Synthetic peptides mimicking protective B- and T-cell epitopes are good candidates for safer, more effective FMD vaccines. Nevertheless, previous studies of immunization with linear peptides showed that they failed to induce solid protection in cattle. Dendrimeric peptides displaying two or four copies of a peptide corresponding to the B-cell epitope VP1 [136–154] of type O FMDV (O/UKG/11/2001) linked through thioether bonds to a single copy of the T-cell epitope 3A [21–35] (termed B2T and B4T, resp.) afforded protection in vaccinated pigs. In this work, we show that dendrimeric peptides B2T and B4T can elicit specific humoral responses in cattle and confer partial protection against the challenge with a heterologous type O virus (O1/Campos/Bra/58). This protective response correlated with the induction of specific T-cells as well as with an anamnestic antibody response upon virus challenge, as shown by the detection of virus-specific antibody-secreting cells (ASC) in lymphoid tissues distal from the inoculation point.
The foot-and-mouth-disease virus (FMDV) causes a highly contagious disease with high morbidity in cloven-hoofed animals, including cattle and swine. FMDV can be controlled by the use of a chemically inactivated whole-virus vaccine; however, some disadvantages are associated with the use of inactivated vaccine. For example, the vaccine provides short-term protection, resulting in the need for revaccination [
The FMD viral particle consists of a positive-strand RNA genome, a single open reading frame (ORF) which encodes four capsid proteins, VP1, VP2, VP3, and VP4, and eleven different mature nonstructural proteins (NSP).
The B-cell binding site located in the G-H loop (around residues 140–160) of FMDV VP1 protein has been identified as a predominant epitope that elicits neutralizing antibodies against this virus in natural hosts and animal models [
The current inactivated FMD vaccines only promote serological protection against a given FMDV serotype, do not confer interserotype protection, and may not, in some cases, confer intraserotype protection given the antigenic variation existing within some serotypes [
Peptide vaccines are an attractive alternative strategy that relies on the usage of short peptide fragments to engineer the induction of highly targeted immune responses, consequently avoiding allergenic and/or reactogenic sequences [
Multiple antigenic peptides (MAPs) are dendrimeric (branched) macromolecules built from a lysine core from which a defined number of epitopes radiate [
The dendrimeric peptide design improves the effectiveness of viral antigenic site presentation to the immune system. Recent studies indicate that vaccination with dendrimeric peptides based on the amino acid sequence of 3A (T-cell epitope) and VP1 GH loop (B-cell epitope) from the type O FMDV O/UKG/11/2001, and branched by means of thioether or maleimide conjugation chemistries, elicits an immune response that achieved protection in up to 100% of the vaccinated pigs [
The aim of this study was to investigate whether dendrimeric peptides elicited protection against heterologous viruses, a relevant issue for efficient vaccine design. To this end, the immune response elicited in cattle by dendrimers containing amino acid sequences of 3A and VP1 GH loop from type O FMDV O/UKG/11/2001, B2T and B4T, and the protection they afforded against the heterologous type O virus O1/Campos/Bra/58, was analyzed.
Our results indicate that B2T and B4T elicited specific humoral responses in cattle and conferred partial protection against the challenge with a heterologous virus O1/Campos/Bra/58. This protective response correlated with the induction of FMDV-specific T-cells as well as with an anamnestic antibody response upon virus challenge, as shown by the detection of virus-specific ASC in lymphoid tissues distal from the inoculation point.
The dendrimeric peptides reproduced the B-cell (PVTNVRGDLQVLAQKAART, residues 136–154 of VP1) and T-cell (AAIEFFEGMVHDSIK, residues 21–35 of 3A) epitopes of FMDV O-UKG 11/01 (Figure
Dendrimeric peptides used in this study.
FMDV O1/Campos/Bra/58 was kindly provided by Biogenesis Bagó SA as binary ethylene-imine (BEI) inactivated (iFMDV). Purified virus was obtained by a sucrose density gradient centrifugation method [
A virus stock derived from FMDV isolated O/UKG/11/2001 (The Pirbright Institute, UK) by two amplifications in swine kidney cells was used in the virus neutralization assays.
Ten Hereford calves serologically negative for FMDV, approximately 6 months old, were used in the experiment. Groups of four animals were inoculated twice (days 0 and 18), by subcutaneous injection in the front left quarter, with 2 mg of B2T or B4T peptide in 2 ml of a water-in-oil single emulsion. The adjuvant included was the same contained in commercial vaccines. At 38 days postvaccination (dpv), the animals were challenged by nasal instillation with 1 ml (0.5 ml for each nostril) of 10000 of 50% bovine infective doses (BID50) of infective FMDV O1/Campos/Bra/58 (determined by titration on cattle tongue) [
Seven days postchallenge (dpc), all animals were checked for FMDV-induced lesions on the feet and tongue. Bovines with the absence of FMDV-induced lesions at the feet were considered as protected to podal generalization (PPG), while, animals with a delay in the onset of symptoms were considered partially protected (PP). At 7 dpc, different lymphoid organs were obtained postmortem from each animal: mandibular lymph nodes (ML), medial retropharyngeal lymph nodes (MRL), and tracheobronchial lymph nodes (TBL). All lymphoid organs were collected aseptically and placed in ice-cold wash buffer (RPMI 1640, 10 mM HEPES, 100 U/ml penicillin G sodium, 100
Another five calves were immunized by subcutaneous injection with a single dose of commercial FMDV vaccine (water-in-oil single emulsion containing FMDV strains A Arg 2000, A Arg 2001, A24 Cruzeiro, and O1 Campos). This vaccine has been approved by the Argentine Animal Health Service (SENASA) with more than 80% of expected percentage of protection against all vaccine strains [
For the estimation of the immune response elicited by the dendrimers, we followed the methods of Soria et al. [
FMDV-specific antibodies were detected by means of an indirect ELISA, as described by Quattrocchi et al. [
The antiviral ELISA detailed above was modified in order to detect FMDV-specific IgG1 and IgG2 (in sera) and IgG1 and IgA (in nasal swabs) antibodies. After incubation with samples, a mouse anti-bovine IgG1, IgG2, or IgA monoclonal antibody was added (kindly provided by Dr. S. Srikumaran, University of Nebraska, USA). Lastly, a (HRP)-labeled goat anti-mouse IgG antibody was added after wash. OPD was used as HRP substrate. Absorbance was recorded at 492 nm (A492) in a microplate photometer (Multiskan FC, Thermo). The cut-off was established as the mean A492 of the negative sera (from all unvaccinated animals) plus two standard deviations (SD). Antibody titres were calculated for IgG1 and IgG2 as log10 of the last reciprocal dilution above cut-off. IgA levels were expressed as the ratio between the OD A492 of the nasal swabs from 22 dpv to 0 dpv. Positive control sera were included in every plate.
The neutralizing index (NI) of serum (variable virus and fixed serum) from cattle immunized with B2T, B4T, or conventional vaccine, at 38 dpv (upon 2 doses of peptide), was measured. A 1/16 serum dilution was incubated with 10-fold dilutions of infective FMDV (1000 to 1 of 50% tissue culture infective dose—TCID50), and the infective virus recovered was determined by a TCID50 assay. The NI of a serum was calculated as the ratio between the titres of the virus in the presence of vaccinated animal serum and in the presence of a negative serum. The results were expressed as log10 of NI.
Serum samples were examined for anti-FMDV neutralizing antibodies (fixed virus and variable serum) as described before [
Peripheral blood mononuclear cells (PBMC) were obtained from cattle as described [
PBMC were cultured with either 50
Mononuclear cell (MNC) suspensions were obtained from lymphoid tissues as previously described [
The InfoStat program was used. One-way analysis of variance (ANOVA) and posttests were used to compare data between three or more groups.
At 38 dpv, all animals inoculated with either B2T or B4T constructs developed specific and pronounced anti-peptide (Figure
Antibody detection by ELISA in vaccinated cattle. Animals were immunized on days 0 and 18 (arrow) with B2T or B4T vaccine. (a) Kinetics of anti-peptide serum antibodies. Bars represent the mean IgG titres from bovines in each group (gray, B2T; black, B4T) throughout the experiment (error marks, SD). (b, c) Kinetics of total IgG and IgG1 anti-FMDV O1/Campos/Bra/58 serum antibodies. Titres were calculated as log10 of the last reciprocal dilution above cut-off. Data points represent the IgG titre (b) or IgG1 titre (c) from each animal represented by different shapes (right legend) throughout the experiment. (d) FMDV-specific mucosal IgG1 and IgA responses. Nasal swabs were collected at 22 dpv. Each point represents the nasal IgG1 anti-FMDV antibody titres (log10) (black) or IgA (gray) anti-FMDV O1/Campos/Bra/58 antibody level of each animal. The cut-off was established as the mean value of mock-vaccinated animals plus twice the SD value (dotted line).
At 38 dpv, high anti-FMDV IgG titres were detected in all animals with an average titre of 3.4 ± 0.4 and 3.3 ± 0.3 in B2T and B4T groups, respectively (Figure
Animals from the B4T group exhibited high levels of anti-FMDV IgG1 in nasal secretions at 22 dpv, with the exception of bovine 166; however, at this time, animals from the B2T group did not present high anti-FMDV IgG1 titres (Figure
The VNT against the homologous virus O/UKG/11/2001 were determined at 32 dpv, and average values of 1.2 ± 0.3 and 1.3 ± 0.3 were found in the B2T and B4T groups, respectively (Table
Virus neutralizing titres prechallenge.
Group | Animal no. | Neutralizing antibodies (38 dpv) | |
---|---|---|---|
VNTa |
log10 NI | ||
B2T | 44 | 1.60 | 2.0 |
170 | 1.20 | 1.3 | |
168 | 1.10 | 0.8 | |
169 | 0.90 | 1.0 | |
B4T | 36 | 1.20 | 1.3 |
431 | 1.75 | 2.7 | |
164 | 1.10 | 2.0 | |
166 | 1.20 | 1.3 | |
Commercial vaccine | 522 | — | 1.8 |
800 | — | 2.1 | |
809 | — | 1.6 | |
810 | — | 2.3 | |
820 | — | 2.8 | |
Negative controls | 167 | <0.9 | 0.0 |
997 | <0.9 | 0.3 |
aTitre of virus-neutralizing antibody at day 38 post vaccination. O/UK/01: FMDV O/UKG/11/2001; O1/C: FMDV O1/Campos/Bra/58.
Before challenge, at 32 dpv, specific
Cellular immune response of cattle 32 days postvaccination analyzed by 3H-thymidine incorporation (a) and IFN-
Group | Animal no. | SI (cpm Ag/cpm medium) | ||||
---|---|---|---|---|---|---|
Ag-B2T | Ag-B4T | Ag-T | iFMDV | |||
B2T | 44 | 1.1 | ||||
170 | 1.0 | 1.2 | ||||
168 | 1.4 | 1.8 | 1.1 | 1.1 | ||
169 | 2.0 | 1.9 | 0.9 | 1.5 | ||
B4T | 36 | 1.4 | ||||
431 | 1.6 | 1.0 | 1.2 | |||
164 | 1.4 | 1.8 | ||||
166 | 0.8 | 1.3 | ||||
Commercial vaccine | 522 | 2.0 | ||||
800 | 1.4 | 2.0 | 1.2 | 1.6 | ||
809 | 1.5 | 1.9 | 1.2 | |||
810 | 1.2 | 1.4 | 0.9 | 0.9 | ||
820 | 0.7 | 0.7 | 0.7 | |||
Negative | 167 | 1.0 | 0.7 | 1.0 | 1.0 | |
controls | 1.0 | 1.4 | 0.9 | 0.9 |
Group | Animal no. | IFN- | ||||
---|---|---|---|---|---|---|
Medium | Ag-B2T | Ag-B4T | Ag-T | iFMDV | ||
B2T | 44 | 7.8 | 7.1 | |||
170 | 14.0 | 12.4 | 11.3 | |||
168 | 13.2 | 11.8 | 13.3 | |||
169 | 12.3 | 14.0 | 14.6 | 15.0 | ||
B4T | 36 | |||||
431 | ||||||
164 | ||||||
166 | 7.9 | 6.0 | 6.5 | 6.8 | 6.8 | |
Commercial vaccine | 522 | 11.6 | ||||
800 | 7.4 | 7.6 | 7.4 | 7.1 | 7.5 | |
809 | ||||||
810 | 14.3 | 13.7 | ||||
820 | 7.3 | 7.3 | 6.5 | 9.4 | 6.9 | |
Negative controls | 167 | 8.4 | 5.2 | 6.5 | 7.2 | 6.1 |
997 | 9.6 | 12.0 | 14.0 | 13.3 | 14.8 |
(a) Lymphoproliferation of PBMC from vaccinated cattle (32 dpv) determined by 3H-thymidine incorporation. Results were expressed as SI. PBMC were stimulated
The levels of IFN-
On the other hand, bovines 169 and 166 did not secrete IFN-
Since the aim of the study was to investigate the protection afforded by the dendrimeric peptides and the infection with FMDV type O other than O1/Campos/Bra/58 was not possible at INTA, bovines were challenged with this virus, an experimental design that allows the assessment of the cross-protection conferred by the dendrimers. Thus, all animals were challenged at 44 dpv by nasal instillation with infective FMDV O1/Campos/Bra/58, and protection was measured by monitoring clinical signs in animals after the challenge. As shown in Table
Clinical scores of vaccinated cattle after challenge.
Group | Animal no. | Clinical score (dpc)a | Protectionb | |||
---|---|---|---|---|---|---|
2 dpc | 3 dpc | 4 dpc | ||||
B2T | 44 | 0 | 0 | 0 | PPG | |
170 | 0 | 0 | 0 | PP | ||
168 | 0 | 0 | 5 | NP | ||
169 | 0 | 0 | 3 | NP | ||
B4T | 36 | 0 | 0 | 0 | 2 | PPG |
431 | 0 | 0 | 0 | PP | ||
164 | 0 | 0 | 3 | NP | ||
166 | 0 | 5 | 6 | NP | ||
Negative controls | 167 | 0 | 0 | 4 | 6 | NP |
997 | 0 | 4 | 4 | 6 | NP |
aClinical score was established after the challenge and was determined by the number of feet presenting FMD lesions plus the presence of vesicles in the snout and/or mouth, 6 being the maximum score. bAnimals with no lesions on the feet were PPG. Animals with a delay in the onset of symptoms of disease were PP, and animals with lesions on their feet before 7 dpc were considered NP.
Animals were euthanized at 7 dpc, and the FMDV-specific mucosal immune responses were studied along the respiratory tract by means of a FMDV-ASC ELISPOT assay (FMDV-ASC ELISPOT). The results showed three profiles of responses (Figure
Profiles of the FMDV-ASC detected in B2T- and B4T-vaccinated cattle after FMDV challenge. (a) Mononuclear cells were purified from mandibular lymph nodes (ML), medial retropharyngeal lymph nodes (MRL), and tracheobronchial lymph nodes (TBL) and characterized by the FMDV-ASC ELISPOT assay, using monoclonal (IgG1 and IgG2) or polyclonal (IgM and IgA) antibodies against bovine immunoglobulin isotypes as probes. (b) Total FMDV-ASC in ML, MRL, or TBL. Results are expressed as the mean number of FMDV-specific ASC per 1 × 106 extracted cells. Each bar represents the mean value of 3 replicates ± SD. PPG: protected against podal generalization; PP: partial protected; NP: non-protected.
Animal 166, which showed delayed humoral response against virus, presented the highest number of IgG1 ASC (>2 × 103 ASC/106 cells) in ML and MRL. Concordantly, bovine 166 was the only animal that showed high numbers of IgM and IgG1 ASC in TBL.
In mock-vaccinated animals (167 and 997), ML and MRL were the most stimulated secondary lymphoid organs at 7 dpc, IgM was the dominant isotype among the FMDV-ASC developed in these organs. In animal 167, IgG1 was the next isotype with regard to the detection level, with levels 10- to 30-fold lower than those detected in the ML of NP vaccinated bovines. When the total FMDV-ASC was calculated, PPG and PP animals presented very low numbers of ASC in comparison with NP vaccinated animals (Figure
In order to determine the memory immunity induced in vaccinated animals after the challenge with the live virus, the level of IFN-
IFN-
Synthetic peptides corresponding to the protective B- and T-cell epitopes can be considered good candidates for FMD vaccines as, among other advantages, they are safe and support a rational design and their production and characterization are simple. The development of successful peptide vaccines has been limited for a number of reasons, including those associated with “in vivo” stability, poor immunogenicity of linear peptides, and lack of adequate T-cell activation due to MHC polymorphism of the host species [
In our experiment, specific antibody responses to virus were observed in all cattle receiving peptide vaccines; however, even when neutralizing antibodies against FMDV O/UKG/11/2001 were detected, their levels were lower than those found in pigs immunized with the same peptides.
The amino acid sequence of the B-cell epitope VP1 (136–154) between FMDV O/UKG/11/2001 and O1/Campos/Bra/58, the virus used for cattle challenge, differs in 3 amino acids. Nevertheless, Wang et al. [
Despite the presence of anti-peptide and anti-FMDV antibodies in sera, they may not have the affinity necessary to effectively neutralize the virus, and only 25% of the B2T- or B4T-vaccinated animals were PPG after the challenge with FMDV O1/Campos/Bra/58. It is possible that when using another adjuvant or other amounts of peptides in the vaccine, the immune response could increase in cattle, achieving the maturation of the antibodies affinity for the viral neutralization of FMDV O1/Campos/Bra/58.
The isotype of antibodies elicited in cattle by the two dendrimers differs from those induced in swine [
Animal-to-animal variation is found in the protective responses evoked by peptide vaccines, including those against FMDV [
Our findings suggest that in some instances, animals showing the highest immunological parameters measured against peptides and iFMDV were better protected against viral challenge. Bovines 44 (B2T) and 36 (B4T) elicited high levels of antibodies against virus (although animal 36 showed levels of neutralizing antibodies of 1.2) and developed high levels of IgA specific against virus in nasal secretions as well as a positive lymphoproliferative response not only against dendrimeric peptides but also against the epitope T 3A (21–40). All these positive parameters in bovines 44 and 36 correlated with a protective immune response. Indeed, these were the only two PPG animals. On the other hand, and despite at the time of challenge the level of antibodies (measured by ELISA) being similarly high for all cattle, animals that showed modest humoral response initially (at 18 or 22 dpv) failed to be protected against viral challenge, which may be due to the lack of antibody maturation. Nonprotected animals 168 (B2T) and 164 (B4T) showed antibody responses against iFMDV only after receiving a second dose of vaccine, and their viral neutralization titres were lower than 1.2 (VNT positive for
Cattle are highly susceptible to FMDV, and the virus usually gains entry through the respiratory tract of these animals [
The study of antibody responses in local lymphoid tissues indicates that the systemic FMD vaccination of cattle with dendrimeric peptides can effectively promote the presence of anti-FMDV ASC in lymphoid tissues associated with the respiratory tract. In addition, the detection of both FMDV O1/Campos/Bra/58-specific ASC and antibodies following vaccination shows that these peptides, encompassing FMDV O/UKG/11/2001 sequences, were able to induce a cross-reactive ASC response.
In peptide-vaccinated unprotected animals, viral challenge by nasal instillation triggered an antibody response compatible with a local anamnestic recall upon contact with replicating FMDV, suggesting that peptide vaccination might induce the circulation of virus-specific B-lymphocytes, including memory B-cells that differentiate into ASC soon after contact with the infective virus. Thus, NP animals showed a strong stimulation of FMDV-specific B-lymphocytes to locally produce antibodies all along the respiratory tract, including in the tracheobronchial lymph nodes (TBL) with frequencies of ASC much higher than those in mock-vaccinated infected animals. In the NP animals, ASC were detected in all studied organs, and the isotype of the antibodies (mainly IgM and IgG1) revealed that even when B2T and B4T peptides elicited specific memory B-cells, the response failed to stop the advance of the challenge virus. Conversely, in peptide-vaccinated PPG and PP animals, no FMDV-ASC were detected in TBL possibly because the virus did not reach that area. Thus, in animals 44 and 36 (PPG) and 170 and 431 (PP), cells producing antibodies against FMDV were not found in TBL, and in general the total number of ASC induced was low.
It has been proposed that structural features lend FMDV capsids towards stimulating B-cells in a T-independent manner [
Overall, our results support that immunization in cattle with dendrimeric peptides B2T and B4T can elicit humoral and cellular immune responses and confer partial protection against a heterologous virus challenge that is associated with the induction of solid T-cell responses as well as of an anamnestic antibody response. Experiments are in progress to address whether modifications such as the replacement of the T-cell peptide by one widely recognized by cattle can result in an improvement of the protective response elicited by these dendrimeric peptides.
The authors declare that there is no conflict of interest regarding the publication of this article.
The authors thank N. Fondevilla for his invaluable work in the BSL-4 OIE facilities at the CICVyA-INTA and J. Leiva, C. Fioroni, D. Franco, and L. Vagnoni for their help in animal handling. The authors also thank E. Rivarola for his administrative assistance and E. Smitsaard (Biogenesis Bagó) for providing the inactivated FMDV. Work at INTA was supported by the National Institute of Agricultural Technology (Grant no. PNSA 1115052) and an INTA-INIA (Spain) cooperation agreement. Work at UPF and CBMSO was supported by MINECO, Spain (Grant no. AGL2014-52395-C2).