Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys

Abstract

Three tetravalent vaccine (TV) formulations of previously described monovalent dengue (DEN) virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses (T-wt) for replication in SCID mice transplanted with human liver cells (SCID-HuH-7) or for replication and immunogenicity in rhesus monkeys. TV-1 consists of recombinant DEN1, -2, -3, and -4, each with a 30-nucleotide deletion in the 3' untranslated region (Delta30). TV-2 consists of rDEN1Delta30, rDEN4Delta30, and two antigenic chimeric viruses, rDEN2/4Delta30 and rDEN3/4Delta30, both also bearing the Delta30 mutation. TV-3 consists of rDEN1Delta30, rDEN2Delta30, rDEN4Delta30, and a 10-fold higher dose of rDEN3/4Delta30. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice with minimal interference in replication among the virus components. TV-1, -2, and -3 were attenuated in rhesus monkeys as measured by duration and peak of viremia. Each monkey immunized with TV-1 and TV-3 seroconverted to the four DEN components by day 28 with neutralization titers ranging from 1:52 to 1:273 and 1:59 to 1:144 for TV-1 and TV-3, respectively. TV-2 induced low antibody titers to DEN2 and DEN3, but a booster immunization after 4 months increased the neutralizing antibody titers to greater than 1:100 against each serotype and elicited broad neutralizing activity against 19 of 20 DEN subtypes. A single dose of TV-2 induced protection against wild-type DEN1, DEN3, and DEN4 challenge, but not DEN2. However, two doses of TV-2 or TV-3 induced protection against DEN2 challenge. Two tetravalent formulations, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials.

FIG. 1.

Serum neutralizing antibody response to TV-2 given in two doses at either 1 or 4 months. Eight rhesus monkeys were inoculated with TV-2 and boosted at 1 month (n = 4) or 4 months (n = 4). Serum was collected on day 0 and at 1, 2, 3, 4, and 5 months postinoculation, and all samples were tested in four individual plaque reduction neutralization assays against a single DEN serotype (DEN1 WP, DEN2 NGC, DEN3 Sleman/78, and DEN4 Dominica/81). The reciprocal dilution of the geometric mean of the PRNT₆₀ for each group is shown. For the group of monkeys that received the second dose of vaccine at 4 months, the titer and ratio of animals with a fourfold further increase in titer above the titer from the 4-month serum sample (in parentheses) are presented. None of the animals receiving a second immunization at 1 month had a further fourfold rise in titer; however, their antibody titers were maintained near peak levels over the subsequent 4-month period of observation.

FIG. 2.

Effect of 4-month boost with TV-3 on neutralizing antibody response. Six rhesus monkeys were inoculated with TV-3 and boosted after 4 months. Serum was collected on day 0 and at 1, 4, and 5 months postinoculation, and all samples were tested in a plaque reduction neutralization assay against DEN1 WP, DEN2 NGC, DEN3 Sleman/78, and DEN4 Dominica/81. The reciprocal dilution of the geometric mean of the PRNT₆₀ against each serotype is shown. Numerical values are indicated for neutralization titers achieved 1 month after booster immunization, and the ratio of animals with a fourfold further increase in titer above the titer from the 4-month serum sample are presented (in parentheses).

FIG. 3.

Two doses of an attenuated tetravalent DEN virus vaccine (TV-2) induce a broadly reactive serum neutralizing antibody response at levels comparable to that of a single dose of tetravalent wild-type virus (T-wt). The 5-month serum samples of the four rhesus monkeys boosted at 4 months with TV-2 (Fig. 1) and the 1-month serum samples from the four monkeys immunized with a single dose of T-wt (Table 3) were evaluated in individual plaque reduction neutralization assays against five DEN subtypes from each serotype. Viruses are described in Table 6. The reciprocal dilution of the geometric mean of the PRNT₆₀ for each group is shown. The dashed line indicates a fourfold increase in neutralizing antibody titer (1:32) above the limit of detection (1:8). Percent seroconversion (percentage of monkeys with greater than fourfold increase in neutralizing antibody titer) was 100% unless indicated numerically above individual bars.