This study evaluated an adenovirus vector (AdaPAscAb) expressing a single-chain antibody against anthrax protective antigen (PA) for passive immunotherapy against anthrax toxin. In vitro, AdaPAscAb expressed and secreted the anti-PA antibody, which specifically bound PA. Mice administered AdaPAscAb produced the antibody in their serum for up to 14 days. Mice treated with AdaPAscAb were fully protected from lethal anthrax toxin challenge 72 hours later, whereas control mice were not protected. While effective, the antibody's half-life was too short for human use; further engineering is needed to extend its circulation time.

1. PASSIVE IMMUNOTHERAPY FOR
ANTHRAX TOXIN MEDIATED BY
ADENOVIRUS EXPRESSING AN ANTI-
PROTECTIVE ANTIGEN
SINGLE-CHAIN ANTIBODY
KAZUHIKO KASUYA, JULIE ET AL, DEPARTMENT OF
GENETIC MEDICINE, 10 DECEMBER
20042005, VOL. 11, N.2, PP. 237-244
JYOTI SOROUT

2. ACKNOWLEDGEMENTS
 Dr Nagendra Singh (Mentor)
 Kazuhiko Kasuya , Julie L. Boyer, Yadi Tan, and D.
Olivier Alipui.
 Neil R. Hackett Ronald G. Crystal

3. TECHNIQUES USED IN THE ARTICLE
 Western blotting
 ELISA

4. INTRODUCTION
Anthrax, a disease caused by infection with Bacillus anthracis,
manifests as cutaneous , gastrointestinal, or pulmonary disease.
All can be fatal, but the inhalational form has the highest mortality,
with a survival rate of only 55% in the recent U.S. bioterrorism
incidents.
Inhalational anthrax is acquired from aerosolized B. anthracis
spores. In the lung, the spores are phagocytosed by alveolar
macrophages and transported to regional lymph nodes, where
they germinate and the bacteria multiply.
The virulence factors of B. anthracis are carried by two plasmids,
one encoding genes for capsule components that limit
phagocytosis and the other for three secreted exotoxin proteins,
protective antigen (PA), lethal factor (LF), and edema factor (EF).

5. •PA plays a central role in virulence in that it enables
LF and EF to function. Lethal toxin, comprising PA and
LF, is responsible for the overwhelming systemic fatal
pathophysiology associated with B. anthracis
infection.
•All available evidence supports the concept that
induction of IgG antibodies against PA can be
protective against B. anthracis.
•An anthrax vaccine composed of an adenovirus
(Ad) vector expressing PA requires only a single
administration to be effective in protecting mice
against the anthrax lethal toxin, but it requires 1
week for anti-anthrax immunity to be effective.

6. METHODS
Adenovirus vectors. AdaPAscAb is a serotype 5, Ad gene transfer
vector that expresses a murine/human single-chain antibody
directed against anthrax protective antigen.
Binding of monoclonal antibodies to the carboxy-terminal domain
of PA inhibits PA binding to cells.
One of these antibodies (14B7) with specificity for PA and lethal
toxin neutralizing activity was engineered by Maynard et al. into a
single-chain antibody (scAb).

7. Using the anti-PA scAb construct, the expression cassette of Ada
PAscAb contains (5V to 3V) the cytomegalovirus promoter/enhancer,
an optimal Kozak sequence (GCCACCATG), the murine Ign-chain
secretion signal sequence, the anti-PA single-chain antibody cDNA
(including the murine anti-PA heavy- and light-chain Fv sequences
and a sequence encoding a human constant n domain), tags to help
identify the construct in preliminary studies (his tag + myc tag), and
the SV40 polyadenylation signal.
Two control Ad vectors were used in the study. Ada CTXscAb is
identical to Ada PAscAb but, instead of the anti-PA single-chain
antibody, the expression cassette encodes a single-chain antibody
reactive with a cobra toxin purified from Naja nigricollis venom.
AdNull is a control vector with identical backbone to AdaPAscAb and
AdaCTXscAb but it contains no transgene .

8. In vitro assessment of AdaPAscAb
In vitro assessment of Ad aPAscA. Expression of the anti-PA
singlechain antibody by Ada PAscAb was assessed in vitro by
Western analysis and by assessment of anti-neutralizing PA
antibodies.
In vivo assessment of anti-PA levels
Following administration of Ada PAscAb. Female BALB/c mice,
4 to 6 weeks of age, were housed under pathogen-free
conditions. BALB/c mice were chosen for this study based on
their sensitivity to lethal toxin.
All administrations of Ad vectors diluted with PBS given
intravenously.

9. Production and purification of lethal toxin
Lethal toxin was generated by combining PA and LF proteins produced
and purified from bacteria. The PA or the LF gene was cloned
downstream of the T7 promoter in the prokaryotic expression plasmid
for expression as 6His fusion proteins. The resulting plasmids were
transformed into the BL21.DE3 strain of Escherichia coli.
Bacteria were grown in Luria broth under ampicillin selection (50 Ag/ml)
to an OD600 of 0.6 to 0.8 and expression of the PA.
RESULTS
In Vitro Characterization of Ad-expressed Anti-PA Single-Chain
Antibody
Ada PAscAb is a serotype 5, Ad gene transfer vector containing a
cDNA encoding a murine /human single-chain antibody directed
against anthrax protective antigen.

10. To examine the expression and secretion of the anti-PA single-
chain antibody by Ada PAscAb, we infected A549 cells with Ada
PAscAb and, as controls, we infected cells with Ada CTXscAb
or AdNull. Supernatants collected from cells infected with
AdaPAscAb at 48 h.
We determined the specificity of the Ad-expressed PA-specific
single-chain antibody for PA by Western analysis.
The anti-PA single-chain antibody recognized PA, but not LF.
Thus, AdaPAscAb expresses a single-chain antibody that has
specificity for PA.
The supernatants from AdaCTXscAb-infected and AdNull-
infected cells had no neutralizing activity

12. In Vivo Expression by AdA PAscAb
We assessed the in vivo expression profile of Ada PAscAb in
mouse serum 0 to 14 days after intra-venous administration by
quantifying human Ign antibody levels.
In contrast, we detected no single-chain antibody over time in mice
receiving
AdNull.

13. AdA PAscAb-Mediated Protection from in Vivo Challenge with
Lethal Toxin.
Mice that had been injected with Ada PAscAb, Ada CTSscAb, or
AdNull with lethal toxin at 72 h post administration, using naive
mice as a negative control.

14. DISCUSSION
The only vaccine approved for use in humans in the United States is
used for military personnel and at-risk first-line responders; it is not
available to the general civilian population.
Using an adenovirus-delivered PA-specific single-chain antibody to
mediate passive immunotherapy against B. anthracis lethal toxin, the
present study presents a strategy to provide rapidly induced (24 h) and
extended (14 days) protection against B. anthracis that can be used for
at-risk individuals as well as those infected with B. anthracis.
While effective, the half-life of the anti-PA single-chain antibody
(identical to that encoded by the cDNA used in the present study) was
only 10.4 min, a time far too short to be of use for protecting humans in
the context of an
anthrax attack.