HEK-293 T, PK15, and IPEC-J2 cells were cultured in DMEM medium (Gibco, United States), supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, United States), and incubated at 37°C in a 5% CO₂ atmosphere. Streptococcus were cultured in Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) media (BD, United States) containing 5% (v/v) FBS (Every Green, China). The strains used in this research are detailed in Supplementary Table S1.

The genomic fragments encoding the endolysins PlyV12 (Genbank ID: AAT01859.1) and Ply2741 (Genbank ID: PQ213358) were synthesized by Tsingke Biotechnology, Co., Ltd. The chimeric endolysin Cly2v was constructed by fusing the Ply2741 CHAP domain (1-155aa) with the PlyV12 CBD domain (146-314aa) via overlap PCR. The Cly2v gene was subsequently cloned into the pCold™ II plasmid (Takara, Japan) following digestion with BamHI and HindIII. The specific primers used in this research are detailed in Supplementary Table S2.

The recombinant plasmid pCold-Cly2v was introduced into E. coli BL21 (DE3) competent cells and grown in LB medium supplemented with 100 μg/mL ampicillin at 37°C until OD_600nm reached to 0.6. After inducing with 0.6 mM Isopropyl β-D-thiogalactoside (IPTG) at 16°C for 18 h, the cells were harvested by ultracentrifugation and resuspended in binding buffer (20 mM Tris, 150 mM NaCl, pH = 7.5). The samples were broken using a cell pressure crusher and centrifuged at 10000 × g for 20 min at 4°C to harvested the crude recombinant protein. And then, the crude endolysin was purified via affinity chromatography (AFC) and size exclusion chromatography (SEC) according to the protocols. Briefly, His-tagged protein was purified using a His-Trap FF column (Cytiva, United States) with elution buffer (20 mM Tris, 150 mM NaCl, 300 mM imidazole pH = 7.5), followed by further purification using a HiLoad Superdex 200pg column (Cytiva, United States) with SEC buffer (20 mM Tris, 150 mM NaCl, pH = 7.5).

The physicochemical properties and hydrophobicity of Cly2v were analyzed using Expasy.¹ The three-dimensional (3D) structure prediction was conducted via the Colabfold webserver (Mirdita et al., 2022), with visualization and analysis performed using PyMOL. The secondary structure was further determined through circular dichroism as described previously (Luo et al., 2020). Briefly, the endolysin was adjusted to a concentration of 0.5 mg/mL in SEC buffer. The experiments were conducted on a J-1500 CD Spectrometer using a 0.1 cm quartz cuvette (JASCO, Japan), measuring wavelengths from 190 nm to 260 nm. The CDNN V2.1 software was used to calculate the secondary structure results.

The effect of temperature and pH on bactericidal activity were determined by turbidity reduction assay as described previously (Gilmer et al., 2013). Briefly, Cly2v was incubated at different temperatures. Then, 100 μL of Cly2v were taken every 10 min and mixed with 100 μL of S. agalactiae ATCC13813, which adjusted with PBS to OD_600nm = 0.8 (approximately 2.5 × 10⁸ CFU/mL). After incubation at 37°C for 30 min, the absorbance at OD_600nm was determined, with PBS serving as the negative control. For the pH stability, Cly2v was mixed with different pH buffers (20 mM sodium acetate buffer (pH 3.0 to 6.0), 20 mM sodium phosphate buffer (pH 7 to 8), and 20 mM Tris–HCl buffer (pH 9.0 to 10.0)) and incubated at 37°C for 30 min. To ensure consistent pH conditions, the bacteria were also resuspended in the respective pH buffers and adjusted to OD_600nm = 0.8 (approximately 2.5 × 10⁸ CFU/mL). Subsequently, Cly2v was mixed with the bacteria and incubated at 37°C for 30 min before measuring the absorbance at OD_600nm. Different pH buffers served as controls to reduce experimental errors from pH effects on bacterial viability. The relative activity of Cly2v under different temperature and pH conditions was expressed as a percentage of the maximum enzyme activity. All experiments were conducted in triplicate.

The thermal stability of Cly2v was assessed using nano differential scanning fluorimetry (nanoDSF) with the Prometheus NT.48 (Nano Temper, Germany). The experiments were conducted over a temperature range of 20°C to 95°C, with a rate of 1°C/min. The first derivative F350/F330 was automatically calculated and the melting temperature (Tm) was determined at the peak of the derivative curve.

To explore the lytic spectrum of Cly2v against Streptococcus species that caused mastitis, the turbidity reduction assay was conducted as described previously (Gilmer et al., 2013; Li et al., 2023). Mid-logarithmic phase bacteria were adjusted with PBS to OD_600nm = 0.6–0.8. Then, 100 μL of the bacteria was mixed with 100 μL of endolysin (50 μg/mL) in a 96-well plate. The absorbance was measured at OD_600nm after a 30-min incubation at 37°C. The turbidity reduction ratio was calculated as described below. Ply2741 was used as the parental endolysin control.

To evaluate the bactericidal efficiency of Cly2v, logarithmic phase bacteria were mixed with Cly2v (100 μg/mL), while PBS served as a negative control. Following a 1 h incubation at 37°C, the mixture was plated on TSA to determine bacterial counts. Moreover, the bactericidal efficiency of Cly2v was evaluated via bactericidal kinetics curve assay. In brief, Cly2v (50 μg/mL) was incubated with bacteria, and bacterial counts were determined by serial dilutions on TSA plates at 5 min intervals for 45 min.

The biofilm-formation ability of Streptococcus was determined via the crystal violet staining method as described previously (Wang et al., 2023). Briefly, overnight cultured bacteria (~5 × 10⁸ CFU/mL) were diluted in TSB medium at a ratio of 1:100. Subsequently, 100 μL of the bacteria and 100 μL of PBS were inoculated into a 96-well plate to form biofilms, while TSB medium served as the control. After incubation at 30°C for 24 h, the wells were washed three times with PBS and dried at room temperature. Each well was stained with 200 μL of crystal violet and incubated at 37°C for 30 min. Finally, after washing the wells with PBS, 33% acetic acid was added to each well to dissolve the stain, and absorbance was measured at 590 nm. The experiments were conducted for three times.

To investigate the inhibitory ability of Cly2v against biofilms, overnight cultures of S. agalactiae X2 and S. uberis 002–1 (~5 × 10⁸ CFU/mL) were diluted at a 1:100 ratio. Subsequently, the bacteria were mixed with different doses of Cly2v (5 μg/mL, 10 μg/mL, and 25 μg/mL) and ampicillin (5 μg/mL) in a 96-well plate to form biofilms after incubation at 30°C for 24 h, with PBS serving as the control. Some wells were washed three times with PBS, resuspended in PBS, and then plated on TSA plates for colony counting. In other wells, biofilm formation ability was determined using the crystal violet staining method.

To investigate the effect of Cly2v on mature biofilms, S. agalactiae X2 and S. uberis 002–1 (~5 × 10⁸ CFU/mL) were diluted at a 1:100 ratio and inoculated into a 96-well plate without treatment to form biofilms. After incubation at 30°C for 24 h, different doses of Cly2v (10 μg/mL, 25 μg/mL, and 50 μg/mL) and ampicillin (5 μg/mL) were added to the wells, and the mixture was incubated at 37°C for 3 h. After incubation, the biofilms were determined by crystal-violet staining method and bacterial counts as described above. All experiments were conducted in triplicate.

The morphological changes of bacterial mature biofilms treated with Cly2v were observed using scanning electron microscopy (SEM) as described previously (Duan et al., 2023). Briefly, mid-logarithmic phase bacteria (~2 × 10⁸ CFU/mL) were inoculated at a 1:100 ratio in a 24-well plate with coverslips and cultured at 30°C for 24 h to form biofilms. Subsequently, Cly2v (100 μg/mL) was added and the mixture were incubated at 37°C for 3 h to remove the biofilms. The samples were then washed with PBS and fixed with 2% glutaraldehyde for 2 h, followed by six washes with ddH₂O. Finally, after spraying with gold, the coverslips were observed using scanning electron microscope (NTC JSM-6390LV, Japan).

The bactericidal activity of Cly2v in milk was evaluated as described previously (Wang et al., 2023). In brief, S. agalactiae ATCC13813 (1 × 10⁵ CFU/mL) was grown in the whole milk at 37°C. After incubation for either 1 h and 2 h, 50 μg/mL of Cly2v was added to the milk and incubated at 37°C for 1 h. Subsequently, samples were serially diluted in ten-fold and plated on the TSA plates for colony counting. All experiments were conducted in triplicate.

To evaluate the toxicity of Cly2v on mammalian cells, a CCK-8 assay was carried out as described previously (Zhao et al., 2018). Cells, specifically PK15, IPEC-J2, and HEK-293 T were seeded at a density of 1 × 10⁵ cells per well. Subsequently, cells were exposed with varying concentrations of Cly2v (200 μg/mL and 400 μg/mL) and incubation for 2 h, with PBS serving as the control. Cells viability was evaluated via the CCK-8 kit (MCE, United States) following the protocols, and the absorbance was measured at 450 nm. All the experiments were conducted for three times.

The toxicity of Cly2v to mouse tissues was further evaluated in a mouse model. Female specific-pathogen-free (SPF) BALB/c mice were divided into 3 groups (n = 5) and each group received an intraperitoneal injection of 400 μg or 800 μg of Cly2v, with PBS serving as the control. The survival rate and physiological condition of the mice were monitored for 5 days, and mouse organs were collected for pathological analysis at 5 d.

A mouse mastitis model was constructed as described previously to explore the efficacy of Cly2v against the bovine mastitis in vivo (Gutiérrez et al., 2020; Li et al., 2023). Briefly, female SPF BALB/c mice were synchronized for pregnancy by circadian cycle adjustment. At 14 to 21 days postpartum, the pups were allowed to nurse for 1 h to fully deplete the milk. Subsequently, the offspring were removed to establish the mastitis model while ensuring that the lactating females remained in the lactation period. Mice were anesthetized with 1.25% avertin, and a small incision of approximately 0.5 mm was made on the L4 and R4 mammary glands to expose the mammary duct. Then, S. agalactiae ATCC13813 (10⁴ CFU/gland) was administered through the mammary duct, except for the control group. After 1 h post-infection, the L4 and R4 mammary glands were injected with PBS, Ply2741 (50 μg/gland), Cly2v (50 μg/gland), or Ceftiofur (25 μg/gland) to establish the PBS-treated group (n = 5), Ply2741-treated group (n = 5), Cly2v-treated group (n = 5), and antibiotic-treated group (n = 5). At 48 h post-treatment, the mice were humanely euthanized, and the L4 and R4 mammary glands were harvested and mixed. The mammary tissue samples were completely homogenized and serially diluted in a ten-fold series for viable cell counting. Inflammatory cytokine levels (TNF-α and IL-6) in the mammary tissues were determined via qRT-PCR assay. Additionally, mammary tissues were collected for histopathological analysis after 48 h of treatment.

The mRNA expression levels of inflammatory cytokine (TNF-α and IL-6) were determined by qRT-PCR assay (Wang et al., 2023). Briefly, TRIpure reagent (Aidlab, China) was used to extracted total RNA from mammary tissues. Subsequently, the extracted RNA was then reverse transcribed into cDNA, and qRT-PCR assay was performed on Applied Biosystems ViiA™7 system (Thermo Fisher Scientific, United States) with Hieff ® qPCR SYBR Green Master Mix (Yeasen, China). The formula 2^-ΔΔCt was used to analyzed the data and the GAPDH gene was served as the internal gene. The primers used in this assay are detailed in Supplementary Table S2.

Five-week-old female SPF mice were randomly divided into 6 groups (n = 6 for each group) and each group injected intraperitoneally with S. agalactiae ATCC13813 at doses of 4 × 10⁹ CFU, 2 × 10⁹ CFU, 1 × 10⁹ CFU, 5 × 10⁸ CFU, and 2.5 × 10⁸ CFU, respectively. Equal volumes of PBS were served as a control. The survival rate of mice was monitored and recorded for 48 h to determine the lethal dose. The minimum challenge dose that resulted in the death of all mice within 48 h was used for the survival rate experiment, while the maximum challenge dose that all mice survived was used for the bacterial load assay.

To explore the therapeutic effect of Cly2v on systemic infection caused by S. agalactiae, mice were randomly divided into 4 groups (n = 6 per group), Group I, II, III, and IV. Group I, II, and III was injected intraperitoneally with 1 × 10⁹ CFU of S. agalactiae ATCC13813, with Group IV received an equal volume of PBS serving as the control. After 1 h post-infection, group II, III, and IV were treated with 200 μg or 400 μg doses of Cly2v, respectively, while the Group I received PBS as the control. The survival rate of mice was monitored for 5 d. Additionally, a survival experiment was established for treatment with Cly2v at 3 h post-infection. Mice were randomly divided into 3 groups (n = 9 per group) and were intraperitoneally injected with 1 × 10⁹ CFU of S. agalactiae ATCC13813. After 3 h of infection, the mice in each group were treated with 200 μg or 400 μg of Cly2v, with an equal volume of PBS serving as the control. The survival rate of the mice was recorded for 5 days.

For bacterial load assay, mice were randomly divided into 2 groups (n = 9 per group), and each mouse was injected intraperitoneally of 2.5 × 10⁸ CFU of S. agalactiae ATCC13813. The treatment group received 200 μg of Cly2v, while the control group was injected with PBS at 1 h post-infection. Mice were humanely euthanized after 6, 18, and 30 h of treatment, and blood, heart, lungs, liver, spleen, and kidneys organs were harvested. After homogenization and ten-fold dilution, the samples were plated on TSA plates for colony counting to determine the bacterial load in the organs.

Data are presented as mean ± standard deviation (SD) and the normality of the data was analyzed using the Anderson-Darling test (N > 6) and Quantile-Quantile plots (N < 6). Statistical significance was analyzed using one- and two-way analysis of variance (ANOVA) with GraphPad Prism (version 8). p values < 0.05 was considered statistically significant (*).

To enhance the bactericidal activity of endolysins, we constructed a novel chimeric endolysin, Cly2v. Cly2v consisted of the CHAP domain from the S. suis endolysin Ply2741 and the CBD domain from the E. faecalis phage endolysin PlyV12. These two domains were connected by a flexible linker (GGSSGS) (Figure 1A). As shown in Supplementary Figure S1A, physicochemical analysis indicated that the theoretical isoelectric point and instability index of Cly2v were 9.52 and 27.69, respectively. Hydropathy analysis revealed that the GRAVY index of Cly2v was −0.413, with a higher number of hydrophilic residues compared to hydrophobic residues, indicating that Cly2v had the potential for soluble expression in vitro. After purification by AFC and SEC, Cly2v exhibited high purity in SDS-PAGE gel with the expected molecular weight (Figure 1B).

Additionally, circular dichroism and colabfold were used to analyze the secondary structure of Cly2v. The results showed that the proportions of α-helices, β-strands, and coils in Cly2v were 6.7, 41.9, and 33.4%, respectively (Supplementary Figure S1B). Specifically, the Cly2v consisted of 5 α-helices and 15 β-strands (Supplementary Figure S1C).

The influence of temperature and pH on the bactericidal activity of the chimeric endolysin Cly2v were further evaluated. The results showed that Cly2v maintained over 90% activity between 4°C and 47°C. However, the bactericidal activity decreased significantly when the temperature increased to 52°C, and Cly2v was completely inactivated after incubation for 40 min at 52°C (Figure 1C). As shown in Figure 1D, Cly2v was able to maintain the bactericidal activity between pH = 3.0–10.0, with an optimal pH of 7.0–8.0. Additionally, thermal stability analysis revealed that the melting temperature (Tm) of Cly2v was 50.7°C, indicating that Cly2v could undergo denaturation and unfolding at temperatures above 50.7°C (Figure 1E). This result was consistent with the observation that Cly2v was inactivated at 52°C.

As shown in Figure 2A, Cly2v exhibited broad-spectrum bactericidal activity against various strains, including S. agalactiae, S. dysgalactiae, and S. uberis. Specifically, Cly2v demonstrated lytic efficiency greater than 40% in 12 out of 22 tested strains. More importantly, Cly2v exhibited higher bactericidal activity against streptococcal species causing mastitis compared with the parental endolysin Ply2741, indicating that Cly2v, a chimeric endolysin constructed based on the Ply2741, had better potential for the treatment of streptococci-induced mastitis in dairy cows.

Additionally, the bactericidal activity of Cly2v was determined by measuring changes in bacterial colony counts after treatment. The results showed that Cly2v significantly reduced the bacterial count in all 10 selected streptococcal strains, particularly in S. agalactiae ATCC13813 strain, where 100 μg/mL of Cly2v led to a reduction from 7.12 log₁₀CFU/ml to 5.57 log₁₀CFU/ml, resulting in a reduction of 97.22% in bacterial count (Figure 2B). Subsequently, we evaluated the bactericidal kinetics curve of Cly2v. The results showed that 50 μg/mL of Cly2v was able to reduce the bacterial count of S. uberis 002–1 from 8.47 log₁₀CFU/ml to 5.82 log₁₀CFU/ml (a reduction percentage of 99.77%) (Figure 2C), S. agalactiae ATCC13813 from 8.24 log₁₀CFU/ml to 6.63 log₁₀CFU/ml (a reduction percentage of 97.56%) (Figure 2D), and S. dysgalactiae T2 from 9.18 log₁₀CFU/ml to 8.44 log₁₀CFU/ml (a reduction percentage of 81.70%) (Figure 2E) within 25 min.

Among the 18 tested strains, S. agalactiae X2, S. uberis 002–1, S.dysgalactiae T1, and S. uberis 018–2 showed the ability to form strong biofilms (Supplementary Figure S2). Due to the poor lytic activity of Cly2v against 018–2 and T1, we selected the strains S. agalactiae X2 and S. uberis 002–1 for subsequent experiments. The results showed that Cly2v significantly inhibited biofilm formation in both S. agalactiae X2 and S. uberis 002–1. At a concentration of 25 μg/mL, Cly2v decreased the OD_590nm absorbance of biofilms by 68% (p < 0.001) and 44% (p < 0.001) for X2 and 002–1 (Figure 3A), respectively, while the bacterial counts decreased from 6.77 log₁₀CFU/ml to 4.07log₁₀CFU/ml and from 6.06 log₁₀CFU/ml to 4.65 log₁₀CFU/ml, corresponding to reductions of 99.80 and 96.09% (Figure 3B). Compared to Cly2v, ampicillin exhibited a stronger inhibitory effect on biofilm formation, with OD_590nm absorbance decreasing by 84 and 68%, respectively. For established biofilms, Cly2v can significantly remove the biofilms in a dose-dependent manner. As shown in Figure 3C, Cly2v (50 μg/mL) significantly reduced the OD_590nm absorbance of biofilms by 58% for X2 (p < 0.001) and 42% for 002–1 (p < 0.001). Additionally, Cly2v decreased the viable cell counts from 6.56 log₁₀CFU/ml to 5.98 log₁₀CFU/ml for X2 and from 5.73 log₁₀CFU/ml to 3.48 log₁₀CFU/ml for 002–1, corresponding to reductions of 73.74 and 99.45% (Figure 3D). Notably, ampicillin demonstrated no effect on mature biofilms.

To observe the morphological changes in bacterial biofilms after Cly2v treatment, we employed scanning electron microscopy (SEM) experiment. The results demonstrated that, compared to the control group, the established biofilms treated with Cly2v exhibited a loose adherence, with no contiguous biofilm formation and a significant reduction in viable cell counts (Figure 3E). These results indicated that Cly2v not only significantly inhibited the formation of bacterial biofilms but also effectively removed mature biofilms.

Streptococci-induced bovine mastitis can adversely impact milk quality. Therefore, we investigated the bactericidal activity of Cly2v against S. agalactiae ATCC13813 in whole milk. As shown in Supplementary Figure S3, after incubating milk with S. agalactiae ATCC13813 for 1 h, the addition of Cly2v led to a significant decrease in bacterial counts, with the counts decreasing from 6.51 log₁₀CFU/ml to 6.09 log₁₀CFU/ml, corresponding to a reduction of approximately 62.0% (p < 0.05).Additionally, Cly2v was added to infected milk at 100 μg/mL after 2 h infection, and the bacterial count decreased from 7.15 log₁₀CFU/ml to 6.49 log₁₀CFU/ml (approximately 74.2% reduction, p < 0.01). These results indicated that Cly2v could significantly reduce streptococci in whole milk.

To investigate the therapeutic effect of Cly2v in bovine mastitis, we established a mouse mastitis model and treated it with Cly2v. Ply2741 and ceftiofur were served as the parental endolysin control and positive control, respectively (Figure 4A). As shown in Figure 4B, Cly2v significantly reduced the bacterial burden in mouse mammary tissues, decreasing the bacterial counts from 5.99 log₁₀CFU/g to 3.83 log₁₀CFU/g, representing a reduction of 99.3% (p < 0.001). Additionally, compared to the parental endolysin Ply2741, the reduction in bacterial burden in mouse mammary tissues after Cly2v treatment was more substantial (p < 0.01). Notably, the ceftiofur-group also reduced bacterial counts to 3.68 log₁₀CFU/g, comparable to the effect observed with Cly2v treatment (p > 0.05). Moreover, Cly2v, Ply2741, and ceftiofur treatments significantly decreased the mRNA levels of inflammatory factors in mouse mammary tissues. After treatment with Cly2v, TNF-α and IL-6 levels decreased by 15.77-fold (Figure 4C) and 2.51-fold (Figure 4D), respectively, with no significant difference compared to the antibiotic group. Histopathological analysis revealed that after infection with S. agalactiae, there was significant thickening of the alveolar wall in the mammary tissue of mice, accompanied by severe edema, congestion, and a large number of inflammatory cells and erythrocyte infiltration, compared to the control group. After treatment with Cly2v, Ply2741, and ceftiofur, the mammary tissues exhibited minimal pathological alterations, with the Cly2v-group showing the least pathological damage (Figure 4E). These results indicated that Cly2v significantly reduced bacterial load in early-stage mouse mastitis models, demonstrating its potential as a treatment for streptococci-induced mastitis.

To evaluate the toxicity of Cly2v on mammalian cells, a CCK8 assay was employed. As shown in Supplementary Figure S4A, the results showed that the survival rates of HEK-293 T, PK15, and IPEC-J2 cells exceeded 100% at concentrations at 200 μg/mL and 400 μg/mL, indicating that Cly2v exhibited no significant toxicity to mammalian cells. Additionally, the toxicity evaluation results indicated that mice receiving intraperitoneal injections of 400 μg, and 800 μg of Cly2v exhibited a 100% survival rate and maintained normal physiological conditions (Supplementary Figure S4B). Pathological analysis indicated that, similar to the PBS group, mice administered 800 μg of Cly2v exhibited no significant pathological damage in their organs (Supplementary Figure S4C).

To investigate the therapeutic effects of Cly2v on systemic infection caused by Streptococcus, we first determined the optimal challenge dose of S. agalactiae ATCC13813. The mice challenged with a dose of 1 × 10⁹ CFU/mouse died within 48 h, while those infected with a dose of 2.5 × 10⁸ CFU survived for 48 h (Supplementary Table S3). Consequently, the optimal infection dose of S. agalactiae ATCC13813 for the survival rate experiment was 1 × 10⁹ CFU, while the challenge dose for the bacterial load assay was 2.5 × 10⁸ CFU.

To further evaluated the therapeutic efficacy of Cly2v against S. agalactiae infection in vivo, we established a systemic infection model in mice and treated with different doses of Cly2v. The results demonstrated that Cly2v exhibited significant protection for mice infected with S. agalactiae ATCC13813. Mice administered an intraperitoneal injection of 200 μg of Cly2v had a survival rate of 83% within 5 days, and those given a dose of 400 μg had a survival rate of 100%, whereas all mice in the control group died within 24 h (Figure 5A). Additionally, the bacterial load in the blood and various organs of mice was determined at different time points following treatment to further evaluate the therapeutic efficacy of the chimeric endolysin Cly2v. The results demonstrated a significant reduction in bacterial load in the blood and organs at 6 h, 18 h, and 30 h post-treatment (Figure 5B). Specifically, at 30 h post-treatment, the bacterial loads in the blood, heart, liver, spleen, lungs, and kidneys were 1.49 log₁₀CFU/mL, 2.22 log₁₀CFU/g, 2.37 log₁₀CFU/g, 3.33 log₁₀CFU/g, 3.68 log₁₀CFU/g, and 2.82 log₁₀CFU/g, representing decreases of 90.1, 99.7, 99.7, 99.7, 96.6, and 99.3%, respectively. Notably, treatment with 400 μg of Cly2v at 3 h post-infection provided a protection rate of 78% (Figure 5C). Although this was less effective compared to treatment at 1 h post-infection, Cly2v still demonstrated efficacy against late-stage sepsis induced by streptococcal infection.