Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak

Laidback Al · Apr 14, 2008

Published online on April 14, 2008
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0801367105

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies

</NOBR>Arun K. Kashyap*, John Steel

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, Ahmet F. Oner

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, Michael A. Dillon*, Ryann E. Swale*, Katherine M. Wall*, Kimberly J. Perry*, Aleksandr Faynboym*, Mahmut Ilhan

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, Michael Horowitz*, Lawrence Horowitz*, Peter Palese

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, Ramesh R. Bhatt*, and Richard A. Lerner,?
*Sea Lane Biotechnologies, 1455 Adams Drive, Menlo Park, CA 94025-1438;

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Departments of Microbiology and Medicine, Mount Sinai School of Medicine One Gustave L. Levy Place, New York, NY 10029;

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Y?z?nc? Yil University, Van TR-65200, Turkey; and ?Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
Contributed by Richard A. Lerner, February 20, 2008 (sent for review February 1, 2008)
Abstract

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

<HR align=left width="50%" noShade SIZE=1>Footnotes

Author contributions: A.K.K., J.S., M.A.D., R.E.S., K.M.W., K.J.P., A.F., M.H., L.H., P.P., R.R.B., and R.A.L. designed research; A.K.K., J.S., M.A.D., R.E.S., K.M.W., K.J.P., A.F., P.P., and R.R.B. performed research; A.F.O. and M.I. contributed new reagents/analytic tools; M.H., L.H., and R.A.L. analyzed data; and A.K.K., M.H., L.H., R.R.B., and R.A.L. wrote the paper.
The authors declare no conflict of interest.

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To whom correspondence may be addressed. E-mail: ramesh.bhatt@sealanebio.com<SCRIPT type=text/javascript></SCRIPT> or rlerner@scripps.edu<SCRIPT type=text/javascript></SCRIPT> ? 2008 by The National Academy of Sciences of the USA

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credits to Shiloh

HenryN · Apr 14, 2008

Re: Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak

Combinatorial antibody libraries from survivors
of the Turkish H5N1 avian influenza outbreak
reveal virus neutralization strategies
Arun K. Kashyap*, John Steel

?, Ahmet F. Oner?, Michael A. Dillon*, Ryann E. Swale*, Katherine M. Wall*,
Kimberly J. Perry*, Aleksandr Faynboym*, Mahmut Ilhan?, Michael Horowitz*, Lawrence Horowitz*, Peter Palese?,
Ramesh R. Bhatt*?, and Richard A. Lerner??

*Sea Lane Biotechnologies, 1455 Adams Drive, Menlo Park, CA 94025-1438;

?Departments of Microbiology and Medicine, Mount Sinai School of Medicine
One Gustave L. Levy Place, New York, NY 10029; ?Yu? zu? ncu? Yil University, Van TR-65200, Turkey; and ?Department of Chemistry, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
Contributed by Richard A. Lerner, February 20, 2008 (sent for review February 1, 2008)

The widespread incidence of H5N1 influenza viruses in bird populations
poses risks to human health. Although the virus has not
yet adapted for facile transmission between humans, it can cause
severe disease and often death. Here we report the generation of
combinatorial antibody libraries from the bone marrow of five
survivors of the recent H5N1 avian influenza outbreak in Turkey. To
date, these libraries have yielded

>300 unique antibodies against
H5N1 viral antigens. Among these antibodies, we have identified
several broadly reactive neutralizing antibodies that could be used
for passive immunization against H5N1 virus or as guides for
vaccine design. The large number of antibodies obtained from
these survivors provide a detailed immunochemical analysis of
individual human solutions to virus neutralization in the setting of
an actual virulent influenza outbreak. Remarkably, three of these
antibodies neutralized both H1 and H5 subtype influenza viruses.

N

ewly emergent, highly pathogenic influenza virus strains pose
a profound threat to man. Three influenza pandemics have
occurred within the past 100 years, each with devastating consequences
(1). The recent emergence of the H5N1 virus sub-type,
although mainly confined at present to avian hosts, has already
demonstrated virulence in humans, causing the death of 200
people (2). Therefore, health care officials, researchers, and governments
are actively considering their options should a pandemic
occur.
One widely considered approach concerns the use of passive
immunization either for the prevention of disease or for treatment
after exposure to virus (3). The potential for passive immunization
against influenza has been evident since the Spanish influenza
outbreak nearly a century ago, where the benefits of transfused
blood, sera, and blood products reduced the risk of mortality by

50% (3). Recently, the benefits of treatment with convalescent
plasma in instances of H5N1 influenza have also been reported (4,
5). Additionally, passive immunization with human and mouse
monoclonal antibodies has been reported to protect animals from
death, even when administered after H5N1 infection (6).
The most logical source of human antibodies for passive therapy
would be patients who have survived infection. With modern
combinatorial antibody library technologies, it is now possible to
capture the entire immunological history of an individual?s response
to an infection (7, 8). Because antibody libraries contain the
complete record of an individual?s response to pathogens, one can
recover the repertoire specific to a given agent by using a laboratory
process of selective enrichment. Such libraries give archival information
about the nature of antibodies made during the infection
and allow recovery of potentially therapeutic human monoclonal
antibodies. Importantly, antibody recovery is independent of
whether an active antibody response is still occurring at the time the
sample is taken. Thus, depending on when the libraries are constructed,
one may obtain antibodies that are currently being made
and/or are part of the individual?s immunological history. For
infections that may be lethal, such analyses carried out on surviving
patients may be particularly important because they chart some of
the immunological mechanisms used during a successful host
defense in the actual clinical setting of an outbreak.
Typically, when libraries are prepared from individuals who have
been infected with a virus, hundreds to thousands of different
antibodies are obtained, as opposed to only a few when other
methods are used (8). A comparative sequence analysis of these
antibodies allows a detailed map of the chemistry of antibody
binding. Similarly, a comparison of neutralizing and nonneutralizing
antibodies can give important information about the nature of
binding interactions that are critical to neutralization.
Here we describe the creation of comprehensive avian influenza
antibody libraries made from survivors of infection with an avian
influenza virus during a confirmed outbreak. We have used these
libraries to obtain large numbers of monoclonal antibodies to the
H5N1 avian influenza virus, some of which have broad reactivity
and are neutralizing across viral subtypes. Ultimately, combinatorial
antibody libraries may hold the key to immunotherapy, such as
passive immunization using one or more member antibodies, or
they may guide the development of vaccines directed at the
antigenic target(s) of the neutralizing antibodies in the library.

Results
The Outbreak and Source of Material.

Between December 2005 and
January 2006, an outbreak of avian influenza H5N1 occurred in
Turkey (9). In total, 12 individuals were infected and only 8
survived. Because bone marrow RNA contains the archived record
of all antibodies made by an individual, we selected it as our source
material. We obtained bone marrow and serum from six of the
Turkish survivors after their recovery and successfully prepared
antibody libraries from five of the six bone marrow samples. In the
sixth sample, the RNA was degraded.

Serological Analysis.

The hemagglutinin (HA) protein is essential
for binding the influenza virus to the cell that is being infected and
is generally considered to be the main target of neutralizing
antibodies (1). Therefore, we tested by ELISA each of the individual
serum samples at high serum dilutions to detect antibodies

against H5 HA proteins

HenryN · Apr 14, 2008

Re: Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak

Table 3. MDCK cells were inoculated with 100 TCID

50 of virus in the presence of 2-fold serial dilutions of monoclonal antibodies

Antibody
Virus
H5 H1 H3
A/Vietnam/
1203/04*
A/Vietnam/
1203/04*
A/Indonesia/
5/05
A/Turkey/
65596/06
A/Egypt/
14725/06
A/New Caledonia/
20/99
A/Hong
Kong/68

Ab 1

? 11?21 2.3?9.3 9.3 9.3 1.2?2.3 9 333
Ab 2? 63 54?217 27 108 7?13 54?108 333
Ab 3? 58 18 16 31 4?8 8?16 333
Ab 4? 1.7?6.3 0.5?2.2 333 Not done Not done 333 333
Mab #8? 2.7 Not done Not done Not done Not done Not done Not done

*Viral neutralization results for two independent experiments are shown.

?

Minimum inhibitory concentrations required to neutralize virus in duplicate samples, in micrograms per milliliter.

?A mouse monoclonal H5N1 neutralizing antibody raised against A/Vietnam/1203/04 (P.P., unpublished data).

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak

Laidback Al

Well-known member

HenryN

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HenryN

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