| The utility of bacteriophage lambda (lambda) phage display, herein coined Mashkiki, has been investigated. Mashkiki phage displaying TAT proved ineffective for delivery of recombinant genetic cargos. However, Mashkiki phage did prove effective for antigen presentation to the immune system. As a subunit vaccine vector, lambda was shown to be a potent stimulus for splenocytes, regardless of immunization. The pro-inflammatory nature of Mashkiki was associated with IFNgamma production. This co-stimulatory signal produced cellular and humoral immunity against lambda and antigen. Antigen was effectively presented with lambda as either a displayed peptide or by genetic immunization.;In order to determine the involvement of TLR4 in adjuvant activity, downstream signalling pathways from TLR4 were assayed for activity after lambda treatment. TLR4 ligation with lambdagfp10-TAT-GFP caused activation of the NF-kappabeta pathway in a TLR4-independent fashion, but lack of MyD88 attenuated activation. In contrast signalling through p38 was largely TLR4-dependent, but MyD88-independent. In the context of TLR signalling, MAPK signalling is therefore TRIF-dependent, and results in IP10 expression. On the other hand NF-kappabeta signalling was largely MyD88-dependent and signalling resulted in IFNgamma and IL-6 expression. The cytokines produced from this response, which differ in timing and amplitude from LPS, the prototypical TLR4 ligand, are: IFNgamma, IL-2, IL-6, IL-10, IL-13, IL17a, IP-10, and MIP-1alpha. The initiation of an adaptive immune response from lambda is therefore multifaceted. The adjuvant activity of lambda is not TLR4 restricted. It is postulated that lambda will provide immune stimulation through TLR9, a NOD-like receptor. (Abstract shortened by UMI.);The adjuvant activity of Mashkiki phage was examined and was found to be intriguing. A study to find the pattern recognition receptor responsible for lambda adjuvant activity ensued. lambda was found to cause TLR4 receptor ligation, therefore a vaccine trial was performed with Mashkiki phage in the TLR4 hypo-responsive mutant C3H/HeJ. The humoral immune response to lambda was found to be TLR4-independent, as titers of lambda recognizing antibodies were equivalent between C3H/HeJ mice and the TLR4 responsive mouse strain C3H/HeOuJ. Interestingly, the adjuvant activity for a lambda displayed antigen was 1 log10 weaker in C3H/HeJ mice than wt C3H/HeOuJ. |
