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

Dengue immune status of host and the quality of cross-reactive anti-envelope antibodies generated 
Wei-Kung Wang

John A. Burns School of Medicine, University of Hawaii at Manoa

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human sera following DENV infection have show a significant proportion of anti-E Abs cross-reacts to all four DENV serotypes and to one or more other flaviviruses, so-called group-reactive (GR), and recognizes the fusion loop (FL) in the E protein, domain II. Studies of mouse anit-E monoclonal antibodies (mAbs) reported that GR mAbs were weakly or non-neutralizing compared with type-specific mAbs. 

First Study:

• A study of epitopes, binding avidity and neutralization potency of 32 human GR mAbs, found that the neutralization potency and binding avidity of GR mAbs derived from secondary DENV infections were significantly higher than those derived from primary infections. Analysis of the repertoire of anti-E mAbs derived from patients with primary DENV infection revealed that the majority were GR, low avidity and weakly neutralizing, whereas those from secondary DENV infection were primarily GR, high avidity and potent neutralizing. The observations suggest that dengue immune status of host affects the quality of cross-reactive FL Abs generated. 

Second Study:

• Quantitative virion-capture ELISA was used to measure the concentration of anti-E Abs in sera from secondary DENV infections in Nicaragua. The proportion of fusion loop (FL) Abs by a capture ELISA using virus-like particles containing mutations in the FL, and calculated the concentration of FL Abs. FL Abs did not correlate with NT50 against the current infecting serotype, however did correlate with NT50 against the serotypes to which patients had likely not yet been exposed to. This suggests that FL Abs may play a protective role against serotypes to which the person has not been exposed to yet, after secondary DENV infection.

It is known that after secondary DENV infection, individuals develop heterotypic neutralizing Abs against serotypes to serotypes they have not been exposed to. These neutralizing Abs are believed to account for protection against subsequent infection by other serotypes (they have not been exposed to). These studies indicate that FL Abs after secondary infection may contribute to such heterotypic neutralizing Abs.

Wei-Kung's Group's Relevant Publications...

1. Lai C-Y, et al. 2013. Analysis of Cross-Reactive Antibodies Recognizing the Fusion Loop of Envelope Protein and Correlation with Neutralizing Antibody Titers in Nicaraguan Dengue Cases. PLoS Negl Trop Dis 7(9): e2451
2. Tsai et al., 2013. High avidity and potent neutralizing cross-reactive human monoclonal antibodies derived from secondary dengue virus infection. JVI ahead of print
3. Lin H-E, et al., 2012. Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay. PLoS Negl Trop Dis 6(1): e1447.

Small Change #3: A single amino acid change

Viral structural dynamics contributing to neutralization

Kimberly Dowd

NIAID-NIH, Ted Pierson's Lab

Flaviviruses explore multiple conformations via dynamic motion of virus structural proteins. The adds complexity to the antigenic surface...in that virus can be referred to as 'breathing'. This 'breathing' varies epitope 'availability' to antibody binding.

The study investigated whether structural dynamics varies among flaviviruses, and how this might contribute to neutralization sensitivity.

• How the virion maturation state governs dynamics:
• Immature virus retains uncleaved prM (think back to Kuhn's talk) which might constrain viral breathing.
• prM+West Nile Virus had a faster decay at rate 1.8x faster than prM- West Nile Virus.
• The intrinsic decay of infectivity is being used as a proxy of dynamics. 
  
• Using a panel of mAbs, it was found that despite having identical backgrounds, prM+ and prM- West Nile virus displayed distinct patterns of time dependent neutralization.
• Therefore, biochemical heterogeneity impacts how neutralization sensitivity changes as a function of dynamics.
•  There was a substantial difference in the neutralization potency between two difference DENV1 genotypes.
• mAb E111 neutralizes strain 16007 over 4000 times better than strain WP.
• Intrinsic decay of WP was 2x faster than 16007. So mutants of WP were generated expressing each of the 13 amino acid differences in the E gene of 16007.
• Mutation of a single residue in WP resulted in that variant behaving identical to strain 16007 (measured by neutralization and decay).
• AND...this residue is located outside the E111 epitope.
• Neutralization susceptibility can be drastically altered in an epitope-independent manner by changing viral dynamics which can affect the landscape of antibody binding as well as virus stability.

Clearly there are a variety of ways in which dengue virus continues to adapt and escape immune pressure and it doesn't take much tip the scales back into viral favor...it definitely keeps us on our toes.