Pentamers Not Found in the Universal Proteome Can Enhance Antigen Specific Immune Responses and Adjuvant Vaccines

<div><p>Certain short peptides do not occur in humans and are rare or non-existent in the universal proteome. Antigens that contain rare amino acid sequences are in general highly immunogenic and may activate different arms of the immune system. We first generated a list of rare, semi-common, and common 5-mer peptides using bioinformatics tools to analyze the UniProtKB database. Experimental observations indicated that rare and semi-common 5-mers generated stronger cellular responses in comparison with common-occurring sequences. We hypothesized that the biological process responsible for this enhanced immunogenicity could be used to positively modulate immune responses with potential application for vaccine development. Initially, twelve rare 5-mers, 9-mers, and 13-mers were incorporated in frame at the end of an H5N1 hemagglutinin (HA) antigen and expressed from a DNA vaccine. The presence of some 5-mer peptides induced improved immune responses. Adding one 5-mer peptide exogenously also offered improved clinical outcome and/or survival against a lethal H5N1 or H1N1 influenza virus challenge in BALB/c mice and ferrets, respectively. Interestingly, enhanced anti-HBsAg antibody production by up to 25-fold in combination with a commercial Hepatitis B vaccine (Engerix-B, GSK) was also observed in BALB/c mice. Mechanistically, NK cell activation and dependency was observed with enhancing peptides ex vivo and in NK-depleted mice. Overall, the data suggest that rare or non-existent oligopeptides can be developed as immunomodulators and supports the further evaluation of some 5-mer peptides as potential vaccine adjuvants.</p> </div

Comparison of 5mer4 with other commercial adjuvants.

<p>Groups of 3–4 BALB/c mice were immunized with H5N1-HA (50 µg) alone, H5N1-HA (50 µg) + 5mer4 (50 µg), H5N1-HA (50 µg) + CpG (10 µg), H5N1-HA (50 µg) adsorbed to Alhydrogel (450 µg), or “All” (H5N1-HA + 5mer4 (50 µg) + CpG (10 µg) + adsorbed to Alhydrogel (450 µg). Cellular immune responses were monitored 10 days post-immunization. Splenocytes were re-stimulated with H5N1-HA peptides pools and detected for IFN-γ secretion. The bars represent total pooled T-cell responses. Error bars represent ± SEM.</p

Evaluation of H1N1-HA DNA vaccine combined with exogenous 5mer4 adjuvant in ferrets.

<p>Ferrets were immunized with 200 µg of the H1N1-HA DNA vaccine with or without the 5mer4 (200 µg) adjuvant. (a) Hemagglutination inhibition antibody responses. Ferrets received immunizations with the H1N1-HA DNA vaccine, administered 3 times at one month intervals. Serum was collected after each vaccination and prior to challenge. HI antibody responses were evaluated at days 55 and day 66 post-immunization and detected using turkey red blood cells. (b) HA-specific T-cell responses. PBMCs were collected by ficoll gradient from all ferrets following challenge with the H1N1-2009 virus. T-cell responses were evaluated following re-stimulation with 3 peptide pools representing the H1N1-2009 HA protein. (c) HA-specific T-cell responses following re-stimulation with 3 peptide pools representing the H1N1-1918 HA protein. (d) Clinical observations of ferrets following H1N1-2009 challenge. Ferrets were scored on a scale of 1–10, where 0 = no signs of disease, 10 = most severe. (E) Infectious and total virus particles. Ferret nasal washes were collected at days 1, 3, and 9 following infection. Left axis: Infectious virus titre from the nasal washes of H1N1-HA + 5mer4 (□), H1N1-HA (▪), and control (♦) ferrets was detected by TCID₅₀ assay. Right axis: Following RNA extraction, nasal wash samples were probed with H1HA specific primers using quantitative real-time RT-PCR. H1N1-HA + 5mer4 (○), H1N1-HA (•), and control (▴) ferrets. Error bars represent the SEM.</p

Evaluation of NK cell responses in response to 5mer4.

<p>(a) NK cells were isolated from the spleen of BALB/c mice using negative selection. Cells were incubated overnight in the presence of 100 U/ml recombinant IL-2, with 2.5 µg/ml of 5mer4, 5mer2 control, DMSO control. Additional groups of cells were incubated with 1000 U/ml IL2 and 1000 U/ml IL2 combined with 5mer4 (2.5 µg/ml). NK cell activation was monitored for PE-CD69 upregulation on APC-DX5⁺ cells by flow cytometry. All cells were CD3⁻. The data is representative of three independent experiments performed on different days. (b) NK depletion in BALB/c mice. Mouse NK cells were depleted by administration of anti-asialo antibody at day -2 and day -1 before vaccination. Whole blood samples were collected from the saphenous vein (50 µl) from NK-depleted and undepleted control animals. All samples were first treated with ACK lysis buffer to remove red blood cells and then stained with PE-CD69 and FITC-DX5 to detect the percentage of NK cells remaining following depletion. (c) Survival of vaccine groups following NK cell depletion. At day 0, NK-depleted BALB/c mice were immunized with H5N1-HA (1 µg) or H5N1-HA (1 µg) + 5mer4 (50 µg), or PBS control. Three groups of mice that were not depleted were immunized with H5N1-HA (1 µg), H5N1-HA (50 ug) or H5N1-HA + 5mer4 (50 µg) as controls. Animals were challenged with 100LD₅₀ of homologous H5N1-H05 virus and survival was monitored over 17 days. Groups of 8–10 BALB/c mice were evaluated.</p