NIAID-DVI: Understanding the E gene Part II: 3 Tales of the power of small changes

Now that we have an appreciation for the dynamic dengue particle back to the ever elusive E gene...

Small Change #1: 2 amino acids

The type-specific neutralizing antibody response elicited by a dengue vaccine candidate is focused on two amino acids of the envelope protein
Ted Pierson
NIAID-NIH

As stated in many of these blog series posts the failure of the Sanofi vaccine has highlighted the limited understanding we still have about dengue and the research continues in many aspects of disease pathogenesis as well as genetic influences on the virus. Ted Pierson (and others) seek to better understand the humoral immune response against DENV infection. They wanted to identify epitopes recognized by serotype-specific neutralizing antibodies elicited by monovalent DENV1 vaccination. To do this they constructed a panel of over 50 DENV1 structural gene variants containing substitutions at surface accessible residues of the envelope protein to match the corresponding DENV2 sequence. They identified two mutations that contribute significantly to type-specific recognition by polyclonal DENV1 immune sera. When they analyzed sera from 24 participants of a phase I clinical study, they found that there was a reduced capacity to neutralize a DENV1 variant which contained both mutations. Sera from 77% of subjects recognized the DENV1 variant and DENV2 equivalently (less than 3 fold difference). The data indicated that the type-specific component of the DENV1 neutralizing antibody response to vaccination was focused on just two regions of the E protein. The amino acids in question? E157 and E126.

Unfortunately the paper hasn't come out specifically on this study that I can find...but Pierson has been involved in numerous studies characterizing aspects of the E gene:

• Shrestha et al., 2012. Complex phenotypes in mosquitoes and mice associated with neutralization escape of a dengue virus type 1 monoclonal antibody. Virology 427: 127
• Austin SK, et al., 2012 Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope. PLoS Pathog 8(10): e1002930
• Sukupolvi-Petty et al., 2013. Functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection. JVI ahead of print.
• Pierson and Kielian. 2013. Flaviviruses: braking and entering. Curr Opin Virol 3:3

Small Change #2: The fusion loop of the E gene

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.