Activation of an innate inflammatory response is a key step in the mechanisms of action of adjuvants. Gene expression profiling of the mouse muscle injected with different adjuvants revealed modulation of a cluster of innate immunity genes, including particularly: cytokines, cytokine receptors, chemokines, adhesion molecules, and proteins involved in host–pathogen interactions (60). In particular the long *PTX3*, a soluble pattern recognition molecule identified in the 1990s (61), was among the over 1,000 genes upregulated by adjuvant treatment. PTX3 is classified as a member of the pentraxins, a family of highly conserved molecules characterized by a unique multimeric structure (62, 63; Garlanda et al., forthcoming2 ). Based on the sequence of the composing promotors, two subfamilies can be distinguished: short and long pentraxins. C-reactive protein (CRP) and serum amyloid P component are prototypic short pentraxins, by contrast PTX3 is a prototypic long pentraxin subfamily member.

Despite their sequence homologies, CRP and PTX3 differ in molecular structure, gene organization and cellular sources. CRP is produced only by the liver in response to IL-6 while PTX3 is produced locally and more rapidly by myeloid and stromal cells in response to primary pro-inflammatory cytokines (IL-1β

and TNF-α) and ligation of TLRs. In addition, PTX3 is stored in neutrophils-specific granules, from which it is promptly released upon microbial recognition (63). From a functional point of view, PTX3 is endowed with multifunctional properties at the crossroad of inflammation, innate immunity, tissue remodeling, and female fertility. Of note, PTX3 plays an essential non-redundant role in resistance to selected microbes. The protein has antibody-like properties, binding selected pathogens (i.e., *A. fumigatus*, *Pseudomonas aeruginosa*, and more) and conserved microbial moieties (outer membrane protein A from *Klebsiella pneumoniae*; HA glycoprotein from influenza virus), and facilitating phagocytosis of recognized microbes in a manner dependent on Fcγ receptors and complement (64, 65). Moreover, PTX3 orchestrates complement activity (66) and, by regulating complement-dependent tumor promoting inflammation, can act as an extrinsic oncosuppressor gene in murine and human tumors (67). Finally, recent results obtained in different models of tissue damage highlighted a non-redundant role of PTX3 in remodeling and repair of tissue *via* its interaction with fibrin (68). This further supports the evidence that the recognition of matrix and microbial components is shared ancestral features of the humoral arm of the innate immune system.

In view of the upregulation of PTX3 by selected adjuvants we investigated the role of this protein in the antibody response using a well-known model of vaccination with OMV from *Nm*. We found that PTX3 binds *Nm* as well as OMV derived from *Nm*, exerting a protective role in a model of infection with *Nm* in the infant rat. *Ptx3*-deficient mice vaccinated with OMV without any adjuvant developed a lower antibody response compared with WT mice. In addition, co-injection of PTX3 enhanced the antibody response, especially in *ptx3*-deficient mice. Recognition of the antigen is essential for the effect exerted by PTX3, since immunization with an antigen not recognized by PTX3, such as ovalbumin, induced similar response in WT and *ptx3−/−* mice (69). This observation was confirmed by further investigations showing that *ptx3−/−* mice produced lower levels of IgM in response to administration of Pneumovax, a human vaccine containing capsular polysaccharide from multiple *Streptococcus pneumoniae* (*Sp*) serotypes (70). Similarly, *ptx3−/−* mice produced less antibodies following immunization with PR8 influenza A virus, which is also recognized by PTX3 (71), and in both cases PTX3 administration restored the response. Searching for an explanation of these effects, it has been found that PTX3 was released by B helper neutrophils (NBh) and bound to B-cells located in the marginal zone (MZ) in the spleen. NBh are an innate-like subset of antibody-producing cells positioned at the interface of the adaptive immune system and the circulation. The interaction between PTX3 and MZ-B-cells activated signals that were independent of FcγR but triggered class-switching from IgM to IgG. PTX3 promoted MZ-B-cell differentiation into extrafollicular plasmablasts and plasma cells, and enhanced IgM and IgG responses to the encapsulated bacterium *Sp*, or after immunization with capsular polysaccharides or bacterial carbohydrates. These results indicate that the humoral pattern recognition molecule PTX3, produced in response to pro-inflammatory cytokines or selected adjuvants, may amplify

2Garlanda C, Bottazzi B, Magrini E, Inforzato A, Mantovani A. PTX3, a key component of humoral innate immunity at the interface between defense and tissue remodeling. *Physiol Rev* (Forthcoming).

effective adaptive antibody responses that are induced by antigens recognized by PTX3, thus serving as an endogenous adjuvant (**Figure 3**).

The data outlined above provide a rationale to evaluate whether PTX3 could be a correlate of the shaping of the immune response induced by adjuvants in humans. In collaboration with D. J. Lewis and G. Del Giudice, PTX3 and CRP plasma levels were measured in a cohort of individuals injected with placebo or with licensed influenza vaccines adjuvanted or not. This study received ethical approval from London—Surrey Borders Research Ethics Committee (REC Ref: 13/LO/0044) and was registered on ClinicalTrials.gov before enrollment (NCT01771367). Preliminary unpublished results evidenced an increase in PTX3 plasma levels at early time points in individuals injected with the adjuvanted vaccine, confirming in humans the effects on PTX3 gene upregulation initially observed in mice (60). In addition, after immunization with an adjuvanted vaccine we observed an earlier induction of PTX3 (peak at 24 h postinjection) compared with CRP (peak at 48 h postinjection), likely due to the local expression of PTX3 versus the systemic production of CRP.

Several single nucleotide polymorphisms (SNPs) have been described in the human *PTX3* gene, mainly located in its non-coding regions with the only exception of one exonic SNP causing an amino acid variation in position 48 (Asp48Ala). *PTX3* haplotypes are associated with increased susceptibility to lung TB (72), *P. aeruginosa* infections in cystic fibrosis Caucasian patients (73), *A. fumigatus* infections in bone marrow transplanted patients (74), and urinary tract infections (75). Results in a large cohort of patients with fungal infections after solid organ transplantation (76) and in 2,609 bone marrow transplanted patients and their donor pairs (77) further reinforced the association of *PTX3* gene SNPs and susceptibility to fungal infection. The protection-associated haplotypes were also associated with higher PTX3 protein expression and circulating levels (74, 76), supporting an active role of PTX3 as non-redundant player involved in the innate defense against recognized pathogens.

In summary PTX3, a molecule of the innate immune system, produced in response to pro-inflammatory mediators, not only acts as an antibody-like molecule, recognizing pathogens and promoting their removal but also helps in antibody production by adaptive immunity, acting as endogenous adjuvant. This is schematically depicted in **Figure 3**.

# CONCLUDING REMARKS

Understanding the factors that are critical for the induction of protective immunity by vaccines is key for the rational development of novel vaccines and to optimize vaccine efficacy. Within the ADITEC work package "Molecular signatures of immunity and immunogenicity," the aim was to identify key molecular mechanisms of innate and adaptive immunity following vaccination and to uncover correlates of protection at the transcriptomic, proteomic and cellular level.

Transcriptomic profiling identified IFN-inducible gene *FCGR1A* as one of the most robust and consistent single biomarkers that could serve as part of reliable predictive signatures discriminating active TB disease from latent infection, identifying individuals at risk of developing TB, and monitoring TB treatment response kinetics. Such signatures are useful in stratifying risk groups for preventive treatment or clinical vaccine testing, as well as minimizing treatment duration and preventing possible *de novo* drug resistance, independent of age, immune status and genetic background. By contrast, when comparing transcriptome profiles between infants vaccinated with parental or recombinant BCG VPM1002 to unravel the mechanisms underlying superior protection against TB by VPM1002, differences at the single gene level could not be detected. Therefore, a new bioinformatics tool (tmod) was used to calculate enrichments in genes, uncovering more pronounced changes in T-cell activation and myeloid/monocyte-associated genes in the VPM1002 vaccinated group. Complementing the work on transcriptomics, HDX–MS was used to determine quantitative proteomic profiles and was successfully applied to define functionally active epitopes and antibodies in polyclonal sera following vaccination for various bacterial vaccines.

At the cellular level, early expansion of vaccine-specific CD4+ Tfh cells in peripheral blood was found to predict long term

# REFERENCES

persistence of neutralizing antibodies against influenza virus, suggesting this to be a possible biomarker of protective vaccine efficacy. In addition, it was observed that vaccine-induced human CD4+ T-cells are functionally heterogeneous and comprise not only clones polarized toward a single fate but also clones that fit the one cell-multiple fates model of CD4 T-cell differentiation. This suggests that inducing broader range of effector and memory T-cells with broad and different functional properties would benefit the host by expanding the number of options to respond to pathogens. Parameters affecting the ability of leukocytes to properly migrate to the site of vaccination and mount an effective immune response were also investigated. Interestingly, persistent immune activation caused impaired T-cell trafficking at mucosal sites, which was due to alterations in the cytoskeleton machinery. Pharmacological intervention acting on the cytoskeleton could restore a proper response to chemokines, calling for novel vaccination strategies in frail individuals. Finally, PTX3 was identified as a molecule produced by the innate immune system in response to pro-inflammatory mediators and was shown to be involved in the innate defense against recognized pathogens. PTX3 functions as an endogenous adjuvant, bridging the humoral arms of the innate and adaptive immune systems.

In summary, within the ADITEC work package "Molecular signatures of immunity and immunogenicity," dissecting the human innate and adaptive immune response to vaccination and infection identified not only novel biomarker signatures at the transcriptomic, proteomic, and cellular level that could serve as potential correlates of risk and of protection but also uncovered novel markers and mechanisms underlying protective immunity. Validating and further expanding on these findings will advance our understanding of protective immunity, which is a key step toward the development of effective novel vaccines.

# AUTHOR CONTRIBUTIONS

All the authors have contributed a section to the manuscript, based on their research in the EC FP7 ADITEC project.

# FUNDING

The authors gratefully acknowledge funding by the European Commission, EC FP7 ADITEC Grant Agreement No. 280873. The text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information.

ethnicity. *Clin Microbiol Infect* (2014) 20:O230–8. doi:10.1111/1469-0691. 12383

and pentamer reveal mutually exclusive cell entry complexes. *Proc Natl Acad Sci U S A* (2015) 112:1767–72. doi:10.1073/pnas.1424818112

[corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. *Pharmacol Rev* (2014) 66:1–79. doi:10.1124/pr.113.007724

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Haks, Bottazzi, Cecchinato, De Gregorio, Del Giudice, Kaufmann, Lanzavecchia, Lewis, Maertzdorf, Mantovani, Sallusto, Sironi, Uguccioni and Ottenhoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Susan L. Baldwin1†, Fan-Chi Hsu1†, Neal Van Hoeven1 , Emily Gage1 , Brian Granger1 , Jeffrey A. Guderian1 , Sasha E. Larsen1 , Erica C. Lorenzo2 , Laura Haynes2 , Steven G. Reed1 and Rhea N. Coler1,3,4\**

*1 Infectious Disease Research Institute, Seattle, WA, United States, 2Center on Aging, Department of Immunology, University of Connecticut School of Medicine, Farmington, CT, United States, 3Department of Global Health, University of Washington, Seattle, WA, United States, 4PAI Life Sciences, Seattle, WA, United States*

### *Edited by:*

*Rino Rappuoli, GlaxoSmithKline, Italy*

### *Reviewed by:*

*Randy A. Albrecht, Icahn School of Medicine at Mount Sinai, United States Katie Louise Flanagan, Monash University, Australia*

### *\*Correspondence:*

*Rhea N. Coler<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 12 September 2017 Accepted: 01 February 2018 Published: 19 February 2018*

### *Citation:*

*Baldwin SL, Hsu FC, Van Hoeven N, Gage E, Granger B, Guderian JA, Larsen SE, Lorenzo EC, Haynes L, Reed SG and Coler RN (2018) Improved Immune Responses in Young and Aged Mice with Adjuvanted Vaccines against H1N1 Influenza Infection. Front. Immunol. 9:295. doi: 10.3389/fimmu.2018.00295*

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/ H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential *in vitro* responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.

Keywords: influenza, vaccine, adjuvant, elderly, T helper 1, H1N1

### INTRODUCTION

Influenza and influenza-related complications are leading causes of death in elderly populations. Older people exhibit increased morbidity and mortality in response to influenza infection due to uncontrolled respiratory inflammation, severe pneumonia, or multi-organ inflammation and failure (1). Although the annual influenza vaccine coverage rate has increased, the CDC estimates that

**256**

individuals older than 65 years of age who have received influenza vaccines in consecutive years are still at high risk of influenza infection (2).

The process of immunological aging, also called immunosenescence, is associated with a progressive loss of functional physiological integrity including collectively increasing DNA damage and genome instability, stem cell exhaustion, cellular senescence, and altered intercellular communication, among other processes [reviewed in Ref. (3)]. Immunosenescence causes the attenuation of both innate and adaptive immune systems including reduced levels and function of TLRs in macrophages and plasmacytoid dendritic cells (4, 5), decreased output of naïve B cell numbers (6), severe reduction of thymopoiesis (7), and insufficient immune synapse generation (8). Furthermore, aged individuals also demonstrate a significant reduction in the quantity, but not quality of antigen-specific antibody responses, and this reduction in antibody quantity is concurrent with a decrease in antigen-specific plasmablasts (9). Indeed, defective antigen presentation and reduced T cell and B cell repertoires in aged individuals results in poor cellular, humoral, and vaccineinduced responses.

As vaccination is the most effective method to prevent infection, ways to improve influenza vaccines for use in the aging human population are continually being explored. These strategies include adjuvant use, intradermal delivery, increased dosage, and altering antigen selection that typically make up seasonal vaccines [reviewed in Ref. (10)]. The first seasonal influenza vaccine containing an adjuvant (MF59), also known as Fluad™, was approved by the FDA in 2015 for use in people over the age of 65 years. Fluzone® High-Dose vaccine (an inactivated, split influenza virus vaccine containing 60 µg of HA for each component of the vaccine, rather than 15 µg) is also approved for use in people ≥65 years of age. Despite advancements in alternative vaccine options for the elderly population, they are still disproportionally affected and continue to exhibit severe morbidity and mortality each year from seasonal influenza infections. Thus, a need still remains for better strategies, including additional adjuvant options, for the influenza vaccine to overcome the challenges of immunosenescence in the elderly.

The disparity of morbidity and mortality burden in elderly individuals is partly due to the incomplete understanding of how adjuvanted vaccines may circumvent immunosenescence in elderly people. Mechanistic details regarding how different adjuvants work in the elderly may reveal ways in which adjuvant formulations can be tailored and adapted for optimal responses that provide enhanced protection for this susceptible population. Administration of adjuvants, including squalene-based oil-in-water emulsions, and those including a TLR4 agonist, have been leveraged in vaccines in order to enhance immune responses in multiple studies (11–18). These adjuvants include MF59 (as described above), and AS04 (comprised of monophosphoryl lipid A and Alum) included in FDA approved vaccines against hepatitis B virus and human papillomavirus (11). In preclinical studies, we have shown that glucopyranosyl lipid A (a synthetic TLR4 agonist) formulated in a stable oil-in-water emulsion (GLA-SE) expands immune responses to Fluzone® (12). GLA has also been included in several completed and ongoing human clinical studies for vaccines against *Leishmania* (NCT01751048), *Mycobacterium tuberculosis* (NCT02508376, NCT0246516, NCT01599897), HIV (NCT01966900, NCT01922284), Schistosomiasis (NCT03041766), malaria (NCT02647489, NCT01540474), and avian influenza (NCT01991561, NCT01147068). In humans, GLA-SE combined with rH5 was considered safe and improved antibody titers compared to the recombinant protein alone (15). Furthermore, no defect has been identified in humans following stimulation with GLA-SE on antigen-presenting cells (APCs) from aged compared to young APCs (16) and enhances T cell responses from older adults following stimulation of peripheral blood mononuclear cells (PBMCs) with live influenza virus (19). Therefore, our approach in this study was to employ the use of different adjuvants combined with an influenza vaccine to overcome the challenge of immunosenescence in aged animals.

In this investigation, one of our main findings reveal that aged mice have dampened cytokine responses to *in vitro* stimulation of DCs and lung homogenates with a T helper 1 (Th1) cytokineinducing agonist. In addition, we show that aged mice require two immunizations with adjuvanted sH1N1 vaccines for robust *in vivo* protection against influenza whereas young mice are protected after a single immunization. Finally, we demonstrate that immunization in young mice with sH1N1 + GLA-SE results in enhanced alveolar macrophage (AM) homeostasis within the lung, increased TLR7 expression within the AMs, and faster clearance of virus after H1N1 challenge compared to mice immunized with sH1N1 + SE or vaccine alone. These results emphasize the age-related differences following immunization, the ability to improve responses to influenza infection in the elderly with two immunizations, and a mechanism of enhanced vaccine protection against influenza with a TLR4 agonist adjuvant.

### MATERIALS AND METHODS

### Mouse Model

Young female C57BL/6 or CB6F1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA) or the Jackson Laboratories (Bar Harbor, ME, USA) and were housed and maintained under specific pathogen free conditions at the Infectious Disease Research Institute. Experiments that included female C57BL/6 or CB6F1 mice aged 18–21 months were acquired with special request from the National Institute on Aging, from the aged rodent colony (National Institute of Health, Bethesda, MD, USA). Mice were housed in a biosafety level 2 (BSL2) environment for the entirety of these studies (including H1N1 challenge studies), and all procedures were performed in accordance with the regulations and guidelines of the IDRI animal care and use committee.

### Adjuvants and Immunization

Vaccines were formulated in saline, SE (2% final v/v oil concentration), an MF59-like adjuvant (2.5% final v/v oil concentration), or GLA-SE (5 µg of GLA in 2% SE). Mice were immunized intramuscularly (i.m.) one or two times 3 weeks apart. The split H1N1 influenza vaccine (sH1N1) (kindly provided by Novartis) and recombinant H1 (rH1) (A/California/4/2009; Protein Sciences Corp., Meriden, CT, USA) was used at 0.01 or 0.1 µg, respectively, for the immunizations. Serum was collected 3 weeks after prime or boost immunizations. Spleens and bone marrow were harvested for immunogenicity studies either 1 or 4 weeks following immunization(s) as described.

### Endpoint Antibody Titers

Sera were analyzed for H1-specific IgG1 and IgG2c endpoint antibody titers by antibody capture ELISA. Polysorp ELISA plates (Nunc-immuno polysorp 96 well plates, VWR) were coated with rH1 (A/H1N1/California/2009) (Protein Sciences Corp., Meriden, CT, USA) at a concentration of 1 µg/ml in 0.1 M bicarbonate coating buffer at 100 µl per well for 4 h at room temperature. Plates were then blocked with a 0.05% PBS-Tween solution plus 1% BSA and incubated overnight at 4°C, followed by five washes in 0.1% PBS-Tween and one PBS wash. Serially diluted mouse sera were added and plates incubated at room temperature, on a shaker, for 2 h. Plates were washed, dried, and secondary antibodies [IgG1-horseradish peroxidase (HRP) and IgG2c-HRP, Southern Biotechnologies, Birmingham, Al, USA] were added at a 1:2,000 dilution. Plates were incubated at room temperature for 1 h, washed, and eBioscience™ TMB solution (Thermo Fisher Scientific) was added to the plates. The enzymatic reaction was stopped with 50 µl per well of 1N H2SO4. Plates were then read on a VERSAmax microplate reader (Molecular Devices) at 450 nm with a reference filter set at 570 nm. Endpoint titers were determined as the last dilution to render a response of greater than 0.1 mean optical density using Prism software (GraphPad Software, La Jolla, CA, USA).

## Hemagglutination Inhibition (HAI) Antibody Responses

Hemagglutination inhibition assays were performed according to World Health Organization guidelines. Briefly, sera was treated with receptor destroying enzyme (from *Vibrio cholerae*, Denka-Seiken, Tokyo, Japan) overnight and heated at 56°C for 30 min to deactivate the enzyme. HAI antibodies were then tested against the vaccine strain (A/H1N1/California/4/2009) with 0.5% turkey red blood cells (Thermo Fisher Scientific). The HI titer was defined as the reciprocal of the highest dilution of sera, which completely inhibited the agglutination of the RBCs. All samples were run in duplicate, and pre-immune titers in all mice were ≤5.

### Weight and Survival Measurement

Ten mice/group were immunized i.m. either once or twice, 3 weeks apart. Three weeks after the prime or boost immunization, mice were infected *via* the intranasal route with 100LD50 A/H1N1/California/4/2009. Survival and clinical signs (weights) were assessed daily, over 14 consecutive days. Animals exhibiting significant weight loss or that were under duress (ruffled fur, weighted breathing, or hunched backs) were sacrificed. All procedures with A/H1N1/California/4/2009-infected mice were performed under BSL2 conditions per IACUC procedures and guidelines.

# *In Vitro* Stimulation of Bone Marrow Dendritic Cells (BMDCs) and Lung Homogenates

Bone marrow was harvested from the femurs of vaccinated mice, and single cell suspensions were prepared at a concentration of 2 × 105 cells/ml in complete RPMI-1640 with 20 ng/ml recombinant mouse GM-CSF (rmGM-CSF; PeproTech). On day 3, additional rmGM-CSF was supplied. BMDCs were harvested on day 6 and were seeded at a concentration of 6.6 × 105 cells per well. Cells were stimulated with a range of 2–20 µg/ml GLA-SE, 0.00008–0.8% SE, or 10 ng/ml Lipid A 506 (a TLR4 agonist as positive control, Peptide Institute Inc., Osaka, Japan) in the presence of 20 ng/ml rmGM-CSF. A sterile single cell suspension was prepared from young and aged mouse lung by passing tissue through a 100 µM cell strainer (Thermo Fisher Scientific). Erythrocytes were lysed using ACK lysis buffer (Gibco by Life Technologies), while remaining leukocytes were enumerated and plated in triplicate wells at a seeding density of 5 × 105 cells per well. Lung homogenates were stimulated with the same conditions as BMDCs described above. Following an 18-h stimulation, cell supernatants from BMDCs or lung homogenates were collected, and cytokine levels in the supernatants were determined by using custom Luminex-based multiplex immunoassay kits (Procarta Cytokine Assay Kit: Affymetrix, Santa Clara, CA, USA). Cell supernatants were incubated with polystyrene beads coated with antibodies corresponding to the different cytokines including: TNF-α, IL-10, IL12p40, and IL-6 and developed according to the manufacturer's instructions. Bead size and fluorescence were measured on a Luminex 200 and data were analyzed using the Masterplex QT software (Miraibio).

# Flow Cytometry

The antigen-specific T cell memory response generated by vaccination was determined by flow cytometry following incubation of splenocytes with 10 µg/ml rH1 (Protein Sciences Corp., Meriden, CT, USA). Cells were cultured at 1 × 106 cells per well in a 96-well plate (Corning Incorporated, Corning, NY, USA) in RPMI-1640 supplemented with 10% heat-inactivated FCS and 50,000 U penicillin/streptomycin (Invitrogen) for 18–20 h in the presence of GolgiStop (BD Bioscience). Each sample was incubated with Fc receptor blocking (clone2.4G2) before incubation on ice for 30 min with the following antibodies: anti-CD4 (clone RM4-5), anti-CD8α (clone 53-6.7), and anti-CD44 (clone IM7). Expression of selected cytokines was determined by incubation with anti-IFN-γ (clone XMG1.2), anti-IL-2 (clone JES6-5H4), anti-CD154 (clone MRI), anti-TNF-α (clone MP6-XT22), and anti-IL-17A (clone TC11-18H10.1). For Tfh cell analysis, singlecell suspension of inguinal lymph nodes (20) were prepared and stained with following antibodies: anti-CD4 (clone RM4-5), anti-CXCR5 (clone L138D7), anti-PD-1 (clone 29F.1A12), anti-B220 (clone RA3-6B2), anti-CD44 (clone IM7), and anti-CD8 (clone 53-5.8), F4/80 (clone BM8), CD11b (clone M1/70) for dump gate. For germinal center (GC) B cell analysis, single-cell suspension of spleen or inguinal LNs were prepared and stained with following antibodies: anti-B220 (clone RA3-6B2), anti-GL7 (clone G7), anti-CD95 (clone SA367H8), anti-IgD (clone 11-26c.2a), anti-IgG1 (clone RMG1-1). For lung immune cell analysis, lungs were chopped and digested in the presence of 70 µg/ml Liberase™ (Roche), 40 µg/ml DNase I (Roche), 10 mM Aminoguanidine (Sigma-Aldrich), and 5 mM KN-62 (Sigma-Aldrich). The homogenates were incubated for 30 min in a 37°C water bath. Single-cell suspensions were prepared by dispersing the tissues through a 70 µm nylon tissue strainer (BD Falcon). Cells were washed with 1× PBS and then stained with a fixable viability dye (Tonbo Biosciences) before being stained with following antibodies: anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD45 (clone 30-F11), anti-NK1.1 (clone PK136), anti-Ly6G (clone 1A8), anti-TLR2 (clone T2.5), anti-TLR3 (clone 11F8), anti-TLR4 (clone SA15-21), and anti-TLR7 (clone A94B10). Antibodies were purchased from BD Biosciences, eBioscience, BioLegend, or Tonbo Biosciences. Samples were analyzed with BD Fortessa or LSRII. Doublets and dead cells were excluded before analysis, and all the data were analyzed with FlowJo software (version 9; FlowJo, LLC).

# Enumeration of Long-Lived Antibody-Secreting Plasma Cells (ASPCs)

A bone marrow ELISPOT was used to determine the induction of vaccine-specific long-lived ASPCs following immunization with sH1N1 vaccine with and without adjuvants. ELISPOTs were performed as previously described (21) with minor revisions. The developed plates were counted by an ELISPOT plate reader (C.T.L. Serie3A Analyzer, Cellular Technology Ltd., Cleveland, OH, USA), and the data were analyzed using ImmunoSpot® software (Cellular Technology Ltd., Cleveland, OH, USA).

## Viral Load Measurement by Real-time Q-PCR

RNA from whole-lung samples was extracted with Trizol® reagent following the manufacturer's instructions (Thermo Fisher Scientific). The mixture of CHCl3 and Trizol was centrifuged at 11,500 *g* for 15 min at 4°C. After centrifugation, the aqueous layer was transferred and mixed with an equal volume of 70% RNase-free ethanol. The RNA extracts were purified with an RNA purification kit (Ambion®, Thermo Fisher Scientific). RNA was then reverse-transcribed with a SuperScript® IV first-strand synthesis system (Invitrogen®, Thermo Fisher Scientific) into cDNA. Expression of H1 was measured using customized TaqMan probes (Applied Biosystem, Thermo Fisher Scientific) including forward primer: 5′-ATTGCCGGTTTCATTGAAGG-3′; reverse primer: 5′-ATGGCATTCTGTGTGCTCTT-3′; probe: 5′-(FAM) ATGAGCAGGGGTCAGGATATGCAGCCGACC (TAMRA)-3′ to detect A/H1N1/California/4/2009 viral load (22). TaqMan probe for GAPDH was used as the internal control (Applied Biosystem, Thermo Fisher Scientific). Samples were analyzed using a Bio-Rad CFX384 real-time PCR detection system (Bio-Rad), and relative gene expression was calculated *via* the 2−ΔΔCT method.

# Statistical Analysis

Statistical analysis of antibody responses (endpoint antibody and HAI titers) and flow cytometry data was performed using one-way ANOVA with the Tukey multiple comparison test unless noted in the figure legend. Statistical analysis of cytokine production from BMDCs and lung homogenates represented in **Figure 4** were performed using two-way ANOVA and Sidak's posttest. A two-way ANOVA with the Tukey multiple comparison test was performed on data represented in **Figure 5** (with the exception of **Figure 5A**; which used one-way ANOVA and the Tukey multiple comparison test). Statistical analysis of survival curves was performed using the Log-Rank/Mantel–Cox test. All statistical analyses were performed using GraphPad Prism version 7 for Windows (GraphPad Software, La Jolla, CA, USA). *p-*Values of <0.05 were considered significant.

# RESULTS

### sH1N1 Vaccine Adjuvanted with GLA-SE Enhances IgG2c:IgG1 Ratios and High HAI Titers in Young CB6F1 Mice

We were particularly interested in determining whether adjuvants could help overcome the immunosenescence observed in elderly populations. The induction of antigen-specific IgG1 antibodies is dependent on Th2-biased immune responses, while class switching to IgG2c in mice is correlated with Th1-biased immune responses and has been well studied (20). We have previously reported that GLA-SE enhances antiviral protection through the induction of Th1-mediated immune responses (14) and, therefore, used this adjuvant formulation in this study. C57BL/6 mice were originally selected as the mouse strain for these studies; however, our data showed no evidence of HA (A/H1N1/California/4/2009) CD4 T cell epitopes in C57BL/6 mice, whereas CB6F1 mice have a confirmed CD4 T cell epitope (manuscript in preparation). These data provided the rationale for switching to the CB6F1 mouse strain for the remainder of the studies. As shown in Figure S1 in Supplementary Material, the recombinant rH1 vaccine combined with adjuvants MF59-like, SE, or GLA-SE in C57BL/6 mice induced both IgG1 and IgG2c responses post boost. An enhanced IgG2c:IgG1 bias was observed in C57BL/6 mice immunized with rH1 + GLA-SE (Figure S1B in Supplementary Material). While HAI titers in young C57BL/6 mice given adjuvanted rH1 were all >1:40 after two immunizations, much lower HAI titers were observed in aged C57BL/6 mice, where only one out of three of the aged animals in the MF59 and GLA-SE adjuvanted groups tested had post-vaccination HAI titers of >1:40 (Figure S1C in Supplementary Material).

We used the experimental design outlined in **Figure 1A** to assess the use of adjuvants in sH1N1 vaccine regimens and evaluate induced adaptive immune responses. Young (1 month old) or aged (18–21 months old) CB6F1 mice were immunized twice with the sH1N1 vaccine control (saline), or adjuvanted with MF59-like adjuvant, SE, or GLA-SE. Three weeks following the boost immunization, H1-specific IgG1 and IgG2c antibodies were evaluated (**Figure 1B**). We found that both young and aged mice receiving adjuvanted sH1N1 vaccines demonstrated measurable induction of IgG1 post boost over that of sH1N1 vaccine alone. However, the induction of IgG2c titers after two immunizations in aged mice were compromised even in adjuvanted groups, suggesting that the Th1-biased antibody response in aged mice,

aged CB6F1 mice. (A) Scheme of immunization procedure: CB6F1 mice were immunized i.m. either once or twice, 3 weeks apart, and antibody analysis was determined on sera collected 3 weeks following immunization. All mice were challenged with 100LD50 A/H1N1/California/4/2009 at day 54, and their physical condition was monitored over 14 days. Data for young mice were color-coded as blue; aged mice were color-coded as red for entire figure. (B) Sera from saline, sH1N1, sH1N1 + MF59-like, sH1N1 + SE, and sH1N1 + GLA-SE groups were analyzed for H1-specific IgG1 and IgG2c endpoint titers. Results are represented as the mean endpoint titer (log10) ± SEM. \*\* indicates *p-*value <0.01; \*\*\* indicates *p-*value <0.001; \*\*\*\* indicates *p-*value <0.0001. (C) Sera harvested from mice after a prime (day 20) or boost (day 42) immunization were analyzed for HAI titers. An HAI titer of five was assigned to responses below the assay detection limit. *p-*Values are denoted as follows: \* indicates <0.05; \*\* indicates <0.01; \*\*\* indicates < 0.001; \*\*\*\* indicates < 0.0001.

with the reduced dose of sH1N1 vaccine (shown to be effective in young mice), is not easily overcome by the action of adjuvants. Next, we tested whether adjuvanted sH1N1 vaccines could induce protective neutralizing antibodies in both young and aged mice. We observed that aged mice, which received MF59-like, SE, and GLA-SE adjuvanted vaccines could induce protective immune responses with two immunizations as compared to saline or sH1N1 vaccine alone, but not with one immunization (**Figure 1C**). These data suggest that adjuvants similar to MF59 like, SE, and GLA-SE could induce protective HAI titers in aged mice with a boost immunization; however, a boost with the TLR4 agonist adjuvant (GLA-SE) was unable to generate a Th1-biased environment capable of inducing IgG2c responses in aged mice with the sH1N1 vaccine.

# Enhanced Body Weights and Survival in Young and Aged CB6F1 Mice Following Two Immunizations with Adjuvanted sH1N1 Vaccines

CB6F1 mice were immunized once or twice with 0.01 µg of sH1N1 vaccine either alone or with MF59-like, SE, or GLA-SE adjuvants and then challenged with 100LD50 A/H1N1/California/4/2009 (**Figure 1A**). All mice that received a single immunization lost weight after viral challenge, yet, we found that young mice immunized with MF59-like, SE, and GLA-SE adjuvants could gain weight back by day 5 postinfection (**Figure 2A**, top left). Conversely, weight loss continued among aged CB6F1 mice until day 7, and only aged mice that received GLA-SE and SE adjuvants gained back weight over time (**Figure 2A**, top right). Both young and aged mice benefited from two immunizations with adjuvanted sH1N1 vaccines. Young mice given a boost with the adjuvanted vaccines quickly controlled the weight loss at day 3, and aged mice given adjuvanted vaccine only lost approximately 5% of starting weight compared to control animals (**Figure 2A**, bottom row). Aged mice given saline or sH1N1 vaccine alone lost approximately 20% of starting weight at day 6 (**Figure 2A**, bottom right). As shown in **Figure 2B**, survival in saline and sH1N1 young mice, and among all aged animals were severely compromised with one immunization. We observed that most of the young mice (with the exception of the saline group)

survived after receiving two immunizations (**Figure 2B**, bottom left). Survival among aged mice given sH1N1 combined with MF59-like, SE and GLA-SE adjuvants was significantly improved with two immunizations (**Figure 2B**, bottom right). Our data suggest that increasing the number of immunizations in aged mice from one to two immunizations could considerably improve clinical outcomes following the early stages of influenza infection. Similar to responses in young and aged mice immunized with adjuvanted sH1N1 vaccines, enhanced survival was observed in both young and aged C57BL/6 mice given two immunizations with adjuvanted rH1 vaccines (Figure S1D in Supplementary Material). This is particularly interesting based on the low HAI titers observed in aged mice following a boost immunization. The enhanced clinical outcome in aged mice with adjuvanted rH1 vaccine in the absence of HAI titers suggest that a compensatory immune response is contributing to the protective responses seen in aged mice.

# The GLA-SE-Based sH1N1 Vaccine Promotes the Generation of Cytokine-Producing T Helper Cells, Long-Lived Bone Marrow Plasma Cells (BMPCs) and GC B Cells in Young Mice

We next examined the cellular components that may contribute to protective immune responses in young and aged CB6F1 mice. Young and aged mice were immunized twice with adjuvanted sH1N1 vaccines using a slightly modified strategy, denoted in **Figure 3A**. The immunogenicity of T cell recall responses and GC B cell responses were determined 7 days following a boost immunization. Splenocytes from young and aged mice with two immunizations were stimulated with rH1 protein, and cytokine production from antigen-specific CD4+ T cells were determined. We found that young mice that received both MF59-like or GLA-SE adjuvanted vaccines generated antigen-specific CD4+ T cells that produce TNF-α, IL-2, and express CD154. However, only young mice given GLA-SE-adjuvanted vaccine induced IFNγ-producing CD4+ T cells. From aged mice, we detected a minimal percentage of antigen-specific CD4+ T cells with increased nonspecific CD154 levels (representing activated T cells); however, some mice were able to produce IL-2 (**Figure 3B**). None of the aged mice induced TNF-α or IFN-γ-producing antigen-specific CD4+ T cells following immunization. These data are consistent with our observation in **Figure 1A**, where aged mice failed to generate Th1-biased IgG2 antibody responses. Also, similar to previous observations (23), we found that aged mice have an increased percentage of non-specific follicular helper T cells (Tfh, CD4+CXCR5+PD-1+) as compared to young mice (**Figure 3C**). It has been shown that the quality and quantity of neutralizing antibodies is crucial for protective immune responses against viral infection (24). Thus, we examined the induction of GC B cells from spleen and inguinal LNs and long-lived BMPCs in both young and aged mice (**Figures 3D,E**). Interestingly, as shown in **Figure 3D**, within the spleen, young mice receiving two immunizations with sH1N1 alone had the highest number of GC B cells, whereas aged mice receiving sH1N1 combined with MF59-like adjuvant and GLA-SE had the highest number of GC B cells (although none of these responses reached statistical significance). The number of B220+CD95+GL7+ GC B cells in the inguinal LN from young mice were enhanced after a boost immunization with all of the adjuvanted sH1N1 vaccines tested, although significant responses were observed only with sH1N1 + SE (**Figure 3D**). In aged mice, the MF59-like adjuvant induced the highest number of GC B cells in the LN (**Figure 3D**). We also found that young mice that received MF59-like or GLA-SE adjuvanted sH1N1 vaccines generated a significant number of antigen-specific BMPCs (**Figure 3E**). Aged mice that received MF59-like and GLA-SE adjuvanted vaccines showed a similar trend to that seen in young mice, although the responses were not statistically significant (**Figure 3E**).

### *In Vitro* Adjuvant Stimulation of BMDCs and Lung Cells from Aged Mice Exhibit Impaired Cytokine-Producing Ability Compared to Responses from Young Mice

Studies have shown that immune responses are reduced in aged animals due to decreased numbers of APCs and stromal cells (25), increased threshold required for TLR signaling (26), reduced B cell repertoire and humoral responses (7), and a phenomenon where immune organs eventually fill with fat or connective tissues (27). Following our observation that the induction of immune responses to sH1N1 vaccines were impaired in aged mice as compared to young mice, we next tested how adjuvants may affect early innate immune responses from both young and aged mice, characterized by *in vitro* cytokine production from BMDCs and lung homogenates. BMDCs, phenotypically and functionally similar to conventional DCs (28), are the surrogate to induction of systemic adaptive immune responses, whereas stimulated lung homogenates illustrate local immune responses. We tested BMDCs and lung homogenates from both young and aged CB6F1 mice in response to various doses of SE and GLA-SE and found that although BMDCs from aged mice are capable of producing innate inflammatory cytokines upon GLA-SE stimulation, aged cells produced significantly less cytokines compared to those from young animals (**Figure 4A**). Strikingly, we could scarcely detect inflammatory cytokines in the cell supernatants from GLA-SE-stimulated aged lung homogenates, especially for IL-12p40 and IFN-γ (**Figure 4B**). Our data suggest that although immunosenescence in aged mice affects innate immune responses both systemically (BMDCs) and locally (lung homogenates), the specific impairment of local immune responses in aged mice could lead to unfavorable and inefficient pathogen clearance at early stages following infection.

# GLA-SE Adjuvanted sH1N1 Vaccine Decreases Viral Load and Prevents Prolonged Lung Inflammation in Young CB6F1 Mice

Since we observed significant weight change and decrease in survival by day 5 after viral challenge in young and aged mice given either saline or sH1N1 vaccine alone (**Figure 2**), we hypothesized that mice immunized with adjuvanted vaccines would be

from aged mice were color-coded as red. \* indicates *p* value < 0.05; \*\* indicates *p* value < 0.01. Error bars indicate mean ± SEM. GC, germinal center; LN, lymph node; Tfh, follicular T helper cells; BMPC, bone marrow plasma cells. Statistical analysis was performed using one-way ANOVA and Tukey's multiple comparison test except 3E, which used one-way ANOVA and Bonferroni's test.

able to control local inflammation in the lung during the early infection phase, preventing subsequent mortality. In order to address this hypothesis, young CB6F1 mice were immunized once with saline, sH1N1, sH1N1 + SE, or sH1N1 + GLA-SE were then challenged with 100LD50 A/H1N1/California/4/2009. Following challenge, viral loads were determined at days 3 or 6 post challenge. Young CB6F1 mice that received GLA-SE adjuvant generated the highest functional neutralizing antibody titers following one immunization as compared to other groups (**Figure 5A**). Consistent with our previous data (**Figure 1C**), mice that received saline or a prime immunization with the sH1N1 vaccine did not generate significant neutralizing antibodies (**Figure 5A**); in addition, we found that the viral loads were 1,000-fold higher in saline and sH1N1 groups compared to responses in the sH1N1 + GLA-SE group at days 3 and 6 following influenza infection (**Figure 5B**). Interestingly, the sH1N1 + SE group had 100-fold higher viral load than the sH1N1 + GLA-SE group at day 3, but no differences in virus titer were observed at day 6, suggesting that sH1N1 adjuvanted with GLA-SE is superior to sH1N1 adjuvanted with SE, and animals immunized with sH1N1 + GLA-SE were able to clear viral infection earlier after influenza challenge (**Figure 5B**).

Next, we examined the inflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF) from young mice at day 3 or 6 after viral challenge. To our surprise, we could not detect any inflammatory cytokines in BALF from mice that received either SE or GLA-SE adjuvants (**Figure 5C**). IL-6, TNF-α, IFN-γ, and IL-12p40 were detected in the BALF from saline and sH1N1 groups at day 3 and 6, indicating that a continuous inflammatory response occurs in the lungs of these mice. Notably, Th2-biased cytokine IL-4, IL-5, and chemokine Eotaxin (data not shown) in the BALF were detected at day 6 from mice that received the sH1N1 vaccine alone. These data corroborate our previous reports that immunization with protein only, or without proper Th1-driving adjuvants, leads to Th2-biased immune responses (29). It has also been reported that alveolar macrophages (AMs) are crucial for lung homeostasis and are the first line cells capable of detecting lung pathogens including bacteria, viruses, in addition to allergens and foreign particles (30). Thus, we examined whether AMs from young mice given a prime immunization were affected after exposure to A/ H1N1/California/4/2009 challenge. Interestingly, we found that unvaccinated, young CB6F1 mice (saline group) had dramatically decreased percentages of AMs at day 3 of infection as compared

\*\* indicates < 0.01; \*\*\* indicates < 0.001; \*\*\*\* indicates < 0.0001. BMDC, bone marrow dendritic cells.

to the other groups. At day 6, both saline and sH1N1 groups had a significantly decreased percentage of AMs as compared to adjuvanted groups, suggesting that AMs are potentially being eliminated by the viral infection (31) (**Figure 5D**). Similarly, a reduction in the percentage of AMs following influenza infection correlate with an increased viral load within the lung. As shown in Figure S2 in Supplementary Material, we found that in young mice given one immunization, only mice that received the sH1N1 + GLA-SE vaccine maintain AM homeostasis and prevent inflammatory cell infiltration (including eosinophils, neutrophils, inflammatory Mϕs, and NK cells) in the lung at both day 3 and 6 post-infection. After influenza infection, cells such as AMs that express TLR7 are able to detect single-strand RNA fragments released from viral particles. We found that expression of TLR7 on AMs were relatively consistent among all groups at day 3; however, the TLR7 levels were significantly decreased in

Figure 5 | GLA-SE adjuvanted sH1N1 vaccine decreases viral load and prevents prolonged lung inflammation in young CB6F1 mice. Young CB6F1 mice (age of 6–8 weeks old) were immunized once with saline, sH1N1, sH1N1 + SE, or sH1N1 + GLA-SE, and sera were harvested 3 weeks after prime. All mice were challenged with 100LD50 A/H1N1/California/4/2009 at day 28 and BALF and lung homogenates were harvested at day 3 and day 6 post-infection. (A) Hemagglutination inhibition (HAI) titer against A/H1N1/California/4/2009 was determined. (B) The relative expression of H1 was determined by real time Q-PCR. The expression of H1 of each sample was normalized to GAPDH (graph on log10 scale). (C) BALF from all infected mice were harvested at day 3 and day 6 postinfection, and the levels of IL-6, IFN-γ TNF-α, IL-12p70, IL-4, and IL-5 cytokines were determined by Luminex. (D) The percentage of AMs was determined by flow cytometry and were normalized to uninfected, naïve mice (as 100%). AMs in the lung homogenates were defined as CD11b−CD11c+Siglec-F+NK1.1− population. (E) The intracellular expression of TLR7 by AMs was determined by flow cytometry and was normalized to uninfected, naïve mice (as 100%). *p* Values are denoted as follows: \* indicates <0.05; \*\* indicates <0.01; \*\*\* indicates <0.001; \*\*\*\* indicates <0.0001. Results are represented as the mean ± SEM. BALF, bronchoalveolar lavage fluid; AM, alveolar macrophages.

saline, sH1N1 and sH1N1 + SE groups by day 6 as compared to sH1N1 + GLA-SE group (**Figure 5E**). Overall, our data suggest that the GLA-SE adjuvanted sH1N1 vaccine provides superior protection in young mice by clearing influenza virus early after infection, and through maintenance of AMs and TLR7 expression levels in the early infection phase.

### DISCUSSION

In this study, the influence of different adjuvants on the efficacy of two types of seasonal influenza vaccine: a rH1 protein vaccine, and a sH1N1 vaccine was evaluated. Adjuvants examined include two squalene-based oil-in-water emulsions [a MF59-like adjuvant and SE (a stable emulsion adjuvant)] and a synthetic TLR4 agonist formulated with SE (GLA-SE). In CB6F1 mice, we showed that both the MF59-like adjuvant and GLA-SE plus sH1N1 vaccine contribute to Th1-biased vaccine-specific IgG2 immune responses following a boost immunization, whereas significantly higher IgG2c titers were not induced in aged mice. HAI titers were, however, induced in both young and aged CB6F1 mice after two immunizations. In contrast, in C57BL/6 mice, we observed an increase in vaccine-specific IgG1 and IgG2c responses following a boost immunization with all of the adjuvanted rH1 vaccines tested. However, whereas HAI titers were above the 1:40 threshold with adjuvanted rH1 vaccines in young C57BL/6 mice [HAI titers above a titer of ≥1:40 in humans is associated with a 50% reduction in seasonal influenza infection and is considered a correlate of protection (32, 33)], HAI titers were not increased in aged mice. Following a boost immunization, aged C57BL/6 and CB6F1 mice induced significant levels of either vaccine-specific IgG2c or HAI titers, respectively, leading to protection against influenza challenge. This observation is of interest as cellular immunity is known to decline with age and compensatory mechanisms induced by adjuvants may aid protection in the elderly. Recently, the induction of IgG2 antibodies have been a focus of interest for protection against infection, including influenza infection. An M2e-specific IgG2c monoclonal antibody was reported to protect mice against influenza infection more effectively than an IgG1 monoclonal due to Fc activation properties (34).

The use of adjuvants as a strategy for enhancing the quality and magnitude of protective antibody responses to influenza vaccines is not a new concept. We have shown previously that the addition of adjuvants such as GLA-SE are able to increase dose-sparing properties and broaden HAI titers against drifted influenza viruses (12). In this manner, applying the GLA-SE adjuvant to influenza vaccine design addresses a noted and significant complication of reduced vaccine-specific antibody responses observed in aged individuals (9). GLA-SE combined with a recombinant H5N1 avian influenza vaccine (rH5) also increases the breadth of the antibody responses and provides protection against a heterosubtypic influenza virus, compared to the vaccine combined with SE (14).

One of the benefits of using GLA-SE as a vaccine adjuvant is the Th1-inducing innate responses that promote protective adaptive Th1-mediated cellular and humoral immunity (12, 29, 35). Characterization of this pure synthetic TLR4 agonist adjuvant in comparison to another TLR4 agonist, monophosphoryl lipid A (MPL), which is derived naturally from the bacterium *Salmonella minnesota*, has been shown following stimulation of DCs from both humans and mice (36). Both agonists were shown to increase DC activation and maturation and induce proinflammatory cytokines and chemokines (12). In elderly humans, the responses of monocyte-derived DCs stimulated with GLA-SE appear intact (16). Here, we add to this paradigm and demonstrate that BMDCs derived from aged mice respond to GLA-SE stimulation; however, cytokine responses are lower than observed following GLA-SE stimulation of young BMDCs.

Peripheral blood mononuclear cells from elderly patients stimulated with GLA-SE and a split virus vaccine have also been shown to increase the ratio of IFN-γ:IL-10, in addition to granzyme B, further supporting this as a potentially effective candidate adjuvant for use in the elderly (19). In our study, MF59-like adjuvant was also capable of promoting vaccine-specific CD4+ T cell responses, GC B cells, and induction of long-lived BMPCs, and like the SE adjuvant, was able to induce HAI titers after a boost immunization leading to protection in mice against challenge with H1N1. The history, safety, and efficacy of the MF59 adjuvant has been recently reviewed (17, 18).

One of the most striking observations in our study was the differential cytokine responses to adjuvant in aged vs. young lung homogenates. The *in vitro* stimulatory response of GLA-SE on lung homogenates from young mice led to the induction of TNF-α, IL-12p40, IFN-γ, and IL-10, whereas only TNF-α and IL-10 were induced in samples from aged mice. This potential local impairment (within the lung) could have substantial consequences following acute infection with influenza virus and other pulmonary infections.

In order to determine the protective mechanism of action of sH1N1 adjuvanted with GLA-SE within the lungs of young mice, we further characterized the pulmonary responses following H1N1 infection, including assessment of viral load and inflammatory responses. We observed less virus in the lung at day 3 post challenge, in mice immunized once with sH1N1 combined with GLA-SE, and decreased levels of virus at day 6 post challenge in mice immunized with SE compared to saline or sH1N1 vaccine only. The magnitude of the neutralizing HAI titer was also highest in mice given the sH1N1 + GLA-SE. Furthermore, a drastic reduction in the inflammatory responses within the lungs of mice immunized with the sH1N1 + GLA-SE was observed compared to the Th2-biased inflammatory responses that were seen in the sH1N1 vaccine alone group. At day 6 of infection, the percentage of AMs was also significantly higher in the sH1N1 + GLA-SE group compared to both saline and sH1N1 immunized mice. Interestingly, TLR7 expression within AMs was also significantly higher in the sH1N1 + GLA-SE-treated mice compared to mice that were immunized with sH1N1 vaccine only and sH1N1 + SE. Recently, Wong et al. reported that AMs from aged mice have an intrinsically impaired function to limit lung damage during influenza viral lung infection and suggested that enhancing the function of AMs may improve outcomes in elderly individuals infected with respiratory viruses (31). It will be of great interest to determine if administration of Th1-inducing vaccines in aged animals can preserve the numbers and functions of AMs during acute phases of infection, and how this might reduce the morbidity and mortality in aged individuals. This significant hypothesis warrants further exploration. We have previously shown, in both mice and non-human primates, that Fluzone® (a trivalent inactivated influenza vaccine) adjuvanted with GLA-SE, increases the magnitude of HAI titers, induces Th1 cellular immune responses, and enhances cross-reactive antibody responses to drifted influenza strains (12). Our current work further addresses additional attributes of GLA-SE-adjuvanted influenza vaccines in the context of increased age.

Finally, the CDC estimates that during the 2015–2016 influenza season, the elderly (making up only 15% of the overall US population) accounted for half or more of hospitalizations associated with influenza and 64% of deaths associated with pneumonia and influenza (37). Our data suggest that the staggering burden of morbidity and mortality due to influenza infection in the elderly population may be alleviated by further detailed and mechanistic selection of proper adjuvants to be included in seasonal vaccines.

## ETHICS STATEMENT

These studies were carried out in accordance with the regulations and guidelines of the IDRI animal care and use committee (IACUC). The animal protocol was approved by the IACUC committee.

# REFERENCES

# AUTHOR CONTRIBUTIONS

SB and FCH wrote and prepared the manuscript; SB, FCH, EG, SL, and RC edited the manuscript; SB, NH, EG, RC, and FCH designed the experiments; NH, EG, and BG did HAI and viral infection; FCH and JG did Luminex assays; FCH did the quantitative PCR and flow analysis; EL and LH did *in vitro* stimulation of BMDCs and lung homogenates; and SB, FCH, and SL did the data analyses.

# ACKNOWLEDGMENTS

We thank Tara Evers, Sharvari Waghmare Joshi, Ghislain Ismail Nana, Tom Hudson, Elyse Beebe, Po-Wei Huang, and Valerie Reese for their technical assistance. We also thank Dr. Chris Fox and the formulation team at IDRI for the adjuvant formulations. In addition, we thank Dr. Winston Wicomb and the vivarium staff for their excellent care of the animals used in these studies. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2011) under grant agreement 280873.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fimmu.2018.00295/ full#supplementary-material.

affects stability, antigen structure, and vaccine efficacy. *Influenza Other Respir Viruses* (2013) 7(5):815–26. doi:10.1111/irv.12031

**Conflict of Interest Statement:** SR is a founder of, and holds an equity interest in, Immune Design Corporation, a licensee of certain rights associated with GLA. All other authors have no financial conflicts of interest.

*Copyright © 2018 Baldwin, Hsu, Van Hoeven, Gage, Granger, Guderian, Larsen, Lorenzo, Haynes, Reed and Coler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model

*Anouk C. M. Platteel1,2†, Natalie E. Nieuwenhuizen2†, Teresa Domaszewska2 , Stefanie Schürer2 , Ulrike Zedler2 , Volker Brinkmann3 , Alice J. A. M. Sijts1‡ and Stefan H. E. Kaufmann2 \*‡*

*1Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands, 2Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany, 3Microscopy Core Facility, Max Planck Institute for Infection Biology, Berlin, Germany*

Tuberculosis (TB), caused by *Mycobacterium tuberculosis* (*Mtb*), remains a global threat. The only approved vaccine against TB, *Mycobacterium bovis* bacillus Calmette–Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the *Mtb* antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8+ T cell responses *in vivo*. As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56\_E, a construct optimized for epitope processing in a mouse model. As *Mtb* antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56\_E cDNA to boost BCG immunization in mice. Dermal H56 and H56\_E cDNA immunization induced H56-specific CD4+ and CD8+ T cell responses and Ag85Bspecific IgG antibodies, but did not reduce bacterial loads, although immunization with H56\_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4+ T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56\_E cDNA induced increased H56-specific CD4+ and CD8+ T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56\_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4+ T cells responding to Ag85B- and ESAT-6-derived epitopes were predominantly IFN-γ+TNF-α+ and TNF-α+IL-2+, respectively. In conclusion, despite inducing appreciable immune responses to Ag85B and ESAT-6, intradermal H56 cDNA tattoo immunization did not substantially enhance the protective effect of BCG under the conditions tested.

### Keywords: tuberculosis, vaccine, vaccination, DNA immunization, *Mycobacterium bovis* bacillus Calmette– Guérin, H56

### *Edited by:*

*Rino Rappuoli, GlaxoSmithKline, Italy*

### *Reviewed by:*

*Luciana Leite, Instituto Butantan, Brazil Juraj Ivanyi, King's College London, United Kingdom*

### *\*Correspondence:*

*Stefan H. E. Kaufmann<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

> *‡ Equal senior co-authors.*

### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 12 September 2017 Accepted: 23 November 2017 Published: 11 December 2017*

### *Citation:*

*Platteel ACM, Nieuwenhuizen NE, Domaszewska T, Schürer S, Zedler U, Brinkmann V, Sijts AJAM and Kaufmann SHE (2017) Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model. Front. Immunol. 8:1744. doi: 10.3389/fimmu.2017.01744*

**Abbreviations:** BCG, *Mycobacterium bovis* bacillus Calmette–Guérin; GC, germinal center; *i.d.*, intradermal; *Mtb, Mycobacterium tuberculosis*; *s.c.*, subcutaneous; TB, tuberculosis; TCM, central memory T cells; TEM, effector memory T cells; TFH, follicular helper T cells; TTFC, tetanus toxin fragment C domain 1.

# INTRODUCTION

Tuberculosis (TB) remains a global health threat, with 10.4 million cases and 1.7 million deaths reported for 2016 (1, 2). It is estimated that approximately a quarter of the world's population has latent TB infection (LTBI) (3). Socioeconomic factors such as poor living conditions, stress and malnutrition, play a major role in susceptibility to developing TB disease, with the HIV pandemic also a major driver (4). Coinfection with *Mycobacterium tuberculosis* (*Mtb*) and HIV leads to accelerated deterioration of immunity, and TB is the most common cause of death in HIV+ individuals. HIV contributes to the increased risk of TB by depleting CD4+ T cells, affecting macrophage effector functions, tipping the Th1/Th1 balance and influencing granuloma formation (5). The risk of those with LTBI developing active disease is approximately 10% over a lifetime, but rises to 5–10% per year in those with HIV infection (6). HIV-exposed uninfected infants are another group at high risk of TB infection, as they have poorer T cell generation and IFN-γ production compared to HIV-unexposed infants (7).

An attenuated form of the causative agent of bovine TB, *Mycobacterium bovis* bacillus Calmette–Guérin (BCG), was introduced as a live vaccine in 1921 and is today the most used vaccine globally (8). BCG provides protection against TB meningitis and other forms of disseminated TB. In addition, it has contributed to a reduction in general childhood mortality by boosting non-specific immunity against common causes of childhood disease (9). However, BCG fails to protect adequately against the pulmonary form of TB (10). Protection also varies geographically; in the UK and Norway, BCG confers 50–60% protection that lasts up to 20 years (11), while the high incidence of TB in developing countries illustrates the need for an improved vaccination strategy. Furthermore, BCG can cause severe adverse effects in immunocompromised individuals, and it is not recommended for HIV-infected individuals, who are thus a target group for vaccination due to their increased risk of developing TB (12). As BCG is given at birth throughout the developing world, a new TB vaccine for children should take into account the fact that the majority of the population has already been BCG-vaccinated. New vaccination strategies aim to improve the efficacy and/or safety of BCG in several ways, including modifying it, combining it with booster vaccines, administering it with different adjuvants or altering the route of vaccination (13–19).

A crucial starting point in improving vaccine strategies is knowledge of immune responses that correlate with protection against TB. Although the precise mechanisms of *Mtb* control are unclear, T cell responses are known to be crucial (19). Successful attempts have been made to enhance the cellular response by modifying BCG; for example, a recombinant BCG strain was generated which expresses the *hly* gene encoding for listeriolysin O (LLO) from *Listeria monocytogenes,* in combination with deletion of urease C, resulting in a pH optimal for LLO activity (20–22). Vaccination of mice with BCG *ΔureC::hly* induced more CD4+ central memory T cells (TCM) than BCG vaccination, which were protective against infection (21). In other studies, Ag85B-specific CD4+ effector memory T cells (TEM) were shown to control infection in the lungs (23), and CD8+ T cells also protect against *Mtb,* particularly during LTBI (24–28).

As most individuals in TB-endemic countries are vaccinated with BCG, another strategy for increasing protection against TB is to boost BCG prime immunization with subunit vaccines containing *Mtb* antigens, using non-live vaccines based on recombinant fusion proteins mixed with adjuvants or non-replicating attenuated viral vectors (13, 14, 16, 18, 19). Boosting with a second dose of BCG itself is currently not recommended by the World Health Organization, as it has not been found to improve protection significantly in earlier studies (29–32). Different *Mtb* antigens have been tested against TB, including Ag85A, Ag85B, TB10.4, HBHA, and others (19). Aagaard et al. (33) created the multistage fusion protein vaccine H56, which is composed of three antigens of *Mtb*: Ag85B, ESAT-6, and Rv2660c. Ag85B and ESAT-6 are both immunogenic proteins secreted early in infection (34, 35), with Ag85B found in both *Mtb* and BCG and ESAT-6 only in *Mtb*. Rv2660c was originally identified as a latency-associated gene of *Mtb* (36). Although it has not yet been detected at the mRNA or protein level (37), recombinant Rv2660c stimulated IFNγ responses in CD4+ T cells from individuals with LTBI and was also recognized by IgG antibodies (38) and increased protection was found after vaccination of mice with H56 compared to H1, a similar fusion protein that lacks only the Rv2660c gene (35). The Rv2660c (Mb2678c) gene was shown to be expressed at the transcript level by BCG *in vitro* (39). Vaccination with H56 or the antigens of H56 administered with adjuvant CAF01 (40, 41) or IC31 (41, 42) protected against *Mtb* infection in mice. Recently, we showed that intradermal (*i.d*.) tattoo administration of H56 cDNA fused to tetanus toxin fragment C domain 1 (TTFC) cDNA elicited vigorous antigenspecific CD4+ T cell and CD8+ T cell responses targeted to H56, without the need for adjuvants (43). The immunogenicity of H56 cDNA was enhanced by fusion to TTFC cDNA, and by substitution of the C-terminal epitope flanking residues, to optimize proteasome-mediated epitope generation. Unlike immunization with live bacterial strains such as BCG, cDNA immunization does not carry a risk of infection and it should, thus, be safer for immunocompromised individuals.

In the present study, we aimed to compare the efficacy of *i.d.* DNA tattoo immunization to that of subcutaneous (*s.c.*) BCG immunization *in vivo*, and to test *i.d*. H56 cDNA tattoo immunization as a booster for *s.c.* injection of BCG*.* We also tested *i.d.* immunization with BCG. Immunization with *i.d.* DNA tattoo alone did not protect against TB. Heterologous vaccination with BCG/DNA improved H56-specific T cell responses, increased antibody titers, and ameliorated lung pathology in our murine TB model. However, bacterial burdens were not reduced when compared to vaccination with BCG alone.

### MATERIALS AND METHODS

### Mice

Six- to eight-week old CB6F1 mice (male C57BL/6 × female BALB/c F1) were purchased from Charles River. All animal studies were ethically approved by the State Office for Health and Social Services, Berlin, Germany. We designed two protocols, one for single vaccination and one for prime-boost vaccination, each consisting of 10 mice per timepoint divided into two groups of five mice for manageability and performed in two separate experiments.

## DNA and Dermal DNA Tattoo Vaccination

The full-length H56 and H56\_E cDNA (33) was codon optimized, and inserted at the 3′end of TTFC cDNA (44, 45) into the pVAX1 vector (Invitrogen) as described previously (43). DNA tattoo immunization was performed with 15 µl cDNA (2 µg/µl) in Tris-EDTA buffer with a 9-needle bar mounted on a tattoo rotary device (Cheyenne) adjusted to 100 Hz at 1 mm depth for 1 min (46), and the mice were under isoflurane anesthesia.

### BCG Vaccination and *Mtb* Challenge

The *Mtb* H37Rv (American Type Culture Collection; catalog no. 27294) and BCG Danish 1331 (BCG SSI) (American Type Culture Collection; catalog no. 35733) were grown in Middlebrook 7H9 broth (BD) supplemented with albumin-dextrose-catalase enrichment (BD), 0.2% glycerol, and 0.05% Tween 80 or on Middlebrook 7H11 agar (BD) containing 10% (vol/vol) oleic acid-albumin-dextrose-catalase enrichment (BD) and 0.2% glycerol. BCG was grown to mid-log phase, washed with phosphatebuffered saline (PBS) and stored at −80°C in PBS/10% glycerol. BCG was washed in PBS before vaccination and administered at a dose of 106 CFU in 100 µl for *s.c.* immunization and 106 CFU in 25 µl for the *i.d.* tattoo. Aerosol challenge with *Mtb* was performed using an inoculum of 20–50 CFU.

# Single Immunization Model

The *s.c*. injection of BCG is known to lead to an approximately 1 log reduction in bacterial burden in mouse models (47, 48) and was included as the standard against which the efficacy of *i.d.* tattoo immunization with BCG or DNA were compared. The DNA vaccines included H56, a DNA vector containing the full-length codon-optimized H56 gene fused to TTFC cDNA, and H56\_E, in which six CD8+ T cell epitope flanking sequences have been optimized to enhance proteasome-mediated processing (43), and were administered at day 0, 3, and 6. BCG was administered *s.c*. at day 0. One group of mice was also immunized by *i.d.* tattoo with BCG (day 0 only), in order to compare *s.c.* and *i.d.* administration of BCG. Immune responses were measured at day 21 and the efficacy of the methods was tested by subjecting the mice to an aerosol challenge with *Mtb* at day 60.

### Prime-Boost Model

Two groups of mice were *s.c.* immunized with BCG at day 0, and one of these was boosted by *i.d.* H56\_E DNA tattoo at day 40 for a heterologous immunization. Another group was primed and boosted *i.d.* with H56\_E cDNA tattoo. Unvaccinated mice were included as controls. Immune responses were measured 2 weeks after the booster vaccination. Mice were infected with *Mtb* at day 100 by aerosol challenge and bacterial burdens and lung pathology were assessed at day 190.

## Vaccine-Efficacy Studies

For measurements of the bacterial load, lungs and spleens were harvested and serial dilutions were performed in PBS-0.05% Tween 80 and plated on Middlebrook 7H11 agar. The percentage of inflammation per lung area was measured in a blinded manner on formaldehyde fixed tissue sections stained in Giemsa. Whole lung sections were scanned with a ZEISS Axioscan Z1 driven by ZEN and lung pathology analysis was performed using Volocity (Perkin Elmer). Lung images shown were chosen based on the image analysis, with the lung having the value closest to the average result per group shown here.

## Peptides

All epitopes, as determined previously (43), were synthesized using Fmoc solid phase chemistry. The sequence enumeration of the synthetic peptides referred to the vaccine H56 (33). CD4+ T cell epitopes included H56242–262 (Ag85B derived) and H56288–307 (ESAT-6 derived). The CD8+ T cell epitopes included H56354–363 (ESAT-6 derived) and five epitopes of Ag85B.

The eighty-five 15mer peptides used as pool to measure the total amount of H56-specific T cells (provided as a kind gift from Dr. Donatella Negri) spanned the entire amino acid sequence of H56, overlapping by 10 amino acid residues, and were synthesized by PRIMM Srl.

### Analysis of Specific CD8**+** and CD4**+** T Cell Responses Using Intracellular Cytokine Staining

For intracellular cytokine production, splenocytes were plated overnight with or without 1 µg/ml peptide or peptide pool containing 1 µg/ml of each peptide at 37°C. During the last 4 h, brefeldin (5 µg/ml, Sigma) was added to wells incubated with peptide or phorbol myristate acetate (50 ng/ml, Sigma) and ionomycin (250 ng/ml, Sigma) or to cells incubated with medium alone. Cells were then stained for the cell surface markers named below, fixed with 2% paraformaldehyde, permeabilized with saponin buffer (saponin, 1 g/l; CaCl2, 0.11 g/l; MgSO4, 0.125 g/l; NaN3, 0.5 g/l; bovine serum albumin (BSA) 1 g/l; 10 mM HEPES in PBS, pH 7.4), and stained for intracellular cytokines as described (43). The cell surface- and intracellular cytokine panel consisted of anti-TCRβ-A700 (Biolegend; clone H57-597), anti-CD4 Pacific Blue (Biolegend; clone GK1.5), anti-CD8 PerCP (Biolegend; clone 53-6.7), anti-IL-2 APC (Biolegend; clone JES6-5H4), anti-IFNγ PE-Cy7 (Biolegend; clone XMG1.2), anti-IL-17 PE (Biologend; clone TC11-18H10.1), and anti-TNF-α FITC (clone MP6-XT-22, grown in-house). Samples were acquired on an LSR II cytometer (BD Biosciences) with BD FACS Diva software and analyzed using FlowJo v10 (TreeStar).

# Analysis of Specific CD8**+** T Cell Responses by IFN-**γ** ELISpot

Multiscreen ELISPOT plates (Millipore) were coated with 2 µg/ ml anti-mouse IFN-γ (clone AN18, grown in-house) in PBS overnight at 4°C. Wells were washed and blocked with 5% BSA/ PBS. 5 × 105 erythrocyte depleted lymph node cells were plated with or without 1 µg/ml synthetic peptide overnight in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal calf serum (FCS) Pen-Strep at 37°C. Plates were washed with PBS plus 0.01% Tween 20 (PBS-T), and IFN-γ was detected with biotinylated anti-IFN-γ (clone XMG1.2), followed by alkaline–phosphatase (AP)-conjugated streptavidin (homemade) in PBS-T supplemented with 2% BSA. The assay was developed with NBT/ BCIP substrate (Thermo Fisher Scientific) and analyzed using an Immunopost S6 Ultra-V Analyzer (Cellular Technology Limited).

### FIGURE 1 | Continued

Immunization with H56 or H56\_E cDNA induces Ag85B- and ESAT-6-specific T cell responses. Mice were immunized *s.c.* with bacillus Calmette–Guérin (BCG) and *i.d*. with BCG, H56, a DNA vector containing the H56 gene fused to TTFC or H56\_E, in which six CD8+ T cell epitope flanking residues in the H56 sequence were optimized to enhance proteasome-mediated processing. Intracellular cytokine staining and ELISpots were performed on splenocytes and inguinal lymph node cells, respectively, harvested at day 21 post vaccination and re-stimulated with different H56 peptides. The peptide pools consisted of eighty-five 15-mer peptides spanning the entire sequence of H56, as a measure of the total percentage of H56-antigen-specific cells. CD4+ T cell epitopes included H56242–262 (Ag85B derived) and H56288–307 (ESAT-6 derived). The CD8+ T cell epitopes included H56354–363 (ESAT-6 derived) and five epitopes of Ag85B. The results show pooled data from two independent experiments (*n* = 10 in total per group). For the unvaccinated group, cells from five mice were pooled into two samples in order to have enough cells. (A) Immunization scheme. (B) Gating strategy for FACS analysis. After gating on live, single cells, cells were gated on CD4+ (R1) or CD8+ (R2). Both R1 and R2 were gated on TCR-β+ cells (R3) and (R4) before gating single IFN-γ, IL-2, IL-17, and TNF-α cells. (C) Heat map showing mean frequency of spleen-derived CD4+ T cells secreting IFN-γ, IL-17, IL-2, or TNF-α after peptide stimulation, normalized to medium incubated cells. (D) Heat map showing mean frequency of spleen-derived CD8+ T cells secreting IFN-γ, IL-17, IL-2, or TNF-α after peptide stimulation, normalized to medium incubated cells. (E) Heat map showing mean number of IFN-γ producing cells per million lymph node cells after stimulation with peptide pools, CD4 epitopes or CD8 epitopes, normalized to medium incubated cells. The statistical differences of values shown in the heatmaps are shown in Tables S1 and S2 in Supplementary Material.

## Analysis of T and B Cell Subpopulations

Spleens and inguinal lymph nodes were collected and singlecell suspensions were generated in IMDM 10% FCS Pen-Strep. Cells were surface stained to quantify cell populations. T cell panel: anti-CD3 Alexa 700 (BioLegend; clone 17A2), anti-CD4 PE-Cy7 (BioLegend; clone RM4-5), anti-CD8 V500 (BD Horizon, clone 53-6.7), anti-CD62L APC (BD Pharmingen; clone MEL-14), anti-CD44 Pacific Blue (clone IM7, grown in-house), anti-CXCR5 PE (BioLegend; clone J252D4), and anti-CCR7 PerCP (BioLegend; clone 4B12). B cell panel: anti-B220 PE (BioLegend; clone RA3-6B2), anti-CD38 APC (BioLegend; clone 90), anti-Fas PE-Cy7 (BioLegend; clone Jo2), anti-GL7 FITC (BioLegend; clone GL7), anti-MHCII Pacific Blue (BioLegend; clone M5/114.15.2), anti-CD19 Alexa 700 (BioLegend; clone 6D5), and anti-CD138 Percp (BioLegend; clone 281-2). CD4+ TCM were CD3+CD4+CD44highCD62high, CD4+ TEM were CD3+CD4+CD44highCD62Llow, T follicular helper cells (TFH) were CD3+CD4+CXCR5+, germinal center (GC) B cells were B220+CD19+GL7+Fas+, and plasma cells B220+CD 19+CD138+CD38+.

### Specific Antibody Responses

Mycobacteria-, Ag85B- and ESAT-6-specific antibodies in serum were measured by indirect enzyme-linked immunosorbent assay using *Mtb* H37Rv lysate (BEI Resources), recombinant Ag85B (BEI Resources), or recombinant ESAT-6 (BEI Resources) to coat and AP-labeled anti-mouse IgG, IgG1, and IgG2c for detection (SouthernBiotech).

### Heat Maps

The scripts for **Figures 1** and **4** are available upon request. FACS data visualization was designed using R-package ggplot2 (v2.1.0; Springer-Verlag New York, NY, USA, 2009.) in R programming language [v3.2.3; R Development Core Team (2008)]. The mean values of percentages of CD4+ or CD8+ T cells producing cytokines after peptide stimulation were normalized to control samples stimulated with medium and visualized using heat maps. ELISpot data visualization was designed using R-package ggplot2 (v2.1.0; Springer-Verlag New York, NY, USA, 2009) in R programming language [v3.2.3; R Development Core Team (2008)]. The mean numbers of IFN-γ-producing cells per million cells after peptide stimulation were normalized to control samples stimulated with medium. For visualization purposes, the mean value for each experimental condition was transformed to square root of mean response signal and displayed as a heat map.

### Statistics

Data were tested for normality with Levene tests. Kruskal–Wallis with Dunn's multiple comparison test was used to analyze differences in bacterial burdens between groups. Differences in immunological parameters between groups were analyzed using a one-way ANOVA with Tukey's multiple comparisons test. *p* < 0.05 was considered significant. The differences in cytokine frequencies were assessed by creating linear regression models with treatment as the predictor and measured cytokine frequency as the dependent variable. The models were created for CD4+ cells, CD8+ cells, all peptide stimulations, and measured cytokines separately. Benjamini–Hochberg method was used to correct for multiple testing. *p*-values <0.05 were considered significant.

# RESULTS

## Immunization with H56 or H56\_E cDNA Induces H56-Specific T Cell Responses

Mice were immunized with BCG (*s.c*. or *i.d*.), with H56, a DNA vector containing the full-length codon-optimized H56 gene fused to TTFC cDNA, or with H56\_E, in which six CD8+ T cell epitope flanking residues in the H56 sequence were optimized to enhance proteasome-mediated processing (43) (**Figure 1A**). Previously, we identified seven novel CD8+ T cell epitopes in the H56 fusion protein (43). In order to compare Ag85B- and ESAT-6-specific T cell responses induced by the cDNA vaccines and BCG, we measured peptide-specific immune responses after the different immunization strategies, including responses to an H56 peptide pool. Splenocytes or lymph node cells from vaccinated mice were re-stimulated *ex vivo* with Ag85B- and ESAT-6-derived CD8+ (43) and CD4+ (49, 50) T cell epitopes and responses in the spleen were measured using intracellular cytokine staining and flow cytometry (**Figures 1B–D**) or in the draining lymph nodes by IFN-γ ELISpot (**Figure 1E**). The mean percentages of cytokine-producing CD4+ or CD8+ T cells after peptide stimulation were normalized to control samples stimulated with medium and visualized using heat maps (**Figures 1C,D**). Results of statistical analysis are shown in Table S1 in Supplementary Material. Incubation of splenocytes with a pool of overlapping 15-mers covering the entire H56 sequence induced increased IFN-γ production by CD4+ T cells from H56 and H56\_E *i.d.* vaccinated mice compared to unvaccinated controls (**Figure 1C**). H56 cDNA immunized mice had increased IFN-γ production in response to both the Ag85B-derived peptide H56242–262 and the ESAT-6 derived peptide H56288–307. No differences in TNF-α, IL-17, or IL-2 secretion by spleen CD4+ T cells were detected between the groups (**Figure 1C**). Differences in CD8+ T cell IFN-γ production were not significant, although there was a trend toward increased recognition of the H56 protein (as measured by responses to the peptide pool) in the H56 or H56\_E cDNA *i.d.* immunized mice (**Figure 1D**). IFN-γ ELISpot analysis of total lymph node cells also demonstrated increased numbers of cells producing IFN-γ in the H56 cDNA *i.d.* vaccinated mice after stimulation with the H56 peptide pool compared to both unvaccinated and BCG *s.c.* vaccinated mice (**Figure 1E**; Table S2 in Supplementary Material). Overall, the H56 cDNA tattoo immunization increased H56-specific IFN-γ-producing T cell responses.

# Increased CD4**+** TEM, TFH, and GC B Cells following Vaccination with BCG

T cell memory populations and B cell responses after vaccination were measured by flow cytometry at day 21 in spleens and draining lymph nodes (**Figure 2A**). Both *s.c*. and *i.d*. BCG immunization resulted in increased percentages of CD4+ TEM among spleen CD4+ T cells compared to unvaccinated mice (**Figure 2B**), with CD4+ TFH showing similar trends (**Figure 2C**). Frequencies of CD4+ TCM were higher in the spleens of DNA *i.d.* vaccinated mice than in BCG *s.c.* vaccinated mice (**Figure 2D**). TEM responses differed in draining lymph nodes in that only H56\_E immunized mice had increased TEM (**Figure 2E**). A similar pattern was observed for TFH in lymph nodes compared to spleen, with all responses increased in all vaccinated groups compared to unvaccinated controls (**Figure 2F**). This pattern was also seen for TCM in draining lymph nodes (**Figure 2G**). There were no significant differences in frequencies of CD8+ TEM or CD8+ TCM between the groups (data not shown).

Humoral responses may contribute to protection against TB (51–53). We measured the frequencies of plasma cells and GC cells among B cells by flow cytometry (**Figure 2H**) in spleens (**Figures 2I,J**) and draining lymph nodes (**Figures 2K,L**). Frequencies of GC B cells were increased in spleens of both groups vaccinated with BCG compared to unvaccinated mice (**Figure 2I**), with a similar trend in plasma cells (**Figure 2J**). In the draining lymph nodes, GC B cells appeared increased in all immunized groups (**Figure 2K**) while plasma cells were similar between groups (**Figure 2L**). Antibody responses were very variable between animals, possibly because of the mixed background of CB6F1 mice, which are first-generation offspring of female BALB/c and male C57/BL6 mice. Although antibody responses varied between mice, the trend was directed toward increased levels of mycobacteria-specific IgG, including IgG1 and IgG2c, in mice vaccinated with BCG, which expresses multiple mycobacterial proteins (**Figure 3A**) and increased levels of Ag85Bspecific IgG, IgG1, and IgG2c in the H56 cDNA vaccinated group (**Figure 3B**). ESAT-6-specific antibodies were not detected (data not shown). Curiously, antibody levels were lower in mice immunized with H56\_E DNA than with H56 DNA, suggesting that B cell epitopes may have been influenced by the sequence modification.

### H56 and H56\_E cDNA Tattoo Immunization Does Not Reduce Bacterial Loads in Mice

To evaluate the protective efficacy of the different vaccines, bacterial loads were measured in lungs and spleens at day 90 after infection with *Mtb* and lung pathology was quantified as cell infiltration per lung area in a blinded manner. Mice vaccinated with BCG *s.c.* benefited from an approximately 1 log reduction in bacterial load in the lung compared to unvaccinated mice, whereas mice vaccinated with BCG *i.d*. did not (**Figure 3C**). Bacterial loads were not decreased after *i.d.* immunization with H56 or H56\_E cDNA. A similar trend was seen in the spleen (**Figure 3D**). Furthermore, mice vaccinated with BCG strains had strongly decreased lung inflammation compared to unvaccinated mice (**Figures 3E,F**). H56 cDNA immunization did not decrease lung pathology compared to unvaccinated mice; however, there was reduced lung pathology in H56\_E cDNA immunized mice (**Figures 3E,F**). Overall, cDNA tattoo immunization *i.d.* with H56 constructs was not as protective as BCG *s.c*. against TB in our model, but H56\_E immunization ameliorated pathology.

### Boosting BCG with *i.d.* H56 cDNA Tattoo Immunization Increases Ag85B and ESAT-6 Specific CD4**+** and CD8**+** T Cell Cytokine Responses

Although *i.d.* H56\_E DNA tattoo vaccination did not reduce *Mtb* burdens, it ameliorated lung pathology, and we hypothesized that it may increase the efficacy of the BCG vaccine in a prime-boost system by boosting responses to Ag85B and inducing responses to the *Mtb-*specific antigens ESAT-6 and Rv2660c found in H56. Therefore, we tested a heterologous prime-boost vaccination regimen (**Figure 4A**). In addition, we tested whether repeating *i.d.* H56\_E DNA tattoo immunization (homologous prime/ boost) could improve its protective efficacy. Spleen H56-specific CD4+ IFN-γ responses were increased at day 14 after boosting of BCG with *i.d.* H56 and H56\_E DNA tattoo immunization as well as by homologous H56\_E cDNA immunization, compared to BCG-only immunized groups (**Figure 4B**; Table S3 in Supplementary Material). In addition, spleen H56-specific CD4+ TNF-α responses were increased in the homologous H56\_E cDNA immunized group compared to the BCG *s.c.* immunized group, and in both groups boosted with H56\_E DNA compared to the unvaccinated controls. The BCG/H56\_E immunized group had increased frequencies of Ag85B-specific CD4+TNF-α+ cells. Homologous cDNA prime-boost induced increased percentages of H56-specific and ESAT-6-specific IFN-γ CD8+ T cells compared to both BCG *s.c.* immunized and unvaccinated groups (**Figure 4C**; Table S3 in Supplementary Material). BCG *s.c.* immunized mice had increased Ag85B-specific CD8+IFN-γ+ responses compared to unvaccinated mice, which were further boosted by H56\_E cDNA compared to BCG alone. ELIspots on lymph node

FIGURE 2 | Differences in T- and B cell sub-populations following immunization with cDNA and BCG. Samples were collected at day 21 post vaccination. Splenocytes and lymph node cells were stained for different surface markers and analyzed by FACS. Pooled data of two experiments (*n* = 10 in total per group) are shown ±SEM. (A) Gating strategy for FACS analysis for T cell sub-populations. After gating on live, single cells, cells were gated on CD3+ (R1). R1 was gated on CD8+ (R2) or CD4+ (R3). CD4+ and CD8+ T cells were then analyzed for expression of CD44, CD62L, and CXCR-5 to identify CD4+ TCM, CD4+ TEM, and CD4+ TFH subsets. (B–D) Percentages of (B) TEM, (C) TFH, and (D) TCM, among spleen CD4+ T cells. (E–G) Percentages of (E) TEM (F), TFH, and (G) TCM, among lymph node CD4+ T cells. (H) Gating strategy for FACS analysis for B cell sub-populations. After gating on live, single cells, B cells were gated as B220+ and CD19+ (R1). Expression of GL7, Fas, CD138 and CD38 to identify plasma cells and germinal center (GC) B cells. (I) Percentages of GC B cells among spleen B cells. (J) Percentages of plasma cells among spleen B cells. (K) Percentage of GC B cells among lymph node B cells. (L) Percentage plasma cells among lymph node B cells. (A–L) Significant differences between groups compared to the unvaccinated group are marked as \* and significant differences between the groups with lines (ANOVA with Tukey's multiple comparison test; \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001; \*\*\*\**p* < 0.0001).

Pooled data of two experiments with (*n* = 10 in total per group) are shown ±SEM. (A) *Mtb* lysate specific total IgG, IgG1, and IgG2c antibody titers as measured at 1:10 dilution. (B) Ag85B lysate specific total IgG, IgG1, and IgG2c antibody titers as measured at 1:10 dilution. (C) Bacterial loads in lungs and (D) spleen. (E) Quantification of percentage of infiltrated area per lung. (F) Giemsa staining of infected lungs. Representative histology slides per group are shown. (A,B,E) Significant differences between antibody titers of treatment groups compared to the unvaccinated group are marked as \* and significant differences between the groups with lines (ANOVA with Tukey's multiple comparison test; \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001; \*\*\*\**p* < 0.0001). (C,D) Significant differences between the groups are shown with \* (Kruskal–Wallis with Dunn's multiple comparison test; \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001).

cells showed increased IFN-γ responses to Ag85B and ESAT-6 CD4 epitopes as well as the H56 peptide pool in mice that had received homologous H56\_E cDNA prime-boost (**Figure 3D**; Table S4 in Supplementary Material). Analysis of T cell subsets demonstrated no differences in frequencies of spleen TEM, TCM, or TFH after DNA boosting (**Figures 4E–G**), with a similar pattern in the draining lymph nodes (**Figures 4H–J**). Frequencies of CD8+ TCM and CD8+ TEM were not significantly different after different immunization strategies, similar to the single vaccination experiments (data not shown).

Frequencies of H56-specific multi-cytokine-producing CD4+ T cells after different vaccination regimes were analyzed (**Figure 5**), since cytokine-producing capacity has been correlated to differentiation state (40, 54, 55). BCG induced much lower frequencies of H56-specific cytokine-producing cells overall. Strikingly, there were differences in cytokine production profiles between groups receiving BCG alone, heterologous prime-boost with BCG and H56\_E cDNA, or homologous H56\_E cDNA prime-boost. Specifically, in the heterologous prime-boosted group, CD4+ T cells responding to Ag85Bderived H56242–262 were primarily IFN-γ+TNF-α+, while in the H56\_E/H56\_E cDNA vaccinated groups they expressed a more mixed cytokine-producing profile. By contrast, CD4+ T cells responding to ESAT-6-derived H56288–307 were TNF-α+IL-2+ in

FIGURE 5 | Frequencies in double or triple cytokine positive CD4+ T cells is correlated to immunization strategy. Intracellular cytokine staining was performed on splenocytes harvested at day 14 after the booster vaccination and re-stimulated with different H56 peptides. The peptide pools consisted of eighty-five 15-mer peptides spanning the entire sequence of H56, as a measure of the total percentage of H56-antigen-specific cells. CD4+ T cell epitopes included H56242–262 (Ag85B derived) and H56288–307 (ESAT-6 derived). Analysis was done using FACS. The results show pooled data from two independent experiments (*n* = 10 in total). Within the CD4+ population, frequencies of peptide-specific single, double, or triple positive cells is shown in bar graphs. The pie graphs denote the proportion of each cytokine-producing subset of the responding cells.

the BCG/H56\_E group and had mixed profiles in the H56\_E/ H56\_E and the BCG-only vaccinated groups. Responses to the peptide pool were different again, with BCG prime/DNA boost mice showing mostly IFN-γ+ cells, while the H56\_E/H56\_E vaccinated group had a mixed profile.

No significant differences were found in lymph node GC and plasma B cells between groups (data not shown), and antibody levels were also similar (**Figures 6A,B**) although the antimycobacteria IgG1 titer was increased in mice receiving a booster immunization with H56\_E cDNA compared to the group vaccinated with BCG alone (**Figure 6A**). Titers of anti-mycobacteria antibodies in H56\_E cDNA/H56\_E cDNA prime-boost mice remained as low as in unvaccinated controls (**Figure 6A**), but Ag85B-specific IgG2c titers were raised (**Figure 6B**).

## Despite Inducing H56-Specific Immune Responses, a Booster Immunization with H56\_E cDNA Does Not Ameliorate Bacterial Loads Compared to BCG Vaccination in Mice

To determine the protective efficacy of the different prime-boost strategies, bacterial loads, and lung pathology were measured 90 days after *Mtb* challenge. BCG and BCG/H56\_E cDNA immunized groups had reduced lung bacterial burdens compared to the unvaccinated mice; however, there was no significant improvement in bacterial loads after boosting with H56\_E cDNA (**Figures 6C,D**). Boosting H56\_E cDNA prime with a homologous immunization did not increase efficacy, and mice were not protected over unvaccinated controls. Bacterial loads in spleens followed a similar pattern (**Figure 6D**), although vaccination was not as protective as in the lung. Quantification of infiltrating cells in the lungs by image analysis also revealed H56\_E homologous prime-boost mice to be the least protected (**Figures 6E,F**). Mice primed with BCG and boosted with H56\_E cDNA were the only group to show significant improvement in lung pathology compared to the unvaccinated group and tended to have less cell infiltration than the BCG-vaccinated mice too, suggesting that the H56-specific T cell responses associated with boosting with H56\_E cDNA did have a beneficial effect in ameliorating disease. Overall, protective efficacy relied on the presence of BCG in the vaccine regimen, suggesting that broader anti-mycobacterial responses may be required for optimal protection in addition to the generation of highly specific responses against particular antigens.

### DISCUSSION

Previously, we demonstrated that the immunogenicity of a DNA vector containing H56 cDNA could be enhanced by fusion of the H56 sequence to cDNA of TTFC and by altering epitope flanking residues to facilitate epitope processing (43). Here, we tested the efficacy of immunization with the optimized constructs against *Mtb* challenge. The results demonstrate that *i.d.* tattoo vaccination with an optimized H56\_E cDNA sequence fused to TTFC cDNA, either as a standalone vaccine or as a booster to BCG, did not reduce *Mtb* burdens in the lung compared to BCG alone, although it showed a tendency to improve lung pathology.

Bacillus Calmette–Guérin is the only licensed vaccine against TB, but it provides variable and inadequate protection against the disease (10). Therefore, recombinant BCG vaccines and other live-attenuated mycobacterial strains are being tested as replacement vaccines, while subunit vaccines containing new antigens, adjuvants, or viral vectors are being investigated for their ability to boost BCG (13–18, 40–42). Boosting of BCG prime vaccination with CAF01-adjuvanted *Mtb* fusion protein H56 (containing Ag85B, ESAT-6, and Rv2660 components) increased BCG-induced protection against TB in a murine model (33) and H56 administered in IC31 adjuvant as a booster following BCG prime vaccination was more protective than BCG alone in macaques (42). In order to test alternative methods of subunit vaccine delivery that could be used in prime-boost regimens without the need for adjuvants, we generated H56 encoding DNA constructs with H56 cDNA fused to TTFC cDNA (43). Several naked DNA vaccine candidates have been tested against TB in mice, with various degrees of immunogenicity and prophylactic efficacy (56), but the protective efficacy of tattoo immunization with constructs containing *Mtb* antigens has not been assessed previously. We tested whether *i.d.* H56 cDNA tattoo immunization was protective against TB in a mouse model, either as a standalone vaccine or as a heterologous boost to BCG prime. We found that *i.d.* tattoo immunization with H56 constructs did not protect against TB in mice, although it increased H56-specific T cell responses, spleen GC B cells, and antibody titers when given as a boost to BCG prime. Overall, our results showed that BCG immunization was more protective than the highly specific response to a few antigens induced by *i.d.* H56 cDNA immunization.

Understanding the underlying mechanisms of protective immunity to *Mtb* will assist in the rational design of effective vaccines (57). Our experiments comparing *i.d.* DNA immunization with H56 constructs and *s.c.* vaccination with BCG demonstrated that the two types of vaccines elicited distinct immune responses. BCG induced increased frequencies of CD4+ TEM, TFH, GC B cells, and mycobacteria-specific antibodies, while H56 cDNA immunization induced H56-specific CD4+IFN-γ+ T cell responses and Ag85B-specific IgG antibodies. BCG shares numerous mycobacterial antigens with *Mtb*, including the immunodominant antigen Ag85B, while the DNA tattoo immunization elicits specific responses against H56, demonstrated by responses against ESAT-6 and Ag85B. Mice vaccinated with BCG accordingly had relatively high levels of mycobacteria-specific antibodies; while mice administered cDNA *i.d.* (particularly the unmodified H56 sequence) developed higher titers of Ag85Bspecific antibodies. Both BCG and the H56 cDNA constructs elicited Ag85B-specific CD4+ and CD8+ T cells, with the peptide H56242–262 being a particularly strong inducer of IFN-γ responses by CD4+ T cells.

Mice immunized with BCG *s.c.* were better protected against *Mtb* challenge than mice that received H56 cDNA *i.d.*, suggesting that broader anti-mycobacterial responses are an important component of vaccine-induced protection. Unlike viruses, which contain only a small number of antigens forming obvious vaccine

FIGURE 6 | Heterologous prime-boost with BCG/H56\_E cDNA decreases lung pathology but not bacterial loads. Serum was harvested at day 14 after the booster vaccination and lungs and spleens were collected at day 90 post *Mycobacterium tuberculosis* (*Mtb*) challenge were plated in serial dilution. Pooled data of two experiments with (*n* = 10 in total per group) are shown ±SEM. (A,B) Total IgG, IgG1, and IgG2c antibodies recognizing (A) *Mtb* lysate or (B) Ag85B were measured in serum of immunized mice at day 40 (total IgG: 1 in 100 dilution, IgG1 and IgG2c, 1 in 10 dilution). (C,D) Lungs and spleens collected at day 90 post *Mtb* challenge were plated in serial dilution. Pooled data of two experiments with (*n* = 10 in total per group) is shown ±SEM. (C) Bacterial loads in lungs and (D) spleen. (E) Quantification of percentage of infiltrated area per lung. (F) Giemsa staining of infected lungs. Representative histology slides per group are shown. (A–E) Significant differences between treatment groups and the unvaccinated group are marked as \* and significant differences between groups with lines. \**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001; \*\*\*\**p* < 0.0001. (A–E) ANOVA with Tukey's multiple comparison test; (C,D) Kruskal–Wallis with Dunn's multiple comparison test.

candidates, bacterial pathogens such as *Mtb* contain thousands of potential antigens, and no single antigen has been identified that is clearly associated with protective responses. This may be why it has been difficult for subunit vaccines to surpass live whole cell vaccines such as BCG in mouse models (10). Live BCG persists in mice for up to 16 months post vaccination and disseminates in the host, with T cell responses peaking around week 32 and waning thereafter (58, 59), whereas T cell responses against DNA vaccines could be measured only up to day 50 (43). Previously, we showed that BCG disseminates to the lungs after *s.c.* vaccination, where it persists for about 55 days (22). *I.d*. tattoo vaccination with BCG showed a trend toward decreased control of lung bacterial burdens compared to *s.c*. vaccination, however, lung pathology and spleen bacterial burdens were similar to that of *s.c.* vaccinated mice. BCG is most commonly administered by *i.d.* injection (not tattoo) in the clinic, and this route was found to be equivalent to percutaneous administration in both efficacy and safety over a 2-year follow-up period (60).

H56 and H56\_E cDNA were not protective as standalone vaccines; hence, we tested whether a homologous prime-boost regimen could improve the efficacy, using H56\_E cDNA. Homologous H56\_E cDNA prime-boost mice were also not protected compared to unvaccinated mice after *Mtb* challenge. These mice had the most vigorous H56-specific CD8+ T cell responses, indicating that this response was not sufficiently effective in preventing murine TB, at least during active infection. Furthermore, mice in this group hardly produced any *Mtb*- and Ag85B-specific IgG, with only a few animals producing Ag85B-specific IgG2c. It is possible that alterations to the H56\_E sequence may have led to a change in B cell Ag85B epitopes or skewing of immune responses away from Ag85B antibody-producing conditions, since in the single-dose vaccination experiments, H56 cDNA also led to increased antibody titers compared to H56\_E cDNA.

Which immune responses are required for optimal protection against TB remains a key question in the attempt to generate a more effective vaccine. In our previous studies with BCG *ΔureC::hly* and BCG *ΔureC::hly ΔnuoG*, increased protection after vaccination against TB coincided with increased TEM, TCM, TFH, GC B cells, and mycobacteria-specific antibody titers as compared to BCG vaccination (22). CD4+ TCM, by virtue of their capacity to generate new CD4+ TEM, are considered important in long-term immunity to *Mtb*. Here, boosting BCG by *i.d.* immunization with H56\_E cDNA did not induce elevated CD4+ TCM over BCG alone. The adjuvant CAF01 induces TCM responses (40) and homing of cytokine-producing cells to the lung parenchyma (61), which could explain why boosting with H56 protein in CAF01 adjuvant was more effective than boosting with the H56 cDNA tattoo immunization. In macaques vaccinated with BCG followed by an H56 booster in IC31 adjuvant, early recall responses to the vaccine antigens were associated with protection (42). A comparison between H56 cDNA and H56 protein/ adjuvant immunization could provide further insights into the type of immunity required for protection against TB. In addition, increased H56-specific CD8+ T cell responses did not coincide with increased protective efficacy in our model. CD8+ T cells play a critical role in vaccine-induced immunity to TB in macaques, whose CD8+ T cells are more similar to humans (62). In mice, CD8+ T cells appear to play a more important role in the chronic phase of TB than in the acute phase (28). Several studies have shown that antibodies and B cells may also play a role in immunity to TB (51–53). Interestingly, a previous study in guinea pigs found that boosting a recombinant BCG expressing ESAT-6 with intramuscular ESAT-6 cDNA injection reduced protection compared to BCG alone, despite inducing increased antigen-specific IFN-γ responses (63). Together with our study, this suggests that cDNA immunization as a whole might not induce the type of immune responses required for immunity to TB.

In summary, *i.d.* DNA tattoo vaccination using H56 and H56\_E constructs alone or in combination with BCG did not significantly improve upon protection induced by BCG vaccination alone in our model, despite inducing strong H56-specific CD4+ T cell and CD8+ T cell responses. Nevertheless, the H56-specific responses induced by H56\_E cDNA vaccination did ameliorate lung pathology, demonstrating the value of adding subunit vaccines containing defined *Mtb* antigens as a booster to BCG. Differences in T cell cytokines and antibody production following the different vaccination regimens as well as the limited number of antigens in H56 compared to BCG may explain the findings. Understanding why the types of immune responses generated by DNA tattoo vaccination does not suffice for protection against TB in contrast to responses generated by BCG or H56 administered with adjuvants such as CAF01 and IC31 will lead to optimized TB vaccination strategies.

### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the GV-SOLAS. The protocol was approved by the State Office for Health and Social Services, Berlin, Germany.

# AUTHOR CONTRIBUTIONS

AP and NN contributed equally to this work. AP, NN, AS, and SK conceptualized the study and wrote the manuscript. AP, NN, UZ, and SS performed experiments. AP, NN, TD, and VB performed data analysis. All authors contributed to the manuscript preparation. AS and SK are joint senior authors.

# ACKNOWLEDGMENTS

Support was by European Union's Seventh Framework Programme [FP7/2007-2013]—Grant No. 280873 ADITEC to AS and SK, Boehringer Ingelheim funds travel grant and EFIS travel grant to AP. We thank Dr. Donatella Negri for providing the 15-mers of H56, Dr. Alexis Vogelzang for technical support, Dr Gesa Rausch for assistance with animal ethics, and the staff of the animal facility.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01744/ full#supplementary-material.

### REFERENCES

Platteel et al. cDNA Tattoo Immunization against *Mtb*

Bacillus Calmette-Guérin ΔureC::hly vaccine's superior protection against tuberculosis. *J Infect Dis* (2014) 210:1928–37. doi:10.1093/infdis/jiu347

**Conflict of Interest Statement:** SK is co-inventor/patent holder of BCG *ΔureC::hly* (VPM1002). The other authors declare to have no conflicts of interest.

*Copyright © 2017 Platteel, Nieuwenhuizen, Domaszewska, Schürer, Zedler, Brinkmann, Sijts and Kaufmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Anne Marit de Groot 1†, Anouk C. M. Platteel1†, Nico Kuijt 2 , Peter J. S. van Kooten1 , Pieter Jan Vos2 , Alice J. A. M. Sijts1 \* and Koen van der Maaden2 \**

*1Department of Infectious Diseases and Immunology, Faculty of Veterinary Sciences, Utrecht University, Utrecht, Netherlands, 2MyLife Technologies, Leiden, Netherlands*

### *Edited by:*

*Peter Andersen, State Serum Institute (SSI), Denmark*

### *Reviewed by:*

*Ed C. Lavelle, Trinity College, Ireland Ji Wang, Harvard Medical School, United States*

### *\*Correspondence:*

*Alice J. A. M. Sijts<EMAIL_ADDRESS>Koen van der Maaden<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 06 September 2017 Accepted: 29 November 2017 Published: 13 December 2017*

### *Citation:*

*de Groot AM, Platteel ACM, Kuijt N, van Kooten PJS, Vos PJ, Sijts AJAM and van der Maaden K (2017) Nanoporous Microneedle Arrays Effectively Induce Antibody Responses against Diphtheria and Tetanus Toxoid. Front. Immunol. 8:1789. doi: 10.3389/fimmu.2017.01789*

The skin is immunologically very potent because of the high number of antigenpresenting cells in the dermis and epidermis, and is therefore considered to be very suitable for vaccination. However, the skin's physical barrier, the stratum corneum, prevents foreign substances, including vaccines, from entering the skin. Microneedles, which are needle-like structures with dimensions in the micrometer range, form a relatively new approach to circumvent the stratum corneum, allowing for minimally invasive and pain-free vaccination. In this study, we tested ceramic nanoporous microneedle arrays (npMNAs), representing a novel microneedle-based drug delivery technology, for their ability to deliver the subunit vaccines diphtheria toxoid (DT) and tetanus toxoid (TT) intradermally. First, the piercing ability of the ceramic (alumina) npMNAs, which contained over 100 microneedles per array, a length of 475 µm, and an average pore size of 80 nm, was evaluated in mouse skin. Then, the hydrodynamic diameters of DT and TT and the loading of DT, TT, and imiquimod into, and subsequent release from the npMNAs were assessed *in vitro*. It was shown that DT and TT were successfully loaded into the tips of the ceramic nanoporous microneedles, and by using near-infrared fluorescently labeled antigens, we found that DT and TT were released following piercing of the antigen-loaded npMNAs into *ex vivo* murine skin. Finally, the application of DTand TT-loaded npMNAs onto mouse skin *in vivo* led to the induction of antigen-specific antibodies, with titers similar to those obtained upon subcutaneous immunization with a similar dose. In conclusion, we show for the first time, the potential of npMNAs for intradermal (ID) immunization with subunit vaccines, which opens possibilities for future ID vaccination designs.

Keywords: nanoporous microneedles, intradermal vaccination, antigen release, humoral immune response, diphtheria, tetanus

**Abbreviations:** IMQ, imiquimod; MNA, microneedle array; npMNA, nanoporous microneedle array; IgG, immune globulin G; DT, diphtheria toxoid; TT, tetanus toxoid; TLR, toll-like receptor; DLS, dynamic light scatter; Lf, limits of flocculation; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid; TMB, 3,3′,5,5′-tetramethylbenzidine; BSA, bovine serum albumin; PBS, phosphate-buffered saline; ACN, acetonitrile; HRP, horseradish peroxidase; OD, optical density; PBST, 0.01% Tween 20 in PBS.

# INTRODUCTION

The skin has great potential for vaccine delivery, because it is a large organ that is easy to reach. Delivery *via* the skin circumvents degradation challenges to biomacromolecules, as posed, for example, by the gastrointestinal delivery route (1, 2). The skin, with the stratum corneum as outer barrier, is designed to keep foreign materials including pathogens out of the body. Besides, the skin is immunologically very potent, with various professional antigen-presenting cells, such as dermal dendritic cells and Langerhans cells (3, 4), present in the dermis and epidermis, respectively. To circumvent the barrier function of the stratum corneum and reach antigen-presenting cells for vaccination purposes, microneedles can be used. Microneedles are needlelike structures with a length in the micrometer range and are a promising tool to deliver drugs and vaccines across the barrier. Furthermore, they represent a possible painless vaccination method (5), they present reduced contamination risks compared with traditional needles, they allow for injection by less trained personnel and even have potential for self-administration (6). However, microneedles need to be sufficiently long and strong enough to pierce the stratum corneum, but also preferably short enough to not reach the nociceptors. Various microneedles are under development, which are hollow-, solid-, dissolving-, or less known porous structured (6–10). For all types, multiple strategies have been investigated for the delivery of vaccine antigens into the skin, as reviewed by van der Maaden et al. (10).

Porous microneedles, which may be used as a single-unit-drug delivery system, can be prepared from pore-forming materials (11), from (nano)particles (12, 13), or by making solid microneedle material porous (14, 15). Porous microneedle arrays (MNAs) can be loaded with a drug, by loading the formulation into the pores of the MNAs. The drug is released when the microneedles are pierced into the skin *via* diffusion from the pores. To date, several materials have been used for the production of porous MNAs. Among these are biodegradable polymeric porous MNAs with a porosity of 75%, which, however, lack the strength to penetrate the skin (11). When using a brittle material, like silicon, the pores that are introduced in the material need to be sufficiently small to provide enough strength for skin piercing (14, 15). The use of porous silicon material, therefore, is limited to the delivery of low-molecular weight drugs (10). Using self-setting ceramics for production of porous MNAs increases MNA strength. However, drug loading into these MNAs requires circumstances that are unfavorable for formulating biomacromolecules, because it involves exothermic reactions or organic solvents (ethanol) (16).

In this study, microneedles composed of a biocompatible ceramic, alumina (Al2O3) (12), were tested for their suitability for intradermal (ID) vaccination. With an average pore size of 80 nm and an estimated porosity of 40%, these microneedles allow for encapsulation of large biomacromolecules before production (10, 13). In previous studies, it was shown that alumina nanoporous microneedle arrays (npMNA) can be successfully loaded with small molecules or nanoparticles with sizes up to 100 nm in solution or dispersion *via* absorption (*via* capillary forces), respectively, and to release these substances *in vitro* by diffusion. The npMNAs had sufficient strength to reproducibly pierce *ex vivo* human skin without breaking (10) and have shown to activate immune cells upon dermal application of peptide-loaded npMNAs in a murine model (17). However, characterization of ceramic alumina npMNAs loaded with larger, more relevant molecules, such as subunit vaccine antigens, had not been performed so far.

In this study, characterization and application of alumina npMNAs are described. Loading of npMNAs with diphtheria toxoid (DT) and tetanus toxoid (TT), antigen release in murine skin *ex vivo*, and *in vivo* immunogenicity of npMNA-delivered antigens were examined. We show that npMNA-mediated vaccine delivery elicits TT- and DT-specific antibody responses in mice, comparable to those induced by subcutaneous (SC) immunization with a similar dose. This is the first report showing the potential of porous microneedles in dermal immunization with subunit vaccines.

### MATERIALS AND METHODS

### Materials

Diphtheria toxoid and TT (for *in vitro* assays) were obtained from Staten Serum Institute (Copenhagen, Denmark) and imiquimod (IMQ) Vaccigrade was obtained from Invivogen. Trifluoroacetic acid (TFA), 3,3′,5,5′-tetramethylbenzidine (TMB) and bovine serum albumin (BSA), and 0.4% (w/v) were obtained from Sigma Aldrich. High-performance liquid chromatography (HPLC)-R grade acetonitrile (ACN) was from Biosolve and phosphate-buffered saline (PBS; pH 7.2, 1.5 mM KH2PO4, 2.7 mM Na2HPO4–7H2O, and 155 mM NaCl) was from Gibco (ThermoFisher Scientific). IRdye800cw carboxylic acid *N*-hydroxy succinimide ester (IRdye800cw-NHS) was purchased from Li-cor (Lincoln, NE, USA). Dexdomitor was purchased from Orion Corporation, Narketan ketamine from Vétoquinol, and Atipam was purchased from Dechra. Goat anti-mouse immune globulin G (IgG) total horseradish peroxidase (HRP) (GaM-IgG total HRP), goat anti-mouse IgG1 HRP (GaM-IgG1 HRP), and goat anti-mouse IgG2a HRP (GaM-IgG2a HRP) were obtained from Southern Biotech and microtiter plates 9610 used for ELISA were from Corning Costar.

### Preparation and Characterization of npMNA

Nanoporous microneedle arrays were produced by using a double replication technology as previously described (12). In brief, from an inverse silicon master a first positive PDMS mold was created, from which a second inverse PDMS mold was produced. Alumina npMNAs were fabricated at LouwersHanique B.V. from the second PDMS mold according to MyLife Technologies' proprietary manufacturing procedure (18), using a slurry that contains alumina nanoparticles with an average size of 300 nm and a plasticizer. After controlled drying, the resulting MNAs were removed from the PDMS mold and were sintered at 1,450°C. This results in removal of the plasticizer and the formation of nanoporous alumina material with an average pore size of 80 nm and a porosity of approximately 40% (10, 12). Microneedles used in this study had a length of 475 µm and a density of 150 microneedles/cm2 on a back plate of 0.7 cm2 (105 microneedles/ array; **Figures 1A,B**). The total volume in the nanopores of only the tips of the microneedles of a single MNA was calculated to be 0.25 µL. Bruker Nano Surface analysis was performed to characterize the geometry of the npMNAs.

### Preparation of npMNAs with Antigen-Loaded Microneedle Tips

To only load the tips of the microneedles, microneedles were pierced through a foil (Parafilm®) by using a UAFM-V1 electrical applicator (uPRAX Microsolutions) at a velocity of 65 cm/s. Next, a drop of 5 µL drug formulation was applied onto the foilpierced npMNAs to absorb a drug/vaccine formulation into the microneedle tips. After 5 s, the surplus drop of drug formulation and the foil were sequentially removed from the npMNAs. To confirm that only the microneedle tips can be loaded with a drug formulation, the tips of npMNAs were loaded with a 0.4% trypan blue solution as described earlier.

### Skin Penetration

To test the piercing ability, a npMNA was applied trice on the dorsal side of *ex vivo* murine ears (Balb/C), which were collected from surplus mice, by using 3D-printed uPRAX impact applicators (**Figure 1C**) having an average holding force of 4.08 ± 0.75 N (mean ± SD, *n* = 19). Subsequently, the three pierced *ex vivo* mouse ears were incubated with 50 µL of a 0.4% trypan blue solution at room temperature. After 30 min, the trypan blue was removed, and the ears were washed in 10 mL PBS. Finally, the blue dots (piercings) were counted, from which the penetration efficiency was calculated.

### Hydrodynamic Diameter and Size Distribution of Antigens

To determine whether the npMNAs are suitable devices to load and release subunit antigens DT and TT, the hydrodynamic diameter and size distribution of DT and TT were determined by using dynamic light scattering on a Zetasizer Nano (Malvern Instruments). For these measurements, DT and TT were at a concentration of 0.8 and 0.4 mg/mL, respectively.

### Release of IMQ and Antigen from Nanoporous Alumina *In Vitro*

Imiquimod has the potential to enhance the immunogenicity of antigens in the skin (19). To evaluate how IMQ is released from IMQ-loaded nanoporous material in the presence of DT and TT, npMNAs were loaded by applying a drop (on the microneedle side of a npMNA that were not pierced through a foil) of 5 µL PBS containing only 2.5 µg IMQ, or 2.5 µg IMQ and 2.5 Lf DT or TT. Such a drop is absorbed into an npMNA within seconds because of capillary forces. Next, the IMQ-loaded npMNAs were incubated in 2.5 mL release buffer (PBS) under shaking at 300 rpm, and samples of 75 µL were taken in duplicate at different time points (1, 5, 10, 30, 60, 120, and 240 min). The IMQ concentration in the release buffer was determined by using HPLC analysis on an Agilent 1100 series HPLC equipped with a UV detector using a Phenomenex Kinetex 150 mm × 4.6 mm 2.6 μm EVO C18 column. A linear gradient of 5% solvent A (ACN with 0.1% TFA) to 68% solvent B (milliQ with 0.1% TFA) from 0 to 12 min was detected at a wavelength of 242 nm at a retention time of 9.8 min.

To assess the release of antigen from antigen-loaded nanoporous material, npMNAs (that were not pierced through a foil) were loaded with 15 µL PBS that contained 5 Lf DT or TT as described earlier. Next, antigen-loaded npMNAs were incubated in 4 mL release buffer while shaking at 300 rpm. At different time points (1, 5, 10, 30, 60, 120, and 240 min), samples of 500 µL were taken in which the released amount of antigen was quantified by measuring the intrinsic fluorescence (emission wavelength of 348 nm) at an excitation wavelength of 280 nm and using standard concentrations of DT and TT ranging from 0.01 to 50 ng/mL on a Tecan Infinite M1000 plate reader. The release of antigen in the presence of IMQ was not investigated, because IMQ is fluorescent at similar wavelengths (excitation at 260 nm and emission at 340 nm) that are used to measure the intrinsic fluorescence of the antigens (20).

### Fluorescently Labeling of Antigens

To quantify the amount of DT and TT that is delivered from DTand TT-loaded npMNAs into skin, DT and TT were labeled with a near-infrared fluorescent dye (IRdye800cw-NHS). To this end, 1 mg/mL solutions of DT and TT in a 100 mM carbonate buffer pH 8.5 were prepared. Subsequently, 1 mL of each of these solutions was added to 500 µg of IRdye800cw-NHS. After 1 h shaking (300 rpm) at 37°C, the free dye was removed and the carbonate buffer was exchanged by PBS using a Zeba™ spin desalting column with a molecular weight cutoff (MWCO) of 7 kDa (Thermo Fisher Scientific). Next, IRdye800cw-labeled antigens were concentrated approximately 50 times by using 0.5 mL Amicon (Millipore) 10 kDa MWCO filters. Finally, the concentration of IRdye800cw-labeled DT and TT was determined by using a calibration curve of non-labeled antigens and measuring the intrinsic fluorescence (as described earlier).

### Quantification of Antigen in *Ex Vivo* Mouse Ears

To quantify the delivered amount of DT and TT into murine skin, npMNAs of which only the tips were loaded (using foil piercing) with fluorescently labeled antigens were prepared by using 5 µL of 12 Lf/μL (DT) and 6 Lf/μL (TT), as described earlier. The microneedles were applied by impact application and retained onto the skin by using a uPRAX 3D-printed applicator. After 30 min at room temperature, the antigen-loaded npMNAs were removed from the ears, and their fluorescence was compared with the fluorescence of standard solutions having known amounts of fluorescently labeled DT and TT, by using a IVIS® lumina II equipped with an ICG filter set. The intradermally delivered amounts of DT and TT were quantified by using Living Image® software (version 4.3.1).

### Preparation of Vaccine Formulations for Loading npMNAs for Immunization