NIAID-DVI: Dengue Cohort Studies in Thailand -- Tim Endy

As a intermittent blogger...or rather any type of blogger, I get an absolute thrill when I see my pageviews jump from a few dozen to a couple hundred at any given time especially since my blog goes "inactive" if I've nothing to say (ie. no conference, no workshop, no training to write about or I'm being lackadaisical about reviewing literature on a blog). On October 9th I hit 244 pageviews...and when I checked today I saw that on Oct 13th I had 488...it was indeed thrilling. The last time I experienced a thrill such as this was when I got into the thousands after I live-blogged the Workshop on Genomics Blog Series in Jan 2013.

It's always nice to know one's blog is proving useful from time to time...

Back to NIAID-DVI!

Human immune responses to dengue infection: lessons from cohort studies in Thailand
Tim Endy
State University of New York Upstate Medical University

Tim Endy like Dr. Kuhn is another one of those individuals in the field I could listen to for hours and not get bored. He's been a long time collaborator of my postdoc mentor, MAJ Jarman and WRAIR, where I work now. He also a retired COL did a stint at AFRIMS in Bangkok and is highly involved and I would contend a pillar in dengue work in Thailand.

Additionally, it's always exciting when someone you look up to in your field uses figures you generated in his presentation...huzzah!

That all being said...

NIAID-DVI: Pediatric Dengue Cohort Study -- Eva Harris

Neutralizing antibody responses in the Nicaraguan Pediatric Dengue Cohort Study
Eva Harris
Division of Infectious Diseases & Vaccinology
SPH, UC Berkley

So I've always enjoyed talks by Eva Harris...she's always very animated and excited about her work and she almost always has an overwhelming amount of information to show--scientific sensory overload at times...you wish you could just have copies of the slides or write faster to catch everything she says. She has a very active group, active collaborations, so she usually has  a lot to say in a short time. But y'know when you've been involved in work in a country for 23+ years...you're going to have a lot to say. 

I first heard Eva talk at ASTMH about Molly OhAinle's work which came out in 2011 in Science and Translational Medicine...

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: The Dynamic Dengue Particle -- Richard Kuhn

New Lessons in dengue virus structure and composition and their influence on vaccine strategies
Richard Kuhn
Purdue University

I had the honor and pleasure of meeting Dr. Kuhn where I work and he is fantastically animated and I very much enjoyed listening to what he had to say.

The structure of dengue has been known for more than 10 years via cryo-electron microscopy; however now with more sophisticated tools we can see the virus in ways unimaginable in the past. His group employed a variety of structure and biochemical tools to probe the structure of the dengue virion as well as conformation, composition and dynamics..

Lets look at some particles...

mmm...pretty. Clustering of dengue particles. Source: MicrobiologyBytes

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.

NIAID-DVI: B-cell responses to dengue infection -- Jens Wrammert

Blog Series: NIAID-DVI

Human B cell responses during dengue infection
Jens Wrammert
Emory University

"Humoral immune responses are thought to play a major role in dengue-induced immuno-pathology, however little is known about the plasmablasts producing these antibodies during an ongoing infection." ~Jens

•  The group analyzed plasmablast responses in patients with acute dengue infection.
• plasmablast responses increased more than 1000 fold over baseline levels.
• These responses made up as much as 30% of the peripheral lymphocyte population
• Responses were dengue specific
• IgG secreting cells that reached high numbers after fever onset coinciding with 'the window' where serious dengue-induced pathology is observed.

Jens' recent paper: Rapid and massive virus-specific plasmablast responses during acute dengue virus infection in humans. 2012. JVI 86: 2911.

 

What is a plasmablast?

"The most immature blood cell that is considered a plasma cell instead of a B cell is the plasmablast. Plasmablasts secrete more antibodies than B cells, but less than plasma cells. They divide rapidly and are still capable of internalizing antigens and presenting them to T cells. A cell may stay in this state for several days, and then either die or irrevocably differentiate into a mature, fully differentiated plasma cell." ~Wikipedia

"A plasmablast is basically a B cell that is actively secreting antibodies. This is in response to acute infection only so it is not 'long term' "~Dr. Friberg-Robertson, Immunologist and Friend Extraordinaire 

Questions raised:

Do these cells have a role in dengue immuno-pathology during an ongoing infection?

Ongoing/Future Research to answer said question:

• Understanding the complete repertoire and specificity of the antibodies that are secreted [en masse] by the plasmablasts.
• Jens' group has/is generated panels of human monoclonal antibodies from dengue infection-induced plasmablasts for 5 patients and have done initial functional analyses...stay tuned into his research for the results.

Other papers that highlight plasmablast responses in dengue infection:

• Simmons et al., 2007. Patterns of host genome--wide gene transcript abundance in the peripheral blood of patients with acute dengue hemorrhagic fever. JID 195: 1097.
• Xu et al., 2012. Plasmablasts generated during repeated dengue infection are virus glycoprotein-specific and bind to multiple virus serotypes. J Immunol 189: 5877.
• Garcia-Bates et al., 2012. Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients. J Immunol 190: 80.

NIAID-DVI: Transcriptional responses to dengue -- Stephen Popper

Blog Series: NIAID-DVI

Early genome-wide host transcriptional responses to dengue that correlate with neutralizing antibody titer following vaccination and natural infection
Stephen Popper
Stanford University School of Medicine

Vaccine development has been typically hampered because we don't have a good understanding of the correlates of protection or the links between innate and adaptive immune responses. We also have a limited understanding of the role of background immune status and it's affect on subsequent infection or vaccination. In order to identify links between early host responses and later adaptive immune responses, Popper's group studies the genome-wide transcriptional response to dengue during vaccine trial (a controlled setting) and in natural infection settings.

• They characterized the temporal dynamics of transcript abundance in subjects vaccinated with rDEN3delta30/31 (TetraVax-DV live vaccine candidate Den3 component developed by NIAID).
• Of Note:
• During early transcriptional response there is an interferon-associated transcript expression pattern that peaked in most subjects between day 6 and day 12 post-immunization and this correlated with the titer of neutralizing antibodies (PRNT60) measured at day 42.

In their newest work...

NIAID-DVI: Systems Vaccinology -- Bali Pulendran

Blog Series: NIAID-DVI

So as I stated in an earlier blog...some things flew well above my head at this meeting so some of these blogs will be a re-print of the abstract and some general links and thoughts as well as a load of definitions and attempts to understand the components that made up said persons abstract with a healthy dose of trying to understand what they do. Also some of the presentations drove me to take lots of notes from which I could draw on while others, I'll admit my eyes glazed over...and the only note I wrote (several times on many talks was)

"Crash Course Immunology?"

It actually became a bit comic with how many presentations I wrote that on. I now have a new found appreciation for what immunologists do AND a new understanding of why I don't do it. So kudos to all vaccinologists and immunologists everywhere!

NIAID-DVI: Sanofi CYD dengue vaccine--Where do we go from here? Jean Lang and Bruno Guy

Blog Series: NIAID-DVI

Sanofi Pasteur CYD dengue vaccine programme update

Jean Lang
Sanofi Pasteur

and

Immunological characterization of the Sanofi Pasteur dengue vaccine candidate: hypotheses and investigations to explain results of the Phase IIb proof of concept efficacy trial in Ratchaburi
Bruno Guy
Sanofi Pasteur

So by way of quick review:

credit: Roehrig's Presentation

• the CYD-TDV vaccine is a yellow fever backbone with the prM and E genes replaced by each of the dengue serotypes.
• There was good response in efficacy trials to dengue 1, 3 and 4 however the vaccine failed in illiciting a response to dengue 2 (response was about 30%) DESPITE having satsifactory PRNT titers...see previous blog post.
• In 2012 there was a Phase IIb efficacy trial in Ratchaburi, Thailand
• It showed the vaccine was safe.
• It showed that it was possible to make an efficacious vaccine against dengue
• It raised questions on the reference PRNT assay
• It challenged some of the dengue vaccine development hypotheses
• It reminds us how complex the disease is

NIAID-DVI: TV003 live attenuated tetravalent dengue vaccine -- Stephen Whitehead

Blog Series: NIAID-DVI

Safety and immunogenicity of the NIH live attentuated tetravalent dengue vaccine candidate TV003

Stephen Whitehead
National Institutes of Allergy and Infectious Diseases, NIH

January 13, 2013, the NIH releases a news statement: NIH-developed candidate dengue vaccine shows promise in early-stage trial where they discuss the new vaccine for dengue that has done well in an early stage clinical trial, the study having been published in the Journal of Infectious disease 17 January 2013. Unfortunately the article is behind a paywall...curses!!! So I will do my best to convey what I can through other numerous links and information garnered from pillaged internet slides about the vaccine and of course what I learned in the meeting.

Falling back on John T. Roehrig's presentation because it's such a nice clear presentation...here is the make up TV003.

NIAID-DVI: Dengue subunit vaccine -- Beth-Ann Coller

Blog Series: NIAID-DVI

Development update - recombinant subunit dengue vaccine

Beth-Ann Coller

Merck & Co., Inc.

So Merck decide to nix the use of the whole genome and opted for focusing in on the Envelope gene; 80% of it.

The current status per the meeting abstract:

• Preclinical studies of this recombinant subunit vaccine have been conducted in non-human primates in order to evaluate immunogenicity and efficacy of tetravalent formulations
• They are doing testing with and without adjuvant
• The formulations have been evaluated in both dengue naive and experienced animals.
• The work has shown that recombinant proteins can induce balanced tetravelent responses without evidence of interference.
• They are in Phase 1 clinical trials with the vaccine in healthy flavivirus naive adults.

So I had one potentially naive question in this presentation which of course I kept to myself because it was a lively bunch and I feared for my life at times given I secretly harbor a copy of immunology for dummies book...

NIAID-DVI: DENVax -- Jorge E Osorio

Blog Series: NIAID-DVI

Pre-Clinical and clinical development of recombinant live attenuated tetravalent dengue vaccine (DENVax)
Jorge E Osorio
Takeda Vaccines

So Takeda Vaccines took over Inviragen and have been developing a tetravalent, live attenuated dengue vaccine called DENVax. It consists of a molecularly characterized, attenuated DENV-2 strain and three chimeras. The chimeras all have the backbone of the attenuated DENV-2 strain, but the prM and E genes have been swapped out with DENV-1, DENV-3 and DENV-4.

DENVax design from Osorio et al., 2011.

NIAID-DVI --A Preamble: The Sanofi Dengue Vaccine Failure (Chimerivax)

Blog Series: NIAID-DVI

A preamble: The Sanofi dengue vaccine failure, ChimeriVax

So given I am not daily entrenched in the world of vaccine development, the most that I ever heard about dengue vaccines was that there were several out there in development and Sanofi's was the furthest along. Then I heard the Sanofi vaccine failed. It illicited good response to dengue 1, 3 and 4 and did a fantastic fail on dengue serotype 2.

I could end this blog there but my curiosity and incessant need to comb the internet and literature revealed just how 'fantastic' of a fail this was:

Still Here...new series of blog posts coming

Greetings to my 6 followers and many others who peruse this blog via google, facebook or other link.

Rest assured I have not fell off the planet; the purpose of this blog was to be a communication, teaching, disseminating tool for when I attend workshops, conferences, develop courses or read literature. Indeed apparently I don't travel as much as some in terms of conferences and workshops so I've had little write about...

That and I got swept away in the bid for grant funds available at my institution...was successful--HUZZAH, 4th year of trying was the charm apparently and now am in the thick of my own research as well as what I do on a daily basis as the Viral Diseases Branch bioinformatician.

However following the Workshop on Genomics this last January (and coming up again this January) I have been teaching a basic class on bioinformatic sequence analysis and recently attended a meeting on the NIAID - Dengue Vaccine Initiative.