Development of a Molecular Adjuvant to Enhance Antigen-Specific CD8+ T Cell Responses

Despite promising progress in malaria vaccine development, an efficacious subunit vaccine against P. falciparum remains to be licensed and deployed. This study aimed to improve on the immunogenicity of the leading liver-stage vaccine candidate (ChAd63-MVA ME-TRAP), known to confer protection by eliciting high levels of antigen-specific CD8+ T cells. We previously showed fusion of ME-TRAP to the human MHC class II invariant chain (Ii) could enhance CD8+ T cell responses in non-human primates, but did not progress to clinical testing due to potential risk of auto-immunity by vaccination of humans with a self-antigen. Initial immunogenicity analyses of ME-TRAP fused to subdomains of the Ii showed that the Ii transmembrane domain alone can enhance CD8+ T cell responses. Subsequently, truncated Ii sequences with low homology to human Ii were developed and shown to enhance CD8+ T cell responses. By systematically mutating the TM domain sequence, multimerization of the Ii chain was shown to be important for immune enhancement. We subsequently identified several proteins from a variety of microbial pathogens with similar characteristics, that also enhance the CD8+ T cell response and could therefore be used in viral vector vaccines when potent cell mediated immunity is required.


Halbroth et al, Supplementary Information
Effect of endogenous signal peptide (eSP) removal from TRAP. C57BL/6 mice were immunised with 10 8 IU ChAd63 vectors encoding ME-TRAP with or without its endogenous signal peptide. Spleens were harvested two weeks later. CD4 + and CD8 + T cell responses to a TRAP peptide pool are shown by measuring intracellular IFN-γ using flow cytometry. Single points represent T cell responses of individual mice and lines indicate the median response per group. Data was analysed with a one-way analysis of variance. No statistical difference was found between the groups. 4 Figure S2 Immune response against Pb9. In the same experiment as described in Figure 1B, CD8 + T cell responses to Pb9 measured by intracellular staining for IFN-γ by flow cytometry is shown. Single points represent T cell responses of individual mice and lines indicate the median response per group. Data was analysed with a one-way analysis of variance with Dunn's multiple comparison post-test. Asterisks denote the level of statistical significance when compared to the control ME-TRAP vaccinated group (**, p<0.01).

Figure S3
MVA induced immune responses. Four C57BL/6 mice per groups were immunised with 10 6 PFU MVA unfused or adjuvanted ME-TRAP as indicated on the x-axis. Spleens were harvested one week later.
CD4 + and CD8 + T cell responses to a TRAP peptide pool are shown by measuring intracellular IFN-γ using flow cytometry. Single points represent T cell responses of individual mice and lines indicate the median response per group. Data was analysed with a one-way analysis of variance with Dunn's multiple comparison post-test. No statistical difference was found between the groups. 5 Figure S4 TRAP-specific response in non-human primates. Male rhesus macaques were vaccinated IM with either ChAd63 encoding ME-TRAP followed eight weeks later by MVA.ME-TRAP, or ChAd63.(tr)human72/Ii-ME-TRAP followed eight weeks later by MVA.(tr)human72/Ii-ME-TRAP. Blood samples were taken on the day of vaccination, weeks, 2, 4, 8 following ChAd63 vaccination on d 1 (9), 3 (11) and 8 (16)

Figure S6
Invariant chain fusion alters the pattern of TRAP expression. A549 cells were transfected with pENTR4-LPTOS plasmids encoding different Ii-ME-TRAP fusion constructs, incubated overnight prior to staining for TRAP expression (green). The nucleus was stained with DAPI (blue). Immunofluorescence images in 100× magnification are shown.

Figure S7
Analyses of (tr)humanQ64A/Ii fused to ME-TRAP. (A) C57BL/6 mice were immunised with 10 7 IU ChAd63 vectors and spleens were harvested two weeks later. T cell responses to a TRAP peptide pool were    Table S1 Homology analysis of full-length Ii sequences. Full-length Ii sequences of various species were compared for homology to the full-length human and mouse Ii sequences. Entitled name, GenBank identifier/NCBI

Table S2
Homology analysis of truncated Ii sequences. Ii sequences from a variety of animal species were truncated corresponding to the length of (tr)human72/Ii and compared for homology to truncated human and mouse Ii sequences. Entitled name, GenBank identifier/NCBI Reference Sequence, and sequence homology with (fl)human/Ii or (fl)mouse/Ii sequence are listed. Homology was analysed using the ClustalW algorithm in DNAStar® Megalign. Highlighted constructs were fused to the N-terminal end of ME-TRAP and produced in ChAd63 vectors.