Dissecting the Influenza Pathogenesis Study Pt. 1

Last night we got our first look at a study that compared the pathogenesis (disease progression) of non-human primates (macaques) infected with the H5N1 virus, seasonal flu, and with two altered viruses carrying genetic material from the 1918 Spanish Flu.

 

The study, entitled Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus by Carole Baskin et. al.  appears in the latest issue of PNAS (The Proceedings of the National Academy of Science).

 

While there is much here to consider (and we’ll get to that), the upshot is that:

 

The avian virus was found to significantly outpace not only run-of-the-mill influenza but even the highly virulent 1918 ressortants, in terms of its relentless pathogenicity.

 

 

I’m not going to try to critique this research, as I’m obviously unqualified to do so.   Someone with more letters after their name than EMT-II (Ret.) will have to take that on.

 

But what I’d like to do is explain, in layman’s language, what the authors are reporting, and some of the implications of what they’ve learned.

 

And here I run into a bit of a dilemma.

 

Just about every facet of this study requires a bit of `background’, to make it understandable.  At least if we’re going to do more than give it a cursory look.

 

Even the title, which refers to the `innate immune response’ requires a bit of explanation.   And the deeper you plunge into this paper, the more `detours‘ – areas that require a good deal of explanation –  you find.  

 

I have to ask myself. 

 

How many of my readers understand the difference between Type I and Type II Pneumocytes?  (I hope the number is low, since I had to look it up)

 

I suddenly realized that, in order to make all of this understandable to the widest number of readers, I’d have to write a multi-part post.   Not really a problem, of course.  After nearly 2800 blogs, what’s a multi-part post?

 

 

So, while I fully expect we can get `there‘ from `here‘, I’ll warn my readers in advance it may require a serpentine route.   As to how many installments it will take to get `there’ – well, we’ll know when we’ve arrived.

 

Given the long, and awkward title of the research paper, once I link to it in at the top of each post, I’m simply going to refer to it as The Baskin Study

 

So, without further ado,  I present Part I of my series Dissecting the Influenza Pathogenesis Study.

 

 

MEET THE PLAYERS

 

 

To perform a systematic comparison of several highly pathogenic influenza viruses, we inoculated cynomolgus macaques (8 animals per group) with either influenza A/Vietnam/1203/2004 (referred to as H5N1), 1918HA/NA:A/Texas/36/91 (1918HANA), 1918HA/NA/NS:A/Texas/36/91 (1918HANANS), or the parental H1N1 A/Texas/36/91 (Texas) virus. – The Baskin Study

 
The hodgepodge of letters and numbers above identify 4 very different influenza viruses, which researchers used to infect macaques in order to study their different disease processes.

 

To understand what these numbers and letters mean, we need to digress briefly. 

 

 

Influenza viruses come in 3 broad types; A, B, and C.  

 

  • Influenza `C’ viruses are relatively rare, generally produce milder symptoms in humans, and are not considered to have epidemic or pandemic potential. 

 

  • `B’ viruses, which are far more common, may produce serious illness and spark regional epidemics, but aren’t viewed as `pandemic strains’.

 

  • It is only the `A’ influenza strain – which is characterized by two surface glycoproteins known as hemagglutinin (HA) and neuraminidase (NA) that mutates rapidly and can cause an influenza pandemic.

 
Influenza `A’ viruses are identified by their HA and NA proteins.  There are 16 known HA subtypes and 9 known NA subtypes.  While many combinations are possible, currently only a few are in circulation.

 

Among humans, the H1N1, H1N2, and H3N2 viruses currently circulate globally.    H5s, H7s, and H9s are commonly found in birds.

 

The H1N1 viruses today are all descendants of the 1918 Spanish Flu.   The H3N2 viruses are descendants of the 1968 pandemic. 

 

The H1N2 virus, which was first detected in 2002, appears to be a hybrid, consisting of the hemagglutinin (HA) from the H1N1 virus and the neuraminidase (NA) from the H3N2 virus.

 

Influenza `A’  viruses are actually complex entities comprised of 10 genes on 8 separate RNA molecules (called: PB2, PB1, PA, HA, NP, NA, M, and NS)

 

 

 

image

 

 

This study compared 4 influenza viruses.

 

  • One wholly avian virus (H5N1), a clade 1 H5N1 virus collected from Vietnam in 2004.

 

  • One seasonal influenza virus (Texas) H1N1 A/Texas/36/91, which circulated widely in the mid and early 1990’s.

 

  • One genetically modified seasonal H1N1 virus (1918HANA) with the HA and NA from the 1918 virus spliced to it.

 

  • And one genetically modified seasonal H1N1 virus (1918HANANS) with the HA, NA, and NS1 from the 1918 virus spliced to it.

 

 

The (HA) and neuraminidase (NA) surface proteins are known conveyors of virulence, and while the resultant reassortant viruses might not have been an exact match to the 1918 pandemic virus, they do represent a reasonable facsimile.

 

The NS1, grafted onto the 2nd 1918-like virus, is known to inhibit the host interferon response.   

 

Interferon is a natural protein produced by the cells of the immune system when they sense a viral or bacterial invader.  Interferons belong to the larger class of glycoproteins known as cytokines. 

 

 

The stage is now set. 

 

Thirty-four macaques were used in this study, four groups of 8 – with 2 `mock infected’ to serve as a reference for host response without viral challenge.  

 

Each group of 8 were macaques were infected with a different strain of the virus via tracheal, nasal, conjunctival, and tonsillar routes.

 

On days 1, 2, 4, and 7 postinfection (PI), 2 animals per group were euthanized.  One macaque died from excessive pulmonary damage between day 6 and 7.

 

On day 7, the two `mock infected’ animals were euthanized.  

 

Necropsies were performed on each animal and tissue and serum samples were subjected to numerous tests. 

 

In the next installment, we’ll look at some of the results.

 

Part 2

This is Part II of a multi-part look at the recent PNAS paper entitled Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus by Carole Baskin et. al., which analyses the pathogenesis of the H5N1 virus compared to seasonal flu, and 1918-like viruses.

 

In part Part I, which appeared on Saturday, we examined the 4 different viruses used in this study, and the methods used by the research team.

 

Today we’ll take a look at the body’s innate immune system and then look at some of the results of this study.

 

Don’t worry, for my own sake I intend to keep this pretty simple.

 

As you’ve probably noticed, `innate immune response’ is a key phrase in the title of this research paper. 

 

Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus.

 

So we know the innate immune response is going to be a big part of this report, but . . .what exactly is it? 

 

And how does it work?

 

All of us are born with what is called an Innate Immune System that can detect, and launch a generic defense against, a wide variety of invading pathogens.

 

And if you think about it, were it not for this built-in immunity, none of us would survive past the first few hours or days of life.  We’d be quickly overrun by opportunistic infections.

 

This innate immune system  not only fights infections on its own, it also buys us time for our Adaptive Immune System to learn to recognize and fight specific pathogens. 

 

It is this adaptive immune system that produces pathogen-specific antibodies that can remember previous encounters with a virus, and can confer long-term immunity. 

 

We call this trait acquired immunity, and that is what keeps us from suffering through viral infections like the measles or mumps over and over again.

 

For our innate immunity to work, it must have a way to recognize an infective agent, even from a pathogen it has never seen before.

 

And that capability comes from PAMPS.

 

PAMPs (Pathogen-Associated Molecular Patterns) are patterns of molecules associated with many types of pathogens.

 

In other words, PAMPs are an easily recognizable signature that tells our innate immune system that we have been infected . . . with something

 

Our immune systems have cells designed to recognize, and react to these signatures, that include:

 

  • phagocytic cells (neutrophils, monocytes, and macrophages);
  • cells that release inflammatory mediators (basophils, mast cells, and eosinophils);
  • natural killer cells (NK cells); and
  • molecules such as complement proteins, acute phase proteins, and cytokines.

 

In other words, our innate immune system throws just about everything but the kitchen sink at an unrecognized infection.  

 

In response our bodies spike a fever while natural killer cells and phagocytes rush to fight the infection. 

 

Our bodies produce protein and cytokine rich fluids at the site of the infection and cells throughout our body release inflammatory mediators.   

 

Generally all of these things are good things, as they help fight the invading pathogen, although they are what make us so miserable when we have an infection.  

 

Unfortunately, it is possible to have too much of a good thing.

 

On very rare occasions the body’s innate immune system can overreact, go into overdrive, and overwhelm and damage the body’s own organs – sometimes resulting in death.

 

This process is commonly called a `Cytokine Storm’, although it is actually a poorly understood phenomenon, and not without controversy.  

 

Cytokines, broadly speaking, are a category of signaling molecules that are used extensively in cellular communication.

 

They are often released by immune cells that have encountered a pathogen, and are designed to alert and activate other immune cells to join in the fight against the invading pathogen.

 

This  `Cytokine Storm’ has been described as a positive feedback loop, where immune cells – encountering a pathogen – secrete  signaling cytokines which call more immune cells to the site of infection – which in turn secrete more cytokines – which call even more immune cells to join in the fight . . .

 

This uncontrolled exuberant immune response has frequently been suggested as being one of the factors leading to the high mortality rate of the 1918 pandemic.   

 

The `other plausible suspect’, which has gained a good deal of support over the past year, being secondary bacterial pneumonia.

 

Of course, neither of these processes need be mutually exclusive.

 

So, with this bare-bones understanding of the innate immune system, and the role of cytokines, we can begin to look at some of the findings of this study.

 

 

The following key points were lifted from the study’s abstract:

 

  1. Among these viruses, HPAI H5N1 was the most virulent.
  2. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions.
  3. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays.
  4. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection.
  5. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.

 

 

The first two points are self-explanatory.

 

The third point, about the H5N1 virus targeting type II pneumocytes, could do with a bit of explanation.  

 

Unlike the 1918-like viruses, which largely targeted type I pneumocytes, the H5N1 virus attacked and damaged type II pneumocytes.

 

image

 

 

 

Pneumocytes are a collective term for the two types of cells lining the alveoli (the air sacs) in the lung; Type I and Type II pneumocytes.

 

  • Type I pneumocytes are responsible for the gas exchange (02 and C02) between the lungs and the blood stream.   Type I pneumocytes are easily damaged and cannot reproduce themselves.

 

  • Type II pneumocytes are responsible for the production of surfactant, which reduces the surface tension of pulmonary fluids and contributes to the elasticity of the lungs.  

 

  • Type II pneumocytes are able to replicate in the alveoli and can create new Type I pneumocytes.

 

 

image

 

 

Since type II pneumocytes are the only repair mechanism available to replace damaged type I pneumocytes, destroying them can have profound effects on the lungs’ ability to recover from injury.

 

 

One of the other big differences between the pathogenesis of the seasonal, 1918-like, and avian viruses was the production of many types of cytokines, which is represented by this heat map below.

 

 

image

 

 

As the press release for this paper stated:

 

The avian virus was found to significantly outpace not only run-of-the-mill influenza but even the highly virulent 1918 ressortants, in terms of its relentless pathogenicity.

 

 

In part III, we’ll take a closer look at this heat map, and some more of the findings of this report.

Part 3

This is third and last part of my extended look at the recent PNAS paper entitled Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus by Carole Baskin et. al., which analyses the pathogenesis of the H5N1 virus compared to seasonal flu, and 1918-like viruses.

 

  • In  Part I, which appeared on Saturday, we examined the 4 different viruses used in this study, and the methods used by the research team.

 

  • In Part II, which appeared on Sunday, we looked at the innate immune system and began looking at the results of this study.

 

This series is not intended to be a scientific review, but instead a layman’s tour of the paper.

 

Today, we’ll finish looking at the findings, and talk about about what it might mean.  In Part II I listed the 5 major findings, and we discussed the first 3.  

 

Today we’ll cover points 4 & 5.

 

4.   The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection.

 

The loss of dendritic cells (named after, but not to be confused with the projections from neurons) through premature apoptosis (programmed cell death) in the lungs and draining lymph nodes is viewed by these researchers as a key finding.

 

image

 

 

Dendritic cells are a special type of immune cell, most commonly found in tissues that are exposed to the outside environment (skin, lungs, digestive tract), that boosts immune responses by showing antigens on its surface to other cells of the immune system.  

 

In the simplest language, dendritic cells help `teach‘ the adaptive immune system how to recognize a virus, and are key in the creation of pathogen-specific antibodies.  

 

A loss of dendritic cells at the site of infection (lungs) could negatively impact the body’s ability to generate post-infection immunity.

 

In other words, catching and surviving the virus may not guarantee future immunity to the virus.

 

 

 

5.   While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.

 

The primates infected with the H5N1 virus began to experience rapid, serious, and potentially permanent tissue damage in the lungs.  

 

Unlike the seasonal, and 1918-like viruses which attacked type I pneumocytes, the H5N1 virus seems to target type II pneumocytes.

 

Type II pneumocytes are not only more numerous in the lungs, they are responsible for producing surfactant with antimicrobial, immunomodulatory, and anti-inflammatory properties.  

 

A loss of type II pneumocytes severely degrades the lungs ability to fight off an infection, and to repair damaged tissue.

 

 

The most widely published graphic from this study has been this dramatic representation of the relative generation of 45 key cytokines in the host animals.

 

Red, as you might imagine, indicates a greater production of cytokines.  Green signals a reduction.

 

 image

 

Seasonal influenza (H1N1-Texas) and the two 1918-like ressortants all generated increased cytokine production – particularly in the first 4 days of infection.   

 

By day 7, however, the production of cytokines was fading.

 

In terms of quantity, the 1918-like viruses produced considerably higher numbers of cytokines than the seasonal virus.

 

But the expression of cytokines by hosts infected with the H5N1 virus elevates very early in the infection, and remains elevated across the board for the entire 7 day observation period

 

Both in terms of quantity, and duration, the H5N1 virus elicits an extreme immune response in the host animals. 

 

The authors refer to this it as  early, sustained, and relentless.   

 

Which pretty well describes it.

 

The authors of this study summarize these findings this way:

 

In conclusion, H5N1 virulence is a multipronged mechanism that causes severe lung pathology with potentially permanent tissue damage within 24 h PI, accompanied by excessive and sustained type I IFN, inflammation, and innate immune induction.

 

          *        *        *        *         *         *        *

 

In November of 2006 I wrote a blog called  Not Your Father’s Influenza , which outlined in very broad terms, some of the differences between seasonal influenza and the (thus far) highly lethal H5N1 virus.

 

After reading WHO (World Health Organization) report entitled Influenza Research At the Human And Animal Interface, I wrote:

 

While H5N1 is indeed an influenza virus, calling it the `flu’ is like calling a Hurricane a patch of bad weather.
It doesn’t even begin to describe it.

 

 

Obviously, with a CFR (case fatality ratio) of 60% among identified human infections, we’ve known for some time that the H5N1 virus isn’t any ordinary flu. 

 

Today, courtesy of this research by Carole Baskin et. al., we now have a much better understanding of  Why the virus is so deadly.

 

And it turns out, there isn’t just one thing, but rather a host of reasons.

 

From a scientific standpoint, all of this is fascinating.  We are not only seeing a unique pathogenesis, we are learning more and more about how the immune system works.

 

But the question I’m sure everyone is asking is: 

 

If the H5N1 virus ever goes pandemic, will it retain this unprecedented lethality?

 

 

We know that the H5N1 virus must undergo some (unknown) number of genetic changes in order to become easily transmissible.   The hope is, these changes will also reduce its virulence.

 

It is a popularly held theory that an outbreak from a virus that is too deadly will burn itself out quickly.  To spread effectively, you need mobile, yet infectious, vectors. 

 

A disease that sickens, incapacitates, and kills too quickly loses too many opportunities to spread.  Many researchers point to the localized, but short-lived outbreaks of Ebola in central Africa as prime examples.

 

But not everyone is convinced that this virulence for transmissibility tradeoff is inevitable.  

 

There are simply no guarantees.

 

Of course, there are no guarantees that the H5N1 virus will ever become a pandemic strain.  

 

The next pandemic could well come from an H2, or an H7, or even an H9 virus, or perhaps from a reassortment of the H5 virus with another strain.

 

But whatever the cause, this research has provided us with a much more detailed look at the pathogenesis of influenza viruses in non-human primates.  

 

And that will hopefully help doctors work on new avenues of treatment.

This comes from FLA_MEDIC’s excellant flu blog, Avian Flu Diary

http://afludiary.blogspot.com/2009/02/dissecting-influenza-pathogenesis-study.html

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