Elsevier

Biotechnology Advances

Volume 29, Issue 2, March–April 2011, Pages 239-247
Biotechnology Advances

Research review paper
Review of dengue virus and the development of a vaccine

https://doi.org/10.1016/j.biotechadv.2010.11.008Get rights and content

Abstract

Dengue viral infection has become an increasing global health concern with over two-fifths of the world's population at risk of infection. It is the most rapidly spreading vector borne disease, attributed to changing demographics, urbanization, environment, and global travel. It continues to be a threat in over 100 tropical and sub-tropical countries, affecting predominantly children. Dengue also carries a hefty financial burden on the health care systems in affected areas, as those infected seek care for their symptoms. The search for a suitable vaccine for dengue has been ongoing for the last sixty years, yet any effective treatment or vaccine remains elusive. A vaccine must be protective for all four serotypes of dengue and be cost-effective. Many approaches to developing candidate vaccines have been employed. The candidates include live attenuated tetravalent vaccines, chimeric tetravalent vaccines based on attenuated dengue virus or Yellow Fever 17D, and recombinant DNA vaccines based on flavivirus and non-flavivirus vectors. This review outlines the challenges involved in dengue vaccine development and presents the current stages of proposed vaccine candidate development.

Introduction

In recent decades, incidences of dengue have grown dramatically, making it a global health concern. To date, no licensed vaccine is available. This has brought together many groups including: The Pediatric Dengue Vaccine Initiative (funded by Bill and Melinda Gates Foundation), the WHO, the US military, as well as industry and governments in many different countries to collaborate in the hopes of accelerating the development of a successful vaccine.

Dengue is a vector-borne virus, transmitted to humans via infected Aedes mosquitoes in tropical and sub-tropical areas. The severity of the disease varies from asymptomatic infections, to a febrile fever, or potentially life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS).

The WHO reports that two-fifths of the world's population is at risk of dengue infection, with an increase in the annual number of cases. The virus is now endemic in more than 100 countries, significantly affecting South-East Asia and the Western Pacific, and in some countries becoming the leading cause of hospitalization and death among children (World Health Organization, 2009a, Simasathien and Watanaveeradej, 2005, Gubler and Meltzer, 1999). Over the past 50 years there has been an apparent 30-fold increase in reported cases of dengue virus infection (Kyle and Harris, 2008). The sharp increase of reported cases of infection after 2000 has been maintained at a reported average annual level of 100 million, although it is understood that that number of actual infections could be even higher. This translates to around 500,000 to 1 million cases of the more severe forms of DHF and DSS, leading to around 22,000 deaths mainly among children (WHO, 2009a). The increase in incidence and disease severity is attributed in part to geographic expansion of the vector, the Aedes aegypti mosquitoes, leading to the increased co-circulation of all four dengue serotypes in urban areas (Eisen and Lozano-Fuentes, 2009). Fig. 1 shows the latest WHO assessment of the geographic distribution of the dengue virus.

There are four antigenically distinct, closely related serotypes of the dengue virus (DENV1–4), exhibiting a 65–70% sequence homology (Rico-Hesse, 1990). All four serotypes are causative agents of dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). DHF and DSS are the more severe effects of dengue virus infection and are more commonly reported in children and adolescents under the age of fifteen. Symptoms include fever, low platelet count, hemorrhagic bleeding, and vascular permeability. The viraemia is 10–100-fold greater than DF (Hammon et al., 1960). DSS is characterized by the shock that ensues when fluid leaks into interstitial space, and it is life threatening. The role of the immune response and the pathogenesis of DHF and DSS are not fully understood, thus leading to another obstacle in the hope of developing a successful vaccine against dengue virus (Halstead, 1988).

Primary infection with any of the four serotypes results in a lifelong immunity to that serotype, and temporary immunity to the others (Sabin, 1952). However, this temporary immunity usually wanes after 6 months, at which point an individual is susceptible to the other three DENV serotypes. The primary infection is most often asymptomatic, but sequential infections in the presence of heterologous dengue antibodies often leads to a more severe secondary infection causing DHF or DSS. This observation was made following an epidemic of DHF/DSS in Cuba in 1981 caused by Dengue 2. One of the major factors attributed to the severity of the dengue infection at this time was a prior outbreak of Dengue 1 some 4 years earlier (Bravo et al., 1987). This is attributed to antibody-dependent enhancement (ADE) when non-neutralizing antibodies form a complex with the virus and infect phagocytes via Fc receptors, causing the enhanced infection involving DHF and DSS (Halstead, 1989). ADE appears to be central to the severe pathogenesis of dengue in infants and may even be associated with sub-neutralizing levels of maternal antibody (Kliks et al., 1988). In order to avoid antibody-dependent enhancement (ADE), a vaccine would need to induce strong neutralizing antibodies against all four dengue serotypes simultaneously (Morens, 1994).

In recent years, research has focused on investigating any correlations between dengue serotypes and the severity of infections. While investigating the outbreaks in endemic areas, such as South East Asia, researchers have found that a primary infection with DENV-1 or DENV-3 results frequently in a more severe disease than if DENV-2 or DEV-4 were the primary infection (Vaughn et al., 2000). A further finding is that a secondary infection with DENV-2 is associated with severe dengue disease (Fried et al., 2010).

Fried et al. (2010) analyzed data collected in Bangkok, Thailand from 1994 to 2006. Their findings include:

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    an association between DHF and secondary infection.

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    DENV-2 is associated with severe dengue disease.

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    DENV-2 and DENV-3 are twice as likely to result in DHF as DENV-4 if acquired as a secondary infection.

A study conducted following the 2001 outbreak in Myanmar is unique in finding that, in an area in which all four serotypes of dengue were prevalent, a single serotype (DENV-1) became predominant, and seemingly displaced another (DENV-4). Myanmar reported 15,361 cases of DHF/DSS and 192 deaths in 2001, and analyses of patient samples revealed 95% of dengue virus isolated was DENV-1. It was found that 46% of the patients in the study had previously been infected, and there was a decrease in DSS cases in 2001 compared to previous years (1998 and 1999) (Thu et al., 2004). This is explained by the lack of DENV-2 serotype, which is associated with DSS, if acquired as a secondary infection (Sangkawibha et al., 1984, Anantapreecha et al., 2005).

Table 1 summarises the association between dengue virus serotype and disease severity based on studies from outbreaks in South-East Asia. The findings are derived from a limited sample size from specific populations, and the results may not be applicable to all populations. However, studies analyzing the serotype-specific associations of dengue disease will help in understanding the factors that influence disease severity.

Section snippets

Virus structure

Dengue is part of the Flaviviridae virus family, which also includes Yellow Fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and Tick-borne encephalitis virus. Dengue is a single stranded, positive-sense RNA genome approximately 10.6 kb long, composed of an open reading frame with genes encoding structural and non-structural proteins. The genome consists of three structural proteins; capsid (C), precursor membrane (prM), and envelope (E). The envelope glycoprotein is

Mechanism of infection

Upon primary infection with DENV, there is an incubation period averaging 4–7 days. In this time the virus replicates in the dendritic cells in close proximity to the bite, also infecting macrophages and lymphocytes, and finally into the bloodstream. Dendritic cells (DCs) are antigen-presenting cells that are integral to inducing an immune response. Wu et al. (2000) have demonstrated that the dengue virus preferentially targets DCs, specifically monocyte-derived DCs (resembling interstitial DCs)

Ideal vaccines

Dengue virus is becoming an increasing concern because of the lack of a licensed vaccine to provide protection against all four dengue serotypes (WHO, 2009b). The increase in dengue infections in recent years, as well as the prevalence of all four circulating dengue serotypes has contributed to the rise in DHF. In order to design a vaccine that is protective against all serotypes but without the potential risk of enhanced disease severity, the molecular mechanisms of dengue pathogenesis must be

Live attenuated vaccines

Live attenuated vaccines (LAV) tend to mimic the natural infection by inducing humoral and cellular responses which induce long lasting immunity, often from a single vaccination. LAVs contain a weakened form of a live virus that allows antibodies to both the structural and non-structural proteins of the virus to be produced. The potential for developing vaccines using live attenuated strains of all four serotypes has been widely accepted, considering this method was successful for yellow fever

Recombinant chimeric vaccines

Another approach to building a DENV vaccine has been to utilize molecular genetics. A recombinant chimeric vaccine is constructed by using the “backbone” of a related flavivirus (yellow fever, attenuated DENV strain), and replacing the prM-E genes with corresponding genes from DENV. The backbone would still contain the capsid and non-structural proteins and the 5′- and 3′-UTRs. The objective is to retain the attenuation properties from the “backbone” viral vaccine, and incorporate dengue

Cell culture bioprocessing for live-attenuated and recombinant chimeric vaccine production

All live attenuated and recombinant chimeric vaccine candidates should be propagated in an approved cell line such as Vero (kidney cells from African green monkey), MRC-5 (normal diploid cells from human lung), or FRhL (normal diploid lung cells from rhesus monkey) for human vaccine production. Since Vero cells are derived from a continuous line from the African green monkey (Cercopithecus aethiops), the cell growth for human vaccine production is restricted to 150 generations due to its

DNA and virus-vectored vaccines

Vaccines based on vectors using recombinant DNA technologies have recently been undergoing animal trials. DNA vaccines have the advantages of inducing intracellular antigen processing for adaptive immunity, being stable (meaning they could not revert to a pathogenic phenotype), and easy manufacture. DNA vaccines do not have the complications associated with live replicating viruses, namely problems of combining monovalent formulations to form tetravalent vaccines, thus inducing serotype

Recombinant protein vaccines

The envelope E protein of DENV has been selected as the major antigen for recombinant protein vaccine development. Expression of the DENV E protein usually requires the expression of prM protein which may act like molecular chaperone to help the correct folding of the E protein. In addition, the prM-E protein expressed intracellularly will be automatically cleaved into prM and E by cellular furin. Studies have also demonstrated that the expression of 80% E (r80E) at the N-terminal can assist

Conclusion

It is clear that a better understanding of immunopathological mechanisms between dengue virus and its target cells (dendritic cells, hepatocytes and endothelial cells) is needed to develop a completely protective vaccine. More research must be conducted to identify the components responsible for the serotype interference/dominance portrayed in the construction of tetravalent vaccines.

Currently, there is no consensus as to how an individual is protected from dengue virus. Generally, strong

Abbreviations

    ADE

    Antibody Dependant Enhancement

    CDC

    Center for Disease Control and Prevention

    CNS

    Central Nervous System

    DC

    Dendritic Cells

    DC-SIGN

    DC-Specific ICAM-3-Grabbing Non-integrin

    DENV 1–4

    Dengue virus serotypes 1–4

    DF

    Dengue Fever

    DHF

    Dengue Hemorrhagic Fever

    DSS

    Dengue Shock Syndrome

    HIV

    Human Immunodeficiency Virus

    ic

    intracerebral

    id

    intradermal

    IFN

    interferon

    ip

    intraperitoneal

    JEV

    Japanese Encephalitis Virus

    LAV

    Live Attenuated Virus

    LC

    Langerhans Cells

    mAb

    monoclonal antibodies

    NIAID

    National Institute of Allergy and Infectious

Acknowledgements

We acknowledge the Natural Sciences Research Council (NSERC) of Canada for funding in the preparation of this review. SCW also acknowledges the dengue research works supported by the National Science Council, Taiwan.

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