Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques

Abstract

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

Figure 1

PET/MRI images (a,b) and TACs (c) for nasal administration of [¹⁸F]-PspA-nanogel or [¹⁸F]-PspA-PBS in a naive rhesus macaque. (a) After nasal administration of [¹⁸F]-PspA-nanogel or [¹⁸F]-PspA-PBS, the macaque's head was scanned for 6 h with a PET scanner. Real-time PET images overlaid on MRI images are shown for the indicated times post-administration. (b) To further check whether [¹⁸F]-PspA accumulated in the CNS or olfactory bulbs (indicated by arrowheads), PET images taken at 6 h post-administration of [¹⁸F]-PspA-nanogel were enlarged. (c) TACs for the nasal cavity for 6-h period after nasal administration of [¹⁸F]-PspA-nanogel or [¹⁸F]-PspA-PBS are presented. The data are expressed as percentages of the dose remaining after nasal administration. a: The same macaque was nasally administered of [¹⁸F]-PspA-nanogel or [¹⁸F]-PspA-PBS with a 1-week interval between administrations. CNS, central nervous system; MRI, magnetic resonance imaging; PET, positron emission tomography; TACs, time-activity curves.

Figure 2

Nasal immunization with PspA-nanogel induced PspA-specific Ab responses in macaques. Each cynomolgus macaque was nasally immunized with PspA-nanogel (macaques #2-#6), PspA alone (#7 and #8), or PBS only (#9) at the times indicated with arrows. Serum, nasal wash, and BALF were collected, and the levels of PspA-specific serum IgG (a), nasal wash IgA (b), and BALF IgG and IgA (c) were determined by ELISA. BALF, bronchoalveolar lavage fluid.

Figure 3

Neutralizing Abs induced by nasal immunization with PspA-nanogel. Serum from each of the macaques was collected 1 week after the final primary nasal immunization with PspA-nanogel, PspA alone, or PBS only. CBA/N mice (10 mice per group) were passively transferred with 100 μl of diluted (1:20) pooled sera via i.p. route. Four hours later, mice were injected i.v. with 1.5 × 10⁴ c.f.u. S. pneumoniae Xen 10 (a) or 1 × 10³ c.f.u. S. pneumoniae 3JYP2670 strain (b). The mice were monitored daily for mortality. Each line represents the median survival time. c.f.u., colony-forming unit; i.p., intraperitoneal; i.v., intravenous.

Figure 4

PspA-nanogel immunization produced CD4⁺ Th2- and Th17-type cytokine responses. CD4⁺ T cells were separated from the PBMCs 1 week after the booster. Purified CD4⁺ T cells were cultured with irradiated APCs and 5 μg ml⁻¹ of PspA with anti-CD28 and CD49d antibodies for 5 days. The levels of the cytokines, IFN-γ (a), IL-4 (b), and IL-17A (c) in the supernatants were measured. This experiment was repeated in triplicate. Values are shown as the means ± s.d. in each experimental group. **P< 0.01 compared between PspA-nanogel and PspA/PBS groups. (d) Serum from macaques was collected 1 week after the final primary nasal immunization with PspA-nanogel (#2-#6), PspA alone (#7, #8), or PBS only (#9). Expression levels of PspA-specific serum IgG subclass Abs were determined by using ELISA. APCs, antigen-presenting cells; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells.

Figure 5

MiRNA expression levels in sera (a), nasal tissues (b), and lung tissues (c) of macaques nasally immunized with PspA-nanogel, PspA alone, or PBS only. Expression levels of the indicated miRNA and Ets-1 mRNA were analyzed by quantitative RT–PCR and normalized to the levels of miR-16 and β-actin, respectively. Values are shown as the means ± s.d. in each experimental group. *P < 0.05, **P < 0.01 when compared between pre-immunization and post-booster groups. b, Compared between PspA-nanogel and PspA/PBS groups in post-booster macaques. MiRNA, microRNA; Pre, pre-immunized serum; Post, post-booster serum.