that were coated with a mixture of anti-gp120 monoclonal antibodies ID6, AD3, and AC4 (12) and cloned. Expression of gpl20 and gp4l was determined by ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4156-4160, May 1993 Immunology
Gene inoculation generates immune responses against human immunodeficiency virus type 1 BIN WANG*, KENNETH E. UGENt, VASANTHA SRIKANTAN*, MICHAEL G. AGADJANYANt, KESEN DANG*, YOSEF REFAELI*, ALICE I. SATO*, JEAN BOYER*, WILLIAM V. WILLIAMS*, AND DAVID B. WEINER*tt *Institute of Biotechnology and Advanced Molecular Medicine in the Department of Medicine, University of Pennsylvania School of Medicine, and tThe Wistar Institute, 3600 Spruce Street, Philadelphia, PA 19104
Communicated by Robert Austrian, January 14, 1993 (received for review October 6, 1992)
ABSTRACT Recently, immunization techniques in which DNA constructs are introduced directly into mammalian tissue in vivo have been developed. In theory, gene inoculation should result in the production of antigenic proteins in a natural form in the immunized host. Here we present the use of such a technique for the inoculation of mice with a human immunodeficiency virus type 1 (HIV-1) envelope DNA construct (pM160). Mice were injected intramuscularly with pM160 and were subsequently analyzed for their anti-HIV envelope immune responses. Antisera collected from inoculated animals reacted with the recombinant HIV-1 envelope in ELISA and immunoprecipitation assays. The antisera also contained antibodies that were able to neutralize HIV-1 infection and inhibit HIV-1-mediated syncytium formation in vitro. Furthermore, splenic lymphocytes derived from pM160-inoculated animals demonstrated HIV-envelope-specific proliferative responses. The gene inoculation technique mimics features of vaccination with live attenuated viruses and, therefore, may ultimately prove useful in the rapid development of safe and efficacious vaccines as it provides for production of relevant antigen in vivo without the use of infectious agents.
MATERIALS AND METHODS Plasmid Construction. The envelope gene of the HIV-1 HXB2 isolate was cloned by polymerase chain reaction (PCR) amplification utilizing a A phage clone obtained from the AIDS Research and Reference Reagent Program (Rockville, MD; ref. 10). The sequences of the 5' and 3' primers are
5'-AGGCGTCTCGQAGACAGAGGAGAGCAAGAAATG-3'
(Xho I site, underlined) and 5'-TTTCCCTCTAGATAAGCCATCCAATCACAC-3' (Xba I site, underlined), respectively, and encompass the gpl60, tat, and rev coding regions. PCR products were digested with Xho I and Xba I, cloned into the Bluescript plasmid (Stratagene), subcloned into the eukaryotic expression vector pMAMneoBlue (Clontech), and designated pM160. The plasmid DNA was purified by CsCl gradient ultracentrifugation. Expression of HIV gpl60 by pM160. Transfection of pM160 into TE671 cells was performed as described (11). Cells expressing the gp160 envelope protein were isolated by binding to M450 magnetic beads (DYNAL, Great Neck, NY) that were coated with a mixture of anti-gp120 monoclonal antibodies ID6, AD3, and AC4 (12) and cloned. Expression of gpl20 and gp4l was determined by Western blot analysis of whole cell lysates of pM160-transfected cells. Gene Inoculation. To enhance muscle cell uptake of plasmid DNA, mice were treated as described by Thomason and Booth (13) with modifications. Specifically, the quadriceps muscles of BALB/c mice were injected with 100 ,ul of 0.5% bupivacaine hydrochloride and 0.1% methylparaben in isotonic NaCl by using a 27-gauge needle. Twenty-four hours after bupivacaine injection, 100 ,ug of the DNA construct was injected into the same site. DNA construct (100 ,ug) was injected biweekly for a total of four inoculations. Recombinant gpl60 Immunization. BALB/c mice were immunized with 1 jig of glycosylated recombinant (r) (HIV1/IIIB) gpl60 (MicroGeneSys, Meriden, CT) in complete Freund's adjuvant followed by three booster injections of 1 ,ug of gpl60 in incomplete Freund's adjuvant at 2-week intervals. Immunoprecipitation. Immunoprecipitation was performed with 1 x 106 cpm of 1251-labeled rgpl60, mouse sera, and protein G-agarose beads (GIBCO) as described (14). The specific precipitates were analyzed by SDS/PAGE on 10% gels and autoradiography on a Kodak XRP film. ELISA Binding to rgpl60 and Peptides. rgpl60 was obtained from MicroGeneSys. Peptides were synthesized as described (14) or obtained from the AIDS Research and Reference Reagent Program. The peptides used in these studies were as follows: HXB2 site implicated in CD4 binding
Recently, direct injection of a normal functional gene into a living animal has been shown to be of great promise for gene therapy (1-3). Direct inoculation of the gene(s) of a pathogen should mimic attenuated vaccines as synthesis of specific foreign proteins would be achieved in the host and would be the target of immune surveillance. A modest level of gene expression was demonstrated as the result of intramuscular injection of the human growth hormone gene and the human dystrophin gene in mice (2, 3), and the gene product in some circumstances can elicit antibody-based immune responses (4). However, it has not been determined whether T-cell responses are elicited or whether relevant immune responses to a human pathogen can be elicited. The human immunodeficiency virus (HIV) is responsible for the AIDS pandemic. Intensive investigation of the immunobiology of HIV has generated a wealth of data regarding the relevant specific immune responses that in vitro appear to correlate with virus inactivation (for review, see refs. 5 and 6). Studies with simian immunodeficiency virus, the related primate retrovirus, have generated insight with regard to relevant protective immune responses in vivo (7-9). Accordingly, the HIV system is a useful model for the analysis of immune responses generated through genetic inoculation techniques. We have analyzed immune responses generated by the intramuscular injection of mice with a HIV-1 envelope gpl60 DNA construct. We demonstrate that this DNA construct is capable of eliciting specific and neutralizing immune responses and appears to include the immune responses desired in a putative vaccine for this human pathogen.
Abbreviations: HIV-1, human immunodeficiency virus type 1; r, recombinant. :To whom reprint requests should be addressed at: Institute of Biotechnology and Advanced Molecular Medicine at the University of Pennsylvania, 570 Maloney, 3600 Spruce Street, Philadelphia, PA
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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(CRIKQFINMWQEVGKAMYAPPISGIRC); BRU/V3 loop peptide (CNTRKRIRIQRGPGRAFVTIGK); MN/V3 loop peptide (YNKRKRIHIQRGPGRAFYTTKNIIC) with the QR sequence from HIV-1/IIIB underlined; Z6/V3 loop peptide (CNTRQSTPIGLGQALYTTRGRTK); gp41(F560) peptide (CIVQQQNNLLRAIEAQQHLLQLTVWGIKQL); and BRU/gp4l immunodominant region peptide (RILAVERYLKDQQLLGIWGCSGKLIC). ELISA were performed as described (12). Neutralization Assay. Neutralization was performed by using 100 tissue culture 50% infective doses (TCID50) of HIV-1/IIIB cell-free virus and the MT-2 cells as the target cells (15). The neutralization value is calculated as described (16). Syncytial Formation Assay. Syncytium inhibition assays were performed as described (17) by cocultivation of the H9/IIIB cell line with MT-2 target cells. Lymphocyte Proliferation Assay. This assay was performed as described (18). RESULTS Inoculation with pM160 Induces Humoral Immunity. Sera collected from mice after four DNA inoculations were compared with sera from mice receiving an equal number of recombinant protein inoculations. Sera from 8 of 10 mice injected with the pM160 construct (Fig. 1) reacted with rgp160 as determined by ELISA (Figs. 2A and 3A). All the mice that responded to pM160 inoculation developed qualitatively similar responses. To evaluate the potential efficacy of pM160 inoculation, the immune responses from the animal with the highest anti-gpl60 titer were analyzed in detail in comparison with preimmune serum from the same animal. Sera of four mice inoculated with the vector control showed no reactivity with rgpl60 in an ELISA; one of these sera was used as an additional control in subsequent experiments. 125I-labeled gpl60 was specifically precipitated with antisera derived from the pM160-injected animal (Fig. 2B, lane 2) and with the positive control anti-gp120 monoclonal antibody ID6 (12) (Fig. 2B, lane 3) and antiserum from the rgpl6Oinoculated mouse (data not shown). In contrast, the preimmune serum (Fig. 2B, lane 1) showed minimal activity. HIV-neutralizing antibodies are specifically targeted to several epitopes of gpl20 and gp4l (for review, see ref. 5). To determine whether the anti-gpl60 antibodies elicited in these mice are reactive with diverse regions of the envelope glycoprotein, ELISA peptide mapping was performed. Fig. 3 shows that antiserum from the mouse injected with pM160 has reactivity to the peptides corresponding to the BRU (C), MN (D), and Z6/V3 (E) loops, the site implicated in CD4 binding (B), F560 (F), and the immunodominant region of gp4l (G) compared with either preimmune serum or sera from mice immunized with rgpl6O protein. Interestingly, the taVrev SV40 pAIP. gpl160 g
X~~~~~~eo
pM160
taVrev
11.00 Kb
SV40 polyA
MMTV LTR
RS
Ori
FIG. 1. HIV gp160 envelope DNA construct (pM160). SV40, simian virus 40; Neo, neomycin-resistance gene; Amp, ampicillinresistance gene; RSV, Rous sarcoma virus; MMTV, mouse mammary tumor virus; LTR, long terminal repeat.
A
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FIG. 2. Humoral immune responses after gene inoculation. (A) Antisera from 10 mice inoculated by injection of pM160 were tested for binding to rgpl60 by an ELISA. Pooled normal mouse sera (NMS) were used as a control. The A450 mean value at a 1:50 dilution of the gene-inoculated group (solid squares connected by the solid line) is 3 standard deviations above the control group (crosses connected by a dashed line). The ELISA readings for individual inoculated mice are also shown. (B) Immunoprecipitation of rgpl60 by pM160-inoculated mouse serum. Lanes: 1, 1 gl of preimmune mouse serum incubated with the 1251-labeled gp160; 2, 1 ,ul of mouse serum inoculated from the pM160-inoculated mice; 3, 1 ,ul of 1:100 dilution of ID6, an anti-gp120 monoclonal antibody (positive control). The arrow indicates the specifically immunoprecipitated 1251labeled gp160 envelope glycoprotein.
antisera from the rgpl6O-immunized mice had higher binding to the rgpl60 and the N-terminal region of gpl20 (Fig. 3 A and H) than the antiserum from pM160-injected mice. This finding supports qualitative and quantitative differences in the anti-HIV envelope humoral immune response(s) elicited by the pM160 inoculation vs. rgpl60 inoculation protocol. Immunoglobulin isotyping studies of the gpl60-specific antibodies elicited by pM160 inoculation showed 19% IgGl, 51% IgG2, 16% IgG3, 10% IgM, and 5% IgA. The predominance of IgG isotypes indicates a secondary immune response and suggests helper T cells are stimulated by gene inoculation. Cellular Immune Responses Elicited by pM160 Inoculation. Immune spleen cell populations were examined for prolifer-
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FIG. 3. Antibody binding to peptides representing rgpl60 epitopes. ELISA assay was as described (12). Antisera were as follows: +, pre-
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serum;
x,
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inoculated serum; o, pM160-inoculated serum; a, serum from mice immunized with the rgpl60 protein. The wells were coated with the following antigens: (A)
0.20-
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ation upon specific antigen stimulation (Fig. 4). Lymphocytes from pM160-inoculated mice proliferated in response to recombinant gp120 glycoprotein (rgpl20). There was no significant proliferative response in the absence of antigen or to an irrelevant antigen (peptide C). Lymphocytes from the vector-inoculated mice also did not respond to the rgp120. Anti-Viral Activity of pM160 Immune Serum. We analyzed the ability of the antiserum dilutions to neutralize HIV-1 infection in vitro (16). Neutralization was not observed in cells infected with cell-free HIV-1/IIIB virus preincubated with preimmune serum (Fig. 5) or antiserum obtained from the vector-injected control mice (data not shown). In comparison, preincubation with four of five sera from pM160inoculated mice neutralized infectivity in vitro. Serum from the pM160-inoculated mouse (but not preimmune serum) demonstrated neutralizing activity at dilutions of up to 1:1280 (Fig. 5B).
1:10000
rgpl60 protein. (B) Peptide derived
from the HXB2 site implicated in CD4 binding. (C) BRU/V3 loop peptide. (D) MN/V3 loop peptide. (E) Z6/V3 loop peptide. (F) Fusogenic region of gp4l(F560) peptide. (G) BRU/gp4l immunodominant region peptide. (H) Peptide derived from amino acids 89-98 of the HXB2 gp120 sequence.
Syncytial inhibition assays were also performed. The preimmune sera (Fig. 6A), antisera from the rgpl60-inoculated mice (Fig. 6B) and antisera from the control vectorinoculated animals (Fig. 6C) failed to inhibit syncytium formation. Antiserum from the pM160-inoculated mouse inhibited HIV-1-induced syncytium formation at a dilution of 1:200 (Fig. 6D). Observations from the neutralization and syncytium inhibition assays demonstrate that the anti-viral activities of the sera correlate with the observed ELISA reactivities against gp160 (Fig. 3). HIV entry into cells is initiated by binding of gp120 to the CD4 molecule (20, 21). Interruption of this binding prevents HIV infection in vitro (5). To test the ability of the pM160inoculated mouse sera to inhibit gpl20 binding to CD4bearing T cells, direct inhibition monitored by flow cytometry was performed exactly as described by Chen et al. (22). A
Immunology: Wang et al. 60000
Proc. Natl. Acad. Sci. USA 90 (1993)
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FIG. 4. Lymphocyte proliferative responses to rgpl2O. Splenocytes pooled from three mice injected with pM160 and two mice injected with control vector were used in proliferative assays. Peptide C, an irrelevant peptide served as a negative control antigen. Anti-CD3 monoclonal antibody 2C11 (19) served as a positive control. The mean cpm incorporated of duplicate samples is shown for each antigen tested.
FIG. 6. Antisyncytial activity. The H9/IIIB cell line was preincubated with a 1:200 dilution of antiserum and then incubated with MT-2 target cells. Fusion was evaluated 3 days later. (A) Target cells cocultivated with HIV-1/IIIB cell line treated with preimmune serum. (B) The same as A but treated with rgpl60-immunized serum. (C) The same as A but treated with vector-control-inoculated serum. (D) The same as A but treated with pM160-inoculated serum.
1:15 dilution of immune serum from the pM160-constructinjected mouse inhibited fluorescein isothiocyanateconjugated gpl20 binding to CD4+ SupTl cells by 22 + 2% in replicate experiments (e.g., the change in mean channel number was decreased from 61.5 to 47.4 by a 1:15 dilution and to 38 by a 1:5 dilution of pM160-inoculated mouse serum in one representative experiment). This indicates that a conformational region for HIV binding to target cells can be functionally recognized by this antiserum.
DISCUSSION Gene Inoculation Elicits Humoral and Cellular Immunity. We observed that gene inoculation by direct injection of DNA into muscle elicited antigen-specific cellular and humoral immune responses. We used bupivacaine pretreatment to stimulate muscle cell regeneration and to increase DNA uptake by the cells (13). In spite of this maneuver, 2 of 10 animals failed to develop a detectable humoral immune response (Fig. 2A). Increased efficiency may be achieved by
A
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FIG. 5. Neutralization by antisera. (A) Virus surviving fraction (V/1/V') vs. the dilution factors of sera from one group of pM160-inoculated animals. The control serum was from preimmune mice. The test sera (G1-G4) were from pM160-injected mice. The mean + SD of triplicate wells is shown. (B) As for A, demonstrating a more complete titration curve for the antiserum with highest reactivity on ELISA (Fig. 2A).
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Immunology: Wang et al.
improved DNA delivery protocols. To evaluate the efficacy of this technique, we analyzed samples from reactive animals that clearly developed immune responses. Cellular immunity to gpl20 was shown by spleen-cell proliferative responses to rgpl20 that typically reflect activation of helper T cells, by the diversity of the humoral response, and by immunoglobulin isotype switching. Preliminary studies of cytotoxic T-cell responses induced by injection of pM160 indicate 33% specific lysis of envelope-expressing target cells at a 15:1 effector to target cell ratio (unpublished data). Epitope Specificity Elicited by Gene Inoculation. The pM160 response was compared with responses elicited by a rgpl60 inoculation protocol. pM160 inoculation elicited different responses to peptide analogs of some epitopes of gpl20/gp41 compared to rgpl60 immunization (Fig. 3). This suggests in vivo production of HIV-1 envelope by pM160 inoculation results in effective presentation of relevant epitopes to the immune system. The observed neutralization and antisyncytial activity may be the result ofreactivity of the elicited antibodies to functionally important diverse regions of the HIV-1 envelope protein, including the V3 loop of gpl20, the conformational CD4 binding site, and regions of gp41. Sera from mice inoculated with pM160 that recognize the homologous V3 loop peptide of HXB2 also appeared to recognize heterologous V3 loop peptides. Whether recognition in this in vitro assay reflects in vivo biological activity needs to be determined. Biological Activity of the Elicited Responses. Evidence of neutralization, inhibition of syncytium formation, inhibition of CD4-gpl20 binding, T-cell proliferation, cytotoxic T-lymphocyte response, and specific reactivity to diverse regions of gpl60 demonstrate that introduction of a DNA construct encoding HIV gp160 membrane bound glycoprotein directly into muscle cells of living animals can elicit cellular and biologically relevant humoral anti-viral responses. The direct introduction of the gene into mouse muscle and its expression in vivo may allow processing of gene products with the cleavage of gp120 and gp41 from the gpl60 and folding into a native structure, leading to presentation of an effective target antigen. The use of DNA as the immunizing agent may circumvent the need for laboratory peptide synthesis or protein production and purification for the production of immunogens. This immunization strategy is rapid and specific; accordingly, it could streamline the development and testing of safe preparations against HIV and other infectious diseases. While further evaluation of the utility and safety of genetic inoculation is required, ultimately, this technology, which combines the positive aspects of immune stimulation inherent in live attenuated vaccines with the safety of recombinant subunit vaccines, could have wide application in humans and animals. We thank V. Ayyavoo, D. Levy, E. O'Donnell, and B. MacDonald for helpful discussion; Dr. Phillipson for colorful and calming discussions; and D. Levy for kindly providing the PCR primers used in this study. We gratefully acknowledge the contribution of Dr. Peter Nowell for help with the preparation of this manuscript. This work
Proc. Natl. Acad. Sci. USA 90 (1993) was supported by grants from the American Foundation for AIDS Research and National Institutes of Health (D.B.W.) and by grants from the National Institutes of Health and the Lupus Foundation (W.V.W.). K.E.U. is an American Foundation for AIDS ResearchPediatric AIDS Foundation Scholar. 1. Nabel, E. G., Plautz, G. & Nabel, G. J. (1990) Science 249, 1285-1288. 2. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. & Felgner, P. L. (1990) Science 247, 1465-1468. 3. Acsadi, G., Dickson, G., Love, D. R., Jani, A., Walsh, F. S., Gurushinghe, A., Wolff, J. A. & Davies, K. E. (1991) Nature (London) 352, 815-818. 4. Tang, D.-C., DeVit, M. & Johnston, S. A. (1992) Nature (London) 356, 152-154. 5. Zolla-Pazner, S. & Gorny, M. K. (1992) AIDS 6, 1235-1248. 6. Nixon, D. F. & McMichael, A. J. (1991) AIDS 5, 1049-1059. 7. Murphey, C. M., Montelaro, R. C., Miller, M. A., West, M., Martin, L. N., Davison, F. B., Ohkawa, S., Baskin, G. B., Zhang, J. Y., Miller, G. B., Putney, S. D., Allison, A. &
Eppstein, D. A. (1991) AIDS 5, 655-662. 8. Carlson, J., McGraw, T., Keddie, E., Yee, J., Rosenthal, A., Langlois, A., Dickover, R., Donovan, R., Luciw, P., Jennings, M. & Gardner, M. (1990) AIDS Res. Human Retroviruses 6, 1239-1246. 9. Hu, S. L., Abrams, K., Barber, G. N., Moran, P., Zarling, J. M., Langlois, A. J., Kuller, L., Morton, W. R. & Benveniste, R. E. (1992) Science 255, 456-459. 10. Fisher, A. G., Collalti, E., Ratner, L., Gallo, R. C. & WongStaal, F. (1985) Nature (London) 316, 262-265. 11. Wang, B., Ugen, K. E., Hall, W., Kaplan, M. H., Dang, K., Srikantan, V., Sato, A. I., Williams, W. V. & Weiner, D. B. (1993) AIDS Res. Human Retroviruses, in press. 12. Ugen, K. E., Ziegner, U., Agadjanyan, M. G., Satre, M. A. R., Srikantan, V., Wang, B., Refaeli, Y., Sato, A., Williams, W. V. & Weiner, D. B. (1993) Vaccine 93 (Cold Spring Harbor Lab., Plainview, NY), in press. 13. Thomason, D. B. & Booth, F. W. (1990) Cell Physiol. 27, C578-C581. 14. Weiner, D., Kokai, Y., Wada, T., Cohen, J., Williams, W. & Greene, M. (1989) Oncogene 4, 1175-1183. 15. Montefiori, D. C., Robinson, W. E., Schuffman, S. S. & Mithell, W. M. (1988) J. Clin. Microbiol. 6, 231-235. 16. Nara, P. (1989) in Techniques in HIV Research, eds. Aldovini, A. & Walker, B. D. (Stockton, New York), pp. 77-86. 17. Weiner, D. B., Huebner, K., Williams, W. V. & Greene, M. I. (1991) Pathobiology 4, 1-20. 18. Williams, W. V., Moss, D. A., Kieber-Emmons, T., Cohen, J. A., Myers, J. N., Weiner, D. B. & Greene, M. I. (1989) Proc. Natl. Acad. Sci. USA 86, 5537-5541. 19. Weiner, D., Liu, J., Hanna, N., Bluestone, J., Coligan, J., Williams, W. & Greene, M. (1988) Proc. Natl. Acad. Sci. USA 85, 6077-6081. 20. Dalgleish, A. G., Beverley, P. C. L., Clapham, P. R., Crawford, D. H., Greaves, M. F. & Weiss, R. A. (1984) Nature (London) 312, 763-767. 21. Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard, D., Hercend, T., Gluckman, J.-C. & Montagnier, L. (1984) Nature (London) 312, 767-768. 22. Chen, Y.-H., Ebenbichler, C., Vormhagen, R., Schulz, T. F., Steindl, F., Bock, G., Katinger, H. & Dierich, M. P. (1992) AIDS 6, 533-539.
