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A recombinant vaccine effectively induces c5a-specific neutralizing antibodies and prevents arthritis.

Nandakumar KS, Jansson A, Xu B, Rydell N, Ahooghalandari P, Hellman L, Blom AM, Holmdahl R - PLoS ONE (2010)

Bottom Line: Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases.Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.

View Article: PubMed Central - PubMed

Affiliation: Medical Inflammation Research, Department of Experimental Medicine, Lund University, Lund, Sweden. Nandakumar.kutty-selva@ki.se

ABSTRACT

Objectives: To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a.

Methods: We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology.

Results: Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.

Conclusions: Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.

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Determination of C5a-specific antibody levels and C5a neutralization.Anti-C5a antibody levels (A) in the sera collected from mice on day 35 immunized for CIA. As described in Fig. 1B, (BALB/c x B10.Q) F1 male mice (8 weeks old) received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA at day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. All the mice were immunized with 100 µg of rat CII in CFA on day 0. Anti-C5a antibody levels (B) and C5a neutralization capacity (C) present in the sera collected from mice on day −1 and day 21. The mice were vaccinated with MBP-C5a or PBS emulsified in CFA/IFA on days −21, −10 and −2 and used in CAIA experiment on day 0, as shown in Fig. 3. Anti-C5a antibody levels (D) present on day 35 in B10.Q mice and the C5 deficient congenic mice strain B10.Q.NOD-Cia2 (NB2). Groups of 8 weeks old mice received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA on day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. Upon vaccination with MBP-C5a protein, C5 deficient mice (NB2) responded with higher anti-C5a levels than C5 sufficient B10.Q mice (p = 0.018). n, indicates number of mice in each group. All the assays were done in triplicates.
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pone-0013511-g004: Determination of C5a-specific antibody levels and C5a neutralization.Anti-C5a antibody levels (A) in the sera collected from mice on day 35 immunized for CIA. As described in Fig. 1B, (BALB/c x B10.Q) F1 male mice (8 weeks old) received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA at day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. All the mice were immunized with 100 µg of rat CII in CFA on day 0. Anti-C5a antibody levels (B) and C5a neutralization capacity (C) present in the sera collected from mice on day −1 and day 21. The mice were vaccinated with MBP-C5a or PBS emulsified in CFA/IFA on days −21, −10 and −2 and used in CAIA experiment on day 0, as shown in Fig. 3. Anti-C5a antibody levels (D) present on day 35 in B10.Q mice and the C5 deficient congenic mice strain B10.Q.NOD-Cia2 (NB2). Groups of 8 weeks old mice received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA on day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. Upon vaccination with MBP-C5a protein, C5 deficient mice (NB2) responded with higher anti-C5a levels than C5 sufficient B10.Q mice (p = 0.018). n, indicates number of mice in each group. All the assays were done in triplicates.

Mentions: All individuals receiving C5a but not control vaccinations developed anti-C5a IgG antibodies (Fig. 4A and B). Subsequently, the ability of serum from C5a vaccinated mice to inhibit C5a-mediated degranulation was determined using a RBL cell line transfected with human C5aR. The amount of released β-hexosaminidase was directly proportional to the amount of C5a added within the range of 2000 ng/ml (240 ng per well) to 2.7 ng/ml (0.3 ng per well). Serial dilutions of serum were added to 5.6 ng of C5a and the degree of inhibition was determined. All the animals receiving C5a but not control vaccination displayed C5a inhibitory capacity (Fig. 4C). Upon vaccination with MBP-C5a protein, C5 deficient mice responded with higher anti-C5a levels than C5 sufficient mice (p = 0.018) and anti-C5a antibodies can be induced in the vaccinated C5 sufficient mice (Fig. 4D).


A recombinant vaccine effectively induces c5a-specific neutralizing antibodies and prevents arthritis.

Nandakumar KS, Jansson A, Xu B, Rydell N, Ahooghalandari P, Hellman L, Blom AM, Holmdahl R - PLoS ONE (2010)

Determination of C5a-specific antibody levels and C5a neutralization.Anti-C5a antibody levels (A) in the sera collected from mice on day 35 immunized for CIA. As described in Fig. 1B, (BALB/c x B10.Q) F1 male mice (8 weeks old) received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA at day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. All the mice were immunized with 100 µg of rat CII in CFA on day 0. Anti-C5a antibody levels (B) and C5a neutralization capacity (C) present in the sera collected from mice on day −1 and day 21. The mice were vaccinated with MBP-C5a or PBS emulsified in CFA/IFA on days −21, −10 and −2 and used in CAIA experiment on day 0, as shown in Fig. 3. Anti-C5a antibody levels (D) present on day 35 in B10.Q mice and the C5 deficient congenic mice strain B10.Q.NOD-Cia2 (NB2). Groups of 8 weeks old mice received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA on day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. Upon vaccination with MBP-C5a protein, C5 deficient mice (NB2) responded with higher anti-C5a levels than C5 sufficient B10.Q mice (p = 0.018). n, indicates number of mice in each group. All the assays were done in triplicates.
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pone-0013511-g004: Determination of C5a-specific antibody levels and C5a neutralization.Anti-C5a antibody levels (A) in the sera collected from mice on day 35 immunized for CIA. As described in Fig. 1B, (BALB/c x B10.Q) F1 male mice (8 weeks old) received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA at day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. All the mice were immunized with 100 µg of rat CII in CFA on day 0. Anti-C5a antibody levels (B) and C5a neutralization capacity (C) present in the sera collected from mice on day −1 and day 21. The mice were vaccinated with MBP-C5a or PBS emulsified in CFA/IFA on days −21, −10 and −2 and used in CAIA experiment on day 0, as shown in Fig. 3. Anti-C5a antibody levels (D) present on day 35 in B10.Q mice and the C5 deficient congenic mice strain B10.Q.NOD-Cia2 (NB2). Groups of 8 weeks old mice received vaccination subcutaneously of 100 µg MBP-C5a or PBS emulsified in CFA on day −21 and were re-vaccinated on days −3 and +28 with 50 µg of MBP-C5a or PBS emulsified in IFA. Upon vaccination with MBP-C5a protein, C5 deficient mice (NB2) responded with higher anti-C5a levels than C5 sufficient B10.Q mice (p = 0.018). n, indicates number of mice in each group. All the assays were done in triplicates.
Mentions: All individuals receiving C5a but not control vaccinations developed anti-C5a IgG antibodies (Fig. 4A and B). Subsequently, the ability of serum from C5a vaccinated mice to inhibit C5a-mediated degranulation was determined using a RBL cell line transfected with human C5aR. The amount of released β-hexosaminidase was directly proportional to the amount of C5a added within the range of 2000 ng/ml (240 ng per well) to 2.7 ng/ml (0.3 ng per well). Serial dilutions of serum were added to 5.6 ng of C5a and the degree of inhibition was determined. All the animals receiving C5a but not control vaccination displayed C5a inhibitory capacity (Fig. 4C). Upon vaccination with MBP-C5a protein, C5 deficient mice responded with higher anti-C5a levels than C5 sufficient mice (p = 0.018) and anti-C5a antibodies can be induced in the vaccinated C5 sufficient mice (Fig. 4D).

Bottom Line: Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases.Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.

View Article: PubMed Central - PubMed

Affiliation: Medical Inflammation Research, Department of Experimental Medicine, Lund University, Lund, Sweden. Nandakumar.kutty-selva@ki.se

ABSTRACT

Objectives: To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a.

Methods: We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology.

Results: Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.

Conclusions: Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.

Show MeSH
Related in: MedlinePlus