A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

Immune stealth-driven O2 serotype prevalence and potential for therapeutic antibodies against multidrug resistant Klebsiella pneumoniae.

Emerging multidrug-resistant bacteria are a challenge for modern medicine, but how these pathogens are so successful is not fully understood. Robust antibacterial vaccines have prevented and reduced resistance suggesting a pivotal role for immunity in deterring antibiotic resistance. Here, we show the increased prevalence of Klebsiella pneumoniae lipopolysaccharide O2 serotype strains in all major drug resistance groups correlating with a paucity of anti-O2 antibodies in human B cell repertoires. We identify human monoclonal antibodies to O-antigens that are highly protective in mouse models of infection, even against heavily encapsulated strains. These antibodies, including a rare anti-O2 specific antibody, synergistically protect against drug-resistant strains in adjunctive therapy with meropenem, a standard-of-care antibiotic, confirming the importance of immune assistance in antibiotic therapy. These findings support an antibody-based immunotherapeutic strategy even for highly resistant K. pneumoniae infections, and underscore the effect humoral immunity has on evolving drug resistance.

Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study.

Several candidate vaccines against Shigella spp. are in development, but the lack of a clear correlate of protection from challenge with the induction of adequate immune responses among the youngest age groups in the developing world has hampered Shigella vaccine development over the past several decades. Bioconjugation technology, exploited here for an Shigella flexneri 2a candidate vaccine, offers a novel and potentially cost-effective way to develop and produce vaccines against a major pathogen of global health importance. Flexyn2a, a novel S. flexneri 2a bioconjugate vaccine made of the polysaccharide component of the S. flexneri 2a O-antigen, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for safety and immunogenicity among healthy adults in a single-blind, phase I study with a staggered randomization approach. Thirty subjects (12 receiving 10 µg Flexyn2a, 12 receiving Flexyn2a with aluminum adjuvant, and 6 receiving placebo) were administered two injections 4 weeks apart and were followed for 168 days. Flexyn2a was well-tolerated, independently of the adjuvant and number of injections. The Flexyn2a vaccine elicited statistically significant S. flexneri 2a lipopolysaccharide (LPS)-specific humoral responses at all time points postimmunization in all groups that received the vaccine. Elicited serum antibodies were functional, as evidenced by bactericidal activity against S. flexneri 2a. The bioconjugate candidate vaccine Flexyn2a has a satisfactory safety profile and elicited a robust humoral response to S. flexneri 2a LPS with or without inclusion of an adjuvant. Moreover, the bioconjugate also induced functional antibodies, showing the technology's features in producing a promising candidate vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT02388009.).

Immunogenicity and safety of a tetravalent E. coli O-antigen bioconjugate vaccine in animal models.

BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are major human pathogens; however, no protective vaccine is currently available. We assessed in animal models the immunogenicity and safety of a 4-valent E. coli conjugate vaccine (ExPEC-4V, serotypes O1, O2, O6 and O25 conjugated to Exotoxin A from Pseudomonas aeruginosa (EPA)) produced using a novel in vivo bioconjugation method. METHODS: Three doses of ExPEC-4V (with or without aluminum hydroxide) were administered to rabbits (2µg or 20µg per O-antigen, subcutaneously), mice (0.2µg or 2µg per O-antigen, subcutaneously) and rats (0.4µg or 4µg per O-antigen, intramuscularly). Antibody persistence and boostability were evaluated in rats using O6-EPA monovalent conjugate (0.4µg O-antigen/dose, intramuscularly). Toxicity was assessed in rats (16µg total polysaccharide, intramuscularly). Serum IgG and IgM antibodies were measured by ELISA. RESULTS: Robust antigen-specific IgG responses were observed in all animal models, with increased responses in rabbits when administered with adjuvant. O antigen-specific antibody responses persisted up to 168days post-priming. Booster immunization induced a rapid recall response. Toxicity of ExPEC-4V when administered to rats was considered to be at the no observed adverse effect level. CONCLUSIONS: ExPEC-4V conjugate vaccine showed good immunogenicity and tolerability in animal models supporting progression to clinical evaluation.

Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella dysenteriae type 1 administered to healthy adults: A single blind, partially randomized Phase I study.

BACKGROUND: Shigellae cause severe disease in endemic countries, especially in children. Several efficacy trials have been conducted with candidate vaccines against Shigellae, but the lack of protection, the safety concerns, or manufacturing challenges hindered successful market approval. Conjugated vaccines have been shown to be safe and effective for different pathogens (i.e., Neisseria meningitidis, Shigella pneumonia, Haemophilus influenzae). The bio-conjugation technology, exploited here for the Shigella dysenteriae candidate vaccine, offers a novel and potentially simpler way to develop and produce vaccines against one of the major causes of morbidity and mortality in developing countries. METHODS: A novel S. dysenteriae bioconjugate vaccine (GVXN SD133) made of the polysaccharide component of the Shigella O1 lipopolysaccharide, conjugated to the exotoxin protein A of Pseudomonas aeruginosa (EPA), was evaluated for immunogenicity and safety in healthy adults in a single blind, partially randomized Phase I study. Forty subjects (10 in each dose group; 2 µg or 10 µg with or without aluminium adjuvant) received two injections 60 days apart and were followed-up for 150 days. RESULTS: Both doses and formulations were well tolerated; the safety and reactogenicity profiles were consistent with that of other conjugated vaccines, adjuvanted or not, independent of the dose and the number of injections. The GVXN SD133 vaccine elicited statistically significant O1 specific humoral responses at all time points in all vaccination groups. Between-group comparisons did not show statistically significant differences in geometric mean titers of immunoglobulin G and A at any post-vaccination time point. CONCLUSIONS: This study demonstrated that the GVXN SD133 vaccine has a satisfactory safety profile. It elicited a significant humoral response to Shigella O1 polysaccharides at all doses tested. The protein carrier also elicited functional antibodies, showing the technology's advantages in preserving both sugar and conjugated protein epitopes. This trial is registered at ClinicalTrials.gov (NCT01069471).

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: preclinical evidence of immunogenicity and protection.

Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb-rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.

Evaluation of protective efficacy of an Actinobacillus pleuropneumoniae serotype 1 lipopolysaccharide-protein conjugate in mice.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The major adhesin of A. pleuropneumoniae has previously been identified as a lipopolysaccharide (LPS), and more recently, we demonstrated that high molecular mass LPS were involved in A. pleuropneumoniae adherence to porcine respiratory tract cells. We postulated that immunization with a LPS-based vaccine may confer a protective immunity. The high molecular mass O-polysaccharides obtained after acid hydrolysis and chromatographic separation were conjugated to bovine serum albumin (BSA) as a protein carrier. Groups of mice were injected twice with the following antigen preparations: whole-cell preparation, outer membrane preparation, O-polysaccharide-BSA conjugate, hydrolyzed LPS and phenol/water extracted LPS. A combination of different adjuvants was also used during these immunization procedures to induce a stronger immunological response to the polysaccharide antigen. Two weeks after the second injection, the mice were challenged intranasally with either homologous A. pleuropneumoniae serotype 1 strain or a serotype 5 strain. The highest survival rate, up to 80%, compared to the control groups (P < 0.05), was recorded when the mice were injected twice with 15 micrograms of carbohydrates of O-polysaccharide-BSA conjugate mixed with the saponin-derived adjuvant Quil A. Survival rates of between 60 and 70%, twice those observed in the control groups immunized with PBS, were recorded in mice injected with the O-polysaccharide-BSA conjugate mixed with other adjuvant preparations such as alhydrogel, peanut oil and Freund's incomplete adjuvant. However, the protection induced by the conjugate antigen preparation was serotype specific, because mice challenged with a serotype 5 strain were killed. Taken together, these results confirm the important role of A. pleuropneumoniae LPS in pathogenesis.