Background The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.Methods Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by West blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of DNA prime plus protein boost and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.Results Response of gp120-specific antibody was relatively low after DNA primes (mean titer=10~(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer=10~(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P <0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.Conclusion Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.