February 4 2010
Parasites and Pestilence
Dengue: Challenges in making a vaccine
i. Introduction- What is dengue
Dengue consists of four positive-stranded RNA viruses, DENV-1, DENV-2, DENV-3, DENV-4, in the genus flavivirus which includes yellow fever, Japanese encephalitis, and tick-borne encephalitis (Williams). Flaviviruses are characterized by common group epitopes on their envelope proteins that result in extensive cross-reaction in serologic tests (Seema et al 1). The virus spreads in the body from uninfected to infected cells like other virus with virus assembly in the infected cell, release of infective virions, from the infected cell into extracellular space, attachment of the virus to receptor structures on the host cell surface, their internalization by endocytosis, penetration through a host cell membrane, uncoating of the viral genome, and its inter-cytoplasmic relocation to the nucleus and or other sites in within the cell (Seema et al 1). Dengue manifests itself through vascular permeability leading to leakage from vascular compartments (WHO), however the exact mechanism in which it does this is not completely understood. A study done by Kanjaksha Ghosh et al demonstrated that exposure to dengue virus of platelets activated them with an increase in P-selectin expression(cell surface receptor present on endothelium and stimulated platelets that is involved with neutrophil migration into inflamed tissue), fibrinogen-binding property(fibrinogen is responsible for coagulation of blood), and caused morphological changes such as altered platelet membrane architecture, degranulation, presence of filopodia, and dilation of the open canalicular system(channels of the platelet system) (Ghosh 1). Flaviviruses as a whole are also shown to cause transient suppression of haematopoiesis (La Russa). A study done by Sharone Greene et al suggests that plasma leakage associated with dengue virus is caused by the circulation of high levels of secreted NS1 in the presence of pre-existing heterologous non-neutralizing antibody (Greene 1) NS1 is one of the 7 dengue structural proteins that are thought to be involved in viral replication (Abcam).
Dengue is an acute febrile disease that in humans cause disease characterized by a four day incubation period, rashes on the skin, vomiting, nausea, headache, loss of appetite, and joint and muscle pain (Dengue). The high fever is usually the initial symptom of the disease and lasts for a duration of a few days. The subsequent symptoms occur following the fever as platelet levels begin to drop. The disease is nicknamed bone break fever because of the intense joint and muscle pain that is often likened to the feeling of bones breaking. Dengue hemmorhagic fever is a dangerous potential complication of dengue. It has similar symptoms and is caused by the same virus but the fever is more severe, the drowsiness and lethargy are more extreme, there is increased vascular permeability, and abnormal hemostasis(the stopping of bleeding) (WHO). There is evidence that getting dengue more than once increases chances of its development into hemorrhagic fever.
The first recorded cases of dengue similar sicknesses occurred in China in 256-420 AD. The first documented case of the disease was in 1780 by Benjamin Rush in Philadelphia.
Dengue is found in tropical and subtropical areas around the globe and is usually in urban or semi urban areas (WHO). It is endemic in 100 countries in Africa, the Americas, the Mediterranean, South-east Asia, and the Western Pacific and there exists the high risk of spreading due to globalization. On February 25, 2010 Tully, Australia had its first locally contracted case of dengue in 20 years (Mawer 1). Cape Verde in Africa reported its first outbreak in 2009 with 12,000 suspected cases and 6 deaths (CDC). The WHO estimates that 2.5 million are at risk of dengue globally and that it infects about 50 million people a year. 500,000 people require hospitalization for dengue hemorrhagic fever yearly with a 2.5% mortality. Without treatment mortality is 20% for hemorrhagic dengue fever.
Dengue is transmitted by the mosquito Aedes Aegepti and vector control is an important method of prevention. Researchers in UCI are actually working on creating a breed of flightless female mosquitos to help curb dengue. The females would not survive in the wild very long and their progeny would either be flightless females or males capable of flight. Male mosquitos do not take blood meals so they are not a problem (“Wingless Mosquitos”). Reservoir hosts for dengue include cattle, bats, and sheep (“Viral Disease”). There are is no cure or vaccine for dengue but replacement of plasma losses with plasma expander or fluid and electrolyte solution drastically decreases mortality and is used to sustain patients until the infection is over(WHO). If leakage is severe blood transfusions may become necessary. Dengue fever is diagnosed by ELISA or PR-PCR in the laboratory (MedicineNet).
Figure 1:Dengue cases by year and number of countries affected
Graph by the World Health Organization
ii. Antibody Dependent Enhancement
As mentioned earlier, dengue has four strains or subtypes. These strains have 60-80% homology. The differences lie in the surface proteins of the virus. Dengue exhibits a lot of diversity and is though to be highly morphogenic as most RNA- dependent RNA polymerases are. The serotypes can be subtyped through RSS-PCR, nucleotide sequencing, restriction enzyme analysis, and RNase Ta oligonucleotide fingerprinting (Marize. 1). The specific differences between the 4 serotypes are not fully understood but in a study done by Angel Balsameda et al it was shown that in Nicaragua DENV-2 was characterized by more shock and hemorrhage. DENV-1 on the other hand was associated with greater vascular permeability (Balmaseda, Angel). A study done in Mexico suggests that serotype also affects the extent of liver damage caused by dengue with DENV-2 being the most severe and then DENV-1 and DENV-3 respectively (Vasquez). A study by Sumalee Jindadmrongwech et al demonstrated that several virus binding proteins are seen for each serotype and this suggests that there is a serotype specific component regulating the entry of the dengue virus into cells (Jindadamrongwech). In a study done in Thailand it was found that DENV-3 caused the most serious infections (Endy).
It is proposed that the large variability between the strains of dengue can be explained by the process of antibody-dependent enhancement (ADE)(Kawaguchi). This process results from a new infection in an individual with acquired immunity to a different sterotype and leads to increased mortality. When a person is reinfected by a second strain the virus tends to grow more rapidly and is more likely to develop into dengue hemorrhagic fever or even dengue shock syndrome which results when dengue hemorrhagic fever causes circulatory collapse. The proposed mechanism of ADE is that virus specific antibodies enhance entry of the virus into macrophages and granulocytic cells through interaction with Fc and receptors. The virus exploits the immune system’s antibodies to previous strains to cause a more acute response. Because, the strains have similar surface proteins, antibodies to previous strains will bind to new strains but not inactivate them. The immune response attracts macrophages which are readily infected since the virus is not inactivated.
Another study performed by Takol Chareonsirisuthigal explored the effects ADE on suppressing the immune response (Caheonsirisithgul). In the study it was found that a second infection with dengue virus resulted in suppression of the transcription and translation of IL-12, IFN-gamma, and TNF- alpha which are pro-inflammatory cytokines. Cytokines are important in the immune response as signaling cells. Dengue was also shown to facilitate the expression of IL-6 and IL-10 which are anti-inflammatory cytokines. This suggests that ADE infection modifies innate and adaptive intracellular antiviral mechanisms resulting in unrestricted DENV replication in cells. Studies done with the T-cell repertoire after secondary DENV infection suggest that expansion of pre-existing memory T- cell populations with greater avidity for the primary but not secondary serotype, may be responsible for sub-optimal viral clearance (Swaminathon 370).
iii. Challenges in developing dengue vaccines
The phenomenon of ADE and its effects on the severity of subsequent dengue infections causes obstacles in the development of a vaccine. It logically follows that if subsequent infections cause worse cases of the disease, the vaccine must be able to address all 4 strains simultaneously or the vaccine may cause dengue infections to be even more severe instead of preventing infection. This is dangerous especially since people who get dengue usually live in endemic areas where they remain at risk to being reinfected. This raises challenges because the 4 strains have many distinct differences.
Another challenge to vaccine development is that the mechanism for protective immunity is not completely understood. It is thought that neutralizing antibodies mediate protection against DENV infection by blocking the attachment of virus to target cells, but this is not proven. Another great challenge is the lack of an appropriate animal model system to evaluate experimental dengue vaccines. Mice and monkeys have been used but they do not manifest ay dengue-like illness so pre-clinical assessment to preface trials done on humans at the moment are difficult to conduct (Swaminathan 370). The best primate model at the moment is the rhesus macaque but its levels of viraemia are substantially lower than in humans. The virus also has difficulties in growing in cultures.
iv. Vaccines in Progress
The WHO has designated the dengue virus as a high-priority target for accelerated vaccine development so that it can expedite its introduction into developing countries (WHO). Currently there are multiple vaccines in the development and clinical trials.
In 1980 a live attenuated tetravalent dengue vaccine was developed by MU’s Center for Vaccine Development at the Institute of Science and Technology with a grant from the WHO. The monovalent attenuated viruses included DENV-1 PDK13, DENV-2,PDJ53, DEN-3 PGMK30/FRhL-3, and DENV-4 PDK48. MU partnered with Aventis Pasteur and they have completed Phase 1 and Phase 2 IIa clinical trials with a 2 dose regimen. The vaccines have an acceptable safety trial and elicit satisfactory immunogenicity in children and adults. They are scheduled to begin Phase 2b in adults and children and hope to enter Phase III in an endemic country (Yokson).
In total,7 vaccines are in the works. Walter Reed Army Institute of Research (WRAIR) and GSK are also developing a live attenuated vaccine that works through cell culture passage and is in Phase II clinical trials as well. Acambis and Sanofi Pasteur are working on a live attenuated vaccine that is a yellow fever- dengue chimera and they have just completed Phase I clinical trials. NIH and Biological E are working on dengue chimeras with deletions and they are in the late stages of development(Almond). The CDC and InViragen/shantha are working on dengue-2 and dengue chimeras and have just passed Phase 1 clinical trials(CDC). HBI and Hawaii Biotech are working on a vaccine utilizing inactivated subunits and are exploiting envelope and NS1 recombinants. and are working on Phase 1 clinical trials (Pediatric Dengue Vaccine Initiative).
Preclinical trials are being performed on a DNA vaccine that utilizes 1 PrM and E dengue virus proteins to induce the production of the neutralizing antibodies without an entire strain. This could prove safer but possibly less effective (WHO). The animal models used in most of the trials were monkeys and mice despite their differences with humans in pathogenesis.
There are many dengue vaccines in the works and they are all currently in clinical trials. There are challenges to making a vaccine but new technologies and human innovation are sure to soon surmount them. Clinical trials usually take years and thousands if not millions of dollars and none of the vaccines are yet at Phase 3 clinical trials. It may not be soon that a vaccine is finally approved and implemented but the successes in previous trials of the multiple potential vaccines are hopeful. Chimeras and other mutated strains of the virus show great potential but long term clinical trials are essential to ensure that reinfection and activation of the virus cannot occur. The OPV polio virus that has been used for years is now being shown to have the ability to reinfect people who have had it for long periods of time and its shedding also contributes to widespread complications. Because infection with more then one strain is suggested to be more severe, it is important that this does not happen. So, it may be years before any of these vaccines are actually used.
Williams, Greg. “Safer Dengue Vaccine.” Medical News Today. 14 February 2009. Web. 24 February 2010.
Seema et al. “Molecular Mechanism of Pathogenesis of Dengue Virus : Entry
and Fusion with Target Cell.” Indian Journal of Clinical Biochemistry. 2005, 20 92-103. Web. 24 February 2010
“Dengue Hemorrhagic Fever.” World Health Organization. World Health Organization. 1997. Web. 24 February 2010.
Kanjaksha Ghosh et al. “Imaging the interaction between dengue 2 virus and human blood platelets using atomic force and electron microscopy” Journal of Electron Microscopy. 8 May 2008 Web. 24 February 2010
La Russa, Vincent et al. “Mechanisms of Dengue Virus Induced Bone Marrow Suppression. ”Baillere's Clinical Haematology. March 1995. Vol. 8 Issue :1 249-270. Web. 24 February 2019.
Greene, Sharone et al. Immunopathological mechanisms in dengue and dengue hemorrhagic fever.” Current Opinion in Infectious Diseases. Vol 19 Issue 5: 429-436. October 2006. Web. 24 February, 2010.
“Dengue Virus NS1 glycoprotein.”Abcam.Web. 24 February 2010
“Dengue Virus Profile.”Web. 24 February 2010.
Mawer, Jessica. “Fears Dengue Fever can Spread.” ABC News. 25 February 2010. Web. 25 February 2010.
“ Dengue, Tropics and Subtropics.” CDC.10 November 2009. Web. 24 February 2010.
“Wingless Mosquitos May Help Control Dengue Fever.” RedOrbit. 23 February 2010. Web 25 February 2010.
“Viral Disease-Dengue.”Preventing Disease, Disability, and Premature Death. Web. 24 February 2010.
Medicine Net. 11 April 2002. Web. 24 February 2010
Marize P et al. “ Rapid Subtyping of Dengue Virus Serotypes 1 and 4 by restriction Site-Specific PCR.” American Society for Microbiology. March 2000. Vol 38 Issue 3:1286-1289. Web. 24 February 2010.
Balmaseda, Angel et al. “ Serotype-Specific Differences in Clinical Mainfestations of Dengue.” Tropical Medicine and Hygiene. 2006. Vol 74 Issue 3:449-456. Web. 24 February 2010.
Vasquez- Pizardo, M et al. “Is Liver Damage Dependent on Serotype of Dengue Virus.” Dengue Bulletin. 2006. Vol 30. Web. 24 February 2010.
Jindadamrongwech, Sumalee et al. Intervirology. 2004. Vol 47 No. 6. Web. 24 February 2010.
Endy, Timothy P. et al. “ Spatial and Temporal Circulation of Dengue Virus Serotpes: A Prospective Study of Primary School Children in KamphaengPhet, Thailand. American Journal of Epidemiology. 2002. Vol 156: 52-29. Web. 24 February 2010.Swaminathan, S. et al. “Dengue Vaccine-current progress and challenges.” Current Science. February 2010. Vol. 98, No. 3. Web. 24 February 2010.
Kawaguchi, Isao. “ Why are dengue virus serotypes so distantly related? Enhancement and Limiting serotype similarity between dengue virus strains.” The Royal Society. 10 September 2003. Web. 24 February 2010.
Chareonsirisuthigal, Takol. “ Dengue Virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti- inflammatory cytokines but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells.” Society for General Microbiology. 2007. Vol 88: 365-375. Web 24 February 2010.
Almond, Jeffrey et al. “ Accelerating the Develpment of a dengue vaccine for Poor Children.” Vaccine. 2002. Vol 20: 3043-3046. 24 February 2010.