NIAID-DVI: Understanding the dengue E gene, Part I -- Human antibody neutralization, Aravinda de Silva

So we're going to spend several blog posts now focusing in on the E gene and understanding it genetically, structurally and it's role immunologically...as there were several topics covering this during NIAID-DVI...

Recent Advances in our understanding of how human antibodies neutralize dengue viruses
Aravinda de Silva
University of North Carolina School of Medicine

From the Abstract: 

Ten years ago it was known that people exposed to dengue virus developed strongly neutralizing antibodies against the homologous serotype but the molecular basis of neutralization was not known. Over the course of the subsequent decade several groups have studied the properties of DENV-specific human serum antibodies (Abs) derived from plasma cells and monoclonal Abs (mAbs) derived from memory B-cells. These studies demonstrated:

• The dominant human Ab response is serotype cross-reactive and non-neutralizing
• While functionally important, neutralizing Abs are a small 'component' to the entire response.
• Human neutralizing Abs bind to complex epitopes centered in the hinge region between domain I and II (DI and DII)  of the dengue E gene.
• Humans also produce strong neutralizing Abs that bind in domain III (DIII) of the E protein.
• Replicating viruses stimulate DI/DII hinge antibodies whereas recombinant antigens trigger DIII directed neutralizing antibody response.

Recent studies have indicated that DENVs in cell culture are dynamic and structurally heterogeneous particles. This heterogeneity is a result of incomplete cleavage of pre membrane (prM) protein during viral release from infected cells. This produces a virus which his a mix of immature, partially mature and mature virions. The extent of heterogeneity is dependent on the cell lines where the virus was grown.

The 'maturation state' of the virus influences the ability to infect cells and antibodies to neutralize DENVs...this is why DENV neutralization titers are notoriously variable.


So there are several papers dealing with the E gene as a target from a genetic and structural perspective as well as role in vaccination response:

•  Smith et al., 2013. Human monoclonal antibodies derived from memory B cells following live attenuated dengue virus vaccination or natural infection exhibit similar characteristics. JID. 207: 1898.
• They generated "a total of 63 new human monoclonal antibodies to compare the B-cell response of subjects who received the National Institutes of Health live attenuated dengue vaccine rDEN1Δ30 to that of subjects following symptomatic primary infection with DENV1."
• "Antigen specificities were very similar, with a slightly greater percentage of antibodies targeting E protein domain I/II than domain III."
• "In most respects, however, the human B-cell response was similar following DENV1 vaccination or natural infection. Results showed that the repertoire in both groups was dominated by cross-reactive antibodies with low or no neutralizing potency and significant potential to enhance infectivity by Fc-mediated mechanisms."
• "The frequency of strongly neutralizing antibodies was similar, albeit very low, making up only 5% of all antibodies tested. Antigen specificities of the response also were similar, with roughly two-thirds of antibodies targeting the E protein and one-third targeting the prM protein."
• The study demonstrated "for the first time that the high frequency of B cells encoding serotype–cross-reactive weakly neutralizing antibodies that has been described for natural DENV infection is also induced following live attenuated virus vaccination."
• Sukupolvi-Petty et al., 2013. Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that imparcts neutralization and protection. JVI Ahead of Print.
• "We observed strain and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domains II (DII) and III (DIII) of the envelope (E) protein."
• "Flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after pre-incubation at 37C."
• "Increasing the time of pre-incubation at 37C or raising the temperature to 40C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains."
• Look at Table 8, I couldn't copy it here because the ahead of print pdf copy looked odd when I tried to insert/copy it over. Table 8. Loss-of-binding residues identified by prM-E protein display on HEK 293T cells. It identifies specific residues that were linked to loss of binding in domains II and III of the E gene.
• Wahala WMPB, et al. (2010) Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses. PLoS Pathog 6(3): e1000821. doi:10.1371/journal.ppat.1000821 has 2 nice tables of E gene antigenic and informative sites as well as a nice figure mapping the sites onto the structure.

 from Wahala et al., 2010.

  from Wahala et al., 2010.

 from Wahala et al., 2010.

Some other interesting things that came out of the discussion/presentation:

1. The presence of escape mutants which they called 'hinge knockouts' as they were located in the hinge region DI/II of the E gene. 
2. These mutants escaped neutralization and yet were able to infect. 
3. Natural infection also seems to direct away from DIII of the E gene and seems to focus on this hinge region. 
• I found this tidbit interesting as in my own quasispecies research I found a surprising amount of mutations focused in on DI/II of the E gene rather than DIII. My samples were from mosquitoes and natural infections (human sera). 

Other articles of interest:

• de Alwis R, Beltramello M, Messer WB, Sukupolvi-Petty S, Wahala WMPB, et al. (2011) In-Depth Analysis of the Antibody Response of Individuals Exposed to Primary Dengue Virus Infection. PLoS Negl Trop Dis 5(6): e1188
• Wahala, et al., 2009. Dengue virus neutralization by human immune sera: Role of envelope protein domain III-reactive antibody. Virology 392: 103.

So I haven't really touched on the second part of Aravinda's talk/abstract which had to do with immature versus mature particle development in different cell cultures. I will deal with this more in Dr. Kuhn's presentation (to come) where they've done crystallography to model the particles and I need time to dig up some really pretty pictures of this.

Essentially though different particles can be visualized in differing ratios depending on cell type where growth occurred. For example: In U937 you have a low ratio of prM/E and you get more 'mature' viral particles whereas in Vero or C6/36 you get more of a mix.

What function do the different particle 'shapes' have? 

What might the particle ratio of immature/mature be in wild type infection (not in cell culture)? 

What are some of the limits and caveats to consider in this work?

We'll address all this coming up...