Why don't we have a #dengue vaccine yet?

This is taken from a recent special issue of the journal Vaccine from September covering the "state-of-the-art" of dengue virus vaccine research. 

The process of generating - and testing -  a dengue vaccine captures the entire field of vaccinology and highlights it's promises and pit-falls and can be used as a model when thinking of any number of potential vaccine initiatives.

See below for my take and summary of this issue:

Dengue versus the epigenome

Dengue virus (DENV) particles. Does dengue interact with the host epigenome? And, why?

Epigenetic modification of chromosomal structure has the ability to rapidly and stably alter gene expression and function within cells, tissues and whole organisms. And, changes have been found to induce a number of diseases in humans, such as cancer and others.

The ability of infecting viruses to influence this process has now been realized with a number of large DNA viruses being shown to take advantage of host epigenetic modifications, including: the histone proteins that are necessary for the structure and modulation of chromatin.

But what hasn't been seen is whether RNA viruses, which replicate within the cytoplasm are able to at least interact with the host chromosome structures. And, more importantly, why would they do so? Researchers have just published evidence suggesting that dengue virus interacts directly with host histone proteins and requires such activity for replication (see PLoS paper here). The reasons why are not understood.

DENV-C specific histone binding (binds to H2A, B, 3 and 4) but not GFP. Does this has an affect on host function?

Dengue Virus (DENV) is a small, single-stranded, positive-sensed RNA virus and belongs within the Flavivirus genus, alongside yellow fever virus and west Nile fever virus. It is spread between humans via the bite of the female Aedes mosquito and can lead to the development of very high fever (41 degrees Celsius), headache and in some cases, a lethal hemorrhagic fever.

It is estimated that two-fifths of the worlds' population are at risk of DENV infection and it is endemic in over 100 countries within tropical or sub-tropical climates. There is currently no clinically proven treatment for dengue fever nor are there any vaccines available, making any research into the basic biology of the virus all the more important.

Using a 'tagged' protein, this group showed that a recombinant DENV C protein (capsid protein forming the outer layers of the virus particle - yellow in the above picture) interacts with the four core histones, H2A, H2B, H3 and H4 found within the nucleus of huh-7 cells, a human hepatocellular carcinoma cell line used a lot in DENV research. See the figure above for the specific binding. DENV is known to infect human liver cells, especially in fatal cases.

GFP-tagged DENV-C co-localizes with huh-7 nuclear and cytoplasmic histones (H2A and H2B)

When over-expressed within these cells, a GFP-tagged C localised to the same areas of the nucleus as each of the histones did and this was extended to DENV-infected cells (see above). C also interacted with the histones while in the cytoplasm as well. In vitro C and histones formed dimers together in the absence of nucleic acids and it was subsequently shown to bind DNA with - or without - histone proteins. See below.
 
This interaction was shown to disrupt the normal oligomerization of histones but not that of histone-DNA binding. Infection up-regulated the expression of the core histones and was essential for normal DENV replication.

DENV-C binds to huh-7 H2A, B, 3 and 4 and causes formation of oligomers (the bands higher up on the gel - compared to lower and smaller bands).

The physical basis of this interaction is currently not understood but is believed to result from the structural similarities between the histones and DENV-C. A common folding pattern - important for oligermization - is shared by both and the authors state they are in the process of crystallizing the two together. The biological significance of this observation was not really addressed here, apart from the fact that histones were required for DENV replication (see graph below). The authors hypothesize the reasons as to why DENV might need this process, but provide no evidence to back it up.

DENV replication requires H2A and H3 proteins. Shown here by siRNA knockowns of the two (columns 1 - 4) when compared to non-specific controls (5 and 6).

It would be interesting to see if this interaction applied to other human cells, including primary cell lines and maybe in vivo. And, if it extended to mosquito cells as well? But, where in the genome does DENV-C bind? Is it specific or more general? What is the basis of the requirement for these histone proteins? Finally, is it possible to inhibit this relationship to develop new anti-DENV therapies.


Colpitts, T., Barthel, S., Wang, P., & Fikrig, E. (2011). Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells PLoS ONE, 6 (9) DOI: 10.1371/journal.pone.0024365

Fighting dengue with mosquito semen

Dengue virus, DENV - an important mosquito-borne virus

Arthropods are important vectors in the transmission of a number of animal and human pathogens. A major vector group are the mosquitoes of which there are over 3,000 species. However, during their life cycle some mosquitoes feed on the blood of other animals - creating an excellent chance for the direct transfer of manymicrobial species. From here the bacteria/viruses/parasites can initiate infection of the new host which then, following another blood feed, may transmit the pathogen to an uninfected insect. This particular lifestyle allows for the development of pathogen control strategies aimed at interfering with the vector species. If we remove or inhibit the vector, we may prevent the spread of the pathogens they carry.

 Why dengue?

Mosquito sex

Another group of mosquito-borne pathogens are the closely related - but distinct - dengue viruses (DENV). DENV is commonly responsible for a 'flu-like- illness' in humans but complications may include a potentially fatal hemorrhagic fever. The incidence is mainly constrained to tropical and sub-tropical areas although in recent decades it has spread to other areas where it may cause massive epidemics. The World Health Organisation states that, "Some 2.5 billion people – two fifths of the world's population – are now at risk from dengue. WHO currently estimates there may be 50 million dengue infections worldwide every year." There is currently no commercially licensed vaccine or antivirals available for the treatment of dengue leaving the only option to prevent transmission through control of mosquito populations.

The most important vector species is the predominantly urban species Aedes aegypti - control of which may aid DENV eradication. There are a number of potential strategies that could be employed to reduce the numbers of this species including chemical poisoning, genetic strategies and biological control. A major goal is therefore to inhibit  mosquito reproduction and feeding behaviour yet this requires intimate knowledge of mosquito reproductive biology. Insects communicate via a number of chemical signals, one mode of communication is via the males ejaculate -what happens to be a convenient opportunity to control a females behaviour. A mated female is behaviourally very different to an unmated one and there is evidence suggesting that this change is initiated by the transfer of male-derived signaling proteins during mating. It may then prove to be useful to identify some of these molecules so as to possibly control mosquito behaviour ourselves.

What is so good about male semen?

Sirot et al recently report, using proteomic analysis, the identificantion of a number of proteins, termed seminal fluid proteins (Spfs) transferred from males (with labelled proteins) to females (non-labelled proteins) during mating. Of which some may be responsible for the male control over female post-reproductive behaviour; this they say, lays the groundwork for future studies investigating the molecular mechanisms behind how they work and their potential use in controlling vector populations. However, care must be taken in interpreting these results as this study does not directly look at the biological effects of these proteins and does not prove that they do have any effect on female behaviour.

Labelled insect sperm

So, what do these semen proteins actually do?

Using this approach they identified 145 proteins transferred from males to females, 17 of which were previously unknown to science - 93, they say, could be assigned as potential biologically active proteins. What function do this proteins have? Well, based on the previous annotation in protein databases, they were able to assign each of their proteins a potential function indicating the potential important roles in female behaviour. These proteins are predicted to be involved in their reproductive biology, specifically protein degradation and hormonal signalling.

This work has identified a number of proteins present in the semen of the dengue vector, Aedes aegypti which are transferred from males to females during mating. This may mean they are involved in the control of female behaviour. Although this work did not look at the function of any proteins directly, it does lay the foundations for future studies. Researchers may now focus there investigations on a set of a now verified smaller set of proteins and genes. This  species is also the vector for a number of other viruses such as chikungunya and yellow fever and so any work on this may aid the control of these important diseases.

  

Gillott C (2003). Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annual review of entomology, 48, 163-84 PMID: 12208817

Sirot, L., Hardstone, M., Helinski, M., Ribeiro, J., Kimura, M., Deewatthanawong, P., Wolfner, M., & Harrington, L. (2011). Towards a Semen Proteome of the Dengue Vector Mosquito: Protein Identification and Potential Functions PLoS Neglected Tropical Diseases, 5 (3) DOI: 10.1371/journal.pntd.0000989

The ‘interactome’ of a host/pathogen triad

In order to survive and replicate within their hosts, viruses must manipulate those pathways and systems in which their host relies upon for its own survival. However, this model gets more complicated with those viruses successfully infecting multiple host species. For example, Dengue virus (DENV) – an emerging pathogen which causes over 50 million cases a year of a mild to deadly disease – infects both humans and mosquito species of the Aedes genus. Thus to accomplish survival, DENV must interact with proteins from these two distantly related hosts. Given this complexity, understanding this dual-host/pathogen system is considerably difficult yet as Doolittle and Gomez (2011) show, computational approaches based on structural predictions of viral and host proteins may allow for the accurate prediction of the complex in vivo ‘interactome’.

Transmission of DENV - the principle mode is direct mosquito to human

The group set out to understand the interactions between both DENV encoded proteins and those of its hosts – humans and Aedes mosquitos. Using previously determined structural information for human and fly (relatively closely related insect to Aedes) and how these proteins interact with each other, they were able to map these back on to host infection. They searched databases for structural similarities between dengue proteins and those from its host (human or fly) – these ‘dengue similar host proteins’ were used to search for host-host protein interactions as a surrogate for host-dengue interactions. 

 “The computational methodology employed to generate this map assumes that proteins with comparable structures will share interaction partners. Therefore, we predict that DENV2 proteins may merge into the host protein interactome at the points normally occupied by structurally similar host proteins, creating an interface for the manipulation of downstream host processes.”

Using this approach, they built up a network of possible host/pathogen interactions, assuming that DENV proteins can participate in the same interactions as host proteins. Of course, this method over estimates interactions so to counter this, they prioritised particular interactions for further study based on previously published, validated in vivo work and those interactions still left hopefully were functionally accurate and important. This approach had previously been used to study human-HIV-1 protein interactions.

DENV capsid structure

Following significantly limiting their map down to those that had been previously validated the biological functions of host target proteins and dengue-similar proteins were analysed to determine whether the predicted functions matched those that would be important for viral infection in both humans and mosquitoes. As shown above, DENV-like proteins participate in interactions involved in diverse processes – importantly including cell death, signalling cascades, immune response and metabolism. They focus the investigation into DENV manipulation of host apoptosis and innate immune signalling and also those proteins which are shared between both insect and human hosts.

They suggest that due to the disparity in the known molecular biology of dengue/host interactions this computational methodology has its limitations in this system yet these data should be used as a springboard for future investigations and hypotheses. This study highlights the importance of global computational analysis in determining basic host/pathogen biology especially in a system which has been poorly studied like DENV.

Doolittle, J., & Gomez, S. (2011). Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts PLoS Neglected Tropical Diseases, 5 (2) DOI: 10.1371/journal.pntd.0000954

Dyer MD, Murali TM, & Sobral BW (2007). Computational prediction of host-pathogen protein-protein interactions. Bioinformatics (Oxford, England), 23 (13) PMID: 17646292