Am. J. Trop. Med. Hyg., 69(6 suppl), 2003, pp. 1-4
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
PROGRESS IN DEVELOPMENT OF A LIVE-ATTENUATED, TETRAVALENT DENGUE VIRUS VACCINE BY THE UNITED STATES ARMY MEDICAL RESEARCH AND MATERIEL COMMAND
BRUCE L. INNIS AND
KENNETH H. ECKELS
GlaxoSmithKline, Collegeville, Pennsylvania; Walter Reed Army Institute of Research, Silver Spring, Maryland
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INTRODUCTION
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Dengue fever is a mounting global public health problem driven by population growth, urbanization, and vastly augmented regional and international travel.1 Since vector control programs have been difficult to sustain, public health authorities have called for development of a dengue virus (DENV) vaccine. Vaccination is a proven method of infectious disease control. The successful development of a live-attenuated vaccine for yellow fever in the mid-20th century encouraged many that a live-attenuated vaccine for dengue fever, which is caused by a complex of four viruses in the same family (Flaviviridae) as yellow fever virus, could be developed. For several decades, the public health vision has been that control of endemic dengue fever could be achieved by routine administration of a live-attenuated DENV vaccine to children residing in affected countries. Because dengue fever is an important disease in some travelers,2 and infected travelers appear to be major means by which DENV spreads between countries, additional disease control could be achieved by vaccination of non-immune travelers to and within the tropics.
Historically, dengue fever has adversely affected military forces deployed from temperate to tropical or sub-tropical locales. Much early research on dengue was conducted or funded by the United States Army, which saw operational effectiveness degraded by epidemic dengue during troop deployments in almost every decade of the 20th century.3–7 Following the successful propagation of dengue virus type 1 (DENV-1) and DENV-2 in mouse brains, the United States Army sponsored more than a decade of research to develop live-attenuated DENV vaccines manufactured in mouse brains.8 This work culminated in a field efficacy trial in Puerto Rico in 1962 in which partial protection of subjects administered DENV-1 vaccine was observed during an outbreak of predominantly DENV-3.9 Ultimately, the United States Army halted development of the DENV mouse brain vaccine when propagation of DENV in cell cultures, a theoretically safer production substrate, was achieved. Bolstered by the preliminary evidence of vaccine efficacy in Puerto Rico, and the observation that low-passage DENV-2 isolates that produced small plaques in cell monolayers were also temperature sensitive and less pathogenic for suckling mice, the United States Army Medical Research and Development Command in 1971 launched a program at the Walter Reed Army Institute of Research (WRAIR) to develop a cloned, temperature-sensitive, live-attenuated vaccine candidate for each DENV type.
Between 1975 and 1985, four vaccine candidates were produced, tested in non-human primates, and then administered to small numbers of volunteers.10–17 Although the DENV-2 candidate, the first to enter clinical trials was attenuated, it was less immunogenic than desired. A DENV-4 candidate was over-attenuated and virus isolated from vaccine recipients had lost the small plaque phenotype. A DENV-1 candidate was under-attenuated and caused illness; the same was true of a DENV-3 candidate. These results demonstrated that temperature sensitivity and plaque morphology were insufficiently predictive of DENV attenuation for humans. A new approach to developing DENV vaccine candidates was required.
The discovery by Halstead at the University of Hawaii that DENV could be propagated serially in primary dog kidney (PDK) cells, an unnatural cell substrate, offered a promising solution.18 Two groups formed to exploit this discovery: one at Mahidol University in Bangkok, Thailand and the other at the WRAIR in the United States. Both received one or more seed viruses that had been adapted to growth in PDK cells from the University of Hawaii. Over the past 18 years, both groups have developed tetravalent DENV vaccines, conducted successful phase I studies, and attracted a major vaccine manufacturer as a co-development partner.19–23 The pre-clinical and early clinical development of the WRAIR live-attenuated tetravalent DENV vaccine candidate is the subject of the following seven papers in this supplement. In this report, we briefly summarize the overall DENV vaccine development effort at WRAIR and collaborating laboratories between 1986 and 2000.
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MODIFYING DENGUE VIRUS PHENOTYPE BY PASSAGE IN DOG CELLS
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Halstead and Marchette19 have summarized evidence that DENV passage in PDK cells resulted in progressive modification of all DENV strains studied. Plaque size, temperature sensitivity (replication shut-off), cytopathic effect (CPE), and mouse neurovirulence were the laboratory markers they used to characterize modification at selected passage levels. Most consistently, plaque size was reduced for all DENV strains with increased passage number. Increased temperature sensitivity of viral replication was also observed with increasing passage, as was reduced CPE and mouse neurovirulence. The DENV strains differed with respect to passage level when these changed occurred. Parent DENV (low-passage virus stock banked before PDK passage) were used as the basis of comparison for all of these studies. These observations lead to rhesus monkey studies of PDK-passaged viruses to assess modification in the animal model considered most predictive for humans.
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PREPARATION OF CLINICAL-GRADE SEEDS AND VACCINE LOTS IN PRIMATE CELLS AND TESTING IN RHESUS MONKEYS
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Infection of rhesus monkeys with DENV produces minimal or no signs of illness; nevertheless, experimental inoculation is followed after several days by approximately 4–6 days of viremia. Viremia generally ends 8–10 days after inoculation when neutralizing antibodies are first detected.24 These events mimic the pattern of infection in humans. Accordingly, one can measure the period of viremia in monkeys following administration of a live DENV vaccine candidate and compare that to the period of viremia in monkeys inoculated with the parent virus strain to evaluate reduced infectivity as a measure of DENV modification or attenuation.
Eckels and others25 present data demonstrating that all PDK-passaged DENV, with the exception of one DENV-1 strain, had reduced infectivity for monkeys with increased passage. For instance, 50 PDK passages of DENV-2 strain S16803 were required to significantly reduce viremia in monkeys, while only six passages of DENV-4 strain 341750 were required for the same effect. Reduced viremia was usually correlated with reduced immunogenicity. A total of 20 PDK-passaged DENV were inoculated into monkeys; 10 of these demonstrated reduced viremia compared with parent viruses and were considered sufficiently modified for Phase 1 clinical studies. The rhesus monkey infectivity model was invaluable in helping to select these vaccine candidates.
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SELECTION OF MONOVALENT DENV COMPONENTS FOR A TETRAVALENT VACCINE
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Kanesa-thasan and others26 describe the preliminary evaluation of 10 monovalent DENV vaccine candidates in 65 non-immune adult volunteers. The vaccine candidates tested were those demonstrated to be attenuated for rhesus monkeys. The purpose of this series of trials, conducted between 1987 and 1996 at the University of Maryland Center for Vaccine Development and the United States Army Medical Research Institute for Infectious Diseases, was to select the least reactogenic and most immunogenic vaccine candidate for each serotype. Trials began with the vaccine candidate prepared at the highest PDK cell passage level and then progressed to the candidates prepared at lower passage levels. Stopping rules were pre-specified to limit administration of under-attenuated vaccine candidates to participants in the clinical trials. There were three vaccine candidates tested for DENV-1 and DENV-2; there were two vaccine candidates tested for DENV-3 and DENV-4. The evaluation of the DENV-1 candidates was reported previously;22 all three DENV-1 vaccine candidates were considered acceptably attenuated. For the other three DENV types, a single vaccine candidate was selected. Immunogenicity, measured as an increase in neutralizing antibodies, ranged from 50% seroconversion (DENV-3) to 100% seroconversion (DENV-1) after one dose. The selection of these monovalent candidates marked an important milestone and validated the decision of the development team 10 years earlier to proceed with a product based on PDK cell passage. The next step was to expand the evaluation of the monovalent vaccines.
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MULTIPLE DOSING TO ACHIEVE TETRAVALENT RESPONSES
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Sun and others27 administered the four monovalent DENV vaccines to 49 additional non-immune adult volunteers at the University of Maryland Center for Vaccine Development or WRAIR to confirm their safety and immunogenicity. Since there were three DENV-1 candidates, the development team chose the candidate with intermediate PDK passage level, PDK-20, for both studies; the others were retained for backup. To assess the frequency and intensity of solicited adverse events over 21 days of follow-up, a continuous measure of composite reactogenicity called a reactogenicity index was created. This scoring system has been used for all subsequent clinical trials of WRAIR DENV vaccine candidates. The initial study confirmed the safety of the four monovalent vaccines but indicated that two doses were probably required for adequate immunization. Therefore, the development team conducted a second study in which volunteers were randomly allocated to receive a second dose of monovalent DENV vaccine at one or three months after the first dose. A second dose of monovalent vaccine achieved additional immunization in some recipients by a variety of serologic measures, but there was no clear difference between administering a second dose at one or three months. Interestingly, there was markedly less reactogenicity following administration of a second dose.
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EXPLORING THE EFFECT OF TETRAVALENT DENV FORMULATION AND SELECTION OF CANDIDATE FORMULATIONS FOR DEVELOPMENT
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Combining several live virus vaccines into one product creates the potential for interference among the component viruses. In developing a tetravalent DENV vaccine, this issue had to be confronted. The development team decided to retain flexibility for pilot studies by formulating tetravalent DENV vaccine by mixing monovalent vaccines of each serotype in equal volumes just prior to administration, resulting in a 1-mL dose. This procedure permitted any monovalent component to be diluted to adjust the ratio of DENV serotypes. The first study of tetravalent DENV vaccine was conducted at WRAIR by Sun and others27 using undiluted, "high dose" monovalent components. A large factorial design study was subsequently conducted at the Center for Vaccine Development by Edelman and others.28 The factorial design assessed safety and immunogenicity of 15 formulations in a small number of volunteers; the results when combined with those from Sun and others allowed selection of tetravalent candidates from all possible high and low dose combinations of DENV monovalent components.
For the pilot study using monovalent components without dilution, the product was administered in an open-label study to 10 volunteers with two doses given one month apart. The safety profile of the tetravalent vaccine candidate was acceptable; the proportion seroconverting to each DENV type after a single dose was 30–70%. Although a second dose one month later elicited no additional seroconversion, a third dose administered four months after dose one elicited high levels of trivalent or tetravalent neutralizing antibody in three of four volunteers. This study was the first to demonstrate the feasibility of a repeated dose, live, tetravalent DENV vaccine. The factorial design experiment by Edelman and others identified three formulations that had acceptable reactogenicity and met the pre-specified immunogenicity criteria.28
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VACCINATION OF INDIVIDUALS WITH PRE-EXISTING FLAVIVIRUS IMMUNITY
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In earlier studies with the DENV-2 S-1 vaccine candidate, vaccine recipients with a history of yellow fever vaccination had an anamnestic immune response to DENV-2 vaccine, resulting in higher seroconversion rates and neutralization antibodies.13 Kanesa-thasan and others29 describe a similar response in yellow fever vaccine-primed volunteers receiving monovalent, PDK-attenuated vaccines. These volunteers were thought to be flavivirus non-immune on the basis of pre-vaccination antibody testing, but their antibody responses to a single vaccine dose were distinctive and characteristic of pre-existing immunity to a flavivirus. As in earlier studies, reactogenicity was similar in primed and non-primed vaccine recipients, but the number of primed subjects followed for safety is too small to predict that the safety profile of the tetravalent DENV vaccine candidate in primed individuals will be acceptable. Well-controlled clinical trials must be designed specifically to address this question. Depending on the profile of the vaccine, targeting vaccination to age groups expected to have been immunologically primed by prior infection could be a useful disease control strategy, especially in regions where there is extensive transmission of DENV such that much of the dengue disease burden is in persons sustaining a secondary dengue infection. Greater than 95% of dengue hemorrhagic fever cases occur in persons sustaining a secondary dengue infection.30
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DEMONSTRATION OF CELLULAR IMMUNE RESPONSES TO TETRAVALENT DENGUE VACCINE
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Gwinn and others31 found that volunteers vaccinated with monovalent or tetravalent DENV vaccines generated cell-mediated immunity with predominant Th1 differentiation as measured by interferon gamma release in vaccine-stimulated peripheral blood mononuclear cells. Results of assays for interleukin-4 (IL-4) and IL-10 were consistently negative, indicating a lack of Th2 response. Indirect evidence suggests that Th1 cells will contribute to protection against dengue by up-regulating cytotoxic responses to destroy virus-infected cells early in infection, limiting the extent of injury and its associated symptoms.32 The Th1 response observed by Gwinn and others was predominantly DENV type specific, but cross-reactive responses also were observed.
Characterization of cell-mediated immunity elicited by vaccination with tetravalent DENV vaccine can provide supportive evidence that a vaccination regimen is immunogenic for all DENV types. Therefore, cell-mediated immunity readouts for DENV vaccine trials should be refined, standardized, and integrated into development programs. Evidence of tetravalent immunity in vaccinated subjects is generally accepted as a prerequisite to launch large clinical trials required to establish vaccine safety and efficacy.
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LOOKING TO THE FUTURE
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The studies summarized in this report concluded in 2000. Since then, clinical development of WRAIR tetravalent DENV vaccine by the US Army Medical Research and Materiel Command in collaboration with GlaxoSmithKline Biologicals has progressed. Presently, there are five monovalent components and two tetravalent DENV formulations that are being evaluated in clinical trials. Studies are planned in children and toddlers as well as in non-immune and flavivirus-immune adults. Two and three dose schedules of immunization are being considered for optimal protection against all four DENV serotypes. The goal of the development program is a vaccine to prevent dengue fever and dengue hemorrhagic fever that can be used in national immunization programs in countries where dengue is endemic and by travelers to these countries at high risk.
Acknowledgments: We thank the many dedicated scientists, technicians, and assistants who devoted themselves to the U.S. Army DENV live-attenuated vaccine project over the period covered by the supplement. These individuals include W. H. Bancroft, C. H. Hoke, Jr., D. R. Dubois, P. L. Summers, D. Barvir, S. Bailey, K. Jones, B. Whitfield, B. Puri, B. Zhao, N. Kanesa-thasan, F. J. Malinoski, G. French, J. Burrous, R. Edelman, C. O. Tacket, S. S. Wasserman, D. W. Vaughn, J. R. Putnak, T. Simms, E. A. Henchal, T. S. Coster, A. D. King, J. M. Scherer, A. Nisalak, M. Gettayacamin, S. B. Halstead, N. J. Marchette, W. Sun, and W. Gwinn.
Authors’ addresses: Bruce L. Innis, GlaxoSmithKline, 1250 South Collegeville Road, Mail Code UP4330, Collegeville, PA 19426-0989. Kenneth H. Eckels, Department of Biologics, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, Telephone: 301-319-9233, Fax: 301-319-9360, E-mail: kenneth.eckels{at}na.amedd.army.mil.
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INTRODUCTION
MODIFYING DENGUE VIRUS PHENOTYPE...
