Journal of Immunology and Vaccination

Carrier Protein Significantly Alters the Magnitude, Duration, and Type of Antibody Response to a Peptide Epitope from a Self-Protein

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Published Date: October 23, 2015

Carrier Protein Significantly Alters the Magnitude, Duration, and Type of Antibody Response to a Peptide Epitope from a Self-Protein

Ryan Taschuk1,2, Philip Griebel1,2 and Scott Napper1,3*

1Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E3, Canada

2School of Public Health, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 5E3, Canada

3Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 5E5, Canada

*Corresponding author: Scott Napper, Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan Canada S7N 5E5, Canada, Tel: 306-966-1546; E-mail:   

Citation: Taschuk R, Griebel P, Napper S (2015) Carrier Protein Significantly Alters the Magnitude, Duration, and Type of Antibody Response to a Peptide Epitope from a Self-Protein. J Imm Vac 1(1): 101.




Vaccines based on peptide epitopes of self-proteins require carrier proteins with T cell epitopes to support B cell responses. We analyzed antibody responses to a synthetic peptide epitope derived from prion protein (PrP) that was expressed as a recombinant chimera with either Mannheimia haemolytica Leukotoxin (Lkt) or truncated rabies glycoprotein (tgG). The tgG-PrP chimera consistently induced epitope-specific serum antibody responses over 10-fold greater and of significantly longer duration than the Lkt carrier protein. Furthermore, similar antibody response kinetics were observed for each carrier when animals received a single immunization. The tgG-PrP chimera induced primarily IgG1 antibody responses, whereas the Lkt-PrP chimera induced both IgG1 and IgG2a antibody responses. The different antibody isotype profiles were associated with distinct cytokine production profiles following splenocyte re-stimulation with carrier proteins. These observations illustrate that carrier proteins alter not only self-peptide epitope immunogenicity but also the magnitude, duration, and isotype of antibody responses.

Keywords: Peptide Epitopes; Carrier Proteins; Vaccine; Leukotoxin; Rabies Glycoprotein G

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The traditional role of vaccines has been to prevent or reduce infection by pathogenic microbes. However, there is increasing interest in both human and veterinary medicine to develop vaccines that induce antibody responses specific to epitopes within self-molecules [1]. Recently, efforts have focussed upon developing vaccines to induce antibodies that may prevent diseases arising from misfolding of a self-protein, such as prion diseases and other neurodegenerative diseases [2].

Peptide-based vaccines provide an attractive approach for inducing highly specific antibody responses to a self-epitope [3,4]. Short peptide sequences behave as haptens, and therefore a carrier protein possessing appropriate T cell epitopes is critical for inducing antibody responses to a peptide vaccine [5,6]. The choice of a carrier protein may be even more critical when delivering peptide epitopes from self-proteins since it may be necessary to overcome immune tolerance.

A modified version of Mannheimia haemolytica leukotoxin has been used as a carrier protein to deliver a variety of peptide epitopes, including self-epitopes from PrP [7,8]. Animal trials with recombinant Lkt-epitope fusion proteins demonstrated that sustained serum antibody responses could be induced in both ruminants and rodents following multiple immunizations.

Glycoprotein G (gG) of rabies virus is the primary antigen responsible for inducing protective virus-neutralizing antibodies. Numerous studies have shown that multiple routes of administration and formulations of gG are capable of inducing rapid, sustained and protective antibody responses in a variety of species [9]. A truncated form of gG (tgG) is produced during normal rabies infections, and lacks carboxy-terminal amino acids coding for transmembrane and cytosolic domains present in the full-length protein [10]. Although this truncation causes release of soluble gG from infected cells, the protein retains the quaternary structural conformation and immunogenicity of full-length gG [11,12].

Others have investigated the ability of full-length and truncated gG to induce humoral response against heterologous antigenic determinants of infectious pathogens [13,14]. Smith and Desmézières reported that immunization with constructs expressing chimeric protein induced strong humoral responses to both heterologous peptide epitopes, as well as the gG carrier. Building upon these results, we hypothesized that the unique immunogenic properties of gG could be exploited to enhance antibody responses to peptide-epitopes derived from a self-protein.

We compared the magnitude, duration and type of antibody responses specific to a self-epitope relevant for prion diseases, the rigid-loop sequence (RL) of PrP [6]. This epitope was expressed as a recombinant fusion with either leukotoxin (Lkt) or a truncated form of rabies glycoprotein G (tgG). The results of this study provide insight into the importance of carrier protein selection for the induction of antibody responses specific to epitopes within a self-protein.

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Materials and Methods


Generation of Leukotoxin constructs

Sequence corresponding to the optimized RL epitope (VDQYSNQNNF) were synthesized (Genscript) [15]. In the context of the final recombinant carrier protein epitopes are in a forward-back-back presentation (with spacers) that is repeated four times (èçç)4. The specific sequence of the resulting construct is (VDQYSNQNNFSSGFNNQNSYQDVSGSFNNQNSYQDV)4. Underlined sequences denote spacer regions. This pattern of inverted repeats had proven successful for the induction of antibody responses against other self-peptide epitopes [16] and has proven similarly beneficial for enhancing the magnitude of antibody responses against prion epitopes [8].” This fragment was subcloned into a modified version of pAA352 to be expressed as a C-terminal fusion to the Leukotoxin protein. Constructs were sequence verified and the resulting Lkt-RL recombinant protein was produced in BL21 cells as described previously [17]. The fusion protein was determined by denaturing polyacrylamide gel electrophoresis to be greater than 85% pure.

Generation of Truncated Rabies Glycoprotein G constructs

 A cDNA library was constructed using Superscript III cDNA Library Construction Kit (Life Technologies) following isolation of total RNA from rabies-positive fox brain tissue. Truncated gG was synthesized by amplifying the open reading frame of gG while excluding the 3’ nucleotides encoding the transmembrane and cytosolic domains. The produced gene was cloned into pEB4.3, an Epstein-Barr based expression plasmid conferring puromycin resistance. The optimized RL peptide-epitope was amplified to facilitate C-terminal his-tag addition and subcloning into the pEB4.3-tgG plasmid. Sequence verified plasmid was transfected into HEK293T cells using X-tremeGENE HP DNA transfection reagent (Roche). Transfectants were selected by the addition of 2 μg/mL puromycin to the culture medium. Cultures were subcultured into HyClone SFM4HEK293 serum-free media (Thermo Scientific), incubated with light shaking, and maintained at a cell density of approximately 4×106 cells/mL. Recombinant his-tagged tgG-RL was purified from conditioned and clarified media by affinity chromatography using TALON Cobalt Affinity Resin (Clontech) following the manufacturers specifications for native purification. Purified protein was identified by western blot and determined to be greater than 85% pure by SDS-PAGE.

Vaccine formulation and delivery

C57/BL6 or BALB/c mice (n = 6 / group) were injected subcutaneously, dorsal to the midline between the scapula, with 10 μg of either Lkt-RL or tgG-RL formulated in a final volume of 100 µl phosphate buffered saline with 30% Emulsigen-D (MVP Technologies) using a 25 G × 5/8 inch needle. Vaccines were administered as either a single dose, or as two immunizations on days zero and 21. All animal trial experimentation protocols were approved by the University of Saskatchewan Animal Care Committee.

Peptide Synthesis: To detect peptide-specific antibody responses, peptides consisting of a single repeat motif for the RL sequence were synthesized and verified as previously described [8].

ELISAs: Serum epitope-specific IgG antibody responses were quantified by ELISA, as previously described [8]. Briefly, Immulon 2HB 96-well polystyrene plates (Nunc) were coated with 0.5 µg RL peptide per well in 100 μl and incubated overnight at 4°C. Plates washed six times in ddH20, blocked for 1 h with 1% skim milk powder (SMP) in TBST and washed as above in preparation for serum addition. Serum samples were initially diluted 1:100 and serial diluted four fold in 1% SMP-TBST. Serially diluted serum samples were incubated with RL peptide for 1 h at room temperature, washed 6x with ddH20, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibody (Kirkegaard and Perry Laboratories) to probe for RL-specific antibody complexed with RL peptide at 1:4000 in 1% SMP-TBST for 1 h. Plates were again washed as above, developed using ρ-nitrophenyl phosphate, and analyzed following 30 min incubation at room temperature. ELISA titres are expressed as the reciprocal of the highest serum dilution resulting in an OD reading exceeding two standard deviations above the value for the pre-immune serum.

IFNγ and IL-5 ELISPOT Assays: Spleens were collected from BALB/c mice (n = 6/group) at five weeks post-immunization. Splenocyte isolation and culture, as well as ELISPOT plate preparation and development were as described previously [18] with appropriate use of splenocyte-stimulating antigens. Stimulating antigens were added to three replicate splenocyte cultures and included: media control; RL peptide (1, 5, 10 μg/ml); tgG (0.1 and 1.0 μg/ml), and Lkt (0.1 and 1.0 μg/ml). Results are expressed as the number of cytokine-secreting cells per million cells in wells containing stimulatory antigen.

Statistical Analysis

The data represents repeated measures of ELISA antibody titres in animals over time and did not adhere to a normal distribution.  To account for the repeated measures study design, data for each animal were first summed over time. The data sums were then ranked to account for their non-normal distribution and then a one-way ANOVA analysis was performed on the ranked sums.  Where appropriate, antibody titer data was normalized by log transformation, and differences between vaccine groups were compared at each time-point. Dunnet’s multiple comparisons test was used to examine differences among treatment groups.  P values less than 0.05 were considered significant.  All analysis met the assumptions of ANOVA. 

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Results & Discussion


Magnitude and Duration of Antibody Responses

Presentation of the peptide-epitope within the context of either the Lkt or tgG carrier resulted in strong peptide-specific antibody responses. Peak titers induced by the tgG construct were over 10-fold higher than those induced by the Lkt-RL fusion (p < 0.0001). Throughout the duration of the trial, peptide-specific antibody titres induced by the tgG carrier were often 100-fold higher than titres induced by the Lkt carrier (p < 0.0001) (Figure 1a). For many vaccines, disease protection correlates directly with the magnitude of the induced immune response.  For example, recent studies evaluating potential ALS vaccines found that animals with the highest antigen-specific antibody titers showed markedly increased survival times, whereas low antibody titers correlated with decreased survival time [19]. Therefore, inducing a sustained 10- to 100-fold increase in antigen-specific antibody titre may be critical for disease protection.

There was a clear difference in the duration of epitope-specific antibody responses following two immunizations with the tgG versus Lkt fusion proteins (Figure 1a). Epitope-specific antibody titers induced by tgG-RL remained at a similar level throughout the trial, and only began to decline following week 43, when compared to the peak titer at week 12 (p < 0.01). In contrast, Lkt-RL immunization induced maximum peptide-specific antibody titers on week four but peptide-specific titres declined significantly (p < 0.0001) within 12 weeks after immunization (Figure 1a). Previous investigators have reported a remarkable duration for gG-specific antibody titres following rabies virus vaccination [20]. We have shown that this remarkable duration of antibody responses to a specific viral protein can be conferred to an associated peptide hapten.

Single Immunization

We further evaluated the capacity of each chimeric construct to induce RL-specific antibody responses following a single immunization. Both carrier systems induced a marked increase in epitope-specific antibody titres three weeks after immunization (Figure 1b). A similar difference in peak antibody tires was again observed, with the tgG-RL fusion inducing a 10-fold greater antibody titer than the Lkt-RL fusion (p < 0.0001). Peptide-specific titers were again maintained at a relatively constant level following a single tgG-RL immunization. A significant decline in titres was delayed until 20 weeks following immunization when compared to the peak antibody titer at week nine (p < 0.02). In contrast, Lkt-RL-induced epitope-specific titers decreased significantly (p < 0.0001) by week 9 post-immunization when compared to peak antibody titer at week six. This experiment confirmed that a single immunization with tgG-RL was sufficient to induce an antibody response that was maintained for at least 44 weeks. From a practical perspective, there is considerable value in a vaccine technology capable of inducing rapid and sustained antibody responses following a single vaccination.


Figure 1: Magnitude and duration of antibody responses to the PrP epitope following immunizations with tgG-RL (tgG) or Lkt-RL (Lkt). C57/BL6 mice (n = 6/group) were injected subcutaneously with either 10 μg tgG-RL or Lkt-RL formulated in 30% Emulsigen D. Animals were immunized (red arrow) twice with a three-week interval (Figure 1A) or received a single immunization (red arrow) on day zero (Figure 1B). Antibody titers were quantified by capture ELISA using RL peptide, and are reported as mean values + 1 SD.


Antibody Isotype Bias

The isotype of epitope-specific antibodies induced by each carrier protein was further evaluated.  Mice injected tgG-RL developed primarily an IgG1 antibody response (Figure 2a), whereas mice injected with Lkt-RL developed both IgG1 and IgG2c antibody responses (Figure 2b). This difference in antibody isotypes was further explored by analyzing splenocyte cytokine production following in vitro re-stimulation with peptide antigen and carrier proteins. Re-stimulation with the RL peptide did not induce detectable cytokine secretion (Figure 3).  Re-stimulation with tgG protein induced primarily IL-5 secretion in mice immunized with tgG-RL protein (Figure 3). In contrast, re-stimulation with Lkt protein induced primarily IFNγ secretion by splenocytes isolated from mice previously with Lkt-RL protein (Figure 3b). These observations support the conclusion that the carrier proteins induced distinct T cell responses that were consistent with the isotype bias observed for epitope-specific antibodies. Therefore, it may be possible to use carrier proteins to influence the isotype of antibodies produced in response to peptide epitopes selected from self-proteins. It has been suggested that vaccines for neurodegenerative diseases may be most effective when inducing primarily an IgG1 antibody response [21,22].

Figure 2: The isotype of RL-specific IgG antibodies was analyzed nine weeks following a single subcutaneous immunization with either 10 μg tgG-RL (Figure 2A) or Lkt-RL (Figure 2B) formulated in 30% Emulsigen D. Data presented are values for individual C57BL/6 mice (n = 5/group) and the horizontal bar represents the mean value for each treatment group. IgG1 and IgG2c epitope-specific serum antibodies were quantified by capture ELISA using RL peptides. 


Figure 3: Cytokine secretion profile of splenocytes isolated from BALB/c mice 5 weeks after a single subcutaneous injection of either 10 μg tgG-RL (A) or Lkt-RL (B) formulated in 30% Emulsigen D. Results are expressed as the number of cytokine-secreting cells per million cells cultured in medium alone or re-stimulated with either RL peptide (peptide), tgG carrier protein (tgG), or Lkt carrier protein (Lkt). Data presented are the mean + 1SD of values from 6 mice per group.

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We thank VIDO animal care for assistance in conducting animal trials, Dr. Brownlie for providing the pEB4.3 expression plasmid, as well as technical expertise. We also thank Dr. Trent Bollinger and the Canadian Cooperative Wildlife Health Centre Western/Northern for supplying rabies infected tissue. This work was funded by support from PrioNet and Alberta Prion Research Institute. Dr. Philip Griebel is supported by a Tier I CRC with funds provided by the CIHR.

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Conflict Of Interest Statement


All authors have no conflicts of interests to disclose.

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Copyright: © 2015 Ryan Taschuk, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.