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Long-acting injectable antiretroviral (LA-ARV) drugs with low toxicity profiles and propensity for drug-drug interactions are a goal for future ARV regimens. C34-PEG4-Chol is a novel cholesterol tagged LA HIV-fusion-inhibitor (FI). We assessed pre-clinical toxicology and first-in-human administration of C34-PEG4-Chol. Pre-clinical toxicology was conducted in 2 species. HIV-positive men were randomised to a single subcutaneous dose of C34-PEG4-Chol at incrementing doses or placebo. Detailed clinical (including injection site reaction (ISR) grading), plasma pharmacokinetic (time-to-minimum-effective-concentration (MEC, 25 ng/mL) and pharmacodynamic (plasma HIV RNA) parameters were assessed. In both mice and dogs, no-observed-adverse effect level (NOAEL) was observed at a 12 mg/kg/dose after two weeks. Of 5 men enrolled, 3 received active drug (10 mg, 10 mg and 20 mg). In 2 individuals grade 3 ISR occurred and the study was halted. Both ISR emerged within 12 hours of active drug dosing. No systemic toxicities were observed. The time-to-MEC was >72 and >96 hours after 10 and 20 mg dose, respectively, and mean change in HIV RNA was −0.9 log10 copies/mL. These human pharmacodynamic and pharmacokinetic data, although limited to 3 subjects, of C34-PEG-4-Chol suggest continuing evaluation of this agent as a LA-ARV. However, alternative administration routes must be explored.

Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high-magnitude, durable CD4+ and CD8+ T-cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV-specific immune responses and determine T-cell cross-reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1-infected patients were vaccinated using heterologous adenoviral vectors (ChAd3-NSmut and Ad6-NSmut) encoding HCV NS proteins in a dose escalation, prime-boost regimen, with and without concomitant pegylated interferon-α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon-γ enzyme-linked immunospot assays. Cross-reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV-specific T-cell responses following vaccination was markedly reduced. CD8+ HCV-specific T-cell responses were detected in 15/24 patients at the highest dose, whereas CD4+ T-cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T-cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. Conclusion: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T-cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections.

Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high-magnitude, durable CD4(+) and CD8(+) T-cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV-specific immune responses and determine T-cell cross-reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1-infected patients were vaccinated using heterologous adenoviral vectors (ChAd3-NSmut and Ad6-NSmut) encoding HCV NS proteins in a dose escalation, prime-boost regimen, with and without concomitant pegylated interferon-α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon-γ enzyme-linked immunospot assays. Cross-reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV-specific T-cell responses following vaccination was markedly reduced. CD8(+) HCV-specific T-cell responses were detected in 15/24 patients at the highest dose, whereas CD4(+) T-cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T-cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. CONCLUSION: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T-cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections.

The isolation of ligands that bind biologically relevant molecules is fundamental to the understanding of biological processes and to the search for therapeutics. Filamentous phage can be used to display foreign peptides and proteins in physical association with their DNA coding sequences. Repertoires larger than 10(8) phage clones expressing different peptide sequences can be prepared using molecular genetic techniques. The strategies utilizing this technology promise to provide not only new binding and possibly catalytic activities, but also lead structures for the development of new drugs and vaccines.

Simian adenoviral and modified vaccinia Ankara (MVA) viral vectors used in heterologous prime-boost strategies are potent inducers of T cells against encoded antigens and are in advanced testing as vaccine carriers for a wide range of infectious agents and cancers. It is unclear if these responses can be further enhanced or sustained with reboosting strategies. Furthermore, despite the challenges involved in MVA manufacture dose de-escalation has not been performed in humans. In this study, healthy volunteers received chimpanzee-derived adenovirus-3 and MVA vaccines encoding the non-structural region of hepatitis C virus (ChAd3-NSmut/MVA-NSmut) 8 weeks apart. Volunteers were then reboosted with a second round of ChAd3-NSmut/MVA-NSmut or MVA-NSmut vaccines 8 weeks or 1-year later. We also determined the capacity of reduced doses of MVA-NSmut to boost ChAd3-NSmut primed T cells. Reboosting was safe, with no enhanced reactogenicity. Reboosting after an 8-week interval led to minimal re-expansion of transgene-specific T cells. However, after a longer interval, T cell responses expanded efficiently and memory responses were enhanced. The 8-week interval regimen induced a higher percentage of terminally differentiated and effector memory T cells. Reboosting with MVA-NSmut alone was as effective as with ChAd3-NSmut/MVA-NSmut. A ten-fold lower dose of MVA (2 × 107pfu) induced high-magnitude, sustained, broad, and functional Hepatitis C virus (HCV)-specific T cell responses, equivalent to standard doses (2 × 108 pfu). Overall, we show that following Ad/MVA prime-boost vaccination reboosting is most effective after a prolonged interval and is productive with MVA alone. Importantly, we also show that a ten-fold lower dose of MVA is as potent in humans as the standard dose.

An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with Interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T- cell responses were rarely detected, and overall the magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFa production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T- cell response. Full-length, next generation sequencing of circulating virus demonstrated that T-cells were only induced by vaccination when there was sequence mismatch between autologous virus and the vaccine immunogen. However, these T cells were not cross -reactive with endogenous viral variant epitopes. Conversely when there was complete homology between immunogen and circulating virus at a given epitope T-cells were not induced. T-cell induction following vaccination had no significant impact on HCV viral load. In vitro T-cell culture experiments identified the presence of T-cells at baseline that could be expanded by vaccination; thus HCV-specific T-cells may have been expanded from pre-existing low-level memory T-cell populations that had been exposed to HCV antigen during natural infection, explaining the partial T-cell dysfunction. In conclusion, vaccination with ChAd3-NSmut and MVA-NSmut prime/boost, a potent vaccine regimen previously optimised in healthy volunteers, was unable to reconstitute HCV-specific T-cell immunity in HCV infected patients. This highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure.

Plasmids containing eukaryotic tRNA genes are faithfully transcribed in the nucleus of Xenopus laevis oocytes [Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) Nucleic Acids Res. 5, 4593-4611]. It has been established that two separated regions within the coding sequence of a tRNA gene are essential and sufficient for promotion of transcription [Hofstetter, H., Kressmann, A. & Birnstiel, M. L. (1981) Cell 24, 573-585; Ciliberto, G., Castagnoli, L., Melton, D. A. & Cortese, R. (1982) Proc. Natl. Acad. Sci. USA 79, 1195-1199]. We have constructed a hybrid tRNA gene containing one essential region from tDNALeu and the other from tDNAPro, both from Caenorhabditis elegans. This hybrid gene is efficiently transcribed, thus showing that the essential regions are independent transcriptional signals regardless of the overall regularities of the structure of tRNA genes. We have also constructed mutants of the tRNAPro gene in which the distance between the two essential regions is changed; optimal transcription occurs when this distance is about 40-50 nucleotides.

We present a novel general method for localized mutagenesis. The DNA segment to be mutagenized is inserted in the beta-galactosidase gene of a M13-lac vector, generally causing loss of beta-galactosidase function by generation of frameshifts or nonsense codons. Mutations in the inserted DNA which restore beta-galactosidase function are readily detected and analyzed. The application of this method to the promoter of an eukaryotic (Caenorhabditis elegans) tRNAPro gene has allowed the isolation of several mutants altered in transcription.

The biosynthesis of transfer RNA (tRNA) in eukaryotes is determined by a variety of enzymatic reactions, which produce the functional mature tRNA molecule (see Figure 1). RNA polymerase III catalyzes the transcription from the double-stranded tDNA gene into an oversized tRNA precursor, which contains 5’-end leader as well as 3’-end trailer sequences and, sometimes, intervening sequences (Altman, 1978). Other protein factors are needed for transcription besides RNA polymerase III: transcription starts with G or A, and control regions have been described, one external to the coding core (De Franco et al., 1980), not always necessary, others within the structural core of the tRNA gene (Hofstetter et al., 1981). In connection to these latter, a series of recent researches, involving genetic manipulation and localized mutagenesis of tRNA genes, indicate at the invariant nucleotides of the I and IV loops of the tRNA molecule the critical positions for transcription (Ciliberto et al., 1981; Soll, 1981). Then, the tRNA precursor molecule undergoes to the exo- and endonucleolytic process which produces the tailoring of the 3’- and 5’-flanking regions to the final tRNA size. The nucleotidyl transferase activity finally refines the aminoacylation site by adding the terminal 3’-triplet.

We have constructed a series of single and double base-pair-substitution mutants in and around the second component (Box B) of the promoter of a tRNA Pro gene from C. elegans. Their analysis in in vivo and in vitro transcriptional systems establishes the importance of single nucleotides in the promotion of transcription. Most mutants in the region coding for the TψCG stem-loop show a reduced gene expression associated with lack of processing of the primary transcriptional products; in the oocytes these are rapidly degraded, with a half-life considerably shorter than that of wild-type tRNA molecules. In contrast, mutations in the DNA region coding for the anticodon stem-loop do not alter the efficiency of transcription or the processing of the transcripts.

Eukaryotic tDNA promoters are composed of two essential regions contained within the coding sequence (Box A and Box B). Due to the highly conserved structure of prokaryotic and eukaryotic tRNA, most prokaryotic tRNA genes are expected to be active templates in eukaryotic transcriptional systems. In this paper we show that Escherichia coli tDNATyr is not transcribed in the nucleus of Xenopus laevis oocytes. By in vitro construction of hybrid molecules between inactive prokaryotic tDNATyr from E. coli, and active eukaryotic tDNAPro from Caenorhabditis elegans, we show that tDNATyr can be made into an active gene if its first third, including the Box A region, is replaced by that of the eukaryotic tDNA . These results suggest that an improper Box A sequence is responsible for the inactivity of the E. coli tRNATyr gene, and argue against the role of secondary and tertiary DNA conformations in RNA polymerase III transcription.