Production of Recombinant Adeno-Associated Virus

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Chapter 4 – Transduction of MoDCs by rAAV

 Introduction

Dendritic cells (DCs) are the key antigen presenting cell for priming naive CD4+ and CD8+ T cell responses (25). In mice selective depletion of DCs inhibits priming of CD4+ and CD8+ T cell responses (441, 442). DCs present endogenous antigens or cross present exogenous antigens on MHC class I molecules, stimulating CD8+ T cell responses (29, 36). DCs stimulate CD4+ T cells by presenting exogenous antigens on MHC class II molecules; intracellular antigens can also be presented on MHC class II molecules (51-53). CD4+ T cell help is required for a robust CD8+ T cell response. CD4+ T cells are important for licensing DCs via CD40L (45), maintaining CD8+ T cell memory (46), and enabling tissue homing of CD8+ T cells (47). Robust antigen-specific CD8+ T cell responses are required for the control of chronic viral infections, such as HBV, HCV, and HIV, and malignancy (6, 443-451). Therefore, dendritic cells are an attractive target for in vivo and ex vivo vaccination strategies.
Efficient priming of T cell responses requires antigen presentation by mature DCs (25, 26). DCs mature in response to danger signals or licensing by CD4+ T cells (25). On maturation DCs upregulate the expression of MHC class I and II molecules, which are loaded with antigenic peptides (signal 1). Mature DCs also up-regulate the expression of co-stimulatory molecules such as CD80 and CD86 (signal 2), which stimulate several rounds of T cell proliferation, and secrete cytokines (signal 3), which program T cells for survival and effector functions (26). Signals 2 and 3 are essential for priming naive T cell responses (26). In contrast, antigen presentation by immature dendritic cells inhibits T cell effector function and induces the expression of antigen-specific IL-10 secreting cells (452). Therefore maturation of DCs is critical in any T cell vaccination strategy.
Distinct subsets of DCs are known to exist, each with variable capability to stimulate T-cell responses and secrete type 1 interferon (453). In the absence of inflammation, conventional DCs (cDCs) and type I interferon-secreting plasmacytoid DCs (pDCs) are found in lymphoid tissues, non-lymphoid tissues, and in blood (454). cDCs found in different lymphoid and non-lymphoid tissues are phenotypically distinct (454). Non-lymphoid tissue resident cDCs include Langerhans cells in the epidermis, dermal DCs in the dermis, mucosa-associated DCs, and interstitial DCs, while lymphoid tissue DCs include splenic marginal zone DCs, T cell zone-associated interdigitating DCs, germinal centre DCs, and thymic DCs (454). Further, several different subsets of DCs can be distinguished in peripheral blood (453). Under inflammatory conditions, monocytes can also differentiate into cDCs, known as monocyte-derived DCs (MoDCs) (453, 455). Cells resembling conventional DCs can be derived in vitro from monocytes or CD34+ haematopoietic stem cells (290, 455); plasmacytoid DCs can be derived from CD34+ haematopoietic stem cells (290). As primary DCs are a rare population in the peripheral blood, in vitro generated DCs are widely used as experimental models and have also been used in ex vivo DC therapy. Various protocols exist for generating monocyte-derived dendritic cells (MoDCs) but most involve culture of monocytes in a cytokine cocktail, usually GM-CSF and IL4 or tumour necrosis factor-α (TNF-α) for between 2-7 days (455, 456). At the end of the culture period the MoDCs phenotypically resemble either immature or mature conventional DCs, depending on the protocol. The process of differentiation of monocytes into DCs in vitro is thought to recapitulate what occurs in vivo at sites of inflammation, suggesting that this in vitro derived cell type has relevance in vivo.
Various viral vectors have been investigated as potential vaccines for stimulating CD8+ T cell responses, including recombinant adenoviruses, poxviruses and lentiviruses (165, 173, 174, 180, 183). Adenovirus, poxvirus and lentivirus vectors are able to infect dendritic cells (115-117, 124, 180). The ability to transduce dendritic cells has been correlated with the ability to stimulate a robust T cell response (115, 378). Adenoviral vectors stimulate strong transgene-specific T cell responses (148, 160). However, anti-vector T cell responses are commonly seen (150, 151), which may be immunodominant and limit the expansion of transgene-specific T cells. Furthermore, preexisting neutralising antibodies to the most frequently used adenoviral serotypes are common, reducing their efficacy (148, 164). Immunodominance is also a problem with the use of poxvirus vectors, which encode multiple viral proteins (152). In addition, infection of dendritic cells by vaccinia virus is abortive, prevents dendritic cell maturation and induces apoptotic cell death (124). While pre-existing immunity is not seen with lentiviral vectors, the risk of insertional mutagenesis may limit their use (183, 185).
Recombinant adeno-associated virus (rAAV) is an alternative candidate T cell vaccine vector and has several advantages over other viral vectors. In contrast to poxviral, lentiviral, and most adenoviral vectors, rAAV lacks sequences encoding viral proteins, reducing the risk of an immunodominant immune response against vector-derived epitopes. Unlike lentiviral vectors the rAAV genome forms an extrachromosomal episome, reducing the risk of insertional mutagenesis (250). Also, there is no clear association between wild-type AAV infection and disease, and there is extensive experience from clinical trials with rAAV as a potential gene therapy vector (312). In vitro experiments in mice and non-human primates have confirmed the ability of rAAV2/2 to prime a CD8+ T cell response, with a reduction in viral set point seen following vaccination in a rhesus macaque SIV model (352, 371). However, a phase I clinical trial of rAAV2/2 as a vector for a prophylactic HIV vaccine demonstrated poor immunogenicity (372). Subsequent studies in mice with rAAV2/7 and 2/8 showed that while large numbers of antigen-specific CD8+ effector T cells were stimulated, there was poor development of T cell memory and an inability to boost rAAV-primed T cell responses. The authors confirmed their results with rAAV2/1, 2/2, 2/5 and 2/9 (373, 374). This phenomenon may reflect poor priming, a lack of CD4+ T cell help, or partial T cell exhaustion due to the persistence of antigen. However, not all pseudotypes prime a dysfunctional T cell response. It was recently shown that rAAV2/rh32.33 primes robust CD4+ and CD8+ T cell responses that can be boosted with a recombinant adenoviral vector (376). Furthermore, the differential ability of various rAAV pseudotypes to prime CD8+ T cell responses has been linked to their ability to transduce DCs (378).
Transduction of human MoDCs was first demonstrated with AAV2/2 (343). Subsequent studies have confirmed the ability of AAV2/2 to transduce MoDCs and to stimulate antigen-specific T cells in vitro and have also found rAAV2/1 to be more efficient than rAAV2 in transducing MoDCs (290, 457). A screen of AAV1-5 and 7-8 revealed the tropism of rAAV5/5 for both human and murine DCs leading to robust immune responses against HIV epitopes in mice (283), while another recent study identified rAAV2/6 as having the highest tropism for murine bone marrow-derived DCs (383). Given that rAAV2/1, 2/2 and 2/5 have been shown to prime dysfunctional T cell responses in a mouse model, the identification of a pseudotype that is more efficient at transducing MoDCs may improve the utility of rAAV as a vaccine vector.
In this study a wide range of pseudotyped rAAV vectors were compared for their ability to transduce human MoDCs at various stages of differentiation. A single exposure of monocytes or immature MoDCs to rAAV2/6 resulted in significantly greater transduction of MoDCs than with other serotypes or clones. Further improvement in transduction efficiency was observed following mutation of tyrosine residues within the VP1 capsid. Transduction by rAAV2/6 caused minimal alteration of immunophenotype of MoDCs which retained their immunostimulatory ability. Furthermore, antigen encoded by rAAV2/6 was correctly processed and presented to T cells by transduced MoDCs. Altogether, these results provide a rational basis for the use of AAV6-based vectors in the development of human vaccines and immunotherapy applications.

Results

Recombinant AAV2/6 is the optimal serotype for the transduction of human monocytes and MoDCs

A limited range of serotypes has previously been assessed for their ability to transduce human monocytes and MoDCs. This study sought to determine whether previously untested serotypes and clones might exhibit a greater tropism for monocytes and human MoDCs. In preliminary experiments human monocytes (day 0) or immature MoDCs were infected with rAAV pseudotyped with capsids from serotypes 1, 2, 5, 6, 8, or 9, or with variants rh8, rh10, or rh13 (Figure 3.6) at a multiplicity of infection (MOI) of 1×105 vg copies per cell. Transduction was assessed 48 hours after infection by detection of expression of the vector-encoded transgene eGFP by flow cytometry (Table 4.1). Only rAAV2/1, rAAV2, rAAV2/5, and rAAV2/6 vectors transduced more than 0.5% of day 6 MoDCs and only rAAV2/6 more than 0.5% of monocytes. Table 4.1 Initial screening study of transduction of monocytes and monocyte-derived dendritic cells by various pseudotypes of rAAV. Results were compared with a one way ANOVA; if p<0.05 then pairwise comparisons were made by the Holm-Sidak method. N=3. a Transduction defined as fluorescence in FL-1 >99.9% of the uninfected control. b AAV6 vs rest, p<0.001, other differences NS. c AAV6 vs rest, p<0.001, other differences NS. The ability of rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/6 to transduce MoDCs and monocytes was examined in more detail using cells isolated from three donors repeated on two different occasions (Figure 4.1). Consistent with the preliminary experiments (Table 4.1) rAAV2/6 was significantly more effective at transducing MoDCs than the next most effective vector, rAAV2/2 (14.2% mean transduction efficiency versus 3.3%, p=0.005), while rAAV2/1 and rAAV2/5 gave transduction efficiencies of 1.0% and 1.6% respectively. Monocytes were considerably more resistant to transduction by all serotypes when compared with MoDCs (Figure 4.1 (B)). However when the assessment of transgene expression in transduced monocytes was delayed until day 8, there was little difference when compared to MoDCs (Figure 4.1 (C)). Only a modest decline in transduction efficiency was observed when the MOI was reduced to 1 x 104 and transduction efficiency with rAAV2/6 at a MOI of 1 x 104 remained higher than with 10-fold more rAAV2/1, rAAV2/2, or rAAV2/5 (Figure 4.1 (A) and Figure 4.1 (D)).
Second strand synthesis has previously been shown to be a rate limiting step in the transduction of MoDC with the use of a self-complimentary rAAV (scAAV) increasing the level of transgene expression (290, 458). In parallel with the experiments described above the effect of a selfcomplimentary genome on the rates of transduction with rAAV2/6 was assessed but no statistically significant difference was seen at the MOIs tested (Figure 4.1 (D)). Notably, eGFP expression was driven by a CMV promoter in the self-complimentary vector while in the single stranded vector expression was driven by the CAG/WPRE promoter enhancer. Of note the CAG-eGFP-WPREbGHpA expression cassette exceeds the packaging capacity of the self-complimentary vector. Figure 4.1 Effect of rAAV serotype, genome configuration and multiplicity of infection on transduction MoDCs. Day 6 MoDCs (A) or day 0 monocytes (B) were infected with the indicated serotypes or variants of rAAV/eGFP at a MOI of 105 and 48 hours later eGFP expression was assessed by flow cytometry. Transduction of MoDCs infected at day 0 was also assessed at day 8 and compared with infection at day 6 at the indicated MOIs (C). To compare self-complimentary and conventional genomes day 6 MoDCs were infected with scAAV2/6 or rAAV2/6 and eGFP expression assessed at day 8 by flow cytometry (D). All experiments were performed in parallel. Monocytes were collected from three donors on two different occasions (n=6) and experiments performed in duplicate. Means are indicated by horizontal lines. Serotypes, variants, and MOIs (A, B, D) were compared with a one way ANOVA with subsequent pairwise multiple comparisons by the Holm-Sidak method. Day of transduction and genome configuration were compared by paired t tests. * AAV6 vs others p<0.01; ‡ AAV6 vs AAV1/5, p<0.01; † AAV6 MOI 105 vs AAV6 MOI 103, p=0.02; †† scAAV6 MOI 105 vs scAAV6 MOI 103 (p<0.001) and MOI 104, p=0.02. There was no significant difference between rAAV2/6 and scAAV2/6 at any MOI.

Transduction of MoDCs by rAAV2/6 is abolished by a lysine to arginine mutation in the capsid

Significantly lower transduction of MoDCs was seen with rAAV2/1 than with rAAV2/6, despite 99.2% sequence similarity between the capsids of AAV1 and AAV6. Wu et al., demonstrated that a single lysine to glutamate mutation (K531E) in the capsid of AAV6 reduced both liver transduction efficiency and the ability to bind heparin while the converse mutation in the AAV1 capsid had the opposite effect (223). To investigate the molecular basis for the distinct tropism of rAAV2/6 for MoDCs the effect of the K531E mutation in the AAV6 capsid and the converse mutation in the AAV1 capsid on the transduction of MoDCs was assessed. Figure 4.2 (A) demonstrates that the K531E mutation significantly reduces the transduction of MoDCs by rAAV2/6 while the converse mutation in AAV1 (E531K) restored a level of transduction comparable to the wild-type rAAV2/6 vector. No inhibition of transduction by rAAV2/6 was seen with soluble heparin except at the highest concentration tested (1mg/ml) where only partial inhibition was seen. By contrast, transduction of MoDCs with rAAV2 was susceptible to inhibition by soluble heparin at a 10-fold lower concentration (Figure 4.2 (B)). These experiments suggest that rAAV2/6 does not utilise heparin as a receptor on MoDCs despite being able to bind immobilised heparin.

Mutation of surface-exposed tyrosine residues improves transduction of MoDCs by rAAV2/6

It has recently been shown that mutation of surface-exposed tyrosine residues in the capsid of AAV2 blocks capsid phosphorylation by epidermal growth factor receptor protein tyrosine kinase (EGFR-RTK) and subsequent ubiquitination and proteasomal degradation (236). Capsid mutants achieved higher levels of transduction of HeLa cells in vitro and murine hepatocytes in vivo than vectors with wild-type capsids and increased nuclear translocation of vector was demonstrated. The amino acid sequences of VP1 from AAV2 and AAV6 are highly conserved; of the three tyrosine residues in the AAV2 sequence that were previously associated with the greatest improvement in transduction efficiency when mutated only Y500 is absent in VP1 from AAV6 (Figure 4.3). To investigate whether mutation of the other two surface-exposed tyrosines would improve transgene expression in rAAV2/6-transduced MoDCs, Y445 and Y731 in the AAV6 capsid were mutated to phenylalanine and an additional double mutant (Y445F + Y731F) was generated. Transduction of HeLa cells with vectors containing these mutations confirmed their ability to increase the rate of transduction albeit at a reduced level compared to that reported previously for rAAV2/2; no synergistic effect was seen with the double mutant (Figure 4.4 (A)). When the effect of tyrosine mutation on the transduction of MoDCs was assessed, a modest (approximately 2-fold) increase in transgene expression was observed with the Y731F mutant. Surprisingly, both the Y445F and double mutant resulted in a significant decrease in transduction (Figure 4.4 (B)). The role of proteasomal degradation in this reduction in transduction was unable to be assessed as proteasome inhibitors were highly toxic to MoDCs (data not shown).

Contents
Abstract
Acknowledgements
Contents
Figures and Tables
Abbreviations
1. Introduction  
1.1 Vaccines
1.2 T cells
1.3 Priming a T cell response
1.4 T cell vaccines
1.5 Adeno-associated virus
1.6 Summary
2. Materials and Methods
2.1 Materials
2.2 Methods
3. Production of Recombinant Adeno-Associated Virus
3.1 Introduction
3.2 Results
3.3 Discussion
4. Transduction of MoDCs by rAAV
4.1 Introduction
4.2 Results
4.3 Discussion
5. Development of a self-adjuvanting rAAV vector
5.1 Introduction
5.2 Results
5.3 Discussion
6. Baculovirus Production System for rAAV6 (Y731F)
6.1 Introduction
6.2 Methods
6.3 Discussion
7. Summary and Perspectives   
7.1 rAAV as a vaccine vector
7.2 Improving immunogenicity by optimising transduction of dendritic cells
7.3 The failure of rAAV to activate DCs
7.4 Self adjuvanting rAAV vectors
7.5 Development of a baculovirus production system
7.6 Use of an alternative vector-encoded adjuvant
7.7 Future assessment of self-adjuvanting rAAV vaccine vectors in vitro
7.8 Future assessment of self-adjuvanting rAAV vaccine vectors in vivo
7.9 The problem of T cell exhaustion
7.10 Conclusions
8. Appendix
9. References

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Improving the Immunogenicity of Recombinant AdenoAssociated Virus

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