ABBS 2005,38(02): Evaluation of Antibody Responses Elicited by Immunization of Mice with a Pneumococcal Antigen Genetically Fused to Murine HSP70 and Murine Interleukin-4

Abstract        The heat shock (stress) protein HSP70
has been shown to be a potent stimulator of cellular immune responses. In order
to determine whether HSP70 has the ability to stimulate antibody responses, we
constructed and expressed fusion proteins consisting of murine HSP70 or murine
interleukin (IL)-4 covalently linked to a pneumococcal cell wall-associated
protein antigen designated PpmA. Immunization of mice with the PpmA-HSP70
fusion protein (PpmA-70) failed to elicit an increased PpmA-specific serum
antibody response. In contrast, mice immunized with PpmA fused to IL-4
(PpmA-IL4), or PpmA fused to both IL-4 and HSP70 (PpmA-IL4-70) fusion proteins
elicited high levels of PpmA-specific antibody responses. These data suggest
that HSP70 has a limited capacity to stimulate immune responses to heterologous
antigens in vivo.

Key words         HSP70; interleukin-4; antibody
response; fusion proteins; PpmA

The heat shock (stress) protein HSP70
has been shown to be capable of stimulating protective immunity against tumors
[1]. The adjuvanticity of HSP70 has been attributed to its ability to associate
with antigenic peptides (chaperone function) and to bind to receptors on the
surface of antigen presenting cells (APCs). These dual attributes of HSP70 ­al­­low
for the efficient presentation of bound peptides to the ­immune system for the
elicitation of peptide-specific T cell immune responses [2,3]. Other studies
have ­sdemonstrated that immunization of experimental animals (mice) with
recombinant proteins consisting of test ­antigens ­covalently linked to HSP70
of mycobacterial origin resulted in dramatic enhancement of humoral and
cellular immune responses directed against the target antigens [4,5]. ­Another
study aimed at characterizing the immunostimulatory ­attributes of HSP70 of
mammalian origin demonstrated that HSP70 has the ability to activate APCs, as
evidenced by enhanced secretion of pro-inflammatory cytokines such as
interleukin (IL)-1b, IL-6, and tumor
necrosis factor (TNF)-a [6]. Subsequent
studies also demonstrated that immunization of mice with previously identified
cytotoxic T-cell epitopes from a variety of sources fused to a closely related
HSP70 homolog of mammalian origin, designated heat shock cognate protein 70
(HSC70) resulted in potent stimulation of peptide-specific cytotoxic-T
lymphocytes (CTL) [7]. These reports have collectively provided ­support for
the notion that HSP70 can be used as a potent adjuvant for the elicitation of protective
immunity against a variety of diseases.

We undertook the experiments described
in this report to examine whether the immunostimulatory attributes of mammalian
HSP70 included the ability to stimulate helper activity for the production of
antibodies. This was prompted by the observation that antibodies can be ­protective
against a number of diseases, including ­bacterial infections [8], viral
infections [9], and cancers [10]. We constructed and purified a series of
fusion proteins consisting of a test antigen, putative protease maturation
protein A (PpmA) of Streptococcus pneumoniae covalently linked to murine HSP70, or
murine IL-4 or linked to both IL-4 and HSP70. We selected PpmA as the test
antigen for these ­experiments because it is poorly immunogenic by itself, and
needs to be administered with an adjuvant in order to elicit high titers of
PpmA-specific antibodies [8]. We elected to use IL-4 as an immunostimulatory
agonist in these experiments because it has previously been shown to be a potent
­activator of humoral immune responses [11].

We used a flow cytometric assay to
demonstrate that the recombinant HSP70 and HSP70-based fusion proteins produced
for these experiments exhibited the ability to bind to primary murine
splenocytes and to a murine monocytic cell line (RAW 264.7). In addition, the
HSP70-based ­recombinant proteins also exhibited the ability to bind to
adenosine triphoshphate (ATP) in the form of ATP-agarose. These observations
allowed us to conclude that our ­­HSP70-based recombinant proteins exhibited
the biological ­activity characteristics of native HSP70 [2,6,12]. ­Contrary to
­previous reports, however, our data indicate that ­biologically active HSP70
lacks the ability to stimulate the secretion ­inflammatory cytokines by APCs
[6,13]. Our data also demonstrate that immunization of mice with a fusion
protein consisting of HSP70 covalently linked to PpmA does not elicit enhanced
production of PpmA-specific antibodies. These ­results support the notion that
HSP70 may have a limited range of immunostimulatory attributes in vitro and in vivo. These factors
should to be taken into account in the ­design of vaccines formulations that
include mammalian HSP70 as an adjuvant.

Materials and Methods

Mice

BALB/c, C3H/HeJ, and C3H/HeOuJ (herein
referred to as C3H/OuJ) mice aged 6–8 weeks were
purchased from Jackson Laboratories (The Boston University School of
Medicine Animal Care and Use Committee approved all immunizations and ­procedures
involving animals, in accordance with ­guidelines from the National Institutes
of Health,

Cloning and expression of
recombinant proteins

Polymerase chain reaction (PCR) was
used to generate gene fragments for this study, using a high fidelity ­thermostable
polymerase (platinum Pfx) from Invitrogen Corporation (

Purification of recombinant
proteins

Recombinant
proteins used in these experiments were purified from lysates of E. coli grown in Luria
broth supplemented with antibiotics. Protein expression was induced by the
addition to log-phase cultures of isopropyl-b–D-thiogalactoside (IPTG) at a final concentration of

Preparation and stimulation of
primary peritoneal macrophages

Peritoneal macrophages were isolated
from peritoneal exudate cells (PEC) of C3H/HeJ and C3H/OuJ mice by plate
adherence, as previously described [16]. Briefly, mice of the indicated strains
were injected intraperitoneally with 3 ml of brewer’s thioglycollate. Four days
later, peritoneal exudates cells were harvested by lavage with 10 ml of RPMI-10
[RPMI 1640 supplemented with 10% fetal ­bovine serum (FBS),

Flow cytometry to evaluate binding
of ­recombinant proteins to cells

Previous reports have demonstrated that
HSP70 can bind to the surface of a variety of cells [3,17]. We therefore tested
the ability of HSP70-containing fusions to bind to either RAW 264.7 macrophages
or to primary spleen cells using a flow cytometric assay. Approximately 2´106 cells were incubated with PpmA, HSP70,
PpmA-70, ­PpmA-IL4, or PpmA-IL4-70, at a final concentration of 0.2 mM in staining buffer (PBS with 0.05% sodium azide and 1%
bovine serum albumin) at 4 ºC for 2 h. Bound ­recombinant proteins were detected by
incubating cells with S-protein conjugated to fluorescein isothiocyanate ­­­(­S-FITC;
Novagen), which binds to the S-Tag domain ­­co-expressed as part of each
recombinant protein. S-FITC was used at a dilution of 1:500 in staining buffer
for 15 min at 4 ºC. Cells were washed with PBS and subjected to flow
cytometry using a bench-top flow cytometer (BD-Coulter,

Immunization of mice and
characterization of ­PpmA-specific serum antibody responses

To test the ability of murine HSP70 to
act as a ­molecular adjuvant for antibody production, fusion proteins ­consisting
of HSP70 genetically linked to the Streptococcus pneumoniae cell wall-associated protein PpmA
(PpmA-70) were injected into BALB/c mice. Mice were also ­immunized with PpmA
alone, PpmA genetically linked to IL-4 ­(PpmA-IL4), or PpmA genetically linked
to both IL-4 and HSP70 (PpmA-IL4-70). For comparative purposes, a group of mice
were also immunized with equimolar concentrations of PpmA emulsified in
incomplete Freund’s adjuvant (IFA). Mice immunized with sterile PBS served as
negative controls. BALB/c mice were immunized at weeks 0 and 3 with 100 pmol
(equivalent to 3.7 mg of PpmA per
immunization) and bled at weeks 2 and 5 to evaluate, respectively, the ­primary
and secondary PpmA-specific serum antibody responses by ELISA. ELISA was
performed using Immulon-1 plates (Dynatech,

Results and Discussion

In order to test the ability of HSP70
to stimulate the secretion of pro-inflammatory cytokines, we used elicited
peritoneal macrophages. In these experiments, we used macrophages from the
endotoxin-resistant C3H/HeJ mouse strain [18], and compared stimulation of
these ­macrophages to that of macrophages obtained from the endotoxin-sensitive
C3H/OuJ mouse strain (a wild-type strain congenic to C3H/HeJ). As shown in Fig. 2, ­recombinant
murine HSP70 prepared by us was unable to elicit secretion of TNF-a, ­­­IL-6, or NO ­release from ­peritoneal ­macrophages
of either C3H/HeJ or C3H/OuJ mice. In contrast, ­incubation of cells with LPS
or HSP70 from Stressgen Biotechnologies (HSP70-SG) was able to stimulate the
secretion of TNF-a, IL-6 and NO.
This was dependent on the presence of an intact LPS ­signaling pathway in the
responding cells (i.e., cells from C3H/OuJ mice, and was not evident with cells
from C3H/HeJ mice). These data are consistent with recent reports showing that
the production by macrophages of TNF-a, IL-6 and NO in
response to HSP70 stimulation was due to endotoxin contamination of the HSP70
preparations used [19–21]. These
observations prompted us to examine ­alternative means of demonstrating the
biochemical ­integrity of our HSP70-based fusion proteins.

The ability of HSP70 to bind to APCs is
believed to be critical to its ability to stimulate adaptive immune responses
[2,12]. We ­­therefore tested the ability of our HSP70-based fusion proteins to
bind to cells. In preliminary experiments we demonstrated that PpmA by itself
did not exhibit binding to mouse spleen cells or to RAW macrophages at the
concentrations tested. Because cells incubated with PpmA exhibited levels of ­binding
to cells equivalent to that of cells stained with ­S-FITC alone (secondary
reagent), the binding of PpmA was used to ­generate all control histograms
against which the binding of HSP70-based fusion proteins were compared. As
shown in Fig. 3(A,B,D,E,G), all HSP70-based recombinant ­proteins
exhibited highly consistent patterns of binding to both RAW 264.7 macrophages
and primary murine splenocytes. In contrast, recombinant ­proteins that did not
contain HSP70 failed to exhibit binding to either RAW 264.7 macrophages or
primary mouse murine splenocytes [Fig. 3(C,F)], indicating that HSP70 was responsible for the binding
to these types of cells types.

We subsequently performed immunization
experiments in BALB/c mice to test whether HSP70 covalently linked to PpmA was
capable of eliciting increased PpmA-specific antibody responses. For these
experiments, mice immunized with PBS served as negative controls, and mice
immunized with PpmA emulsified in incomplete Freund’s adjuvant (IFA) served as
positive controls. Immunization of mice with PpmA covalently linked to HSP70
(PpmA-70) failed to elicit increased primary [Fig. 4(A)] or ­secondary [Fig. 4(B)] PpmA-specific
serum antibody responses, compared to the primary and secondary PpmA-specific
antibody responses elicited by immunization with PpmA alone. We also performed
immunizations with PpmA covalently linked to either IL-4 alone (PpmA-IL4), or ­covalently
linked to both IL-4 and HSP70 (PpmA-IL4-70) in order to obtain a better picture
of the adjuvant properties of HSP70 when compared to equimolar ­concentrations
of a known immunostimulant (IL-4). ­Immunization of mice with PpmA-IL4 or
PpmA-IL4-70 elicited robust PpmA-specific responses after a single ­immunization
[Fig. 4(A)], which were
further enhanced following a second immunization with the respective ­immunogens
[Fig. 4(B)]. The secondary
PpmA-specific subclass ­responses elicited by immunization with PpmA-IL4 were
characterized by elevated levels of PpmA-specific IgG1 and IgG2a, whereas the
secondary PpmA-specific ­responses following immunization with PpmA-IL4-70 were
characterized by elevated levels of IgG2a, but ­relatively low levels of IgG1.
The results of these ­immunization experiments demonstrate that HSP70 by ­itself
is a poor stimulant of T cell helper activity, as ­defined by the ability to
enhance the elicitation of PpmA-specific antibody responses.

Activation of naive T cells to undergo
clonal expansion and develop effector functions requires the presentation of
antigen by APCs that are activated to express ­co-stimulatory molecules and
secrete pro-inflammatory cytokines [22,23]. Currently available data indicate
that HSP70 from mice and humans can enhance the efficiency of ­presentation of
associated antigens for the stimulation of both CD8+ and CD4+ T cells [24,25]. However, ­mammalian HSP70
appears to have a limited capacity to activate APC, which is consistent with
Bausinger and Tsan [19,26]. The poor adjuvanticity of HSP70 demonstrated in our
­experiments is consistent with the results described in ­another recent
report, which showed that the ability of HSP70-peptide complexes to stimulate
effective CD8+ T cell responses
required additional stimulation with either peptide pulsed dendritic cells or
with CD4+ T helper cells
[24]. It may therefore be worthwhile to consider the use of other
immunostimulants capable of enhancing APC activation, such as IL-4 [27], in
conjunction with HSP70-associated antigens, for the elicitation of more potent ­antigen-specific
immune responses.

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