Hepatic adeno-associated virus (AAV)-serotype 2 mediated gene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid-specific CD8(+) T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.

Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles. They are attractive vaccine vectors as they induce both innate and adaptive immune responses in mammalian hosts. Currently, adenovirus vectors are being tested as subunit vaccine systems for numerous infectious agents ranging from malaria to HIV-1. Additionally, they are being explored as vaccines against a multitude of tumor-associated antigens. In this review we describe the molecular biology of adenoviruses as well as ways the adenovirus vectors can be manipulated to enhance their efficacy as vaccine carriers. We describe methods of evaluating immune responses to transgene products expressed by adenoviral vectors and discuss data on adenoviral vaccines to a selected number of pathogens. Last, we comment on the limitations of using human adenoviral vectors and provide alternatives to circumvent these problems. This field is growing at an exciting and rapid pace, thus we have limited our scope to the use of adenoviral vectors as vaccines against viral pathogens.

IntroductionAdeno-associated viral (AAV) vectors are widely used for stable in vivo gene transfer to terminally differentiated or quiescent cells such as muscle fibers, hepatocytes, neurons, retinal cells, and others. These vectors, derived from a nonpathogenic, replicationdefective parvovirus with a small single-stranded (ss) DNA genome, have recently been successfully used in clinical gene transfer for inherited blindness and also show promise for other diseases. 1,2 Eight years ago, Zaiss et al 3 found that ssAAV serotype-2 vectors caused only a weak and highly transient innate immune response in the liver, suggesting that inflammatory reactions to AAV are negligible. Numerous animal studies have shown stable correction of genetic diseases by hepatic AAV gene transfer that may, in part, be because of the low innate immune profile of the vector, avoiding inflammatory signals. 4 In humans, hepatic gene transfer with AAV2 has been hampered by pre-existing adaptive immunity after natural infection in the form of neutralizing antibodies and capsid-specific CD8 ϩ T cells. 5 Numerous changes to capsid and vector genomes have been developed in recent years in attempts to improve gene transfer efficacy and possibly evade immunity. For example, AAV8 shows substantially higher transduction efficiency in mouse liver and reduced activation of capsid-specific T cells, and it facilitates tolerance induction to transgenes. 6,7 Furthermore, prevalence for neutralizing antibodies in humans is markedly lower to AAV8 than to AAV2. 8 In another set of investigations, replacing surfaceexposed tyrosine residues to phenylalanine has been shown to improve gene transfer for several serotypes. The resulting reduction in capsid phosphorylation in turn reduces accumulation in the cytoplasm (in favor of trafficking to the nucleus) and ubiquitination of capsid. 9 AAV2 gene transfer to hepatocytes was most improved by a combination of 3 Tyr-Phe changes in amino acid residues 444, 500, and 730. 10 Modifications of the recombinant AAV genome also can improve transduction rates. Being ss, the ssAAV genome has to be converted to a double-stranded form in the nucleus of an infected cell for transgene expression to occur. To overcome this ratelimiting step, self-complementary (sc)AAV vectors were developed by elimination of the terminal resolution site in one of the inverted terminal repeats (ITRs). 11 For such a genome to be packaged into capsid, the size of the expression cassette has to be further reduced to not exceed the packaging limit. Two groups reported optimized scAAV vectors for treatment of the X-linked bleeding disorder hemophilia B (coagulation factor IX deficiency) by liver gene transfer. 12,13 The hepatic microenvironment is more tolerogenic than that of many other tissues. 14 For example, we were able to tolerize hemophilia B mice to human factor IX (hF.IX) by hepatic ssAAV2 gene transfer. This protocol was successful in several strains, with the exception of C3H. 15 Nonetheless, AAV8 and AAV2(Y444/500/ 730F) vectors were able to...

The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (≥30 min), cognate antigen–dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.