Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa

Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa.

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections

Galectin CvGal2 from the Eastern Oyster (<i>Crassostrea virginica</i>) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric <i>Perkinsus</i> Species

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is hijacked by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. Screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). Phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. Glycan array analysis, however, revealed that, unlike CvGal1 which preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Furthermore, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and β-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for Perkinsus chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of <i>Perkinsus</i> species by the oyster galectins is consistent with their relative prevalence in oyster and clam species and supports their role in facilitating parasite entry and infectivity in a host-preferential manner

¹H and ¹³C values for different <i>S</i>. Typhimurium COPS residues.

<p>¹H and ¹³C values for different <i>S</i>. Typhimurium COPS residues.</p

Chemical structure of the OPS used for computational simulations and chemical models and sampled volumes of the most prevalent conformational cluster “11111111”.

<p>(A) Structure of the base (O:4,12) 3-repeat STm polysaccharide unit used for computational analyses. Individual monosaccharide units are indicated numerically, with the reducing end rhamnose (Rha) designated as (1, blue). The representative O-acetylated polysaccharide was constructed with the hydroxyl group substituted by an acetyl at the C2 position of both α-D-Abequ<i>p</i> and α-L-Rha<i>p</i> (-OH, red). (B,C) Structural models of GL cluster “11111111” displaying 90° of rotation around the carbohydrate stem for the base (B) and O-acetylated base (C) polysaccharides. The acetyl group is shown in brown, glucose in blue, mannose in green, galactose in yellow, abequose in purple, and rhamnose in cyan. For clarity, all hydrogen atoms are omitted except those on the acetyl group. (D,E,F,G) 3D spatial distributions (wire frame) displaying 90° of rotation around the carbohydrate stem for the base (D,E) and O-acetylated base (F,G) polysaccharides for all non-hydrogen atoms. The reducing end Rha (1, blue), indicated at the base, is approximated as linked to the core polysaccharide or preceding OPS repeat. Conformational flexibility increases progressively relative to the reducing end anchor point. The base polysaccharide backbone is identified by a transparent grey surface overlaid with the individual O-acetyl groups in color for monosaccharides (1) [blue], (4) [red], (5) [orange], (8) [tan], (9) [green] and (12) [purple]. Contour levels are set to 10⁻⁶ for the full polysaccharide and to 10⁻⁵ for the individual acetyl groups.</p

STm COPS size characterization and the effect of pH on rhamnose and abequose O-acetylation in 1925wzzB-COPS.

<p>(A) SDS-PAGE and Pro-Q staining for LPS from CVD 1925 (lane 1) and CVD 1925 (pSEC10-<i>wzzB</i>) (lane 2). (B) Chromatogram of purified D65 COPS (dashed line) and 1925wzzB-COPS (solid line) assessed by HPLC-SEC with detection by refractive index. (C) ¹H NMR analysis of 1925wzzB-COPS after exposure to different pH levels. Peaks for rhamnose C2/C3 and abequose C2 O-acetyls are indicated. (D) Hestrin analysis of residual O-acetylation of 1925wzzB-COPS after incubation at different pH levels. (E) ELISA reactivity of 1925wzzB-COPS and dOAc-1925wzzB-COPS with monoclonal antibodies against O4 (α-STm O4) and O5 (α-STm-O5) or polyclonal sera recognizing O1,12 and core epitopes (α-SE). Error bars represent s.d. and were derived from technical replicates from one experiment.</p

Immunogenicity, protective efficacy and functional analyses of vaccine-induced antibody for mice immunized with conjugates of native or dOAc-1925wzzB-COPS with CRM₁₉₇.

<p>(A) Serum IgG titers for 1925wzzB-COPS (black), dOAc-1925wzzB-COPS (grey) or SE COPS (open) from mice (<i>n</i> = 40/group) immunized with PBS, STm-COPS^KDO:CRM₁₉₇ or dOAc-STm-COPS^KDO:CRM₁₉₇ as indicated. Each point represents an individual mouse. Red squares indicate mice that succumbed after challenge. Solid bars indicate the GMT; comparisons between groups were accomplished by either a two-tailed Mann-Whitney U test (PBS vs. conjugate, conjugate 1 vs. conjugate 2) or two-tailed Wilcoxon signed-rank test (paired serological analyses). (B) Kaplan-Meier survival curves of mice immunized with STm-COPS^KDO:CRM₁₉₇ (circle), dOAc-STm-COPS^KDO:CRM₁₉₇ (square) or PBS (diamond) after challenge (<i>n</i> = 20/group) with 1x10⁶ CFU (open symbol, dashed line) or 5x10⁶ CFU (closed symbol, solid line) of STm D65. Adjustments for multiple comparisons were not made. *<i>P</i> ≤ 0.0001 for indicated comparisons or for vaccinated mice relative to the respective PBS challenge group. ^#<i>P</i> ≤ 0.05, ^##<i>P</i> ≤ 0.005 for STm-COPS^KDO:CRM₁₉₇ vaccinated mice relative to the respective challenge dose group immunized with dOAc-STm-COPS^KDO:CRM₁₉₇. (C) Representative serum anti-COPS epitope profiles (identified by symbol) from mice immunized with either STm-COPS^KDO:CRM₁₉₇ or dOAc-STm-COPS^KDO:CRM₁₉₇. Selected sera were chosen to assess serum bactericidal antibodies (“SBA”, D) and opsonophagocytic antibodies (“OPA”, E) and were diluted such that each sample contained equivalent anti-1925wzzB-COPS IgG EU. Curves were fitted to each serial dilution of serum and were compared using nonlinear regression analysis. Dashed line indicates 0% killing. Results are representative of two independent assays, error bars represent s.d. and were derived from technical replicates; n.s., not significant.</p

Biophysical and biochemical attributes of STm COPS conjugates used in this study.

<p>Biophysical and biochemical attributes of STm COPS conjugates used in this study.</p

Percent of total simulation time occupied by the accessible conformations of each STm OPS^a.

<p>Percent of total simulation time occupied by the accessible conformations of each STm OPS<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005493#t003fn001" target="_blank">^a</a>.</p

¹H and ¹³C NMR analysis of native and de-O-acetylated 1925wzzB-COPS and native D65 COPS.

<p>¹H and ¹³C spectra obtained by 800 MHz NMR are shown for 1925wzzB-COPS, dOAc-1925wzzB-COPS, and D65 COPS. NMR shifts for O-acetylated and native monosaccharides within the COPS polymer are differentiated by letter: α-D-Abequ<i>p</i> [A], α-D-Abequ<i>p</i> + OAc at C2 [B], α-L-Rha<i>p</i> [C], α-L-Rha<i>p</i> + OAc at C2 [D], α-L-Rha<i>p</i> + OAc at C3 [E].</p