Biology of E1-deleted adenovirus vectors in nonhuman primate muscle.

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.

Adenoviral-mediated suicide gene therapy for ovarian cancer.

The purpose of this phase I study was to determine the potential efficacy of adenoviral-mediated suicide gene therapy in women with recurrent ovarian cancer. Fourteen patients were treated intraperitoneally with herpes simplex virus-thymidine kinase (HSV-TK)-encoding adenovirus (AdHSV-TK) in dosages ranging between 1x10(9) and 1x10(11) pfu. Beginning 2 days later, ganciclovir (GCV) was administered intravenously at a dose of 5 mg/kg bid for 14 days. Transient vector-associated fever was experienced by 4 of 14 (29%) treated patients. Other possible vector-associated constitutional symptoms, abdominal pain, and gastrointestinal symptoms were experienced by 6 of 14 (43%) treated patients. No other dose-limiting vector-specific side effects were noted. Of the 13 patients evaluable for response, 5 (38%) had stable disease and 8 (62%) had evidence of progressive disease. Molecular analysis of evaluable ascites samples demonstrated the presence of transgene DNA and RNA in most patients 2 days following Ad HSV-TK administration. Ten of 11 evaluable patients had an increase in anti-adenovirus antibody titer. These results suggest that treatment with AdHSV-TK in combination with GCV is feasible in the context of human ovarian cancer and tolerated at the dosages studied.

Route of administration determines induction of T-cell-independent humoral responses to adeno-associated virus vectors.

Vectors based on adeno-associated viruses (AAV) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. This study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. Route of vector administration in mice had a qualitative effect on antivector B cell responses. Administration of vector into the tail vein resulted in T-cell-dependent (TD) B cell responses that were completely inhibited with depleting CD4 antibody. Delivery of vector into the portal circulation via the spleen yielded B cell response that were partially T cell independent (TI) rendering strategies based on T cell inhibition ineffective in allowing vector readministration. The TI B cell response was short lived in comparison to the TD response. Rhesus monkeys produced a B cell memory response to intraportal vector which appeared to be T cell dependent based on Ig isotypes. Gene therapy strategies that require AAV vector readministration should consider vector biodistribution and its impact on B cell activation.

Readministration of adenovirus vector in nonhuman primate lungs by blockade of CD40-CD40 ligand interactions.

The interaction between CD40 on B cells and CD40 ligand (CD40L) on activated T cells is important for B-cell differentiation in T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus monkeys in the presence or absence of a transient treatment with a humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated with hu5C8 at the time vector was instilled into the lung. Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM) levels comparable to control animals, but did not secrete IgA or develop neutralizing antibodies; consequently, the animals could be readministered with adenovirus vector expressing alkaline phosphatase. A second study was designed to examine the long-term effects on immune functions of a short course of hu5C8. Acute hu5C8 treatment resulted in significant and prolonged inhibition of the adenovirus-specific humoral response well beyond the time hu5C8 effects were no longer significant. These studies demonstrate the potential of hu5C8 as an immunomodulatory regimen to enable administration of adenoviral vectors, and they advocate testing this model in humans.

Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle.

Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis.

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.

Evaluation of an E1E4-deleted adenovirus expressing the herpes simplex thymidine kinase suicide gene in cancer gene therapy.

Studies with first-generation adenoviral vectors have uncovered limitations that include finite transgene persistence, potential hepatotoxicity, and contamination with replication-competent adenovirus (RCA). To address these limitations within the context of cancer suicide gene therapy, a new adenoviral vector was developed containing the herpes simplex virus type 1 thymidine kinase (HSV tk) gene inserted in the E1 region of a recombinant vector containing deletions in the E1 and E4 regions of the Ad5 genome. The HSV tk minigene was placed under transcriptional control of a Rous sarcoma virus (RSV) promoter. This new E1E4-deleted vector was compared with the first-generation E1E3-deleted Ad.RSVtk vector. Generation of replication-competent adenovirus during production was eliminated. Using semiquantitative immunoblotting, the two vectors produced equivalent amounts of the expected 44-kDa tk-encoded protein in three different cell lines tested. The ability of the E1E4-deleted vector to sensitize tumor cells to ganciclovir (GCV) using in vitro assays and mixing studies was comparable to that of the E1E3-deleted vector. In vivo bystander effects were investigated using mixing studies in a syngeneic flank tumor model and demonstrated no difference between vectors in either immunocompetent or immunodeficient mice. To test the efficiency of these vectors in treating tumors in clinically relevant models, virus was injected intraperitoneally into tumor-bearing SCID mice and intrapleurally in a syngeneic rat mesothelioma model. After treatment of animals with ganciclovir, both vectors were roughly equivalent in their ability to increase mean survival (from approximately 40 to approximately 70 days) and markedly reduce tumor burden. Finally, formal toxicology studies were performed and showed similar amounts of local inflammation without systemic toxicity. In summary, this series of in vitro and in vivo experiments indicates that the performance of the recombinant E1E4-deleted adenoviral vector was virtually identical to that of the E1E3-deleted vector. Since the E1E4 vector has a much lower rate of recombination during production and has been shown to be less hepatotoxic in animal models, this new vector should prove superior to the first-generation Ad.HSVtk vectors in clinical cancer gene therapy trials.

Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer.

Stable delivery of a therapeutic protein under pharmacologic control was achieved through in vivo somatic gene transfer. This system was based on the expression of two chimeric, human-derived proteins that were reconstituted by rapamycin into a transcription factor complex. A mixture of two adeno-associated virus vectors, one expressing the transcription factor chimeras and one containing erythropoietin (Epo) under the control of a promoter responsive to the transcription factor, was injected into skeletal muscle of immune-competent mice. Administration of rapamycin resulted in 200-fold induction of plasma Epo. Stable engraftment of this humanized system in immune-competent mice was achieved for 6 months with similar results for at least 3 months in a rhesus monkey.

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus.

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.

Role of E4 in eliciting CD4 T-cell and B-cell responses to adenovirus vectors delivered to murine and nonhuman primate lungs.

Adenovirus vectors delivered to lung are being considered in the treatment of cystic fibrosis (CF). Vectors from which E1 has been deleted elicit T- and B-cell responses which confound their use in the treatment of chronic diseases such as CF. In this study, we directly compare the biology of an adenovirus vector from which E1 has been deleted to that of one from which E1 and E4 have been deleted, following intratracheal instillation into mouse and nonhuman primate lung. Evaluation of the E1 deletion vector in C57BL/6 mice demonstrated dose-dependent activation of both CD4 T cells (i.e., TH1 and TH2 subsets) and neutralizing antibodies to viral capsid proteins. Deletion of E4 and E1 had little impact on the CD4 T-cell proliferative response and cytolytic activity of CD8 T cells against target cells expressing viral antigens. Analysis of T-cell subsets from mice exposed to the vector from which E1 and E4 had been deleted demonstrated preservation of TH1 responses with markedly diminished TH2 responses compared to the vector with the deletion of E1. This effect was associated with reduced TH2-dependent immunoglobulin isotypes and markedly diminished neutralizing antibodies. Similar results were obtained in nonhuman primates. These studies indicate that the vector genotype can modify B-cell responses by differential activation of TH1 subsets. Diminished humoral immunity, as was observed with the E1 and E4 deletion vectors in lung, is indeed desired in applications of gene therapy where readministration of the vector is necessary.

Validation of retroviral detection for rodent cell-derived products and gene therapy applications.

The availability of sensitive assays for detecting infectious murine retroviruses has become critical for the development and acceptance of a number of biopharmaceuticals, including monoclonal antibody-derived products and gene therapy vectors. Comparative studies demonstrated that the PG4 S+L- retrovirus infectivity test routinely yields higher titres than the mink cell test for xenotropic, amphotrophic and MCF murine retroviruses. A validation study for the PG4 S+L- assay demonstrated very good linearity (r2 of 0.95 to 0.99), reproducibility within a study (+/-0.35 log10 units), and precision between tests (+/-0.45 log10 units). Interference (or selectivity) in the presence of a non-specific antibody was insignificant (less than 0.2 log10 units). Sensitivity levels established from measurements as virus titres approach zero demonstrated a threshold value of 2-3 focus forming units (FFU)/ml. Two methods for increasing assay sensitivity were used including: (i) increased product samplings combined with a Poisson distribution analysis, and (ii) a 14-day co-cultivation with Mus dunni cells. Each of these methods was shown to increase sensitivity by at least one log10 unit. Murine retroviruses may also be detected by a less sensitive immunofluorescence assay (IFA) using specific monoclonal antibodies; this assay is essential for detecting certain recombinant ecotropic MuLVs. In summary, murine retroviral detection ranked by sensitivity is mink S+L- < IFA with monoclonal antibodies < PG4 S+L- < Mus dunni co-cultivation followed by PG4 S+L-.

Allograft safety: viral inactivation with bone demineralization.

A study was performed to validate the effectiveness of a bone demineralization process with respect to its inactivation of viruses. The viruses selected for study included human immunodeficiency virus (HIV), duck hepatitis B virus (a model for human hepatitis B), bovine viral diarrheal virus (a model for human hepatitis C), human cytomegalovirus, and human poliovirus (a model for small nonenveloped viruses, e.g., hepatitis A). This study was performed in compliance with Good Laboratory Practice regulations using validation methodology similar to that used to ensure the safety of blood derivatives and other products. Use of the bone demineralization process described in this report resulted in a reduction in infectivity of greater than one million (10(6)) for all viruses and as much as one trillion (10(12)) for the poliovirus.

Keto/enol epoxy steroids as HIV-1 Tat inhibitors: structure-activity relationships and pharmacophore localization.

Inhibition of the HIV-1 nuclear regulatory protein tat could potentially yield particularly useful drugs because it functions as an activator of transcription. It has no known cellular counterpart, and deletions in the tat gene destroy the ability of HIV-1 to replicate. We recently reported that a structurally unique class of tat inhibitors, 3-keto/enol 4,5-alpha-epoxy steroids bearing electron-withdrawing substituents at position 2, specifically inhibit tat-induced gene expression in virus free transfected SW480 cells. In this paper, we report on additional SAR (structure-activity relationships) for the steroid series and the localization of the pharmacophore to the A-ring functionality. There is a weak enantioselective preference for the natural steroid stereochemistry and hints of additional SAR in the electron-withdrawing group. Compound 34a is of particular interest in that it inhibits HIV replication in H9 cells at a concentration equivalent to its inhibitory level in the primary tat assay.

An inactivated hepatitis A viral vaccine of cell culture origin.

Hepatitis A virus (HAV) strain CR326, adapted to grow in LLC-MK2 cells, was highly purified, inactivated with formalin, adsorbed to alum, and tested for capacity to induce antibody to HAV in both mice and marmosets. The minimum dose of HAV antigen necessary to produce antibody in 50% of mice was 10 ng. As little as three doses of 1 ng each produced antibody in 50% of marmosets. Further, all marmosets with any detectable antibody to HAV, as a result of vaccination, were protected against virulent infection on challenge with HAV. Thus a highly efficacious, inactivated hepatitis A vaccine can be produced from virus grown in cell culture. Although LLC-MK2 cells are unacceptable for use in human vaccine preparation, HAV can also be prepared in a similar manner in MRC-5 cells, which are acceptable for human vaccine manufacture.

Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide.

Comparative surface feature analyses of the VP1 sequences of hepatitis A virus (HAV) and poliovirus type 1 allowed an alignment of the two sequences and an identification of probable HAV neutralization antigenic sites. A synthetic peptide containing the HAV-specific amino acid sequence of one of these sites induced anti-HAV-neutralizing antibodies. It is concluded that a structural homology exists between the two viruses, despite minimal primary sequence conservation.

Isolation and immunizations with hepatitis A viral structural proteins: induction of antiprotein, antiviral, and neutralizing responses.

An immune affinity purification procedure for hepatitis A virus (HAV) was designed which yielded milligram quantities of the virus with greater than 95% purity. The major structural proteins VP-1, VP-2, and VP-3 were isolated from the purified virus by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to immunize Lewis rats (three to four doses, 10 to 15 micrograms per dose). The two Lewis rats immunized with VP-1 developed a strong antibody response to VP-1, as determined by Western blot analysis and immune precipitation of the denatured protein. These animals also developed a good antibody response to the whole virus, as demonstrated by a positive response in a competitive radioimmunoassay (HAV antibody test) and by precipitation of the whole virus. In addition, both animals developed a low titer neutralizing antibody to HAV, as demonstrated by an in vitro cell culture assay. While the two rats receiving VP-2 developed only minimal responses to the protein and to the virus by the same assays described above, one of the two developed a significant neutralizing antibody to HAV. The immunization of one Lewis rat with VP-3 induced a good antibody response to both denatured protein and the whole virus. This serum sample was also demonstrated to neutralize the viral infectivity. Finally, two rabbits that had received inoculations of sodium dodecyl sulfate and heat-disrupted HAV (containing 20 to 30 micrograms of each protein per dose) developed good antiprotein responses to all of the proteins and good antiviral responses, including a consistently significant neutralizing activity. The neutralizing antibody responses suggest that the structural proteins of HAV, or a portion of them, could provide the basis for a subunit vaccine for HAV.

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.

Hypothermia-inducing peptide promotes recovery of vesicular stomatitis virus from persistent animal infections.

A single injection of the hypothermia-inducing neuropeptide bombesin resulted in an excellent recovery system for reisolating viruses from Swiss albino mice infected with vesicular stomatitis virus even up to 90 days after infection. The virus was recovered from a cell homogenate prepared from whole brain tissue 24 h after intracerebral injection of bombesin; brain cells were cocultivated with BHK-21 cell monolayers and then plaqued on BHK-21 cells at 31 degrees C. All of the recovered viruses were identified as vesicular stomatitis virus by antibody neutralization and peptide analyses of some of the structural proteins. However, some of the recovered viruses were altered with regard to tryptic peptide maps, temperature sensitivity, and central nervous system disease induced compared with the viruses used to initiate the infection. Most of the recovered viruses induced a similar disease when reinoculated intracerebrally into mice, characterized by hind-leg paralysis 4 to 6 days after infection. Two of the recovered viruses were lethal, however, resulting in a relatively rapid generalized wasting disease and death in 3 to 4 days.