Novel Whole-Cell Inactivated Neisseria Gonorrhoeae Microparticles as Vaccine Formulation in Microneedle-Based Transdermal Immunization

Abstract

Neisseria gonorrhoeae is a strict human pathogen responsible for more than 100 million new sexually transmitted infections worldwide each year. Due to the global emergence of antibiotic resistance, the Center for Disease control (CDC) recently listed N. gonorrhoeae as an urgent threat to public health. No vaccine is available in spite of the huge disease burden and the possibility of untreatable gonorrhea. The aim of this study is to investigate the immunogenicity of a novel whole-cell-based inactivated gonococcal microparticle vaccine formulation loaded in dissolvable microneedles for transdermal administration. The nanotechnology-based vaccine formulation consists of inactivated whole-cell gonococci strain CDC-F62, spray dried and encapsulated into biodegradable cross-linked albumin matrix with sustained slow antigen release. The dry vaccine nanoparticles were then loaded in a dissolvable microneedle skin patch for transdermal delivery. The efficacy of the whole-cell microparticles vaccine formulation loaded in microneedles was assessed in vitro using dendritic cells and macrophages as well as in vivo mouse model. Antibody titers were measured using an enzyme immunosorbent assay (ELISA) and antigen-specific T lymphocytes were assessed in spleens and lymph nodes. Here we report that whole-cell-based gonococcal microparticle vaccine loaded in dissolvable microneedles for transdermal administration induced significant increase in antigen-specific IgG antibody titers and antigen-specific CD4 and CD8 T lymphocytes in mice compared to gonococcal antigens in solution or empty microneedles. Significant increase in antigen-specific IgG antibody levels was observed at the end of week 2 in groups that received the vaccine compared to the group receiving empty nanoparticles. The advantages of using formalin-fixed whole-cell gonococci that all immunogenic epitopes are covered and preserved from degradation. The spherical shaped micro and nanoparticles are biological mimics of gonococci, therefore present to the immune system as invaders but without the ability to suppress adaptive immunity. In conclusion, the transdermal delivery of microparticles vaccine via a microneedle patch was shown to be an effective system for vaccine delivery. The novel gonorrhea nanovaccine is cheap to produce in a stable dry powder and can be delivered in microneedle skin patch obviating the need for needle use or the cold chain.

Keywords: Neisseria gonorrhoeae; antigen-specific CD4 T lymphocytes; antigen-specific antibody; gonorrhea; microneedle; nanotechnology; skin patch; vaccine.

Figure 1

Characterization of formalin fixed whole-cell N. gonorrhoeae. (A) Scanning electron microscopic (SEM) image of formalin fixed whole-cell N. gonorrhoeae, which is the antigen for the vaccine. (B) SEM image of spray-dried microparticles containing the N. gonorrhoeae vaccine antigen. (C) N. gonorrhoeae whole-cell vaccine particle uptake by RAW264 macrophages induced autophagic vacuoles visualized using fluorescence microscopy. Green puncta (arrow) are autophagic vacuoles and blue are nucleus stained with DAPI. (D) Unstimulated RAW264 macrophages.

Figure 2

Nitric oxide (NO) production of murine dendritic cells DC 2.4 treated with N. gonorrhoeae microparticles. Nitric oxide release from dendritic cells was assessed in murine dendritic cells (DC 2.4) (2.5 × 10⁵) after pulsing with gonorrhea vaccine microparticles for 16 h. Nitrite accumulation in the supernatants was determined using the Greiss reagent. Error bars represent the standard deviation from the average of two independent determinations. The data shown are representative of three independent experiments. Blank MP: empty microparticles; GC-MP: N. gonorrhoeae-loaded micropraticles. * p ≤ 0.05.

Figure 3

Dosage dependent toxicity test of microparticles on RAW264 murine macrophages cell line. Albumin based microparticles are not toxic to macrophages. RAW264 murine macrophages treated with increasing doses of microparticles and incubated overnight. RAW264 murine macrophages treated with increasing doses of microparticles and incubated overnight. The cytotoxicity was analyzed by the Alamar Blue assay that uses the reducing power of living cells to quantitatively measure cell viability. Atropine sulphate is the positive control and cell only serves as the negative control. Vaccine-loaded microparticles were not cytotoxic for the studied concentrations ranging between 50 to 500 µg. Results are expressed as mean ± standard error (n = 3).

Figure 4

Scanning electron microscopy (SEM) image of the N. gonorrhoeae vaccine microparticles loaded in dissolvable microneedles. (A) Formulated microneedle is 600 µm in length. (B) N. gonorrhoeae microparticles loaded in microneedle. (C) dissolvable microneedles after transdermal delivery. (D) Microneedle skin patch “Band-Aid”. (E) Microchannels created by the microneedles patch on the skin using methylene blue dye. (F) Z-stack of calcein-stained microchannels as seen on mouse skin using confocal microscopy. Calcein was observed in microchannels to up to 600 ± 60 µm depth.

Figure 5

N. gonorrhoeae-specific antibody measurement in serum using ELISA. The mice received one prime dose subcutaneously at week 0 followed by 2 booster doses at week 1 and 2. The vaccinated mice groups receiving either GC suspension or GC microparticles vaccines showed significantly higher serum IgG levels from week 4 till week 10, when compared to the unvaccinated mice group (p < 0.05). At week 8 and 10, the group receiving microparticulate vaccine showed significantly higher response compared to the subcutaneous vaccine group (* p < 0.05). GC-susp: N. gonorrhoeae antigen in suspension; GC-MP: N. gonorrhoeae antigen-loaded in microparticles.

Figure 6

N. gonorrhoeae-specific antibody measurement in mice serum immunized transdermally using microneedles patch. Mice were vaccinated at day 0 followed by two booster at week 1 and 2 and immune response was monitored for 10 weeks. ELISA method was used to measure induced serum IgG levels in vaccinated mice and control groups. The groups receiving vaccine showed significantly higher serum IgG titers when compared to the controls i.e., blank microparticles and blank microneedles (MN) after week 2. The group which received the gonorrhea vaccine microparticles in microneedles (GC-MP-MN) showed significantly higher antibody titers than the other 2 vaccine groups at week 6 and 8 (n = 6) (* p < 0.001; # p < 0.05). GC-susp SubQ: N. gonorrhoeae antigen in suspension administered subcutaneously (GC-susp SubQ); GC-susp MN: N. gonorrhoeae antigen in suspension administered transdermal via microneedle; GC-MP-MN: N. gonorrhoeae antigen loaded in nanoparticles administered transdermal via microneedles.

Figure 7

N. gonorrhoeae microparticle vaccine induced antigen-specific CD4⁺ and CD8⁺ T-cells counts in the splenocytes at week 10 after immunization. Groups receiving the vaccine showed significantly higher CD4⁺ and CD8⁺ T cells than compared to the controls i.e., unvaccinated and blank microneedles (p < 0.05). GC-susp SubQ: N. gonorrhoeae antigen in suspension administered subcutaneously; GC-susp MN: N. gonorrhoeae antigen in suspension administered transdermal via microneedle; GC-MP-MN: N. gonorrhoeae antigen loaded in microparticles administered transdermal via microneedles.