Xu, D. antibody in rabbits. Lastly, antisera from both mice and rabbits vaccinated with DNA expressing sgp120-C3d3 elicited higher titers of neutralizing antibody than did nonfused forms of Env. These results indicate that C3d, conjugated to sgp120, enhances the antibody reactions to Env compared to non-C3d fused forms of Env, and this approach may be one method to overcome the poor ability of DNA vaccines to generate antibodies to Env. DNA vaccination (genetic vaccination) induces protecting immunity against a variety of pathogens (for evaluations, see referrals 20, 32, 53, and 54). These genetic vaccines consist of eukaryotic manifestation plasmids that are inoculated into target cells and translated into proteins (20). Previous studies have shown that DNA vaccination efficiently induces both humoral and cellular immune reactions to immunogens from varied infectious providers (20, 32, 53, 54). However, DNA immunizations have been less successful at generating neutralizing antibodies against human being immunodeficiency disease type 1 (HIV-1) (54). Unlike most immunogens, multiple DNA immunizations are required to elicit even moderate titers of neutralizing antibody to the HIV envelope (Env) glycoprotein (4, 5, 11, 18, 33, 34, 43, 59). In addition, the antibody CC-115 reactions raised by DNA vaccination, like those to Env (gp120) subunit immunizations, are transient, rising and falling with each successive immunization (26, 42, 51). In HIV-infected individuals or in experimentally SIV-infected rhesus macaques, specific antibodies require 6 to 8 8 months to accomplish affinity maturation and may be associated with the appearance of neutralizing antibody (17). Consequently, we sought to increase the effectiveness of DNA vaccines expressing HIV Env by using a component of the innate immune system, C3d, in order to enhance antibody titer, the affinity maturation, and the virus-neutralizing ability of the elicited antibody. In the human being immune system, C3d is one of the final degradation products of the third complement protein, C3. The C3d receptor, CD21 or CR2, is located on B cells, follicular dendritic cells (FDC), and possibly some epithelial cells (33, 34). One result of match activation is the covalent attachment of the C3d to antigen. On B lymphocytes, C3d-CD21 connection stimulates B cells amplifying lymphocyte activation (19). Recently, our laboratory while others have shown that C3d can enhance antibody responses directed toward a specific antigen encoded by a DNA vaccine (26, 38, 55, 56, 62). A DNA vaccine expressing a fusion of hemagglutinin (HA) from influenza disease or measles disease fused to three copies of the murine homologue of C3d (mC3d) accomplished an earlier and more efficient immune response (26, 38, 55). These results shown that mice vaccinated with DNA expressing a secreted, soluble HA-mC3d3 immunogen elicited antibody that underwent more rapid affinity maturation than antibody generated by non-C3d fused forms of secreted or transmembrane of HA. This resulted in more rapid appearance of hemagglutination inhibition activity, neutralizing titers, WNT-4 and protecting immunity (26, 38, 55). In addition, our laboratory offers used a similar approach with the HIV-1 envelope (gp120) fused in the carboxyl terminus with C3d3 (56). By using DNA vaccination, BALB/c mice were inoculated and assayed for enhanced immune reactions. The fusion constructs induced higher antibody reactions to Env and a faster onset of affinity maturation than did the respective wild-type gp120 sequences (56). The present study differs from our past studies in three significant elements. (i) In order to determine the number of C3d genes necessary to elicit the maximum immune response by DNA CC-115 vaccination, mice were vaccinated with DNA expressing sgp120 fused to one, two, or three copies of mC3d. (ii) Even though mice vaccinated with sgp120-mC3d3-DNA elicited enhanced antibody reactions, these same antibodies were unable to neutralize HIV illness. Consequently, in the present study, vaccination with DNA was followed by protein boosts of gp120 in order to enhance the neutralizing antibody titers to Env. (iii) The human being homologue of C3d was fused to sgp120 and examined for anti-Env immunogenicity and neutralizing ability. MATERIALS AND METHODS Plasmid DNA. pTR600, a eukaryotic manifestation vector, has been explained previously (27). Briefly, the vector was constructed to contain the cytomegalovirus immediate-early promoter plus intron A for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation transmission for termination of transcription (Fig. ?(Fig.1).1). The vector contains the ColE1 source of replication for prokaryotic replication and the kanamycin resistance CC-115 gene for selection in.