and aged in-house. Male and female μMT mice (strain B6.129S2-Ighmtm1Cgn/J) were originally purchased from The Jackson Laboratory and bred in house for experiments. All mice were housed in sterile set-up with filter-top cages. Rodents were fed normal chow, with addition of weekly supplemental wet diet gel 76 A (ClearH2O, Portland, ME) for aged mice experiments. Mice were euthanized by CO 2 inhalation and exsanguination (immunization studies) Olfactory habituation-dishabituation test & nasal cavity histology. Mice were individually housed and IN treated or left naive and tested by olfactory habituation-dishabituation 24 h later as previously described 23 . Treatments included 30 μg LTA1 (0.5 mg/ml) or 1-30 μg CT (1 mg/ml). Trials were performed with 2cmx2cm filter paper freshly infused with 20 μl mineral oil (Millipore Sigma), cheese trout bait (Berkley, Columbia, SC, diluted 1:2 in ethanol), or shrimp oil (Atlas Mike's, Fort Atkinson, WI) and by recording times investigating paper within ≤1 mm. After testing, select mice (n = 3/group) were euthanized using ketamine/xylazine and nasal cavities dissected according to established guidelines 27 for formalin fixation, paraffin embedding, sectioning, H&E staining, and blindly analyzed by a pathologist using a score added for all three nasal sections per mouse (0 = normal, 1 = mild neutrophil inflammation and/or edematous changes, 2 = moderate degenerative or proliferative lesion, 3 = severe lesions with extensive damage to tissue architecture). Mast cells were analyzed using standard toluidine blue staining of paraffin embedded sections, counting total number purple cells per 100x microscopic field area added for two nasal sections (0 = <5 cells per area, 1 = 5-15/area, 2 = 15-25/area; 3 = 25-35/area; 4 = >35/area).

Animal vaccination & H1N1 challenge.
Groups of mice (n = 4-8) were immunized with 3.6 μg FZ (or 1.2 μg of H1N1) containing 0-10 μg LTA1 diluted in PBS (20-30 μl total volume) IN by pipetting directly into nares of mice anesthetized with ketamine/xylazine or (aged mice only) inhaled isoflurane. Two weeks after the last immunization, mice were euthanized for sample collection or anesthetized again and challenged IN with mouse-adapted influenza A/California/07/2009 (H1N1) diluted in PBS (50 μl total volume) at median lethal (1xLD50) or lethal (2xLD50) doses optimized for Balb/c or C57Bl/6 mouse strains. Mice were monitored for weight daily with loss of 30% initial body weight the endpoint for euthanasia. Some surviving mice were euthanized and perfused by cardiac puncture with saline, prior to inflation of lungs with 10% formalin for paraffin embedding, sectioning and H&E staining.