The adjuvant BcfA activates antigen presenting cells through TLR4 and supports TFH and TH1 while attenuating TH2 gene programming

Introduction Adjuvants added to subunit vaccines augment antigen-specific immune responses. One mechanism of adjuvant action is activation of pattern recognition receptors (PRRs) on innate immune cells. Bordetella colonization factor A (BcfA); an outer membrane protein with adjuvant function, activates TH1/TH17-polarized immune responses to protein antigens from Bordetella pertussis and SARS CoV-2. Unlike other adjuvants, BcfA does not elicit a TH2 response. Methods To understand the mechanism of BcfA-driven TH1/TH17 vs. TH2 activation, we screened PRRs to identify pathways activated by BcfA. We then tested the role of this receptor in the BcfA-mediated activation of bone marrow-derived dendritic cells (BMDCs) using mice with germline deletion of TLR4 to quantify upregulation of costimulatory molecule expression and cytokine production in vitro and in vivo. Activity was also tested on human PBMCs. Results PRR screening showed that BcfA activates antigen presenting cells through murine TLR4. BcfA-treated WT BMDCs upregulated expression of the costimulatory molecules CD40, CD80, and CD86 and produced IL-6, IL-12/23 p40, and TNF-α while TLR4 KO BMDCs were not activated. Furthermore, human PBMCs stimulated with BcfA produced IL-6. BcfA-stimulated murine BMDCs also exhibited increased uptake of the antigen DQ-OVA, supporting a role for BcfA in improving antigen presentation to T cells. BcfA further activated APCs in murine lungs. Using an in vitro TH cell polarization system, we found that BcfA-stimulated BMDC supernatant supported TFH and TH1 while suppressing TH2 gene programming. Conclusions Overall, these data provide mechanistic understanding of how this novel adjuvant activates immune responses.


Introduction
Many approved vaccines are comprised of purified antigens admixed with an adjuvant, referred to as subunit vaccines.The inclusion of immune stimulatory adjuvants in vaccine formulations bolsters immune responses versus antigens alone, supports dose sparing, reduced frequency of administration (1,2), and improves the stability and pharmacokinetics of the antigens leading to an increased in vivo half-life (2).Another notable advantage is the ability of the adjuvant to shape the phenotype of the resulting cellular and humoral responses (1).
Alum (aluminum hydroxide or aluminum phosphate) was the first adjuvant to be licensed for human use more than a century ago.For seven decades, it was the only adjuvant used in FDA-and EMAapproved vaccines administered to protect against a multitude of infectious diseases including hepatitis, pertussis, and diphtheria (3,4).In the late 1990s, other adjuvants were approved for addition to previously licensed human vaccines (3).The first of these was the oil-in-water emulsion MF59 added to a trivalent influenza vaccine, FLUAD (5,6).The more recently described AS01-AS04 family of adjuvants are TLR4 ligands and aim to maximize the immune response while maintaining tolerability using a mix of classical adjuvants and other immunostimulatory molecules (3).These are included in vaccines against shingles (7), malaria (8), pandemic influenza (9), HBV and HPV (10).The oligonucleotide cytosine phosphoguanosine 1018 (CpG 1018), is a TLR9 agonist included in a hepatitis B vaccine (11-13).
These approved adjuvants have different mechanisms of action and are safe and effective at generating T cell and antibody-mediated responses.Notably, however, they all elicit mixed T H 1/T H 2-polarized immunity (14).In contrast, natural immune responses to infection with most viral and bacterial pathogens elicit T H 1/T H 17-polarized T cell and antibody responses, which are correlated with sustained protection against disease and infections in both humans and mouse models (15)(16)(17)(18)(19). Recent studies (20) also highlight the importance of tissue-resident memory T (T RM ) and B cell responses which provide sustained protection at barrier sites (21-24).Therefore, there is a need for safe novel adjuvants that elicit T H 1/T H 17 -polarized immune responses, and also generate tissue-resident memory.To rationally design such adjuvants, it is critical to understand their mechanism of action.We previously identified Bordetella colonization factor A (BcfA), a bacterial protein of the Gram-negative pathogen Bordetella bronchiseptica, and we have reported that it functions as an adjuvant in vivo (25).Systemic administration of BcfA-containing vaccines elicited T H 1-and T H 17-polarized CD4 + and T H 1-polarized CD8 + T cell responses (25), T follicular helper (T FH ) cells, and T H 1-skewed antibody responses (20) alone and in combination with alum.Mucosal immunization with antigens mixed with BcfA elicited CD4 + IL-17 + T RM cells in the lungs and nose (20, 26).Importantly, when BcfA was mixed with alum-containing approved and experimental vaccines, T H 2 responses were attenuated while T H 1 and T H 17 responses were sustained or amplified (20,25), suggesting that BcfA can override the T H 2 polarized responses elicited by alum.This unique property therefore supports the potentially broad applicability of BcfA as an adjuvant for bacterial and viral pathogens where T H 1 and T H 17 responses are important for protection against infection and disease.
Here, we investigated the mechanism of BcfA activation of immune responses.We report that BcfA activates bone marrowderived dendritic cells (BMDCs) through the pattern-recognition receptor TLR4.We observed dose-dependent upregulation of costimulatory molecules CD40, CD80 and CD86 on wildtype (WT) BMDCs and the production of innate cytokines IL-6, TNFa and IL-12/23 p40.BcfA-activated BMDCs more efficiently processed and presented the model antigen DQ-OVA.Furthermore, BcfA-stimulated BMDC conditioned medium supported differentiation of T FH -like and T H 1 CD4 + T cells in an in vitro culture system, demonstrating the ability of BcfA to shape therapeutically beneficial T cell responses.These results provide insight regarding the TLR-dependent mechanism of action of this novel adjuvant and suggest that inclusion of BcfA in nextgeneration vaccine formulations may represent a promising avenue to enhance protective long-term immunity.

Mice
All experiments were reviewed and approved by The Ohio State University (OSU) Institutional Animal Care and Use Committee (Protocol number 2017A00000090).C57BL/6J, TLR4 knockout mice on the C57BL/6 background were obtained from Jackson Laboratories and bred in our facility.Tail DNA from mice was genotyped by Transnetyx, Inc. (Cordova, TN) using validated probe sets to confirm genotypes.

Differentiation of murine bone marrow derived dendritic cells
Murine BMDCs were prepared according to published protocols (28).Briefly, bone marrow was isolated, dissociated into a single cell suspension, and red blood cells were lysed with ACK lysing buffer (Gibco Ref A10492).The cell suspension was resuspended in RPMI1640 (Gibco) + 10% fetal bovine serum (FBS, Sigma-Aldrich F4220), 10µg/mL gentamicin, 5x10 -5 M ßmercaptoethanol and 40 ng/mL GM-CSF (R&D Systems, Minneapolis, MN, catalog no.415-ML-020)) and seeded in 10 cm 2 non-tissue culture treated petri dishes (VWR catalog no.25384-342) at a density of 5-10 x10 6 cells/plate.Half of the medium was replaced every 2 days with the addition of fresh GM-CSF.On day 6-7 post-differentiation, BMDCs were transferred to 6-well tissue culture-treated plates (Falcon catalog no.353046) at a density of 0.5-1 x10 6 cells/well.The next day, the cells were stimulated with various concentrations of BcfA for 20-24hr.Supernatant was collected for ELISA analysis and the cells were harvested for flow cytometry.

Collection of human PBMCs and stimulation with BcfA
Peripheral blood was collected from adult human donors under a protocol approved by The Ohio State University Institutional Review Board (Protocol numbers 2020H0404 and 2021H0179).Whole blood was collected in EDTA treated tubes.PBMCs were separated from whole blood on a Percoll gradient (Cytiva, catalog no.17144003), and cryo-preserved at -80°C.Cells were thawed and cultured in RPMI + 10% human AB serum (Sigma Aldrich, catalog no.H4522).To determine TLR4 expression, PBMCs were stained with a-human TLR4 (Thermo Fisher Scientific catalog no.12-9041-80) antibody.Cells were analyzed on a Cytek Aurora spectral flow cytometer.Fluorescence minus one (FMO) controls were used for gating.To test cytokine production, 1-2x10 6 cells from individual donors were stimulated for 20-24hr and the supernatant was tested by ELISA.

Enzyme-linked immunosorbent assay for innate cytokines
The production of murine TNF-a, IL-6, IL-12/23 p40 common g chain and IL-12 was quantified by a sandwich ELISA according to the manufacturer's instructions (Life Technologies or BioLegend).Human cytokines IL-6 and TNF-a were quantified using Quantikine ELISA kits (R&D Systems).Plates were read at A 450 on a SpectraMax i3x ® plate reader and concentrations were calculated based on the standard curve.

DQ-OVA uptake and processing
BMDCs were plated in 6 well plates at 1 x10 6 cells/well and stimulated with BcfA or cultured with medium alone.At 24 hr post-stimulation, DQ-OVA (InvivoGen, Inc. catalog no.D12053) was added at a concentration of 10 µg/mL for 45 min.The cells were then washed and harvested to quantify expression of CD11c, MHC-Class II and DQ-OVA by flow cytometry.

Immunization of mice
Mice were lightly anesthetized with 2.5% isoflurane/O 2 for immunization.To stimulate APCs in the lungs, mice were immunized intranasally with 10 µg BcfA or LPS-EB VacciGrade ™ (InvivoGen, catalog no.vac-3pelps) (a TLR4 agonist used as a positive control) in 50 µL divided between both nares.Lungs were harvested 24 hr post-inoculation.
Fluorescence minus one or isotype control antibodies were used as negative controls.After two washes, cells were resuspended in PBS/1% FBS and samples were collected on or BD FACS Symphony or Cytek Aurora spectral flow cytometer (Cytek Biosciences).
Analysis was performed using FlowJo software version 10.8.0.The number of cells within each population was calculated by multiplying the frequency of live singlets in the population of interest by the total number of cells in each sample.

Statistical analysis
Data were analyzed using GraphPad Prism by the methods described in each figure legend.

Activation of murine BMDCs by BcfA is dependent on TLR4
We hypothesized that BcfA activates antigen-presenting cells (APCs) through a PRR and conducted an empirical PRR screening using the HEK-Blue system, where human embryonic kidney (HEK) cells were transfected with the SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of a promoter inducible by NF-kB and activator protein-1 (AP-1) and expressing murine TLR (mTLR) or NOD receptors.SEAP reporter production was quantified using a colorimetric assay.The screen showed activation of mTLR4 and mTLR2 (Supplementary Figure S1A).We then stimulated cells expressing TLR4 (HEK-mTLR4) and TLR2 (HEK-mTLR2) with 1 or 5 µg/mL BcfA.Maximal SEAP release was detected from HEK cells expressing murine TLR4 (HEK-mTLR4) following stimulation with 1 µg/mL BcfA (Supplementary Figure S1B) at levels comparable to purified LPS used as a positive control.SEAP production from HEK-mTRL2 cells was nearly maximal at 5 µg/mL BcfA stimulation (Supplementary Figure S1C) with lower SEAP production detected with 1 µg/mL BcfA stimululation.To mitigate any confounding effects of BcfA produced in E. coli as a bacterial recombinant protein, we utilized a stringent purification procedure to remove LPS (27).HEK-mTLR4 or HEK-mTLR2 cells incubated with this minimal LPS (≤150 pg/µg protein) (min LPS) did not produce SEAP.
We then evaluated the BcfA stimulatory activity in a more physiologically relevant system.We isolated bone marrow from WT C57BL/6 mice and TLR4 KO mice and differentiated BMDCs in vitro.The immature DCs were treated with BcfA (1 or 5 µg/mL) for 24 hr and analyzed for the expression of costimulatory molecules by flow cytometry.BMDCs were identified as a CD11c + MHC-II hi population (Supplementary Figure S2) (29) and were evaluated for the expression of costimulatory molecules by flow cytometry.Representative flow plots are shown in Figure 1A.The mean fluorescence intensity (MFI) expression of CD40 (Figure 1B), CD80 (Figure 1C), and CD86 (Figure 1D) and percentage of cells expressing CD40 (Figure 1E) CD80 (Figure 1F) and CD86 (Figure 1G) was upregulated in response to BcfA stimulation compared to no stimulation (NS) control.LPS-EB (de-Oacelylated lipooligosaccharide) (5 µg/mL) that has been used as an adjuvant in vivo (30) and purified E. coli LPS (100 ng/mL) were included as positive controls for TLR4, and PAM3Csk was used as a positive control to confirm that TLR4 KO BMDCs respond to stimulation.Both the MFI and percentage of cells that upregulated costimulatory molecule expression was reduced to background in BcfA-stimulated TLR4 KO BMDCs, suggesting that BcfA primarily functions through this PRR.We also tested activation of TLR2 KO BMDCs which showed that costimulatory molecule expression was not reduced by the absence of TLR2 (Supplementary Figure S3), suggesting that BcfA primarily functions through TLR4.Thus, we focused on TLR4 mediated activity of BcfA in the following studies.

BcfA induces the production of T FH /T H 1/ T H 17-polarizing cytokines by BMDCs
To determine the T cell responses that may be supported by APCs activated by BcfA, we quantified cytokine production from BcfA-stimulated BMDCs by ELISA.WT BMDCs produced IL-6 (Figure 2A), TNF-a (Figure 2B), IL-12/23 p40 (Figure 2C) and IL-12 (Figure 2D) at levels comparable to the positive control LPS.Production of all four cytokines was significantly reduced in TLR4 KO BMDCs.These results show that innate immune responses elicited by BcfA are mediated through TLR4 and may support T H 1/ T H 17-polarized immune responses.
We then tested whether BcfA activated human PBMCs.First, we confirmed expression of TLR4 on adult human PBMCs by flow cytometry (Figure 3A).We then stimulated PBMCs in vitro with BcfA and measured the levels of innate cytokines IL-6 and TNF-a present in the cell culture supernatant after 24 hr.We found that IL-6 was significantly induced in response to stimulation with 5 µg/mL or 25 µg/mL of BcfA (Figure 3B).Although TNF-a production was substantially increased upon stimulation with BcfA, differences were not statistically significant compared to unstimulated cells (Figure 3C).These data show that BcfA stimulation elicits T H 1/T H 17-polarizing innate cytokines by human cells.

BcfA stimulation of BMDCs supports antigen uptake and processing
Next, we evaluated whether BcfA stimulation of BMDCs would increase antigen uptake and processing of DQ-OVA, a chicken ovalbumin (OVA) conjugate that displays a bright green fluorescence only after proteolytic processing.We stimulated BMDCs with 5 µg/mL BcfA or media alone as a negative control for 24 hr at 37°C.The next day, cells were treated with DQ-OVA for 60 min.at 37°C.The level of green fluorescence was then quantified by flow cytometry as a proxy for antigen uptake and processing.BcfA-treated BMDCs took up more DQ-OVA than unstimulated BMDCs (Figure 4A).We also observed an increase in the percentage of BMDCs with green fluorescence (Figure 4B) compared to NS controls.Together, these data suggest that BcfA-activated BMDCs have an increased ability to uptake and process antigen.

BcfA upregulates costimulatory molecule expression on CD11b + CD11c + cells via TLR4 in vivo
To test whether BcfA stimulates lung cells, we administered 10 µg BcfA or LPS-EB intranasally to C57BL/6 and TLR4 KO mice.Lungs were harvested 24 hr later and evaluated for the expression of MHC Class II, CD40, CD80, and CD86 on the CD11b + CD11c + population (31) (Figure 5A).The gating strategy is shown in Supplementary Figure S4.MFI expression of MHC Class II (Figure 5B), CD40 (Figure 5C), CD80 (Figure 5D) and CD86 (Figure 5E) and the percentage of cells that upregulated expression of MHC Class II (Figure 5F), CD40 (Figure 5G), CD80 (Figure 5H) and CD86 (Figure 5I) increased on this cell population, demonstrating that BcfA efficiently activates putative antigen presented cells in the lungs.The MFI of costimulatory molecule expression was not upregulated on CD11b + CD11c + cells in TLR4 KO lungs (Figures 5B-E).The percentage of cells expressing MHC Class II compared to no stimulation increased in WT and TLR4 KO lungs (Figure 5F).In contrast the percentage of TLR4 KO lung cells expressing the costimulatory molecules CD40 (Figure 5G), CD80 (Figure 5H) and CD86 (Figure 5I) did not increase compared to no stimulation.These data further confirm TLR4 as the primary bona fide PRR for BcfA mediated activity.

BcfA-stimulated BMDC-conditioned medium supports T FH and T H 1 cell programming
As part of pathogen-specific immune responses, naïve CD4 + T cells differentiate into effector subsets which perform specialized activities to orchestrate immune-mediated clearance of infection.Particularly critical for responses to intracellular pathogens like SARS-CoV-2 and primarily extracellular pathogens like B. pertussis are T helper 1 (T H 1) and T follicular helper (T FH ) populations.Of these, T H 1 cells activate other immune populations by secreting proinflammatory cytokines such as IFN-g, while T FH cells produce IL-21 and provide help to B cells to promote antibody generation and humoral immunity (32,33).We previously reported that intramuscular (i.m.) immunization with an acellular pertussis vaccine (aPV) containing BcfA elicited systemic T H 1 responses in vivo (25).In our recent work (20), we established that immunization of mice with BcfA-adjuvanted SARS-CoV-2 spike protein supported production of IFN-g-driven IgG2c spike protein-specific antibodies.Thus, we hypothesized that BcfA may support both T H 1 and T FH cell differentiation programs, and consequently productive anti-bacterial and anti-viral cell-mediated and humoral immune responses.
Effector CD4 + T cell differentiation is directed by a coordinated interplay between cell-intrinsic transcriptional networks and environmental cytokine signals that are often produced by antigen- presenting cells (34).To test whether the IL-6, IL-12, and IL-12/23 common chain cytokines BcfA-stimulated supernatants would be sufficient to induce T FH and/or T H 1 differentiation, we stimulated naïve CD4 + T cells on plate-bound a-CD3/a-CD28 in the presence of cell-free supernatant from untreated BMDCs (NS) or those treated with 5 µg/mL BcfA for 72 hr.We then assessed the expression of key T FH and T H 1/ genes via qRT-PCR.Consistent with our hypothesis, expression of genes for lineage-defining transcription factors and effector cytokines associated with T FH (Bcl6, Il21; Figure 6A) and T H 1 (Tbx21 (T-bet), Ifng; Figure 6B) cells were elevated in cells cultured in the presence of BcfA-conditioned medium relative to NS controls.
As IL-6/STAT3 signaling is an established driver of T FH differentiation, we determined whether the IL-6 in the culture medium was responsible for driving T FH differentiation.We cultured cells in BcfA-treated or NS control medium in the presence or absence of IL-6-neutralizing antibody.Consistent with a role for IL-6 in promoting T FH gene expression, we observed a significant reduction in the expression of both Bcl6 and Il21 when IL-6 was neutralized (Figure 6C).
As standard T FH -like and T H 1 culture conditions include the addition of anti-IL-4 neutralizing antibody which prevents inherent T H 2 polarization (35), we also tested whether BcfA elicited T FH or T H 1 gene programming in the absence of IL-4-neutralization. Naïve T cells were differentiated in vitro without the addition of anti-IL-4 blocking antibody.We evaluated the expression of T cell subsetspecific lineage-defining transcription factors and key cytokines by qRT-PCR and flow cytometry.Interestingly, BcfA reduced the RNA (Figure 7A) expression of the T H 2 lineage-defining transcription factor Gata-3, while the protein level was unchanged (Figure 7B).Similarly, the RNA (Figure 7C) level of the T H 2 effector cytokine IL-4 was reduced, while the protein (Figure 7D) expression was unchanged.In contrast, while transcript abundance for the T FH lineage-defining transcription factor Bcl-6 was unchanged (Figure 7E), its protein expression was elevated (Figure 7F).Yet, transcript levels for the T FH cytokine IL-21 were significantly elevated (Figure 7G), while protein expression was not significantly increased (Figure 7H).Finally, T H 1 differentiation was elevated, as the RNA expression of the T H 1-defining factor Tbx21 (T-bet) was increased (Figure 7I), and the protein level was slightly but not significantly elevated (Figure 7J).RNA for the T H 1 cytokine IFN-g was unchanged (Figure 7K) while protein expression was increased (Figure 7L).Overall, these data suggest that BcfA both negatively regulates T H 2 differentiation and positively regulates T FH and T H 1 programming.

Discussion
The development and characterization of novel adjuvants is an active area of research, with goal of defining adjuvants that elicit strong, sustained vaccine-mediated immune responses.Despite the decades-long use of alum as an adjuvant and the recent development of newer formulations, there remains a need for safe and effective adjuvants that elicit systemic and mucosal immunity.
Here, we investigated the mechanism of action of the adjuvant BcfA, which was discovered by our group and has demonstrated adjuvant activity in experimental vaccines against respiratory pathogens B. pertussis (25,26) and SARS CoV-2 (20).The hallmark feature of BcfA that distinguishes it from FDA-approved and other experimental adjuvants is the absence of T H 2 responses when BcfA is used as the single adjuvant (25) and strong attenuation of T H 2 responses when BcfA is added to alum-adjuvanted vaccines (20,26).
Furthermore, BcfA elicits T H 1 and T H 17 responses which are critical for protection against both bacterial and viral pathogens.Attenuation of T H 2 immune responses may also reduce the risk of vaccine related adverse events such as antibody dependent enhancement of disease (ADE) and vaccine associated enhancement of respiratory disease (VAERD).
As BcfA is a protein, we hypothesized that it may activate immune responses through PRRs expressed on APCs.A PRR/NOD receptor screen identified both murine TLR4 and TLR2 as the receptors triggered on HEK-293 reporter cell lines following BcfA stimulation (Supplementary Figure S1).When the activity of BcfA was tested in murine BMDCs as a more physiological system, upregulation of costimulatory molecule expression (Figure 1) and production of innate cytokines (Figure 2) was significantly reduced in TLR4 KO BMDCs compared with WT BMDCs, but not in TLR2 KO BMDCs (Supplementary Figure S3).These data show that BcfA acts primarily through TLR4.BcfA-stimulated BMDCs showed uptake and processing of the model antigen DQ-OVA (Figure 4), suggesting that BcfA may also support T cell activation by amplifying antigen presentation.We pretreated BMDCs with BcfA prior to testing uptake of DQ-OVA.Coadministration of antigen and adjuvant may change the kinetics or magnitude of antigen uptake and processing and is a limitation of our experimental design.
To determine the potential utility of BcfA as an adjuvant for human vaccines, we tested cytokine production by human PBMCs stimulated with BcfA for 24 hr (Figure 3).Production of IL-6 was detected in supernatants of all samples tested, suggesting that human PBMCs stimulated with BcfA may support T FH polarization of CD4 + T cells.Expression of the inflammatory TNF-a was variable between donors, and not significantly increased compared with unstimulated cells.It is important to note that we collected supernatant at 24 hr post-stimulation, which may be beyond the time of maximal TNF-a production in this context.Alternately, there may be autocrine consumption of TNF-a produced by the monocytes (36).Thus, the ability of BcfA to activate human cells suggests that BcfA will have adjuvant function when included in human vaccines.
Although bacterial LPS is the canonical ligand for TLR4, this PRR may also be activated by a wide array of bacterial proteins (37).Interestingly, triggering of the same receptor may elicit disparate immune phenotypes.The pneumococcal proteins DnaJ and Ply (38,39) and the M. tuberculosis derived proteins RpfE and Rv0652 are TLR4 ligands that activate DCs and polarize the immune response towards the T H 1/T H 17 phenotype (40, 41), but elicit different innate cytokines.Collectively, these findings suggest that the type of immune response elicited by adjuvants depends both on the responding PRR as well as the properties of the ligand.
Adjuvants also activate the adaptive immune response by directly triggering PRRs on lymphocytes (42).Murine and human T cells express TLR4 (43).Thus, BcfA may directly activate T cells when delivered in vivo as part of a vaccine formulation, and thereby amplify the vaccine-elicited response.This potential function of BcfA is an area of future study for our group.
Using an in vitro T cell polarization system, we showed that the innate cytokines produced by BcfA-stimulated BMDCs support the differentiation of T H 1 and T FH cells while preventing T H 2 differentiation in parallel (Figure 6 and Figure 7).This result is in accordance with our previous report of the ability of BcfA to attenuate alum-induced T H 2 responses when combined in vivo (20, 26).In addition, the IL-6-dependent polarization of T FH cells we observed also provides a potential mechanistic explanation for the induction of IgG2c antibody production we observed in the serum and lungs of mice immunized with BcfA-containing vaccines.Generation of mucosal immunity to respiratory pathogens is critical for providing sustained protection against infection (44, 45) and subsequent transmission (46).For B. pertussis, the CD4 + IL-17 + T cells generated during natural infection are essential for reducing bacterial colonization of the nose (45,46).Immunization of baboons with the whole cell pertussis vaccine (wPV) also clears the infection from the nose (47).In contrast, acellular pertussis (aPV) vaccine immunization does not reduce nasal bacterial burden (48).Our recent paper (26) showed that mucosal delivery of BcfA-adjuvanted acellular pertussis vaccines elicited CD4 + IL-17 + tissue-resident memory T cells (T RM ) in the nose and reduced nasal bacterial burden.Although we detected production of the IL-12/23 p40 common g chain by BcfA-stimulated BMDCs, suggesting that IL-17 may also be produced, we did not detect T H 17 polarization in vitro (data not shown), suggesting that either the amount of IL-12/23 produced in BcfA supernatant is insufficient, or that additional factors such as such as TGF-b (49) that support T H 17 generation are not replicated in the culture system.
In summary, this work provides a mechanistic understanding of the function of BcfA as an adjuvant and supports the utility of BcfA for use in human vaccines.Continued identification and validation of novel adjuvants that are safe, effective, and generate mucosal immunity is key to improving vaccine-mediated protection against infectious diseases.