PASSIVE IMMUNOTHERAPY FOR ANTHRAX TOXIN MEDIATED BY ADENOVIRUS EXPRESSING AN ANTI-PROTECTIVE ANTIGENSINGLE-CHAIN ANTIBODY

KAZUHIKO KASUYA, JULIE ET AL, DEPARTMENT OF GENETIC MEDICINE, 10 DECEMBER 20042005, VOL. 11, N.2, PP. 237-244 

JYOTI SOROUT

ACKNOWLEDGEMENTS Dr Nagendra Singh (Mentor) Kazuhiko Kasuya , Julie L. Boyer, Yadi Tan,

and D. Olivier Alipui. Neil R. Hackett Ronald G. Crystal

TECHNIQUES USED IN THE ARTICLE

Western blotting ELISA

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).

•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.

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).

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 .

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 levelsFollowing 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.

In vitro assessment of AdaPAscAb

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.

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

In Vivo Expression by AdA PAscAbWe 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 receivingAdNull.

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.

DISCUSSIONThe 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 ananthrax attack.