午夜福利1000集合

THERE鈥檚 something you should know about vaccines. Given that immunisation is
one of medicine鈥檚 greatest success stories, and vaccines save millions of lives
every year, you might reasonably expect scientists to know exactly how they
work. Well, bad news. Two hundred years after Edward Jenner first immunised
people against smallpox, the function of a key ingredient of modern vaccines
remains an almost total mystery. It has been called the immunologist鈥檚 dirty
little secret.

Vaccines are supposed to work by giving the immune system a sneak preview of
future enemies in the form of weakened or killed disease-causing microorganisms,
bits of those microorganisms, or even pieces of their DNA. A quick look at this
pale imitation, called an immunogen, is enough to get the immune system stocking
up its armouries ready to retaliate when the microorganism strikes for real.

However鈥攁nd here鈥檚 the secret鈥攖he vast majority of vaccines do
not work unless they also contain something called an adjuvant, a fancy-sounding
name for a nonspecific mix of some very weird ingredients. Vaccines based on
whole viruses, such as the oral polio vaccine, can wake up the immune system
single-handed. But all the bacterial vaccines, and the more modern viral
vaccines, such as the hepatitis B one, that for safety reasons use only parts of
a virus, are doomed to fail without an adjuvant.

At one time or another, the mysterious adjuvants have included any one of a
list of suspicious-sounding substances: detergent, oil and water, aluminium
hydroxide, dead bacteria that have nothing to do with the disease the vaccine
protects against, bits of those dead bacteria, or some mix of these (see
Table).

This eye of newt, toe of frog approach to vaccine design makes some
immunologists uneasy. Not least because if they are honest, they know as much
about witches鈥� spells as they do about how adjuvants work. Early adjuvants were
merely by-products of the vaccine production process. But vaccine developers
soon realised that adjuvants caused inflammation, and the worse the
inflammation, the more effective the vaccine. The very strongest immune
responses鈥攖hose provoked in laboratory animals by researchers studying
immune cells and antibodies鈥攁re triggered by an adjuvant called Complete
Freund鈥檚 that is so inflammatory it cannot be used on humans. 鈥淚t blows a hole
in your arm,鈥� explains Polly Matzinger, an immunologist at the National
Institutes of 午夜福利1000集合 near Washington, DC. Immunologists who accidentally inject
themselves with it can need surgery to remove the damaged tissue.

Now, after years of being quietly pushed under the carpet, the mysterious
adjuvants are finally coming under the full glare of modern molecular biology.
And although it has yet to completely explain how the adjuvants work, some new,
effective adjuvants have already been developed. For example, an experimental
flu vaccine that uses an adjuvant of carefully crafted microscopic spheres of
human oil induces such a strong immune response, and with so few side effects,
that it could save a significant number of lives among the elderly.

In part, the new approach to the adjuvants is driven by the vaccine
manufacturers who, keen to avoid lawsuits, are determined to make their products
squeaky-clean. None of the old adjuvants used in human vaccines are exactly
life-threatening, but they have been blamed for causing alarming fevers,
persistent crying in babies, and scarring. The new interest has also been
spurred on by a handful of incongruous new results.

This spring, for example, Paul Lehmann鈥檚 team at Case Western Reserve
University in Cleveland, Ohio, showed that, far from being nonspecific additives
that just trigger inflammation, different adjuvants can provoke entirely
different types of immune response. When Lehmann and his colleagues injected
mice with an experimental immunogen (a protein found in eggs) in Complete
Freund鈥檚 adjuvant, made up of oil, water and dead tuberculosis bacteria, it
activated one subset of immune cells called inflammatory T cells. The same
immunogen injected in Incomplete Freund鈥檚 adjuvant, the emulsion minus the dead
bacteria, triggered a different subset of T cells that also stimulate the
production of antibodies by B cells.

But perhaps the most curious puzzle is how the new generation of DNA vaccines
that contain part of the genetic code of a microorganism and not much else
manage to provoke a truly vigorous immune response in the absence of
adjuvant.

It was immunologist Charlie Janeway at Yale University in New Haven,
Connecticut, who first called adjuvants a 鈥渄irty little secret鈥� at a prestigious
symposium at Cold Spring Harbor Laboratory in New York in 1989. Then, as now, he
argued that the mystery that immunology seemed determined to ignore could in
fact be explained. Adjuvants alert the body鈥檚 immune system to the presence of
the immunogen, he says, by mimicking some key characteristic of a bacterium that
is removed when the microorganism is rendered harmless enough to be used in a
vaccine. Without the adjuvant鈥檚 clear message that the body鈥檚 barricades have
been breached, the immune system will not wake up, and the components of the
vaccine that trigger the specific immune response will be ignored.

To understand what it takes to wake up the immune system, spend a minute
thinking about the design of its two arms. The primitive, or innate, part of the
immune system responds rapidly, if rather bluntly, to molecules that are only
found in bacteria, not in vertebrates: lipopolysaccharides, for example. The
primitive immune system controls the macrophages and other types of white blood
cells that will guzzle up any stray bacteria and viruses, but will miss those
that are already inside the cells.

The other arm is the adaptive immune system. It minutely tailors its response
to each new invading microorganism, and creates a memory of that microorganism
for future use. At the heart of this plasticity are two types of immune cell, T
cells and B cells, that can recognise and respond to an almost infinite variety
of proteins, including those that make up bacteria and viruses. It is this
second part of the immune system that the vaccine鈥檚 immunogen pumps up,
expanding the numbers of highly specific T and B cells that then wait in the
wings ready to attack a bacterial or viral invader.

Fail-safe trigger

But the very flexibility of the adaptive system means that it must be tightly
controlled to stop it wreaking havoc on the body鈥檚 tissues. Most T cells and B
cells that attack the body鈥檚 own proteins are killed off as a fetus develops in
the womb, but some survive. To ensure that they only destroy potentially
dangerous proteins, there鈥檚 a fail-safe mechanism. The T cells need not one
signal, but two, before they will go on the attack themselves or tell B cells to
start producing antibodies (see
Diagram). First, the cell must bind to the
specific protein it recognises, which has already been partially processed by
the immune system and is presented on the surface of an antigen-presenting cell.
(The antigen-presenting cells are cells such as macrophages which also play a
role in the primitive arm of the immune system.) Next, they need a second signal
from the surface of the same antigen-presenting cell that says: 鈥淭his is
serious, do something.鈥� This second signal comes in the form of two specific
molecules known, prosaically, as B7.1 and B7.2.

So far, so good. But what tells the antigen-presenting cell to send the
second signal in the first place? The answer is both highly controversial, and a
major clue to how adjuvants work. Janeway believes that specific bacterial
molecules trip the second switch. These molecules, such as the bacterial
lipopolysaccharides, are recognised by receptors on the surfaces of the
antigen-presenting cells. Those receptors not only send the second signal, but
also tell the antigen-presenting cells to produce chemicals known as cytokines,
such as interleukin 1, which cause inflammation. The adjuvant, says Janeway,
probably trips the second signal by standing in for the bacterial molecules.

And there is certainly evidence to support this view. Molecules called
muramyl dipeptides that make up part of the bacterial cell wall have turned out
to be the vital ingredient in the crude mush that is Complete Freund鈥檚 adjuvant.
What is more, even harmless proteins can be made to look threatening to the
immune system if it sees them in the presence of dead bacteria. A mouse鈥檚 immune
system will chew up one of the body鈥檚 most vital proteins鈥攖he myelin
sheath that surrounds the animal鈥檚 nerve cells鈥攊f that protein is injected
together with Complete Freund鈥檚 adjuvant.

But the critics are quick to point out two weak points in Janeway鈥檚 theory.
First, the immune system sometimes reacts to apparently harmless proteins that
are mammalian not bacterial鈥攅ither 鈥渘on-self鈥� proteins in the case of the
immune system鈥檚 rejection of transplanted organs, or 鈥渟elf鈥� proteins in
autoimmune diseases like rheumatoid arthritis. Second, some adjuvants
鈥攕uch as oil-and-water emulsions, aluminium hydroxide and the detergent
saponins鈥攈ave nothing remotely bacterial about them.

Robert Bomford, an expert in adjuvants at the company Peptech (UK) in
Cirencester, has one answer to the last criticism. He says the modern adjuvants
may mimic the physical, rather than the biochemical, effects of the bacteria and
viruses. For example, Incomplete Freund鈥檚 adjuvant makes soluble immunogens
solid for the macrophages to eat. 鈥淭he mere act of having to go round and gobble
them up may be enough,鈥� says Bomford, to fool the macrophages into thinking a
bacterium is present. Similarly, he says, the detergent adjuvants may poke holes
in the membranes of antigen-presenting cells, perhaps allowing the immunogen to
get inside, where they are made to look more 鈥渞eal鈥� to the immune system.

But while Bomford is content to envisage a scenario in which adjuvants are
either microorganisms themselves or just good physical mimics, Matzinger claims
that Janeway鈥檚 theory is indeed fundamentally flawed. She argues that the
critical factor that triggers the second signal is not the mere presence of
microbial molecules, but a more generic danger signal that comes from any tissue
damage that exposes the antigen-presenting cells to molecules that normally
live only inside cells. The damage can be done by bacteria, by a surgeon鈥檚 knife
during an organ transplant, or by any other substance or procedure that ruptures
cell membranes.

Adjuvants in vaccines create a danger signal simply by damaging tissue,
Matzinger argues, and, conversely, 鈥渄anger [is] the body鈥檚 own natural
adjuvant鈥�. In her view, the 鈥渄anger鈥� signal is all, and there is certainly no
need to think in traditional terms of an immune system that operates on the
basis of distinguishing 鈥渟elf鈥� from 鈥渘on-self鈥� (New Scientist, Science,
30 March, p 14) 鈥攁 charge that is ardently opposed by many
immunologists.

But, while debates about self and non-self are likely to remain arcane, more
practically minded immunologists are keen to pinpoint the exact mechanisms and
molecules that trip the all-important second signal that activates the T and B
cells. That way they hope they will be able to develop new adjuvants that
trigger a hefty immune response with none of the unpleasant side effects. At the
pharmaceuticals company Chiron Biocine in Emeryville, California, Tyler Martin
thinks that day may not be so far off.

Chiron has been experimenting with an adjuvant called MF59. It consists of an
emulsion of tiny 鈥渕icrofluidised鈥� particles of a type of oil called squalene
that is found in large quantities in shark livers, and in smaller quantities in
human blood. The company has tested MF59 in a flu vaccine, and discovered that
it stimulates a strong antibody response against the flu virus even in elderly
people, whose immune systems are less efficient and who are more susceptible to
the virus. The results of the Chiron trials, announced at a meeting in Bergen in
Norway this summer, showed that elderly people vaccinated with the flu vaccine
containing MF59 were 60 per cent less likely to die over one winter than people
vaccinated with the conventional flu vaccine lacking the adjuvant.

Naked DNA

Martin is not sure how MF59 works, but, he says, the size of the oil
particles is crucial: the smaller the better. Bomford speculates that, since the
most effective particles are as small as viruses鈥攂etween 30 and 150
nanometres across鈥攖hey may fool macrophages into thinking the body is
being attacked by viruses.

But it is the arrival of DNA vaccines that has provoked the freshest new take
on adjuvants. Back in 1993, scientists at the pharmaceuticals company Merck in
Philadelphia immunised mice against flu virus with nothing more than the naked
DNA encoding a very few of the virus鈥檚 proteins. Since then, mice have been
immunised against numerous other microorganisms in the same way. Most of the
time, the DNA has been injected straight into the animals鈥� muscle cells where
the proteins it encodes are manufactured. Those proteins activate the T cells
and B cells鈥攁ll without a hint of an adjuvant. At first, this prompted
some researchers to argue that adjuvants鈥� days were numbered.

It turns out, though, that the obituaries were premature. In July, Eyal Raz
at the University of California, San Diego, reported that DNA vaccines only work
well when they contain certain sequences of DNA called CpG repeats. CpG repeats
are the genetic signature of bacteria, and they rarely occur in mammals. In DNA
vaccines, the CpG repeats lay not in the piece of DNA that encodes the
immunogen, but in the plasmids鈥攔ings of bacterial DNA that carry the naked
DNA into the mouse cells. Raz and his team call these sequences the vaccines鈥�
鈥渁djuvant units鈥�. And the CpG repeats do the trick without causing inflammation,
providing 鈥減roof that inflammation is not necessary鈥� for an effective vaccine,
says Raz.

In some shape or form, adjuvants, it seems, are here to stay. Cleaner,
certainly; better understood, perhaps. Either way, the immunologist鈥檚 dirty
little secret is no secret any more.