Dengue vaccines that induce a T-cell response
A new opportunity
Daniëlle Voshart
Supervised by I. A. Rodenhuis-Zybert Medical Microbiology
June 2015
Index
Abstract 3
Introduction 4
Dengue virus 4
Adaptive immune response to DENV 4
Vaccine development 6
Research question 7
Arguments against inducing a T-cell response with a dengue vaccine 9 Original antigenic sin impairs T-cell response to DENV infection 9 T-cell produced cytokines contribute to pathogenesis of dengue 10 Arguments for inducing a T-cell response with a dengue vaccine 11
T-cells have a protective role in DENV infection 11
T-cells can prevent ADE in DENV infection 11
Vaccines directed at T-cell induced immunity can prevent ADE 13 Original antigenic sin does not have a negative effect on the immune
response to DENV infections 14
Summarizing discussion 16
A protective or pathogenic role of T-cells in DENV infection? 16
Original antigenic sin 16
How to prevent ADE? 16
Conclusion and implications 17
References 18
Abstract
Dengue virus (DENV) is endemic in a large part of South Asia, the America’s and Africa. The virus has four serotypes and each of them can cause plasma leakage, shock and even death. Vaccine research focuses primarily on vaccines that induce an antibody response, but the most important vaccine candidate doing this, recently failed to induce immunity against all serotypes. This thesis investigates whether dengue vaccine research should focus more on eliciting T-cell mediated immunity. T-cells might contribute to disease pathogenesis, but overall, the T-cell response against DENV is beneficial for the individual. Furthermore, although original antigenic sin of T-cells occurs during a secondary T-cell infection, this has proven to be non-pathogenic. It also does not reduce the efficiency of the T-cell response to DENV infection. Lastly, with a vaccine that only induces a T-cell response, the antibody-dependent enhancement of infection can be avoided. In conclusion, dengue vaccine research should focus more on T-cell response for both a vaccine that only evokes a T-cell response and a vaccine that elicits a T-cell and an antibody response.
Introduction
Dengue virus
Half of the world’s population is now at risk of being infected with dengue virus (DENV). The mosquito-borne viral disease that was seen in only nine countries before 1970, is currently seen in 96 million individuals per year primarily living in the America’s, south-east Asia, the Pacific and Africa1. Outbreaks of DENV also occurred in Europa and might be more prevalent when the vectors of this disease, Aedes aegypti and Aedes albopictus, enlarge their territory2. DENV is a member of the Flavivirideae family, genus Flavivirus. It is an enveloped virus with a single-stranded positive RNA genome. The viron consist of three structural proteins, 2 of them: precursor membrane (preM) and envelope (E), are present of the of the viral envelope while capsid (C) and RNA encoding seven non- structural proteins involved in viral replication (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) comprise the nucleocapsid3,4. There are four serotypes of the dengue virus (DENV-1 to 4) that all cause dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS).
However, the majority of cases are in fact asymptomatic. While DF is a mild disease, DHF and DSS, who can develop from DF, are more severe and cause haemorrhagic bleeding and vascular permeability5. Half a million people are diagnosed with DHF or DSS each year, which causes death in 2,5% of those affected1. The first infection with DENV is usually asymptomatic or results in DF and mounts an immune response that protects the individual against secondary infection with the same serotype (homotypic infection) but only temporarily against secondary infection with one of the other serotypes (heterotypic infection). In fact, individuals with secondary heterotypic infections are at risk of developing DHF or DSS. This is due to the immune response against DENV infection5.
Adaptive immune response to DENV
Cellular T-cell response against DENV is primarily focused against the non-structural proteins of the virus, especially NS3 and NS5. These proteins contain immunodominant T-cell epitopes, resulting in a higher quantity of T-cells for these immunodominant epitopes6,7. In reaction to these proteins, serotype-specific and cross-reactive memory T-cells are produced5. Furthermore, CD4+T- cells respond to DENV infection primarily with the production of T-helper 0 (th0) and th1 cytokines, though they also have the ability to lyse infected cells. Cytokine response shifts from th0 and th1 response to th2 cell response when the condition of the patient worsens. CD8+ T-cells produce less cytokines, mainly TNFα and IFNγ. Their primary function is to lyse infected cells8,9. Individuals with DHF and DSS were found to have elevated levels of pro-inflammatory and vasoactive cytokines, such
as TNF and IL-10, in comparison to individuals with DF. This so-called cytokine storm induces vascular permeability8.
Humoral immunity against DENV is primarily directed against structural DENV proteins E and preM and against soluble non-structural protein NS1. NS1 can be secreted by the infected cell or can be found on the cell membrane. The secreted NS1 causes an antibody response that can lead to activation of the complement system and as a result lysis of the infected cell 5,8. In primary infections, antibody levels rise when viremia is already declining and peak two weeks after the onset of infection when vireamia is undetectable. Therefore, antibodies in primary infection do not control vireamia10 (fig. 1). However, this is not the case during a secondary heterotypic infection11. Within a few weeks after the start of the DENV infection, affinity maturation occurs8. Most of the memory B-cells produced will later generate cross-reactive antibodies. Only a 5% of dengue specific memory B-cells produces serotype specific antibodies12. During a secondary heterotypic infection, these cross- reactive antibodies will bind to the E protein of the virus, but instead of neutralizing the virus, these antibodies are thought to enhance viral uptake. This occurs through the binding of this complex to the Fcγ receptor after which the virus enters the cell via phagocytosis. Because of this so-called the antibody-dependent enhancement of infection (ADE), more cells are infected with DENV during a secondary heterotypic infection then during a primary or homotypic infection. This results in a higher vireamia and therefore a more severe disease11 (fig. 2).
Accordingly, a safe and efficacious dengue vaccine is sought to induce immunity against all serotypes. When a vaccine induces immunity to, for example, only one serotype, infection with another would probably induce more severe dengue in comparison to a primary DENV infection in someone who is not immune to a serotype. However, inducing a balanced immune response have proven very difficult, making it one of the main reasons no licensed vaccine against dengue is on the market yet5.
Figure 1. Antibody response to DENV. Reprinted from: Guzman, et al., 2010, Nature Review Microbiology, S7-S16. Copyright 2010 by WHO/TDR.
Figure 2. Mean dengue virus titer (log mosquito infectious doses/ml [log MID50/mL]) by fever day for dengue 2 virus patients experiencing DF (thin dashedline, n = 16), DHF (solid thin line, n = 26), and DSS (heavy solid line, n = 5). Defervescence occurred on fever day 0. Adapted from: Vaughn, et al., 2000, The Journal of Infectious Diseases, 118(1), p. 7. Copyright 2015 by Infectious Diseases Society of America.
Vaccine development
Vaccine development for DENV has proven to be very difficult. Not only should this vaccine induce protection against all DENV serotypes, but the response against each of them should also be very well balanced. A response that is too low will not induce immunity, while a response that is too high might cause cytokine storms and pathogenicity4.
Different approaches can be taken in the development of a vaccine. One of the most used methods is to use inactivated or attenuated virus to induce a response. Life attenuated vaccines are made by weakening the viruses by serial passage through non-human cells or by introducing mutations in the virus that limit its ability to replicate4. Another method is the inducement of genes of a heterologous virus into the genome of a harmless virus. This chimeric virus will induce an immune response against antigens of the inserted genes and the backbone virus. A vaccine can also be produced with only some parts of the virus. This is the case in antigen vaccines. These antigens can be purified or synthetically produced. These antigens are often adapted using recombinant DNA techniques. Lastly, DNA can be used as a vaccine. This DNA is then presumably transcribed and translated by antigen presenting cells and presented as antigens. Although this kind of vaccine is never tested in humans, it is thought to induce a very strong, broad and lasting immune response13.
One of the vaccines currently tested for dengue is DENVax. This is a chimeric vaccine with preM and E genes of all serotypes into the backbone of a live-attenuated DENV-2 strain. Therefore, the vaccine consists of 4 live attenuated viruses, one live attenuated DENV-2 and three live- attenuated DENV-2 viruses, each inserted with proteins of DENV-1, DENV-3 or DENV-4. This vaccine currently undergoes phase II testing. Live attenuated vaccines have proven to be successful for other flaviviruses, such as yellow fever(YF) and Japanese encephalitis4. Therefore this method looks very promising for DENV. Another live-attenuated vaccine undergoing phase II is TV003/TV005. This vaccine is composed of live-attenuated DENV-1, DENV-3 and DENV-4 vaccines and a chimeric DENV-2 vaccine. Furthermore a tetravalent inactivated vaccine, a synthetic antigen vaccine and a DNA vaccine are presently tested in phase I clinical trials14.
Lastly, one of the most promising vaccine currently tested is made of the live attenuated YF vaccine. Sanofi Pasteur used this vaccine as a backbone for a chimeric dengue vaccine. To elicit a response against DENV, preM and E genes were inserted into YF 17D virus. To prevent ADE, chimeric vaccines were made for all 4 serotypes and combined in a so-called tetravalent CYD vaccine15. The vaccine was shown to mount neutralizing humoral responses against all four DENV serotypes, however in phase IIb and III trials the vaccine failed to protect against DENV-2 infection and only partly protected against this serotype in another phase III trials (42%). Moreover, although significant, protection against the other serotypes was never optimal. This suggests an insufficient immune response to all four DENV serotypes16,17.
Research question
Important lessons can be learned from the CYD clinical trials for further DENV vaccine development. For instance, further research might have to be alert on interference from the four vaccine viruses18. More importantly, the vaccine only induced a response against structural proteins
preM and E15. This causes an antibody, but not a T-cell response7,19. T-cell response to DENV is primarily focused on the non-structural proteins of the virus, such as NS37. The failed CYD clinical trials showed that only inducing an antibody response might not be enough to gain immunity against dengue16. This raises a question whether dengue vaccine research should focus more on inducing a T- cell response. The aim of my thesis is to address this question by reviewing the current knowledge on the pros and cons of T-cells mediated immunity. To answer this question, I will discuss both the positive and negative views on inducing T-cell response for vaccination purposes. After this, I will compare both and then share my own opinion on the subject.
Arguments against inducing a T-cell response with a dengue vaccine
Recent vaccine development is primarily focused against inducing antibody-mediated immunity, instead of T-cell mediated immunity. T-cell response is often seen as pathogenic instead of protective. Here below, I will summarise the arguments against inducing a T-cell response with a dengue vaccine .
Original antigenic sin impairs T-cell response to DENV infection
Just as antibodies, T-cell response differs in secondary infections as opposed to primary infection. Cross-reactive memory T-cells react and proliferate faster than naïve T-cells to DENV infection. Because of this, cross-reactive T-cells with a high avidity for a non-infecting DENV serotype are abundantly present in contrast to T-cells specific for the infecting DENV serotype. This is called original antigenic sin8,20. Mongkolsapaya et al.20 showed that original antigenic sin occurs in T-cells during a DENV infection in humans. They saw a high concentration of CD8+ with a low avidity for the currently infecting serotype and a high avidity for a non-infecting serotype and a much lower concentration of T-cells with a high avidity for the currently infecting DENV serotype. Although a pathogenic result of original antigenic sin during a DENV infection was not proven during this study20, multiple observations strengthen to the idea that T-cells contribute to the pathogenesis of DHF and DSS via original antigenic sin. Firstly, DHF and DSS occur mostly after a secondary heterotypic infection5. During a such an infection, cross-reactive T-cells with a high avidity for a non-infecting DENV serotype are present20. This is also an argument for pathogenesis caused by ADE, as cross- reactive antibodies are also present during secondary heterotypic infection11. However, severe symptoms of DHF and DSS do not occur until virus loads are diminishing fast, suggesting that another immune response than ADE contributes to the development DHF and DSS21. Lastly, Studies with other viruses do show impairment of viral clearance or even pathology due to original antigenic sin or a similar phenomenon22,23. In mice infected with lymphocytic choriomeningitis virus (LCMV), original antigenic sin was shown to impair viral clearance by CD8+ cells in comparison to a primary infection23. Other studies with murine cytomegalovirus, vaccina virus and influenza A virus demonstrated that heterologous T-cell immunity causes pathology22,23. Heterologous immunity is a phenomenon like original antigenic sin, but in this case the secondary infection is unrelated to first infection24. However, with -25-40% difference at the amino acid level, dengue serotypes are also diverse5.
T-cell produced cytokines contribute to plasma leakage in DHF and DSS
One of the more severe symptoms of DHF and DSS is plasma leakage, which can lead to shock5. Although the reasons for this increased vascular permeability are not entirely clear, DENV infection of endothelial cells does not seem to be the primary cause. Plasma leakage is induced at the end of the fever when the virus is almost cleared. This suggest that plasma leakage is a result of the pathogen but of the host response25. More specifically, the result of a cytokine storm caused by T-cell response. In patients with DHF and DSS more CD8+ and CD4+ T-cells are present. There are also more Th2 cytokines produced which are associated to more severe disease9. Some of the cytokines produced by T-cells, such as IL-2 and TNF, are known to produce vascular permeability26-28. Increased levels of TNFα, IL-6 and IL-8 are linked to disturbed coagulation. Likewise IL-6 and IL-8 probably mediate fibrinolysis. Lastly, increased levels of IL-10 are known to reduce platelet concentration. This is all thought to contribute to plasma leakage in patients with DHF or DSS22.
In conclusion, these studies show that T-cell response to DENV infection might be harmful to the patients. In secondary infection low avidity T-cells can cause impaired or pathogenic reactions to DENV. Furthermore, cytokines produced by T-cells are associated with plasma leakage.
Arguments for inducing a T-cell response with a dengue vaccine
Although T-cell response to DENV infection was previously often seen as pathogenic, recent studies have shown a more protective role29. Here below I will summarize these studies.
T-cells have a protective role in DENV infection
The cytokine storm during DHF and DSS contributes to plasma leakage and thereby worsen the condition of the patient8. However, these cytokines also help clearing the virus and modulate immune response. TNF mediates the recruitment of leukocytes, IL-10 modulates the innate immune system and IL-2 is essential for regulatory T-cells survival and activity30. This indicates that the cytokines produced upon DENV infection are not only pathogenic, but can also be protective. This protective role is further strengthened when HLA alleles associated with an increased risk of severe dengue were also associated with a weaker CD8+ response and vice versa31. For example, patients with the HLA-A*01 allele are more likely to develop severe dengue in comparison to other HLA alleles, recent studies showed that patients with this allele also showed a very weak T-cell response32. T-cells have also been proven to protect against harmful effects of DENV infection in multiple mouse models33-35. In one of those studies, vaccine induced cellular and humoral response against DENV were compared. When isolated from the rest of the immune system, T-cells always reduced viral load, while antibodies sometimes reduced viral load and sometimes increased it34. Another study by An et al.35 showed that DENV specific CD8+ T-cells partially protected mice from an otherwise lethal DENV infection. This in contrast to mice without any DENV specific CD8+ T-cells, indicating a protective role for T-cells. Furthermore, a mouse model of DENV-2 infection was also used to study the effects of CD8+ T-cells33. This study showed that CD8+ T-cells enhanced viral clearance. When mice were immunized with viral peptides an infection with DENV caused a 350-fold lower viral load than without immunisation. This was confirmed to be caused by T-cell response. This proves that vaccines focused on inducing T-cell mediated immunity against DENV could work33. All of these studies together show an overall protective role for T-cells.
T-cells can prevent ADE in DENV infection
A infection with DENV mostly passes without any symptoms. When symptoms do occur it is almost always in the form of DF. In less than 3% of the cases DF is followed by DHF or DSS. 90% of the cases of DHF of DSS occur during a second infection with DENV25. Cases of DHF or DSS during primary infection with DENV occur mostly in infants with DENV immune mothers, were about 20% of the cases DF is followed by DHF or DSS36,37. This is significantly more than in children or adults25. The severity of the disease in infants is associated with ADE, just as secondary infections with DENV25,37.
In infants ADE is mediated through maternal antibodies from the placenta protects neonates until six months after their births38(fig. 3). Although T-cells are present in infants, in contrary to adults their functionality is poor of even absent39. This could indicate that, if functional, T-cells might help to prevent ADE. This hypothesis is strengthened by the fact that in the case of a secondary infection with DENV, infection does not always result in DHS of DSS, although antibodies against a previously infecting serotype are present40. It was recently proved that CD8+ T-cells could prevent ADE in mouse infected with DENV. Zellweger et al.41 discovered this by first priming mice with an adjuvant containing inactivated DENV-2 (al-UV-DENV-2). This induced an non-neutralizing antibody response, but no significant CD8+ T-cell response. Before infection with DENV-2, DENV-2-primed CD8+ T-cells were injected into one of the two groups of mice. The primed CD8+ T-cells prevented ADE and reduced viral concentration in the mice (fig. 4). Although this may not directly apply to humans, it is very likely that T-cell response does reduce the risk of DHF and DSS mediated by ADE41. This is supported by the finding that HLA alleles associated with weak T-cell response are also associated with a bigger risk of getting severe dengue31.
Figure 3. Relationship between the age distributions of infants hospitalized for dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and the protective and infection-enhancing effects of maternal dengue antibodies. Shown are mean age specific hospitalization rate/1,000 for Bangkok and Thonburi, 1962–1964. At birth, antibodies are at protective concentrations. With the passage of time, maternal immunoglobulin G antibodies are catabolized to concentrations that result in antibody-dependent enhancement (ADE) of infections. By the end of the first year of life, ADE antibodies are catabolized to concentrations below the ADE threshold, and DHF/DSS cases disappear. Reprinted from: Halstead, et al., 2002, Emerging Infectious Diseases, 8(12), p. 356. Copyright 2015 by EBSCO.
Figure 4. Model summarizing the effect of al-UV-DENV2 priming on subsequent DENV2 infection with or without transfer of exogenous DENV2-primed CD8+ T cells. Reprinted from: Zellweger, et al., 2014, The Journal of Immunology, 193(8), p. 3122. Copyright 2015 by The American Association of Immunologists.
Vaccines that only induce a T-cell response can avoid ADE
As previously stated, adaptive immune response in infants with DENV immune mothers consists only of maternal antibodies38. In the case of dengue, these antibodies are more a curse then a blessing. Maternal IgG antibodies are only protective against DENV in high concentration. When the antibodies are catabolised to a titer less than 1:8, these antibodies can enhance the uptake of viral particles, thereby worsening the symptoms (fig. 3). Vaccines must therefore induce a high antibody response or none at all, because a low antibody response will more likely result in ADE upon infection with DENV42. Furthermore, ADE might have caused failure in live-attenuated and recombinant vaccines as is elegantly describes by Halstead42.
With a vaccine that only induces T-cell response, ADE can be avoided. One of these vaccines consists of recombinant nucleocapsid-like particles from DENV-243. This vaccine does not induce an antibody response that could evoke ADE, but does induce a T-cell response that results in a reduced viral load in mice and vervet monkeys. The vaccine could not induce full immunity against DENV-2.
This is probably due to the low quantity of T-cell epitopes that the C protein contains43,44. However, this method would probably work with a protein that evokes a bigger T-cell response, such as NS3 or with the use of adjuvants7,44. Such vaccine will not only avoid ADE, but also other negative effects associated with antibody response against DENV. One of the other negative effects of antibody response against DENV is the inducement of the complement system, which is associated to plasma leakage. Furthermore, antibodies against DENV also react to host proteins such as plasminogen and endothelial cell proteins 8.
Original antigenic sin does not have a negative effect on the immune response to DENV infections In these previously discussed studies, T-cell response in secondary infections remained a mechanism that could have a negative effect on the immune response or even harm the patients due to original antigenic sin. However, a study by Weiskopf et al.31 showed that original antigenic sin does not decrease the quality or quantity of T-cell mediated immune response. They studied naturally occurring DENV infection in Sri Lankan, a country in which DENV-2 infection has been the most common serotype for years. Recently DENV-1 and DENV-3 have also been introduced in the population. Researchers therefore presumed that in the case of a secondary heterotopic infection, DENV-2 caused the primary infection. Next they analysed CD8+ T-cells response ex-vivo from conserved T-cells against the primary DENV infection to T-cells specific for the secondary infecting serotype. They found that CD8+ T-cells were more prone to have a response against the primary infecting serotype, but this did not quantitative of qualitative impair viral clearance31 (fig 5.). This study changed the way we think about original antigenic sin and T-cell contribution to dengue vaccine development29,31. Furthermore, Weiskopf et al.29 greatly improved the number of characterized DENV T-cell epitopes. This resolves one of the limitations regarding T-cell research in DENV. DENV vaccine development concentrated on T-cell mediated will benefit from this enlarged database.
Figure 5. Avidity of responding T-cells was determined by incubating peripheral blood mononuclear cells with ascending concentrations op peptide pools (0.001, 0.01, 0.1, 1, 10µg/mL). The peptide concentration necessary to induce 50% of the maximum responses (EC50) was calculated, and the average EC50 was compared between the conserved and serotype-specific epitopes. Reprinted from: Weiskopf, et al., 2013, Proceedings of the National Academy of Sciences, 110(22), p. E2050. Copyright 2015 by National Academy of Sciences.
As discussed, multiple studies show that T-cell response in DENV infection is more helpful than harmful. It was also shown that T-cells could prevent ADE. Furthermore, it was discussed how DENV-specific antibodies can induce ADE if their concentration falls below a certain level and that this could be averted by producing a vaccine that only induces a T-cell response. Lastly, original
antigenic sin was shown to have no negative effect on viral clearance in DENV infection. Combined, these arguments form a solid case for the inducement of a T-cell immunity with a dengue vaccine.
Summarising discussion
A protective or pathogenic role of T-cells in DENV infection?
Cytokines produced by T-cells during a dengue infection are associated with increased vascular permeability and other damage leading to plasma leakage9,22,25,26,28
. However, these cytokines can also be protective30. Little evidence that shows an overall negative effect of T-cells on the host during a dengue infection exists and even in this case, both protective and pathogenic roles are assigned to T-cells in DENV infection35. This in contrary to the positive effects of T-cells during a DENV infection. Not only did T-cells reduce viral load33,34, it was also shown that a stronger T-cell response reduced the possibility of severe dengue31. Furthermore, the ability from T-cells to reduce the risk of ADE clearly suggests a protective role. In conclusion, components of the T-cell response to DENV infection might be pathogenic, however, in total the T-cell response is protective and even reduces the risk of DHF or DSS.
Original antigenic sin
In the case of original antigenic sin, cross-reactive memory T-cell response outnumbers the serotype specific response in a secondary DENV infection8. Original antigenic sin can impair viral clearance, as is shown with LCMV23. Heterologous immunity, a phenomenon similar to original antigenic sin, is even proved to be pathogenic in some viruses22,23. Original antigenic sin of T-cells is confirmed to exist during a secondary dengue infection in mice20. Although existing, original antigenic sin was proven not to be pathogenic or even impairing to the viral clearance during a DENV infection in humans31. Although this was only proven in one study ex-vivo, the results will probably stand in further studies. Negative effects of original antigenic sin are only seen for other viruses than DENV, but never during a dengue infection. To conclude, original antigenic sin of T-cells exist in secondary dengue infections but does cause any harmful effects.
How to prevent ADE?
Because only a high concentration of DENV specific antibodies will cause antibody-mediated immunity against dengue virus and a low concentration of antibodies will cause ADE42, the question arises if DENV vaccine research should aim for antibody-mediated immunity or if antibody-mediated immunity should be avoided altogether. Vaccines that only induce an antibody response have recently failed to induce full immunity16,17. This could be due to ADE42. A vaccine that only evokes T- cell mediated immunity could prevent ADE. Unfortunately, such a vaccine would not prevent infection with DENV, but it would quickly reduce viral load and prevent DHF and DSS44. Both of these
methods are flawed. A third possibility is a vaccine that mounts both a T-cell and an antibody response. With such a vaccine, induced antibody levels should be high enough to avoid ADE.
Conclusion and implications
T-cell response seems to have a protective role in DENV infection and is not impaired by original antigenic sin. A vaccine that only elicits a T-cell response would avoid ADE. Such a vaccine will not stop DENV infection immediately. However, because it does not evoke any antibody reaction, an imperfect serotype coverage of the vaccine is not dangerous for the people that get vaccinated.
When immunity against a certain serotype is not induced, infection with this specific serotype will not result in a higher change of severe dengue in comparison to someone that is not vaccinated.
Therefore, I conclude that it would be wise to focus on a vaccine that only evokes a T-cell response.
Such a vaccine would be safe and effective. Furthermore, research should also continue with vaccines that induce a strong humoral response, but this in combination with a T-cell response. A combination of both will induce a stronger response against the serotypes and could potentially eradicate DENV. This could for instance be done by changing the YF NS3 in the Sanofi Pasteur vaccine for the DENV NS3. In conclusion, dengue vaccine should focus more on inducing a T-cell.
References
1. World Health Organisation. Fact sheet: Dengue and severe dengue.
http://www.who.int/mediacentre/factsheets/fs117/en/. Updated 2015. Accessed June/7, 2015.
2. Schaffner F, Mathis A. Dengue and dengue vectors in the WHO european region: Past, present, and scenarios for the future. The Lancet Infectious Diseases. 2014;14(12):1271-1280.
3. Perera R, Kuhn RJ. Structural proteomics of dengue virus. Current Opinion in Microbioly.
2008;11(4):369-377.
4. Murrell S, Wu S, Butler M. Review of dengue virus and the development of a vaccine.
Biotechnology Advances. 2011;29(2):239-247.
5. Guzman MG, Harris E. Dengue. The Lancet. 2014;385(9966):453-465.
6. Appanna R, Huat TL, See LLC, Tan PL, Vadivelu J, Devi S. Cross-reactive T-cell responses to the nonstructural regions of dengue viruses among dengue fever and dengue hemorrhagic fever patients in malaysia. Clinical and Vaccine Immunology. 2007;14(8):969-977.
7. Duangchinda T, Dejnirattisai W, Vasanawathana S, et al. Immunodominant T-cell responses to dengue virus NS3 are associated with DHF. Proceedings of the National Academy of Sciences.
2010;107(39):16922-16927.
8. Rothman AL. Immunity to dengue virus: A tale of original antigenic sin and tropical cytokine storms. Nature Reviews Immunology. 2011;11(8):532-543.
9. Bäck AT, Lundkvist Å. Dengue viruses - an overview. Infection Ecology & Epidemiology. 2013;3:1- 21.
10. Guzman MG, Halstead SB, Artsob H, et al. Dengue: A continuing global threat. Nature Reviews Microbiology. 2010;8(120):S7-S16.
11. Flipse J, Wilschut J, Smit JM. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus infection in humans. Traffic. 2013;14(1):25-35.
12. Smith SA, de Alwis AR, Kose N, Jadi RS, de Silva A,M., Crowe JE. Isolation of dengue virus-specific memory B cells with live virus antigen from human subjects following natural infection reveals the presence of diverse novel functional groups of antibody clones. Journal of Virology.
2014;88(21):12233-12241.
13. Abbas AK, Lichtman AH, Pillai S. Strategies for vaccine development. In: Gruliow R, ed. Cellular and molecular immunology. 7th ed. Philadephia, USA: Elsevier Saunders; 2012:361-362.
14. Schwartz LM, Halloran ME, Durbin AP, Longini Jr. IM. The dengue vaccine pipeline: Implications for the future of dengue control. Vaccine. 2015;33(29):3293-3298.
15. Monath TP, Seligman SJ, Robertson JS, et al. Live virus vaccines based on a yellow fever vaccine backbone: Standardized template with key considerations for a risk/benefit assessment. Vaccine.
2015;33(1):62-72.
16. Capeding MR, Tran NH, Hadinegoro SRS, et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in asia: A phase 3, randomised, observer-masked, placebo- controlled trial. The Lancet. 2014;384(9951):1358-1365.
17. Sabchareon A, Wallace D, Sirivichayakul C, et al. Protective efficacy of the recombinant, live- attenuated, CYD tetravalent dengue vaccine in thai schoolchildren: A randomised, controlled phase 2b trial. The Lancet. 2012;380(9853):1559-1567.
18. Halstead SB. Identifying protective dengue vaccines: Guide to mastering an empirical process.
Vaccine. 2013;31(41):4501-4507.
19. Rivino L, Kumaran EAP, Jovanovic V, et al. Differential targeting of viral components by CD4(+) versus CD8(+) T lymphocytes in dengue virus infection. Journal of Virology. 2012;87(5):2693-2706.
20. Mongkolsapaya J, Dejnirattisai W, Xu X, et al. Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nature Medicine. 2003;9(7):921.
21. Mongkolsapaya J, Duangchinda T, Dejnirattisai W, et al. T cell responses in dengue hemorrhagic fever: Are cross-reactive T cells suboptimal? The Journal of Immunology. 2006;176(6):3821-3829.
22. Martina BEE, Koraka P, Osterhaus ADME. Dengue virus pathogenesis: An integrated view. Clinical Microbioly Reviews. 2009;22(4):564-581.
23. Klenerman P, Zinkernagel RM. Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature. 1998;394(6692):482-485.
24. Rehermann B, Shin E. Private aspects of heterologous immunity. Journal of Experimental Medicine. 2005;201(5):667-670.
25. Mathew A, Rothman AL. Understanding the contribution of cellular immunity to dengue disease pathogenesis. Immunological Reviews. 2008;225(1):300-313.
26. Green S, Vaughn DW, Kalayanarooj S, et al. Early immune activation in acute dengue illness is related to development of plasma leakage and disease severity. Journal of Infectious Diseases.
1999;179(4):755-762.
27. Yang G, Hamacher J, Gorshkov B, et al. The dual role of TNF in pulmonary edema. Journal of Cardiovascular Disease Research. 2010;1(1):29-36.
28. Dutcher J, Atkins M, Margolin K, et al. Kidney cancer: The cytokine working group experience (1986-2001). Medical Oncology. 2001;18(3):209-219.
29. Zompi S, Harris E. Original antigenic sin in dengue revisited. Proceedings of the National Academy of Sciences. 2013;110(22):8761-8762.
30. Abbas AK, Lichtman AH, Pillai S. Functions of TH1 cells. In: Gruliow R, ed. Cellular and molecular immunology. 7th ed. Philadephia, USA: Elsevier Saunders; 2012:229-230,231.
31. Weiskopf D, Angelo MA, de Azeredo EL, et al. Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells. Proceedings of the National Academy of Sciences. 2013;110(22):E2046-E2053.
32. Monteiro SP, Brasil, Pedro Emmanuel Alvarenga Americano do, Cabello GMK, et al. HLA-A*01 allele: A risk factor for dengue haemorrhagic fever in brazil's population. Memórias do Instituto Oswaldo Cruz. 2012;107(2):224-230.
33. Yauch LE, Zellweger RM, Kotturi MF, et al. A protective role for dengue virus-specific CD8(+) T cells. Journal of immunology. 2009;182(8):4865-4873.
34. Zellweger RM, Miller R, Eddy WE, White LJ, Johnston RE, Shresta S. Role of humoral versus cellular responses induced by a protective dengue vaccine candidate. PLoS Pathogens. 2013;9(10):1- 13.
35. An J, Zhou D, Zhang J, Morida H, Wang J, Yasui K. Dengue-specific CD8+ T cells have both
protective and pathogenic roles in dengue virus infection. Immunology Letters. 2004;95(2):167-174.
36. Hung NT, Lei H, Lan NT, et al. Dengue hemorrhagic fever in infants: A study of clinical and cytokine profiles. Journal of Infectious Diseases. 2004;189(2):221-232.
37. Simmons CP, Chau TNB, Thuy TT, et al. Maternal antibody and viral factors in the pathogenesis of dengue virus in infants. Journal of Infectious Diseases. 2007;196(3):416-424.
38. Basha S, Surendran N, Pichichero M. Immune responses in neonates. Expert review of clinical immunology. 2014;10(9):1171-1184.
39. Ygberg S, Nilsson A. The developing immune system ? from foetus to toddler. Acta Paediatrica.
2012;101(2):120-127.
40. Guzman M, Alvarez M, Halstead S. Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: An historical perspective and role of antibody-dependent
enhancement of infection. Arch Virol. 2013;158(7):1445-1459.
41. Zellweger RM, Eddy WE, Tang WW, Miller R, Shresta S. CD8+ T cells prevent antigen-induced antibody-dependent enhancement of dengue disease in mice. The Journal of Immunology.
2014;193(8):4117-4124.
42. Halstead SB, Nguyen TL, Thein TM, et al. Dengue hemorrhagic fever in infants: Research opportunities ignored. Emerging Infectious Diseases. 2002;8(12):1474.
43. Gil L, Bernardo L, Pavón A, et al. Recombinant nucleocapsid-like particles from dengue-2 induce functional serotype-specific cell-mediated immunity in mice. J Gen Virol. 2012;93:1204-1214.
44. Gil L, Izquierdo A, Lazo L, et al. Capsid protein: Evidences about the partial protective role of neutralizing antibody-independent immunity against dengue in monkeys. Virology. 2014;456–
457(0):70-76.
