DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice

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Vaccine, Vol. 15, No. 8. pp. 849-852, 1997 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264410X/97 $17+0.00

DNA-based immunization against hepatitis B surface antigen (HBsAg) in normal and HBsAg-transgenic mice Heather L. Davis*jjj, Cynthia L. Brazolot Millan*, Maryline Mancinia, Michael J. McCluskie*, Michelle Hadchouelg, Lacrimioara Comanita*, Pierre Tiollaisf, Robert G. Whalen1 and Marie-Louise Michell Hepatitis B virus (HBV) remains a serious worldwide health problem and the possibility to control it will depend on the availability of safe, eflective and afordable vaccines. Recombinant protein or plasma-derived vaccines containing HBV surface antigen (HBsAg) are safe and generally eficacious, however, they are too expensive for widespread use in areas of HBV endemicity and are only partially effective for treatment of HBV chronic curriers. Immunization of mice by injection of HBsAg-expressing plasmid DNA results in rapid induction of strong and long-lasting humoral and cell-mediated immune responses. Here we report optimization of the humoral response with the use of necrotizing agents, co-expression of cytokines or co-stimulatory molecules and formulation of the DNA with cationic liposomes. DNA-based immunizution of HBsAg-transgenic mice can also overcome non-response to HBsAg. Thus, DNA vaccines against HBV may be useful for both prophylactic and therapeutic purposes. 0 1997 Elsevier Science Ltd. Keywords: hepatitis

B; DNA vaccine;

mouse;

chimpanzee:

antibody;

cytotoxic

Hepatitis B virus: epidemiology and inununogenicity

Hepatitis B virus (HBV) remains an important worldwide health problem and the prospects for control of infection and disease depend on the availability of safe, effective and affordable vaccines. Currently available antigen-based vaccines contain empty subviral particles composed solely of envelope protein’.‘. These are safe and generally effective but are expensive to produce and this has limited their use in less developed areas of the world. As well, there is an urgent need for an effective treatment against the chronic carrier state since the estimated 250 million chronically infected individuals in the world today carry an increased risk of developing cirrhosis and hepatocellular carcinoma”. DNA-based immunization against HBV is potentially useful for both prophylactic and therapeutic applications. The structural gene for the HBV surface or envelope protein is a single large open reading frame containing *Loeb Medical Research institute, Ottowa Civic Hospital, 1053 Carling Avenue, Ottawa, Kl Y 4E9, Canada. fFaculties of Health Sciences and Medicine, University of Ottawa, Ottawa, Canada. IUnite de Recombinaison et Expression G&&ique, lnstitut National de la Sante et de la Recherche MBdicale (INSERM), U163 and Dkpartement de Biologie Mokulaire, lnstitut Pasteur, 28, rue du Docteur Roux, 75724, Paris, cedex 15, France. §Centre de Recherche INSERM, U 347, 80 rue du G&&al Leclerc, 94276 Le Kremlin, Bic&tre, cedex, France. IBiochimie Cellulaire, Collkge de France, 11 place Marcelin Berthelot, 75231, Paris, cedex 5, France. IITo whom correspondence should be addressed.

T cells: non-response:

HBV chronic

carrier

three in-frame ATG start codons that divide the gene into three domains designated pre-Sl, pre-S2 and S (from 5’ to 3’), and a single stop codon. The different sized polypeptides produced are known as small or major (S), middle (M=pre-S2+S) and large (L=preSl+pre-S2+S) envelope protein*. Several B and T cell epitopes have been identified which are known collectively as HBV surface antigen (HBsAg) and these are found on both S and pre-S domains. Antibodies against HBsAg (anti-HBs) alone are sufficient to confer protection. The common clinical standard for anti-HBs antibody levels is milli-International Units ml - ’ (mIU ml-‘) and in humans a level of lOmIUml_’ is considered sufficient to confer protection4. Cellmediated immunity (CMI) may also result from natural HBV infection, but the exact role of cytotoxic T lymphocytes (CTL) in protection is not known. DNA-based immunization against HBsAg Intramuscular (i.m.) injection of HBsAg-expressing DNA into the TA muscle of the mouse results in transfection of l-2% of the muscle fibers which have a normal histological appearance 5 days after DNA injection, but are seen to fragment at about 10 days and to have disappeared completely by 30 days. This is probably due to attack by HBsAg-specific CTL since similarly transfected fibers are spared in mice with severe combined immunodeficiency’6. Very strong CTL responses are detected with spleen cells of DNAimmunized mice by 6 days after injection of DNA and these are maintained for at least several months’.

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Low levels of circulating antigen (5 1 ng ml- ‘) are detected prior to the muscle fiber fragmentation, indicating secretion of HBsAg-containing particles from intact muscle cell@. Anti-HBs antibodies are detected as early as 1 week after injection of HBsAg-expressing DNA. The kinetics, antibody isotype and fine-specificity of the humoral response is comparable to that seen following natural infection. The precocity and strength of the humoral response is dose-de endent, although not in a directly proportional manner ?. Antibodies detected at the earliest time point are principally of the IgM isotype but they soon undergo a class shift to the IgG isotype. With the pCMV-S2.S vector, there are also high levels of antibodies to the pre-M domain which may be important for clearance of virus from the liver’ and owing to their precocious appearance, may be advantageous for vaccination of infants born to chronically infected mothers. Peak antibody titers are reached by 4-8 weeks after injection of DNA and are maintained at near maximal levels for up to 17 months without boost’ even though there is no detectable synthesis of new antigen after about 2 weeks. Despite the high sustained response, antibody levels may be further increased ca tenfold with a second injection of DNA. A boost with 4 mg recombinant HBsAg protein will also increase antibody titers, however, in this case the increase is only about fivefold, even though 1000 times more antigen was injected than would have been synthesized in situ in the DNA-immunized animals’. We have also vaccinated two chimpanzees against HBV by i.m. injection of pCMVS2.S DNA. A high dose (2 mg DNA) in one chimpanzee induced high titers of anti-HBs (1000 mIU ml-‘), however, at least two booster injections of DNA were required to prevent the levels of antibody from diminishing over time. Nevertheless, the extremely high titers of anti-HBs which were attained and sustained for at least 1 year, compared very favorably with the best results induced by the commercial recombinant subunit vaccine. A lower dose in a second chimpanzee (4OOpg DNA), induced no detectantibodies until after the able HBsAg-specific second injection of DNA, and although antibody titers of 60 mIU ml-’ were eventually attained, these were transient even after three booster injections of DNA. Nevertheless, in spite of the loss of detectable anti-HBs, this chimpanzee showed a strong anamnestic response to injection of recombinant HBsAg given 1 year after the initial DNA injection”.

MATERIALS

AND METHODS

DNA vectors and direct gene transfer DNA-based immunization against HBV was carried out by i.m. injection of eukaryotic expression vectors which express one or more of the HBV envelope proteins under the control of the immediate early promoter of cytomegalovirus (CMV), one of which also includes the CMV intron A (CMV,,). The CMV promoter drives expression of the S @CMV-S and pCMV -S) or the S+M (pCMV-S2.S) HBV envelope proteins ?DNA was purified using anion exchange chromatography columns (Qiagen GmbH, Hilden, Germany). Plasmid DNA was injected into normal or regenerating tibialis anterior (TA) muscles of mice as previously described6.

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Time (weeks) Figure 1 Groups of female BALE/c mice &lo) were immunized with 1Opg pCMV,,,-S DNA (5pg bilaterally in TA) with and without the use of necrotizing agents. The DNA was injected as follows: 13, in phosphate buffered saline (PBS) into normal muscle; +, in PBS with 0.25% Marcaine@ into normal muscle; or n , in PBS into regenerating muscle (5 days after cardiotoxin-induced degeneration). Anti-HBs levels in pooled sera taken at different times after injection of DNA were determined by ELISA assay and expressed in values of mlU ml-‘, based on comparison with WHO-defined standards. A level of 10 mlU ml-’ is considered sufficient to confer protection against HBV infection in humans

Assay of immune responses Plasma was collected and assayed for anti-HBs antibodies as previously described’ ‘. Anti-HBs levels were expressed in mIU ml-’ by comparison with WHOdefined standards, or as end-point dilution titers, which were defined as the highest plasma dilution resulting in an absorbance value two times greater than that of control plasma with a cut-off of 0.05. Plasma HBsAg was quantitated using a commercial ELISA kit (Monolisa AgHBs, Diagnostics Pasteur).

RESULTS AND DISCUSSION Optimization of humoral response in mice We have previously shown that stronger humoral immune responses are obtained if the DNA is injected into regenerating vs normal muscle, which is obtained by inducing degeneration by injection of necrotizing agents17. This may be due to the improved efficiency of transfection of the muscle fibers, although the presence of macrophages in the regenerating muscle may also play a role. Here we show that co-injection of another necrotizing agent, the local anaesthetic bupivacaine (Marcainefm) along with HBsAg-expressing plasmid DNA also results in better humoral responses and this is likely due to similar reasons (Figure I). The anti-HBs humoral response can also be significantly improved by co-injection of DNA encoding a cytokine or one of the B7 co-stimulatory molecules. For example, co-transfection with vectors expressing HBsAg (pCMV,,+, 1 pg) and murine granulocyte-macrophage colony stimulating factor (pRSV-GM-CSF, 200 pug) resulted in higher anti-HBs titers (Figure 2). The humoral response was also improved by i.m. co-injection of a vector coding for the B7.1 co-stimulatory molecule (pCMV-B7.1, 20 pg) along with the HBsAg-encoding DNA (pCMV,,,-S, 1 pug) (Figure 2). This is possibly

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Time (weeks) Figure 2 Groups of female BALB/c mice (n=lO) were immunized with 1 pg pCMV,,,-S DNA (0.5 pg bilaterally in normal TA). The DNA was injected: c), alone (in PBS); +, mixed with 20 pg DNA expressing the murine 87.1 molecule (pCMVB7.1); or n , mixed with 2OObg DNA expressing murine GM&F. Anti-HBs levels in pooled s&a taken at different times after injection of DNA were determined by ELISA assay and expressed in values of mlU ml-’

Time (weeks) Figure 3

Female BALBlc mice (n=lO) were immunized with 1 fig pCMV,,,-S DNA (0.5pg bilaterally in normal TA). The DNA was injected dissolved in PBS (0) or formulated with a cationic lipid (Gibco BRL experimental lipid No. 302) in DNA:lipid w/w ratios of 1:l (+), 1:lO (0) and 1O:l (W). Anti-HBs levels in pooled sera taken at different times after injection of DNA were determined by ELISA assay and expressed in values of mlU ml-’

because expression of the B7 molecule makes the transfected muscle fiber behave somewhat like an antigen presenting cell (APC). Anti-HBs antibodies are also induced if the DNA is formulated with cationic lipids (Figure 3), but this appears to be due solely to the presence of free DNA as DNA:lipid ratios where all the DNA is complexed with the lipid is not able to induce immune responses in mice (Figure 3)“. DNA overcomes non-response to HBsAg in HBsAg-transgenic mice

We have used transgenic (Tg) mice that constitutively express HBsAg in the liver as a model of chronic HBV carriers in order to study the potential of genetic immunization to induce an immune response which could break tolerance to the viral antigens and thus control the

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Time (weeks) Figure 4 Female transgenic mice (Tg; n=5) and non-transgenic littermates (non-Tg; n=6) were immunized once by i.m. injection of 100 ,ug of pCMVS2.S DNA. Mice were bled at weekly intervals and their plasma was assayed for HBsAg (ng ml-‘) and anti-HBs by serial end-point dilution ELISA. Representative results are shown for Tg and non-Tg mice injected with DNA

infection. The transgene in these mice consists of one copy of the HBV genome with the core gene deleted. Sequences encoding the HBV envelope proteins are under the control of an endogenous HBV promoter and are expressed from before birth, principally in the liver. As a consequence, the plasma of these mice contains between 200-10000 ng mll ’ of HBsAg, however, they exhibit no accumulation of HBsAg within hepatocytes nor liver pathology12. We have previously shown that B cell non-response to HBsAg, which is in fact due to lack of response to T-cell epitopes, can be overcome in these mice by multiple injections of HBsAg protein which has been modified by addition of a heterologous epitope, HBsAg of a different HBV subtype or plasma derived particles in Freund’s adjuvant”. Furthermore, in a pilot study, we have found that therapy using the standard recombinant HBsAg vaccine reduced HBV replication and cancelled immunotolerance to HBsAg particles in about half of the patients treated14. Remarkably, a single injection of pCMV-S2.S DNA into HBsAg-Tg mice provoked an appearance of antiHBs antibodies, which increased over time, and a concomitant decrease in titers of HBsAg (Figure 4). In some of the mice, antigen was undetectable in the plasma as early as 2 weeks after injection of the DNA and remained so for at least 12 weeks without a booster injection of DNA. In the remaining mice antigen levels fell to, and were maintained at low levels. The antibodies induced were of the IgG isotype and such isotype switching of auto reactive B cells strongly suggests that the DNA-mediated immunization triggered CD4+ T helper cells. Histological examination of the vaccinated Tg mice liver showed no evidence of pathology, with neither necrosis nor inflammation being observed, indicating that the DNA immunization had not triggered any detectable HBsAg-specific cytopathic response. Thus these results show that a single i.m. injection of DNA encoding HBsAg sufficiently changes the mode of

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antigen presentation and the nature of APCs such that non-response to HBsAg in Tg mice is overcome. This in turn results in clearance of circulating antigen as well as induction of T-cell responses. In chronic carriers of HBV, the absence of an immune response able to resolve the viral infection may be due to a lack of helper function from HBV-specific CD4+ T cells, which are found in all atients who clear the virus but not in those who do not’. P. On the other hand, B cells from HBV chronic carriers are able to produce antibodies when stimulated in vitro with low doses of antigen. Thus, in individuals with chronic hepatitis, there may be a deficit in the T-cell repertoire which results in non-response to the HBV envelope protein. Nevertheless, we have shown here tbat by using DNAmediated immunization, possibly recruiting more professional APC, B- and T-cell non-response can be overcome. This raises the possibility of designing more effective ways of treating HBV chronic carriers.

REFERENCES 1

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Potential for HBV DNA vaccine for humans

The results obtained to date in animals indicate that DNA may be better than recombinant protein for immunization against HBsAg with respect to rapidity, strength and longevity of humoral and cellular immune responses. If equally efficacious for humans, DNA vaccines could be useful for prophylactic and/or therapeutic immunization. Combined with the many economical and practical advantages of DNA, this would be particularly true for use in less developed areas of the world. As a prophylactic vaccine, the DNA approach may be superior to the use of recombinant HBsAg, especially for the induction of a very rapid humoral response in neonates born to chronic carrier mothers. In addition, a DNA vaccine may be effective, possibly because of the induction of strong CTL, for therapeutic immunization of HBV chronic carriers. Nevertheless, despite the promising results in animals, it will be desirable to further optimize either the efficiency of direct gene transfer or the strength of the immune response to the expressed antigen, so that the lowest possible doses can be used in humans.

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ACKNOWLEDGEMENTS We gratefully acknowledge the generous gifts of Dr Hildegund Ertl (Wistar Institute, Philadelphia, PA) for the murine GM-CSF expression vector, Dr David Curie1 (University of Alabama, Birmingham, AL) for the murine B7.1 expression vector, Dr Joel Haynes (Agracetus, Middleton, WI) for the pCMV,,,-S vector and Dr Joel Jessee (Gibco BRL, Gaithersburg, MD) for the cationic lipids. This work was supported by operating grants from the MRC (Canada), WHO and the University of Ottawa to H.L.D., from the AFM and CNRS to R.G.W., from INSERM and the Pasteur Institute to M.L.M.

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Valenzuela, P., Medina, A., Rutter, W.J., Ammerer, G. and Hall, B.D. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 1982, 298, 347-350 Michel, M.-L., Pontisso, P., Sobczak, E., Malpiece, Y., Streeck, R.E. and Tiollais, P. Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin. Proc. Nat/ Acad. Sci. USA 1984, 81, 7708-7712 Tiollais, P. and Buendia, M.A. Hepatitis B virus. Sci. Am. 1991, 284,40-54 Centers for Disease Control. Recommendations of the Immunization Practises Advisory Committee. Update on hepatitis 6 prevention. Morbid. Mortal. Wklv Rep. 1987. 38. 353 Davis, H.L., Schirmbeck, R., deimann, J. and’whalen, R.G. DNA-mediated immunization in mice induces a potent MHC class-l restricted cytotoxic T lymphocyte response to the hepatitis B envelope protein. Human Gene Ther. 1995, 6, 1447-1456 Davis, H.L., Michel, M.-L. and Whalen, R G. DNA based immunization for hepatitis B induces continuous secretion of antigen and high levels of circulating antibody. Human Molec. Genet. 1993, 2, 1847-1851 Davis, H.L., Michel, M.-L., Mancini, M., Schleef, M. and Whalen, R.G. Direct gene transfer in muscle with plasmid DNA for the purpose of nucleic acid immunization. Vaccine 1994, 12, 15031509 Alberti, A., Cavalletto, D., Pontisso, P., Chemello, C., Tagariello, G. and Belussi, F. Antibody response to preS2 and hepatitis B virus induced liver damage. Lancet 1988, 1421-l 424 Davis, H.L., Mancini, M., Michel, M.-L. and Whalen, R. G. DNA-mediated immunization to hepatitis B surface antigen: Longevity of primary response and effect of boost. Vaccine 1996, 14, 910-915 Davis, H.L., McCluskie, M.J., Gerin, J.L. and Purcell, R.H. DNA vaccine for hepatitis B: immunogenicity in chimpanzees and comparison with other vaccines. Proc. Nat/ Acad. Sci. USA (in press) Michel, M.-L., Davis, H.L., Schleef, M., Mancini, M., Tiollais, P. and Whalen, R.G. DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B viral infection in humans. Proc. NatlAcad. Sci. USA 1995, 92, 5307-5311 Farza, H., Salmon, A.-M. and Hadchouel, M. et a/. Hepatitis B surface antigen gene expression is regulated by sex-steroids and glucocorticoids in transgenic mice. Proc. Nat/ Acad. Sci. USA 1987, 84, 1187-1191 Mancini, M., Hadchouel, M., moHais, P., Pourcel, C. and Michel, M.-L. Induction of anti-hepatitis B surface antigen (HBsAg) antibodies in HBsAg producing transgenic mice: a possible way of circumventing “nonresponse” to HBsAg. J. Med. Viral. 1993, 39, 67-74 Pol, S., Driss, F., Michel, M.-L., Nalpas, B., Berthelot, P. and Brechot, C. Specific vaccine therapy in chronic hepatitis B infection. Lancet 1994, 344, 342 Ferrari, C., Penna, A. and Bertolettr, A. et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J. fmmunoi 1990, 145,

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Davis, H.L., Brazolot Millan, C.L. and Watkins, S.C. Immunemediated destruction of transfected muscle fibers after direct gene transfer with antigen-expressing plasmid DNA. Gene Ther. 1997, (in press). McCluskie, M.J. and Davis, H.L. DNA-based immunization of mice against HBV by intramuscular injection requires free DNA. In: Vaccines 97: Molecular Approaches to the Control of infectious Diseases, Eds Brown, F., Burton, D., Doherty, P., Mekalanos, J. and Norrby, E., Cold Sprtng Harbour Laboratory Press, New York, 1997 (in press)