Timos Papagatsias

Adenovirus vector vaccination induces expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1

In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α4β7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.

Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs.

Langerhans cells are more efficiently transduced than dermal dendritic cells by adenovirus vectors expressing either group C or group B fibre protein: Implications for mucosal vaccines

Vaccines against viruses need to target dendritic cells (DC) and stimulate mucosal immunity. Most vaccine studies have focussed on monocyte‐derived or dermal DC (dDC) but recent evidence suggests that Langerhans cells (LC) may stimulate mucosal immunity more effectively. New chimeric adenovirus vectors expressing fibre protein from group B adenoviruses (rAd5/11), which utilise CD46 rather than the Coxsackie adenovirus receptor (CAR), have been developed as vaccines to improve transduction and overcome problems of pre‐existing vector immunity. Transduction of LC and dDC by rAd5/11 and standard rAd5 expressing green fluorescent protein (GFP) showed that both DC types were more efficiently transduced by rAd5/11 than by rAd5. Although expression of CD46 and the integrins αvβ3 and αvβ5, which recognise the adenovirus penton base and mediate virus internalisation, was similar in LC and dDC, LC expressed higher levels of GFP. Transduction by electroporation of plasmid also resulted in higher GFP expression in LC, suggesting differences between the two DC populations at a post‐entry stage. Transduction with either vector did not induce maturation of LC or dDC and did not affect their ability to stimulate T cells. These findings suggest that vaccine strategies that target LC with adenovirus vectors may be worthy of exploration.

Use of adenovirus in vaccines for HIV.

The best hope of controlling the HIV pandemic is the development of an effective vaccine. In addition to the stimulation of virus neutralising antibodies, a vaccine will need an effective T-cell response against the virus. Vaccines based on recombinant adenoviruses (rAd) are promising candidates to stimulate anti-HIV T-cell responses. This review discusses the different rAd vector types, problems raised by host immune responses against them and strategies that are being adopted to overcome this problem. Vaccines need to target and stimulate dendritic cells and thus the tropism and interaction of rAd-based vaccines with these cells is covered. Different rAd vaccination regimes and the need to stimulate mucosal responses are discussed together with data from animal studies on immunogenicity and virus challenge experiments. The review ends with a discussion of the recent disappointing Merck HIV vaccine trial.

Fragmentation of SIV-gag Vaccine Induces Broader T Cell Responses

Background High mutation rates of human immunodeficiency virus (HIV) allows escape from T cell recognition preventing development of effective T cell vaccines. Vaccines that induce diverse T cell immune responses would help overcome this problem. Using SIV gag as a model vaccine, we investigated two approaches to increase the breadth of the CD8 T cell response. Namely, fusion of vaccine genes to ubiquitin to target the proteasome and increase levels of MHC class I peptide complexes and gene fragmentation to overcome competition between epitopes for presentation and recognition. Methodology/Principal Findings Three vaccines were compared: full-length unmodified SIV-mac239 gag, full-length gag fused at the N-terminus to ubiquitin and 7 gag fragments of equal size spanning the whole of gag with ubiquitin-fused to the N-terminus of each fragment. Genes were cloned into a replication defective adenovirus vector and immunogenicity assessed in an in vitro human priming system. The breadth of the CD8 T cell response, defined by the number of distinct epitopes, was assessed by IFN-γ-ELISPOT and memory phenotype and cytokine production evaluated by flow cytometry. We observed an increase of two- to six-fold in the number of epitopes recognised in the ubiquitin-fused fragments compared to the ubiquitin-fused full-length gag. In contrast, although proteasomal targeting was achieved, there was a marked reduction in the number of epitopes recognised in the ubiquitin-fused full-length gag compared to the full-length unmodified gene, but there were no differences in the number of epitope responses induced by non-ubiquitinated full-length gag and the ubiquitin-fused mini genes. Fragmentation and ubiquitination did not affect T cell memory differentiation and polyfunctionality, though most responses were directed against the Ad5 vector. Conclusion/Significance Fragmentation but not fusion with ubiquitin increases the breadth of the CD8 T vaccine response against SIV-mac239 gag. Thus gene fragmentation of HIV vaccines may maximise responses.

Fusion of ubiquitin to HIV gag impairs human monocyte-derived dendritic cell maturation and reduces ability to induce gag T cell responses

The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs.

OA07-02. Adenovirus vectors induce expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1 infection.

Open Access Oral presentation OA07-02. Adenovirus vectors induce expansion of memory CD4 T cells with a mucosal homing phenotype that are readily susceptible to HIV-1 infection A Benlahrech*1, J Harris2, A Meiser1, T Papagatsias1, J Hornig1, P Hayes1, A Lieber3, T Athanasopoulos2, V Bachy4, R Daniels5, K Fisher6, F Gotch1, L Klavinskis4, L Seymour6, K Logan1, R Barbagallo1, G Dickson2 and S Patterson1