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So Anaplasma disguises itself. Can we help the immune
system see past the Msp2 mask? Is there some other surface
protein—one that doesn’t change every few weeks—that could be used
as a vaccine? Msp2 garners most of the attention from the immune
system, but there are dozens more, present in small amounts and not
well understood. Most intriguing of all, vaccinating with Msp2
alone does not protect against Anaplasma infection—but
vaccinating with a mixture of outer membrane proteins does.
“So there’s something in the outer membrane that’s
important,” says immunologist Wendy Brown. Unfortunately, she says,
a vaccine made of membrane preparations is “impractical. You’d have
to pay $500 a shot, probably.” She set out to discover which
proteins in the outer membrane mixture confer protection. Perhaps
they could be the basis for an economical vaccine.
Analyzing membrane proteins is not a new idea, but until
recently the experiments weren’t feasible. Getting enough of any
one protein to use as a test vaccine was difficult, and testing any
vaccine in cattle takes months. Testing multiple combinations of
proteins was simply unmanageable.
Brown invented a way to test proteins on cow cells rather than
in a whole cow. She first immunizes a cow with the mixture of
membrane proteins. After the cow’s immune system has had a chance
to respond, she takes a blood sample, and from that she extracts T
cells. Those are the immune system’s “memory cells”; if the
vaccinated cow were to be bitten by an Anaplasma-carrying
tick, its T cells would recognize antigen(s) on the
Anaplasma surface and would trigger antibody production by
other immune system cells. Brown presents the T cells with
individual Anaplasma proteins in a way that mimics what
happens in the body. If the T cells recognize a protein, they start
dividing and making interferon—which tells her the cow’s immune
system had responded to that particular protein. It’s a nifty way
to rapidly screen a large number of proteins for their potential as
vaccines.
With help from WSU chemist Bill Siems, Brown identified more
than 20 proteins from Anaplasma’s outer membrane that had
never been described before and that were recognized by T cells
from vaccinated cows. Now the group is testing whether any of the
proteins make an effective vaccine. They’ve found that a
combination of some of the proteins does protect against infection
by Anaplasma. Will the mixture lead to a commercially viable
vaccine? Brown and her colleagues don’t know yet; but after years
of chasing the shape-shifting Msp2, it’s an encouraging result.
Like Anaplasma, Babesia is studded with proteins,
at least two of which change over time. Because Babesia’s
genome is larger and more complex than Anaplasma’s, the team
hasn’t yet figured out how the proteins change. The one thing
that’s clear is that Babesia doesn’t use the same process of
gene conversion as Anaplasma.
In addition to working on variable proteins, Brayton and McElwain
have teamed with The Institute for Genomic Research to take a
closer look at Babesia’s genome. They’ve discovered a group
of DNA sequences they call “SmORFs,” for “small open reading
frames.” (“We took some heat on that name from reviewers, but we
stuck to it,” says McElwain. “We’ve got to have some fun with
this.”) Since all 44 SmORFs lie next to genes for a variable
surface protein, McElwain suspects they’re involved with immune
escape, but work on them is still preliminary.
Although its variable proteins remain puzzling, Babesia
might be outmaneuvered another way. In the 1960s, scientists in
Australia discovered that if they infected a calf with Babesia, let
the infection develop for about a week, drew some blood from that
calf and used it to infect another calf, and did that 25 to 30
times, the pathogen would lose its ability to cause disease. It
still provoked an immune response when inoculated into a new host,
but the host no longer got sick; instead, it gained protection. In
other words, the so-called attenuated strain worked as a
vaccine.
It worked so well that attenuated strains have been used in
Australia and Israel—both fertile grounds for Babesia—for
many years. Attenuated Babesia vaccines have been banned in
many other countries, including the United States, because they are
blood-based and might carry other pathogens. For countries like the
U.S. where Babesia is not a big threat, they’re not worth
the risk. Even in countries where Babesia is a threat, their
value is limited because they require a cold chain. They’re also
not a permanent solution; every few years, they change so they no
longer protect the host. When that happens, the whole attenuation
process has to be repeated.
In 2005, the WSU team won a $1.8 million grant from the Wellcome
Trust to figure out why attenuated Babesia protects against
further infection, yet does not cause disease itself. Can we
design strains of Babesia that will mimic the
attenuation effect, and that can be used as vaccines? Such a
vaccine would avoid the threat of blood-borne pathogens and the
tedious and expensive attenuation process.
Colleagues in Argentina have already produced an attenuated
strain the old-fashioned way. While they test it on herds there,
Brayton and molecular biologist Audrey Lau in Pullman are comparing
the genome of the attenuated strain with that of the original
strain. Since they know the sequence of the original, they can
trace any changes that occurred during the attenuation process.
With luck, they’ll identify what change(s) turned a killer into a
life-preserving vaccine.
“The clearest-cut scenario is that a gene’s missing,” says
Brayton. “You’re usually not that lucky. But [if that happens], you
could [ask], what is that gene in the virulent strain? What do we
know about it?” Even if nothing is missing, they might find that
one or more genes have changed. If Lau and Brayton find clear
differences between the strains, the next step will be to create a
copy of the attenuated pathogen in the lab and test its
effectiveness as a vaccine.
The field tests will be done by the group’s collaborators in
Mexico and Argentina, working with herds under natural conditions.
Palmer says translating lab results into real-world applications is
central to what the WSU group is trying to do—and essential to
developing a vaccine that will work against the form of the
pathogen that cattle actually encounter.
“People don’t like to do it, because nature’s messy, you know,
and the lab is not,” he says. “For a long time there’s been a
tendency to rely on strains [of pathogens] that are well
established in the laboratory. But when you get out into the field,
you find that isn’t necessarily representative of what’s out there
in the natural situation.”
No one in the group is predicting when they’ll have a vaccine
for either disease. Anaplasma and Babesia have
confounded expectations before. But the team members share a sense
of excitement about their progress—and an admiration for their
elusive adversaries. The most rewarding aspect of their work so
far, says McElwain, has been gaining an understanding of the
complex, elegant strategies that enable these pathogens to persist
in their supposedly more sophisticated hosts.
“That’s been something that we started out, 25 years ago, not
appreciating,” he says. “And that is not only something that you
can appreciate, if you can find beauty in these organisms. It’s
also the challenge, the huge challenge that we have.”
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