Elsevier Vaccine, Vol. 15, No. 8. pp. 874-878, 1997 0 1997 Elsevier Science Ltd. All rights resewed Printed in Great Britain zyxwvuts ELSEVIER PII: SO264-41OX(96)00257-5 026&410X/97 $17+0.00 zyxwvutsrq Nucleic acid vaccination with HIV regulatory genes: a combination of HIV-l genes in separate plasmids induces strong immune responses Jorma Hinkula*, Peter Lundholm and Britta Wahren zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK The concept of combining several genes in order to immunize against a microbial agent has been tested, W e selected human immunodeficiency virus (HIV) genes that individually have been shown to mediate immune responses against HIVproteins. These zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ proteins w ere the regulating geneslproteins of HIV-l rev, tat and nef as well as structural genes for gp160 under the control of rev, and the capsidp24 represented by the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML larger precursor gene ~37. Two findings were of particular interest. The combination of these jive gene constructs gave strong reactivity to all of them, compared with previous results using each one in single injections. The intranasal immunization route gave good mucosal reactivity by inducing IgG, IgA and T-cell proliferative responses. 0 1997 Elsevier Science Ltd Keywords: DNA vaccine; HIV-l regulatory genes; intranasal immunization Immunity can be induced to human immunodeficiency virus (HIV-l) regulatory proteins by vaccination with genes1,2. Such responses may establish resistance to early viral replication events if it were to be induced in individuals subjected to risk of infection. To obtain control of primary HIV infection it will be necessary to create mucosal immunity, and mucosal vaccine delivery systems. HIV transmission occurs primarily via vaginal and rectal routes during intercourse. The induction of mucosal immunity at intestinal, vaginal and as in our case the nasal/pulmonary sites may contribute to protec- tion against HIV by controlling viral infection and dissemination at these portals of entry. HIV-l contains two transactivating proteins, rev and tat, which regulate gene expression by binding to specific regions of viral nucleic acids3. Rev is a small basic protein that is located in the cell nucleus and is essential for the expression of the viral structural genes gag, pal, and env. These two proteins occur in low levels and only around 2WO% of HIV-infected individuals develop humoral or cellular responses to these proteins. Nef on the other hand is highly immunogenic, and has been shown to give both antibody and potent cytolytic T-cell reactivity in 6&70% of infected individuals. Both struc- tural p24 and gp160 induce strong immune reactivities early, of which the functional immune p24 reactivity acts through cytolytic cells and the anti gp160 reactivity by neutralization of envelop proteins and possibly anti- body dependent cytolytic cells. Induction of all these Microbiology and Tumorbiology Center, Microbiology, Pathology and Immunology Center, Karolinska Institute, S-171 77, Stockholm, Sweden. *To whom correspondence should be addressed. 874 Vaccine 1997 Volume 15 Number 8 reactivities in the absence of infectivity might give an immune protection similar to that of an attenuated live virus. The present data demonstrate that it is feasible to induce concomitant immune reactivities in experimental animals to all or most of these proteins by immunizing with the gene combination. MATERIALS AND METHODS Experimental animals C57Bl/6 x dba Fl mice transgenic for HLA-A.2 were inoculated with pDNA representing the genes for HIV LA1 rev, tat, nef, gp160 and p37(p24 gag), all under the control of the CMV IE promoter. The selected genes of HIV-l LA1 tat, nef, rev, and gp160 were transactivated by the human cytomegalovirus promoter IE and named pHCMVsrev, pHCMVtat, pHCMVnef and pCM- Vgpl60. Intramuscular and intradermal injections of plasmid DNA were combined2,4. This strain of mice was used in order to pave the way for later use of such mice in immunizing HLA-A.2 bearing cells. Assays were made with proteins produced in baculovirus/ lepidopteran and Escherichia coli expression systems4. Three other strains of mice were used for comparative purposes: dba/2, Balb/c and scid/scid C.B.-17 mice repopulated with human lymphoid cells2. The plasmid mixtures contained zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR 10 pug of each DNA in the first inoculation, thereafter 5,ug for two boosts days 0, 30 and 90. Each plasmid mixture was aimed to induce mucosal immunity in the oral cavity by intranasal (i.n.), tongue injection, Accell device’, or a dental injector. Each group contained four animals. Serum was collected for immunoglobulin determinations, spleen and lymph