3.1. Glycosyl composition and linkage analyses of LOS from O35Elgt5 or O35EgalE mutant
Glycosyl composition and linkage analyses showed that the OS from the parental O35E contained Gal, Glc, GlcNAc, and Kdo. The O35E LOS had the presence of 4-substituted Glc (4-Glc) with terminal substituted Glc (t-Glc) and Gal (t-Gal) in 1:1 molar ratio. However, the O35Elgt5 LOS lacked the 4-subtituted Gal (4-Gal), indicating one Gal-residue loss when compared with the O35E LOS. On the other hand, the O35EgalE LOS lacked detectable levels of Gal, suggesting two Gal-residues were missing (; ).
Glycosyl linkage analysis of OSs isolated from O35E, O35Elgt5, and O35EgalE
3.2. Characterization of the dLOSs
Because LOS is too toxic to be used as an antigen for humans, an alkaline anhydrous hydrazine was employed to detoxify the LOSs. After the treatment, the dLOSs and their LOSs were characterized with a silver-stained SDS-PAGE assay, which displayed a single band for 200 ng of each LOS from strains O35E, O35Elgt5, and O35EgalE (). In contrast, as much as 10 μg of each dLOS did not show a band at the migration of each LOS, indicating that the residual LOS was less than 2%. By the LAL assay, each LOS from O35E, O35Elgt5 or O35EgalE displayed the endotoxin content of 24,400, 36,650 or 32,475 EU/μg, respectively. But each corresponding dLOS showed only 1.32, 0.07 or 0.91 EU/μg, accounting for an 18,538, 479,690 or 40,497-fold reduction of toxicity, respectively ().
Fig. 2 Silver-stained SDS-PAGE patterns (A) and the toxicity (B) of the LOSs and dLOSs from M. catarrhalis O35E, O35Elgt5, and O35EgalE. Each of the LOSs (200 ng/lane) and the dLOSs (10 μg/lane) was loaded with LPSs from Salmonella minnesota Ra (200 (more ...)
3.3. Composition and antigenicity of the conjugates
After the synthesis reaction of each dLOS derivative with a carrier to form a dLOS-TT conjugate, the contents of carbohydrate and protein in the conjugate were analyzed (). For the conjugates derived from strains O35E, O35Elgt5, and O35EgalE, the molar ratios of dLOS to TT were 45:1, 62:1, and 56:1, respectively. The yields of the dLOS-TT derived from strains O35E, O35Elgt5, and O35EgalE were 30%, 41%, and 36%, respectively, on the basis of carbohydrate content. The antigenicity of all conjugates was similar to that of the O35E LOS ().
Composition, yield, and antigenicity of conjugate vaccines
3.4. Conjugate-induced rabbit serum IgG level evaluated with the homologous LOS and O35E LOS
The immunogenicity of the conjugates was studied by immunizing rabbits with different conjugates and detecting rabbit antiserum IgG with an ELISA assay (). A control mixture of dLOSs and TT with the adjuvant elicited a very low level of anti-LOS IgG after injections. In contrast, O35E dLOS-TT, a positive control, induced significant increases of anti-O35E LOS IgG after the second and the third injections (2187- and 6561-fold above the preimmune serum levels, respectively). Mutant O35Elgt5 dLOS-TT induced high levels of anti-LOS IgG after two and three injections (729- and 2187-fold against the homologous O35Elgt5 LOS, and 243- and 140-fold against O35E LOS, above the preimmune serum levels). Mutant O35EgalE dLOS-TT also enhanced the anti-LOS IgG level after two and three injections (16- and 27-fold against homologous O35EgalE LOS, and 16- and 140-fold against O35E LOS, above the preimmune serum levels). The adjuvant enhanced the levels of anti-LOS IgG in all conjugate groups. Notably, O35Elgt5 or O35EgalE dLOS-TT with the adjuvant all elicited high levels of serum IgG against O35E LOS after two and three injections (729- and 729-fold or 243- and 1263-fold above the preimmune serum levels, respectively).
ELISA reactivity of conjugate-induced rabbit serum IgG evaluated with corresponding homologous LOS and the wild type O35E LOS
3.5. Bactericidal activity of rabbit sera against the conjugates
To explore the biological functions of the rabbit antisera, a complement–mediated bactericidal assay was used for measurement. 50% to 100% of rabbit sera against each conjugate with or without the adjuvant displayed bactericidal activity toward the wild type strain O35E after two and three injections (). There was a correlation between anti-O35E LOS IgG ELISA units and the bactericidal titers among 14 rabbits (r =0.420, P =0.0261), suggesting that the serum IgG might be a potential factor for the complement-mediated bactericidal activity of the rabbit sera.
Bactericidal activity against M. catarrhalis O35E for rabbit sera elicited by conjugate vaccines
Two rabbit sera with higher bactericidal titers against each conjugate, O35E dLOS-TT (both with three injections), O35Elgt5 dLOS-TT (rabbit 1 with three injections and rabbit 2 with two injections), and O35EgalE dLOS-TT (both with two injections), plus the adjuvant were then chosen. They were further tested for bactericidal activities toward heterologous-prototype strains. The sera against O35E dLOS-TT displayed titers of 20 and 40, <5 and <5, and <5 and 20 against strains 25238 (A), 26397 (B), and 26404 (C), respectively. The sera against O35Elgt5 showed titers of 5 and 5, 5 and 5, 5 and <5 against strains 25238 (A), 26397 (B), and 26404 (C), respectively. The sera against O35EgalE dLOS-TT revealed titers of 5 and <5, <5 and <5, and 10 and <5 against strains 25238 (A), 26397 (B), and 26404 (C), respectively. As positive controls, previous rabbit sera against 26397 (B) or 26404 (C) dLOS-TT plus the adjuvant with three injections both showed bactericidal titers of 20 toward their homologous strains.
Since the rabbit sera induced by O35Elgt5 dLOS-TT showed broad bactericidal activity against 3 prototype strains of M. catarrhalis, a serum (rabbit 1) against O35Elgt5 dLOS-TT plus the adjuvant with three injections was selected to test its bactericidal activity toward additional heterologous M. catarrhalis strains. It displayed bactericidal titers of 10, 5, 5, 10, 5, 10, 5, 10, 10, 5, 5, 10, 5, 5, 10, and 5 against strains 26395, 26394, M1, M3, M6, M9, 26391, 3292, 26400, 25239, 49143, 43627, TTA24, M4, M7, and M10, respectively. Thus, O35Elgt5 dLOS-TT-elicited rabbit sera exhibited a broad bactericidal spectrum against all tested M. catarrhalis three-serotype strains and clinical isolates.
3.6. Cross-reactivity of rabbit sera against conjugates from O35Elgt5 or O35EgalE
A whole-cell ELISA was further used to explore the cross-reactivity of rabbit sera against conjugates from O35Elgt5 or O35EgalE by comparing them with positive rabbit sera toward each conjugate from strains O35E (A), 26397 (B), and 26404 (C) (). Two rabbit sera elicited by each conjugate plus the adjuvant with three injections were tested. An O35E LOS-deficient mutant, O35ElpxA, had very low cross-reactions (geometric mean units ≤3) with all the rabbit sera. This observation demonstrated that the whole-cell ELISA reactions between the rabbit sera and all the strains were mainly on M. catarrhalis LOS antigen. As for binding with serotype A and C strains, rabbit sera against O35Elgt5 dLOS-TT showed higher reactivity than that against 26397 (B) dLOS-TT. Rabbit sera toward O35EgalE dLOS-TT also displayed better binding activity than that against 26397 (B) dLOS-TT for most serotype A and C strains (). As for binding with serotype B strains, rabbit sera against O35Elgt5 dLOS-TT had a higher reactivity than that toward O35E (A) dLOS-TT or 26404 (C) dLOS-TT while rabbit sera induced by O35EgalE dLOS-TT showed low binding activity (). Therefore, O35Elgt5 dLOS-TT-elicited rabbit sera demonstrated the broadest spectrum of cross-reactivity against the M. catarrhalis strains among all the tested sera.
Fig. 3 Whole-cell ELISA binding activity of rabbit sera elicited by dLOS-TT conjugates from O35Elgt5, O35EgalE, O35E (type A), 26397 (type B), and 26404 (type C) among M. catarrhalis clinical strains. Two rabbits were injected subcutaneously (1 dose every 4 (more ...)
3.7. Immuno-electron microscopy of M. catarrhalis binding with O35Elgt5 dLOS-TT-vaccinated rabbit serum IgG
To determine if rabbit serum IgG induced by the O35Elgt5 dLOS-TT recognizes surface-exposed epitopes on the intact M. catarrhalis, immuno-electron microscopy was conducted on fixed whole cells. Rabbit serum IgG bound to the surface of all representative strains O35E (type A, parental), 25238 (type A, heterologous), 26397 (type B, heterologous), and 26404 (type C, heterologous), as indicated by the electron dense gold particles scattered across the surface of the cells (). This confirmed the broad cross-reactivity of the O35Elgt5 dLOS-TT-induced rabbit serum binding with all three serotypes of M. catarrhalis strains.
Fig. 4 Binding of the rabbit serum against O35Elgt5 dLOS-TT to the surface of M. catarrhalis as visualized by immuno-electron microscopy. Rabbits were injected subcutaneously (1 dose every 4 weeks, 3 doses total) with O35Elgt5 dLOS-TT (50 μg of carbohydrate (more ...)