1 Institut für Immunologie, Bundesforschungsanstalt für Viruskrankheiten der Tiere, Paul-Ehrlichstr. 28, D-72076 Tübingen, Germany
2 EMC microcollections GmbH, Sindelfinger Str. 3, D-72070 Tübingen, Germany
3 Bayer Animal Health, Building 6210, D-51368 Leverkusen, Germany
Correspondence
Armin Saalmüller
armin.saalmueller{at}tue.bfav.de
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ABSTRACT |
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Present address: Miltenyi Biotec, Friedrich-Ebert-Str. 68, D-51429 Bergisch Gladbach, Germany.
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INTRODUCTION |
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Current FMD vaccines are produced by infecting cultured cells with virulent FMDV, followed by inactivation and purification of the newly synthesized virus particles. Whereas the resulting conventional vaccines have been applied successfully for decades, they have a number of disadvantages. Handling FMDV is inherently hazardous and necessitates high security facilities (Barteling & Vreeswijk, 1991). To ensure reliable inactivation, FMD vaccine plants need a special design and complex operating procedures that have to be strictly monitored. A conventional FMD vaccine is a biological product that is very difficult to standardize. To ensure the required potency, animal experiments are necessary, not only for vaccine registration but also for batch control (Barteling & Vreeswijk, 1991
). In addition, vaccines will quickly lose potency without a consistent cold chain from the producer to the veterinarian in the field. Furthermore, protection induced by conventional vaccines is limited mostly to one or a few topotypes within one serotype (Pereira, 1981
). New strains are constantly evolving in endemically infected countries, making it difficult for vaccine producers to keep pace. Protection is short-lived, often making frequent revaccination indispensable for an effective disease control (Barteling & Vreeswijk, 1991
). Whereas conventional vaccines can prevent clinical signs and further spread of disease in vaccinated populations, they are not able to protect virus-exposed animals from becoming persistently infected virus carriers.
To overcome these problems, a lot of work was performed on alternatives to conventional vaccines. Synthetic peptides were early found to be promising vaccine candidates (Pfaff et al., 1982; DiMarchi et al., 1986
; Doel et al., 1990
). Major antigenic regions of the virus were found on the structural protein 1D (Pfaff et al., 1982
; Strohmaier et al., 1982
; Acharya et al., 1989
; Bittle et al., 1982
). The antigenic site comprised of aa 140160 of 1D (containing the receptor-binding site of the virus) is usually referred to as site A, whereas site C contains aa 200213 of 1D. In initial experiments, peptide vaccines consisting of site A or of constructs combining sites A and C were used. Whereas these vaccines induced considerable titres of neutralizing antibodies in guinea pigs, cattle and swine and sometimes also good protection in vivo (Pfaff et al., 1982
; Bittle et al., 1982
; DiMarchi et al., 1986
; Morgan & Moore, 1990
), in general, their immunogenicity was lower than that of conventional vaccines. This may be due to the now widely accepted thesis: that the immune response against FMDV is not only B-cell but also T-cell dependent (Domingo et al., 1990
; Collen et al., 1991
; Brown, 1992
; Collen, 1994
; Sobrino et al., 1999
). Because sites A and C contain mainly B-cell epitopes, in a recent study these immunogenic sites were combined with a further 1D peptide containing a T-cell epitope (Taboga et al., 1997
). This resulted in a minor increase in protection rates.
The aim of this study was to evaluate the potential of lipopeptides containing FMDV-specific B-cell epitopes from structural and non-structural (NS) proteins to induce an FMDV-specific immune response in cattle. A special focus was laid on the variance of the immune response of different randomly selected outbred cattle against the peptides used for the immunizations. In addition, the potency of the B-cell epitopes in the induction of a peptide and/or FMDV-specific T-cell reaction was studied. Furthermore, the capacity of the lipopeptides to induce protection against a homologous challenge infection was investigated.
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METHODS |
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Viruses.
FMDV strain O1Kaufbeuren (O1K) was grown on monolayers of BHK cells, clone Tübingen. Virus stocks used for proliferation assays were stored as clarified tissue culture harvest material (3000 g for 20 min) at -70 °C. For LPBE (Hamblin et al., 1986), virus was inactivated with binary ethyleneimine and additionally incubated at 32 °C overnight.
Virus used for challenge infections was passaged in cattle and each animal was infected with 2x5000 p.f.u. intralingually.
Peptides.
Peptides were synthesized by conventional solid-phase methodology (Merrifield & Stewart, 1965) and dissolved in a concentration of 10 µg µl-1 in DMSO. Overlapping peptide amides (14 or 15 aa in length, overlapping each other by 10 aa) were synthesized based on the sequence of FMDV O1K (Forss et al., 1984
) using Fmoc chemistry (Multiple Peptide Synthesizer SyRo, MultiSyntech). Synthesized peptides were analysed by analytical high-pressure liquid chromatography (System Gold, Beckman Coulter) on a Nucleosil C18 column (Grom) and ion-spray mass spectroscopy (VG Quattro II Triple-Quatrupol Ionspray-MS, Micromass). The purities of crude peptides were, in general, higher than 70 %. For immunization purposes, peptides were synthesized as Pam3Cys peptides, as described previously (Wiesmüller et al., 1989
).
Vaccination and infection of animals.
Vaccination was carried out intramuscularly with 2 mg peptide per animal. Lipopeptides were dissolved in PBS and emulsified with an equal volume of Montanide ISA 9. A 1·5 ml volume per dose was administered. Challenge infections were carried out according to the methodologies described by the European Pharmacopoeia for the determination of the potency of conventional vaccines. A total of 104 p.f.u. FMDV O1K was inoculated intradermally into two sites on the upper surface of the tongue (0·1 ml per site). After 8 days, animals were sedated with Rompun and examined carefully for FMD-specific lesions. Animals that showed lesions at sites other than the tongue (secondary lesions) at 8 days post-infection were considered non-protected. To control the challenge infection, two control animals were included in the experiment. Both control animals developed lesions: one animal showed FMDV-caused lesions on all four extremities, the other one only on two extremities.
LPBE.
LPBE was performed as described (Hamblin et al., 1986), with slight modifications. Briefly, ELISA plates (Nunc) were coated overnight with rabbit anti-FMDV O1K serum. Twofold dilution series of each test serum were mixed with equal volumes of antigen (cell culture supernatant) of a predetermined dilution and kept at 4 °C overnight in a dummy plate. After washing, each well of the coated ELISA plates received 50 µl antigen/serum mixture. Control wells received antigen/buffer mixture (antigen control) or antigen/standard serum mixture, respectively. After incubation for 1 h at 37 °C, plates were washed and incubated with guinea pig anti-FMDV O1K serum. After incubation for 1 h at 37 °C, plates were washed again and incubated for a further 1 h at 37 °C with goat anti-guinea pig gamma-globulin-peroxidase conjugate (Biozol). After the final washing step, the chromogen orthophenylenediamine (OPD) (Sigma) with H2O2 was added. The reaction was stopped after 15 min with H2SO4 and plates were read in an automatic microplate reader at 492 nm. Antibody titres were expressed as the dilution at which the test sera showed greater than 50 % inhibition of the absorbance value recorded for the antigen control wells. Titres below 1 : 40 (1·6 log10) were considered positive.
Peptide ELISA.
Plates (Immunoplate Maxisorb, Nunc) were coated with 10 µg peptide ml-1 in 100 µl H2O per well and air-dried at 37 °C. Peptide-coated plates were blocked for 2 h at 37 °C with 3 % BSA/PBS and then incubated with sera diluted in 3 % BSA/PBS at a 1 : 100 dilution for 1 h at 37 °C. Specific antibodies were detected with horseradish peroxidase-conjugated goat anti-bovine immunoglobulin antibodies (Dianova). Colour development was obtained after addition of the substrate (OPD with 0·2 µl H2O2 ml-1). The reaction was stopped with 2 M H2SO4. All plates were read in an automatic microplate reader (Titertek).
Results are expressed as indices (average absorbance value of duplicate wells of the sample/average absorbance value of the duplicate wells of the negative controls). As negative controls, three FMDV O1K peptides that did not contain linear B-cell epitopes were used. The indices of all animals were below 2 before the first vaccination.
Lymphocyte proliferation assay.
Bovine PBMCs were isolated by Histopaque (Histopaque 1083, Sigma) centrifugation of heparinized blood. Cultivation of PBMCs (5x105 ml-1) was performed in MEM- (Gibco-BRL) supplemented with 10 % FCS, 2 mM L-glutamine, 5x10-5 M 2-mercaptoethanol, 10 mM HEPES buffer, 100 IU penicillin ml-1 and 0·1 mg streptomycin sulphate ml-1. For the proliferation assay, PBMCs were incubated for 5 days at 37 °C with 5 % CO2 in 200 µl microcultures in round-bottomed microtitre plates (Greiner). PBMCs from vaccinated animals were restimulated with either FMDV O1K (log2 titration, starting with 5x106 p.f.u. per well) or synthetic peptides (25 and 2·5 µg ml-1). As negative controls, cell culture supernatants from mock-infected cells or irrelevant 15-mer peptides derived from hepatitis C virus dissolved in DMSO (25 and 2·5 µl ml-1) were used.
To quantify the proliferative response, 1 µCi [3H]thymidine (Amersham) per well was added for an additional 1618 h. After the microplates were freeze-thawed, the cells were harvested using a multi-harvester system (Wallac) and [3H]thymidine uptake was measured in a -counter (Wallac).
All results are expressed as stimulation indices (SI), which can be defined as geometric mean c.p.m. of the triplicate microcultures of the sample/geometric mean c.p.m. of the triplicate microcultures of the respective negative controls. The SI determined before vaccination was below 2 for all animals.
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RESULTS |
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Immunization of cattle with lipopeptides and determination of their antibody responses
To determine the ability of the identified linear B-cell epitopes to induce an in vivo immune response and to protect immunized animals from challenge infection, peptides identified as linear B-cell epitopes (Fig. 2) were selected, synthesized and N-terminally elongated by the immunostimulating lipoamino acid Pam3Cys. To reduce the number of peptides for the immunization experiments, only seven peptides were selected out of the 15 identified as linear B-cell epitopes (Figs 1 and 2
), namely peptides 1, 3, 4, 6, 7, 11 and 15. Peptide 1 derived from the 1D structural protein contained the RGD region responsible for the attachment of the virus to the cellular receptor. The epitope corresponding to peptide 3 is located in the C-terminal part of NS protein 2B. Peptides 4 and 6 describe two B-cell epitopes located in the N terminus of NS protein 2C. Peptide 7 was derived from protein 3A and peptide 11 from the first tandem repeat of protein 3B. Peptide 15 was part of the N terminus of protein 3D. The sequences of all these peptides used for the immunization experiments are summarized in Fig. 2
. As mentioned above for the vaccination experiments, these peptides were synthesized as lipopeptides and applied in an oil emulsion. Seven cattle were immunized with a dose of 2 mg for each peptide per animal. Blood samples were taken from each animal 21 days later and the induction of specific antibodies against FMDV and against the synthetic peptides used for the immunization was determined. Animals were challenged with FMDV O1K, from which the sequence of the synthetic peptides had been derived. In this experiment, four of seven animals were protected (Fig. 4
b).
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Investigating the sera derived after the first immunization prior to the challenge infection for antibodies against peptides (Fig. 4, closed bars), the sera of most animals showed a very low reaction in the peptide ELISA and only against a few of the peptides that were included in the vaccine (A492>2). The serum of animal #69064 showed a response to peptide 15 and the serum of animal #99219 showed a response against peptides 1 and 7. However, there were also animals that did not elicit a detectable amount of antibodies against any of the peptides, for example, the protected animal #66671.
In short, the humoral immune response after vaccination revealed no conclusive pattern. About 3 weeks after challenge (Fig. 4, open bars), the sera of most animals showed an increased antibody level not only against the peptides they had already responded to after the vaccination but also against peptides they did not show any response to before the vaccination. Protected animal #15879 had developed antibodies against peptides 7 and 15 after vaccination and additionally against peptides 1, 3, 6 and 11 after challenge infection. An exception was animal #43912, which showed, in contrast to all other animals, almost no detectable antibody response against any peptide in the peptide ELISA. This animal had also a very low antibody titre in the LPBE after the first immunization, but after the challenge infection the antibody titre was quite high (titre>1 : 2400).
A comparison of the antibody responses of the four protected and the three non-protected animals showed no clear difference, neither with regard to the reactivity of the sera to FMDV in the LPBE nor in the peptide ELISA. A minor point might be that the protected animals showed on average a higher antibody level against the peptides after challenge than the non-protected animals, but animal #71775 was not protected in spite of a good antibody response to several peptides.
A summary of the data about the humoral immune response of lipopeptide-vaccinated cattle against FMDV and against synthetic peptides as described in detail above appears to be difficult and it seems to be impossible to make a clear correlation between antibody response and protection against a challenge infection. Within the group of protected and also within the group of non-protected animals, animals with high and low antibody levels against peptides as well as FMDV could be found.
In vitro analysis of the cellular immune response
Because no clear correlation between the humoral immune response and protection could be recognized in the experiments described above, further investigations were directed towards the second branch of the immune system the cellular immune response mediated by antigen-specific T-lymphocytes. Therefore, PBMCs of all vaccinated animals were examined in lymphoproliferation assays for their virus- and peptide-specific T-cell response after lipopeptide vaccination and after challenge infection.
Most of the cattle showed a virus-specific T-cell response after immunization (Fig. 5, closed bars). Both groups, protected and non-protected animals, contained high (e.g. animals #69064 and #15879) as well as weak (e.g. animals #43912 and #99219) responding animals.
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No positive correlation could be found between the peptide-specific reaction pattern of the respective T-cells and protection. But concerning the intensity of the T-cell reactions a correlation with protection can be suggested. All protected animals showed a very high proliferative response (SI>15) against at least one of the peptides. Animals #15879 and #38715, for example, showed a very high response to peptide 3, whereas animals #99219 and #66671 reacted with a high proliferative response towards peptides 7 and 15, respectively. None of the non-protected animals showed such a high T-cell activity against any of the peptides before challenge infection (Fig. 5a).
A strategy to overcome confusion about immune response and protection might be a combinatorial view on B- and T-cell responses of the protected and non-protected animals. Maybe a summary of the B- and T-cell responses, as presented in Fig. 6, can give an explanation for a correlation between the FMDV-specific immune response and protection.
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DISCUSSION |
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These results indicate that if it should be possible to formulate a protective vaccine containing these B-cell epitopes, a good chance would exist that this vaccine would show protection against several or most of the FMDV sero- and subtypes.
The idea to use peptides for the development of a FMDV vaccine already existed in the eighties of the last century. Different B- and T-cell epitopes were used for protection experiments with cattle (DiMarchi et al., 1986; Morgan & Moore, 1990
; Taboga et al., 1997
; Volpina et al., 1999
). The results varied strongly. The ratio of protected animals in some experiments was quite high in spite of the fact that in most cases no more than two different peptides were used for vaccination. But there were already hints that the use of only few B- and T-cell epitopes supports the selection of escape variants (Taboga et al., 1997
). As in all of these early experiments, all peptides were derived from the structural proteins of FMDV, a heterotypic protection against different FMDV serotypes appeared to be highly unlikely.
In contrast to these experiments, the study presented here used a vaccine with seven different epitopes (six of seven) derived mainly from the NS protein region.
Regarding the peptide-specific B-cell response, vaccinated animals showed a reaction pattern against single peptides that was highly variable. Such variations in antigen recognition are already known for FMDV immunizations (McCullough et al., 1992). Even if a B-cell response is not MHC-restricted, quite often animal-to-animal variations are observed for the same antigen (Riley, 1996
).
In contrast to peptide-specific antibodies that could be detected in the sera of most vaccinated cattle, only in few animals could antibodies against FMDV be determined with the ELISA technique used commonly for FMDV diagnosis (Hamblin et al., 1986).
When looking at the cellular immune response, most cattle showed a good reaction against single peptides after vaccination. Astonishing was the extremely good proliferative response that could be shown for some of the protected animals (SI>15). As mentioned above, a correlation with the antibody titre could not be presented in these animals. But despite the absence of detectable antibody titres against FMDV, these animals were protected from challenge infection. This is most probably due to an effective T-cell reaction that was able to support the formation of very effective neutralizing antibodies. On the other hand, this protection by T-lymphocytes might be explained by an effective cellular immune response, including the activity of T-helper cells as well as cytolytic T-lymphocytes. Further studies might elucidate these speculations.
It was also interesting to note with regards to the T-cell response against the synthetic peptides that five of the seven peptides used for the immunization experiments seemed to contain T-cell epitopes responsible for the in vitro restimulation of the respective T-lymphocytes. Lipopeptides are synthetic analogues of membrane lipoproteins of bacteria and activate cells for cytokine release by their interaction with Toll-like receptors, which indicates a molecular link between host defence mechanisms and microbial products. (Aliprantis et al., 1999). Synthetic analogues of the N-terminal part of these lipoproteins constitute potent immunoadjuvants in vitro and in vivo (Wiesmüller et al., 1992
). One might also speculate that the Pam3Cys-coupled peptides were caught by the specific B-cell receptors, the surface immunoglobulin molecules, internalized into the B-cells and presented by B-cell MHC molecules to the respective T-cell receptors of the specific T-lymphocytes. At this stage, it is not clear whether the presentation is due mainly to MHC class II molecules as it might be expected or whether MHC class I molecules are involved also. Nothing is known to date whether the Pam3Cys-anchor molecule can be involved in a kind of cross-presentation. These questions will be the content of further studies.
Another peculiarity without clear explanation was the reduced proliferation against peptides after challenge infection. The reason for this decrease of the proliferative capacity is most probably due to the fact that the ability of the T-cells to become restimulated was inhibited due to the strong stimulation before. This could be a momentary effect, as described similarly for the mouse (De Mattia et al., 1999), but such a decreased reactivity of antigen-specific T-helper cells after restimulation could also occur after restimulation with the same antigen after consecutive vaccinations.
In general, it can be concluded that it seems that animals were protected (four of seven) when a high T-cell activity (SI>15) could be shown for at least one peptide.
This information is important for a further optimization of a potential peptide vaccine, and after a detailed analysis of this ex vivo data, it might be possible in future experiments to avoid challenge infections. This would considerably reduce the cost and time needed for further investigations.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 22 May 2003;
accepted 19 August 2003.
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