Development of a nano-emulsion based multivalent protein subunit vaccine against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

The L-DBF vaccine cross protects mice against different Shigella serotypes after prior exposure to the pathogen.

IMPORTANCE: Shigellosis is endemic to low- and middle-income regions of the world where children are especially vulnerable. In many cases, there are pre-existing antibodies in the local population and the effect of prior exposure should be considered in the development and testing of vaccines against Shigella infection. Our study shows that L-DBF-induced immune responses are not adversely affected by prior exposure to this pathogen. Moreover, somewhat different cytokine profiles were observed in the lungs of vaccinated mice not having been exposed to Shigella, suggesting that the immune responses elicited by Shigella infection and L-DBF vaccination follow different pathways.

Impact of the TLR4 agonist BECC438 on a novel vaccine formulation against Shigella spp.

Shigellosis (bacillary dysentery) is a severe gastrointestinal infection with a global incidence of 90 million cases annually. Despite the severity of this disease, there is currently no licensed vaccine against shigellosis. Shigella's primary virulence factor is its type III secretion system (T3SS), which is a specialized nanomachine used to manipulate host cells. A fusion of T3SS injectisome needle tip protein IpaD and translocator protein IpaB, termed DBF, when admixed with the mucosal adjuvant double-mutant labile toxin (dmLT) from enterotoxigenic E. coli was protective using a murine pulmonary model. To facilitate the production of this platform, a recombinant protein that consisted of LTA-1, the active moiety of dmLT, and DBF were genetically fused, resulting in L-DBF, which showed improved protection against Shigella challenge. To extrapolate this protection from mice to humans, we modified the formulation to provide for a multivalent presentation with the addition of an adjuvant approved for use in human vaccines. Here, we show that L-DBF formulated (admix) with a newly developed TLR4 agonist called BECC438 (a detoxified lipid A analog identified as Bacterial Enzymatic Combinatorial Chemistry candidate #438), formulated as an oil-in-water emulsion, has a very high protective efficacy at low antigen doses against lethal Shigella challenge in our mouse model. Optimal protection was observed when this formulation was introduced at a mucosal site (intranasally). When the formulation was then evaluated for the immune response it elicits, protection appeared to correlate with high IFN-γ and IL-17 secretion from mucosal site lymphocytes.

Immunogenicity and protective efficacy of nanoparticle formulations of L-SseB against Salmonella infection.

Salmonella enterica, a Gram-negative pathogen, has over 2500 serovars that infect a wide range of hosts. In humans, S. enterica causes typhoid or gastroenteritis and is a major public health concern. In this study, SseB (the tip protein of the Salmonella pathogenicity island 2 type III secretion system) was fused with the LTA1 subunit of labile-toxin from enterotoxigenic E. coli to make the self-adjuvanting antigen L-SseB. Two unique nanoparticle formulations were developed to allow multimeric presentation of L-SseB. Mice were vaccinated with these formulations and protective efficacy determined via challenging the mice with S. enterica serovars. The polysaccharide (chitosan) formulation was found to elicit better protection when compared to the squalene nanoemulsion. When the polysaccharide formulation was used to vaccinate rabbits, protection from S. enterica challenge was elicited. In summary, L-SseB in a particulate polysaccharide formulation appears to be an attractive candidate vaccine capable of broad protection against S. enterica.

A protein subunit vaccine elicits a balanced immune response that protects against Pseudomonas pulmonary infection.

The opportunistic pathogen Pseudomonas aeruginosa (Pa) causes severe nosocomial infections, especially in immunocompromised individuals and the elderly. Increasing drug resistance, the absence of a licensed vaccine and increased hospitalizations due to SARS-CoV-2 have made Pa a major healthcare risk. To address this, we formulated a candidate subunit vaccine against Pa (L-PaF), by fusing the type III secretion system tip and translocator proteins with LTA1 in an oil-in-water emulsion (ME). This was mixed with the TLR4 agonist (BECC438b). Lung mRNA sequencing showed that the formulation activates genes from multiple immunological pathways eliciting a protective Th1-Th17 response following IN immunization. Following infection, however, the immunized mice showed an adaptive response while the PBS-vaccinated mice experienced rapid onset of an inflammatory response. The latter displayed a hypoxic lung environment with high bacterial burden. Finally, the importance of IL-17 and immunoglobulins were demonstrated using knockout mice. These findings suggest a need for a balanced humoral and cellular response to prevent the onset of Pa infection and that our formulation could elicit such a response.

Effect of Two Unique Nanoparticle Formulations on the Efficacy of a Broadly Protective Vaccine Against Pseudomonas Aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections in humans. In addition to its innate antibiotic resistance, P. aeruginosa is very effective in acquiring resistance resulting in the emergence of multi-drug resistance strains and a licensed vaccine is not yet available. We have previously demonstrated the protective efficacy of a novel antigen PaF (Pa Fusion), a fusion of the type III secretion system (T3SS) needle tip protein, PcrV, and the first of two translocator proteins, PopB. PaF was modified to provide a self-adjuvanting activity by fusing the A1 subunit of the heat-labile enterotoxin from Enterotoxigenic E. coli to its N-terminus to give L-PaF. In addition to providing protection against 04 and 06 serotypes of P. aeruginosa, L-PaF elicited opsonophagocytic killing and stimulated IL-17A secretion, which have been predicted to be required for a successful vaccine. While monomeric recombinant subunit vaccines can be protective in mice, this protection often does not transfer to humans where multimeric formulations perform better. Here, we use two unique formulations, an oil-in-water (o/w) emulsion and a chitosan particle, as well as the addition of a unique TLR4 agonist, BECC438 (a detoxified lipid A analogue designated Bacterial Enzymatic Combinatorial Chemistry 438), as an initial step in optimizing L-PaF for use in humans. The o/w emulsion together with BECC438 provided the best protective efficacy, which correlated with high levels of opsonophagocytic killing and IL-17A secretion, thereby reducing the lung burden among all the vaccinated groups tested.