Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horses

Abstract

Horses that have undergone infection caused by Streptococcus equi subspecies equi (strangles) were found to have significantly increased serum antibody titers against three previously characterized proteins, FNZ (cell surface-bound fibronectin binding protein), SFS (secreted fibronectin binding protein), and EAG (alpha2-macroglobulin, albumin, and immunoglobulin G [IgG] binding protein) from S. equi. To assess the protective efficacy of vaccination with these three proteins, a mouse model of equine strangles was utilized. Parts of the three recombinant proteins were used to immunize mice, either subcutaneously or intranasally, prior to nasal challenge with S. equi subsp. equi. The adjuvant used was EtxB, a recombinant form of the B subunit of Escherichia coli heat-labile enterotoxin. It was shown that nasal colonization of S. equi subsp. equi and weight loss due to infection were significantly reduced after vaccination compared with a mock-vaccinated control group. This effect was more pronounced after intranasal vaccination than after subcutaneous vaccination; nearly complete eradication of nasal colonization was obtained after intranasal vaccination (P < 0.001). When the same antigens were administered both intranasally and subcutaneously to healthy horses, significant mucosal IgA and serum IgG antibody responses against FNZ and EAG were obtained. The antibody response was enhanced when EtxB was used as an adjuvant. No adverse effects of the antigens or EtxB were observed. Thus, FNZ and EAG in conjunction with EtxB are promising candidates for an efficacious and safe vaccine against strangles.

FIG. 1.

Schematic presentation of the three S. equi proteins EAG, SFS, and FNZ. Abbreviations: S, the signal sequence; W, the cell wall-associated domain; M, the membrane-spanning region. For protein EAG the albumin-binding region Alb is indicated. For proteins FNZ and SFS different repetitive regions are indicated by R. Arrows indicate the part of the respective protein that was used for antigen preparation. The deduced molecular mass for proteins EAG4B, SFSC1, and FNZN are indicated.

FIG. 2.

Antibody titers in sera from horses. The log₁₀ dilution of sera required to give an absorbance value at a cutoff of 1.0 was calculated for each individual serum sample. Mean values with standard errors are shown. P values for comparing normal (n = 16) versus strangles (n = 10) sera against FNZ, SFS, and EAG are <0.001, 0.02, and <0.0001, respectively.

FIG. 3.

Antibody titers in sera from mice. The log₁₀ dilution of sera required to give an absorbance value at a cutoff of 0.5 was calculated for each individual serum sample. Mean values with standard errors are shown. Samples taken before challenge, which at the lowest dilution (20 times) gave an absorbance value of <0.5, were assigned a value of 1.30. P values for comparing sera taken before (pre) challenge and during infection against all three antigens are <10⁻⁴.

FIG. 4.

Antibodies in immunized mice. Seventeen mice were immunized i.n. with FNZ, SFS, and EAG, 11 of the mice with EtxB and 6 without EtxB. Panel A shows the mean values and standard error of IgA levels against EAG in BAL fluid (P = 0.02). Panel B shows the mean values and standard error of serum dilutions giving an absorbance of 1.0 for the three antigens by an ELISA detecting all classes of antibodies (n = 4 for animals where EtxB was included; n = 3 for animals without EtxB). Panel C shows IgA levels in sera (same animals as described in the legend to 4B) at 40 dilutions. White bars, preimmunization; gray bars, after immunization.

FIG. 4.

Antibodies in immunized mice. Seventeen mice were immunized i.n. with FNZ, SFS, and EAG, 11 of the mice with EtxB and 6 without EtxB. Panel A shows the mean values and standard error of IgA levels against EAG in BAL fluid (P = 0.02). Panel B shows the mean values and standard error of serum dilutions giving an absorbance of 1.0 for the three antigens by an ELISA detecting all classes of antibodies (n = 4 for animals where EtxB was included; n = 3 for animals without EtxB). Panel C shows IgA levels in sera (same animals as described in the legend to 4B) at 40 dilutions. White bars, preimmunization; gray bars, after immunization.

FIG. 4.

Antibodies in immunized mice. Seventeen mice were immunized i.n. with FNZ, SFS, and EAG, 11 of the mice with EtxB and 6 without EtxB. Panel A shows the mean values and standard error of IgA levels against EAG in BAL fluid (P = 0.02). Panel B shows the mean values and standard error of serum dilutions giving an absorbance of 1.0 for the three antigens by an ELISA detecting all classes of antibodies (n = 4 for animals where EtxB was included; n = 3 for animals without EtxB). Panel C shows IgA levels in sera (same animals as described in the legend to 4B) at 40 dilutions. White bars, preimmunization; gray bars, after immunization.

FIG. 5.

Protection against infection by S. equi subsp. equi. Mice (n = 12) were immunized with FNZ, SFS, and EAG with EtxB (filled symbols), and control groups (n = 12) were given only EtxB (open symbols). Mean values with standard errors are shown. Panels A and B show results after i.n. immunization; panels C and D show results after s.c. immunization. Panels A and C show growth of S. equi subsp. equi in noses. The nasal growth of S. equi subsp. equi was determined by placing the noses against BG plates, and the bacteria were streaked out to single colonies. The number of bacteria was given a value from 0 to 3, where 0 means no growth or <8 colonies, 1 means 9 to ca. 100 colonies, 2 means more than ca. 100 colonies, and 3 means confluent growth. Panels B and D show the daily percentage weight loss. Significance values are given in the text.

FIG. 6.

IgA antibodies in nasal washings of immunized horses. Mean absorbance values (n = 3) in ELISAs are shown for groups A to D. The horses were divided into four groups: group A received the antigens plus EtxB, given both s.c. and i.n.; group B received the same immunizations as group A but given only i.n.; group C received the same treatment as group A but without EtxB; and group D (control group) was given only EtxB both i.n. and s.c. Open bars, samples from day 0; and filled bars, samples from day 56.

FIG. 7.

IgG antibodies in sera of immunized horses. The log dilution of sera required to give an absorbance value at a cutoff of 1.0 was calculated for each individual serum sample. Mean values (n = 3) with standard errors are shown. Samples taken before (day 0) and after (day 56) immunizations are shown. The horses, the same as described in the legend of Fig. 6, were divided into four groups: group A (white bars) received the antigens plus EtxB, given both s.c. and i.n.; group B (striped bars) received the same immunizations as group A but given only i.n.; group C (gray bars) received the same treatment as group A but without EtxB; and group D (black bars; control group) was given only EtxB both i.n. and s.c..