Comparing Pooled Peptides with Intact Protein for Accessing Cross-presentation Pathways for Protective CD8+ and CD4+ T Cells

Abstract

To better understand the mechanisms of intracellular trafficking and presentation of exogenous peptides by antigen-presenting cells (APC), we compared the handling of overlapping 24-mer peptides from HIV Nef either mixed or covalently linked in tandem in one protein. Once internalized, peptides trafficked not only to endosomes but also to cytosol, and activated CD8+ and CD4+ T cells. In contrast, whole protein was found to traffic only to the endosomal compartments, and primarily activated CD4+ T cells. Finally, with adjuvant, overlapping peptides were capable of protecting against lethal viral challenge, whereas the intact protein was less protective. These data suggest that overlapping long peptides are cross-presented through more varied intracellular routes and are more efficient in priming protective immunity than the whole protein. To better understand the mechanisms of intracellular trafficking and presentation of exogenous peptides by antigen-presenting cells (APC), we compared the handling of overlapping 24-mer peptides from HIV Nef either mixed or covalently linked in tandem in one protein. Once internalized, peptides trafficked not only to endosomes but also to cytosol, and activated CD8+ and CD4+ T cells. In contrast, whole protein was found to traffic only to the endosomal compartments, and primarily activated CD4+ T cells. Finally, with adjuvant, overlapping peptides were capable of protecting against lethal viral challenge, whereas the intact protein was less protective. These data suggest that overlapping long peptides are cross-presented through more varied intracellular routes and are more efficient in priming protective immunity than the whole protein. Despite much progress in vaccine development, there are still several challenges for design of subunit vaccines. Against intracellular pathogens, it is especially important to immunize protective CD4+ and CD8+ T cell responses. The former recognize epitopes presented by major histocompatibility complex (MHC) 3The abbreviations used are: MHC, major histocompatibility complex; pAPC, professional antigen-presenting cell; CFA/IFA, complete/incomplete Freund's adjuvant; ROP, recombinant overlapping-peptide protein; ppROP, protease-processed ROP (peptides); DC, dendritic cells; vv-Nef, vaccinia virus expressing Nef; HIV, human immunodeficiency virus; MALDI, matrix-assisted laser desorption ionization; IFN, interferon; Ni-NTA, nickelnitrilotriacetic acid. class II molecules that are normally loaded mainly with peptides derived from exogenous antigens. Natural processing and loading are catalyzed by HLA- or H2-DM in acidic lysosomes (1Kropshofer H. Hammerling G.J. Vogt A.B. Immunol. Rev. 1999; 172: 267-278Crossref PubMed Scopus (74) Google Scholar). By contrast, CD8+ T cell responses typically depend on epitope loading via the class I pathway. This “endogenous” route starts from the cytosol of any cells naturally infected by the relevant virus, which may not include professional antigen presenting cells (pAPCs). Once processed by cytosolic proteases, viral peptide fragments are transported into the endoplasmic reticulum, and loaded onto MHC class I molecules, which then traffic to the surface where they can present to CD8+ T cells (2Norbury C.C. Hewlett L.J. Prescott A.R. Shastri N. Watts C. Immunity. 1995; 3: 783-791Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 3Huang A.Y. Bruce A.T. Pardoll D.M. Levitsky H.I. Immunity. 1996; 4: 349-355Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). For exogenous antigens, “cross-presentation” by pAPCs is essential for priming CD8+ as well as CD4+ T cells. Although they must be crucial for most CD8+ T cell vaccination strategies, the mechanisms underlying cross-presentation are not understood. First, internalized antigens are known to enter the cytoplasm, although the mechanisms remain unknown. Once in the cytoplasm, they can follow the conventional MHC class I pathway, i.e. processing by cytosolic proteasomes or proteases, transport into the endoplasmic reticulum, loading onto MHC class I molecules, and then trafficking to the surface for recognition by T cells (2Norbury C.C. Hewlett L.J. Prescott A.R. Shastri N. Watts C. Immunity. 1995; 3: 783-791Abstract Full Text PDF PubMed Scopus (357) Google Scholar, 3Huang A.Y. Bruce A.T. Pardoll D.M. Levitsky H.I. Immunity. 1996; 4: 349-355Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Second, antigens internalized into endocytic compartments could be degraded by the local proteases into peptides and then loaded onto MHC class I molecules in the endocytic compartments that are recycled to and from the cell surface (4Gromme M. Uytdehaag F.G. Janssen H. Calafat J. van Binnendijk R.S. Kenter M.J. Tulp A. Verwoerd D. Neefjes J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10326-10331Crossref PubMed Scopus (239) Google Scholar). Third, endosomes may fuse with the endoplasmic reticulum, allowing direct access of such peptides for loading onto nascent MHC class I molecules there before presentation at the cell surface (5Gagnon E. Duclos S. Rondeau C. Chevet E. Cameron P.H. Steele-Mortimer O. Paiement J. Bergeron J.J. Desjardins M. Cell. 2002; 110: 119-131Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar); however, this theory remains controversial (6Touret N. Paroutis P. Terebiznik M. Harrison R.E. Trombetta S. Pypaert M. Chow A. Jiang A. Shaw J. Yip C. Moore H.P. van der Wel N. Houben D. Peters P.J. de Chastellier C. Mellman I. Grinstein S. Cell. 2005; 123: 157-170Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 7Groothuis T.A. Neefjes J. J. Exp. Med. 2005; 202: 1313-1318Crossref PubMed Scopus (59) Google Scholar). It has been hypothesized that endocytic and cytosolic proteases process antigens differently. If so, exogenous antigens that have been processed in the endocytic compartments in un-infected APC might yield epitopes distinct from those processed in the cytosol of infected cells (8Cresswell P. Ackerman A.L. Giodini A. Peaper D.R. Wearsch P.A. Immunol. Rev. 2005; 207: 145-157Crossref PubMed Scopus (338) Google Scholar). It is not yet clear how to optimize antigens for cross-presentation. Although intact proteins are preferred to nonamer epitopes (9Norbury C.C. Basta S. Donohue K.B. Tscharke D.C. Princiotta M.F. Berglund P. Gibbs J. Bennink J.R. Yewdell J.W. Science. 2004; 304: 1318-1321Crossref PubMed Scopus (249) Google Scholar), there is recent evidence that long 15–20-mer peptides are even more suitable (10Jiang S. Borthwick N.J. Morrison P. Gao G.F. Steward M.W. J. Gen. Virol. 2002; 83: 429-438Crossref PubMed Scopus (35) Google Scholar, 11Serna A. Ramirez M.C. Soukhanova A. Sigal L.J. J. Immunol. 2003; 171: 5668-5672Crossref PubMed Scopus (48) Google Scholar). However, it is not known whether long peptides and intact proteins access the same intracellular route(s) for cross-presentation. Here we designed constructs to enable us to address that issue and test whether these differences are important for eliciting protective immune responses in vivo. Because an intact protein usually contains multiple epitopes, it would not be fair to compare a whole protein with a single peptide. It is also not ideal to compare a set of overlapping peptides with their correspondent native protein because overlapping peptides, which include extra overlapped sequences, may contain relatively more epitopes than their correspondent native protein. Consequently, we engineered and expressed a protein with 20 tandem-linked overlapping long peptides that could be separated by clipping with protease Factor Xa. We compared the recombinant protein with the enzymically cleaved overlapping peptides from the same protein. Previous publications have shown that overlapping synthetic peptides are capable of generating immune responses (12Jiang S. Song R. Popov S. Mirshahidi S. Ruprecht R.M. Vaccine. 2006; 24: 6356-6365Crossref PubMed Scopus (15) Google Scholar, 13Vambutas A. DeVoti J. Nouri M. Drijfhout J.W. Lipford G.B. Bonagura V.R. van der Burg S.H. Melief C.J. Vaccine. 2005; 23: 5271-5280Crossref PubMed Scopus (93) Google Scholar). Our recombinant overlapping peptide protein (ROP) covers the complete sequence of HIV Nef. We compared its uptake and immunogenicity either intact or clipping into its constituent 24-mers, and tested whether the resulting responses can protect animals against lethal viral challenge. DNA Design-A synthetic gene was designed to include a His tag and a series of 20-mer peptides, each overlapping by 10 amino acids, and separated by IEGR clip sites for the protease Factor Xa, it covers the full 206 amino acids of Nef from the Lai strain of human immunodeficiency virus (HIV) (Fig. 1A). The resulting construct was optimized for Escherichia coli expression, and restriction enzyme sites NdeI and BamHI were added at the ends for subsequent subcloning and expression in the pET16b vector. The gene was commercially synthesized by GeneArt, Regensburg, Germany. Subcloning of the Target Gene into the pET16b Expression Vector-The synthesized target gene, provided in cloning vector pPCR-Script, was restriction-digested with NdeI and BamHI. The DNA was gel-purified using QIAquick® Gel Extraction kit (Qiagen, Germany). Meanwhile, the pET vector was digested with the same restriction enzymes, and the ends were de-phosphorylated with shrimp alkaline phosphatase (Roche). The vector was then gel-purified as described above. The target gene was ligated into vector DNA with T4 DNA ligase (New England Biolabs, UK), and the ligation product was transformed into DH5α bacteria. Colonies were inoculated from overnight LB agar plate cultures and incubated in LB medium with 100 mg/ml ampicillin. After overnight culture, plasmid DNA was extracted by the QIAprep Spin Miniprep Kit (Qiagen, Germany), and sequenced to confirm the presence of the correct insert. Protein Expression-Miniprep DNA was transformed into BL21(DE3) bacteria, and colonies were inoculated and grown in 30 ml of low salt LB medium overnight at 37 °C. The culture was 1:30 diluted in low salt LB medium and grown at 37 °C with shaking for 2–3 h, when the bacteria reached exponential growth stage, as determined by A600 = 0.6–1.0. 1 mm Isopropyl 1-thio-β-d-galactopyranoside was added to induce protein expression. After a 4-h shaking at 28 °C, bacteria were pelleted at 4000 rpm, and frozen at -70 °C until further extraction and purification, when they were re-suspended in cold phosphate-buffered saline and lysed by sonication. The inclusion bodies, which contain the expressed protein, were washed with 1:100 B-Per solution (Pierce), pelleted at 15,000 × g for 20 min, and extracted with 8 m urea in 50 mm Tris buffer (pH 7.8) containing 500 mm NaCl. Protein Purification-The protein was further purified by nickel affinity chromatography using Ni-NTA purification kit (Qiagen, Germany) according to the manufacturer’s operation manual. After the purification, the eluting buffer containing imidazol was exchanged to 25 mm ammonium bicarbonate buffer by extensive dialysis overnight at 4 °C. The purified protein (1.5–2 mg/ml) was either lyophilized for further use or digested into peptides. Protease Digestion-The purified protein in 25 mm ammonium bicarbonate buffer containing 5 μm CaCl2 was incubated with protease Factor Xa (Qiagen, UK) at 1 mg of protein/1 unit of protease at 4 °C for around 120 min. The digestion was stopped by adding 1 μm phenylmethylsulfonyl fluoride, and the protease was removed in the YM10 filter tube (Millipore, Bedford, MA). The free His tag and the trace salts were removed on Ni-NTA-agarose and C18 columns (Waters, Milford, MA). The resulting mixture of the proteinase-processed recombinant overlapping peptides (ppROP) was lyophilized for mass spectrometry analysis and future experiments. Mass Spectrometer Analysis-The protease-digested sample was analyzed on a Bruker Daltonics Ultraflex TOF/TOF mass spectrometer to confirm the identity of the digested peptides. All MALDI spectra were obtained in reflectron mode. A nitrogen laser, emitting 337 nm light in 3-ns pulse, was the ionization source. The accelerating voltage in the ion source was 30 kV. For MALDI analysis, the peptide mixture was purified and desalted on a C18 column. About 10 μm peptide solution was premixed with the matrix: α-cyano-4-hydroxycinnamic acid (10 mm in 35% aqueous acetonitrile, 0.1% trifluoroacetic acid) at a 1:1 ratio and 1 μl of mixture to the sample The was before analysis in the mass were using kit the in the than in the is and or were with of Nef antigens with 100 μl of complete or or were at with the same vaccine with the were for intracellular challenge, and were with All were in with local and or were also with of Nef antigens with an of A cell However, this is from the of in or cells from and were washed and in 10 ml of medium containing UK) in culture for at 10 ml of medium was added to the 10 ml of culture medium were by 10 ml of containing medium until they were on and were using were overnight with 10 μm ppROP, ROP, or peptides in (Millipore, Bedford, and with overlapping synthetic peptides the Lai or Nef for were and were added for at by 1 of at with with After the was stopped by with and were were with an Germany). were expressed as cells. For intracellular were at 5 × with or 10 μm overlapping synthetic peptides in culture for before cells were with according to the were then with or or an for 20 min. were then to intracellular using the kit and according to the manufacturer’s were on a and data were analyzed using or cells were with 5 μm or ROP at The were by before with or at a ratio of overnight at 37 °C in was on the The were incubated 10 of A 1 before and with or and cell was provided by of J. Immunol. PubMed Google Scholar). For of or ROP, we used 500 μl mg/ml) of or ROP were incubated with μl of at for 30 min. The and ROP can then be at °C before To the uptake of or ROP by the we incubated 1 × ml of in culture Bedford, overnight at 37 °C, before the with or ROP, 50 μl of diluted was added to the cell The culture was washed 5 with phosphate-buffered saline and to μl of with before adding μl of or was added as a After of at 37 °C in cells were washed and with The cells were analyzed either by or as by on a and with a from of C. M. PubMed Scopus Google Scholar), were grown in cells. were with or as above. the were with × of The of the was for 8 and of the ROP was designed to yield cleaved 20-mer peptides by the complete HIV Nef sequence each peptide is a clip for Factor Xa protease (Fig. which to be each with a IEGR sequence not found in Nef the there is a tag by the clip to in protein of overlapping peptides used in this in a The gene ROP was transformed into E. coli cells and expressing were After expression, the ROP protein was extracted from inclusion and purified by affinity chromatography (Fig. It then as a on but as a and on (Fig. the protein is present as and of the ROP by Factor Xa the constituent overlapping peptides, as by mass spectrometry (Fig. In there were that may have from by Factor Xa. For (Fig. may have from of the Factor Xa may have cleaved from its were for and in in the uptake ROP and by they were incubated with the dendritic cell J. Immunol. PubMed Google in First, they were with the cell kit from which covalently to free amino with a and the was The cells were with to then the cells were with Finally, the ROP or were incubated with the cells that were for and ppROP, was only 5 were found and lysosomes before (Fig. 120 these peptides were in the lysosomes (Fig. but only of peptides were with lysosomes a of by 120 These data that there is a of peptides not in the ROP, by contrast, intracellular was only min, whereas the protein even with (Fig. h, the ROP was still in cells (Fig. This was complete from until at ROP is internalized much more than ppROP, and of it to the whereas to access more compartments, the The more intracellular of may a than with The uptake of was when incubated with not whether and ROP can be presented by cells were at 37 °C with or ROP for 1 by After the cells were incubated overnight with from or that and from and only responses from (Fig. was the were incubated with A to and with (Fig. This was with and a was not These data are further evidence that for efficient with in in of tested whether were to T cell responses in and against overlapping synthetic peptides or (Fig. were by In with responses than of the mixture of overlapping synthetic peptides was in (Fig. A and but their epitope were and were the peptides by whereas cells only peptides and (Fig. A and The of was for the Lai and peptides in but were much in even the amino acids differences were and to the of peptides and (Fig. and a in and it with at one viral T cells from to ROP in to the native HIV Nef protein in as in This that with overlapping peptides is to immunity against epitopes processed from the protein. To whether or T cells from to the CD4+ or CD8+ we tested T cells to peptides and by intracellular (Fig. CD4+ and CD8+ T cells from responses (Fig. and for where the was only for CD4+ T cells (Fig. In CD4+ T cell responses were also against and than in the (Fig. A and but they were less than in For CD8+ they were only against (Fig. of with against whether the immunity in by is we as with a lethal of Because HIV, and not we used × of vaccinia virus expressing Nef of the with the of the or and only of those = (Fig. The in the was not than This that immunity by vaccination with could protect against a lethal viral challenge, and that this was on the in the We that recombinant overlapping peptides (ppROP) can access the intracellular for cross-presentation much more than the intact ROP protein. the of priming in with the overlapping peptides CD8+ and CD4+ T cell responses that protect animals from lethal viral challenge. there is the cytosol of viral antigens epitopes presented through the MHC I to be expressed on the surface of the same However, in not which are essential for priming CD8+ and CD4+ T cells. pAPCs have to antigens in from exogenous such as infected or peptides in these antigens would be into the cytosol to access the pathway, the of antigen presentation would that of infected cells. 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We found that they intracellular peptides were in into the endosomes and compartments the cytosol, and proteins were in only in the and (Fig. A and and The of this is that long peptides may through MHC class I and class II to CD8+ and CD4+ T whereas protein may mainly CD4+ T cells. with these peptides immune responses that CD8+ and CD4+ T cells (Fig. whereas with the protein not immune responses. can present and ROP to whereas This that the protein antigen is not efficient in priming T cells (Fig. and but not priming with in immune responses in of the of the responses in the of was but the epitope were distinct (Fig. A and The T cells also to Nef or ROP These the recombinant overlapping peptides as vaccine that can be to animals and with for of MHC with responses to epitopes processed naturally from the Nef protein that against viral challenge. 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