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Abstract: Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.

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Abstract: Many aldehydes such as furfural are present in high quantities in lignocellulose lysates and are fermentation inhibitors that make biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinkii. Rational engineering of this enzyme could enhance the furfural tolerance of this organism thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75 Å. Docking studies identified residues involved in substrate binding and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggests that the active site of the enzyme is optimally configured for catalysis with the substrate tested.

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Abstract: Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.”

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Abstract: There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.

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Abstract: Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte epitope produce the SLFNTIAVL triple mutant “ultimate” escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent-exposed residues in the peptide destabilise the peptide–HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate re-evaluation of this exemplar model of HIV TCR escape.