Cross-protective effect of Strangvac during a natural episode of Streptococcus zooepidemicus respiratory disease in ponies

Cross-protective effect of Strangvac during a natural episode of Streptococcus zooepidemicus respiratory disease in ponies | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 25 February 2026 V1 Latest version Share on Cross-protective effect of Strangvac during a natural episode of Streptococcus zooepidemicus respiratory disease in ponies Authors : Romain Paillot 0000-0003-3081-2037 [email protected] , J. Richard Newton , Sonia Gonzalez-Medina 0000-0001-6854-7879 , Sara Frosth 0000-0002-4080-5183 , Lars Frykberg , Margareta Flock , Bengt Guss , Jan-Ingmar Flock , and Andrew Waller 0000-0002-7111-9549 Authors Info & Affiliations https://doi.org/10.22541/au.177206235.50501700/v1 410 views 138 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Streptococcus equi subspecies zooepidemicus is a highly diverse opportunistic pathogen of horses, associated with respiratory disease and endometritis. Objectives: To characterise S. zooepidemicus isolates recovered from young ponies during a natural episode of respiratory disease, and to determine if vaccination with Strangvac, a vaccine against Streptococcus equi subspecies equi , conferred cross-protection. Study design: Retrospective analysis of a double-blinded placebo-controlled study involving 32 ponies (16 vaccinates and 16 placebo controls) that experienced a natural episode of respiratory disease around the time of second vaccination. Methods: Ponies were monitored daily for clinical signs of respiratory infection. Nasopharyngeal swabs were taken from affected animals to identify equine pathogens by qPCR and culture. The genomes of 23 S. zooepidemicus isolates were sequenced. Clinical and serological differences between vaccine and placebo groups were determined. Results: The clinical samples were positive for equine herpes virus-4 or S. zooepidemicus (1/15 (7%) and 13/15 (87%), respectively). Seven different S. zooepidemicus sequence types, which encoded between four and seven of the eight antigens in Strangvac with ≥70% amino acid identity, were recovered from affected horses. The most conserved antigens were CNE, EAG, Eq5 and IdeE. The number of days that ponies had a cough were significantly associated ( p =0.005) with the vaccination status, with fewer days in the vaccinated compared to co-mingled control ponies (1.8±1.9 days vs. 3.4±3.5 days). A higher antibody titre to IdeE immediately prior to second vaccination correlated with a lower cumulative coughing score ( p =0.03). A reduced number of days with abnormal temperature correlated with increased antibody levels against Eq85 ( p =0.023-0.028). Main limitations: Small group size. Conclusions: This study provides evidence in support of a cross-protective effect of Strangvac for the reduction of clinical signs associated with natural infection with S. zooepidemicus , which correlated with the presence of higher antibody titres to IdeE and Eq85. Cross-protective effect of Strangvac during a natural episode of Streptococcus zooepidemicus respiratory disease in ponies SUMMARY: Background: Streptococcus equi subspecies zooepidemicus is a highly diverse opportunistic pathogen of horses, associated with respiratory disease and endometritis. Objectives: To characterise S. zooepidemicus isolates recovered from young ponies during a natural episode of respiratory disease, and to determine if vaccination with Strangvac, a vaccine against Streptococcus equi subspecies equi , conferred cross-protection. Study design: Retrospective analysis of a double-blinded placebo-controlled study involving 32 ponies (16 vaccinates and 16 placebo controls) that experienced a natural episode of respiratory disease around the time of second vaccination. Methods: Ponies were monitored daily for clinical signs of respiratory infection. Nasopharyngeal swabs were taken from affected animals to identify equine pathogens by qPCR and culture. The genomes of 23 S. zooepidemicus isolates were sequenced. Clinical and serological differences between vaccine and placebo groups were determined. Results: The clinical samples were positive for equine herpes virus-4 or S. zooepidemicus (1/15 (7%) and 13/15 (87%), respectively). Seven different S. zooepidemicus sequence types, which encoded between four and seven of the eight antigens in Strangvac with ≥70% amino acid identity, were recovered from affected horses. The most conserved antigens were CNE, EAG, Eq5 and IdeE. The number of days that ponies had a cough were significantly associated ( p =0.005) with the vaccination status, with fewer days in the vaccinated compared to co-mingled control ponies (1.8±1.9 days vs. 3.4±3.5 days). A higher antibody titre to IdeE immediately prior to second vaccination correlated with a lower cumulative coughing score ( p =0.03). A reduced number of days with abnormal temperature correlated with increased antibody levels against Eq85 ( p =0.023-0.028). Main limitations: Small group size. Conclusions: This study provides evidence in support of a cross-protective effect of Strangvac for the reduction of clinical signs associated with natural infection with S. zooepidemicus , which correlated with the presence of higher antibody titres to IdeE and Eq85. Keywords: Streptococcus zooepidemicus , coughing, respiratory disease, vaccine 1. Introduction (400 words): Streptococcus equi subspecies zooepidemicus ( S. zooepidemicus ) is associated with a wide range of diseases in horses and other animals [1–6]. The presence of S. zooepidemicus is significantly associated with respiratory disease and poor performance in young Thoroughbred racehorses [7–9] with a reported incidence of 8.9 cases/100 horses/month [8,10]. Infections occur as different peer-groups mix when they enter training or competitions [7] with some episodes leading to the death of the affected horse [2,11]. The prevalence of respiratory disease associated with S. zooepidemicus decreases in association with increasing age, consistent with exposure to different strains and the development of natural cross-protective immunity [8,12,13]. The development of a multilocus sequence typing (MLST) scheme has facilitated the identification of different groups of genetically related strains [1] and has been utilised to identify particular sequence types (STs) of S. zooepidemicus recovered from outbreaks of respiratory disease in horses [14,15]. The increased discriminatory capacity of whole genome sequencing was also used to differentiate strains of S. zooepidemicus , and to identify the cause of an epidemic of equine respiratory disease in Iceland, which had affected almost the entire geographically-isolated national population of 77,000 horses and led to a self-imposed ban on the export of horses for a four-and-a-half-month period [11]. The MLST scheme currently defines 565 STs, including ten STs of the S. zooepidemicus biovar, Streptococcus equi subspecies equi ( S. equi ) [1]. S. equi is the causative agent of the disease known as ‘strangles’ in horses, which is characterised by abscessation of the lymph nodes of the head and neck. Analysis of MLST and genome sequencing data identified that S. equi evolved from, and is actually an equine-adapted subspecies of, S. zooepidemicus [1,16]. S. equi shares virulence factors, with strains of S. zooepidemicus [16], suggesting that the vaccination of horses against strangles might provide cross-protection against S. zooepidemicus . A multicomponent fusion protein vaccine, Strangvac, provided 94% protection to ponies against experimental challenge with S. equi in one of the clinical trials [17]. Recent publications have reported the effectiveness of Strangvac for the protection of horses against natural exposure to S. equi [18,19]. The high levels of protection conferred by Strangvac against natural infection with S. equi raise the possibility that this vaccine may confer significant levels of cross-protection against natural exposure to S. zooepidemicus subspecies. Here we report the retrospective analysis of an episode of respiratory disease, characterised by coughing, that occurred during one of the clinical trials of Strangvac. 2. Materials and Methods: 2.1. Vaccine production . Strangvac is based on CNE, SclC, SclF, SclI and EAG (fused as CCE), Eq8 and Eq5 (fused as Eq85) and IdeE. The recombinant antigens were produced in E. coli BL21, purified and formulated with Matrix V adjuvant (Novavax) as described previously [17]. 2.2. Immunisation of ponies . The study was previously described as study IV by Robinson et al. (2020) [17]. Data considered for the current report cover the period day 0 (D0) to D70 (42 days post-V2). Ponies between 4 and 6 months of age (5.3±0.3 months, n = 32) were assigned randomly into two groups of 16. The ponies were then administered 2 ml of Strangvac, or a placebo vaccine that contained 326 µg of Matrix C, a saponin-derived adjuvant (Novavax AB, Uppsala, Sweden), via intramuscular injection into the neck on D0 and D28 using a double-blinded protocol such that the vaccination status of all ponies was blinded to study personnel and attending veterinarians throughout the experimental phases. Ponies were maintained together in barns and clinical signs were recorded daily. The detail of the clinical examination scoring is presented in supplementary Table S1. 2.3. Respiratory disease outbreak and sample collection. The ponies experienced an episode of respiratory disease, probably caused by S. zooepidemicus infections, which was first identified on D21 post-V1. Ten of the 32 ponies (six controls and four vaccinates) were identified as suffering from mild respiratory disease with clinical signs of coughing and elevated body temperatures. Nasopharyngeal swabs were taken by the attending veterinarian from 12 ponies on D21 and tested by the diagnostic laboratory at the Animal Health Trust for the presence of equine herpes virus type-1 and -4 (EHV-1 and EHV-4), and S. zooepidemicus using qPCR, but not for equine influenza virus (EIV) at this stage. On D22, 23 ponies (13 controls and ten vaccinates) received a five-day treatment with trimethoprim and sulfadiazine (for 150 kg body weight, once daily per os ). The clinical signs of coughing reduced in response to treatment and ponies were deemed fit to receive second vaccination on D28. However, clinical signs of coughing returned five days after the second vaccination. Nasopharyngeal swabs were taken by the attending veterinarian from another three ponies (two controls and one vaccinate) on D34 and submitted to the diagnostic laboratory at the Animal Health Trust for analysis. Among these samples, all tested negative for EHV-1 and EIV, one tested positive for EHV-4 and all three samples tested positive for S. zooepidemicus by qPCR. Further antibiotic treatment was given (five-day course of penicillin via intramuscular route; details are presented in supplementary Table S2). Nasopharyngeal swab samples were collected from all ponies as part of the study protocol on D28 and D35. Surplus samples collected from these swabs were available from 10 and eight ponies that were collected on D28 and D35, respectively, which were used to isolate S. zooepidemicus by culture on COBA streptococcal selective agar (BioMérieux). Isolates were stored at -20 o C prior to genome sequencing. Blood samples were also collected from all participants as part of the study protocol every week between D0 and D42, and then every two weeks until D70. Samples were allowed to clot at room temperature for two hours and the serum was then removed and stored at -20 o C. 2.4. DNA sequencing and phylogenomic analysis A single colony of each isolate of S. zooepidemicus was inoculated onto bovine blood (5%) agar plates ({masked for review}, Uppsala, Sweden) and incubated overnight at 37 o C in a humidified atmosphere containing 5% CO 2 . DNA was prepared using the Qiagen XL Advanced nucleic acid extraction system (Qiagen, Hilden, Germany) and the EZ1 DNA Tissue Kit (Qiagen). Approximately one-third of a 20 µL inoculation loop of bacterial culture was suspended in 180 µL G2 lysis buffer (EZ1 Tissue Kit, Qiagen). To assist lysis of the S. zooepidemicus cells, 15 μL of mutanolysin (5000 U/mL) (Sigma−Aldrich, St. Louis, MO, USA) and 15 μL of lysozyme (100 mg/mL) (Sigma−Aldrich) were added and the samples were incubated in a ThermoMixer (Eppendorf, Hamburg, Germany) at 37 o C and 300 rpm for 4 h. The temperature was then raised to 54 o C, 15 μL of proteinase K (EZ1 Tissue Kit, Qiagen) was added, and incubation continued for another 4 h. The samples were then frozen at -70 o C and after thawing at 20 o C, 2.5 μL of Ambion RNase Cocktail Enzyme Mix (Invitrogen, Carlsbad, CA, USA) was added and samples incubated for 10 min. After a final incubation at 50 o C for 30 min, DNA was extracted according to the manufacturer’s instructions. The elution volume used was 100 μL and the DNA concentration was measured using a Qubit 2.0 instrument (Invitrogen). DNA libraries were prepared using Illumina’s Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Normalization of libraries was done manually using the DNA concentrations obtained by a Qubit dsDNA High Sensitivity Kit and average library sizes from a High Sensitivity DNA ScreenTape Analysis D1000 Kit (Agilent Technologies, Santa Clara, CA, USA) and a 4150 TapeStation instrument (Agilent Technologies). Sequencing of 23 S. zooepidemicus isolates was performed using an Illumina NextSeq 500 benchtop sequencer (Illumina) with 2 x 150 bp paired-end reads. The raw sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1420222, with BioSample accession numbers SAMN55212160–SAMN55212182. Obtained sequencing reads were analysed using the MBioSEQ Ridom Typer software (Ridom GmbH, Münster, Germany). The quality of the sequenced reads was examined using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adapter content removed by Trimmomatic [20]. Fastq files were assembled de novo using SKESA [21] with default settings. Mash Screen [22] was used to check the assemblies for possible contamination. For comparison, Fastq files from 72 diverse S. zooepidemicus isolates previously reported by [23] were downloaded and processed using the same bioinformatic workflow as described above. In addition, genome assemblies of strain H70 ( Sz H70) [16] and MGCS10565 [24] were included in the analyses. Genome assemblies for all 97 isolates of S. zooepidemicus were uploaded into the Pathogenwatch bioresource for S. equi (https://cgps.gitbook.io/ pathogenwatch/), which assigns phylogeny based on the sequences of 1286 alleles in the core genome of S. equi as described previously [25]. The collection in Pathogenwatch can be accessed at: https://pathogen.watch/collection/gq17xsdkwark-97-s-zooepidemicus. The DNA sequences encoding the eight antigen fragments in Strangvac of CNE (SEQ_0935), EAG (SEQ_0721), SclF (SEQ_0855), SclI (SEQ_1817), SclC (SEQ_2101), Eq8 (SEQ_0402), Eq5 (SEQ_0256) and IdeE (SEQ_0999) were extracted in silico from the genome sequencing data using the MBioSEQ Ridom Typer software (Ridom GmbH). Differences in the predicted amino acid sequences of the antigens were identified using MEGA 12 [26]. MLST alleles were extracted using the BIGSdb software [27]. 2.5. Quantification of the immune response to vaccination . Antibody responses to the vaccine antigens (CCE, IdeE and Eq85) were measured by conventional iELISA as previously described [28]. Samples were diluted serially two-fold and the log10 value of the dilution required to obtain an absorbance value below a cut off threshold of 1.5 was determined. 2.6. Statistical methods . Assumptions of the normal distributions of data were measured with the Shapiro-Wilk test. The association between the vaccination status and clinical scores was tested with the ꭕ² and the Fisher exact tests. The parametric independent student’s t-test or the non-parametric Mann-Whitney U test were used to measure differences in clinical scores between vaccinates and controls. The repeated ANOVA test (with Greenhouse-Geisser correlation when required) was used for primary analyses of the kinetics of antibody responses to the vaccine antigens. Post-hoc pairwise comparisons were performed using Bonferroni-corrected tests. To measure the significance and direction of the relationship between clinical scores and antibody titres, the Pearson and the Spearman’s Rank tests were used. Multiple linear regression analysis was used to assess the combined effects of the antibody titres to the different vaccine antigens relative to the clinical scores. Cumulative cough scores measured between D28 and D46 were square root transform to maintain homogeneity of variance. Statistical significance for all analyses was set at p ≤ 0.05. 2.7. Ethical considerations . This work was conducted under the conditions of a Home Office Project License and following ethical review and approval by the Animal Health Trust’s Animal Welfare and Ethical Review Body (RPP 01_08). The ponies ultimately received a third vaccination at three months after the second dose and were challenged two weeks later by spraying 100 million cells of S. equi strain 4047 into the nasopharynx of each pony. All of the control ponies developed clinical signs of strangles compared with one of the ponies vaccinated with Strangvac as reported previously (EXP IV in Robinson et al ., 2020) [17]. 3. Results: 3.1. Genetic relationships between the isolates of S. zooepidemicus. The genetic relationships of the isolates were defined by Pathogenwatch and MLST and viewed in Microreact (https://microreact.org/project/mFAsN4XhqVsqGhjUZzqPyS-newton-et-al, Figure 1). The overall collection of S. zooepidemicus was diverse, differing by an average of 27,988 SNPs across the core genome defined by Mitchell et al ., 2021 [25]. The 23 isolates of S. zooepidemicus that were recovered from the study were differentiated into seven different STs. Two ST-49 isolates (5305 and 8457.3) differed by 11 SNPs from each other and by 26 and 23 SNPs from ERR14109311, which shared the same ST. Six ST-118 isolates (2156, 2434.1, 7411.1, 8320, 8457.1 and 8457.2) were genetically identical and clustered most closely to the ST-81 isolate, ERR14109354, although the genome of this isolate differed by 6,555 SNPs relative to the ST-118 isolates. Three ST-300 isolates (1819.1, 1819.2 and 8490) differed from each other by 0 and 6 SNPs, and from the ST-386 isolate, ERR14109266, by up to 3,767 SNPs. The two ST-43 isolates (7411.2 and 9756) were genetically identical and clustered most closely to the ST-15 isolate, ERR14109383, which differed by 8,000 SNPs. The four ST-103 isolates (929, 2943.1, 2943.2 and 6030) differed by up to 1 SNP, and from the ST-471 isolate (ERR14109124) by 1,615 SNPs. The ST-366 isolate 2434.2 did not cluster with any other isolates that were recovered from coughing ponies and differed from the closest isolate (ERR14109498, ST-485) by 24,932 SNPs. Finally, the five ST-418 isolates (114.1, 114.2, 363.1, 363.2 and 2448) were genetically identical and differed from the ST-62 isolate (ERR14109312) by 12,206 SNPs. 3.2. Sequence conservation between Strangvac antigens and S. zooepidemicus isolates Homologues of the antigens in Strangvac were encoded by all of the 97 isolates of S. zooepidemicus used for comparison, including the 23 isolates recovered from ponies in this study (Table 1 and supplementary Table S3). A conservative requirement of ≥70% amino acid identity was applied for the purpose of identifying homologues that were most likely to be targeted by cross-protective immune responses induced by vaccination. Across the 97 genomes of S. zooepidemicus , the homologues of EAG and IdeE had the highest levels of amino acid conservation relative to the Strangvac antigens with averages of 96.1% and 97.7%, amino acid sequence identity in 99.0% and 86.6% of isolates, respectively (Table 1). One isolate, UBA1577-2, which was recovered from a horse in Argentina encoded only two of the eight antigens in Strangvac with ≥70% amino acid identity (Figure 1). Twelve, 41, 31, seven and five isolates encoded three, four, five, six or seven of the Strangvac antigens, respectively. Overall 84/97 (86.6%) of the S. zooepidemicus isolates encoded at least four of the antigens in Strangvac with ≥70% amino acid identity. All of the 23 isolates recovered from coughing ponies encoded homologues of CNE, EAG, Eq5 and IdeE with ≥70% amino acid identity. The four ST-103 isolates that were recovered from coughing horses encoded antigens with the greatest homology to those in Strangvac, sharing CNE, EAG, SclF, SclI, SclC, Eq5 and IdeE at ≥70% amino acid identity. 3.3. Strangvac vaccination and protection against clinical respiratory disease Thirteen control and 10 vaccinated ponies received treatment with trimethoprim and sulfadiazine between D22 and D26. Therefore, only results from D28 (which was 2 days after the cessation of antibiotic treatment and the day of second vaccination, V2) were considered for the following analyses. The antibiotic treatment administered between D22 and D26 had no statistically significant effect on the clinical signs of disease observed between D28 and D46. Statistically significant associations were evident between the vaccination status (i.e. control or vaccinated) and the number of days with cough and marked cough (score = 2) post-V2 (measured from D28 to D46; p = 0.0048 and p = 0.0037, respectively). The proportions of days with any cough recorded during this period were 17.8% (3.4 ±3.5 days per individual) in the control group and 9.9% (1.8 ±1.9 days per individual) in the vaccinated group. When only marked cough was considered, the proportions were 4.6% (0.9 ±1.1 days per individual) in controls and 0.66% (0.1 ±0.3 days per individual) for the vaccinates. Whilst the individual cumulative cough scores (measured from D28 to D46) were not significantly different ( p =0.14) between the two groups (4.25 ±4.4 and 1.9 ±1.9, respectively), the individual cumulative marked cough scores measured during this period were significantly higher in control ponies when compared with vaccinated ponies (1.75 ±2.3 and 0.25 ±0.7, respectively; p = 0.035, Figure 2). No other significant effects of the vaccination status were measured for the other clinical signs of disease recorded during this period. 3.4. Relationship between antibody titres induced by Strangvac and clinical signs of respiratory disease. 3.4.1. Antibody titres in serum. Only two vaccinated ponies showed marked cough and both were only affected for one day. Pony #3121 had significantly lower antibody titres to CCE and IdeE when measured after V2 (D35, D42 and D56 responses combined) than the rest of the vaccinated group ( p = 0.011 and p = 0.009, respectively). The antibody titres of this pony to Eq85 were not significantly different from other vaccinated animals ( p = 0.84). While lower antibody titres to the most conserved vaccine antigens may provide an explanation for the susceptibility of this pony, Pony #2997 had antibody titres to CCE and IdeE that were close to the average for the vaccinated group. It was hypothesised that protective antibody titres measured prior to, or during, S. zooepidemicus infection would be negatively correlated to clinical signs of disease. The Spearman rank test showed a statistically significant negative correlation between IdeE titres measured immediately prior to V2 (D28) and the cumulative coughing score measured in vaccinated ponies from D28 to D46 (i.e. high antibody titres to IdeE correlated with low coughing scores; r(14) = -0.54, p = 0.031). Multiple linear regression indicated that there was a moderate collective significant effect between antibody titres to CCE, IdeE, Eq85 and the cumulative cough score (F(1, 14) =5.8, p = 0.030, R 2 = 0.29, R 2 adj = 0.24), with the antibody titre to IdeE identified as a significant predictor. No significant correlation was measured between antibody titres to CCE or Eq85 and the cumulative coughing score ( p >0.82). When the analysis was adjusted to D35 (for both measured antibody titres and cumulative cough scores) to consider the boosting effect of V2, the results of the multiple linear regression analyses indicated that there was also a moderate collective significant effect between the antibody titres to CCE, IdeE, Eq85. However, individual assessments showed that the cumulative cough score correlated with the antibody titre to IdeE on D35 (F(1, 14) = 7.19, p = 0.018, R 2 = 0.34, R 2 adj = 0.29) and there was no significant correlation between the antibody titres to CCE or Eq85 on D35 and the cumulative coughing score (P >0.52; Figure 3). When analysing other clinical signs, results of the multiple linear regression analysis indicated that there was a moderate collective significant effect between the antibody titres to CCE, IdeE and Eq85 that were measured on D35 or D42 and the individual cumulative number of days with abnormal temperature (i.e. >38.8°C) measured from D35 to D46 (F(1, 14) = 6.03-6.55, p = 0.023-0.028, R² = 0.3-0.32, R² adj = 0.25-0.27). Antibody titres to Eq85 were identified as the significant predictor at both time points, with no significant correlation identified between the antibody titres to CCE or IdeE, individually (P >0.12) and the cumulative abnormal temperature (Figure 4). There was no significant collective effect identified with D28 antibody titres ( p = 0.6) and abnormal temperatures measured from D28 to D34 were considered to be potential post-vaccination adverse events and excluded from the analysis [17]. No other significant relationships were identified between the antibody titre to the vaccine antigens and other markers of disease. 3.4.2. Antibody titres in nasopharyngeal secretions. Statistically significant positive correlations were identified between serum and nasopharyngeal secretion antibody titres measured between D28 and D45 (Table 2). Results of the multiple linear regression indicated that there was a strong collective significant effect between the antibody titres to CCE, IdeE, Eq85 in nasopharyngeal swab samples collected on D35 and D42 and the cumulative coughing score measured in vaccinated ponies from D35 to D46 (F(2, 13) = 4.68-8.32, p = 0.029-0.005, R² = 0.42-0.56, R² adj = 0.33-0.49), with antibody titres to IdeE and Eq85 identified as the significant predictors at both time points. Results of the multiple linear regression analysis indicated that there was a strong collective significant effect between the antibody titres to CCE, IdeE and Eq85 that were measured in nasopharyngeal swab samples collected on D35 and the individual cumulative number of days with abnormal temperature (i.e. >38.8°C) measured from D35 to D46 (F(1, 14) = 7.96, p = 0.014, R 2 = 0.36, R 2 adj = 0.32), with antibody titres to IdeE identified as the significant predictor. A statistically significant negative correlation was also measured between IdeE antibody titres in nasopharyngeal swab samples collected on D35 and abnormal temperature (D35-D46; r(14) = -0.602, p = 0.014; Figure 5). 3.5. Impact of S. zooepidemicus infection on CCE, IdeE and Eq85 antibody titres. 3.5.1. Fluctuation of background CCE, IdeE and Eq85 antibody levels in the control group. While the overall antibody titres to IdeE remained below the level defined as post-vaccination-positive in control unvaccinated ponies, the repeated measures ANOVA test (with Greenhouse-Geisser correlation used) of background IdeE antibody titres measured from D28 to D70 indicated a significant difference between the time points ( F (1.77, 24.84) = 12.31, p <0.001). The pairwise comparison with application of a strict Bonferroni correction that set the significance level at p ≤0.005, reported that the D28 IdeE titres (2.85 ±0.7) and D35 IdeE titres (2.89 ±0.38) were significantly lower than D42 titres (2.97 ±0.41; p = 0.0016 and p = 0.0016, respectively), D56 titres (3.00 ±0.44; p = 0.0022 and p = 0.0032, respectively) and D70 titres (3.07 ±0.47; p = 0.0013 and p = 0.0012, respectively). D28 and D35 IdeE titres were not significantly different ( p = 0.025) and D42 and D56 IdeE titres were not significantly different ( p = 0.07). IdeE titres returned to D28 and D35 titres levels on D126 and D132 ( F (1.1, 15.38) = 6.29, p = 0.022 with significance set at p ≤0.008 with application of a strict Bonferroni correction). The repeated measures ANOVA test (with Greenhouse-Geisser correlation) of background CCE antibody titres measured from D28 to D70 also indicated a significant difference between the time points ( F (2.48, 34.74) = 6.37, p = 0.003). The pairwise comparison with Bonferroni correction (significance set at p ≤ 0.005) reported that D28 CCE titre (2.5 ±0.145) was significantly lower than D35 CCE titre (2.55 ±0.15; p = 0.0009), borderline non-significantly lower than D42 titres (2.55 ±0.14; p = 0.007), significantly lower than D56 titres (2.57 ±0.13; p = 0.003) and D70 titres (2.58 ±0.11; p = 0.002). Titres measured at later sampling time points (D84 and D98) returned to D28 titres levels ( F (1.45, 20.33) = 2.84, p = 0.095). D35, D42 and D56 titres were not significantly different ( p >0.09). No significant differences were measured in antibody titres to Eq85 when measured from D28 to D70 ((1.99, 27.81) = 1.95, p = 0.161). 3.5.2. Impact of S. zooepidemicus infection on CCE, IdeE and Eq85 antibody titres in vaccinates. The seroconversion induced by V2 and subsequent decrease of antibody titres was likely obscuring a modification of CCE, IdeE or Eq85 antibody titres due to S. zooepidemicus infection itself. As a result, the relationship between the antibody titres and the extent of S. zooepidemicus infection was investigated. It was hypothesised that antibody titres after S. zooepidemicus infection would be positively correlated to the disease severity (i.e. a higher level of disease would result in higher immune stimulation and lead to higher antibody titres). The cumulative score of nasal discharge between D28 and D46 was used as a clinical marker of likely S. zooepidemicus infection. Nasal discharge was the most commonly observed clinical sign during this episode of S. zooepidemicus infection, with severity scores ranging from 0 to 4 (supplementary Table S1). Its clear association with respiratory tract pathology makes it less likely to be influenced by vaccination‑related adverse effects, in contrast to systemic manifestations such as fever. The relationship between clinical score and antibody titres was measured prior to S. zooepidemicus infection (D14, D21 or early phase of infection D28) and after S. zooepidemicus infection (D56, D70 and D84). The multiple linear regression analyses reported no significant effect between D-1, D21 or D28 antibody titres and the cumulative nasal discharge score (D28-D46): D-1 ( F (1, 14) = 0.48, p = 0.499, R 2 = 0.03, R 2 adj = -0.04), D21 (( F (1, 14) = 0.67, p = 0.428, R 2 = 0.05, R 2 adj = -0.02) and D28 ( F (1, 14) = 0.34, p = 0.570, R 2 = 0.02, R 2 adj = -0.05). However, after S. zooepidemicus infection the multiple linear regression analyses identified a significant positive effect between D56, D70 and D84 antibody titres and the cumulative nasal discharge score (D28-D56): D56 ( F (1, 14) = 15.75, p = 0.001, R 2 = 0.53, R 2 adj = 0.5); D70 ( F (1, 14) = 21.12, p <0.001, R 2 = 0.6, R 2 adj = 0.57) and D84 ( F (1, 14) = 19.47, p <0.001, R 2 = 0.58, R 2 adj = 0.55) with CCE antibody titre being the most affected. Representative data (D21 and D70) are shown in Figure 6. 4. Discussion: Streptococcus zooepidemicus is an important opportunistic pathogen of horses and other animals. Genome sequencing was used previously to investigate an epidemic of respiratory disease in Iceland during 2010 [11]. The 81 ST-209 isolates of S. zooepidemicus that were recovered from 45 horses across 21 farms over a period of 152 days, a cat, and the blood of a lady who had suffered a miscarriage following contact with infected horses, differed by less than 25 SNPs, thereby confirming that this strain had spread rapidly through the Icelandic horse population and was the cause of the epidemic. Similarly, the six ST-118 isolates recovered from five ponies, two ST-43 isolates recovered from two ponies, four ST-103 isolates recovered from three ponies and five ST-418 isolates recovered from three ponies were either genetically identical or differed by only one SNP. The ST-118 isolates, ST-43 isolates and ST-103 isolates currently listed on the MLST database (Streptococcus zooepidemicus | PubMLST) were recovered from the respiratory tract of horses. Whilst no isolates of ST-418 are currently listed on the MLST database, a single locus variant, ST-24, was reported previously as the cause of an outbreak of respiratory disease in Swedish horses [14]. Therefore, the ST-118, ST-43, ST-103 and ST-418 isolates of S. zooepidemicus that were recovered from ponies with respiratory disease are proposed to have contributed to the observed clinical signs. The 32 ponies enrolled in this study originated from four geographically distinct areas in the UK, likely contributing to the observed diversity of the S. zooepidemicus strains recovered. Examination of the genomes of the collection identified that 86.6% of the S. zooepidemicus collection, including all 23 of the isolates recovered from the coughing ponies in this study, encoded homologues of at least four of the eight antigens in Strangvac with ≥70% amino acid identity. The homologues of CNE, EAG, Eq5 and IdeE were encoded by 97.9%, 97.9%, 100% and 85.5% of S. zooepidemicus isolates with an average amino acid identity of 85.7%, 97.2%, 81.5% and 99.1%, respectively. Our data indicate that the immune responses to these antigens that are generated by vaccination with Strangvac are expected to cross-react with strains of S. zooepidemicus . In agreement with the generation of a cross-reactive immune response to S. zooepidemicus , the administration of 2 doses of Strangvac was significantly associated with fewer days with a cough, or marked cough. Moreover, the vaccinated ponies that had higher antibody titres to IdeE had significantly lower cumulative cough scores between D28 and D46. This effect was enhanced on consideration of the antibody titres on D35, which was seven days after second vaccination. The antibody titres towards IdeE remained the significant predictor of reduced clinical signs of coughing and the responses to Eq85 being the significant predictor of reduced fever. The critical role of IdeE and Eq85 in conferring protection was further supported by the analysis of nasopharyngeal antibody titres, which is consistent with the fact that S. zooepidemicus respiratory infection remains predominantly localised at the mucosal surface [29]. Our data are the first report evidencing a cross-protective effect of vaccination with Strangvac against respiratory disease caused by S. zooepidemicus . Whilst SclC, SclF, SclI, Eq8 and Eq5 undoubtedly contribute to the protection provided by vaccination with Strangvac [17], the biological function of these sortase-processed surface proteins remains unknown. CNE is a collagen-binding surface protein [30]. EAG is a protein G-related cell surface protein that binds to alpha 2 macroglobulin, albumin and IgG [22] and antisera against EAG enhanced opsonophagocytic activity in vitro [31]. IdeE is an IgG endopeptidase and the vaccination of ponies with Strangvac induced an antibody response that neutralised the ability of IdeE to cleave IgG in vitro [32]. We propose that the responses to the components of Strangvac both enhance the phagocytosis of S. zooepidemicus and neutralise its ability to degrade IgG and cause disease. The correlation of higher antibody titres to IdeE with reduced coughing is consistent with this antigen being highly conserved across different strains of S. zooepidemicus and the primary correlate of protection against experimental infection with S. equi (Paillot et al ., in preparation). Cross-protection between different streptococcal species is not without precedent. McCabe et al ., (2023) reported that the Group A Streptococcus (GAS, S. pyogenes ) vaccine candidate VAX-A1 conferred protection against Group B streptococcus (GBS, Streptococcus agalactiae ) in mice [33]. Although GAS and GBS belong to distinct species within the pyogenic group of Streptococcus [34], VAX-A1 contains the GAS surface-expressed C5a peptidate (ScpA) as an antigen, which shares 97-98% amino acid sequence homology with ScpB, the corresponding virulence factor produced by GBS [35]. Like IdeE, both ScpA and ScpB facilitate bacterial immune evasion. They both cleave and inactivate C5a, which contributes to the virulence of GAS and GBS by impeding neutrophils recruitment and the activation of phagocytic cells [36,37]. Antibodies induced by VAX-A1 were shown to cross-react with ScpB, to increase the bactericidal activity of human neutrophils against GBS and to protect immunised mice against GBS infection [33]. Cross-protection has also been reported in mice vaccinated with EndoSe from S. equi against experimental challenge with S. zooepidemicus or S. pyogenes . EndoSe is a glycosyl hydrolase that inhibits the opsonic function of IgG in vitro and is thought to contribute to immune evasion by S. equi . EndoSe shares 86-88% amino acid sequence identity with EndoSz from S. zooepidemicus and 70% with EndoS from S. pyogenes [38]. The antibody titres to IdeE and CCE measured in control ponies showed a modest but statistically significant transient increase following the episode of respiratory disease. However, these titres remained below the positivity threshold of the vaccine antigen iELISA, which indicates that the assay retains high specificity to S. equi and/or that natural exposure to S. zooepidemicus was insufficient in these animals to prime a strong immune response to these antigens . The vaccine antigens used in our study are not among the dominant antigens identified after natural infection with S. equi [39]. Cross-reactive epitopes are typically subdominant and often fail to robustly prime an immune response during natural infection. For example, repeated exposure is required to induce an immune response to the subdominant J8 peptide from the conserved domain of the GAS M-protein in humans, whereas J8-specific memory B cells generated through vaccination respond rapidly upon infection and confer protection in mice [40]. There was also a significant positive correlation between the CCE and Eq85 antibody titres in vaccinated ponies following exposure to S. zooepidemicus and the observed level of nasal discharge. This suggests that pre-existing immunity induced by vaccination was potentiated by S. zooepidemicus infection. The prevalence of respiratory disease associated with S. zooepidemicus declines in parallel with age, consistent with the exposure of young horses to successive strains of S. zooepidemicus (and potentially S. equi ) and the development of natural cross-protective immunity [8,12,13]. Our data are consistent with and support these observations. This study is constrained by the absence of S. zooepidemicus -specific assays and reagents, which limits our ability to fully characterise the immune response induced by S. zooepidemicus and the level of cross-reactivity with the iELISA used in this study. The small number of ponies restricted the statistical power and the extent to which these findings can be generalised to the wider horse population. Several observations emerged serendipitously rather than through a predefined experimental design and should therefore be interpreted with caution. However, our conclusion supports the anecdotal reports of protection against S. zooepidemicus infection in the field mentioned by Rendle et al ., (2025) [41]. Further clinical investigations and controlled trials are warranted to substantiate and expand these preliminary observations. 5. Conclusions: The conservation of the antigens in Strangvac encoded by a diverse collection of S. zooepidemicus strains, and IdeE, CNE, EAG and Eq5 in particular, is encouraging towards the development of cross-protective immune responses against this important opportunistic pathogen of horses. A significant reduction in the level of coughing and fever was observed in ponies vaccinated with Strangvac suggesting that vaccination may accelerate the development of natural cross-protective immune responses in young horses against S. zooepidemicus . Tables Table 1: Conservation of the Strangvac antigens encoded by 97 isolates of S. zooepidemicus , including 23 isolates from ponies in the current study. % encoding a homologue with >40% amino acid identity % encoding a homologue with >70% amino acid identity (%) Average amino acid identity in isolates encoding a homologue (%) CCE CNE (304) 100 97.9 85.1 EAG (161) 99.0 97.9 96.1 SclF (50) 100 20.6 59.3 SclI (56) 11.3 11.3 86.9 SclC (58) 51.5 33.0 85.1 Eq85 Eq8 (196) 100 4.3 52.7 Eq5 (440) 100 100 81.5 IdeE IdeE (315) 86.6 85.5 97.7 Table 2: Correlations between serum and nasopharyngeal secretion CCE, IdeE and Eq85 antibody titres. The Pearson correlation coefficient (r), the coefficient of determination (R²) and the p value are reported; significant results are in bold text. D28 r = 0.457 R² = 0.209 p = 0.075 r = 0.592 R² = 0.35 p = 0.016 r = 0.834 R² = 0.696 p < 0.0001 D35 r = 0.791 R² = 0.624 p = 0.0003 r = 0.755 R² = 0.569 p = 0.0007 r = 0.464 R² = 0.215 p = 0.07 D45 r = 0.474 R² = 0.225 p = 0.06 r = 0.433 R² = 0.187 p = 0.09 r = 0.508 R² = 0.281 p = 0.044 List of Figure legends Figure 1: Genetic relationships of the S. zooepidemicus isolates. The genetic relationships of isolates collected from coughing ponies are shown by the teal circles with the ST indicated. The genomes from Wilson et al ., 2025 are depicted by the blue circles [23]. The S. zooepidemicus H70 and MGCS10565 genomes are depicted by the yellow and orange circles, respectively. The ST of the groups of isolates identified in this study are indicated. The presence of homologues of the Strangvac antigens with ≥70% amino acid identity is depicted by the teal metadata blocks. The total number of antigens encoded by each genome with ≥70% amino acid identity is indicated by the coloured bars. Figure 2: Individual cumulative marked cough score (= 2) measured from D28 to D46 for control and vaccinated ponies. Level of statistical significance was set at p ≤0.05. Figure 3: Relationship between the individual cumulative cough score measured in vaccinated ponies from D35 to D46 and the antibody titres to CCE, IdeE or Eq85 that were measured on D35. The correlation was measured with the Pearson test with significance set at p ≤0.05. Significant results are in bold text and the coefficient of determination (R²) is reported. Figure 4: Relationship between the individual number of days with abnormal temperature (T°C <38.8°C) measured in vaccinated ponies from D35 to D46 and the antibody titres to CCE, IdeE or Eq85 that were measured on D42. The correlation was measured with the Pearson test with significance set at p ≤0.05. Significant results are in bold text and the coefficient of determination (R²) is reported. Figure 5: Relationship between the individual number of days with abnormal temperature (T°C <38.8°C) measured in vaccinated ponies from D35 to D46 and the nasopharyngeal antibody titres to CCE, IdeE or Eq85 that were measured on D35. The correlation was measured with the Pearson test with significance set at p ≤0.05. Significant results are in bold text and the coefficient of determination (R²) is reported. Figure 6: Representative relationship between the individual cumulative nasal discharge score as a measure of S. zooepidemicus infection (measured from D28 to D46) and the CCE, IdeE or Eq85 antibody titres measured in vaccinated ponies prior to S. zooepidemicus infection (D21, in red) and after (D70, in blue). Correlation was measured with the Pearson test with significance set at p ≤0.05. Significant p values are in bold text and the coefficient of determination (R²) is reported. List of legends for Supplementary items Supplementary Table S1 : Clinical examination scoring. 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Authors Affiliations Romain Paillot 0000-0003-3081-2037 [email protected] Intervacc AB View all articles by this author J. Richard Newton University of Cambridge Department of Veterinary Medicine View all articles by this author Sonia Gonzalez-Medina 0000-0001-6854-7879 CVS Equine Division View all articles by this author Sara Frosth 0000-0002-4080-5183 Swedish University of Agricultural Sciences View all articles by this author Lars Frykberg Swedish University of Agricultural Sciences View all articles by this author Margareta Flock Karolinska Institutet Institutionen for mikrobiologi tumor- och cellbiologi View all articles by this author Bengt Guss Swedish University of Agricultural Sciences View all articles by this author Jan-Ingmar Flock Karolinska Institutet Institutionen for mikrobiologi tumor- och cellbiologi View all articles by this author Andrew Waller 0000-0002-7111-9549 Intervacc AB View all articles by this author Metrics & Citations Metrics Article Usage 410 views 138 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Romain Paillot, J. Richard Newton, Sonia Gonzalez-Medina, et al. Cross-protective effect of Strangvac during a natural episode of Streptococcus zooepidemicus respiratory disease in ponies. Authorea . 25 February 2026. DOI: https://doi.org/10.22541/au.177206235.50501700/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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