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

New Data on Vaccine Antigen Deficient Bordetella pertussis Isolates

Evolution of Bordetella pertussis is driven by natural and vaccine pressures. Isolates circulating in regions with high vaccination coverage present multiple allelic and antigenic variations as compared to isolates collected before introduction of vaccination. Furthermore, during the last epidemics reported in regions using pertussis acellular vaccines, isolates deficient for vaccine antigens, such as pertactin (PRN), were reported to reach high proportions of circulating isolates. More sporadic filamentous hemagglutinin (FHA) or pertussis toxin (PT) deficient isolates were also collected. The whole genome of some recent French isolates, deficient or non-deficient in vaccine antigens, were analyzed. Transcription profiles of the expression of the main virulence factors were also compared. The invasive phenotype in an in vitro human tracheal epithelial (HTE) cell model of infection was evaluated. Our genomic analysis focused on SNPs related to virulence genes known to be more likely to present allelic polymorphism. Transcriptomic data indicated that isolates circulating since the introduction of pertussis vaccines present lower transcription levels of the main virulence genes than the isolates of the pre-vaccine era. Furthermore, isolates not producing FHA present significantly higher expression levels of the entire set of genes tested. Finally, we observed that recent isolates are more invasive in HTE cells when compared to the reference strain, but no multiplication occurs within cells

The diversity of lipopolysaccharide (O) and capsular polysaccharide (K) antigens of invasive klebsiella pneumoniae in a multi-country collection

Klebsiella pneumoniae is a common cause of sepsis and is particularly associated with healthcare-associated infections. New strategies are needed to prevent or treat infections due to the emergence of multi-drug resistant K. pneumoniae. The goal of this study was to determine the diversity and distribution of O (lipopolysaccharide) and K (capsular polysaccharide) antigens on a large (\u3e500) global collection of K. pneumoniae strains isolated from blood to inform vaccine development efforts. A total of 645 K. pneumoniae isolates were collected from the blood of patients in 13 countries during 2005-2017. Antibiotic susceptibility was determined using the Kirby-Bauer disk diffusion method. O antigen types including the presence of modified O galactan types were determined by PCR. K types were determined by multiplex PCR and wzi capsular typing. Sequence types of isolates were determined by multilocus sequence typing (MLST) targeting seven housekeeping genes. Among 591 isolates tested for antimicrobial resistance, we observed that 19.3% of isolates were non-susceptible to carbapenems and 62.1% of isolates were multidrug resistant (from as low as 16% in Sweden to 94% in Pakistan). Among 645 isolates, four serotypes, O1, O2, O3, and O5, accounted for 90.1% of K. pneumoniae strains. Serotype O1 was associated with multidrug resistance. Fifty percent of 199 tested O1 and O2 strains were gmlABC-positive, indicating the presence of the modified polysaccharide subunit D-galactan III. The most common K type was K2 by both multiplex PCR and wzi capsular typing. Of 39 strains tested by MLST, 36 strains were assigned to 26 known sequence types of which ST14, ST25, and ST258 were the most common. Given the limited number of O antigen types, diverse K antigen types and the high multidrug resistance, we believe that an O antigen-based vaccine would offer an excellent prophylactic strategy to prevent K. pneumoniae invasive infection

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

Distribution of serotypes and antibiotic resistance of invasive Pseudomonas aeruginosa in a multi-country collection

Background: Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections and is frequently associated with healthcare-associated infections. Because of its ability to rapidly acquire resistance to antibiotics, P. aeruginosa infections are difficult to treat. Alternative strategies, such as a vaccine, are needed to prevent infections. We collected a total of 413 P. aeruginosa isolates from the blood and cerebrospinal fluid of patients from 10 countries located on 4 continents during 2005-2017 and characterized these isolates to inform vaccine development efforts. We determined the diversity and distribution of O antigen and flagellin types and antibiotic susceptibility of the invasive P. aeruginosa. We used an antibody-based agglutination assay and PCR for O antigen typing and PCR for flagellin typing. We determined antibiotic susceptibility using the Kirby-Bauer disk diffusion method.Results: Of the 413 isolates, 314 (95%) were typed by an antibody-based agglutination assay or PCR (n = 99). Among the 20 serotypes of P. aeruginosa, the most common serotypes were O1, O2, O3, O4, O5, O6, O8, O9, O10 and O11; a vaccine that targets these 10 serotypes would confer protection against more than 80% of invasive P. aeruginosa infections. The most common flagellin type among 386 isolates was FlaB (41%). Resistance to aztreonam (56%) was most common, followed by levofloxacin (42%). We also found that 22% of strains were non-susceptible to meropenem and piperacillin-tazobactam. Ninety-nine (27%) of our collected isolates were resistant to multiple antibiotics. Isolates with FlaA2 flagellin were more commonly multidrug resistant (p = 0.04).Conclusions: Vaccines targeting common O antigens and two flagellin antigens, FlaB and FlaA2, would offer an excellent strategy to prevent P. aeruginosa invasive infections

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

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