Eur. J. Immunol. 1992. 22: 1365-1372

Heat-shock proteins as carrier molecules

1365

Christy Barriosov, Alexander R. Lussowoe, Jan Van Embden., Ruurd Van der Zee., Rino Rappuolia, PaoIo Costantinon, Jacques A . Louisov, Paul-Henri LambertO and Giuseppe Del GiudiceO

Mycobacterial heat-shock proteins as carrier molecules. 11: The use of the 70-kDa mycobacterial heat-shock protein as carrier for conjugated vaccines can circumvent the need for adjuvants and Bacillus Calmette Gubrin priming"

World Health Organization-Immunology Research and Training Center, Department of Pathologyo, University of Geneva, Geneva, Institute of Biochemistryv, University of Lausanne, Epalinges, National Institute of Public Health and Environmental Protection., Bilthoven and Research Centern, Sclavo, Siena

In a recent work, we have shown that mycobacterial heat-shock proteins (hsp) of 65-kDa (GroEL-type) and 70-kDa (DnaK-type) acted as carrier molecules in mice, previously primed with Mycobacterium tuberculosis var. bovis (bacillus Calmette-Gukrin, BCG), for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP)4o. Anti-peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial hsp, was given in the absence of any adjuvants (LLISSOW et al., Eur. J. Immunol. 1991. 87: 2960). In this report, we show that mice immunized with peptides or oligosaccharides conjugated to the 70-kDa hsp produced high titers of IgG antibodies in the absence of any previous priming with BCG. The anti-peptide antibody response persisted for at least 1 year.This adjuvant-free carrier effect of the 70-kDa hsp wasTcell dependent, since no anti-peptide nor anti-70-kDa IgG antibodies were induced in athymic nulnu mice. Previous immunization of mice with the 65-kDa or 70-kDa hsp did not have any negative effect on the induction of anti-peptide IgG antibodies after immunization with hsp-peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65-kDa hsp could substitute for BCG in providing an effective priming for the induction of anti-(NANP) antibodies. Finally, both the 65-kDa and 70-kDa hsp acted as carrier molecules for the induction of IgG antibodies to group C meningococcal oligosaccharides, in the absence of adjuvants. These findings strongly suggest that the use of hsp as carriers in conjugated constructs for the induction of anti-peptide and anti-oligosaccharide antibodies could be of value in the design of new vaccines for eventual use in humans.

1 Introduction Development of vaccines consisting of defined recombinant or synthetic epitopes from selected antigens is now being pursued by several investigators for viral, bacterial, and parasitic diseases [l, 21. Such an approach is particularly needed when the preparation of classical vaccines (via inactivation or attenuation of the pathogen) is not achievable. However, many of the developed constructs exhibit a [I 10205] ~~

*

~

This work was supported by the World Health OrganizationTransdisease Vaccinology Programme, the UNDPiWorld BanWWHO Special Programme for Research and Training in Tropical Diseases, and the Swiss National Science Foundation. Supported by a fellowship from the Commission Federale des Bourses pour Etudiants Etrangers, Switzerland. Present address: Ludwig Institute for Cancer Research, Laus a m e Branch, CH-1066 Epalinges, Switzerland.

Correspondence: Giuseppe Del Giudice, WHO-Immunology Research and Training Centre, Department of Pathology, University of Geneva, rue Michel Servet 1, CH-1211 Geneva 4, Switzerland Abbreviations: hsp: Heat-shock protein hspR65: Recombinant M . bovis 65-kDa IGroEL-type) hsp hspR70: Recombinant M . tuberculosis 70-kDa (DnaK-type) hsp MenC: Group C meningococci T T Tetanus toxoid 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

low immunogenicity. With the ultimate aim of increasing the immunogenicity of synthetic and recombinant preparations, several attempts have been made towards the development of new safe and strong adjuvants acceptable for human use [3 ].Along the same lines, in the development of polysaccharide vaccines against bacterial diseases much work is being devoted in the preparation and evaluation of polysaccharides conjugated to protein carriers, with the aim of increasing the protective immune response to the polysaccharide moiety in 5 18-month-old children [4]. To enhance the immune response to a synthetic malaria polypeptide, (NANP)40, consisting of 40 Asn-Ala-Asn-Pro repeats from the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and considered as a basis for the eventual development of a malaria vaccine [ 5 ] ,we applied a system originally reported by Lachmann et al. [6]. It was shown [7] that high and long-lasting anti-peptide IgG antibody titers could be induced in the absence of adjuvants, and irrespective of their genetic (MHC) background [S-101, when the mice were first primed with BCG (Bacillus Calmette-GuCrin, Mycobacterium tuberculosis var. bovis, currently used for human vaccination against tuberculosis) and then immunized with the (NANP)do peptide conjugated t o tuberculin PPD (currently used to measure the Tcell reactivity in people previously exposed to tuberculosis or vaccination against tuberculosis) [7]. More recently 1111, the same effect was demonstrated using some mycobacterial heat0014-298019210606-1365$3.50+ ,2510

1366

C. Barrios, A. R. Lussow, J.Van Embden et al.

Eur. J. Immunol. 1992. 22: 1365-1372

shock proteins (hsp), i.e. proteins whose level of synthesis growth the culture was centrifuged and filtered. CRM 197 is regulated in response to heat shock or a variety of was precipitated by adding ammonium sulfate (43% w/v) to environmental stimuli [12]. High titers of ~ I I ~ ~ - ( N A N P )the ~ " cell-free culture supernatant and further purified by IgG antibodies were induced in mice primed with live BCG DE52 ion exchange chromatography [21], and subsequent and then immunized with the (NANP)4o peptide conju- hydroxylapatite column chromatography. The preparation gated to the recombinant M . bovis BCG 65-kDa (GroEL- was analyzed for flocculation units content (Lf) against a type) hsp (hspR65) or to the recombinant M . tuberculosis standard anti-diphtheria toxin antiserum. Protein nitrogen 70-kDa (DnaK-type) hsp (hspR70) in the absence of was determined by the micro-Kyeldahl method [22]. The adjuvants [ l l ] . purity of the preparation resulted of 2500 Lf/mg N. The group C meningococcal (MenC) polysaccharide was puriIn the present report, we discuss experiments aimed at fied as described [23]. The purified polysaccharide investigating the relative requirements for BCG priming in (10 mg/ml) was depolymerized by hydrolysis in acetate our model of immunization, the positive or negative effects buffer 0.01 M pH 5 , at 100°C for 8 h. The resulting product of previous immunization of mice with mycobacterial hsp was analyzed by analytical chromatography on Sephadex alone on the induction of anti-peptide antibodies using G-50 and showed a Kd (partition coefficient) of 0.27. hsp-petide conjugates, and the possible application of this model of immunization for the induction of antibodies to hsp-conjugated oligosaccharide antigens in the absence of 2.4 Introduction of primary amino groups into the end groups of the oligosaccharide adjuvants.

2 Materials and methods 2.1 BCG The M . tuberculosis var. bovis used was the BCG-F strain obtained from the BCG laboratory of the Institut Pasteur (Paris, France) [13]. Aliquots of 0.5 ml at lo9 CFU/ml were stored at -70°C to increase their stability [14]. For priming, mice were infected with lo6 CFU given i.p.

The solution derived from the hydrolysis of the MenC polysaccharide was made 0.5 M in ammonium chloride and 0.15 M in sodium cyanoborohydryde. The pH was adjusted to 7 and the resulting solution was kept at 35 "Cfor 1 week. The oligosaccharide was then purified by Sephadex G-15 chromatography; fractions at void volume containing chemical activity for the carbohydrate and amino groups were pooled, leaving behind monomeric sugars and excess of reagents. Characterization of MenC amino-oligosaccharide involved chemical determination of amino groups [24], sialic acid [25], and 0-acetyl [26].

2.2 Peptides, mycobacterial hsp, and hsp-peptide conjugates

2.5 Production of the CRM 197-, hspR65-, and hspR7O-MenC oligosaccharide glycoconjugates

The (NANP)40 synthetic peptide, from the P fulciparum CS protein, and the (QGPGAP)4 synthetic peptide, from the P yoelii CS protein, were produced according to the methods described in detail elsewhere [9, 151. Both peptides behave as bothTand B cell epitopes only in H-2b mice [ 8 , 9, 161. Lyophilized material was resupsended in sterile distilled water at 10 mg/ml, aliquoted, and stored at - 70 "C until use. Tetanus toxoid (TT) was from the Schweiz, Serum- and Impfinstitut (Bern, Switzerland). M . bovis BCG GroEL-type 65-kDa hsp (hspR65) was expressed from a recombinant E. coli K12 strain harboring plasmid pRIB1300 [17, 181, and p u r i k d as described [19]. Recombinant M . tuberculosis DnaK-type 70-kDa hsp (hspR70) was obtained and purified by ATP-agarose chromatography according to Mehlert and Young [20].

MenC amino-oligosaccharide was solubilized in dimethylsulfoxide with 10% water and then reacted with a 12-fold excess of the N-hydroxysuccinimide diester of adipic acid (prepared according to Hill et al. [27]), as compared to amino groups. After purification by 1-4 dioxane precipitation, the activated oligosaccharide was dried under vacuum and analyzed for its content in N-hydroxysuccinimide ester. A 16-fold molar excess of oligosaccharide-active ester was reacted with CRM 197 in 0.1 M phosphate buffer, pH 7. hspR65 and hspR70 at 5 mg/ml of 0.1 M phosphate buffer, pH 7,were reacted with a 300-fold molar excess of activated oligosaccharide. The resulting glycoconjugates were purified from unreacted oligosaccharides by phenyl Sepharose (Pharmacia, Uppsala, Sweden) column chromatography, filtered, and stored at 4°C. The ratio between their sialic acid content and the percent of sialic acid in the starting MenC polysaccharide, determined as described [25], represented the amount of coupled oligosaccharide. The protein content of the preparation was determined according to Lowry [28].

One milligram each of TT, hspR65, and hspR70 was coupled to 1 mg of (NANP)4o or (QGPGAP)4 peptide in the presence of 0.002% glutaraldehyde for 2 h, and then dialyzed against PBS overnight [7]. Aliquots of each conjugate were stored at - 70°C until use.

2.6 Mice and immunizations 2.3 Purification of CRM 197 and of group C meningococcal (MenC) polysaccharide, and production of MenC oligosaccharide CRM 197 was purified from culture filtrates of the Corynebacterium diphtheriae strain C7P197 M8. The strain was grown in a 40-1 fermentor using a CY medium [21]. After

Female 8-12-week-old BALB/c (H-2d), C57BL/6 (H-2b), and CBA/J (H-2k) mice were bred in our animal facilities. The original breeding pairs were obtained from the Jackson Laboratory (Bar Harbor, ME). Athymic BALB/c nulnu mice were purchased from Iffa Credo (EArbresle, France).

Heat-shock proteins as carrier molecules

Eur. J. Immunol. 1992. 22: 1365-1372

According to the immunization schedule already established [7, 111,on day 0 each mouse received i.p. lo6 CFU of BCG (or PBS: control group), and then 20 pg of hsppeptide conjugate i.p. in 0.5 ml PBS on days 14 and 35. In some experiments, groups of mice received 100 pg of hspR65 or hspR70 in incomplete Freund’s adjuvant (IFA) i.p. on day-1. followed by immunization with conjugates in PBS. The same immunization schedule was followed in groups of mice that received (in PBS) the hsp conjugated with MenC oligosaccharide. Control groups received the MenC oligosaccharide alone, or the CRM197-MenC oligosaccharide conjugate vaccine absorbed onto aluminium hydroxide (1 mg/dose in 0.5 ml). In each immunization, mice received 2 pg of MenC oligosaccharide, which corresponded to 8.7 pg of CRM197-MenC oligosaccharide conjugate vaccine, 8.4 pg of hspR70-MenC oligosaccharide conjugate, or 3.7 pg of hspR65-MenC oligosaccharide conjugate, respectively. 2.7 Detection of antibodies by ELISA Mice were bled weekly from the retroorbital plexus, the serum was separated, and antibodies were titered by ELISA. Anti-(NANP),o and anti-(QGPGAP), IgG antibodies were titered using the ( N A N P ) J ~ or the (QGPGAP)J peptide a\ solid phase (1 pg/ml), as described in detail [S, 16, 291. Serum samples giving an absorbance at 492 nm < 0.2 at the first dilution tested (1 : 100) were considered as negative. A similar ELISAwas carried out for the detection ot IgG antibodies specific for the hspR65 or hspR70, using hspR65 or hspR70 as solid phase ( I pglml).

1367

TT on day 0 no

1 I~

2

3

4

Log 10 anti-(NANP) IgG titers

Figure 1. Epitope-specific suppression by preimmunization with TT. O n e group of five B A L B k mice was immunized o n day 0 with 100 pg of T T (given in IFA i.p.): another group received IFA alone. Fourteen and twenty-one days later mice were immunized i.p. with 20 pg of the TT-(NANP)do conjugate in IFA. Two (hatched bars) and four weeks after (solid bars), blood samples were taken, and anti-(NANP)j(,IgG antibody titers were measured by ELISA using the peptide as solid phase. Results are expressed as t h e geometrical mean of the Log10 reciprocal of the last serum dilution positive for each group of mice. i.e. giving an absorbance 2 0 . 2 at 492 nm.

fact, using the T T as a carrier molecule conjugated to the (NANP)lo peptide, and given in IFA, it was observed, in agreement with previous reports from other groups [30], that mice previously immunized with T T alone produced much less anti-peptide IgG antibodies than mice not pretreated with T T (Fig. 1).

Based on these data, the question was raised as to whether the adjuvant-free carrier effect of the mycobacterial For the detection of anti-MenC IgG antibodies, 96-well hspR65 would be exerted in BALB/c mice with high titers flat-bottom plates (Nunc lmmunoplate I , Nunc, Roskilde, of anti-hspR65 antibodies at the time of immunization Denmark) were coated with MenC polysaccharide (days 14 and 35) using the hspR65-(NANP)lo peptide (5 pg/ml) in PBS, pH 7.4, by overnight incubation at 37°C. conjugate. As shown in Fig. 2 B,when mice were pretreated After extensive washing with PBS containing 0.05% with a high dose of hspR65 (100 pg in IFA), primed with Tween-20 (PBS-T) and 1 h incubation at 37°C with 200 p1 BCG, and then immunized twice with the hspR65PBS-Tcontaining 5% FCS, wells were incubated overnight (NANP)4o conjugate in the absence of adjuvant, antiat 4°C with 100 pl of mouse sera diluted in PBS-T (NANP)40 IgG antibodies were produced following the containing 5% FCS. After more washing, plates were again same kinetics and reaching the same high titers as in the incubated for 3 h at 37°C with 100 pl of an appropriate group of BALB/c mice not treated with the hspR65 on dilution of an anti-mouse IgG antiserum conjugated to day-1, but primed with BCG on day 0 (Fig. 2 A ) . In the horseradish peroxidase. The presence of specific antibodies same series of experiments, it was interesting to observe was revealed by addition of 2,2’-azinobis(3-ethylbenzthia- that the group of mice treated on day-1 with the hspR65, zoline-sulfonic-acid) (ABTS; Kirkegaard and Perry Labo- but not primed with BCG on day 0 (Fig. 2 C). mounted an ratories, Inc., Gaithersburg, MD) as a substrate. Results anti-peptide IgG antibody response comparable to that were read in absorbance at 414 nm. Serum samples with an observed in groups of BCGprimed BALB/c mice. As absorbance < 0.2 at the first dilution tested (1 : 50) were expected from our previous data [ I l l , mice immunized with the hspR65-(NANP)~oconjugate without adjuvants, but considered as negative. not previously primed with BCG or hspR65 alone, produced negligible amounts of anti-peptide and anti-hspR65 antibodies (Fig. 2 D). Similar results were also obtained in 3 Results C57BL/6 mice (H-2h)(not shown). 3.1 Requirement for BCG priming and lack of epitope suppression using the hspR65 as a carrier molecule

A series of experiments were carried out to see whether previous immunization of mice with hsp could affect the induction of anti-peptide antibodies after immunization with hsp-peptide con,jugates in the absence of adjuvants. In

These data strongly suggest that pretreatment of mice with the hspR65 does not induce any detectable epitope-specific suppression on the anti-peptide antibody production. Furthermore, such a pretreatment can favorably substitute for BCG priming in the induction of anti-peptide antibodies after immunization with the hsp-peptide conjugate without adjuants.

1368

C. Barrios, A. R. Lussow, J.Van Embden et al.

Eur. J. Immunol. 1992. 22: 1365-1372 hrpR70 BCG on day -1 on day 0

hopR65 BCG on day -1 on day 0

41

P

A)

P

31

4i

-

2

0

7

14 21

28

35 42 49

Days post-BCG priming

2-

0

7

14 21

28

35 42 40

Days post-BCG prlmlng

Figure2. Effect of preimmunization on the carrier effect of hspR65 in the absence of adjuvants. Groups of five BALBk mice wereimmunizedornot with hspR65 onday-1 (100 pginIFAi.p.), primed or not with live BCG on day 0 (lohCFU i.p.). On days 14 and 35 all the mice were immunized with 20 pg of the hspR65(NANP)40peptide conjugate in PBS (arrows). Blood samples were were taken weekly, and anti-(NANP)4" (0)and anti-hspR65 (0) measured by ELISA using the peptide or the hspR65 as solid phase. Results are expressed as the geometrical mean of the LoglO reciprocalof the last serum dilution positive for each group of mice, i.e. giving an absorbance 2 0.2 at 492 nm.

Figure3. The adjuvant-free carrier effect of hspR70 does not require previous priming with BCG. Groups of five BALBk mice wereimmunizedornotwithhspR7Oonday-1(100 pginIFAi.p.), primed or not with live BCG on day 0 (loh CFU i.p.). On days 14 and 35 all the mice were immunized with 20 pg of the hspR70(NANP)40peptide conjugate in PBS (arrows). Blood samples were taken weekly, and anti-(NANP)do (0)and anti-hspR70 ( 0 )IgG antibody titers were measured by ELISA using the peptide or the hspR70 as solid phase. Results are expressed as the geometrical mean of the LoglO reciprocal of the last serum dilution positive for each group of mice, i.e. giving an absorbance 2 0 . 2 at 492 nm.

3.2 The adjuvant-free carrier effect of hspR70 does not require previous priming with neither BCG nor hsp

titers were boosted after the second immunization (day 35). Surprisingly, when control mice, neither primed with hspR70 nor BCG, were immunized with the hspR70(NANP)40 conjugate without adjuvants, anti-(NANP) and anti-hspR70 IgG antibodies were detected at titers similar to those obtained in primed animals (Fig. 3 D ) .

To see whether o r not these findings were specific of the carrier effect mediated by the hspR65, similar experiments were carried out in BALB/c mice using the hspR70. A s one can see in Fig. 3 B, mice primed on day- 1with hspR70 and on d a y 0 with BCG, and then immunized with hspR70(NANP)40 peptide conjugate without adjuvant, produced IgG antibody titers similar t o those obtained in mice not treated with hspR70, but primed with B C G (Fig. 3A). Mice receiving hspR70 on day-1, but not primed with BCG on day 0 produced anti-(NANP), IgG antibody titers (Fig. 3 C ) similar to those observed in groups of mice primed with BCG. I n all groups, anti-peptide IgG antibodies were already detectable after the first immunization (day 14) with the hspR70-(NANP)40 conjugate, and their

In experiments aimed at investigating the persistence of anti-peptide antibodies induced after immunization of mice with hspR70-peptide conjugates, it was observed that anti-(NANP)40 peptide IgG antibodies were still detectable after 1year at titers of about 1: 1000,irrespective of the fact that mice were o r not previously primed ith BCG (Fig. 4). These findings suggest that, as for the hspR65, no epitopespecific suppression occurred after pre-immunization with

Eur. J. Immunol. 1992. 22; 1365-1372

Heat-shock proteins as carrier moleculcs

Days PoSt-BCG prlming

1369

-f 44 c

49

175

237

0

7

14

21

28

35

Days post immunization

291

370 7

2

-

1

3 4 5 LoglO antl-(NANP) igG titers

Figure 4. Presistence of the anti-peptide IgG antibodies in mice immunized with hspR70-(NANP)4o peptide conjugate without adjuvant. Groups of five BALBk mice, primed (hatched bars) or not primed (solid bars) with BCG (loh CFU i.p.) on day 0, were immunized with 20 pg of the hspR70-(NANP),o peptide conjugate in PBS on days 14 and 35. Not primed, not immunized mice served as a control (empty bars). Blood samples were taken periodically, and anti-(NANP)40 IgG antibody titers were measured by ELISA using the peptide as solid phase. Results are expressed as the geometrical mean of the Log10 reciprocal of the last serum dilution positive for each group of mice, i.e. giving an absorbance 2- 0.2 at 492 nm.

Figure 6. Adjuvant-free carrier effect of hspR70 for anti(QGPGAP), IgG antibody production in the absence of a previous priming with BCG. Groups of five BALB/c mice werc immunized with 20 pg of the ~ S ~ R ~ O - ( Q G P G peptide A P ) ~ conjugate in PBS o n days 0 and 21 (arrows). Blood samples were taken weekly, and anti-(QGPGAP)4 (0) and anti-hspR70 (0) IgG antibody titers were measured by ELISA using the peptide or the hspR70 as solid phase. Results are expressed as the geometrical mean of the LoglO reciprocal of the last serum dilution positive for each group of mice, i.e. giving an absorbance 2-0.2 at 492 nm.

As shown in Fig. 5 , when CBA/J (H-2k)and C57BL/6 mice (H-2b), not previously primed with BCG, were immunized with the hspR7O-(NANP)do conjugate in the absence of adjuvants, anti-(NANP)40 IgG antibodies were detected already after the first immunization and their titers were boosted after the second immunization (CBA/J mice). These results strongly suggested that the adjuvant-free carrier effect of the mycobacterial hspR70 in the absence of previous BCG priming was not peculiar to one particular strain of mice.

the hspRO alone. However, unlike the hspR65, the adjuvant-free carrier effect of the hspR70 was exerted without requiring priming with BCG. To inestigate whether or not the results obtained in the absence of BCG priming were due to the mouse strain employed (BALB/c) or to the The same carrier effect was observed in BALB/c mice peptide present in the conjugates [(NANP)40], other strains immunized with a conjugate consisting of the hspR70 and of mice were immunized with the ~ S ~ R ~ O - ( N A N of P )the ~ ~(QGPGAP)4 synthetic peptide from the R yoelii CS conjugate and another hspR70-peptide conjugate was protein [16]. In fact, in the absence of previous priming utilized as an immunogen. with BCG, an anti-(QGPGAP) and anti-hspR70 IgG

s

'J 5{

CBAlJ

I

CS7BU6 I

.is' 0

7 14

Days post immunlzatlon

Figure 5. Adjuvant-free carrier effect of hspR70 in the absence of a previous priming with BCG in CBAD and C57BL/6 mice. Groups of five mice were immunized with 20 pg of the h~pR70-(NANp)~,, peptide conjugate in PBS on days 0 and 21 (CBAN) or on day 0 only (C57BL/6) (arrows). Blood samples were taken weekly, and anti-(NANP)40 (0)and anti-hspR70 (0) IgG antibody titers were measured by ELISA using the peptide or the hspR70 as solid phase. Results are expressed as the geometrical mean of the Log10 reciprocal of the last serum dilution positive for each group of mice, i.e. giving an absorbance 2 0.2 at 492 nm.

BALB/c

BALB/c nu/nu

BALB/c

BALB/c nu/nu

Figure 7. Tcell dependence of the adjuvant-free carrier effect of hspR70 in the absence of previous BCG priming. Groups of five BALBJc and athymic BALB/c nulnu mice were immunized with 20 pg of the hspR65-(NANP)4() (A) or the ~ s ~ R ~ ~ - ( N A N P ) ~ , ) peptide conjugate (B) in PBS on days 0 and 21. Blood samples were taken 10 days after the second immunization, and anti-(NANP)40 (solid columns) and anti-hsp (hatched columns) IgG antibody titers were measured by ELISA using the peptide as solid phase. Results are expressed as the geometrical mean of the LoglO reciprocal of the last serum dilution positive for each group of mice, i.e. giving an absorbance 2- 0.2 at 492 nm.

1370

C. Barrios, A. R. Lussow, J.Van Embden et al.

antibody response was observed after two immunizations with the conjugate (Fig. 6).These results suggested that the adjuvant-free carrier effect of the mycobacterial hspR70 in the absence of previous BCG priming was not the specific characteristic of one particular peptide sequence present in the conjugate. To investigate whether or not the adjuvant-free carrier effect of mycobacterial hsp in BCG non-primed mice was T cell dependent, BALB/c and athymic BALBk nulnu mice, not primed with BCG, were immunized twice with the hspR70-(NANP)do conjugate without any adjuvant, or with the ~ S ~ R ~ ~ - ( N conjugate, A N P ) ~ as ~ ~a control. Fig. 7 shows that no antibody response was ever detectable in nulnu mice with either conjugate. This suggests that the carrier effect mediated by the mycobacterial hspR70 for the production of anti-peptide antibodies, in the absence of adjuvants and of a previous BCG priming, depended on the presence of an intact Tcell compartment.

Eur. J. Immunol. 1992. 22; 1365-1372

carrier effect would be mediated by mycobacterial hsp for the induction of an antibody response to an oligosaccharide antigen (MenC) from group C meningococci. To this aim, hspR65- and hspR70-MenC oligosaccharide conjugates were prepared, and used to immunize BALBk and CS7BL/6 mice, previously primed or not primed with BCG, Control groups of mice received the MenC alone, or a CRM 197-MenC conjugate candidate vaccine in aluminium hydroxide. Fig. 8 shows that, indeed, anti-MenC oligosaccharide IgG antibodies were induced after immunization with hsp-MenC conjugates, at titers comparable to or higher (C57BL/6 in Fig. 8 A) than those obtained with the CRM 197-MenC conjugate vaccine in aluminium hydroxide. Interestingly, this adjuvant-free carrier effect was, again, observed with the hspR70-MenC conjugate in the absence of a previous priming with BCG (Fig. 8B). Taken together, these results show that the approach of immunization using mycobacterial hsp in the absence of adjuvants can also be successfully applied to oligosaccharide antigens.

3.3 The carrier effect of hspR65 and hspR7O is also exerted after conjugation with oligosaccharide antigens

4 Discussion

Previous [11] and present data have shown that mycobacterial hsp behave as potent carrier molecules for the induction of strong antibody responses to peptides in the absence of adjuvants, and, as in the case of the hspR70, even in the absence of a previous BCG priming. The question was then asked whether a similar adjuvant-free

In this report we have shown that: (a) a potent carrier effect is exerted by the hspR70 molecule even in mice not previously primed with BCG; (b) hsp act as potent carrier molecules for the induction of specific IgG antibodies to oligosaccharide in the absence of adjuvants and (c) the carrier effect of the mycobacterial hspR65 and hspR70 for the production of anti-peptide antibodies in the absence of adjuvants is not affected by the presence of high titers of antibodies directed against the hsp.

BALB/c

"i/, -

1

1.59 0

0

d

I

A I

A I

C57BU6

A

U I

I

I

I

d I

I

0

7 14 21 28 36 42 49

A I

A I

f I

i I

A I

A I

I

7 14 21 28 35 42 49

51

,

,

,

,

,

,

,

,

": ..-

16

0

7 14 21 28 36 42 49

0

7 14 21 28 35 42 49

Days post-BCO priming

Figure8. Mycobactcrial hsp act as carrier molecules for the induction of anti-oligosaccharide IgG antibodies in the absence of adjuvants. Groups of five BALBk and C57BL/6 mice were primed on day 0 i.p. with lo6 CFU of BCG (A) or not primed with BCG (B), and then immunized on days 14 and 35 (arrows) with in PBS. hspR65-MenC (0) or hspR70-MenC conjugate (0) Control groups of mice were immunized with the MenC oligosaccharide alone (A), or with the CRM 197-MenC conjugate vaccine (0)in aluminium hydroxide. Blood samples were taken weekly, and anti-MenC IgG antibody titers were measured by ELISA using the MenC as solid phase. Results are expressed as the geometrical mean of the Log10 reciprocal of the last serum dilution positive for each group of mice, i.e. giving an absorbance 2 0.2 at 414 nm.

In our previous report, it was shown that priming with live BCG was required in order to obtain an anti-(NANP)4o IgG antibody response after immunization with PPD(NANP)4o conjugate without adjuvants [ll].It was hypothesized that this was possibly due to the activation of hsp-specific T cells at high frequency after infection by BCG. Here, it has been shown that an effective priming is achieved using the hspR65 alone, given at high dose (100 kg) in IFA, instead of BCG. One hypothesis could be that the priming of mice with the hspR65 in IFA allowed the persistence of the antigen, and the activation of specific T cells able to mediate the helper effect required for the induction of anti-peptide antibodies, in a manner similar to that achieved after priming with live BCG. One intriguing finding reported here is that anti-(NANP)4o, anti-(QGPGAP)A, and anti-oligosaccharide IgG antibodies were induced, in the absence of any adjuvant, in different strains of mice immunized with hspR70 conjugates, and not previously primed with BCG nor hspR70. Since this was not the case of the hspR6S carrier molecule (that necessarily required previous BCG priming for anti-peptide antibody induction), it is reasonable to hypothesize that the helper effect provided by the hspR70 molecule was achieved through mechanisms different from those triggered by the hspR65. Even though it is not possible to completely rule out that the carrier effect mediated by the hspR70 molecule was due to bacterial products (e.g. LPS) possibly contamitaning its prepration, this seems to us to be unlikely since: (a) no detectable antibody response (against the peptide or the hsp molecule) was observed in athymic

Eur. J. Immunol. 1992. 22: 1365-1372

Heat-shock proteins as carrier molecule\

1371

BALB/c nulnu mice immunized with the hspR70- vaccines, especially against Haemophilus influenzae type b, (NANP)40 conjugate; (b) the antibodies induced in have proven to be particularly effective [36]. Our approach euthymic mice were mainly of the IgG isotype and (c) their to immunization offers the advantage of not requiring any titers were significantly boosted by a second injection of the adjuvant to be added to the conjugate preparation, and of conjugate. All this suggests that the helper effect triggered inducing long-lasting antibody response. by the hspR70 was mediated by Tcells, and that it was not triggered in a T cell-independent manner, via a polyclonal One of the problems encountered in the development of activation of B cells. One explanation for the carrier effect subunit vaccines consisting of defined epitopes conjugated of the hspR70 molecule in the absence of a previous to carrier molecules, is represented by the possibility that priming with BCG, might be that a priming of Tcells anti-carrier antibodies induced by previous immunizaspecific for the hsp 70-kDa molecule had already naturally tion(s) with the carrier alone (e.g. as a vaccine) may occurred in mice at the moment of immunization with the interfere with the induction of an effective antibody hsp conjugates, for example, through hsp derived from the response to a given epitope, when this is given as an natural intestinal flora. However, should this be the case, it immunogen conjugated to the same carrier. This phenomis not clear why this natural priming of Tcells occurred for enon, the so-called epitope-specific suppression, has been the hsp 70-kDa, but not for the hsp 65-kDa molecule. In mainly described with carrier molecules, such as T T and this respect, it would be very interesting to investigate our diphtheria toxoid, that are commonly used as vaccines in model of immunization in germ-free animals. Alternative- humans [37-391. Our results using TT as a carrier for the ly, it may be that activation of specificTcells (or particular induction of anti-(NANP)40 antibodies in BALB/c mice Tcell subsets) at high frequency was directly triggered by previously immunized withTTalone, are in agreement with the immunization with the conjugates containing the hsp these reports. However, in some instances, preimmuniza70-kDa. but not the hsp 6.5-kDa molecule. It is not known tion of mice withTT [40] or T T fragments [30]was shown to whether the mycobacterial hsp 70-kDa molecule could bind enhance the antibody response to (NANP) peptides conjuimmunogenic peptides in antigen-presenting cells in a gated to TT. Both suppression and enhancement of the manner similar to that of hsp-like molecules (belonging to antibody response by previous priming with TT, depending the hsp70 heat-shock family; [31]), possibly facilitating the on the dose employed, have recently been reported using association between processed antigens and MHC class 11 conjugates containing MenC or other oligosaccharides [41]. molecules. One could also hypothesize that the peculiar In the experiments shown in this report, preimmunization carrier effect provided by the mycobacterial hsp 70-kDa, in of mice with hspR65 or hspR70, and the following high the absence of adjuvants and of previous priming with anti-hsp antibody response, did not affect the induction of BCG, might be exerted through the specific chaperone high titers of anti-peptide IgG antibodies after immunizafunctions of this class of stress proteins, i.e. their partici- tion with hsp-peptide conjugates in the absence of adjupation in the binding, folding, and unfolding of proteins vants. This finding is interesting because antibodies to hsp [32], and through their participation in the translocation of molecules have been reported to occur in individuals proteins through the membranes and targeting to cellular naturally exposed to a wide variety of infections, such as organelles [33,34], after endocytosis of the hsp-peptide and malaria 142, 431, filariasis 144, 451, schistosomiasis 1461, hsp-oligosaccharide conjugates. It remains to be deter- American trypanosomiasis 1471, leprosy [48, 491, candidiamined whether or not these (and/or other) mechanisms sis [SO], and others. Our data suggest that the presence of intervene in the helper effect provided by the hspR70 in the these anti-hsp antibodies, even in the case they recognize absence of adjuvants in this model of immunization. the conserved regions of the hsp molecules 1511,would not However, regardless of the underlying mechanisms, the interfere with the carrier effect provided by hsp, if they helper effet of anti-peptide and anti-oligosaccharide IgG were employed in vaccine constructs, at least under the antibody production, the long persistence of the antibodies conditions used in the present work. induced, and the lack of requirement for a previous priming with live BCG, make this approach of immunization While keeping in mind the risk of inducing immune particularly interesting in the potential development of responses against self antigens, due to homologies existing between microbial and human hsp [.52],the potent carrier vaccine strategies. effect of mycobacterial hsp, which is exerted in the absence The potential use of hsp as carrier molecules was further of adjuvants, makes this approach of immunization particsupported by the finding that the adjuvant-free carrier ularly interesting for the development o f vaccine strategies. effect of mycobacterial hsp was also exerted on the Data presented in this report, together with the successful production of specific antibodies against oligosaccharide immunization of non-human primates with hsp-peptide antigens. In fact, anti-MenC oligosaccharide antibodies conjugates given intradermally (manuscript in preparawere induced in the absence of adjuvants at titers similar to tion), the widespread exposure to microbial hsp (through those obtained with a conjugate vaccine given in aluminium natural infections or BCG vaccination), which may trigger hydroxide, and consisting of the MenC oligoccharide the priming of specific Tcells, and the high frequency of conjugated to CRM 197, representing the nontoxic form of hsp-reactive T cells even in apparently healthy individuals the diphtheria toxin 13.51. It is known that in infections due [.53], may speak in favor of the feasibility of such an to capsular bacteria protective immunity is mediated by approach. antibodies directed against capsular polysaccharides. Much work has recently been done towards the development of polysaccharidic vaccines, consisting of the polysaccharide moiety chemically conjugated to protein molecules, such as TT, diphtheria toxoid or others, in order to increase their Receivcd Deccmber 13, 1991: in revised form February 10. immunogenicity in very young children [4]. Some of these 1992.

1372

C. Barrios, A. R. Lussow, J.Van Embden et al.

Eur. J. Immunol. 1992. 22: 1365-1372

5 References 1 Ada, G. L., in: Paul, W. E. (Ed.), Fundamental Immunology, 2nd edn., Raven Press, New York, 1989, p. 985. 2 Milich, D. R., Adv. Immunol. 1989. 45: 195. 3 Lussow, A. R., Aguado, M. T., Del Giudice, G. and Lambert, I?-H., Immunol. Lett. 1990. 25: 255. 4 Robbins, J. B. and Schneerson, R., J. Infect. Dis. 1990. 161: 821. 5 Nussenzweig,V and Nussenzweig, R. S., Adv. Immunol. 1989. 45: 283. 6 Lachmann, I? J., Strangeways, L.,Vyakarmam, A. and Evan, G., Ciba Found. Symp. 1986. 119: 25. 7 Lussow, A. R., Del Giudice, G. , RCnia, L., Mazier, D., Verhave, J. I?, Verdini, A . S., Pessi, A., Louis, J. A. and Lambert, P.-H., Proc. Natl. Acad. Sci. USA 1990. 87: 2960. 8 Del Giudice, G., Cooper, J. A. , Merino, J.,Verdini, A . S., Pessi, A.,Togna, A . R., Engers, H. D., Corradin, G. and Lambert, I?-H., J. Immunol. 1986. 137: 2952. 9 Togna, A. R., Del Giudice, G . ,Verdini, A. S., Bonelli, F., Pessi, A , . Engers, H. D. and Corradin, G., J. Immunol. 1986. 137: 2956. 10 Good, M. F., Berzofsky, J. A , , Maloy,W. L., Hayashi,Y., Fujii, N., Hockmeyer, W. and Miller, L. H., J. Exp. Med. 1986.164: 655. 11 Lussow, A. R., Barrios, C. ,Van Embden, J. ,Van der Zee, R., Verdini, A. S., Pessi, A , , Louis, J. A. , Lambert, I?-H. and Del Giudice, G., Eur. J. Immunol. 1991. 21: 2297. 12 Young, D. B. and Garbe, T. R., Infect. Immun. 1991. 59: 3086. 13 Gheorghiu, M., Chambon, L., Serie, C. and Juvin-Calmette, D., Dev. Biol. Stand. 1977. 38: 29. 14 Gheorghiu, M. and Lagrange, I?, Ann. Immunol. (Inst. Pasteur) 1983. 134: 125. 15 Esposito, G., Pessi, A. and Verdini, A. S., Biopolymers 1989. 28: 225. 16 Grillot, D., Michel, M., Miiller, I., Tougne, C., RCnia, L., Mazier, D., Corradin, G. , Lambert, I?-H., Louis, J. A. and Del Giudice, G . , Eur. J. Immunol. 1990. 20: 1215. 17 Thole, J. E., Dauwerse, H. G. , Das, I? K., Groothuis, D. G ., Schouls, L. M. and Van Embden, J. D., Infect. Immun. 1985. 50: 800. 18 Van Eden, W., Thole, J.,Van der Zee, R., Noordzij, A ., Van Embden, J., Hensen, E . and Cohen, I., Nature 1988. 331: 171. 19 Thole, J. E.,VanSchooten,W. C., Keulen,W. J.,Hermans, RW., Janson, A. A., DeVries, R. R., Kolk, A. H. and Van Embden, J. D., Infect. Immun. 1987. 55: 1466. 20 Mehlert, A. and Young, D. B., Mol. Microbiol. 1989. 3: 125. 21 Pappenheimer, A. ML., Jr., Uchida, T. and Harper, A. A , , Immunochemistry 1972. 9: 123. 22 United States Pharmacopeia, The National Formularx XXII edn., United States Pharmacopeial Convention, Inc., Rockville 1990, p. 1542. 23 Frasch, C. E. in: Mizrahi, A. (Ed.), Advancesin Biotechnolog-

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

ical Processes: Bacterial Vaccines Wiley-Liss, Inc., New York 1990, p. 123. Habeeb, A. F. S. A., Anal. Biochem. 1966. 14: 328. Svennerholm, L., Biochim. Biophys. Acta 1957. 24: 604. Hestrin, S. J., J. Biol. Chem. 1949. 180: 249. Hill, M., Bechet, J. J. and d’Albis, A ., FEBS Lett. 1979. 102: 282. Lowry, 0. H., J. Biol. Chem. 1951. 193: 265. Del Giudice, G . ,Verdini, A. S., Pinori, M., Pessi, A.,Verhave, J. P. ,Tougne, C., Ivanoff, B., Lambert, I?-H. and Engers, H. D., J. Clin. Microbiol. 1987. 25: 91. Etlinger, H. M., Gillessen, D., Lahm, H.-W., Matile, H., Schonfeld, H.-J. and Trzeciak, A , , Science 1990. 249: 423. VanBuskirk, S., Crump, B. L., Margoliash, E. and Pierce, S. K., J. Exp. Med. 1989. 170: 1799. Pelham, H. R. B., Cell 1986. 46: 959. Deshaies, R. J., Koch, B. D.,Werncr-Washburne,M., Craig, E. A. and Schekman, R., Nature 1988. 332: 800. Chirico,W. J.,Wasters, M. G . and Blobel, G., Nature 1988.332: 805. Giannini, G., Rappuoli, R. and Ratti, G ., Nucleic Acids Res. 1984. 12: 4063. Ward, J., Vaccine 1991. 9 (Suppl.): 17. Herzenberg, L. A., Tokuhisha, T. and Herzenberg, L. A.. Nature 1980. 285: 664. Schutze, M. P., Leclerc, C.. Jolivet, M., Audibert, F. and Chedid, L., J. Immunol. 1985. 135: 2319. Jacob, C. O., Arnon, R. and Sela, M., Mol. Immunol. 1985.22: 1333. Lise, L. D., Mazier, D., Jolivet, M., Audibert, F., Chedid, L. and Schlesinger, D., Infect. Immun. 1987. 55: 2658. Peeters, C. C. A. M.,Tenbergen-Meekers, A.-M., Poolman, J. T., Beurret, M., Zegers, B. J. M. and Rijkers, G. T., Infect. Immun. 1991. 59: 3504. Mattei, D., Bensaude, O., Dubois, I? and Alexandre, C. 0. I?, Bull. SOC.Franc. Parasitol. 1990. 8: 615. Kumar, N., Zhao,Y., Graves, I?, Perez Folgar, J., Maloy, L. and Zheng, H., Infect. Immun. 1990. 58: 1408. Rothstein, N. M., Higashi, G.,Yates, J. and Rajan,T.V., Mol. Biochem. Parasitol. 1989. 33: 229. Selkirk, M. E., Denham, D. A , , Partono, F. and Maizels, R. M., J. Immunol. 1989. 143: 299. Moser, D., Doumbo, 0. and Klinkert, M.-Q., Parasite Immunol. 1990. 12: 341. Engman, D. M., Dragon, E. A. and Donelson, J. E., J. Immunol. 1990. 144: 3987. Britton, W. J., Hellqvist, L., Basten, A . and Raison, R. L., J. Immunol. 1985. 135: 4171. McKenzie, K. R., Adams, E., Britton,W. J., Garsia, R. J. and Basten, A., J. Immunol. 1991. 147: 312. Ivanyi, L. and Ivanyi, J., J. Infect. Dis. 1990. 162: 519. Mazier, D. and Mattei, D., Springer Semin. Immunopathol. 1991. 13: 37. Winfield, J. B. and Jarjour, W. N., Curr. Top. Microhiol. Immunol. 1991. 167: 161. Munk, M., Schoel, B., Modrow, S., Karr, R.,Young, R. and Kaufmann, S., J. Immunol. 1989. 143: 2844.