WHEN

IMMUNE
SYSTEMS
ATTACK
How one Stanford researcher is
combating multiple sclerosis with
DNA vaccines

10 Stanford Scientific ©iStockphoto.com/geopaul

 W
e are surrounded by microbes
bent on using us as their own incubators.
Our only defense is our immune
system. Without this intricately woven
meshwork of defenders, our bodies would helplessly
succumb to the myriad of bacteria, viruses, fungi,
and other pathogens that surround us every moment
without our slightest awareness. Yet even something
so utterly vital for our existence has its shortcomings.

by
One such flaw is autoimmunity, a condition in which
the immune system attacks the body’s own tissues.
Perhaps one of the most debilitating autoimmune
diseases is multiple sclerosis, a neurological disorder
Jonathan Frohnmayer affecting more than half a million people in the United
States. The disease’s forefront researcher, Dr. Lawrence
& Tammy Doukas

Layout designed by Stephanie Le Volume IV 11

When Immune Systems Attack

Neuron diagrams Steinman, has published over 325 scientific articles and book
chapters on MS in his career at Stanford, and although much
progress has been made since the disease was first discovered
Neuron in the 1970’s, Steinman admits that much, including the very
cause of the disease, remains shrouded in mystery.
http://www.nlm.nih.gov/medlineplus/ency/imagepages/9682.htm

Nerve
How MS Inflicts Its Damage
Multiple sclerosis is a disease that affects the fatty white
matter of the central nervous system (CNS). CNS cells — known
as neurons — are supported and maintained by a variety of other
types of cells called glial cells, from the Greek word meaning
“glue”. The most important glial cell is the oligodendrocyte.
Neurons operate by propagating action potentials—electrical
signals in the form of positive ions—to other neurons along rod-
like structures called axons. Like the electrical transmission in
Myelin sheath your car or house, they require insulation so that the charge
does not weaken or disperse between its origin and destination.
Oligodendrocytes form a synergistic relationship with neurons.
They wrap around axons and secrete an insulating layer of protein
^ Bundles of neurons make up the nerves which are insulated by the myelin
Myelin
sheath.and nerve structure

}
oligodendrocyte axons http://science-education.nih.gov/nihHTML/ose/snapshots/multimedia/ritn/spinal/myelin2.html
Identical twins
contract the
illness at a 33%
node of
Ranvier
higher rate than
fraternal twins.
myelin

myelin called the myelin sheath. The myelin sheath is analogous to the
sheath rubber that surrounds an electrical wire, allowing electricity to
travel inside. Without this sheath, axons are unable to conduct
electricity, and their neurons are unable to communicate.
^ The CNS consists of neurons supported by glial cells including oligodendro- During the course of multiple sclerosis, the immune system
cytes which form the myelin sheath that wraps around neurons, insulating attacks the myelin sheath and surrounding proteins including the
them. The gaps are known as nodes of Ranvier. protective oligodendrocytes. Without both the myelin sheath
and the cells that produce it, nerves are left bare. This process
normal inflamed detached scarred
of demyelination is analogous to the rubber being stripped off a
wire. The transmission of nerve impulses slows, stops, or jumps
to other cells, and eventually the neuron dies.
Neuron death can be concentrated anywhere in the CNS,
and the specific location determines the symptoms the victim
suffers. Multiple sclerosis can destroy virtually any function
that the CNS controls, including movement, vision, cognition,
and a host of other conscious and unconscious functions. A
http://www.nlm.nih.gov
victim dies from multiple sclerosis when a sufficient portion of
^ In multiple sclerosis, the myelin sheath is destroyed in a process known as neurons that regulate the body’s autonomic functions such as
demyelination. breathing, heart rate, and digestion are destroyed.

12 Stanford Scientific
 When Immune Systems Attack

• Multiple sclerosis is a disease in which the insulation of the nerves – myelin - is attacked by the
body’s own immune system in a process known as demyelination. The affected nerves do not func-
tion properly, leading to gradual paralysis and death.
OVERVIEW

• The cause is unknown, but one theory is based on the fact that many common viruses produce a
protein sequence that closely matches a portion of the myelin. The immune system – on the look-
out for the invading viral sequence – may attack the myelin in a case of mistaken identity. Although
this cause has not yet been proven, researchers at Stanford continue to try to identify the causes
and develop treatments for multiple sclerosis.

• Dr. Lawrence Steinman may be close to a human treatment with a “DNA vaccine” – injection of
DNA encoding altered forms of the body’s proteins that are attacked by the immune system. In
mice models of multiple sclerosis as well as early human trials, his vaccines have decreased the
MS

frequency of attacks and severity of the disease.

How Viruses May be Involved in MS
Now, the elusive problem: what could possibly cause our
immune system to suddenly and brutally destroy the myelin
sheaths that are so critical to our CNS function? According to
Steinman, some researchers like to believe it isn’t our immune
system at work. They contend there is an MS virus, possibly
something akin to the measles virus, that is responsible for the
damage. The idea is so alluring that researchers periodically
claim to have found this enigmatic pathogen, only to realize
months later that their discovery was in vain.
Steinman does believe that pathogens of some sort may be
involved in MS, but not in the traditional way. The pathogen
itself may not cause direct inflammation of the myelin sheath,
nor does it kill the oligodendrocytes. Instead, the pathogen
may trigger the body’s own immune system to attack itself in a
process known as molecular mimicry.
Molecular Mimicry: Viruses That Resemble
Our Nerve Insulation
There is evidence that this molecular mimicry may be one
cause of multiple sclerosis. A portion of a protein sequence of
many viruses—including influenza, adenovirus (a virus that can
cause the common cold), papillomavirus (common warts), and
Epstein Barr virus (a virus that is present in 90% of all adults)—
is highly similar to a portion of the myelin basic protein (MBP)
sequence. So, if a person is infected by one of those viruses
or, theoretically, any pathogen that contains this particular
sequence, his or her immune system has a chance of recognizing
the normal MBP sequence as the invading enemy sequence.
Studies have shown that autoantibodies and self-reactive T
cells in some MS patients recognize amino acids 84-103 of
MBP, including a specific five amino acid sequence VHFFK.
Interestingly, many viral proteins have peptide sequences that
are similar to this 84-103 region of MBP, and especially to the
sequence VHFFK.
Immunology 101
So how would this homology between MBP and viral proteins
induce MS? Basic immunology can explain this phenomenon.
When foreign agents enter the body, white blood cells including
macrophages and B cells engulf and cleave the foreign proteins
into short peptide sequences typically nine amino acids or so

Dr. Lawrence Steinman

}
http://cmgm.stanford.edu/med/steinman/

Without both the

myelin sheath
and the cells that
produce it, nerves
are left bare.
Volume IV 13
When Immune Systems Attack
in length. This cleavage can also occur inside other cells that
become infected with foreign agents. Inside the cells, the
cleaved foreign peptides associate with molecules known as
the major histocompatibility complex (MHC) to form peptide-
MHC complexes. These complexes are brought to the cell
clue, noting that MS, while not transmitted genetically by any
known pattern, does seem to occur slightly more frequently
in certain families. Additionally, identical twins contract the
illness at a 33% higher rate than fraternal twins. The most likely
explanation is genetic variability.
This genetic factor behind MS can be
explained by MHC restriction. The MHC

{
complex encodes three different types of

The pathogen may Class I genes known as Human Leukocyte

Antigens, HLA-A, HLA-B, and HLA-C,
which are expressed on the surface of all

trigger the body’s cells. The MHC complex also encodes

Class II genes known as HLA-D that are
expressed on the surface of macrophages

own immune system and B cells. Each individual has two

copies of each of these genes, but there
are many different alleles, or variations,

to attack itself. of these genes among the population.

There are approximately 309 different
alleles of HLA-A, 563 alleles of HLA-B,
167 alleles of HLA-C, and thousands of
surface where they are presented to T cells. If the peptide-MHC different combinations of HLA-D alleles. Each of these alleles
complexes are recognized by T cells as “self”, then the T cells is restricted to presenting specific peptides.
do not attack. However, if a complex is detected as “foreign”, In an example of MHC restriction, if you have two individuals
then it is attacked, and other immune cells are signaled to be on who each have contracted a papillomavirus containing the
high alert for this sequence. Consequently, these events prime sequence TRSGIRIGGRVHFFKDISPIATQESIELQP, but each
the immune system to become very sensitive to the detection individual has a different set of MHC alleles, then one of them
of that particular sequence. The more this foreign sequence is may present for destruction the nine amino acid sequences
detected, the more aggressive the immune response becomes IRIGGRVHF and FKDISPIAT, while the other may present for
to it. destruction the sequences GRVHFFKDI and SPIATQESI. The
Thus follows how a rare event can become more likely. The second individual may be more likely to produce an immune
immune cells are on a seek-and-destroy
mission and search so actively that they GFP-transfected oligodendrocyte
mistakenly target a peptide sequence, such
as that in MBP, that closely but imperfectly
resembles the foreign sequence. Once the
“self” sequence is initially identified as
“foreign”, other immune cells will target
that sequence. The status of the “self”
peptide becomes increasingly recognized
as a “foreign” epitope—a sequence that
triggers an immune response. Once a
foreign epitope is identified by the immune
system, adjacent peptides are increasingly
likely to also become targeted for
destruction, changing their safe self status
to foreign, in a process known as epitope
spreading.

So Why Don’t We All Get MS?

Over the course of a lifetime, we all get
the common cold, flu, and other viruses at
some point, and typically many more times
than we like. So if this theory of virus-
induced MS is true, then why is it that most
people do not develop MS? The reason The oligodendrocyte secretes myelin. This particular one was engineered
has been eluding researchers for years. to express green fluorescent protein (GFP).
However, recent research has pointed to a http://en.wikipedia.org/wiki/Image%3AOligodendrocyte.png

14 Stanford Scientific
 When Immune Systems Attack

Molecular Mimicry One of the most promising discoveries

for treating MS is the DNA vaccine.

modified from a slide by Steinman

Myelin Basic Protein peptide: ENPVVHFFKNIVTP Steinman’s team has been developing DNA
tolerizing vaccines, or injection of DNA
Viral peptide examples: encoding proteins that program the immune
>> Human papillomavirus 7 IGGRVHFFKDISPIA cells to tolerate specific proteins rather than
13 IGGRVHFFKDISPIS attack them. Using data obtained from
40 IGGRVHFFRDISPIG microarray analysis to find the targets of the
>> Cytomegalovirus DRHPVYFFKSACPPN autoimmune response in sick EAE mice,
>> Dhori virus SDDFIHFFKAKSYDD the team created a tolerizing DNA vaccine
>> Herpes simplex type 1 GGRRLFFVKAHVRES that delivered four of the targeted proteins,
>> Epstein-Barr virus TGGVYHFVKKHVHES including self-myelin antigens. When the
>> Influenza type AKDMTKEFFKNKSETW DNA vaccine was injected into these mice,
>> Epstein-Barr virus VSGFISFFKNPFGGM the desired proteins were produced and a
>> Hepatitis A virus EVKPASFFKNPHNDM programmed tolerization process began,
>> Human adenovirus LATYHIFFKNQRIPL reducing both relapse rates and epitope
Many viral proteins contain a sequence that matches or closely mimics the spreading in the mice. Although EAE is
peptide sequence VHFFK in myelin basic protein.
not an exact replica of MS because MS is
more heterogeneous and the primary target
in MBP, making it more likely for an autoimmune response antigen(s) have not been definitively found, the treatment is
against the myelin sheath to occur. promising for humans.
Alternatively, both individuals may present the identical To apply this strategy to humans, the idea is to put blood
peptide sequence GRVHFFKDI, but their different MHC alleles serum from an MS patient onto an autoantigen microarray
may have different grooves that fit the peptides in slightly containing the subset of proteins that is typically attacked by T
different conformations. In this example, one individual cells, including MBP. Autoantibodies from the serum that bind
presents the nine amino acid sequence to the T cells with the to specific proteins on the microarray identify key antigenic
portion FFKDI exposed, while the other individual presents the proteins involved in the disease. The genes encoding each of
sequence with VHFFK as the exposed portion that is accessible these proteins are inserted into plasmids and injected into the
to the T cells. patient. Injection of the DNA makes the immune system more
Under the theory of genetic predisposition to MS, the reason tolerant of the encoded proteins.
that correlation between identical twins is not 100 percent is
because the chain of events that leads to the disease is still
caused by foreign agents. Thus the number of times a virus Altered peptide ligand
infects someone, as well as the “viral load”—the amount of viral T-Cells
molecules one is exposed to—play a decisive role. The more
a person is exposed to a virus with a similar protein sequence
as his MBP, and the more frequent and aggressive the immune
response, the more likely he will develop MS.
So, then, is multiple sclerosis contraction totally dependent
on circumstance, happening or not happening solely as a result
of random chance and a person’s DNA? Perhaps, but that has
clearly not stopped researchers from searching for a cure. For Native Altered
instance, the National MS Society has spent over $500 million Peptide Peptide
in grants dedicated to MS research. Ligand Ligand
modified from a slide by Steinman

Promising Cures for MS

Although there is no cure yet for multiple sclerosis,
Steinman and his colleagues are making progress. They found
that if the epitope is slightly different than the one normally
recognized, the T cell begins to secrete alternate molecules that
reduce the immune response to the original epitope and prompt
other immune molecules to cease their attack. Epitopes that Antigen Presenting Cells
have this down-regulating effect are known as “altered peptide ^ If the peptide sequence of the epitope is slightly different than
ligands”. Steinman’s group has created altered peptide ligands the one normally recognized, the T cell secretes alternate factors
of dominant myelin basic protein epitopes in which one or more that reduce the immune response to the original epitope and
prompt other immune molecules to cease their attack. The sev-
amino acids are substituted. Administration of these ligands
enteen amino acid peptide designed around native Myelin Basic
inhibited development of disease in a mouse model of MS Protein (MBP) has four key amino acid substitutions.
known as experimental autoimmune encephalomyelitis (EAE).

Volume IV 15
When Immune Systems Attack

The Design of Personalized DNA Vaccines

Autoantigen microarray contains the

subset of proteins that is typically
attacked by T cells, including MBP

Probe arrays with autoimmune

serum from MS patients

Detection with fluorescently labeled

secondary antibodies that recognize
autoimmune molecules
Y
Y

modified from a slide by Steinman

Genes encoding the
proteins attacked in the
MS patient are identified
Scan

^ Blood serum from an MS patient is put onto an autoantigen microarray containing the subset of proteins that is typically attacked
by T cells, including MBP. Autoantibodies from the serum bind to specific proteins on the microarray, identifying key antigenic pro-
teins involved in the disease. The genes encoding each of these proteins are inserted into plasmids and injected into the patient.

One advantage of DNA vaccines over other methods of
tolerization is that they allow for multiple autoimmune targets
to be tolerized simultaneously rather than one at a time.
Another benefit is that DNA vaccines can be customized for
each patient. The specific antigenic proteins can be identified
for each patient, and patient-specific vaccines can be made.
This increases the effectiveness of the vaccine while reducing
the likelihood of side effects caused by targeting the wrong
proteins. Human DNA vaccines are now in clinical trials,
and the MS vaccine—tested on ten patients during an early
trial—so far appears to be safe and effective.
While we don’t know for certain what causes multiple
sclerosis, and even less so how to prevent it, we do know
that MS is a debilitating illness that strikes at the core of
our understanding of immunology. As more light is shed
on this illness, then we will also gain more insight into
other autoimmune disorders affecting millions worldwide,
including rheumatoid arthritis and insulin-dependent
diabetes. The task of curing multiple sclerosis is an arduous
one for Steinman and other leading researchers, but it is a
critical endeavor that will benefit millions of people suffering
from autoimmune disease. S

{ One of the most

promising discover-
ies for treating MS
is the DNA vaccine.

16 Stanford Scientific
Jonathan Frohnmayer is a junior double majoring in his-
tory and human biology. He enjoys running, tennis, yoga,
debilitating brain diseases, and barbecued pork chops. He
hopes to attend law school, and considers “happiness” his
singular goal in life.

Tammy Doukas has been in SSR since its conception, and

believes in spreading science knowledge to the Stanford
community as well as the general public. She has worked
at Genentech and NASA Ames, and holds an MS in Biologi-
cal Sciences from Stanford. Currently she is earning a PhD
in Microbiology and Immunology from the Stanford School
of Medicine.