Vaccine 22 (2004) 1576–1585 Gene immunization may induce secondary antibodies reacting with DNA Maria G. Isaguliants a,b,∗ , Konstantin Iakimtchouk a,b , Natalia V. Petrakova b , Marina A. Yermalovich a , Anne Kjerrström Zuber a , Vladimir I. Kashuba a , Sergey V. Belikov a,c , Sören Andersson a,d , Sergey N. Kochetkov e , Dennis M. Klinman f , Britta Wahren a a Swedish Institute for Infectious Disease Control and Microbiology and Tumour Biology Center, Karolinska Institute, SE-171 82 Solna, Sweden b D.I. Ivanovsky Institute of Virology, Gamaleja Str. 16, 123098 Moscow, Russia c Department of Cell Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden d Department of Clinical Microbiology and Immunology, Regionsjukhuset, Örebro Läns Landsting, Örebro SE-70185, Sweden e W.A. Engelhardt Institute of Molecular Biology, Vavilova Str. 32, 117984 Moscow, Russia f Division of Viral Products/CBER, US Food and Drug Administration, Rockville, MD 20902, USA Received 18 June 2002; received in revised form 12 May 2003; accepted 18 September 2003 Abstract The fear of autoimmunity in DNA-vaccine recipients initiated screening for anti-DNA antibodies in rabbits immunized with genes of viral nucleic acid-binding and adapter proteins. Of 11 DNA/protein-immunized rabbits, seven had developed secondary antibodies against DNA detected at weeks 11–50 from the on-start of immunization. Two rabbits immunized with HIV-1 reverse transcriptase gene developed transient anti-double-stranded DNA antibodies of high avidity that recognized DNA in the kinetoplasts of Crithidia luciliae. Others developed antibodies reacting with DNA in ELISA and targeting nuclear-associated antigens in the immunofluoresence test. No anti-DNA antibodies were detected at these time-points in any of the controls (P = 0.036). Induction of anti-DNA antibodies by epitope spreading from protein domains involved in nucleic acid-binding versus maturation of anti-protein antibodies to dual protein-DNA specificity is discussed. (126 words). © 2003 Published by Elsevier Ltd. Keywords: DNA immunization; Anti-DNA antibodies; HIV-1 reverse transcriptase; nef; HCV core 1. Introduction Genes carried by plasmids are now widely available for vaccine development. The technique has shown promise for improving the existing vaccines and development of the new ones [1]. The initial fear of autoimmunity in DNA vaccine recipients based on the ability of bacterial double-stranded DNA (dsDNA) to elicit autoantibodies (reviewed in [2]) has so far not come true. No anti-DNA antibodies were re- ported in human DNA vaccine trials [3,4]. A single case Abbreviations: HIV-1, human immunodeficiency virus type 1; HCV, human hepatitis C virus; SLE, systemic lupus erythematosus; aa, amino acid(s); OD, optical density; SI, stimulation indice; ss- and dsDNA, single- and double-stranded DNA; RNP, ribonuclear protein; nDNA, native human placental DNA; RT, reverse transcriptase of HIV-1 strain HXB-2; core152, core protein of HCV truncated at aa 152; CMV IE promoter, immediate early cytomegalovirus promoter ∗ Corresponding author. Tel.: +46-8-457-2524; fax: +46-8-33-7272. E-mail addresses: isaguliats@hotmail.com, maria.isaguliants@smi.ki.se (M.G. Isaguliants). with anti-DNA antibodies induced after gene vaccination, was a subject with a borderline antinuclear antibody level prior to the on-start of vaccination [5]. Plasmid injections were reported to induce transient production of anti-DNA antibodies in mice [6]. However, the initial modest rise in their levels was insufficient for the development of systemic autoimmunity [1,6]. Viral nucleic acid-binding proteins, such as capsids, regulatory genes and enzymes, are attractive targets for DNA-vaccination [4,7–9]. Both DNA-binding proteins and ribonucleoprotein (RNP) complexes are, however, known as potent autoimmunogens [10,11]. Antibody against the protein component of these complexes may then directly bind, or undergo clonal expansion and differentiation to bind host DNA, histones, and RNPs [12–14]. These ex- perimental findings have relevance to human disease. In patients with autoimmune syndroms, antibodies against DNA-binding polyoma virus T antigen correlate with reactivity against single-stranded DNA (ssDNA) [15]. Thus, a possibility exists that some proteins endogenously 0264-410X/$ – see front matter © 2003 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2003.09.033