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Short Communication |
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden, and Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden
Correspondence
Gunilla B. Karlsson Hedestam
Gunilla.Karlsson.Hedestam{at}ki.se
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ABSTRACT |
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Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94270, USA.
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TOPABSTRACTMAIN TEXTREFERENCES |
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, 1999; Fleeton et al., 1999). However, like many other viral vectors, rSFV-based vaccines are relatively poor at inducing humoral immunity in the absence of subsequent booster immunizations with purified protein antigen (Forsell et al., 2005). Heterologous regimens based on priming with viral vectors and boosting with purified protein therefore represent an attractive means to induce both cellular and humoral immune responses (Lubeck et al., 1997; Malkevitch & Robert-Guroff, 2004; Montefiori et al., 1992; Patterson et al., 2004; Shu et al., 2006).
We showed recently that rSFV vectors can drive the expression of soluble HIV-1 envelope glycoprotein (Env) monomers and trimers and that these molecules are recognized by conformation-sensitive antibodies, suggesting that native folding is retained (Forsell et al., 2005). We also showed that rSFV-Env vectors prime Env-directed antibody responses efficiently when followed by a boost with purified matched Env protein antigen (Forsell et al., 2005). One likely explanation for the need of a protein boost is that the amount of antigen produced by replication-defective viral vectors is suboptimal. A vector designed to combine viral adjuvant properties with the ability to elicit cellular responses and to produce high levels of B-cell antigens would therefore be desirable and may reduce the need for subsequent protein boosts. To produce such a vector and to investigate the effect of antigen-expression levels for antibody elicitation, we designed a novel rSFV vector, rSFV-Eiss, that encodes the SFV translation-enhancer element (Sjöberg et al., 1994) upstream of and in frame with an internal signal sequence (iss) to drive the secretion of soluble HIV-1 gp120. The enhancer element has previously been shown to promote translation of downstream sequences under highly restrictive conditions, such as during rSFV-induced host-cell translational shut-off (McInerney et al., 2005; Sjöberg et al., 1994). However, it promotes the increased expression of heterologous antigens only when situated upstream of and in frame with those antigens (Sjöberg et al., 1994), thus preventing the use of standard N-terminal signal sequences to direct the antigen into the endoplasmic reticulum (ER). To overcome this obstacle, we constructed a vector encoding an iss placed downstream of the enhancer element (E) and upstream of gp120. This way, the increased expression provided by the translation-enhancer element is combined with a mechanism allowing native gp120 to be processed correctly in the ER membrane prior to its secretion. Specifically, the rSFV-Eiss-gp120 vector was created by inserting the enhancer element, the first 103 nt of the SFV subgenomic RNA capsid-coding region, into the vector downstream of the subgenomic promoter. The iss, derived from the SFV E1 spike protein (Liljeström & Garoff, 1991) (Fig. 1a), was inserted in frame between the regions encoding the enhancer and gp120 from the primary HIV-1 isolate YU2 (Fig. 1b) (Li et al., 1991).
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), which is more efficient than the native HIV-1 Env signal sequence (Grundner et al., 2005). rSFV-gp120 and rSFV-Eiss-gp120 particles, prepared by using the rSFV split helper system (Smerdou & Liljeström, 1999), were used to infect BHK-21 cells (ATCC) at an m.o.i. of 20. At 12 h post-infection (p.i.), the cells were incubated in methionine-free (starvation) medium for 15 min; they were then pulsed by incubation in starvation medium supplemented with 50 µCi (1.85 MBq) [35S]methionine ml–1 for 15 min and chased in complete medium according to standard procedures (Karlsson & Liljeström, 2004). At various chase times, supernatants from infected cells were collected, and radioactively labelled proteins were separated by SDS-PAGE and analysed by autoradiography. Analysis of the supernatants showed that gp120 was detected after 240 min chase from cells infected with rSFV-gp120. In contrast, secreted gp120 expressed from rSFV-Eiss-gp120 was detected already after a 60 min chase time. After the 240 min chase, cells infected with rSFV-Eiss-gp120 secreted significantly more gp120 than cells infected with rSFV-gp120 (Fig. 1c). For a more thorough comparison of the amounts of gp120 present in the supernatant of the infected cultures, we diluted supernatants from cells infected with rSFV-Eiss-gp120 twofold, fivefold and tenfold and compared them with undiluted supernatant from rSFV-gp120-infected cells (Fig. 1d). Densitometry analysis of the bands (data not shown) demonstrated that approximately tenfold more gp120 was secreted by rSFV-Eiss-gp120-infected cells than by cells infected with rSFV-gp120 after a 240 min chase, consistent with the increase in expression of intracellular proteins reported previously from vectors encoding the SFV enhancer element (Berglund et al., 1998, 2007; Huckriede et al., 2004; Karlsson & Liljeström, 2004; Sjöberg et al., 1994). To examine the difference in gp120 levels secreted by the two vectors by another method, we also analysed non-labelled supernatants harvested at 12 h p.i. for gp120 by using Western blot analysis. This experiment confirmed that rSFV-Eiss-gp120-infected cells produced considerably higher levels of gp120 than cells infected with rSFV-gp120 (Fig. 1e). Furthermore, N-terminal sequencing of the unlabelled secreted gp120 product from rSFV-Eiss-gp120-infected cells confirmed that proteolytic cleavage had occurred after the C-terminal Ala–Arg–Ala (ARA) signal peptidase cleavage motif present in the iss, creating the expected -GNLWVTYYG- N terminus of YU2gp120 (Fig. 1b).
To examine the biosynthesis of gp120 produced from the two vectors in more detail, we analysed the supernatants and cell lysates from an independent pulse–chase experiment after endoglycosidase H (Endo H; Roche) treatment of the labelled proteins. Whereas only one major protein form was detected in the untreated lysates from rSFV-gp120-infected cells (Fig. 2a), two major protein forms were detected in lysates from rSFV-Eiss-gp120-infected cells (Fig. 2b). The lower-mobility form was consistent with fully glycosylated gp120, whilst the higher-mobility form migrated with an apparent molecular mass of about 55 kDa, corresponding to non-glycosylated gp120. The presence of the 55 kDa form is likely because the cells are unable to direct the translocation of all overexpressed nascent polypeptides across the ER membrane. Endo H treatment of cell-associated proteins taken 15 or 60 min after chase indicated that most of gp120 was retained in an immature (high-mannose), fully Endo H-sensitive form, which migrated with an apparent molecular mass of 125 kDa. After 240 min chase, gp120 migrated as a 120 kDa protein, suggesting that some of the glycans had been processed to smaller complex-type oligosaccharides. At this time point, most of the gp120 had been transported into the supernatant and the secreted protein was partially Endo H-resistant, consistent with the presence of both high mannose- and complex-type oligosaccharides on mature gp120, as reported previously (Leonard et al., 1990; Sanders et al., 2002a) (Fig. 2a, right-hand panel). The kinetics of maturation and secretion of gp120 from rSFV-Eiss-gp120-infected cells were similar (Fig. 2b, right-hand panel), demonstrating that even when gp120 is highly overexpressed from the rSFV-Eiss vector, the secreted product retains a biosynthetically mature phenotype.
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; Brimnes et al., 2003; Hidmark et al., 2006; Hutchings et al., 2005; Thompson et al., 2006). BALB/c mice were immunized twice subcutaneously with 100 µl containing 1x107 infectious units (IU) either rSFV-gp120 or rSFV-Eiss-gp120 in PBS, at an interval of 3 weeks. The mice were bled 12 days after the second immunization and the serum was analysed for anti-gp120 reactivity by using an ELISA, as described previously (Forsell et al., 2005). The results show that six of six BALB/c mice immunized with rSFV-Eiss-gp120, but only one of six of mice immunized with rSFV-gp120, had detectable serum antibody responses (Fig. 3a). To quantify the antibody induction in responder sera, we determined ELISA end-point titres. Serum from the only responder in the group immunized with rSFV-gp120 had an end-point titre of 450, whereas the sera from the six rSFV-Eiss-gp120-immunized mice had end-point titres of 12 150, 36 450, 36 450, 109 350, 109 350 and 984 150, respectively (Fig. 3b). Repeat studies confirmed that rSFV-gp120 only induced detectable anti-gp120 antibody responses in a minority of immunized animals (two of a total of 14 mice), whereas rSFV-Eiss-gp120 consistently induced anti-gp120 antibodies upon immunization (13 of a total of 14 mice) and these responses had a titre 1–3 logs higher than the responses in rSFV-gp120-immunized animals.
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). We have shown previously that rSFV immunization stimulates a Th1-biased response, with detectable CD4+ T-cell gamma interferon (IFN-
) production upon antigen restimulation in vitro, but with no detectable production of interleukin-4, as determined by ELISPOT analysis (Forsell et al., 2005). In this study, we found that both vectors induced HIV-1 Env-specific IFN- CD4+ T-cell responses upon immunization and there was no significant difference in the magnitude of the response induced by the two vectors (Fig. 3c). This suggests that antigen levels produced by rSFV-gp120 are not limiting for induction of CD4+ T-cell help. Further analysis of the quality of the immune responses induced by the enhanced rSFV-Eiss vector, including the ability of the vectors to elicit broadly neutralizing antibodies against HIV-1, will require the use of Env immunogens that mimic the functional viral spike better than the monomeric gp120 used here (Barnett et al., 2001; Binley et al., 2000; Earl et al., 1994, 2001; Farzan et al., 1998; Sanders et al., 2002b; Schulke et al., 2002; Yang et al., 2000, 2001, 2002). Thus, neutralizing-antibody responses were not analysed in this study. In conclusion, we show that the rSFV vector system can be modified to encode a translation enhancer inserted in frame with an iss, allowing enhanced expression and secretion, respectively, of mature HIV-1 Env glycoproteins. When the enhanced vector was used to induce anti-Env antibody responses in mice, a significant improvement in antibody titres was observed compared with the responses elicited by the conventional rSFV vector. These data encourage the use of rSFV-Eiss to overcome some of the limitations of the rSFV vector system to induce humoral immune responses. Furthermore, the SFV enhancer element inserted in frame with the iss used here could also be used in some other well-selected viral vector systems to enhance secretion of soluble antigens. Thus, this vector design could be a more broadly applicable means to enhance immune responses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Berglund, P., Quesada-Rolander, M., Putkonen, P., Biberfeld, G., Thorstensson, R. & Liljeström, P. (1997). Outcome of immunization of cynomolgus monkeys with recombinant Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein and challenge with a high dose of SHIV-4 virus. AIDS Res Hum Retroviruses 13, 1487–1495.[Medline]
Berglund, P., Smerdou, C., Fleeton, M. N., Tubulekas, I. & Liljeström, P. (1998). Enhancing immune responses using suicidal DNA vaccines. Nat Biotechnol 16, 562–565.[CrossRef][Medline]
Berglund, P., Fleeton, M. N., Smerdou, C. & Liljeström, P. (1999). Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice. Vaccine 17, 497–507.[CrossRef][Medline]
Berglund, P., Finzi, D., Bennink, J. R. & Yewdell, J. W. (2007). Viral alteration of cellular translational machinery increases defective ribosomal products. J Virol 81, 7220–7229.
Binley, J. M., Sanders, R. W., Clas, B., Schuelke, N., Master, A., Guo, Y., Kajumo, F., Anselma, D. J., Maddon, P. J. & other authors (2000). A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol 74, 627–643.
Boudet, F., Chevalier, M., Jourdier, T. M., Tartaglia, J. & Moste, C. (2001). Modulation of the antibody response to the HIV envelope subunit by co-administration of infectious or heat-inactivated canarypoxvirus (ALVAC) preparations. Vaccine 19, 4267–4275.[CrossRef][Medline]
Brimnes, M. K., Bonifaz, L., Steinman, R. M. & Moran, T. M. (2003). Influenza virus-induced dendritic cell maturation is associated with the induction of strong T cell immunity to a coadministered, normally nonimmunogenic protein. J Exp Med 198, 133–144.
Earl, P. L., Broder, C. C., Long, D., Lee, S. A., Peterson, J., Chakrabarti, S., Doms, R. W. & Moss, B. (1994). Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities. J Virol 68, 3015–3026.
Earl, P. L., Sugiura, W., Montefiori, D. C., Broder, C. C., Lee, S. A., Wild, C., Lifson, J. & Moss, B. (2001). Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140. J Virol 75, 645–653.
Farzan, M., Choe, H., Desjardins, E., Sun, Y., Kuhn, J., Cao, J., Archambault, D., Kolchinsky, P., Koch, M. & other authors (1998). Stabilization of human immunodeficiency virus type 1 envelope glycoprotein trimers by disulfide bonds introduced into the gp41 glycoprotein ectodomain. J Virol 72, 7620–7625.
Fleeton, M. N., Sheahan, B. J., Gould, E. A., Atkins, G. J. & Liljeström, P. (1999). Recombinant Semliki Forest virus particles encoding the prME or NS1 proteins of louping ill virus protect mice from lethal challenge. J Gen Virol 80, 1189–1198.[Abstract]
Forsell, M. N., Li, Y., Sundbäck, M., Svehla, K., Liljeström, P., Mascola, J. R., Wyatt, R. & Karlsson Hedestam, G. B. (2005). Biochemical and immunogenic characterization of soluble human immunodeficiency virus type 1 envelope glycoprotein trimers expressed by Semliki Forest virus. J Virol 79, 10902–10914.
Grundner, C., Li, Y., Louder, M., Mascola, J., Yang, X., Sodroski, J. & Wyatt, R. (2005). Analysis of the neutralizing antibody response elicited in rabbits by repeated inoculation with trimeric HIV-1 envelope glycoproteins. Virology 331, 33–46.[CrossRef][Medline]
Hidmark, A. S., Nordström, E. K., Dosenovic, P., Forsell, M. N., Liljeström, P. & Karlsson Hedestam, G. B. (2006). Humoral responses against coimmunized protein antigen but not against alphavirus-encoded antigens require alpha/beta interferon signaling. J Virol 80, 7100–7110.
Huckriede, A., Bungener, L., Holtrop, M., de Vries, J., Waarts, B. L., Daemen, T. & Wilschut, J. (2004). Induction of cytotoxic T lymphocyte activity by immunization with recombinant Semliki Forest virus: indications for cross-priming. Vaccine 22, 1104–1113.[CrossRef][Medline]
Hutchings, C. L., Gilbert, S. C., Hill, A. V. & Moore, A. C. (2005). Novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity. J Immunol 175, 599–606.
Karlsson, G. B. & Liljeström, P. (2004). Delivery and expression of heterologous genes in mammalian cells using self-replicating alphavirus vectors. Methods Mol Biol 246, 543–557.[Medline]
Leonard, C. K., Spellman, M. W., Riddle, L., Harris, R. J., Thomas, J. N. & Gregory, T. J. (1990). Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J Biol Chem 265, 10373–10382.
Li, Y., Kappes, J. C., Conway, J. A., Price, R. W., Shaw, G. M. & Hahn, B. H. (1991). Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes. J Virol 65, 3973–3985.
Liljeström, P. & Garoff, H. (1991). Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor. J Virol 65, 147–154.
Lubeck, M. D., Natuk, R., Myagkikh, M., Kalyan, N., Aldrich, K., Sinangil, F., Alipanah, S., Murthy, S. C., Chanda, P. K. & other authors (1997). Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization. Nat Med 3, 651–658.[CrossRef][Medline]
Malkevitch, N. V. & Robert-Guroff, M. (2004). A call for replicating vector prime-protein boost strategies in HIV vaccine design. Expert Rev Vaccines 3, S105–S117.[CrossRef][Medline]
McInerney, G. M., Kedersha, N. L., Kaufman, R. J., Anderson, P. & Liljeström, P. (2005). Importance of eIF2
phosphorylation and stress granule assembly in alphavirus translation regulation. Mol Biol Cell 16, 3753–3763.
Montefiori, D. C., Graham, B. S., Kliks, S. & Wright, P. F. (1992). Serum antibodies to HIV-1 in recombinant vaccinia virus recipients boosted with purified recombinant gp160. NIAID AIDS Vaccine Clinical Trials Network. J Clin Immunol 12, 429–439.[CrossRef][Medline]
Patterson, L. J., Malkevitch, N., Venzon, D., Pinczewski, J., Gomez-Roman, V. R., Wang, L., Kalyanaraman, V. S., Markham, P. D., Robey, F. A. & Robert-Guroff, M. (2004). Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting. J Virol 78, 2212–2221.
Sanders, R. W., Venturi, M., Schiffner, L., Kalyanaraman, R., Katinger, H., Lloyd, K. O., Kwong, P. D. & Moore, J. P. (2002a). The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J Virol 76, 7293–7305.
Sanders, R. W., Vesanen, M., Schuelke, N., Master, A., Schiffner, L., Kalyanaraman, R., Paluch, M., Berkhout, B., Maddon, P. J. & other authors (2002b). Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol 76, 8875–8889.
Schulke, N., Vesanen, M. S., Sanders, R. W., Zhu, P., Lu, M., Anselma, D. J., Villa, A. R., Parren, P. W., Binley, J. M. & other authors (2002). Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein. J Virol 76, 7760–7776.
Shu, Y., Winfrey, S., Yang, Z. Y., Xu, L., Rao, S. S., Srivastava, I., Barnett, S. W., Nabel, G. J. & Mascola, J. R. (2006). Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs. Vaccine 25, 1398–1408.[CrossRef][Medline]
Sjöberg, E. M., Suomalainen, M. & Garoff, H. (1994). A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Biotechnology (N Y) 12, 1127–1131.[CrossRef][Medline]
Smerdou, C. & Liljeström, P. (1999). Two-helper RNA system for production of recombinant Semliki Forest virus particles. J Virol 73, 1092–1098.
Thompson, J. M., Whitmore, A. C., Konopka, J. L., Collier, M. L., Richmond, E. M., Davis, N. L., Staats, H. F. & Johnston, R. E. (2006). Mucosal and systemic adjuvant activity of alphavirus replicon particles. Proc Natl Acad Sci U S A 103, 3722–3727.
Yang, X., Farzan, M., Wyatt, R. & Sodroski, J. (2000). Characterization of stable, soluble trimers containing complete ectodomains of human immunodeficiency virus type 1 envelope glycoproteins. J Virol 74, 5716–5725.
Yang, X., Wyatt, R. & Sodroski, J. (2001). Improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers. J Virol 75, 1165–1171.
Yang, X., Lee, J., Mahony, E. M., Kwong, P. D., Wyatt, R. & Sodroski, J. (2002). Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin. J Virol 76, 4634–4642.
Received 5 April 2007;
accepted 6 June 2007.
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), whereas the Endo H-treated immature gp120 migrates with an apparent molecular mass of 55 kDa. After 240 min chase, some of the gp120 is mature and migrates with an apparent molecular mass of 120 kDa (
), which is partially Endo H-sensitive (
). Most of the mature gp120 is found in the culture supernatant.
) or rSFV-Eiss-gp120 (