Background: Vaccine development against Chlamydia, a prevalent sexually transmitted infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccines based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP). Methods: Cell-free protein synthesis (CFPS) technology is an attractive approach to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provide a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade, we have been working toward the production and characterization of MOMP-NLP complexes for vaccine testing. Results: The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scattering (DLS), which confirm the formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1. Conclusion: Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential to elicit robust immune responses. However, further research is warranted to optimize vaccine formulations further, validate efficacy against Chlamydia trachomatis, and better understand the underlying mechanisms of immune response.

Developing novel strategies that can exploit a cell's endogenous machinery to selectively release toxic compounds in diseased cells, while leaving healthy cells unaffected, is a challenging task to accomplish. An established approach involves antigen recognition on tumor cells, enabling targeted delivery of prodrugs in which a cytotoxic compound is appended to an antibody by a cleavable linker. However, this strategy depends on the presence of highly expressed surface markers for which antibodies can be developed, limiting its applicability to cancers that display these markers. A new approach that could rely on disease-specific expression of an intracellular enzyme, which can be more broadly targeted, could expand the therapeutic potential of prodrugs clinically. Here, we demonstrate that the immunoproteasome, an isoform of the standard proteasome that is upregulated under conditions of inflammation, has the potential to be harnessed as a prodrug release enzyme. We conjugate the highly toxic and widely used therapeutic cargo MMAE onto an immunoproteasome-selective peptide and demonstrate its selective release in cancerous cells at low nM to pM concentrations, while healthy cells remain viable. We also establish the first translational validation that using immunoproteasome prodrugs in vivo leads to a significant reduction in tumor volume without significant toxicities in small cell lung cancer. We anticipate this approach to be broadly applicable, as the immunoproteasome is upregulated in a variety of cancers and does not require antibody recognition to be effective.