(190af) Simulating Bacterial Infection to Trick Neutrophils into Enhancing Vaccine-Induced Immune Response

ABSTRACT

BACKGROUND:

Neutrophils are part of the innate immune system, and are amongst the first to extravasate from the blood circulation to the site of bacterial infection to help clear the pathogen. Upon reaching the infected site, neutrophils phagocytose the bacterial pathogen and secrete cytokines and chemokines to communicate with other cells of the immune system to mount an adaptive immune response. Neutrophils are attracted to the site of infection by chemoattractants released by the bacteria. N-formylmethionyl-leucyl-phenylalanine (fMLP), a tripeptide released by bacteria is an especially potent neutrophil chemoattractant. We hypothesized that fMLP could be used as a vaccine adjuvant, and that addition of fMLP to the vaccine formulation should enhance the immune response against the antigen.

METHODS:

To test our hypothesis, we delivered ovalbumin (OVA) as a model antigen to mice with and without fMLP. Since neutrophils are highly phagocytic, we also asked the question if incorporation of particles in the formulation could boost the immune response. Thus, in some formulations we mixed poly (lactic-co-glycolic) acid (PLGA) particles. Since the site of vaccine delivery can affect the level of immune response, we compared the immune responses after hypodermic injection of the vaccine into the thigh muscle (IM) or the tongue. We selected tongue for comparison because we postulated that its presence in the oral cavity might result in a better mucosal immune response. Mice were vaccinated twice at three-week intervals with various formulations. A control group was given OVA+Alum via the intramuscular route. At various time points, serum, fecal matter, saliva and vaginal wash samples were collected. At the end of the study, spleen, lung lavage, and mesenteric lymph nodes were also collected. Serum IgG, IgG1, IgG2a, and mucosal IgA antibody responses were measured using Enzyme Linked Immunosorbant Assay (ELISA). Splenocytes were restimulated to get a better understanding of the cellular responses. In a separate study, immunohistochemistry (IHC) of thigh and tongue tissues was done 24 h after vaccine injection to study neutrophil chemotaxis.

RESULTS:

Statistical analysis (n=5 mice/group) of ELISA data showed that injection of the formulation comprising of OVA+PLGA+fMLP in the tongue generated a significantly greater (p<0.0001) fecal mucosal IgA response than that of IM. Similarly, delivery of formulations to the tongue generated significantly higher (p<0.0001) systemic IgG responses. Inclusion of PLGA to the Ova+fMLP formulations significantly increased the IgG response in IM (p<0.005) and tongue groups (p<0.0001) suggesting an important role of particles in the formulation. IHC imaging of tissue cross-section illustrated increased neutrophil and macrophage infiltration at injection sites with use of fMLP.

CONCLUSION:

Our data shows that the OVA+PLGA+fMLP formulation when delivered in the thigh or the tongue of a mouse results in a strong immune response, and inclusion of both fMLP and PLGA is required for significant enhancement in immune response. Thus, use of fMLP is unique and it can simulate bacterial infection to attract neutrophils to the site of vaccination, which in turn can help to generate a stronger immune response against the antigen. These studies thus lay the foundation of investigating fMLP and other neutrophil attractants as a novel approach for enhancing vaccine-induced immunity.