BACKGROUND: We performed human immunodeficiency virus type 1 (HIV-1) transmitted/founder (T/F) virus analysis of the VAX003 vaccine efficacy trial participants to characterize the transmission bottleneck and test for vaccine-associated reduction or enhancement of infection in this injection drug user (IDU) cohort. METHODS: We performed single genome sequencing of plasma vRNA from 50 subjects sampled in early HIV infection. Sequences were analyzed phylogenetically, T/F viruses enumerated, and a sieve analysis performed. RESULTS: Eight of 19 (42%) placebo recipients were productively infected by more than 1 virus (range 1-5, median 1, mean 1.7). This frequency of multiple virus transmission was greater than reported for heterosexual cohorts (19%, P = .03) but not statistically different from vaccine recipients (22.6%, P > .05), where the range was 1-3, median 1, and mean 1.3 (P > .05 for all comparisons). An atypical sieve effect was detected in Env V2 but was not associated with reduction or enhancement of virus acquisition. CONCLUSIONS: The number of T/F viruses in IDUs was surprising low, with 95% of individuals infected by only 1-3 viruses. This finding suggests that a successful vaccine or other prevention modality generally needs to protect against only one or a few viruses regardless of risk behavior. T/F analysis identified an atypical genetic sieve in the V2 region of Envelope and found no evidence for vaccine-mediated enhancement in VAX003.