An oligonucleotide encoding the amino acids 731-752 of the gp41 envelope protein of the human immunodeficiency virus type 1 strain IIIB, which is known to induce cross-reactive neutralizing antibodies in humans, was inserted into a full-length clone of the RNA encoding the coat proteins of cowpea mosaic virus (RNA 2 of CPMV). When transfected together with RNA 1 of CPMV, transcribed RNA 2 was able to replicate in plants and form infectious virions (CPMV-HIV). Purified virions were injected subcutaneously with alum adjuvant into adult C57/BL6 mice to determine their ability to stimulate ELISA and neutralizing antibody specific for HIV-1. Antisera to CPMV-HIV obtained after only two injections gave a strong ELISA response (mean of 1:25,800) using the free gp41 peptide as antigen, showing that the gp41 peptide incorporated into the chimera was immunogenic. The same antisera gave 97% neutralization of HIV-1 IIIB at 1:100 dilution, with a highly uniform response in all (six of six) animals tested. A third injection barely increased the neutralization titer. Normal mouse serum had no neutralizing activity. Antisera also strongly neutralized the HIV-1 strains RF and SF2. ELISA and neutralizing activity to HIV-1 IIIB declined after the second injection and were undetectable after 7 weeks, but were restimulated to the same level after the third injection. Neutralization was marginally more stable after the third injection. Antibody specific for CPMV epitopes was equally short lived. A bonus of this system was unexpected neutralizing activity specifically stimulated by unmodified CPMV virions, although this amounted to no more than 10% of the neutralizing activity stimulated by the CPMV-HIV chimera.(ABSTRACT TRUNCATED AT 250 WORDS)

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The Kennedy peptide, (731)PRGPDRPEGIEEEGGERDRDRS(752), from the cytoplasmic domain of the gp41 transmembrane envelope glycoprotein of HIV-1 contains a conformationally dependent neutralizing epitope (ERDRD) and a linear nonneutralizing epitope (IEEE). No recognized murine T cell epitope is present. The peptide usually stimulates virus-specific antibody, but this is not always neutralizing. Here we show that IEEE (or possibly IEEE plus adjacent sequence) is immunogenically and antigenically dominant over the ERDRD neutralizing epitope. Thus rabbits immunized in a variety of routes, doses, and adjuvants with a chimeric cowpea mosaic virus (CPMV) expressing the Kennedy peptide on its surface (CPMV-HIV/1) synthesized IEEE-specific serum antibody but no ERDRD-specific or HIV-1-neutralizing antibody. To test if this resulted from immunodominance or from a hole in the antibody repertoire, we immunized rabbits with chimera CPMV-HIV/29, which expresses the GERDRDR part of the Kennedy sequence. This chimera readily stimulated ERDRD-specific, neutralizing antibody. In mice the situation was less extreme, but individual animals with low neutralizing titers had a high ratio of IEEE-specific:ERDRD-specific antibody. Data are consistent with immunodominance of IEEE over ERDRD in the Kennedy peptide. IEEE-specific antibody was also antigenically dominant and prevented ERDRD-specific antibody from binding to its epitope and from neutralizing HIV-1. It may be that HIV-1 has evolved a nonneutralizing immunodominant epitope that allows it to possess a neutralizing epitope without suffering the consequences, and this idea is supported by the covariance of both epitope sequences. To our knowledge this is the first example of a defined sequence that controls the activity of an adjacent epitope.