Liposome-encapsulated antigens induce a protective CTL response against Listeria monocytogenes independent of CD4+ T cell help.

Abstract

Protection against intracellular pathogens is usually mediated by cytotoxic T lymphocytes (CTL). Induction of a protective CTL response for vaccination purposes has proven difficult because of the limited access of protein antigens or attenuated pathogens to the MHC class I presentation pathway. We show here that pH-sensitive PE/CHEMS liposomes can be used as a vehicle to efficiently deliver intact proteins for presentation by MHC class I. Mice immunized with listerial proteins encapsulated in such liposomes launched a strong CTL response and were protected against a subsequent challenge with L. monocytogenes. Remarkably, the CTL response was induced independently of detectable CD4(+) T cell help.

Full text

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This is a postprint of an article published in
Grenningloh, R., Darj, A., Bauer, H., Zur Lage, S., Chakraborty, T.,
Jacobs, T., Weiss, S.
Liposome-encapsulated antigens induce a protective CTL response against
Listeria monocytogenes independent of CD4+ T cell help
(2008) Scandinavian Journal of Immunology, 67 (6), pp. 594-602.

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Liposome-encapsulated antigens induce a protective CTL response
against Listeria monocytogenes independent of CD4+ T cell help

Roland Grenningloh1,3, Ayub Darj1,4, Heike Bauer1, Susanne zur Lage1, Trinad
Chakraborty2, Thomas Jacobs1,5 and Siegfried Weiss1,6


1


Molecular Immunology
Helmholtz Centre for Infection Research
Innhofenstrasse 7, D-38124 Braunschweig, Germany

Institute for Medical Microbiology,
University of Giessen
Frankfurterstr. 107, D-35392 Giessen, Germany


2


?Short title: Protective CTL response induced by liposomes
Keywords: Listeria monocytogenes/liposomes/cytotoxic T lymphocytes


6
corresponding author:
Tel: ++49-(0)531-6181-5100
Fax: ++49-(0)531-6181-5002
e-mail: siw@gbf.de


3 present address: Brigham and Women’s Hospital
Department of Medicine
Division of Rheumatology, Immunology, and Allergy
Boston MA 02115 USA

4 present address: Centre de Recerca en Sanitat Animal
Universidad Autonoma de Barcelona
Campus de la UAB
E-08193 Bellaterra, Spain

5 present address: Department of Immunology
Bernhard Nocht Institute for Tropical Medicine

Bernhard-Nocht-Str. 76
D-20359 Hamburg, Germany

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Abstract
Protection against intracellular pathogens is usually mediated by cytotoxic T lymphocytes
(CTL). Induction of a protective CTL response for vaccination purposes has proven
difficult because of the limited access of protein antigens or attenuated pathogens to the
MHC class I presentation pathway. We show here that pH-sensitive PE/CHEMS liposomes
can be used as a vehicle to efficiently deliver intact proteins for presentation by MHC class
I. Mice immunized with listerial proteins encapsulated in such liposomes launched a strong
CTL response and were protected against a subsequent challenge with L. monocytogenes.
Remarkably, the CTL response was induced independently of detectable CD4+ T cell help.

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Introduction
Protective immunity against intracellular pathogens is usually mediated by cytotoxic T
cells. However, the generally used method of immunization using inactivated pathogens or
purified proteins mostly fails to elicit a protective CTL response since these antigens only
have limited access to the MHC class I presentation pathway. Several experimental
approaches have been described that circumvent this problem. For example, attenuated
intracellular bacteria like Salmonella and Listeria have been used to deliver heterologous
antigens into the cytosol of host cells [1-5] although living vaccine carriers might be
considered problematic in terms of safety and general applicability. Alternatively, peptides,
either in adjuvant or loaded onto dendritic cells, could be used to induce a CTL response [6,
7]. However, peptide vaccines would have to comprise a large number of different epitopes
to ensure presentation by the excessive number of HLA alleles. Bacterial toxins have also
been used to deliver intact proteins or peptides into the cytosol for MHC I-restricted
presentation [8]. Nevertheless, questions remain with regard to the safety of bacterial toxins
for the use in humans.

A safe alternative to deliver intact proteins to the cytosol of professional APC would be to
entrap subunit vaccines into liposomes that have the property to target the MHC class I
presentation pathway. Liposomes are artificial membrane vesicles with an enclosed
aqueous compartment. Many different forms of liposomes are known depending on their
chemical formulation and some are commercially available [9, 10]. Using liposome-
encapsulated antigens, effective induction of antibody production, T cell proliferation and

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subsequent protection have been demonstrated in different infection models, including viral
[11-13], protozoal [14-17] and bacterial [18-21] pathogens. In these cases the immune
response was driven by CD4+ T cells. On the other hand, efficient induction of CTL
responses in vitro and in vivo has been achieved by the use of pH-sensitive liposomes [22-
27]. These vesicles are stable at neutral pH but disintegrate under mild acidic conditions
e.g. in endosomes and lysosomes [28]. Such liposomes have been demonstrated to deliver
encapsulated antigens into the cytosol of host cells, thus targeting them into the MHC class
I presentation pathway [29, 30]. Immunization with tumor associated antigens encapsulated
in acid labile liposomes partially protected mice against subsequent tumor challenge [31].
However, it remained to be shown that immunization with liposome-encapsulated proteins
can effectively protect the host against intracellular pathogens. Therefore, we used Listeria
monocytogenes as a model for an intracellular pathogen. Protection against L.
monocytogenes is normally conferred by CD8+ T cells. CD4+ T cells provide only limited
protection [32-34], and a protective role for antibodies is controversial [35-37]. Thus,
efficient vaccination against this pathogen requires the induction of a strong CTL response.

We immunized mice with listeriolysin O (LLO), p60, or a combination of both entrapped in
PE/CHEMS liposomes. Both antigens elicited strong CTL responses. Mice immunized this
way were protected against a subsequent lethal challenge with L. monocytogenes.
Interestingly, immunization with PE/CHEMS liposomes did not induce a detectable CD4+
T cell response. In addition, a CTL response could also be induced in mice deficient for
MHC class II. Consistent with findings described earlier no CTL memory response was

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