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Laboratori di Ricerca-Area
*
Infettivologica and
Biotecnologie e Tecnologie Biomediche,
Dipartimento di Malattie Infettive, and
Servizio di Virologia, IRCCS Policlinico San Matteo and University of Pavia, Pavia, Italy;
¶ Istituto di Ricerche di Biologia Molecolare "P. Angeletti," Pomezia, Italy; and
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Liver Diseases Section, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
"Modelli per lo sviluppo di vaccini contro il virus dell’epatite C ed il micobatterio della tubercolosi," and Progetto di Ricerca Corrente 3180/98. Grant support was also provided by Schering-Plough (Italy), Fondazione Oretta Bartolomei Corsi (Firenze, Italy), and Ministero dell’Università e della Ricerca Scientifica e Technologica-Cofin no. MM06261448_003.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The importance of the environmental selection pressure has been recently confirmed by comparing the stability of HVR1 as a function of time in humans and primates. In agreement with the weak anti-HVR1 responses detected in chimpanzees, HVR1 sequence diversity was significantly lower in this setting compared with those in humans (12)
That being stated, the biological relevance of HVR1 is still unclear.
Several studies identified binding sites for allegedly neutralizing Ab
in this region (13, 14), and it is therefore conceivable
that HVR1 expressed on integral HCV particles could represent a major
target for immune responses that may play an important role in the
outcome of HCV infection. In this study, we have generated mAbs by
immunizing mice with peptides derived from an HVR1 consensus profile,
accounting for 
80% of the total sequence variability
(15), and we characterized such mAbs in terms of fine
specificity and ability to bind to HVR1 sequences expressed in the
context of correctly folded chimeric E2-HVR1 proteins and on
recombinant and natural bona fide viral particles. The long-term
objective was to analyze in detail the features of the humoral immune
responses elicited by peptides that are thought to represent efficient
antigenic and immunogenic mimics of natural HVR1 sequences
(15) and that may be considered as potential components of
a synthetic vaccine inducing broad specificity for several HCV
variants.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HVR1 surrogate peptides (mimotopes) were obtained by
constructing an HVR1 library by back-translation of an HVR1 consensus
profile, accounting for 80% of the total sequence variability, into
the corresponding nucleotide sequence (15). A degenerate
synthetic oligonucleotide was cloned as a fusion to the gene encoding
for the major coat protein (pVIII) in a phagemid vector for display on
M13 bacteriophage. Phage were subjected to two rounds of affinity
selection using sera from anti-HCV-positive patients. A number of
clones reacting exclusively with HCV sera were identified, with the
best clones reacting with 80% of the sera (15). Nine
HVR1 mimotopes were synthesized as multiple antigenic peptides (MAPs)
(16) and used for immunization as described below (Table I
).
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Immunization
Four- to 7-wk-old female BALB/c mice (Charles River Italia, Como, Italy) were immunized i.p. with 100 µl immunogen at wk 0, 3, and 7 and bled on day 0 (prebleed) and 10 days after each injection. MAPs used as immunogens were dissolved in PBS at a concentration of 400 µg/ml and injected as a 1/2 dilution in either CFA (first injection) or IFA (booster).
Splenocytes were taken from the immunized mice with the highest titers
3 days after a booster injection (i.v., without any adjuvant) at wk 10.
Two mice, M18 and M22, were used for the production of mAbs. M18 was a
control mouse immunized with MAP 313, which in preliminary experiments
was found to be reactive with <15% of anti-HCV positive sera. M22
was instead immunized with a pool of all nine MAPs (Table I
), one of
which (MAP 455) was recognized by >65% of anti-HCV-positive sera,
to maximize the repertoire of the Ab response to HVR1.
Construction and expression of chimeric glycoproteins
Natural HVR1 sequences were selected from databases as
previously described (15). They were cloned into
V1JnsTPA-
E2 plasmid, containing the E2 coding region of a genotype
1b isolate (N strain) (17) devoid of HVR1 (amino acids
411–684) as previously described (18). Briefly, for each
clone, a forward primer (with a PacI site at the 5' end) and
a reverse primer were synthesized. Together, these primers encompassed
all the HVR1 sequence to be cloned and had a central overlapping region
of 19 nucleotides. The forward and reverse primers were diluted in 1x
Klenow buffer (Biolabs, Northbrook, IL) at a final concentration of 20
pmol/µl. For the annealing, the mixture of primers was boiled for 2
min and then left at room temperature until cool. Fill-in was performed
by adding 1 U DNA polymerase I Klenow fragment (Biolabs, 210S) per
microgram of DNA and incubating for 20 min at room temperature. The
fragment was purified, digested with PacI, and cloned into
the PacI site of V1JnsTPA-E2 vector.
Large quantities of DNA were prepared using Qiagen 2500-Tip columns (Plasmid Mega kit, catalog no. 12183; Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
The different clones were expressed by transient transfection of human
embryonal kidney 293 cells using the calcium phosphate method (Calcium
Phosphate Mammalian Cell Transfection kit, catalog no. 2-463335;
5'
3', Boulder, CO). Cell extracts were harvested, and the amount of
E2 was evaluated as described by Yagnik et al. (19).
Production of HVR1-specific mAbs
The mAbs were essentially produced as originally described by Köhler and Milstein (20) by fusing splenocytes from immunized animals with the non-Ig-secreting, hypoxanthine guanine phosphoribosyltransferase-deficient murine myeloma cell line Sp2/0-Ag14. Following selection on hypoxanthine-aminopterin-thymidine, cells were expanded, and anti-HVR1 secreting hybrids were cloned by limiting dilution at 5, 1, and 0.5 cell/well in complete medium (RPMI 1640, 4 mM L-glutamine, 2 mM sodium pyruvate, 1% nonessential amino acids, and 10% FBS) containing 20% hybridoma cell growth supplement (ICN Biomedicals, Irvine, CA). After positive identification of specific Ab-secreting cultures, hybridomas were subjected to at last two subcloning cycles at 0.5 cell/well and further characterized in terms of Ig production, subclass, and fine specificity as described below.
ELISA
Two different MAPs were used for screening hybrid cultures. MAP
313 was used to screen cultures derived from M18 (immunized with the
same MAP), whereas MAP 455, which was previously shown to be highly
cross-reactive (15), was used for screening M22 (immunized
with a pool of MAPs including 455). Cultures testing negative upon
screening with MAP 455 were also examined for binding to the remaining
eight mimotopes not used for preliminary analysis. Assay conditions
were essentially as previously described (9) with
modifications as a function of the Ag used. Specifically, MAPs were
coated onto 96-well microplates at a concentration of 10 µg/ml in 50
mM bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. To
detect binding to peptides derived from natural HVR1 isolates or to
determine the fine specificity of mAb binding, biotinylated 27-, 16-,
and 14-mers were added to streptavidin-coated ELISA plates exactly as
previously described (9). Coating of ELISA plates with
Galantus nivalis lectin (GNA; Sigma, St. Louis, MO) was
required for adsorption of E2 chimeric glycoproteins. Briefly, GNA (1
µg/well in PBS) was incubated overnight at 4°C, and after
saturation of nonspecific binding sites with PBS, 2.5% BSA, and 0.1%
Tween 20 followed by several washing steps, 5 µl supernatant from
cells transiently transfected with the V1JnsTPA-E2 vector described
above was added in a 1/20 dilution to GNA-coated wells. E2 chimeras
were allowed to bind for 2 h at room temperature under
agitation.
After washing, 100 µl hybridoma culture supernatants were added to Ag-coated microplate wells and incubated for 2 h at room temperature. After several washing steps with PBS-0.05% Tween 20 and an additional 1-h incubation at room temperature with appropriately diluted HRP-conjugated goat anti-mouse Ig (Sigma) followed by washing, the reaction was developed with o-phenylenediamine (DAKO, Copenhagen, Denmark) as substrate. Absorbance values were read at 492 nm.
Characterization of Ig secreted by HVR1-specific hybridomas
To determine the Ig class secreted by hybridoma cultures, mAbs
were captured with HVR1 MAPs bound to solid phase and identified with
anti-IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 H chains and
anti-
and anti-
L chains (Sigma) as previously described
(21). Ig concentrations in hybridoma supernatants were
determined by quantitative ELISA as previously described
(21). Standards were obtained from commercially available
mouse myeloma cell lines (Sigma).
Competitive inhibition of mAb binding to HVR1 peptides by anti-HCV-positive human sera
Hybridoma supernatants were diluted in PBS-BSA to adjust the
specific mAb concentration to give an A492
reading of 1.0. Streptavidin-coated ELISA plates were incubated with
a number of biotinylated 16-mers derived from naturally occurring HVR1
sequences as previously described (9). Human sera
(n = 4) from patients with chronic HCV infection were
diluted 1/10 in PBS containing 0.05% Tween 20 and added to the wells
for 5 min at 37°C as previously described (22). Murine
mAb 3C7-C3, which displayed promiscuous binding to several HVR1
peptides (this article), was subsequently added and incubated for
1 h at 37°C. Following extensive washing, goat anti-mouse Ig
was dispensed in each well, and the reaction was developed as described
above. Twelve sera from HCV-seronegative healthy subjects served as
controls. Competition was considered significant if reduction of mAb
binding to HVR1 peptides was >28%, 3 SD above the mean (9%)
percentage of inhibition exerted by control sera.
Binding of mAbs to rHCV-like particles (LP)
HCV-LP were synthesized in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural region, including core, E1, and E2, and partially purified as previously described (23). HCV-LP were prepared from strains H (genotype 1a) (24) and J (genotype 1b) (25). The binding of mAbs to HCV-LP was determined by ELISA as previously described (26) with minor modifications. Briefly, HCV-LP were incubated overnight at 4°C on GNA-coated 96-well microplate (Maxisorp, Nalge-Nunc International, Roskilde, Denmark) prepared as described above at a concentration of 100 µg/ml in PBS and 10% glycerol to avoid possible denaturation. In some experiments, HCV-LP were denatured by boiling for 5 min in PBS-glycerol containing 0.1% SDS. Wells were blocked with PBS containing 4% goat serum (Sigma), 10% glycerol, and 5% skimmed dry milk (Sigma) for 3 h at room temperature. After several washing steps with PBS and 10% glycerol, mAb supernatants were added undiluted to the wells and incubated at 37°C for 1 h. After being washed with PBS glycerol, the wells received 100 µl HRP-conjugated anti-mouse Ig (Sigma) diluted 1/10,000 in PBS-glycerol-skimmed dry milk. The reaction was then developed with o-phenylenediamine, and OD was determined at 492 nm.
Binding of HVR1-specific mAbs to bona fide HCV particles
Magnetic beads coated with rat anti-mouse IgG1 mAb (Dynabeads M-450, Dynal, Oslo, Norway) were used to capture circulating bona fide HCV particles as follows. Beads were washed three times with PBS and 0.1% BSA, and 2 mg was incubated with 2 µg purified mAb 3C7-C3 in 2 ml PBS-BSA. Control beads received buffer alone. Following incubation for 45 min at 4°C, beads were exposed to a magnet for 10 min, and excess mAb was removed by aspiration and washing five times with 3 ml PBS-BSA after exposure to a magnet each time for 7 min. Serum from a viremic (HCV RNA titer, 3.7 x 107 GEq/ml by branched chain DNA assay; Quantiplex HCV RNA 2.0 assay; Chiron, Emeryville, CA) immunosuppressed patient infected with genotype 2c and containing no detectable Abs to several HVR1 peptides was diluted 1/20 in PBS-BSA and incubated with the beads at 37°C for 90 min under rotation. Beads were again washed five times with PBS-BSA by exposing the mixture each time to 1 ml PBS- BSA, transferred to an Eppendorf tube, and centrifuged at 10,000 x g for 20 min at room temperature. The supernatant was discarded, and the pellet containing magnetic beads was processed for HCV RNA detection by PCR as follows. Viral RNA was extracted using the QIAmp Viral RNA kit (Qiagen) with minor modifications. Specifically, after addition of AVL buffer (containing guanidinium thiocyanate), samples were left on the bench for 10 min at room temperature and then vortexed. The procedure was repeated three times. After centrifugation at 10,000 x g for 1 min, the supernatant was subjected to a standard extraction procedure and amplification by a two-round nested RT-PCR as reported previously (27). First- and second-round PCR products were subjected to electrophoresis on a 2% agarose gel containing ethidium bromide. The lengths of the first and second round PCR products were 289 and 235 bp, respectively.
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Results |
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, four secreted IgM, two
secreted IgA, and in one, the Ig isotype could not be characterized. To
avoid the possibility that screening with a single mimotope (455) could
select for a dominant epitope present on HVR1, cultures negative upon
initial screening with MAP 455 were tested for binding to the remaining
eight MAPs included in the immunizing pool. Only two IgM-secreting
hybridomas showing weak or no significant reactivity for MAP 455 were
reactive with MAP 441 and 443, further attesting to the promiscuous
reactivity of MAP 455 synthesized on the basis of the HVR1 consensus
profile (15). MAP 313 was only weakly reactive with the
majority of mAbs as previously described for human sera
(15). The mAb titer was determined by end-point dilution
for selected IgG- and IgA-secreting hybridomas after normalizing the Ig
concentration to 0.5 µg/ml. Typical titers determined on MAP 455
varied from 1/8 to 1/65,000. Although there was a trend for low titer
mAbs to show limited reactivity with the different 16- and 27-mers used
in the experiments, there was no correlation between mAb titers and
cross-reactivity for HVR1 variants (data not shown). Murine mAb reactivity with peptides derived from natural HVR1 isolates
To evaluate the potential of HVR1 mimotope-specific mAbs to
recognize natural HVR1 sequences we used panels of biotinylated
peptides synthesized from the deduced amino acid sequences of several
HCV isolates obtained from patients with acute and chronic HCV
infection (9, 15). The analysis was restricted to IgG- and
IgA-secreting hybridomas, because IgM showed high background binding to
irrelevant peptides (data not shown). As shown in Fig. 1, mAbs showed significant
cross-reactivity with a number of 16- and 27-mers, confirming the
findings reported previously using polyclonal sera from patients
(9) and animals (3) and providing additional
evidence in favor of recognition of residues with conserved
hydropathicity profile that confer a remarkable conformational
conservation to HVR1, as recently suggested (28). The
relevance of the sequence recognized by our murine mAb panel was
further supported by competitive inhibition studies in which mAb
binding to HVR1 peptides was almost always significantly reduced by
preincubation with sera from patients with HCV infection, but not with
control sera (Fig. 2).
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To determine the fine specificity of our mAbs, we used a panel of
overlapping 14-mers, scaled by one amino acid residue, and covering the
entire HVR1 sequence (amino acids 384–410). Peptides were synthesized
on the basis of the HVR1 consensus profile previously identified
(15). As shown in Fig. 3, all but one of those mAbs for which a PEPSCAN analysis could be
performed (15 IgG and two IgA) recognized a C-terminal subregion of
HVR1 broadly defined by amino acid residues 390–410. One mAb (1D5-F10)
showed restricted binding to residues 384–398 at the amino-terminal
end. These findings are entirely compatible with previous studies from
our group suggesting the existence of an immunodominant epitope on the
HVR1 C-terminal subregion recognized by human sera (9).
The inability to map the fine specificity of four of our mAbs may have
several explanations. First, epitope mapping of these mAbs may be
difficult using shorter peptides. Second, the overlapping 14-mers were
designed on the basis of a highly cross-reactive HVR1 sequence
expressed in MAP 455 and linear 27-mer 539, which, however, cannot be
representative of all HVR1 variants. Third, the peptides showing little
or no reactivity in the fine-mapping experiments were also poorly
reactive with the panel of 16- and 27-mers, and this may be the most
plausible explanation.
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To examine whether our mAbs could bind to HVR1 expressed in its
correct conformation as part of the complete E2 envelope glycoprotein
we constructed chimeric E2-HVR1 glycoproteins in which the HVR1 moiety
was identical with that expressed as linear peptide (27-mer). In these
constructs the E2 moiety was derived from the HCV N strain (genotype
1b). In addition, several other soluble E2 proteins derived from
natural isolates containing HVR1 and truncated at position 661 (Table II) were used in binding experiments as
described above. As illustrated in Fig. 4, virtually all mAbs were able to bind
to peptide 539 and its matched chimera E2-F78. This was not unexpected,
because 539 is a broadly reactive linear peptide version of MAP 455
used for the immunization protocol. One mAb (3C7-C3) efficiently bound
to several E2-HVR1 chimeras/linear peptides pairs, including
peptide 730 and H661 (a native truncated E2 protein derived from HCV
strain H), suggesting promiscuous recognition of a
conformation-independent, conserved sequential epitope expressed in the
context of a correctly folded E2 glycoprotein (Fig. 4). Such an epitope
was also present on the natural E2 glycoprotein H661 derived from HCV
strain H (24) (genotype 1a), but not on E2 from genotype
1b strains BK (29), J (25), and N
(17) (a linear peptide from N was recognized by mAbs
2B4-B6 and 2B4-G6). Recognition was limited to the linear peptide only
in three instances, whereas three chimera/peptide pairs were
nonreactive. Three additional mAbs showed only limited reactivity for
the 267/E2–267 peptide/chimera pair.
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To determine whether our mAbs could recognize HVR1 expressed on
viral particles we used HCV-LP, constituted by HCV structural proteins
assembled in insect cells, which provide a good surrogate model of
native HCV particles. Care was applied to avoid denaturing of such
particles, which could expose hidden epitopes that would normally not
be accessible to Ab. The entire panel of IgG- and IgA-secreting mAbs
was screened for binding to HCV-LP derived from genotype 1a and 1b
isolates. The mAb 3C7-C3, which showed promiscuous binding to several
E2-HVR1 glycoproteins, showed consistent and reproducible binding
to HCV-LP from genotype 1a, but not to HCV-LP from genotype 1b (Fig. 6). Binding was completely abolished by
preabsorption of 3C7-C3 to magnetic beads coated with linear 27-mer 539
derived from the HVR1 consensus sequence but not when beads were coated
with an irrelevant 27-mer. A control mAb (7C2) specific for
anti-apolipoprotein A1 (a gift from Dr. V. Bellotti, University of
Pavia, Pavia, Italy) failed to bind to HCV-LP. None of the remaining
mAbs were able to bind to either HCV-LP 1a or 1b. Interestingly,
denaturation significantly increased the binding efficiency of 3C7-C3
to HCV-LP 1a, probably as a result of better exposure of the relevant
epitope, but had no effect on binding to HCV-LP 1b (Fig. 6). None of
the mAbs that failed to bind to integral HCV-LP was able to recognize
HCV-LP 1a or 1b under denaturing conditions (data not shown).
Collectively, these findings indicate that HVR1 is expressed on rHCV
particles and that such region may be accessible to Ab, although the
relevant epitope may often not be completely exposed on integral viral
particles in a conformation recognizable by Ab.
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As an extension of the experiment described above, we asked
whether mAb 3C7-C3 could capture bona fide circulating HCV particles by
determining the presence of HCV RNA bound to magnetic beads coated by
HVR1-specific mAbs or control mAbs. As shown in Fig. 7, only mAb 3C7-C3 could efficiently
capture HCV RNA, whereas control beads coated with control mAb 7C2
showed no specific signals, providing further evidence in support of
the contention that HVR1 is indeed expressed on native HCV
particles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility to generate broadly reactive Abs may represent a useful approach to overcome the natural diversity of a virus such as HCV, suggesting that mimotope-based vaccines can be used as potentially effective HCV immunogens. This assumption is based on evidence indicating that Abs to HVR1 can prevent HCV infection in the chimpanzee model (13, 14). However, exposure of HVR1 on complete viral particles has not been formally proven, and in principle, the role of this sequence in binding neutralizing Abs is far from being established. A recent preliminary study suggested that mAbs obtained by immunization with peptides derived from natural HVR1 isolates were able to capture bona fide viral particles only from homologous HCV isolates and could also prevent infection of an allegedly susceptible cell line in vitro (32). These findings are in partial agreement with our data, in that one of our mAbs was also able to capture bona fide and recombinant viral particles, although there was no apparent genotype- or isolate-specific recognition. Indeed, mAb 3C7-C3 bound to rHCV-LP 1a and was able to specifically capture HCV RNA-positive material from serum of a patient infected with genotype 2c. Although the HVR1 sequence(s) could not be determined in this patient, this finding strongly suggests that the HVR1 sequence(s) from the 2c isolate shared significant homology with the HCV-H (genotype 1a) sequence recognized by the 3C7-C3 capture Ab. This observation fits with the idea of a significant structural conservation of HVR1 as discussed above. Instead, we found that mAb binding to serum HCV particles was difficult to demonstrate using sera from immunocompetent patients with persistent HCV infection, in whom anti-HVR1 Abs are invariably detected (3), presumably as a result of competitive inhibition by circulating Ab.
It may be argued that mAbs raised against HVR1 peptides are unable to recognize the same sequence when expressed in the context of a correctly folded complete E2 glycoprotein that included HVR1. The correct conformation of E2-HVR1 chimeras was previously demonstrated by binding to a conformation-sensitive mAb specific for an epitope expressed on a native prebudding form of the HCV envelope (18, 33), by the presence of a small quantity of disulfide-bridged aggregates (18), and by binding to CD81, which is known to be conformation dependent (18, 34). In this study, we showed that the overwhelming majority of our mAbs could recognize a chimeric E2 polypeptide that expressed the same HVR1 sequence synthesized as linear peptide, which was promiscuously recognized by several human sera and murine mAbs. However, cross-reactivity was confined to a limited number of E2-HVR1 chimeric proteins, and in some instances it was restricted to the homologous linear peptide only, suggesting recognition of a sequential epitope. To this effect, it is interesting to note that only one mAb of our entire panel bound to HCV-LP, and that recognition of the HVR1 region could be improved under denaturing conditions. This finding corroborates the hypothesis of the existence of a major sequential epitope at the C terminus of HVR1, which may, under certain circumstances, be exposed on integral viral particles. The binding pattern of mAb 3C7-C3, which, among other proteins, recognized a truncated natural E2 protein (H661) as well as HCV-LP 1a, both derived from strain H, suggests a possible role for HVR1 in virus neutralization. Evidence in support of this hypothesis comes from experiments demonstrating that a hyperimmune serum raised against an HVR1 peptide from strain H was able to prevent HCV infection in chimpanzees following either in vitro incubation with a pedigreed HCV inoculum (13) or passive immunization in vivo (14).
The mechanism by which anti-HVR1 can modulate HCV infection is a matter of speculation. Current evidence suggests that mammalian cell-derived E2 glycoproteins (35) and, to a lesser extent, HCV-LP 1a (M. Triyatni and T. J. Liang, unpublished observations) can bind human CD81, a candidate receptor molecule for HCV. Yet HCV-LP can penetrate into HepG2 cells via alternative receptor(s) which are constitutively expressed on hepatocytes (M. Triyatni and T. J. Liang, unpublished observations), supporting the hypothesis that HCV requires a second receptor molecule for internalization (35). Preliminary evidence suggests that HVR1 is not involved in binding to CD81 (36, 37), although no information is yet available on HCV envelope regions interacting with the putative alternative receptor(s) expressed on susceptible cells. The unique properties of the structure of HVR1, which contains several positively charged conserved residues, suggest that it is most likely involved in binding to negatively charged compounds, such as glycoaminoglycans, of which cell surface receptors are particularly rich, and/or phospholipids. Therefore, high affinity anti-HVR1 Ab elicited by immunization could modulate HCV infection by inhibiting binding of viral particles to a cellular receptor(s) and/or preventing interaction with plasma low density lipoproteins, which have been shown to bind HCV (38). This approach may have important implications for immunotherapy or prophylaxis of HCV infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mario U. Mondelli, Laboratori di Ricerca-Area Infettivologica, IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. E-mail address: m.mondelli{at}smatteo.pv.it
3 Abbreviations used in this paper: HVR, hypervariable region; HCV, hepatitis C virus; MAP, multiple antigenic peptide; GNA, Galantus nivalis lectin; -LP, -like particles.
Received for publication April 5, 2001. Accepted for publication July 9, 2001.
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