Emulsified Phosphatidylserine, Simple and Effective Peptide Carrier for Induction of Potent Epitope-Specific T Cell Responses

<div><p>Background</p><p>To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) <i>in vivo</i>. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability.</p> <p>Methodology/Principal Findings</p><p>Data from an epitope-specific <i>in vivo</i> CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c⁺CD11b⁺MHCII⁺ conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells <i>in vivo</i>.</p> <p>Conclusions/Significance</p><p>This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses <i>in vivo</i>. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.</p> </div

Mice immunized with PS-conjugated peptide induced epitope-specific CTL effectively <i>in vivo</i>.

<p>(A) B6 mice (3 to 4 mice per group) were immunized s.c. with each carrier-conjugated NP_366–374 (A/HK483) peptide or peptide without carrier in the presence of poly(I:C). Seven days after the immunization, bright CFSE-labeled target cells pulsed with peptide used for the immunization and dim CFSE-labeled target cells pulsed with an irrelevant peptide were injected i.v. as an <i>in vivo</i> cytotoxicity assay. Viability of the target cells in the spleen was examined 20 h after injection. Reduction ratios of epitope-specific target cells were calculated using the formula described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060068#s2" target="_blank">Materials and Methods</a>. (B) A24Tg mice (3 mice per group) were inoculated with PS- or liposome-conjugated NP_257–264 (A/HK483) peptide. The <i>in vivo</i> cytotoxicity assay was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060068#pone-0060068-g001" target="_blank">Figure 1A</a>. n.s. indicates not significant. *p<0.01, **p<0.0001.</p

Proliferation assay of epitope-specific helper T cells.

<p>CD4⁺ T cells from mice immunized with PS-conjugated or unconjugated peptide were co-cultured with activated BMDCs for 2 days in complete RPMI medium containing the indicated concentration of NP_311–325 peptide. Proliferation of NP_311–325-specific CD4⁺ Th cells was measured by BrdU uptake. The experiment was repeated three times with similar results. *p<0.05, **p<0.005</p

Frequency of epitope-specific CD8⁺ T cells in immunized mice.

<p>(A) PS-conjugated NP_366–374 (A/PR8) peptide, PS-conjugated OVA_257–264 peptide or unconjugated peptide was inoculated into B6 mice (3 to 4 mice per group). The <i>in vivo</i> cytotoxicity assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060068#pone-0060068-g001" target="_blank">Figure 1</a>. *p<0.01, **p<0.0001. (B) Splenocytes from naïve mice and mice immunized with PS-conjugated or unconjugated peptide in the presence of poly(I:C) were stained with tetramer and anti-mouse CD8 Ab. The percentage indicates the tetramer-positive cells in total CD8⁺ cells. The experiment was repeated three times with similar results.</p

PS-conjugated Ags were captured by professional APCs effectively.

<p>(A) Splenocytes were classified into five subpopulations (I to V) based on the expression pattern of CD11b and CD11c. I:CD11b⁻CD11c⁻ cells, II:CD11b^intCD11c⁻ cells, III:CD11b^highCD11c⁻ cells, IV:CD11b⁺CD11c⁺ cells, V:CD11b⁻CD11c⁺ cells. (B, C) Each isolated population was co-cultured with sfGFP, sfGFP-PS or sfGFP-liposome for 60 min, and then the amount of uptake was analyzed by flow cytometry. (D, E) Each population of isolated cells was co-cultured with DQ-OVA or DQ-OVA-PS for 60 min, and then the efficiency of antigen degradation processing was analyzed by flow cytometry.</p

Confocal laser scanning microscopy analysis of splenocytes co-cultured with PS-conjugated antigens.

<p>(A, B) CD11b⁺ or CD11c⁺ cells were cultured with sfGFP, sfGFP-PS, DQ-OVA or DQ-OVA-PS plus Hoechst33342 for 60 min at 37°C. After the incubation, cells were washed with PBS, and then analyzed under a LSM780 confocal laser scanning microscope system. Blue: cell nucleus, Green: sfGFP or DQ-OVA, Red: CD11b or CD11c.</p

CD11c⁺CD11b⁺ cells are main APCs in peptide vaccines.

<p>The five kinds of sorted splenocytes were activated with CpG5002 <i>in vitro</i> and cultured with CD8⁺ T cells from NP_366–374-PS immunized mice with serial dilutions of PS-conjugated NP_366–374 peptide for 2 days. Proliferation of NP_366–374-specific CD8⁺ cells was measured by BrdU uptake. The experiment was repeated twice with similar results. *p<0.05, **p<0.01.</p

Relative abundances of different bacterial genera in the salivary glands of (A) <i>I. ovatus</i>, (B) <i>I. persulcatus</i> and (C) <i>H. flava</i>.

<p>All genera with less than 1.0% contribution were pooled into one group and labelled “others”.</p

Phylogenetic analysis of the 16S rDNA sequences of unclassified bacteria from IPf2, IPf3, IPf4, and IPf5 using maximum likelihood method.

<p>The tree is rooted with the <i>Escherichia coli</i>. All bootstrap values from 1000 replications are shown on interior branch nodes.</p

Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens

<div><p>Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (<i>Ixodes ovatus</i>, <i>I. persulcatus</i> and <i>Haemaphysalis flava</i>) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. <i>Rickettsia-</i>specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in <i>I. persulcatus</i> female samples. The numbers of bacterial genera identified for the tick species were 71 (<i>I. ovatus</i>), 127 (<i>I. persulcatus</i>) and 59 (<i>H. flava</i>). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was <i>Coxiella</i>. <i>Spiroplasma</i> was detected in <i>Ixodes</i>, and not in <i>H. flava</i>. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female <i>I. persulcatus</i> samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.</p></div