Bruce C. Schnepp

Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys

The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine.

Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.

Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues

ABSTRACT Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

Novel adeno-associated virus vector vaccine restricts replication of simian immunodeficiency virus in macaques.

ABSTRACT Gene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.

Molecular Characterization of Adeno-Associated Viruses Infecting Children

ABSTRACT Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (∼98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.

Infectious Molecular Clones of Adeno-Associated Virus Isolated Directly from Human Tissues

ABSTRACT Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.

Adeno-Associated Virus Delivery of Broadly Neutralizing Antibodies

Purpose of reviewIn the present review, we will discuss the emerging field of vector-mediated antibody gene transfer as an alternative HIV vaccine. This approach is an improvement over classical passive immunization strategies that administer antibodies to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, resulting in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Recent findingsLarge numbers of very potent and broadly neutralizing HIV antibodies have recently been isolated and characterized. Vector-mediated antibody gene transfer allows one to immediately use these antibodies as a vaccine. Gene transfer studies in both mice and monkeys demonstrate long-term antibody expression in serum from a single injection at concentrations that provide sterilizing immunity. SummaryVector-mediated antibody gene transfer can rapidly move existing, potent anti-HIV molecules into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown little promise.

Prevalence of human bocavirus in human tonsils and adenoids.

To the Editor: Recently, Longtin et al. (1) reported a high rate (43%) of human bocavirus (HBoV) infection in a group of children chosen to serve as controls in a study of HBoV prevalence among hospitalized children and adults. In contrast, previous reports had found low HBoV prevalence rates of 0%–1% in control groups (2,3). Attempting to explain this surprising difference in rates, Lu et al. (4) suggested that selection of control patients may be related to the difference in rates. The control group used in the Longtin study were primarily (71%) children undergoing elective surgery; previous studies had selected control groups from other sources, including well children on routine visits and outpatients with nonrespiratory symptoms. Because the Longtin study surgeries were mainly tonsillectomies, adenoidectomies, and myringotomies, Lu et al. examined the possibility that tonsillar tissues, which include the adenoids, are sites of persistent HBoV infection. When these researchers extracted DNA from tonsillar lymphocytes obtained from pediatric patients who had undergone tonsillectomies or adenoidectomies, they detected HBoV DNA in 32% of the samples (4). These findings strongly suggest a connection between HBoV and tonsillar tissue. Therefore, we tested a number of tonsillar samples for the presence of HBoV DNA. Sample acquisition was approved by the Nationwide Children’s Hospital Institutional Review Board. Tonsils and adenoids were obtained from 91 patients who underwent elective surgery at Nationwide Children’s Hospital from June through September 2004. Patients’ ages ranged from 1–16 years (median 5.9 years; age was unknown for 4 patients). Samples consisted of surgically removed tonsil or adenoid tissues. DNA was extracted and its concentration was determined as previously described (5). Two primer sets were used for HBoV detection by using real-time PCR with SYBR Green detection and melting-point determination. We designed primers 3097F (5′-GTC-CAA-TTA-CAT-GAT-CAC-GCC-TAC-TC) and 3420R (5′-TGC-GTC-CAC-AGT-ATC-AGG-TTG-TTG) that targeted the viral protein ½ (VP1/VP2) region of HBoV. The nonstructural protein 1 (NP1) region was targeted by using primers 188F and 542R from Allander et al. (6) Each 20-μL reaction contained SYBR Green JumpStart Taq ReadyMix (Sigma, St. Louis, MO, USA), 4 mmol/L MgCl2, 250 nmol/L primers, double-distilled H2O, and 2 μL of DNA (50–200 ng) cycled on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA) instrument at 94oC for 2 min, followed by 40 cycles of 94°C for 20 s, 60°C (NP1 primers) or 68°C (VP1/VP2 primers) for 20 s, and 72°C for 14 s. Amplification and melting curves were analyzed with 7900HT 2.2.1 software (Applied Biosystems); positive samples were verified by sequence analysis. Sequenced VP- and NP1-generated amplicons were 99%–100% identical to HBoV strain ST1 (6). The detection sensitivities of the VP and NP1 assays, determined by using a plasmid construct containing the full HBoV genome, were 1–5 and 5–10 gene copies/reaction, respectively. Our testing identified HBoV DNA in 5 (5.5%) of the 91 children who underwent elective tonsillectomy/adenoidectomy. Ages ranged from 1.9–4.6 years, with a median age of 3.4 years. The reason for the much lower HBoV prevalence in this group of children, compared with prevalences found in studies by Longtin et al. (1) and Lu et al. (4), is unclear. Lu et al. (4) reported a much higher HBoV rate of lymphocytes from adenoids (56%) than from tonsils (16%). Although we did not know the exact tissue type of each sample, only that tonsils, adenoids, or both combined could be present, the 5.5% rate we found was about one third the rate found in tonsil lymphocytes and about one tenth the rate Lu et al. found in adenoid lymphocytes. A seasonal effect may contribute to the large discrepancies found in HBoV prevalences. Apparently, viruses can persist in tonsillar tissue well after the symptomatic phase of illness. In children with no signs of acute respiratory infection, Drago et al. (7) reported that 45.5% of samples contained viral nucleic acid. Depending on the duration of persistence, asymptomatic children, sampled shortly after the season of the virus in question, would be more likely to have detectable virus in their tonsillar tissue. The Longtin study samples were collected from December through April; our study samples were collected from June through September. If HBoV is seasonal as has been suggested (3), it may have been circulating in the target population before samples were taken and persisted only in tonsillar tissues. Thus, if tonsillar tissue from asymptomatic children was obtained within the persistence period after the HBoV season, samples would be HBoV positive; those obtained shortly after the persistence period would have a much lower rate. Differences in patient age in the 3 studies may also have contributed to the different rates observed. The Longtin group was substantially younger (median age, 23 months) than the Lu group (median, 5 years) or our group (median, 5.9 years). Preliminary seroepidemiology reports indicate the presence of HBoV antibodies in >50% of children by 2–3 years of age (8,9). The detection of HBoV in the tonsillar tissues we tested showed a higher rate of infection than would be expected in an asymptomatic population. However, the rate was far lower than that previously reported for tonsillar tissues (1,4).

Recombinant Adeno-Associated Virus Vector Genomes Take the Form of Long-Lived, Transcriptionally Competent Episomes in Human Muscle

Gene augmentation therapy as a strategy to treat alpha-1 antitrypsin (AAT) deficiency has reached phase 2 clinical testing in humans. Sustained serum levels of AAT have been observed beyond one year after intramuscular administration of a recombinant adeno-associated virus (rAAV) vector expressing the AAT gene. In this study, sequential muscle biopsies obtained at 3 and 12 months after vector injection were examined for the presence of rAAV vector genomes. Each biopsy sample contained readily detectable vector DNA, the majority of which existed as double-stranded supercoiled and open circular episomes. Episomes persisted through 12 months, although at slightly lower levels than observed at 3 months. There was a clear dose response when comparing the low- and mid-vector-dose groups to the high-dose group. The highest absolute copy numbers were found in a high-dose subject, and serum AAT levels at 12 months confirmed that the high-dose group also had the highest sustained serum AAT levels. Sequence analysis revealed that the vast majority of episomes contained double-D inverted terminal repeats ranging from fully intact to severely deleted. Molecular clones of vector genomes derived directly from the biopsies were transcriptionally active, potentially identifying them as the source of serum AAT in the trial subjects.

Engineered Expression of Broadly Neutralizing Antibodies Against Human Immunodeficiency Virus

This review discusses recent progress made in developing a vaccine and novel treatments for human immunodeficiency virus (HIV). It highlights the shortcomings of the RV144 vaccination trial [ALVAC-HIV (vCP1521) and AIDSVAX B/E] and the current standard of care and proposes that engineered expression of broadly neutralizing antibodies (bNAbs) against HIV-1 could overcome these shortcomings. Current developments in three major lines of research on HIV prevention and treatment using bNAbs are reviewed: firstly, the use of sequential immunogens to activate B cells to express bNAbs; secondly, the delivery of novel and extremely potent bNAbs through passive administration; and finally, the use of gene transfer using adeno-associated viral vectors to deliver bNAbs.