Multivalent mRNA-DTP vaccines are immunogenic and provide protection from Bordetella pertussis challenge in mice.

Acellular multivalent vaccines for pertussis (DTaP and Tdap) prevent symptomatic disease and infant mortality, but immunity to Bordetella pertussis infection wanes significantly over time resulting in cyclic epidemics of pertussis. The messenger RNA (mRNA) vaccine platform provides an opportunity to address complex bacterial infections with an adaptable approach providing Th1-biased responses. In this study, immunogenicity and challenge models were used to evaluate the mRNA platform with multivalent vaccine formulations targeting both B. pertussis antigens and diphtheria and tetanus toxoids. Immunization with mRNA formulations were immunogenetic, induced antigen specific antibodies, as well as Th1 T cell responses. Upon challenge with either historical or contemporary B. pertussis strains, 6 and 10 valent mRNA DTP vaccine provided protection equal to that of 1/20th human doses of either DTaP or whole cell pertussis vaccines. mRNA DTP immunized mice were also protected from pertussis toxin challenge as measured by prevention of lymphocytosis and leukocytosis. Collectively these pre-clinical mouse studies illustrate the potential of the mRNA platform for multivalent bacterial pathogen vaccines.

An mRNA-based platform for the delivery of pathogen-specific IgA into mucosal secretions.

Colonization of the gut and airways by pathogenic bacteria can lead to local tissue destruction and life-threatening systemic infections, especially in immunologically compromised individuals. Here, we describe an mRNA-based platform enabling delivery of pathogen-specific immunoglobulin A (IgA) monoclonal antibodies into mucosal secretions. The platform consists of synthetic mRNA encoding IgA heavy, light, and joining (J) chains, packaged in lipid nanoparticles (LNPs) that express glycosylated, dimeric IgA with functional activity in vitro and in vivo. Importantly, mRNA-derived IgA had a significantly greater serum half-life and a more native glycosylation profile in mice than did a recombinantly produced IgA. Expression of an mRNA encoded Salmonella-specific IgA in mice resulted in intestinal localization and limited Peyer's patch invasion. The same mRNA-LNP technology was used to express a Pseudomonas-specific IgA that protected from a lung challenge. Leveraging the mRNA antibody technology as a means to intercept bacterial pathogens at mucosal surfaces opens up avenues for prophylactic and therapeutic interventions.

Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: A new approach to prevent and treat bacterial infections.

The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.

Advancements in mRNA Encoded Antibodies for Passive Immunotherapy.

Monoclonal antibodies are the fastest growing therapeutic class in medicine today. They hold great promise for a myriad of indications, including cancer, allergy, autoimmune and infectious diseases. However, the wide accessibility of these therapeutics is hindered by manufacturing and purification challenges that result in high costs and long lead times. Efforts are being made to find alternative ways to produce and deliver antibodies in more expedient and cost-effective platforms. The field of mRNA has made significant progress in the last ten years and has emerged as a highly attractive means of encoding and producing any protein of interest in vivo. Through the natural role of mRNA as a transient carrier of genetic information for translation into proteins, in vivo expression of mRNA-encoded antibodies offer many advantages over recombinantly produced antibodies. In this review, we examine both preclinical and clinical studies that demonstrate the feasibility of mRNA-encoded antibodies and discuss the remaining challenges ahead.

Characterization of an Antibody Recognizing the Conserved Inner Core of Pseudomonas aeruginosa Lipopolysaccharides.

Bacterial infections are a growing public health threat with carbapenem-resistant Pseudomonas aeruginosa being classified as a Priority 1 critical threat by the World Health Organization. Antibody-based therapeutics can serve as an alternative and in some cases supplement antibiotics for the treatment of bacterial infections. The glycans covering the bacterial cell surface have been proposed as intriguing targets for binding by antibodies; however, antibodies that can engage with high affinity and specificity with glycans are much less common compared to antibodies that engage with protein antigens. In this study, we sought to characterize an antibody that targets a conserved glycan epitope on the surface of Pseudomonas. First, we characterized the breadth of binding of VSX, demonstrating that the VSX is specific to Pseudomonas but can bind across multiple serotypes of the organism. Next, we provide insight into how VSX engages with its target epitope, using a combination of biolayer interferometry and nuclear magnetic resonance, and verify our results using site-directed mutagenesis experiments. We demonstrate that the antibody, with limited somatic hypermutation of the complementarity-determining regions (CDRs) and with a characteristic set of arginines within the CDRs, specifically targets the conserved inner core of Pseudomonas lipopolysaccharides. Our results provide important additional context to antibody-glycan contacts and provide insight useful for the construction of vaccines and therapeutics against Pseudomonas aeruginosa, an important human pathogen.

A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins.

Conjugates between proteins and small molecules enable access to a vast chemical space that is not achievable with either type of molecule alone; however, the paucity of specific reactions capable of functionalizing proteins and natural products presents a formidable challenge for preparing conjugates. Here we report a strategy for conjugating electron-rich (hetero)arenes to polypeptides and proteins. Our bioconjugation technique exploits the electrophilic reactivity of an oxidized selenocysteine residue in polypeptides and proteins, and the electron-rich character of certain small molecules to provide bioconjugates in excellent yields under mild conditions. This conjugation chemistry enabled the synthesis of peptide-vancomycin conjugates without the prefunctionalization of vancomycin. These conjugates have an enhanced in vitro potency for resistant Gram-positive and Gram-negative pathogens. Additionally, we show that a 6 kDa affibody protein and a 150 kDa immunoglobulin-G antibody could be modified without diminishing bioactivity.

Antibody-Bactericidal Macrocyclic Peptide Conjugates To Target Gram-Negative Bacteria.

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off-target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody-bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram-negative bacteria. We used cysteine SN Ar chemistry to synthesize and systematically study a library of large (>30-mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.

Automated solid-phase synthesis of hyaluronan oligosaccharides.

Well-defined fragments of hyaluronic acid (HA) have been obtained through a fully automated solid-phase oligosaccharide synthesis. Disaccharide building blocks, featuring a disarmed glucuronic acid donor moiety and a di-tert-butylsilylidene-protected glucosamine part, were used in the rapid and efficient assembly of HA fragments up to the pentadecamer level, equipped with a conjugation-ready anomeric allyl function.

Combinatorial chemistry in glycobiology.

The application of combinatorial chemistry to glycobiology historically has proven challenging due to numerous synthetic hurdles. The advent of novel methodologies has enabled the production of natural as well as mimetic analogues for proof-of-concept experiments and SAR. This review highlights some of the recent synthetic advances in combinatorial carbohydrate synthesis. The application of carbohydrate libraries in glycobiology is also discussed.

Recent advances in automated solid-phase carbohydrate synthesis: from screening to vaccines.

The past year has witnessed significant advances in a new technology for the synthesis of complex carbohydrates. Solid-phase methods have been applied to the construction of previously inaccessible carbohydrates. Furthermore, the application of automated solid-phase carbohydrate synthesis is promising. New methods for the synthesis of carbohydrates and potential applications are described in this review.