Mucosal Immunogenicity of Genetically Modified <i>Lactobacillus acidophilus</i> Expressing an HIV-1 Epitope within the Surface Layer Protein

<div><p>Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified <i>Lactobacillus acidophilus</i> strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the <i>Lactobacillus</i> S-layer protein for development of oral vaccines targeting specific peptides.</p></div

Induction of MPER-specific antibody production by long-term immunization.

<p>Mice received GAD19 orally every 2 weeks for 14 weeks. (a) Diluted serum (1/100) was analyzed by ELISA at each time point. Arrows represent timing of the gavage. (b) Endpoint titers (or absorbance at 450 nm) of MPER-specific serum IgG, cecal IgA, vaginal IgA, and vaginal IgG. Each symbol represents an individual mouse.</p

Validation of genetically modified <i>L</i>. <i>acidophilus</i> producing MPER-displaying S-layer proteins.

<p>The <i>L</i>. <i>acidophilus slpA</i> gene in NCK1909 was replaced with the modified <i>slpA</i> gene including MPER-encoding sequences by homologous recombination in NCK2208. (a) The gene replacement of <i>slpA</i> with the modified <i>slpA</i> was confirmed by PCR. L, DNA ladder marker. Amplified DNA fragments using primers, AK_62 and AK_65 (lane 1 and 4), AK_62 and AK_57 (lane 2 and 5), or AK_56 and AK_65 (lane 3 and 6). (b) Detection of the MPER epitope in S-layer (SlpA) protein using 2F5 mAb. Total cell proteins and purified S-layer proteins of NCK1909 and NCK2208 were separated by SDS-PAGE. The gels were stained with CBB or blotted onto PVDF membrane followed by western blot analysis using 2F5 (anti-MPER monoclonal human IgG). (c) The exposed MPER epitope was detected by flow cytometry. The <i>L</i>. <i>acidophilus</i> strains labeled with 2F5 and Alexa Fluor 488-conjugated anti-human IgG were analyzed. Relative fluorescence intensity of each strain was shown as histogram plot.</p