Dengue vaccine trial guidelines and role of large-scale, post proof-of-concept demonstration projects in bringing a dengue vaccine to use in dengue endemic areas

Abstract

In this review, we consider the issues impacting conduct and design of dengue vaccine trials with reference to the recently published World Health Organization "Guidelines for Conduct of Clinical Trials of Dengue Vaccines in Endemic Areas." We discuss logistic, scientific and ethical challenges concerning evaluation and introduction of dengue vaccines; these range from randomized trials that establish "proof of concept" of vaccine efficacy, to post-"proof of concept" trials, particularly demonstration projects likely to be required for licensure or for the introduction of an already licensed vaccine into public use. We clarify and define the meaning of "proof of concept" in the clinical trial context and the meaning of terms "phase 2b", "phase 3b" and "demonstration project", which are commonly used but have not been defined well in the clinical literature.

Background

Dengue is a group of viruses with complex transmission and disease characteristics. Mosquito borne transmission of this group of viruses makes disease control and prevention more complex. Transmission can be erratic, moving its focus from one adjacent community to another in sequential years.1

There are four dengue viruses which act as independent infectious agents,1 i.e., immunity to any one type does not provide long-term immunity to the other three types. The distribution and relative proportion of the 4 virus types varies by year and geographic location. Asia is considered “hyperendemic” with all four viruses circulating simultaneously in most years, but in varying proportion.1,2 Generally type 4 accounts for a lower proportion of disease in the neighborhood of 5–10%. Latin American patterns to date have exhibited varying circulation of dengue 1–3 and a paucity of type 4 virus.2–4 Although there is cross protective immunity between viruses for a brief period after infection with a single virus, that cross protective immunity wanes over a period of 4–6 months.5 As cross-protective immunity wanes, a point is reached where there is a hypothetical potential for increased severity of dengue disease with second infections, owing to a phenomenon called antibody dependent enhancement (ADE).6,7 A brief description of this process is that pre-existing, non-protective, sub-neutralizing antibody from prior infection with a dengue virus type enhances viral binding to Fc receptors on dendritic monocytes leading to increased viral replication within these cells. This is accompanied by complement activation and memory T cell activation; the latter has been shown capable of inducing a cytokine cascade that ultimately targets vascular endothelial cells, causing them to leak plasma and protein. This ‘plasma leakage’ is the pathogenic mechanism of serious dengue infection that has been called dengue hemorrhagic fever (DHF) and can lead to dengue shock syndrome (DSS) by virtue of extravasation of significant fluid volume from the intravascular spaces.8 Although ADE is a theoretical concern with regard to dengue vaccination, there is no evidence that vaccines actually promulgate the effect. There is one study that offers reassurance of the absence of vaccine induced ADE.9,10 In that study, 400 patients followed 3 to 8 years after immunization with an attenuated tetravalent vaccine experienced multiple subsequent infections with dengue viruses, without apparent clinical or serologic enhancement. In support of this vaccine trial's conclusions, pre-existing in vitro ADE levels have not correlated with disease severity upon natural infection.11 Clinical studies of antibodies in dengue infected infants have provided evidence suggestive of viremic enhancement with waning maternally acquired antibody, but a clear effect on disease severity is less clear.12,13 The numbers of patients in these studies is too small for statistically reliable conclusions. In addition, the risk of acquiring DHF is dramatically reduced as soon as responses have been induced against at least two serotypes.14 Nevertheless, there is a need for thoughtful planning of clinical evaluation of vaccine candidates to elucidate the short and long term efficacy and safety of the vaccines.

The complexity of this disease and its large impact on the world's tropical and sub-tropical populations has led to a need to craft specific guidelines for best practices (GCP) in carrying out vaccine efficacy trials with dengue vaccine candidates. The concern that a vaccine could theoretically enhance dengue severity further prompted the need for such a document.

These characteristics and complexity of dengue make demonstration projects (see Table 1 under Phase 3b) more important than with some other vaccines. However, there is an additional reason to consider demonstration projects for vaccines such as those against dengue, which will be applied in the developing world, where the introduction of new vaccines presents considerable economic and logistic challenges due to the scarcity of resources. For example, annual per capita expenditures on health may be only a few dollars. This makes the issue of balancing costs of vaccine purchase and delivery against benefits of vaccine use an important consideration. The exigencies of vaccine delivery in the developing world result in the need for studies that establish effectiveness of a vaccine under field conditions at an affordable cost.15

In this chapter, we consider the issues impacting conduct and design of dengue vaccine trials with reference to the recently published World Health Organization (WHO) guidelines for conduct of clinical trials of dengue vaccine candidates.16,17 We discuss challenges in evaluation of dengue vaccines ranging from randomized trials that establish ‘proof of concept’ to post proof of concept trials, particularly demonstration projects.

Definition of Terms (see Table 1).

Vaccine development proceeds through several phases of development beginning with preclinical testing which is carried out in various animal models to establish basic safety and immunogenicity before testing in humans. Human clinical evaluation begins with Phase 1 trials in small numbers of adults (∼20–30) to establish the lack of serious immediate reactions to the vaccine, to quantitate common reactions and to establish the likelihood of a favorable immunogenic response to the vaccine antigen(s). This is followed by Phase 2 testing in target populations and likely vaccination age groups. The number of subjects ranges from 30–300 (or more) in a single trial. These studies establish greater levels of safety and immunogenicity in varied ages, with dose ranging to determine the optimal response.18,19 When dosing, immunogenicity and safety at this level have been established, a Phase 3 trial is usually undertaken. These trials are generally randomized and placebo controlled to further evaluate safety and clinical efficacy in a minimum target population of 1,000–5,000 depending on the expected disease incidence in the given population, expected vaccine efficacy and a power computation to detect differences between vaccine and control groups. Safety concerns may require phase 3 trials numbers in excess of 10,000 with multisite trial design. If acceptable levels of efficacy and safety are established, the vaccine may be licensed and further safety data are gathered via active and passive reporting of adverse events following mass distribution in Phase 4 studies.15,20–22

Terminology that refers to levels of development in clinical vaccine testing has become progressively more complex with the addition of terms such as Phases 2b and 3b (defined below) in addition to the more standard Phases 1 to 4. A Phase 2b trial is generally smaller than a Phase 3 trial and as such, lacks the size and statistical power required of a Phase 3 trial. Nevertheless, a Phase 2b trial may be able to demonstrate vaccine efficacy, but with a broad confidence interval or alternatively may show a ‘trend’ toward efficacy, but not at a statistically significant level.16 Such a trial may not offer proof of efficacy, but is large enough to provide a strong suggestion of efficacy that justifies continued investment in the vaccine candidate under study as opposed to another candidate and thus large Phase 3 efficacy trials. This has been described as ‘proof of concept’, meaning that there is a reasonable suggestion from a 2b trial that the vaccine will prevent disease.

Proof of concept justifies further study of a vaccine candidate, but does not achieve all of the necessary elements required for a ‘pivotal trial’ that would make a candidate eligible for licensure. These pivotal requirements include statistical significance with a confidence interval that does not overlap zero, and the use in the trial of a final vaccine manufacturing production lot that is available for a scale-up to either a second efficacy trial or a large effectiveness trial. The requirement for use of final manufacturing production lot might be waived if there are established correlates of protection, but no such correlates are yet available for dengue.

Phase 3b trials are generally larger than Phase 3 trials and may take the form of large ‘demonstration’ trials that may or may not be randomized. These trials may involve tens of thousands of subjects, and can also assess vaccine efficacy but have a much greater ability than smaller Phase 2b and Phase 3 trials to assure safety of a vaccine because of their much larger size. Phase 3b trials can be especially useful if there are concerns about rare adverse events related to a vaccine. The large size of a Phase 3b trial allows a fuller measurement of safety and can provide assessment of effectiveness in the more practical circumstances in which vaccines are administered. As a result of the size they can be very expensive and industry is not likely to undertake them before vaccine licensure.

Multi-year demonstration projects can be conducted in a controlled fashion to provide critical information on efficacy, effectiveness or extended safety early in the execution of the projects. A recent example of phase 3/3b demonstration size projects addressing efficacy and safety were carried out with two candidate rotavirus vaccines, each being conducted in over 60,000 infants in multiple locations. The trials were designed to detect possible vaccine-associated intussusception, a rare adverse event which had occurred with a prior licensed rotavirus vaccine.22 These trials provided reasonable assurance that the new rotavirus vaccines did not cause intussusception.23,24

Effectiveness is distinguished from efficacy in that it is a measure of how well a vaccine protects against disease under conditions that are not as idealized and controlled as in Phase 3 efficacy trials where confounding variables have been tightly controlled. Phase 3b and 4 trials can also assess effectiveness measures such as herd immunity and compatibility with co-administered EPI vaccines that would not likely be evident in a Phase 3 trial.15 Recent examples of Phase 4 effectiveness are experiences with Haemophilus influenzae type b (Hib) and pneumococcal conjugate vaccines. These polysaccharide-protein conjugated vaccines have been shown to reduce disease in non-immunized child20,25–27 and adult populations.28–31 Finally, Phase 3b demonstration projects can measure effectiveness in countries other than the one where the vaccine was first licensed. An example would be hepatitis B vaccines, licensed in the United States and later demonstrated to be effective in a quite different setting, The Gambia.32,33

Phase 2b, 3 and 3b trials can establish proof of concept since each may be the first type of trial used to demonstrate that a vaccine will be effective in disease prevention. Proof of concept is, however, more likely to come from smaller Phase 2b or 3 trials. The large, multisite rotavirus trials noted above might be examples of controlled Phase 3b trials that yielded proof of concept, but in addition provided sufficient data to qualify as a pivotal trial for licensure. The rotavirus trials also provided enhanced safety and effectiveness data as well, by using multiple sites where conditions varied from one site to the next.23,24 The larger Phase 3b trials, if they occur, more often follow Phase 2b or 3 trials, justified by proof of concept established by the smaller trials and the need to assess rare adverse events or effectiveness through staged introduction. For example, the Gambian hepatitis B vaccine trial used a ‘stepped wedge’ vaccine introduction design (randomized design involving sequential roll-out of an intervention to individuals or clusters over a number of time periods) allowing for comparison of disease rates in immunized and non-immunized groups. This method allows for the determination of vaccine effectiveness on a large scale while ensuring that eventually all participants receive the vaccine, which has been shown to be safe and efficacious in one or more locations but has not been tested in the particular environment and population of the trial.32,33

Status and Challenges of Dengue Vaccine Development

The last decade has seen dramatic advancement in the development of tetravalent dengue vaccine candidates being tested for their safety, immunogenicity and protective efficacy. The Pediatric Dengue Vaccine Initiative (PDVI) is a program of the International Vaccine Institute (IVI) and is a product development partnership (PDP) that has identified five reasonably advanced vaccine candidates for its portfolio, four of which are live viruses attenuated by a variety of mechanisms. The remaining candidate is a subunit vaccine. These vaccine constructs are briefly described in Table 2. The WRAIR/GSK36 and Acambis/s-p40 vaccine candidates have undergone Phase 2 trials in humans. The sanofi pasteur vaccine is currently being evaluated in a Phase 2b trial in Thailand. For the National Institutes of Health (NIH) vaccine, monovalent components and some multivalent combinations have been administered in Phase 1 trials, and a tetravalent formulation has been prepared and is ready to go into Phase 1 trials. Of the remaining two candidates, CDC/InViragen has been tested in non-human primates in preparation for Phase 1 trials and the Hawaii Biotech subunit vaccine is undergoing monovalent and is being prepared for tetravalent Phase 1 trials.

Evaluation of dengue vaccines for efficacy and safety will require large-scale clinical trials conducted in dengue endemic areas, most located in developing countries. The recently revised WHO guidelines for conducting dengue vaccine trials specify that the primary end-point should be all virologically-confirmed dengue illness irrespective of severity, and that trials must be conducted in sites where fever surveillance has accurately established dengue incidence. In addition, the determination of long-term vaccine safety will require large numbers of participants (3,000–5,000) followed for a minimum of three to five years. In a developing country, following this number of subjects for this long can be a daunting task.

Other challenges in clinical evaluation of dengue vaccines include:

1. The need to conduct trials in both Asia and the Americas because of apparent different dengue disease outcomes.

2. Temporal variation of circulating dengue virus serotypes, the proportion of any single serotype circulating in a given population, and the need to determine vaccine efficacy or effectiveness against each serotype. The variability in the proportion of dengue virus type occurrence in a given area by year poses a special challenge in determining adequate sample size to achieve desirable statistical power to reliably detect vaccine efficacy against any singe virus type.

3. Potential confounding of vaccine outcomes by serum cross-reacting antibodies to other flaviviruses, especially Japanese encephalitis and yellow fever viruses and

4. The need to identify an immune correlate for protection.

Future Challenges

(A) Temporal variation in circulation of the four dengue virus types, lack of long-standing heterologous immunity and uneven exposure to all of the virus types over time suggest a need for large trial populations to determine vaccine efficacy and effectiveness for each serotype: Trial sample size should ideally be large enough to detect uncommon vaccine adverse events (AEs), long-term dengue complications and effectiveness against at least one serotype, or a composite of several circulating serotypes encountered during the trial. It is unlikely all 4 serotypes will be circulating simultaneously in one site during a pivotal trial. Multiple trials or trial sites may be required to confirm protection against each of the four serotypes. At a minimum, the sample size should be large enough to show a targeted efficacy with 90% or 95% power. In practical terms, adequate sample size for this purpose may be prohibitively expensive in some situations. In addition, a Phase 3 trial or trials must enroll 3,000–5,000 individuals for safety analysis before licensure, which assumes a sample adequate to detect AEs occurring at a rate of 1/1,000. As stated in the Background section, the estimated number of enrollees for a dengue trial will be higher than estimated for most other vaccines for several reasons. First, we must detect possible late enhancement of dengue infections as a consequence of waning vaccine-induced immunity and second, trials must be conducted in multiple sites to maximize the chance of establishing efficacy against each virus type. Alternatively, post proof-of-concept demonstration projects could meet this need, but multiple sites would still be required.

There are several options to accelerate the progression from efficacy trials to effectiveness studies, including conducting effectiveness studies in specially developed sites not previously used for efficacy trials. Another option is to progress from a smaller efficacy trial to a larger effectiveness trial at the same site. This could be done by continuing to conduct dengue surveillance in populations in the same catchment area as the vaccine efficacy trials. An advantage to this approach would be that it would minimize the resources and lead-time required to establish accurate dengue surveillance, and could also provide important information regarding extended effects of vaccination, such as herd protection. Such trials could help establish serologic correlate of protection to each of the four dengue virus types, an outcome not likely to come from a single efficacy trial. Regulatory agencies may require expanded trials to establish clear protective correlates for all four viruses. Such correlates, in turn, would help to accelerate licensure of ‘second-comer vaccines’ on the basis of their ability to evoke these correlates of protection, even if differences in design of these vaccines required efficacy studies as well. Having a correlate of protection to measure against could be quite useful in dose ranging and save time and expense as a result.

(B) Another challenge is the possibility of rare adverse events, such as vaccine-induced DHF, presumably via ADE triggered by vaccine virus rather than wild-type virus infection. Even though vaccine-induced ADE seems unlikely, it has yet to be ruled out: As discussed in the Background section, if all four virus types in a vaccine induce high titers of long-circulating neutralizing antibody the likelihood of vaccine-induced ADE may be negligible. If on the other hand, one of the vaccine virus types induces low neutralizing antibody titers, or there is rapid waning of such antibody leaving a residual of high, non-neutralizing ADE antibody, there may be potential for vaccine-induced ADE to the poorly performing vaccine virus types (attenuated vaccines) or antigen(s) (subunit vaccines) in some individuals. For this reason it seems likely that large, post proof-of-concept studies may be needed to determine dengue vaccine effectiveness against multiple dengue virus serotypes while simultaneously providing for broader assessment of safety including low probability events such as vaccine-induced ADE. Bridging studies using post proof-of-concept demonstration models employing proven, immune correlates of protection (discussed in the next paragraph) could also be used to extend conclusions regarding protective efficacy among different populations, or between different vaccines. These types of trials and Phase 4 follow-up studies subsequent to licensure would provide considerably more data on rare adverse events.

(C) The need for a correlate of protection: It would be ideal to identify at least one correlate of protection in early vaccine trials so that subsequent vaccine candidates might be licensed on the basis of their safety profile and ability to produce an immune response measured through the established correlate of protection. Affordability of dengue vaccines may also be enhanced if second-comer vaccines produced by developing country manufacturers could be licensed on the basis of comparability to previously proven vaccine candidates, thus avoiding the expense of additional large-scale Phase 3 efficacy trials.

If an immunologic correlate of clinical protection is verified via a Phase 3 field trial, it can serve to establish non-inferiority between a licensed vaccine and a new vaccine candidate. Because there have been no such field trials to date, no proven immunologic correlate or correlates of protection currently exist against dengue. Serum neutralizing antibodies measured by the plaque reduction neutralization test (PRNT), is the most likely and most frequently utilized assay for protection. Assay procedures should be carefully standardized, following the WHO guidance document on PRNT.41 Control sera for assay validation are currently being produced. A micro-neutralization assay is being developed by industry to help standardize and facilitate the high throughput needed for large-scale vaccine trials. Although it appears unlikely that cell mediated immunity (CMI) will provide a single correlate of protection, specific CMI assays may be suitable for characterizing important vaccine features, such as detection of immunological memory and durability of protection,42 CMI data may also help to corroborate the safety profile of a vaccine, including potential for induction of immunopathology.

In summary, if a correlate of protection is identified, a bridging study hypothesis could be confirmation of “no clinically meaningful difference in the seroprotection rate between groups”. Bridging studies may also assess any changes in manufacturing processes for dengue vaccines, new dosing schedules and new target populations based on age, genetic or environmental characteristics.16,17,43

(D) Securing necessary information to license and introduce dengue vaccines shown to be efficacious into developing country immunization programs: Phase 3 trial results do not provide all the information needed to mount a dengue vaccine program. Such commitment depends on many political, economic and public health factors. For example, registration/licensing by the relevant national authority and optionally, by an internationally recognized registration authority (such as the WHO prequalification process for assessing manufacturers and national regulatory authorities), is expected if Phase 3 trials demonstrate protection at some predetermined level, and all relevant safety data support a favorable risk-benefit outcome. At this point, several criteria should be fulfilled before moving forward into a regular vaccination program:

1. A rigorous Phase 4 trial is feasible and likely to be conducted immediately after licensure.

2. The vaccine is likely to yield a measurable public health benefit.

3. The political will to use the vaccine by the government and commitment by the manufacturer to provide vaccine for the national program.

4. The vaccine is likely to generate advance orders and economic return to the vaccine developer/manufacturer.

This all presupposes adequate regulatory authorities and processes in place at a country level or at least a regional level that countries within a region could access. The regulatory process should exist not only for vaccine trials at Phase 1–4 stages, but also for manufacturing processes where countries are engaged in vaccine manufacturing. Unfortunately these requirements are not uniformly available in the developing world. There is need for assistance from WHO and others in fostering development of such processes. WHO has begun to address these issues through a Developing Country Vaccine Manufacturers Network, a Developing Countries' Vaccine Regulatory Network and other initiatives.

(E) Potential confounding of vaccine outcomes by cross-reacting antibodies to other flaviviruses: There is potential for confounding of vaccine outcomes by cross-reacting antibodies to other flaviviruses induced by vaccination or by wild virus infection, especially by Japanese encephalitis virus in Asia and yellow fever and West Nile viruses in Latin America and Africa. Since these infections vary by region, there may be a need for demonstration trials to be carried out in multiple regions to verify effectiveness after a successful efficacy trial in another part of the world. In addition, there is the intriguing prospect of measuring enhancing or detrimental effects of prior exposure to other flaviviruses on dengue vaccine performance. This would likely require trials of a larger scope than standard Phase 3 trials.

(F) There may be a need to demonstrate vaccine efficacy in differing areas of the world: There are several apparent differences in the epidemiology of dengue in Latin America and Asia. Dengue was very much reduced in the Americas in the 1970s by the combined use of several vector control strategies such as covering water containers and widespread use of DDT. Once the latter stopped, dengue gradually made its return, but there are usually only one or two virus types in circulation at one time, unlike the situation in Asia where all four virus types can co-circulate, although generally in unequal proportion. These epidemiological variations may lead to differing risks for severe disease and may also result in differences in population age strata at risk. There are suggestions in the literature that there is more adult disease in parts of Latin America than in Asia. There is also documentation of different genotypes of dengue virus types circulating in the Americas and Asia. When introduced from Asia, a genotypic variant of virus type 2 led to increased pathogenicity in the Americas. These factors may lead to the need for post proof of concept trials to show equivalent effectiveness of a vaccine in the two major regions where dengue is best understood, i.e., Asia and Latin America, where simultaneously trials could be conducted over 3–5 year time frames as was done with the recent Rotavirus vaccine trials.23,24 Africa may be eligible for such trials if reliable dengue incidence or even prevalence data can be obtained.

(G) Measurement of herd protection: A dengue vaccine with sufficiently high vaccine efficacy might well reduce the number of viremic individuals from which mosquitoes can obtain and subsequently transmit the virus. This could lead to a reduction of dengue transmission and so enhance vaccine effectiveness in a region not completely immunized. Such herd protection has occurred with conjugate Hib and pneumococcal vaccines that reduce nasopharyngeal carriage of the respective bacteria and thus reduce the transmission of disease to non-immunized individuals.20–22,25–29 Oral polio vaccine achieves a similar effect by virtue of transmission of vaccine virus shed in infant stools, resulting in enhanced community immunity in non-immunized individuals. It would probably require large numbers of exposed community members in both vaccinated and non-vaccinated cohorts to determine this herd effect. The lower the actual vaccine efficacy, the larger the number of non-vaccinated individuals needed to determine a herd effect. It may require a vaccine of 80% efficacy to reduce dengue virus transmission to a point where the herd effect is detectable and a high vaccine coverage rate (as much as 85%) may be necessary to achieve herd immunity, depending on vaccine efficacy.44

Resources Needed to Meet the Challenges

The challenges cited above are largely scientific and the technology exists to resolve them if financial and logistic resources could be made available. Questions involving correlates of protection and interfering flavivirus antibodies will require laboratory supported clinical studies in Phase 3 trials. Many of the additional challenges cited above will require demonstration Phase 3b and Phase 4 trials to obtain suitable answers. These trials are far more expensive than Phase 3 trials and could amount to several hundred million dollars.

Large scale trials that approximate the type of demonstration projects anticipated here were recently conducted for two new rotavirus vaccine candidates. The concerns about dengue vaccine-induced ADE and multiple dengue serotypes are analogous in some ways to the rotavirus experience, where rare adverse events and a need to demonstrate effectiveness against multiple infecting serotypes of rotavirus were also a concern. The trials each enrolled more than 60,000 subjects in various parts of the world and were very costly. If needed, it is likely that the institutional structures needed for the dengue effort are largely in place or can be built in a reasonably short period of time. Pieces of that infrastructure reside in country Ministries of Health and Regulatory Authorities, pharmaceutical companies, academic institutions and field sites with sufficient laboratory capacity and clinical trial experience to carry such a large effort forward. Infrastructure building by the PDVI through its field site Consortium and regional Dengue Prevention Boards, have already provided some essential building blocks for future clinical trials and dengue surveillance. Funding for these efforts has thus far come from private philanthropic foundations, vaccine developers and to a limited extent, dengue endemic countries. A unique resource challenge is that faced by developing country manufacturers who do not have the same development resources at their disposal as large western pharmaceutical companies to carry out expensive and burdensome Phase 4 trials. Part of the solution to this may be for developing country manufacturers to use phase 3b demonstration projects such as the step wedge introduction of hepatitis B vaccine in the Gambia to reduce expense of post introduction monitoring.32,33

Summary and Conclusions

We have summarized and highlighted some of the issues discussed in greater detail in the recently published WHO guidelines for the clinical evaluation of dengue vaccines in endemic areas.16,17 The overall conclusion is that dengue is a complex disease leading to substantial clinical evaluation challenges:

1. It is caused by four serologically distinct viruses, and there is only short-lived cross-protection. The epidemiology of disease varies between Asia and Latin America, posing a need for trials in multiple sites in these regions in differing age groups with differing exposure over time to different virus types.

2. Infection with a sequence of dengue serotypes may lead to greater risk of severe disease, thus requiring simultaneous administration of all four serotypes in a tetravalent vaccine. Additionally, there is an unproven theoretical concern that vaccine might predispose to severe disease if a vaccine were insufficiently immunogenic against one or more dengue serotypes and

3. The diagnosis of dengue infection and evaluation of dengue illness and vaccines is confounded by poor diagnostics, lack of correlates of protection, and the fact that dengue virus often co-circulates with other flaviviruses, which cross-react serologically and possibly biologically. The special safety concerns and unpredictable circulation of different dengue serotypes may require large, long term and expensive field trials to assure vaccine licensure for the large populations at risk for dengue infection and disease. All this is complicated by the lack of good regulatory structures in many areas of the developing world where dengue vaccines would be utilized.

To adequately address these issues it will be necessary to strategize comprehensively, taking into account all of the challenges noted above in order to avoid some of the difficulties that have occurred with vaccines such as hepatitis B. After first licensure, hepatitis B vaccine was not widely available to highest risk populations for over twenty years, until it was made available through developing country manufacturers. Recent experience with rotavirus vaccines illustrates the utility of large multisite trials to address many of the concerns outlined here, but comes up short of a mechanism for providing affordable vaccines via developing country manufacturers. This means that even if dengue vaccine or vaccines were proven safe and efficacious, they may not be available to many developing countries in need, mimicking the decades' long delay attending availability of hepatitis B vaccines.

Figures and Tables

Table 1 The clinical trial phases used in the development and evaluation of vaccine candidates34

Phase	Type of study	Purpose	Sample size	Comment
Phase 135	non-randomized, may be a blinded comparator	to examine safety and immune responses, viral shedding for live virus vaccines, dose evaluation	small number, 15–50 depending on trial design, e.g., multiple doses or formulations usually healthy adult volunteers not from the target population, may later include children	While establishing lack of *AEFI is the primary goal of Phase 1, human immunogenicity can also be demonstrated on a limited basis
Phase 236,37 (Phase 2a)	may involve comparator vaccines, may include dose ranging, may include age variation	to further examine safety and immunogenicity, and establish optimal dose and immunization schedule	30–100 s; in target populations and likely vaccination age groups	trial vaccine may be administered at the same time as other routinely scheduled vaccines
Phase 2b38	randomized, doubleblind, placebo or comparator vaccine—controlled	to obtain a preliminary estimate of vaccine efficacy (but with wide confidence interval), and to further examine safety and immunogenicity	larger than Phase 2, but limited by power to detect a significant difference, absence of production lot for scale-up studies	proof of concept: that the vaccine candidate protects or shows a trend toward protection against target disease in vaccinated persons in the final target population, using the final vaccine formulation
Phase 339	randomized, doubleblind, placebo (or another vaccine)—controlled	to determine statistically significant vaccine efficacy with a production lot that can be scaled-up, and to examine safety and correlates of protection	established − priori; depends on endpoints, and may involve several thousand participants (in some cases even >10,000), usually from the target population	usually considered as ‘pivotal’ for licensure. Achieves expanded safety assessment and lot consistency.
Phase 3b32,33	various designs: randomized controlled, ‘stepped wedge’ introduction, geographically focused introduction	to examine rare AEFIs, or to examine the efficacy or effectiveness of the vaccine in a different population from the Phase 3 study	larger than Phase 3 (>10,000); may be a staged introduction of the vaccine over a geographical region/country	these are sometimes termed ‘demonstration projects’
Phase 420–22, 25–28	non-randomized; observational surveys of vaccine uptake, surveillance for target disease and rare AEFI*	post-licensure studies to assess rare adverse events and overall effectiveness under routine conditions	entire regional or country populations	can also examine herd immunity effects

* AeFi, adverse event following immunization.

Table 2 Current PDVI portfolio of dengue vaccine candidates

Developer	Commercial partner	Approach
Live Attenuated
WRAIR*	GSK**	Cell culture passage
Acambis (acquired by s-p, 2008)	Sanofi pasteur (s-p)	17D Yellow fever—dengue chimera
NIH/NIAID/LID¶	Biological E, Butantan, Panacea, Vabiotech	Dengue 4—dengue chimeras or attenuation by nucleotide deletion
CDC§	Inviragen	Attenuated Dengue 2—Dengue chimera
Subunit
Hawaii Biotech	Hawaii Biotech	Envelope +/− NS1 recombinant proteins

* WRAIR, Walter Reed Army Institute of Research.

** GSK, GlaxoSmith-Kline.

¶ NIH/NIAID/LID, US National Institutes of Health/National Institute for Allergy and Infectious Diseases/Laboratory of Infectious Diseases.

§ CDC, US Centers for Disease Control and Prevention.

Acknowledgements

The Pediatric Dengue Vaccine Initiative is a Bill and Melinda Gates funded project.