The control vaccine, inactivated hepatitis A vaccine (Havrix, GlaxoSmithKline), was formulated as 720 enzyme-linked immunosorbent assay (ELISA) units of inactivated hepatitis A virus combined with 0.5 mg of alum, in a volume of 0.5 ml. For the study to be blinded, the control vaccine was given at 0, 1, and 6 months in doses containing one half the usual volume and one half the usual amount of antigen.

Study Design

The double-blind, randomized field trial was designed in collaboration among the trial sponsors, the National Institutes of Health (NIH) and GlaxoSmithKline; the study chair; the executive committee; and the scientific leadership group. Data were collected with the use of the GlaxoSmithKline remote data-entry system and were monitored by GlaxoSmithKline. All the authors and the trial sponsors vouch for the accuracy and completeness of the data. Data were electronically transferred to EMMES, a contract research organization, where they were analyzed according to the analysis plan prepared by biostatisticians at GlaxoSmithKline and EMMES, with input to the analysis plan from the NIH, the study chair, and the executive committee. The manuscript was drafted by the first author, with input from the biostatisticians at EMMES and from the executive committee.

Study End Points

The primary end point of the study was prevention of genital herpes disease caused by HSV-1, HSV-2, or both from month 2 (1 month after vaccine dose 2) through month 20. Genital disease was defined as clinically compatible signs and symptoms confirmed by viral culture, seroconversion, or both within 6 months after disease onset. Secondary end points included prevention of HSV-1 or HSV-2 infection (with or without disease) from month 2 through month 20 (two-dose efficacy) or month 7 through month 20 (three-dose efficacy) and prevention of genital herpes disease caused by individual HSV types. Cases of infection and disease were determined centrally by an independent, blinded end-point review committee with the use of documented criteria.

Substudy of Viral Shedding

Subjects identified as having acquired genital HSV-2 disease or HSV-2 infection during the study were invited to participate in an evaluation of viral shedding. Subjects were instructed to collect daily swabs from the anogenital area for 60 consecutive days and to maintain a diary of genital signs and symptoms, as previously described, beginning 3 to 6 months after HSV-2 seroconversion or disease onset.

Laboratory Studies

Western blot analysis (University of Washington Clinical Virology Laboratory at Seattle Children’s Hospital) was used to confirm HSV-1– or HSV-2–seronegative status at study entry and seroconversion during the follow-up period.6 Seroconversion to HSV-1 or HSV-2 was defined as a positive Western blot analysis in a subject with a previously negative analysis for the corresponding HSV type.

Serum specimens from a random subset of 611 subjects in the HSV-vaccine group and 223 subjects in the control group were assessed for the development of antibodies to vaccine antigens with the use of ELISA4,7 for gD-2 and virus neutralization of HSV-2 at 0, 2, 6, 7, 12, 16, and 20 months. Long-term genital shedding of HSV-2 DNA was assessed with the use of a quantitative, real-time, fluorescence-based polymerase-chain-reaction assay, as described previously, with a positive result defined as 150 copies per milliliter.