p64k meningococcal protein as immunological carrier for weak immunogens
TRANSCRIPT
P64k Meningococcal Protein as Immunological Carrier for Weak
Immunogens
S. GONZAÂ LEZ,* A. ALVAREZ,* E. CABALLERO,* L. VINÄ A,² G. GUILLEÂ N* & R. SILVA*
*Center for Genetic Engineering and Biotechnology, Havana, Cuba
(Received 21 February 2000; Accepted in revised form 5 June 2000)
GonzaÂlez S, Alvarez A, Caballero E, VinÄa L, GuilleÂn G, Silva R. P64k Meningococcal Protein as
Immunological Carrier for Weak Immunogens. Scand J Immunol 2000:52:113±116
Previously, the P64k meningococcal protein, an antigen of 64 kDa expressed in Escherichia coli, has been
extensively characterized. We have successfully conjugated several synthetic peptides and meningococcal
group C polysaccharide to P64k. In three out of four model peptides, the murine humoral immune response
against the homologous peptide, evaluated after three doses of conjugate, was higher in the animals
immunized with the coupled peptide than in those that received free peptide. The fourth and largest was
immunogenic by itself. Similarly, the antigroup C polysaccharide levels reached by conjugated polysacchar-
ide were signi®cantly higher than those produced against unconjugated polysaccharide. As a carrier for one of
the peptides, P64k was compared with bovine serum albumin (BSA) and tetanus toxoid (TT), being able to
induce slightly higher or similar antipeptide antibody levels than these well-establish protein carriers. Our
results suggest that recombinant P64k protein could be a readily available immunological carrier, as ef®cient
as other commonly used large carrier molecules.
Dr S. GonzaÂlez, DivisioÂn de Vacunas, Centro de IngenieriÂa GeneÂtica y BiotecnologiÂa, Apdo 6162, C. Habana
10600, Cuba. E-mail: [email protected]
INTRODUCTION
Frequently, there is a need of conjugating synthetic peptides and
microbial polysaccharides to protein carriers, to increase their
immunogenicity [1] and/or to turn them in T-cell dependent
antigens [2]. Large proteins, like bacterial toxoids, BSA and
keyhole limpet hemocyanin, that contain suf®cient reactive
groups are widely used for chemical conjugation of peptides
and polysaccharides.
The high molecular weight meningococcal protein P64k has
been expressed as a soluble antigen in Escherichia coli, account-
ing for 20% of the total cell proteins [3]. Pure recombinant
protein, that has been extensively characterized [4,5], was used to
immunize mice, rabbits and monkeys. The protein was found to
be immunogenic in all these species [6]. Moreover, the antigen
seems to elicit antibodies in humans who suffered meningococ-
cal disease [3]. As a result of all these features we decided to
evaluate it as a carrier for weak immunogens and to compare its
performance with the one shown by known protein carriers, like
BSA and tetanus toxoid (TT).
MATERIALS AND METHODS
Immunogens. Recombinant P64k protein was obtained as described
earlier [3]. BSA (Fraction V Powder) was purchased from Sigma
Chemical Co. (St. Louis, MO, USA). TT was kindly donated by the
Division of Formulations of our centre. Four peptides were synthesized
at the Peptide Synthesis Unit of our institute. The peptide sequences are
the following: SP1 from human immunode®ciency virus (HIV) 1 gp120
protein, RQSTPIGLGQALYTT; SP2 from HIV 1 p24 protein, IRQGP
KEPFRDYVDRFYK; SP3 from HCV core protein, PKPQRKTKRN
TNRRPQDVKFPGGGQIVGGVY; and SP4 from HCV NS4 protein,
SGRPAVIPDREVLYQEFDEMEECASHLPYIEQGMQLAEQFKQKA
LGL. Meningococcal serogroup C polysaccharide (Men C) was supplied
by the Instituto Finlay, Havana, Cuba.
Conjugation. The peptides were conjugated to protein carriers by the
glutaraldehyde method, as previously described [7]. After coupling,
peptide-protein conjugates were examined by Sodium Dodecyl Sulfate
ÿ Polyacrylamide Gel Electrophoresis (SDS-PAGE) [8] and protein
concentration was determined using the Lowry's method [9]. Addition-
ally, the conjugates were analyzed by Immunoblot [10], using mono-
clonal or polyclonal antibodies that recognized each particular peptide.
SP1 was conjugated either to BSA, TT or P64k. SP2 to SP4 were coupled
to P64k protein only. Men C was conjugated to P64k via adipic acid
dihydrazide, by using the carbodiimide method, as previously described
[11]. The polysaccharide content in the samples was determined using
the method reported by Svennerholm [12].
Scand. J. Immunol. 52, 113±116, 2000
q 2000 Blackwell Science Ltd
² Present address: Center of Molecular Immunology, PO Box 16040, Havana
11600, Cuba.
Immunizations. In all experiments 5 to 10 female BALB/c mice (8±
10-week-old) per group were immunized. In a ®rst experiment, three
doses (20 mg each) of either SP1-BSA, SP1-P64k, BSA, P64k, or free
peptide, emulsi®ed with Freund's Adjuvant were subcutaneously (s.c.)
administered, at two-week intervals, to mice divided in ®ve experimental
groups. In a second experiment, mice received three doses (10 mg each)
of SP1-TT, SP1-P64k or free peptide, absorbed to 400 mg of aluminium
hydroxide (Superfos Biosector, Vedbaek, Denmark). The doses were
given s.c. at two-week intervals. A third immunization schedule was
performed with the same antigen amount, adjuvant, route, and time
intervals. Mice were injected with three doses of SP2, SP3 or SP4,
respectively, either free or coupled to P64k. In a fourth experiment, the
animals were s.c. immunized with three doses of either Men C (2,5 mg),
P64k (3 mg) or Men C-P64k (2,5 mg Men C and 3 mg P64k/dose)
absorbed to 100 mg of aluminium hydroxide. In all cases, the immuniza-
tions were given in a total volume of 100 ml on days 0, 14 and 28. Serum
samples were obtained from blood extracted from mice at days 0, 14, 28,
35 and 42.
ELISA. Antibody levels in sera were determined by Enzyme Linked
Immunosorbent Assay (ELISA). To detect antipeptide antibodies, 96-
well plates (High Binding, Costar, USA) were coated with 100 ml/well of
SP1 (20 mg/ml), SP2 (10 mg/ml), SP3 (0.8 mg/ml) or SP4 (1.6 mg/ml) in
carbonate buffer (0.05 M Na2CO3, pH 9.6). Skim-milk powder (3%) was
used as a blocking reagent. Plates were processed as published elsewhere
[13]. Anti-Men C antibodies were measured as previously described
[14]. All sera were analyzed in duplicate. Serum antipeptide and
antipolysaccharide antibody levels were expressed as their absorbance
(492 nm) values in ELISA and used for statistical analysis. Moreover,
pooled sera from each group were titrated by serial dilution, being the
cut-off values calculated as twice the mean absorbance of preimmune
serum.
Statistical methods. The signi®cance of differences between data in
the second and third immunization experiment was determined with the
Student's t-test. Conversely, in the ®rst experiment, the signi®cance of
differences between antibody levels was analyzed by using a split plot
design (simple) with mice as plots, protein carrier as the between
subjects factor (factor A) and time point as within blocks factor (factor
B). In the last immunization routine, Newman±Keuls multiple compar-
ison test was used to determined differences between the data. A P-value
of < 0.05 was considered statistically signi®cant. In the ®gures, bars
represent the mean of antibody levels 6 the standard deviation for each
experimental group. Reciprocal antipeptide or antipolysaccharide titers
of pooled sera corresponding to each experimental group are shown on
the bars.
RESULTS
P64k as a carrier, compared to BSA and TT
In a parallel coupling reaction, the peptide SP1 (15 mer) was
linked ®rst to P64k and BSA. Both conjugates migrated in SDS-
PAGE as a smear of multiple bands, which are mainly concen-
trated in two zones (data not shown). The upper zone included
bands of molecular weight higher than 116 kDa and the lower
zone was composed by molecules weighing between 97 kDa and
66 kDa, respectively. A peptide-speci®c Immunoblot suggested
similar peptide substitution rates for both conjugates. Protein
carriers, conjugates, and free peptide were employed to immunize
the mice. Figure 1(A) re¯ects the time course of antipeptide
antibody levels observed in the groups that received SP1 coupled
to the two protein carriers. As it can be seen, both conjugates
were immunogenic in mice. The level of anti-SP1 antibodies was
slightly higher (with statistical signi®cance) in the group immu-
nized with SP1-P64k than in the group injected with SP1-BSA.
The antibody levels remained negligible, after three doses of
antigen, for the animals immunized either with carrier protein or
free peptide (data not shown).
In a second experiment, the carrier capacity previously found
for P64k was demonstrated by comparing it with that showed by
TT. The conjugate SP1-TT migrated in SDS-PAGE like the
previously described conjugates, but the upper zone contained
most of the molecules. Again, the peptide substitution rates were
estimated to be similar for both conjugates by Immunoblot. Mice
were immunized with SP1 either conjugated to P64k, to TT or
114 S. GonzaÂlez et al.
q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 113±116
Fig. 1. Anti-SP1 antibody levels. (A) Mice (n� 5) were
subcutaneously (s.c.) immunized with three doses (20 mg/dose) of
SP1-P64k or SP1-BSA. (B) Mice (n� 10) were s.c. immunized with
three doses (10 mg/dose) of SP1-P64k or SP1-TT. Anti-peptide
antibody levels in sera are expressed as their absorbance (492 nm)
values in ELISA (Serum dilution: 1: 250). Reciprocal antipeptide titers
of pooled sera corresponding to each experimental group are shown
on the bars.
free, using aluminium hydroxide as an adjuvant. Figure 1(B)
shows the time course of anti-SP1 antibody levels for the two
experimental groups that received conjugates. Both conjugates
elicited similar antipeptide antibodies after the second dose
(P < 0.05), that remained true after a third one. The uncoupled
peptide was not immunogenic in mice, even after three injections
(data not shown).
P64k as a carrier for peptides of variable length
To further study the carrier ability of the recombinant meningo-
coccal protein, we chemically coupled peptides 18 amino acid
residues (aa) (SP2), 31 aa (SP3) and 47 aa long (SP4), respec-
tively, to P64k and injected mice either with the conjugates or
free homologous peptide. Two weeks after the second dose, there
was a signi®cant statistical difference between the group that
received the conjugate and the group immunized with free
peptide (data not shown). Fifteen days after the third dose, the
same difference was observed for SP2 and SP3 (Fig. 2), whereas
similar levels of antipeptide antibodies were found for SP4 free
or coupled to P64k.
P64k as a carrier for Men C polysaccharide
Men C polysaccharide conjugated to P64k elicited antipolysac-
charide antibodies in mice after immunization (Fig. 3). Even
after the ®rst dose, the conjugate was immunogenic. The anti-
body levels continued to increase after the second dose and
remained stable after the third one. There was no signi®cant
statistical difference between data obtained after the second and
third dose of conjugate.
DISCUSSION
In the present study, we have coupled P64k to four peptides and
Men C polysaccharide and administered three doses of conju-
gates to mice. After two doses of peptide coupled to P64k, most
of the mice seroconverted; having the expected response, con-
sidering its high molecular weight and observed immunogeni-
city. Even after three doses of antigen, three of the uncoupled
peptides failed to elicit a signi®cant antibody response. Only SP4
(47 mer), induced antibody levels similar to those produced by
the conjugated peptide, that could be expected owing to its
length. Most probably it contains both T-cell and B-cell epitopes,
having itself the ability to induce a humoral immune response
after repeated immunizations. Owing to the encouraging results
achieved in the ®rst immunization experiment, we decided to
half the antigen amount in the second and third one, obtaining
positive results as well. It is worth noting that P64k can exert its
carrier function absorbed on aluminium hydroxide, one of the
few adjuvants widely used in human vaccines. The antibody
levels against the meningococcal group C polysaccharide were
signi®cantly increased after its conjugation to P64k. Similar
results were obtained by Costantino and coworkers [15], who
coupled Men C to CRM 197 and injected the conjugate in mice
and rabbits. However, MenC±P64k could prime the host for a
T-cell memory response to the pathogen, by employing a carrier
protein derived from the same bacterium.
BSA, as a carrier, is widely used at the laboratory scale
because of its availability and reduced cost. However, its
mammalian origin, has been reported to reduce its ef®ciency
for that purpose [16]. The foreignness of meningococcal P64k
can contribute to its performance in this respect. Frequently, the
haptens have been coupled to TT or cholera toxoid in human
vaccines, because these proteins are readily available and have
been used in humans without side-effects [17]. Nonetheless,
P64k as a Carrier 115
q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 113±116
Fig. 2. Anti-peptide antibody levels. Mice (n� 8) were s.c. immunized
with three doses (10 mg/dose) of free SP2, SP3, or SP4, or the same
amount of the respective P64k conjugate. Antibody levels in sera were
measured against the homologous peptide and are expressed as their
absorbance (492 nm) values in ELISA (Serum dilution 1 : 100).
Reciprocal antipeptide titers of pooled sera corresponding to each
experimental group are shown on the bars.
Fig. 3. Anti-meningococcal group C polysaccharide (Men C)
antibodies. Mice (n� 7) were immunized with three doses of Men C
(2,5 mg/dose), P64k (3 mg/dose), or Men C-P64k (2,5 mg Men C/dose).
Anti-Men C antibody levels in sera are expressed as their absorbance
(492 nm) values in ELISA (Serum dilution 1 : 100). Reciprocal anti-
Men C titers of pooled sera corresponding to each experimental group
are shown on the bars.
owing to the successful development of new conjugate vaccines
and the limited availability of protein carriers, concern is
increasing regarding the epitope overload and suppression that
can take place when the same molecule is used in several
vaccines [18,19]. The meningococcal protein employed by us
can circumvent these drawbacks of known carriers. Recently, a
Phase I clinical trial conducted in healthy human volunteers
showed that P64k is safe and immunogenic after a three-dose
immunization protocol [20].
The recombinant P64k meningococcal protein could be used
in future conjugate vaccines. The T-cell epitopes present in this
protein are under investigation.
ACKNOWLEDGMENTS
We are grateful to Ms Dagmara Pichardo for her excellent work
in the animal care and immunization. We also acknowledge the
help of Dr Carlos Duarte for providing us with peptide SP1 and
helpful advice. Dr Lidia I. Novoa kindly provided us with
peptides SP3 and SP4.
REFERENCES
1 Carter JM. Techniques for conjugation of synthetic peptides to
carrier molecules. In: Dunn BM, Pennington MW eds. Peptide
Analysis Protocols. Totowa, NJ: Humana Press, Inc., 1994:155±91.
2 Robbins JB, Schneerson R. Polysaccharide-protein conjugates: a
new generation of vaccines. J Infect Dis 1990;161:821±32.
3 GuilleÂn G, Alvarez A, Silva R et al. Expression in Escherichia coli of
the lpdA gene; protein sequence analysis and immunological char-
acterization of the P64k protein from Neisseria meningitidis. Bio-
technol Appl Biochem 1998;27:189±96.
4 Li I, Pernot L, Prange T et al. Molecular structure of the lipoamide
dehydrogenase domain of a surface antigen from Neisseria menin-
gitidis. J Mol Biol 1997;269:129±41.
5 GoÂmez R, Madrazo J, GonzaÂlez J et al. Functional and structural
characterization of the recombinant P64k protein of Neisseria
meningitidis. BiotecnologiÂa Aplicada 1999;16:83±7.
6 GuilleÂn G, Silva R et al. Cloning and expression in E. coli of a high
molecular weight outer membrane protein (PM6) from the Neisseria
meningitidis strain B. 4: P1.15 (Cuban isolate). Evaluation of the
immunogenicity and bactericidal activity of antibodies raised against
the recombinant protein. In: Conde-Glez CJ et al. eds. Pathobiology
and Immunobiology of Neisseriaceae. Cuernavaca: Instituto Nacio-
nal de Salud PuÂblica, 1994:834±40.
7 Hancock D, Evan G. Synthesis of peptides for use as immunogens.
In: Manson M. eds. Immunochemical Protocols. Totowa, NJ:
Humana Press, Inc., 1992:23±32.
8 Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature (London) 1970;227:680±5.
9 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measure-
ment with the Folin phenol reagent. J Biol Chem 1951;193:265±75.
10 Towbin H, Gordon J. Immunoblotting and dot immunobinding ÿ
current status and outlook. J Immunol Meth 1984;72:313±40.
11 Bartoloni A, Norelli F, Ceccarini C, Rappuoli R, Costantino PA.
Immunogenicity of meningococcal B polysaccharide conjugated to
tetanus toxoid or CRM197 via adipic acid dihydrazide. Vaccine
1995;13:463±7.
12 Svennerholm L. Quantitative estimation of sialic acids. Biochim
Biophys Acta 1957;24:604±11.
13 ExpoÂsito N, Mestre M, Silva R et al. Preformulation study of the
vaccine candidate P64k against Neisseria meningitidis. Biotechnol
Appl Biochem 1999;29:113±9.
14 Andersen J, Berthelsen L, Lind I. Measurement of antibodies against
meningococcal capsular polysaccharides B and C in enzyme-linked
immunosorbent assay: towards an improved surveillance of menin-
gococcal disease. Clin Diag Lab Immunol 1997;4:345±51.
15 Costantino P, Viti S, Podda A, Velmonte MA, Nencioni L, Rappuoli
R. Development and phase 1 clinical testing of a conjugate vaccine
against meningococcus A and C. Vaccine 1992;10:691±8.
16 Geerligs HJ, Weijer WJ, Welling GW, Welling WS. The in¯uence of
different adjuvants on the immune response to a synthetic peptide
comprising amino acid residues 9±21 of herpes simplex virus type 1
glycoprotein D. J Immunol Meth 1989;124:95±102.
17 Peeters CCAM, Tenbergen-Meekes AM, Poolman JT, Beurret M,
Zegers BJM, Rijkers GT. Effect of carrier priming on immuno-
genicity of saccharide-protein conjugate vaccines. Infect Immun
1991;59:3504±10.
18 Fattom A, Cho YH, Chu C, Fuller S, Fries L, Naso R. Epitopic
overload at the site of injection may result in suppression of the
immune response to combined capsular polysaccharide conjugate
vaccines. Vaccine 1999;17:126±33.
19 Herzenberg LA, Tokuhisa T, Herzenberg LA. Carrier-priming leads
to hapten-speci®c suppression. Nature 1980;285:664±7.
20 Silva R, GonzaÂlez S et al. Safety and preliminary immunogenicity
data of the recombinant protein P64k of Neisseria meningitidis in
human volunteers. In: Gavilondo JV, Ayala M, Acevedo B. eds.
Proceeding of BiotecnologiÂa Havana 99, Medical Applications of
Biotechnology. La Habana: Elfos Scientiae, 1999:O18.
116 S. GonzaÂlez et al.
q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 113±116