In this first clinical evaluation of the new WRAIR-GSK TDEN live vaccine candidate that is the successor to the live tetravalent DENV vaccine previously in development, we found that the new candidate had a clinically acceptable safety profile in a small number of healthy adults. In addition, we found that it was moderately immunogenic after two doses were administered 6 months apart in persons with no baseline neutralizing antibodies to DENV.
The new TDEN vaccine candidate was re-derived by transfection and amplification of the WRAIR's attenuated DENV strains used in the precursor vaccine to improve their quality characteristics. Although non-clinical testing conducted before this clinical trial identified no phenotypic or genotypic changes relative to the precursor vaccine, the trial was designed to explore whether there were important clinical differences between the new candidate and its precursor. The intent to assess potential clinical differences between the new TDEN candidate and the F17/Pre candidate vaccine, while controlling for responses that might be affected by the trial's environment, mandated our use of two control groups: F17/Pre with a virus content adjusted to the level of the new TDEN F17 vaccine and a saline placebo.
This clinical trial was also intended to explore whether the response to the TDEN vaccine was conditioned by its DENV-4 content. The DENV-4 strain had the fewest cell culture passages compared with the other three strains; moreover, in clinical trials of the precursor vaccine F17/Pre, the DENV-4 strain was the most common cause of documented viremia. Consequently, we blended and finished two lots of the TDEN vaccine, F17 and F19, which differed in their DENV-4 content. Although F19 was intended to have 10-fold less DENV-4, the potency testing at release showed that the measured reduction in F19 DENV-4 content was fourfold. Nevertheless, we believed this difference was sufficient to potentially affect interactions between the DENV strains and the clinical responses in terms of fever and other symptoms related to virus replication, viremia, and the humoral immune response to each DENV type.15
Lastly, with respect to design of this clinical trial, we retained the two-dose schedule for primary vaccination used in previous trials of the precursor F17/Pre vaccine. These previous trials had clearly shown the need for two vaccine doses to generate tetravalent antibody responses in unprimed subjects. We also retained the 6-month interval between doses, because pilot experiments conducted with the precursor vaccine at the WRAIR had suggested that intervals of 1 or 3 months between doses was ineffective (Gibbons R and others, unpublished data), whereas a 6-month interval had allowed the second vaccine dose to be consistently immunogenic.
Because this study was the first clinical evaluation of a new vaccine candidate, it was conducted in healthy adults. In contrast to our preceding early-phase live DENV vaccine clinical trials, we elected to enroll subjects without first screening to exclude those individuals with baseline antibodies to DENV or other flaviviruses. This enrolment better represents the profile of potential vaccine recipients who may be at highest risk for dengue, while acknowledging that the responses of primed and unprimed subjects may be different in terms of both reactogenicity and immunogenicity. We did not know whether priming increased or reduced reactogenicity, but we did know that priming increased immunogenicity. In light of our study location in metropolitan Washington, DC, we expected the proportion of enrollees who were primed to DENV to comprise less than 20% of the total enrolled cohort.
The total vaccinated cohort had a median age of 35.5 years, with a maximum age of 45 years. African Americans comprised 57% of the cohort. Although the effects of age and race on response to live DENV vaccines are not fully understood, it is important to note that increasing age in general diminishes responses to many vaccines, and others have hypothesized that persons of African ancestry may have less clinically overt responses to wild-type DENV infection.16
The prevalence of priming to DENV in the ATP immunogenicity cohort with MN50 data was 18%; this prevalence allowed us to have 15–16 unprimed subjects and 3–4 primed subjects per treatment group, a minimally adequate sample size to preliminarily assess the potential of the vaccine candidate to offer effective immunization based on its immunogenicity in unprimed adults.
The most important endpoints for safety were injection site and general symptoms that we solicited from subjects by asking them to maintain a diary for 21 days after each vaccination. Of these symptoms, we considered the absence of fever to be the most objective and reliable measure of overall attenuation.17
Although the group sizes in the total vaccinated cohort for each treatment were small (21 or 22 subjects in study stage 1), they were appropriate for an initial clinical trial.
In study stage 1, most solicited injection site symptoms reported in the first stage of the study were mild to moderate and lasted 2 days or less. Their frequency within the DENV vaccine groups did not seem to differ from placebo or increase when a second dose was administered. The F17/Pre group had the highest proportion of subjects reporting pain, particularly after dose 2 (43.8%; acceptable for a parenterally administered vaccine); although this result may have been a chance finding, the presence of human serum albumin in the F17/Pre formulation and its absence in the other formulations may have been contributory. In study stage 2, when a small number of subjects received a third TDEN dose, the occurrence of solicited injection site symptoms was very low.
We found no major differences among treatment groups in the incidence of solicited general symptoms in study stage 1. Grade 3 solicited general symptoms were reported by < 5% (zero to one subjects per group; the one exception was in the placebo group). The incidence of fever by group after dose 1, dose 2, and overall did not suggest any difference among treatments. Rash was the only symptom reported exclusively in DENV groups (31.8% F17/Pre, 13.6% F17, and 14.3% F19); its occurrence in recipients of live DENV vaccines has been noted previously.18,19
In addition to soliciting subjects for the occurrence of symptoms that could be expected in response to a live DENV vaccine, we also collected all spontaneous reports of adverse events (unsolicited symptoms) for 31 days after each vaccination and SAEs throughout the study. There were no clinically important differences in the incidence of unsolicited AEs among treatment groups in either study stage. Only one grade 3 causally related unsolicited symptom was reported during stage 1 (back pain in placebo group). In study stage 2, the occurrence of solicited general symptoms was low. Throughout the entire study, there were only four SAEs; none were assessed by an investigator as related to the study treatment. Overall, the findings with respect to solicited and unsolicited symptoms after vaccination support that the new TDEN vaccine candidate as either formulation is attenuated and has a reactogenicity profile that compares favorably with the precursor vaccine and saline placebo.
To extend the evaluation of clinical responses to vaccination during the 2 weeks after administration, the period when clinical responses to replication of the vaccine viruses were expected to be maximal, we evaluated all members of the study cohort on 2 randomly selected days (from the set of days 2, 5, 8, 10, 12, and 14) and 1 month after vaccination during study stage 1 for dengue-like physical examination finding, hematologic and serum chemistry tests, DENV viremia, and AEs of potential cardiac origin. We performed these same observations on the subset of subjects who received a third dose of TDEN vaccine during study stage 2.
During the entire study, there were no important differences among groups in terms of dengue-like physical examination signs; the incidence of rash and generalized rash was low in the DENV groups, but no rash was observed in the placebo group. Of all dengue-like signs, rash seems to be the one with the most specific potential association with receipt of a live DENV vaccine relative to placebo.18,19
Our clinical impression is that these rashes are not bothersome to vaccine recipients unless they are accompanied by pruritis. With respect to hematologic or serum chemistry laboratory results that differed from baseline, there were few abnormal results during the entire study; consequently, there were no notable differences among groups in the proportion of subjects who reached this endpoint.
In a previous clinical trial of the F17/Pre vaccine candidate, one adult subject reported transient palpitations approximately 3 weeks after vaccination (Gibbons R and others, unpublished data). This report, in conjunction with reports of myocarditis after dengue and fatal myopericarditis after smallpox vaccination, prompted us to include clinical evaluations designed to detect AEs of potential cardiac origin in this study. During the entire study, there were no AEs of cardiac origin confirmed by a Walter Reed Army Medical Center cardiologist. This type of evaluation has been included in subsequent expanded phase II trials of the TDEN vaccine candidate.
The screening for DENV viremia in this trial was more intensive than we have performed in earlier trials of the F17/Pre vaccine candidate; all subjects in this trial were tested two times on randomly assigned days during the 2 weeks after each vaccine dose, whereas formerly, we tested subjects only on day 10 or when they developed illness suspected to be dengue. During study stage 1, we detected DENV-4 viremia in five recipients of F17/Pre vaccine and one recipient of F17 vaccine (from 7 to 15 days after vaccination). The single instance of viremia occurring in the TDEN recipient was subclinical, because it was not associated with symptoms, dengue-like physical signs, or clinical laboratory abnormalities. However, four of five subjects in the F17/Pre group with DENV-4 viremia did have symptoms, although only one had fever (on a single day). None of these four subjects had a clinically notable illness; none met the pre-specified case definition for dengue. The levels of DENV4 viremia in these F17/Pre vaccine recipients were in line with those levels reported in children who received F17/Pre vaccine9,10
; however, these levels of viremia were substantially less than those levels determined by bioassay in log median mosquito infectious doses per milliliter in patients with clinically overt dengue caused by wild-type infection.20
Overall, the findings with respect to dengue physical examination signs, clinical laboratory determinations, and viremia after vaccination further support the acceptable safety profile of the new TDEN vaccine candidate. The detection of DENV-4 viremia in one TDEN vaccine recipient in contrast to five F17/Pre vaccine recipients also suggests that the re-derivation and serial passage of the DENV-4 seed virus for the TDEN vaccine candidate may have created a more attenuated DENV-4 strain. More clinical experience with the TDEN vaccine candidate will be required to confirm this observation.
We assessed vaccine immunogenicity as a surrogate for clinical benefit. In subjects who were seronegative for a DENV type at baseline, we considered that the detection of neutralizing antibodies at or above the assay positive threshold indicated activation of an adaptive immune response. In subjects who were already seropositive for a DENV type, we assessed whether there was an increase in antibody titer and whether primed subjects with less than tetravalent antibody responses were promoted to acquire tetravalent neutralizing antibodies. A dengue vaccine will ideally elicit protection against disease caused by any of the four DENV types; nevertheless, as with other multivalent live vaccines, there is a potential for interference between the DENV types in any tetravalent live DENV vaccine, resulting in an incomplete or diminished tetravalent immune response profile.2,15
Although we do not know if generation of sustained tetravalent neutralizing antibodies after vaccination is associated with protection, we assume that such responses acutely after vaccination are favorable, whereas their absence is not.
In this trial, we used a microneutralization assay to quantitate the 50% effective dose of neutralizing antibodies to each DENV type in contrast to the 50% plaque reduction neutralization test (PRNT) that we have used in all previous work. Access to a qualified, high-throughput, serotype-specific neutralization assay is essential for development of a DENV vaccine.2,21
The microneutralization test that we used was developed at the WRAIR Pilot Bioproduction Facility to overcome many of the PRNT's limitations, such as its low throughput, unacceptable labor-intensive nature, and high degree of interassay variability.22
This new assay was shown to be specific and sensitive for the detection of anti-DENV neutralizing antibodies (e.g., limit of the blank < 1:3.3; limit of detection < 1:7; limit of quantification ≤ 1:10 for all four serotypes). The precision of the assay was estimated to range from 39% to 59% depending on serotype. Performance characteristics of this assay are described in another publication under preparation (De La Barrera R and others, unpublished data).
We found that the TDEN vaccine candidates were modestly immunogenic across all DENV types after a single dose and moderately immunogenic across all types after two doses. Overall, dose 2 expanded and broadened the immunogenicity responses (seropositivity rates, GMTs, and tetravalent seroconversion) elicited after dose 1. There were no clear differences in the immunogenicity of any of the three DENV vaccine candidates. As has been the case in all prior studies of the F17/Pre vaccine candidate and as seen with other live dengue vaccines in development,23
we found that most vaccine recipients failed to develop tetravalent responses after the first vaccine dose. Among unprimed subjects in this trial who received a TDEN vaccine formulation, the tetravalent vaccine response increased from 37.5–40.0% after dose 1 to approximately 60.0–66.7% after dose 2. In a previous adult study of F17/Pre vaccine candidates, Sun and others8
observed a 63% tetravalent antibody rate 1 month after dose 2 measured by the PRNT. In two subsequent studies of the F17/Pre vaccine candidate in children9
100% of the DENV unprimed children and 54% of the DENV unprimed infants had tetravalent antibody 1 month after the second dose.
We found that a third dose administered 5–12 months after the second dose was ineffective at boosting the immune responses to any of the four DENV types. Tetravalent response rates and GMTs did not increase from 1 month after dose 2 to 1 month after dose 3; however, they did increase from pre-dose 3 to 1 month after dose 3 among the small number of subjects tested. In a study of the F17/Pre vaccine candidate administered on the same two-dose schedule given 6 months apart to infants, it was found that, by 1 year after the second dose, seropositivity rates decreased to all but one of the DENV types. This study suggested that DENV neutralizing antibody titers determined 1 year after administration of vaccine dose 2 may underestimate immunity based on the observed loss of detectable anti-DENV-4 antibody in subjects who had antecedent DENV-4 vaccine viremia.10
The impact of declining levels of DENV neutralizing antibodies on protective immunity is unknown and warrants additional study.