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Presented in part: 63rd Annual Meeting of the American Society of Tropical Medicine and Hygiene, New Orleans, Louisiana, November 2014.
The ideal dengue vaccine will provide protection against all serotypes of dengue virus and will be economical and uncomplicated in its administration. To determine the ability of a single dose of the live attenuated tetravalent dengue vaccine TV003 to induce a suitable neutralizing antibody response, a placebo-controlled clinical trial was performed in 48 healthy adults who received 2 doses of vaccine or placebo administered 12 months apart. Evaluation of safety, vaccine viremia, and neutralizing antibody response after each dose indicated that the first dose of vaccine was capable of preventing infection with the second dose, thus indicating that multiple doses are unnecessary.
Clinical Trials Registration.NCT01782300.
Dengue remains the most significant mosquito-borne viral disease throughout the world. Four serotypes of dengue virus (dengue virus serotype 1 [DENV-1], DENV-2, DENV-3, and DENV-4) are transmitted by Aedes mosquitoes, and the geographical spread of both mosquito vectors and the 4 viruses has led to an increasing number of countries experiencing epidemic dengue disease and an associated public health concern. Up to 3 billion people are at risk of infection in tropical and subtropical countries, and an estimated 390 million infections occur each year, with approximately 1 in 4 infections resulting in apparent signs or symptoms of disease . The spectrum of DENV disease ranges from subclinical disease or undifferentiated febrile illness to classic dengue fever to life-threatening severe dengue . Although long-term homotypic immunity is induced by a single infection with DENV , immunity to a single DENV serotype has been identified as a risk factor for more-severe disease upon secondary, heterotypic infection [4, 5]. In addition, a waning or unbalanced immune response from either natural infection or inadequate vaccination may pose a similar risk. Thus, an effective and safe DENV vaccine needs to simultaneously induce long-lived protective immunity against all 4 DENV serotypes.
A wide variety of dengue vaccine platforms are currently in development, and live attenuated vaccines are furthest along the development pathway . The catalyst for this progress may be recognition of the distinct advantages afforded by live dengue vaccines: live vaccines currently in use for other flavivirus diseases, including yellow fever (YF-17D) and Japanese encephalitis (SA14-14-2), are effective and can be very economical to produce ; live viruses replicate and therefore induce both humoral and cellular immune responses, assisted by the presentation of epitopes in their native conformation; and live attenuated vaccines have been successful against numerous other nonflavivirus pathogens and, when administered parenterally (subcutaneous and intramuscular routes), are effective after a single dose, often eliciting antibody responses that persist for >30 years . For these reasons, we sought to develop a single-dose live attenuated tetravalent vaccine for dengue. Over the course of developing the TV003 and TV005 vaccines, we evaluated many monovalent vaccine components with regard to safety, infectivity, and immunogenicity in clinical trials to select suitable strains to include in a tetravalent mixture . To determine whether a second dose of vaccine would increase the frequency of seroconversion to multiple DENV serotypes or boost the magnitude of the neutralizing antibody response, we conducted numerous clinical studies with both monovalent components and tetravalent mixtures of vaccine candidates. The overall conclusion is that a second vaccine dose administered 6 months after the primary dose does not provide significant enhancement of neutralizing antibody titers and, in the case of tetravalent admixtures, does not increase the frequency of multivalent antibody responses .
In the current study, the timing of a second vaccine dose was extended to 12 months following administration of the primary dose. Forty-eight DENV-naive adult subjects (40 vaccine recipients and 8 placebo recipients) were enrolled between January and September of 2013 under study protocol CIR283 (clinical trials registration NCT01782300; Supplementary Figure 1). This phase 1 randomized, double-blinded, placebo-controlled trial was performed under an investigational new drug application reviewed by the Food and Drug Administration and was approved by the institutional review boards at the University of Vermont and Johns Hopkins University. Informed consent was obtained in accordance with federal and international regulations (21CFR50, ICHE6). External independent monitoring was performed, and the National Institute of Allergy and Infectious Diseases Data Safety Monitoring Board reviewed all safety data every 6 months. To determine the effect on safety, viremia, and immunogenicity, a second dose of the same vaccine was administered 12 months after the first dose. As in previous clinical evaluations, the study addressed vaccine safety, vaccine-associated viremia (characterized by mean peak titer, day of onset, and duration), antibody response (characterized by 50% plaque-reduction neutralization titer [PRNT50]), and frequency and distribution of seroconversion. The tetravalent vaccine, designated TV003, was administered subcutaneously as a combination of 1000 plaque-forming units (PFU) of each serotype component and has been described previously . Subjects were followed as outpatients and self-recorded oral temperature 3 times daily for 16 days. Clinical assessments and physical examinations were performed on study days 0, 3, 8, 10, 14, 21, 28, 56, 90, and 180. The schedule of study visits was the same for both doses. Serum samples collected through day 14 were tested for infectious virus by amplification and direct titration on Vero cell monolayers . Serum neutralizing antibody response was measured by the PRNT50 assay in accordance with other live attenuated DENV vaccine evaluations . PRNT50 assays used an initial serum dilution of 1:5, and seroconversion after the first dose was defined as a PRNT50 of ≥1:10 by study day 90. A boost in neutralizing antibody from a second dose of vaccine was defined as a ≥4-fold rise in neutralizing antibody titer by study day 270, compared with study day 180.
The frequency and significance of adverse events following the first dose of vaccine confirm the favorable safety profile of the vaccine and were similar to those observed previously for TV003 (Supplementary Table 1), with the only statistically significant event being the mild vaccine-associated rash evident in 63% of vaccinees. The average time of rash onset was at 10.8 days (range, 9.6–12.0 days) after vaccination, and the rash lasted for an average of 7.7 days (range, 6.6–8.8 days). Only a single vaccinee was observed with a vaccine-associated rash following the second dose, indicating that the prior dose of vaccine limited the rash reaction following the second dose, presumably because of neutralization of vaccine virus replication. Following the first dose of vaccine, 43% of subjects had infectious vaccine virus isolated from their serum during clinical follow-up (Table (Table1).1). Recovery of DENV-1 and DENV-3 was 3–5 times more frequent than recovery of DENV-2 and DENV-4. Although titers of observed viremia were relatively low (0.5–1.6 log10 PFU/mL) and viremia was generally limited to a single day following dose 1, viremia was undetectable following the second dose, further confirming the neutralization of vaccine virus replication during the second dose, which is typical of other successful live attenuated viral vaccines.
The reasons for considering administration of additional vaccine doses as part of the primary vaccination schedule include the need to increase the magnitude of the immune response and to effectively immunize individuals who did not respond to the first dose. Therefore, careful monitoring of vaccine immune responses is required to assess the effectiveness of multidose vaccine strategies. In the current study, serum samples for antibody analysis were collected on the day of each dosing and at 3 time points after each dose, enabling the evaluation of several important serological parameters: mean peak titers of neutralizing antibody, frequency of seroconversion, and fold-change in antibody titers after the second dose. Because the trial was conducted in a non–dengue-endemic area, we did not screen subjects for seropositivity to DENV prior to enrollment, and at enrollment only 2 of 40 subjects were marginally seropositive to ≥1 DENV serotype. After the first dose of TV003, the reciprocal mean peak titer of neutralizing antibody ranged from 80 to 255 among the serotypes, with the highest titer against DENV-4 (Table (Table2).2). One year after vaccination, the mean neutralizing antibody titers had uniformly declined, as expected, to resting levels of 23–51, with the highest titer against DENV-2. This resting level of antibody was not significantly boosted after the second dose. The majority of vaccinees achieved a small increase in antibody titer; however, the fold-change in mean titer was ≤2-fold for each serotype (data not shown). Although the mean antibody titers before and after the second dose of vaccine were increased less than the boost threshold (4-fold), there were titer increases within the mean calculation that exceeded 4-fold, although this occurred at a very low frequency of 3%–10% across all serotypes (Table (Table2).2). Antibody titers for some vaccinees decreased after the second dose, with the following frequency: 14% for DENV-1, 47% for DENV-2, 16% for DENV-3, and 10% for DENV-4. Although we do not know what titer of neutralizing antibody is required for protection against DENV infection or apparent disease, the mean peak titers indicate that the antibody response across serotypes is relatively uniform, demonstrating that the immunogenicity is not dominated by ≥1 serotype. Rather than indicating a lack of interference among the vaccine serotypes, we believe that the even immunogenicity is a result of the similar infectivity of each vaccine component, as demonstrated earlier in individual phase 1 studies.
In the absence of a known immune correlate of protection, the magnitude of the neutralizing antibody response may be less informative than the frequency of seroconversion, especially when considering the breadth of the response across all 4 serotypes. We have defined both seroconversion and boost as a 4-fold rise in antibody titer as compared to preexisting levels. Since the initial dilution of serum in the PRNT assay is 1:5, we assigned a titer of 2.5 to samples with undetectable titers at the initial serum dilution tested. Therefore, seroconversion is considered to occur when the reciprocal antibody titer reaches ≥10 at any time point following vaccine administration. A subsequent boost in antibody titer occurs when preexisting titers are increased by ≥4-fold. The high frequency of seroconversion following a single dose of TV003 administered to dengue-naive subjects is unprecedented. After the first dose of TV003, the frequency of seroconversion to each serotype was 89%–100% and did not change following the second dose, similar to previous observations following a 6-month dosing interval (Table (Table2).2). In addition, following the first dose of vaccine, 86% of vaccinees seroconverted to all 4 DENV serotypes, with 98% seroconverting to ≥3 serotypes. Again, the multivalency of the neutralizing antibody response did not change following the second dose. It should be noted that the 4 individuals who lacked antibodies to ≥1 serotype and received a second vaccination did not seroconvert to the missing serotypes.
As expected following administration of live vaccines, the first vaccine dose elicited sterilizing immunity capable of neutralizing a second vaccine dose, as indicated by the lack of detectable viremia and the lack of an increase in antibody titer. By this definition, a single dose of TV003 elicited sterilizing immunity to all 4 serotypes for at least 1 year in 80% of vaccinees. Vaccine viremia was not detected in any vaccinee following the second dose, and of the 6 vaccinees in whom neutralizing antibody titers were boosted, 5 experienced a boost for only a single serotype (DENV-2, DENV-3, or DENV-4), and 1 experienced a boost for 3 serotypes (excluding DENV-3).
Data from this study strongly suggest that a second dose of TV003 is unnecessary and provides only minimal benefits. Accordingly, phase 2 studies in Thailand and Bangladesh are proceeding with a single dose of vaccine. However, it is not known how durable the immune response to a single dose of TV003 will be. Because it is not possible to unequivocally equate resistance to revaccination with protection against natural infection, future studies will be needed to validate the effectiveness of a single dose. The ongoing phase 3 study in Brazil includes a single dose of vaccine and a 5-year follow-up period. As is the case with the measles-mumps-rubella vaccine, which is given in the second year of life, followed by a second immunization when a child enters primary school, a second dose of TV003 may be needed, although the timing of the second dose will need to be determined.
Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.
Acknowledgments.We thank the dedicated volunteers, as well as the University of Vermont Medical Center Clinical Research Center and the Johns Hopkins University Center for Immunization Research and their excellent research nurses and staff.
Financial support.The work was supported by the National Institute of Allergy and Infectious Diseases Intramural Research Program, National Institutes of Health (contract HHSN272200900010C).
Potential conflicts of interest.All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.