. 2024 Jan 29;36(1):33-43.

doi: 10.1093/intimm/dxad045.

Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue

Xiao Sun^{1

2}, Koji Hosomi¹, Atsushi Shimoyama^{3

4}, Ken Yoshii^{1

5}, Azusa Saika¹, Haruki Yamaura³, Takahiro Nagatake^{1

6}, Hiroshi Kiyono^{7

8

9

10}, Koichi Fukase^{3

4}, Jun Kunisawa^{1

2

3

4

5

11

12

13

14}

Affiliations

¹ Laboratory of Vaccine Materials and Laboratory of Gut Environmental System, Microbial Research Center for Health and Medicine, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085, Japan.
² Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
³ Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
⁴ Collaborative Research Between NIBIOHN and Graduate School of Science, Forefront Research Center, Osaka University, Osaka 560-0043, Japan.
⁵ Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
⁶ Department of Life Sciences, Laboratory of Functional Anatomy, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan.
⁷ Division of Gastroenterology, Department of Medicine, University of California San Diego (UCSD), San Diego, CA 92093, USA.
⁸ Chiba University (CU)-UCSD Center for Mucosal Immunology, Allergy and Vaccines (cMAV), UCSD, San Diego, CA 92093-0063, USA.
⁹ Future Medicine Education and Research Organization, Chiba University, Chiba 260-8670, Japan.
¹⁰ Department of Human Mucosal Vaccinology, Chiba University Hospital, Chiba 260-8677, Japan.
¹¹ International Vaccine Design Center, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
¹² Department of Microbiology and Immunology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
¹³ Research Organization for Nano and Life Innovation, Waseda University, Tokyo 162-0041, Japan.
¹⁴ Graduate School of Dentistry, Osaka University, Suita 565-0871, Japan.

PMID: 38006376
PMCID: PMC10823578
DOI: 10.1093/intimm/dxad045

Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue

Xiao Sun et al. Int Immunol. 2024.

. 2024 Jan 29;36(1):33-43.

doi: 10.1093/intimm/dxad045.

Authors

Affiliations

¹ Laboratory of Vaccine Materials and Laboratory of Gut Environmental System, Microbial Research Center for Health and Medicine, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085, Japan.
² Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
³ Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
⁴ Collaborative Research Between NIBIOHN and Graduate School of Science, Forefront Research Center, Osaka University, Osaka 560-0043, Japan.
⁵ Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
⁶ Department of Life Sciences, Laboratory of Functional Anatomy, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan.
⁷ Division of Gastroenterology, Department of Medicine, University of California San Diego (UCSD), San Diego, CA 92093, USA.
⁸ Chiba University (CU)-UCSD Center for Mucosal Immunology, Allergy and Vaccines (cMAV), UCSD, San Diego, CA 92093-0063, USA.
⁹ Future Medicine Education and Research Organization, Chiba University, Chiba 260-8670, Japan.
¹⁰ Department of Human Mucosal Vaccinology, Chiba University Hospital, Chiba 260-8677, Japan.
¹¹ International Vaccine Design Center, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
¹² Department of Microbiology and Immunology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
¹³ Research Organization for Nano and Life Innovation, Waseda University, Tokyo 162-0041, Japan.
¹⁴ Graduate School of Dentistry, Osaka University, Suita 565-0871, Japan.

PMID: 38006376
PMCID: PMC10823578
DOI: 10.1093/intimm/dxad045

Abstract

We previously demonstrated that Alcaligenes-derived lipid A (ALA), which is produced from an intestinal lymphoid tissue-resident commensal bacterium, is an effective adjuvant for inducing antigen-specific immune responses. To understand the immunologic characteristics of ALA as a vaccine adjuvant, we here compared the adjuvant activity of ALA with that of a licensed adjuvant (monophosphoryl lipid A, MPLA) in mice. Although the adjuvant activity of ALA was only slightly greater than that of MPLA for subcutaneous immunization, ALA induced significantly greater IgA antibody production than did MPLA during nasal immunization. Regarding the underlying mechanism, ALA increased and activated CD11b+ CD103- CD11c+ dendritic cells in the nasal tissue by stimulating chemokine responses. These findings revealed the superiority of ALA as a mucosal adjuvant due to the unique immunologic functions of ALA in nasal tissue.

Keywords: Alcaligenes; MPLA; chemokines; lipid A; nasal vaccine; stromal cells.

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Figures

**Figure 1.**
For both intranasal and subcutaneous vaccination, *Alcaligenes*-derived lipid A (ALA) induced higher levels of antigen-specific antibodies than did monophosphoryl lipid A (MPLA). In particular, ALA was superior for intranasal vaccination. (A) OVA-specific IgG in the serum of mice vaccinated subcutaneously was evaluated by ELISA (n = 4). (B) OVA-specific IgA in the nasal wash of mice vaccinated intranasally was evaluated by ELISA (n = 4). (C) OVA-specific IgG in the serum of mice vaccinated intranasally was evaluated by ELISA (n = 4). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by two-way analysis of variance (ANOVA; *P < .05, **P < .01, ***P < .001, ****P < .0001)..

**Figure 2.**
Intranasal administration of ALA increased the generation of GCs and the numbers of GC B cells, IgA⁺ GL7^hi B cells, and T_fh cells in NALT. (A) GCs (arrows) in NALT were analyzed through immunohistochemistry. PNA: GC marker; B220: B cell marker (n = 4) (scale bar = 200 μm). (B) The populations of GC GL7^hi B cells (gated on: CD3ε⁻ B220⁺ GL7^hi) and IgA⁺ GL7^hi B cells (gated on: CD3ε⁻ B220⁺ GL7^hi IgA⁺) in NALT were analyzed by flow cytometry (n = 4). (C) The population of T_fh cells (gated on: CD3ε⁺ CD8α⁻ CD4⁺ PD-1^hi) in NALT was analyzed by flow cytometry (n = 4). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by one-way ANOVA (*P < .05, **P < .01, ****P < .0001).

**Figure 3.**
Intranasal administration of ALA increased the generation of GCs and the numbers of GC B cells, IgA⁺ GL7^hi B cells, and T_fh cells in CLNs. (A) GCs (arrows) in CLNs were analyzed by immunohistochemistry. PNA: GC marker; B220: B cell marker (n = 4) (scale bar = 50 μm). (B) The populations of GC GL7^hi B cells (gated on: CD3ε⁻ B220⁺ GL7^hi) and IgA⁺ GL7^hi B cells (gated on: CD3ε⁻ B220⁺ GL7^hi IgA⁺) in CLNs were analyzed by flow cytometry (n = 4). (C) The population of T_fh (gated on: CD3ε⁺ CD8α⁻ CD4⁺ PD-1^hi) in CLNs was analyzed by flow cytometry (n = 4). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by one-way ANOVA (*P < .05, **P < .01, ***P < .001).

**Figure 4.**
Intranasal administration of ALA induced greater infiltration of type 2 conventional DCs (cDC2s) in nasal tissue than did MPLA. (A) The numbers of CD103⁺ CD11b⁻ cDC1s and CD11b⁺ CD103⁻ cDC2s due to intranasal adjuvant administration were evaluated by flow cytometry (n = 3). (B) The density of CD103⁺ CD11b⁻ cDC1s and CD11b⁺ CD103⁻ cDC2s due to subcutaneous adjuvant administration were evaluated by flow cytometry (n = 3). (C) The populations of CD11b⁺ CD103⁻ CLEC10A⁻ cDC2As (*) and CD11b⁺ CD103⁻ CLEC10A⁺ cDC2Bs (^#) in nasal tissue were analyzed by flow cytometry (n = 4). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by one-way ANOVA (*,^#P < .05, **P < .01; ns, not significant).

**Figure 5.**
Intranasally administered ALA induced greater expression of CD40 and CD80 than did MPLA. The expression of (A) CD40 and (B) CD80 on cDC2s after intranasal adjuvant administration was evaluated by flow cytometry (n = 3). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by one-way ANOVA (**P < .01, ***P < .001, ****P < .0001).

**Figure 6.**
The gene expression of *Csf2*, *Ccl2*, *Ccl3*, *Ccl4*, and *Tlr4* in sorted cells from nasal tissue. (A) The sorting strategy for CD45⁺ immune cells, EpCAM⁺ CD45⁻ epithelial cells, and double-negative stromal cells (n = 4). (B) The gene expression of *Tlr4* on sorted cells was evaluated by quantitative PCR (qPCR) analysis (n = 4). (C) The gene expression of *Csf2*, *Ccl2*, *Ccl3*, and *Ccl4* from CD45⁺ cells due to stimulation by ALA or MPLA was evaluated by qPCR (n = 4). (D) The gene expression of *Csf2*, *Ccl2*, *Ccl3*, and *Ccl4* from double-negative cells due to stimulation by ALA or MPLA was evaluated by qPCR (n = 4). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by one-way ANOVA (**P < .01, ***P < .001, ****P < .0001).

**Figure 7.**
Intranasal administration of ALA with anti-CCL2 or anti-CCL3 antibodies inhibited the infiltration of cDC2s in nasal tissue. The numbers of CD11b⁺ CD103⁻ cDC2s in nasal tissue were analyzed by flow cytometry (n = 4). The data are shown as the mean ± 1 SD from two independent experiments; differences were analyzed by two-way analysis of variance (ANOVA; *P < .05, **P < .01; ns, not significant).

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Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue

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Alcaligenes lipid A functions as a superior mucosal adjuvant to monophosphoryl lipid A via the recruitment and activation of CD11b+ dendritic cells in nasal tissue

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