CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

Hexon-modified recombinant E1-deleted adenovirus vectors as dual specificity vaccine carriers for influenza virus.

To determine if an ordered and repetitive display of an epitope promoted induction of superior antibody responses, we compared B-cell responses to an influenza A virus epitope that was either encoded as a transgene by an adenovirus (Ad) vector or expressed on the vector's surface. To this end, we constructed a panel of influenza A virus vaccines based on chimpanzee-derived replication-defective adenovirus (AdC) vectors of serotype SAd-V25 also called AdC68. AdC68 vectors were modified to express a linear B-cell epitope of the ectodomain of matrix 2 (M2e) within variable regions 1 (VR1) or 4 (VR4) of the adenovirus hexon. Additional vectors with wild-type or M2e-modified hexon encoded M2e fused to the influenza A virus nucleoprotein (NP) as a transgene product. Hexon-modified vectors were tested for immunogenicity and efficacy in mice in comparison to vectors with native hexon expressing the M2e-NP fusion protein. Upon priming, vectors expressing M2e within VR1 of hexon induced M2e-specific antibody responses of higher magnitude and avidity than those carrying M2e within VR4 or vectors expressing the M2e as part of a transgene product. CD8(+) T-cell responses to the transgenic NP were comparable between vectors. M2e-specific antibody responses could be boosted by a second dose of the VR1 hexon-modified vector but not by repeated immunization with the VR4 hexon-modified vector.

Adeno-associated virus vectors serotype 2 induce prolonged proliferation of capsid-specific CD8+ T cells in mice.

Using adoptive transfer models we determined that an adeno-associated viral vector of serotype 2 (AAV2) induces in mice proliferation of CD8(+) T cells that recognize an epitope within the viral capsid. Proliferation to an endogenous epitope within viral protein (VP)3 could be observed for at least 3 weeks while a foreign epitope placed at multiple copies within VP2 elicited CD8(+) T cell expansion for at least 10 weeks. These data show that capsid antigens of AAV2 degrade slowly over a period of weeks and during this period provide targets to CD8(+) T cells.

Vaccine-induced T cells provide partial protection against high-dose rectal SIVmac239 challenge of rhesus macaques.

Despite enormous efforts by the scientific community, an effective HIV vaccine remains elusive. To further address to what degree T cells in absence of antibodies may protect against simian immunodeficiency virus (SIV) disease progression, rhesus macaques were vaccinated intramuscularly with a chimpanzee-derived Ad vector (AdC) serotype 6 and then boosted intramuscularly with a serologically distinct AdC vector of serotype 7 both expressing Gag of SIVmac239. Animals were subsequently boosted intramuscularly with a modified vaccinia Ankara (MVA) virus expressing Gag and Tat of the homologous SIV before mucosal challenge with a high dose of SIVmac239 given rectally. Whereas vaccinated animals showed only a modest reduction of viral loads, their overall survival was improved, in association with a substantial protection from the loss of CD4(+) T cells. In addition, the two vaccinated Mamu-A*01(+) macaques controlled viral loads to levels below detection within weeks after challenge. These data strongly suggest that T cells, while unable to affect SIV acquisition upon high-dose rectal infection, can reduce disease progression. Induction of potent T-cell responses should thus remain a component of our efforts to develop an efficacious vaccine to HIV-1.

A universal influenza A vaccine based on adenovirus expressing matrix-2 ectodomain and nucleoprotein protects mice from lethal challenge.

A universal influenza vaccine, designed to induce broadly cross-reactive immunity against current and future influenza A virus strains, is in critical demand to reduce the need for annual vaccinations with vaccines chosen upon predicting the predominant circulating viral strains, and to ameliorate the threat of cyclically occurring pandemics that have, in the past, killed tens of millions. Here, we describe a vaccine regimen based on sequential immunization with two serologically distinct chimpanzee-derived replication-defective adenovirus (Ad) vectors expressing the matrix-2 protein ectodomain (M2e) from three divergent strains of influenza A virus fused to the influenza virus nucleoprotein (NP) for induction of antibodies to M2e and virus-specific CD8(+) T cells to NP. In preclinical mouse models, the Ad vaccines expressing M2e and NP elicit robust NP-specific CD8(+) T-cell responses and moderate antibody responses to all three M2e sequences. Most importantly, vaccinated mice are protected against morbidity and mortality following challenge with high doses of different influenza virus strains. Protection requires both antibodies to M2e and cellular immune responses to NP.

Immune barriers to successful gene therapy.

Immune responses to viral vectors pose one of the main obstacles to successful human gene replacement therapy, unless gene transfer vectors are applied to immune-privileged sites. Both innate and adaptive immunity work in concert against sustained gene transfer, but the functions of patients' regulatory T cells (Tregs) and tolerogenic dendritic cells (DCs) could potentially be harnessed to reduce these immune responses. Over the last few years, immunologists have gained an ever-increasing knowledge of immunoregulatory pathways, especially those that prevent or dampen adaptive immune responses. The gene therapy community is now in a position to use this expanding knowledge in basic immunology to overcome the so far nearly unsurpassable obstacles posed by the immune system to the long-term replacement of missing or faulty genes by the use of viral vectors. Here, we discuss the current challenges in overcoming immune barriers to gene therapy. In addition, we point out potential strategies that might allow circumvention of cellular or humoral immune responses against the vector or the transgene product.

Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

Viral delivery for gene therapy against cell movement in cancer.

Viral delivery for cancer gene therapy is a promising approach, where traditional radiotherapy or chemotherapy to limit proliferation and movement of cancer cells has met resistance. Based on the new understanding of the biology of the viral vectors, therapeutic viral vectors for cancer gene therapy have been improved for greater safety and efficacy as well as transitioned from being non-replicating to replication-competent. Traditional oncolytic vectors have focused on eliminating tumor growth, while novel vectors simultaneously target epithelial-to-mesenchymal transition (EMT) in cancer cells, which could further prevent and reverse the aggressive tumor progression. In this review, we highlight the illustrative examples of cancer gene therapy in clinical trials as well as preclinical data and include proposals on methods to further enhance the safety and efficacy of oncolytic viral vectors in cancer gene therapy.

A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients.

Many studies report the level of total viral DNA in HIV-infected patients, but few studies report the level of integrated DNA. It is important to measure integrated DNA in HIV-infected patients because the information could shed light on the effectiveness of antiretroviral therapy, especially intensified therapy, when viral loads may remain undetectable. In order to develop an integration assay for patient samples, we enhanced the sensitivity of our prior integration assay. To do this, we exploited a technique that we developed, called repetitive sampling, and optimized reaction conditions for rare event detection, rather than large dynamic range. We also designed our primers to match more conserved regions of HIV. The result is a new, sensitive, quantitative assay that allows us to measure integrated DNA in HIV-infected patients. When we applied our integration assay to patient PBMCs, we found that the use of HAART is associated with reduced levels of integrated DNA.

Efficacy and safety of statins in hypercholesterolemia with emphasis on lipoproteins.

Information of the effect of statin on lipoproteins such as apolipoprotein (apo) A-I, lipoprotein (a) [Lp (a)], or apolipoprotein B levels is limited. This investigation was a crossover study designed to evaluate the efficacy and safety of atorvastatin and simvastatin in patients with hyperlipidemia. Sixty-six patients were involved in the study. Group I consisted of 32 patients, who were first treated with atorvastatin (10 mg) then switched to simvastatin (10 mg). Group II consisted of 34 patients, who were first treated with simvastatin then switched to atorvastatin. Each regimen was used for 3 months (phase I), stopped for 2 months, and then restarted for another 3 months (phase II). Both statins effectively reduced total cholesterol, low-density lipoprotein cholesterol (LDL-C), apo B, and Lp (a) (P < 0.001 in all comparisons). A significant increase in the high-density lipoprotein cholesterol (HDL-C) was noted after both statin treatments (P < 0.05 in all comparisons). Both statins caused an increase in the apo A-I levels, and the extent of changes in apo A-I revealed no difference between the two drugs. Compared to the simvastatin group, there were more patients in the atorvastatin group achieving the National Cholesterol Education Program ATP-III LDL-C goal (P < 0.05) and European LDL-C goal (P < 0.001). Both treatments were well tolerated; no patient was withdrawn from the study. This study demonstrates that both statins can effectively improve lipid profiles in patients with hyperlipidemia. Atorvastatin is more effective in helping patients reach the ATP-III and European LDL-C goals than simvastatin at the same dosage.