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...


They've started to look at relating these findings (described above) to natural infection by examining the relationship between transcriptional response and DENV-specific antibody development in Nicaraguan children with acute primary DENV3 infection.

• Of Note: Data suggests that there are early correlates of subsequent protective adaptive immune responses.

Papers of note linked to their work:

• Popper SJ, Gordon A, Liu M, Balmaseda A, Harris E, et al. (2012) Temporal Dynamics of the Transcriptional Response to Dengue Virus Infection in Nicaraguan Children. PLoS Negl Trop Dis 6(12): e1966. doi:10.1371/journal.pntd.0001966
•  Waddell SJ, Popper SJ, Rubins KH, Griffiths MJ, Brown PO, et al. (2010) Dissecting Interferon-Induced Transcriptional Programs in Human Peripheral Blood Cells. PLoS ONE 5(3): e9753. doi:10.1371/journal.pone.0009753 (nothing to do with dengue per say but an interesting look at their work with interferons and microarrays, I'm on my third read through attempting to follow all the interferon types and immunology terms...once I got to the microarray stuff I was much more comfortable)

For those like myself who are learning immunology as they go...Waddell's paper introduction has a nice short explanation of interferons (the linked references will take you to the references as cited in Waddell's paper):

"Interferons are a class of cytokines first identified in 1957 as having a protective effect against viral infection [1]. Interferons can be divided into three groups; type I (IFNα/β/ε/κ/ω) that engage the IFNAR1/2 receptor, type II (IFNγ, the sole member) that signal through the IFNGR1/2 receptor [2], and type III (IFNλ) that utilize IFN-λR1 and IL-10R2 receptors [3], [4]. 

The type I interferons, IFNα (of which there are 13 subtypes), IFNβ and IFNω are secreted by most cell types in response to viral infection [5]. Mice lacking intact interferon receptors are highly susceptible to viral infection [6]. Type I IFN stimulation induces a number of different systems involved in the activation of the immune response, cell growth and the control of apoptosis, in addition to the PKR (dsRNA-dependent protein kinase), 2-5A synthetase and Mx antiviral systems [7], [8]. Type I interferon subtypes have also been reported to have distinct activities [9], [10]; these IFN subtype-specific effects are influenced by factors such as receptor binding efficiencies [11], constitutive levels of IFN expression [12], and the specific viral-target cell combination [13]. By contrast type II interferon (IFNγ), secreted by activated NK cells and T lymphocytes, has been implicated primarily in the activation of macrophages and has been demonstrated to be important for the protection of the host against intracellular pathogens such as Leishmania, Toxoplasma and Mycobacterium species [14]. Mutations in the IFNγ receptor have been associated with increased susceptibility to mycobacterial infection [15]. Interferons are involved in a wide range of clinically important phenomena, ranging from activation of immune responses to infection [14] to cancer suppression [16] to depression [17]. Recombinant interferon therapy has been approved for a spectrum of conditions such as hepatitis B and C infections, Kaposi's sarcoma, multiple sclerosis and chronic granulomatous disease [5]."

Right...we're all experts now. Note: I'm still looking up things as I read through Waddell's article! 

On 'Correlates of Protection'...

I've heard this a lot throughout the talks and the current status or general consensus for dengue virus is, we don't really know what the true 'correlates of protection' are; though Popper's group (above) and many others have been attempting to study this. 

The term seems very nebulous to me. As with all things nebulous or undefined in my mind...I go to Wikipedia then I go to the literature to put it in context more:

Correlates of immunity/protection to a virus or other infectious pathogen are  measurable signs that a person (or other potential host) is immune, in the sense of being protected against becoming infected and/or developing disease. For many viruses, antibodies serve as a correlate of immunity. However this isn't always to case. In the example of rubella, if you have antibodies you are protected. However, this isn't true for HIV because antibodies can be measurable and yet the individual is not protected from the disease.

In the case of the Sanofi failed vaccine, individuals were shown to have dengue 2 titers and yet were not protected from the circulating diversity. There could be any number of reasons why...the most popular one circulating is that the vaccine is tetravalent and you may have competition between the serotypes once they've entered the human body. As mentioned in a previous blog the human 'dominant' response is to Dengue 4 for their vaccine. So antibody may not be the only correlate that can be measured and 'prove' an individual is protected from dengue.

Stanley Plotkin has two very nice articles, an older one from 2008 and a newer 2010 article, both on 'correlates of protection in vaccination'. They do very well to define what correlates for protection are/can be and how they are obtained/quantified:

TABLE 1.

Definitions of terms used in this article

Term	Definition
Correlate	An immune response that is responsible for and statistically interrelated with protection
Absolute correlate	A specific level of response highly correlated with protection; a threshold
Relative correlate	A level of response variably correlated with protection
Cocorrelate	One of two or more factors that correlate with protection in alternative, additive, or synergistic ways
Surrogate	An immune response that substitutes for the true immunologic correlate of protection, which may be unknown or not easily measurable

from: 2010 Plotkin article.


Correlates of vaccine-induced immunity

from: 2008 Plotkin article.

Plotkin mentions in his articles that all these can be correlates or 'surrogates' (see above table) of protection:

• Antibodies
• Antibodies on mucosal surfaces (Mucosal antibodies)
• Organ-specific correlates (very little research done in this area)
• Anamnesis: long term immunological memory...antibodies can decline over time, the body's 'memory' becomes integral in mounting a response. Plotkin gives the example of Hepatitis B: 

"Despite the loss of antibodies, B cell central memory is prolonged and protective efficacy is maintained at a high level [59, 60]. B cell memory to hepatitis B virus acts as a surrogate of protection, which is actually mediated through the antibodies evoked by antigenic stimulation of memory cells." (Plotkin, 2008)

• Cellular responses: CD4+ and CD8+

TABLE 2.

Quantitative correlates and surrogates of protection after vaccination

Vaccine	Test	Level required	Reference(s)a
Anthrax	Toxin neutralization	1,000 IU/ml	87, 136, 149,170, 191
Diphtheria	Toxin neutralization	0.01-0.1 IU/ml	14, 92
Hepatitis A	ELISA	10 mIU/ml	45, 110
Hepatitis B	ELISA	10 mIU/ml	66
Hib polysaccharides	ELISA	1 μg/ml	74
Hib conjugate	ELISA	0.15 μg/ml	73
Human papillomavirus	ELISA	NDb	140
Influenza	HAI	1/40 dilution	50, 171
Japanese encephalitis	Neutralization	1/10 dilution	63
Lyme disease	ELISA	1,100 EIA U/ml	128
Measles	Microneutralization	120 mIU/ml	24, 120, 158
Meningococcal	Bactericidal	1/4 (human complement)	96
Mumps	Neutralization?	ND	189
Pertussis	ELISA (toxin)	5 units	25, 173, 180.
Pneumococcus	ELISA; opsonophagocytosis	0.20-0.35 μg/ml (for children); 1/8 dilution	68, 81, 167
Polio	Neutralization	1/4-1/8 dilution	41, 95, 139
Rabies	Neutralization	0.5 IU/ml	196,
Rotavirus	Serum IgA	ND	49, 67, 104,199, 200
Rubella	Immunoprecipitation	10-15 mIU/ml	2, 27, 53, 99,141, 169
Tetanus	Toxin neutralization	0.1 IU/ml	13, 37,
Smallpox	Neutralization	1/20	89, 93, 139,160
Tick-borne encephalitis	ELISA	125 IU/ml	77
Tuberculosis	Interferon	ND	46
Varicella	FAMA gp ELISA	≥1/64 dilution; ≥5 IU/ml	195
Yellow fever	Neutralization	1/5	79, 97
Zoster	CD4+ cell; lymphoproliferation	ND	190

• ↵a Also see the text.
• ↵b ND, not defined.

from: 2010 Plotkin article.

Plotkin mentions dengue vaccine correlates of protection as well stating that they are becoming more important and aren't fully defined yet:

"Dengue vaccines are in advanced clinical trials, and therefore, the definition of a correlate of protection has become important. Whereas neutralizing antibody clearly protects against a homologous serotype (157, 175), controversy has centered on what function gives heterologous serotype protection in the face of the enhanced disease that occurs in the presence of antibody-dependent enhancement (ADE) (57). It appears from studies in a mouse model that heterotypic neutralizing antibodies do give heterotypic protection if they are present in concentrations sufficient to protect but insufficient to cause ADE (8,78, 186)."

Humans as 'Fuzzy Networks'...

Timing, dosage, age, gender, host genetics, viral genetics, immune background, cellular targets and responses...

Humans are just complicated. The virus is a predator, humans it's prey...in the constant 'battle' within our bodies the balance is repeatedly tipped back and forth:


Ok so it's not 'classic' Lotka-Volterra dynamics but the idea is there. Essentially, we as humans adapt as much as the virus--perhaps not as quickly as the virus on the genetics level, but we all have our immune predispositions shaped by our geographical location, our diet, our previous exposures...

Those in dengue endemic areas where the immune system has seen flaviviruses and dengue specifically, perhaps multiple times, are going to have a different immune network of responses to infection than someone from the Midwest  U.S. whose immune system has never seen flaviviral infection of any type let alone dengue. Therefore it's reasonable to assume that their response to vaccine might be different as well.

As dengue clinical trials push forward (Sanofi in a phase III and many others in phase II) hopefully the correlates of protection will come to light and we can tip the balance back in our favor.