Background/Objectives: Strangles, caused by Streptococcus equi subspecies equi (S. equi), remains a common and severe equine infectious disease. Strangvac®, a recombinant fusion protein vaccine licenced in Europe, contains the antigens (Ag) CCE, Eq85, IdeE and a saponin adjuvant. Although its efficacy is high (94% in clinical trials and 100% in some natural outbreaks), immune correlates of protection have not been defined. This study determined the antibody (Ab) thresholds predictive of protection against clinical disease following high-dose experimental S. equi infection and the expected levels of protection at 6 and 12 months after V2. Methods: This study was a retrospective analysis of six independent double-blinded placebo-controlled experimental infection studies involving 129 ponies (80 vaccinated controls and 49 placebo controls) and a serology study (12 vaccinated ponies). Ponies received two to five vaccine doses before being experimentally challenged with S. equi strain Se4047. Ponies in the serology study were not experimentally infected. The onset of pyrexia (≥39 °C for at least 2 of 3 consecutive days, OOT) was used as a disease marker. Serology to IdeE, Eq85 and CCE was analysed with standardised clinical outcomes to define protective thresholds through correlation and Receiver Operating Characteristic (ROC) analyses. The predicted level of protection up to one year after V2 was then calculated (duration of immunity: DOI). Results: A protection threshold of ≥10 days to OOT, derived from the control distribution, was used for ROC modelling. Predictive performance (e.g., accuracy, precision, specificity) was calculated for individual and combined Ab thresholds. All controls developed pyrexia (median 6 days, IQR 5–7), with 46 out of 49 (93.9%) within 9 days of the challenge. Vaccinated ponies showed significantly delayed or absent OOT compared with controls (p < 0.0001), with 37 vaccinated ponies (46.25%) reaching the end of the studies without developing pyrexia. The Ab titre to all antigens was significantly associated with the level of protection (p < 0.0001). ROC analyses demonstrated high discriminative power (AUC 0.86–0.88). Optimal Ab titre boundaries yielded high precision (≥80%) for all Ags (IdeE: 3.5–4.3; Eq85: 2.65–3.7 and CCE: 2.66–3.2). Both precision and accuracy remained above 80% for levels of IdeE and Eq85 Ab titres superior or equal to those measured up to one year after V2, with an estimated level of protection of 78.9% to 81.2% in vaccinated animals. Conclusions: Ab titres to all three Ags represent robust correlates of protection against pyrexia following high-dose experimental S. equi challenge in Strangvac®-vaccinated ponies. Ab titres measured up to one year after V2 were estimated to continue to provide significant protection in vaccinated animals. These findings support the observed levels of protection conferred by Strangvac® against natural infection with S. equi.

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 was 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.

This focus article has been prepared by Romain Paillot, Francesco Righetti, Carl Robinson, Lars Frykberg, Margareta Flock, Olof Zachrisson, Bengt Guss, Jan‐Ingmar Flock and Andrew S Waller . It reflects on a recently published study investigating correlations between strangles vaccine‐induced antibody titres and levels of protection and duration of immunity in horses.

Background: Streptococcus equi subspecies equi (S. equi) is the cause of strangles, one of the most prevalent diseases of horses worldwide. The disease is characterised by fever and the formation of abscesses in the lymph nodes of the head and neck, which can restrict the airway. A multicomponent subunit vaccine, Strangvac, has been shown to effectively reduce clinical signs of strangles and to reduce its incidence. Objective: The aim of this study was to determine the immune response against the immunoglobulin-cleaving endopeptidase IdeE, a key protective component within the vaccine and the ability of antibodies to neutralize the proteolytic activity of IdeE. Methods: An in vitro assay was developed to measure the functional inhibition of recombinant IdeE by horse sera pre- and post-vaccination. The IdeE-neutralising titres were compared to the corresponding IdeE-specific antibody titres measured by iELISA (indirect Enzyme-Linked Immunosorbent Assay). Results: A significant IdeE-specific antibody response in blood serum collected from ponies was induced after Strangvac vaccinations. Concomitantly, significant increases in the neutralising activity of IdeE occurred, persisting for at least 12 months post-second vaccination. IdeE-neutralising activity was further increased significantly after a third vaccination, even when the third dose was administered 12 months after the second dose, demonstrating that immunological memory to the vaccine persisted for 12 months. There was a significant correlation between the IdeE-neutralising activity of blood sera and the level of IdeE-specific antibodies. Conclusions: These data provide insights into one potential mechanism by which this vaccine protects Equids against or during S. equi infection.

The equine disease strangles, caused by Streptococcus equi, remains a major cause of welfare and economic cost to the global horse industry. Here we report the safety, immunogenicity and efficacy of a novel multi-component chimeric fusion protein vaccine, called Strangvac, when administered to ponies via the intramuscular route. Across the four studies, Strangvac was safe and induced robust antibody responses towards the vaccine components in blood serum and the nasopharynx, which were boosted by revaccination up to 12 months after a primary course of 2 vaccinations 4 weeks apart. The vaccine response did not cross-react with a commercial strangles iELISA, which identifies horses that have been exposed to S. equi, demonstrating that it was possible to differentiate infected from vaccinated animals (DIVA). Following challenge with S. equi strain 4047 (Se4047), all 36 control ponies that had received an adjuvant-only placebo vaccine developed clinical signs of strangles. In contrast, intramuscular vaccination with Strangvac protected ponies significantly from challenge with Se4047 at two weeks (5 of 16 ponies protected (31%), P = 0.04) and two months (7 of 12 ponies protected (58%), P = 0.0046 (including pooled control data) after second vaccination. Optimal protection (15 of 16 ponies protected (94%), P < 0.0001) was observed following challenge at two weeks post-third vaccination. Our data demonstrate that Strangvac is safe, has DIVA capability and provides a rapid onset of protective immunity against strangles. We conclude that Strangvac is a valuable tool with which to protect horses from strangles, particularly during high-risk periods, whilst maintaining the mobility of horse populations as required by the global equine industry. (C) 2020 The Authors. Published by Elsevier Ltd.

The host-restricted pathogen Streptococcus equi causes strangles in the horse, which is characterised by abscessation of the lymph nodes of the head and neck. The disease is endemic throughout the world causing considerable welfare and economic cost to the horse industry. Here we report the results of three studies where ponies were vaccinated with combinations of recombinant fusion proteins to optimise vaccine production and the level of protection conferred. Optimal protection was conferred by a prototype multicomponent subunit vaccine, Strangvac 4, which contained eight proteins CNE, SclC, SclF, SclI, EAG (fused as CCE), SEQ_402, SEQ_0256 (fused as Eq85) and IdeE. Across the three experiments only three of 16 ponies vaccinated with Strangvac 4 became pyretic compared to all 16 placebo-vaccinated control ponies (P < .001). S. equi was recovered from the lymph nodes of eight Strangvac 4-vaccinated and 15 control ponies (P = .016). None of the ponies vaccinated with Strangvac 4, or the other prototype vaccines developed adverse reactions following vaccination. Our data provide evidence in support of the further clinical development of the Strangvac 4 vaccine.

EndoSe from Streptococcus equi subsp. equi is an enzyme hydrolyzing glycosyl groups on IgG, analogous to EndoS from Streptococcus pyogenes. We here show that the activity of EndoSe leads to an antiphagocytic function and may thus be a contributory factor to immune evasion of S. equi. Despite the damaging effect that EndoSe has on IgG, antibodies against EndoSe can neutralize its function. Antibodies against EndoSe restored the opsonic activity of specific opsonizing antibodies. Mice infected with either S. equi subsp. equi or subsp. zooepidemicus or S. pyogenes could be protected by vaccination with EndoSe. It is speculated that EndoSe could be a suitable vaccine candidate against streptococcal infections.

Equine granulocytic anaplasmosis (EGA) is a seasonal rickettsial disease of horses transmitted by Ixodes spp. ticks. The etiological agent is Anaplasma phagocytophilum, a coccobacillary gram-negative organism with a tropism for granulocytes. Clinical manifestations include fever, partial anorexia, depression, distal limb edema, petechiation, icterus, ataxia, and reluctance to move. Hematologic changes observed are thrombocytopenia, decreased packed-cell volume and marked leukopenia involving first lymphocytes and then granulocytes. Diagnosis is based on awareness of geographic area for infection, typical clinical signs, abnormal laboratory findings, and visualization of characteristic morulae in the cytoplasm of neutrophils and eosinophils in a peripheral blood smear. Treatment consists of the administration of tetracycline. The disease is being diagnosed with increasing frequency in the United States, Canada, Brazil and northern Europe.

Streptococcus equi ssp. equi causes strangles, a highly contagious and serious disease in the upper respiratory tract of horses. Streptococcus equi ssp. zooepidemicus, another subspecies of this genus, is regarded as an opportunistic commensal in horses. The present study describes the characterization of two novel immunoglobulin G (IgG) endopeptidases of these subspecies, IdeE2 and IdeZ2. Both enzymes display sequence similarities with two previously characterized IgG endopeptidases, IdeE of S. equi ssp. equi and IdeZ of S. equi ssp. zooepidemicus. IdeE2 and IdeZ2 display high substrate-specificity in comparison with IdeE and IdeZ, as they both completely cleave horse IgG, while the activity against IgG from mouse, rabbit, cat, cow, sheep and goat is low or absent. The potential use of IdeE and IdeE2 as vaccine components was studied in a mouse infection model. In this vaccination and challenge study, both enzymes induced protection against S. equi ssp. equi infection.

Streptococcus equi subspecies equi ( S. equi ) is a clonal, equine host-adapted pathogen of global importance that causes a highly contagious suppurative lymphodendopathy of the head and neck, more commonly known as Strangles. The disease is highly prevalent, can be severe and spread easily by visibly infected animals or by carrier animals that show no clinical signs of disease. Antibiotic treatment is usually ineffective. However, the majority of horses develop immunity to re-infection, suggesting that vaccination should be a feasible way to prevent the infection. Live attenuated vaccine strains of S. equi are available but adverse reactions have been reported and they suffer from a short duration of immunity. Thus, a safe and effective vaccine against S. equi is highly desirable. In this report, Welsh mountain ponies vaccinated with a combination of seven recombinant S. equi proteins, were significantly protected from experimental infection by S. equi , resembling the spontaneous disease. The protective antigens consisted of five surface localized proteins and two IgG endopeptidases. The results from a second vaccination trial indicate that the endopeptidases were important for good protection. The similarity of S. equi to other pyogenic streptococci suggests that our findings have broader implications for the prevention of streptococcal infections.

Streptococcus equi subspecies equi (S. equi) is a clonal, equine host-adapted pathogen of global importance that causes a suppurative lymphodendopathy of the head and neck, more commonly known as Strangles. The disease is highly prevalent, can be severe and is highly contagious. Antibiotic treatment is usually ineffective. Live attenuated vaccine strains of S. equi have shown adverse reactions and they suffer from a short duration of immunity. Thus, a safe and effective vaccine against S. equi is highly desirable. The bacterium shows only limited genetic diversity and an effective vaccine could confer broad protection to horses throughout the world. Welsh mountain ponies (n = 7) vaccinated with a combination of seven recombinant S. equi proteins were significantly protected from experimental infection by S. equi, resembling the spontaneous disease. Vaccinated horses had significantly reduced incidence of lymph node swelling (p = 0.0013) lymph node abscessation (p = 0.00001), fewer days of pyrexia (p = 0.0001), reduced pathology scoring (p = 0.005) and lower bacterial recovery from lymph nodes (p = 0.004) when compared with non-vaccinated horses (n = 7). Six of 7 vaccinated horses were protected whereas all 7 non-vaccinated became infected. The protective antigens consisted of five surface localized proteins and two IgG endopeptidases. A second vaccination trial (n = 7+7), in which the IgG endopeptidases were omitted, demonstrated only partial protection against S. equi, highlighting an important role for these vaccine components in establishing a protective immune response. S. equi shares >80% sequence identity with Streptococcus pyogenes. Several of the components utilized here have counterparts in S. pyogenes, suggesting that our findings have broader implications for the prevention of infection with this important human pathogen. This is one of only a few demonstrations of protection from streptococcal infection conferred by a recombinant multi-component subunit vaccine in a natural host.

Strangles is an upper respiratory tract infection in horses, which is highly contagious and one of the more costly diseases of the horse. Three recombinant antigens were used to vaccinate horses, which were then experimentally challenged with Streptococcus equi, the causative agent for strangles. The vaccinated horses showed significantly reduced bacterial growth (p=0.02) and nasal discharge (p=0.0004), a typical symptom of strangles. Other clinical signs of strangles were also reduced and at post mortem examination, lower rate of empyaema or scarring of the guttural pouches was found in the vaccinated group (p=0.01). The antigens used were EAG (α2-macroglobulin, albumin, and IgG-binding protein), CNE (a collagen-binding protein), and SclC (a collagen-like protein). The adjuvant used was Abisco, a saponin derived matrix. No adverse effects were observed following vaccination with the antigens and adjuvant.

A mouse model resembling Streptococcus equi subspecies equi infection in the horse, strangles, was used to assess the protective effect of vaccination with selected recombinant proteins from S. equi subsp. equi. After challenge the infection was monitored by weight loss and by nasal colonisation with S. equi subsp. equi. Vaccination with a collagen-binding protein (CNE) and a collagen-like protein (SclC) resulted in protective antibodies, whereas a novel fibronectin-binding protein (FNEB) did not. Co-administration of CNE with EAG, a poorly immunogenic alpha2-macroglobulin-, albumin- and immunoglobulin G-binding protein, resulted in a significant synergistic effect and enhanced the protective immune response against EAG.

ABSTRACT Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in S. equi subsp. equi , FNE and SFS. Assays of antibody titers in horses and in experimentally infected mice indicate that the protein is immunogenic and expressed in vivo during S. equi subsp. equi infection. Using Western ligand blotting, it was shown that FNEB binds to the N-terminal 29-kDa fragment of fibronectin, while SFS and FNE both bind to the adjacent 40-kDa fragment. S. equi subsp. equi is known to bind fibronectin to a much lower degree than the closely related S. equi subsp. zooepidemicus , but the binding is primarily directed to the 29-kDa fragment. Inhibition studies using S. equi subsp. equi cells indicate that FNEB mediates cellular binding to fibronectin in this species.