RESUMO
This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies 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 limited promise.
Assuntos
Anticorpos Neutralizantes/genética , Doenças Transmissíveis/terapia , Técnicas de Transferência de Genes , Genes de Imunoglobulinas , Vetores Genéticos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/genética , Infecções por HIV/terapia , HIV-1/imunologia , Humanos , Imunização Passiva/métodos , Vacinas de DNA/administração & dosagemRESUMO
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.
Assuntos
Dependovirus/patogenicidade , Adolescente , Sequência de Bases , Western Blotting , Criança , Pré-Escolar , Clonagem Molecular , Primers do DNA , Dependovirus/genética , Dependovirus/isolamento & purificação , Células HeLa , Humanos , Reação em Cadeia da Polimerase , VirulênciaRESUMO
Antibody-like molecules were evaluated with potent simian immunodeficiency virus (SIV) neutralizing properties (immunoadhesins) that were delivered by a recombinant adeno-associated virus (rAAV) vector in the SIV-infected rhesus macaque model. When injected intramuscularly into the host, the vector directs in vivo production of the transgenes with antibody-like binding properties that lead to serum neutralizing activity against SIV. To extend the half-life of the immunoadhesins, rhesus cluster of differentiation 4 (CD4) and a single-chain antibody (4L6) were fused with albumin molecules, and these constructs were tested in our model of SIV infection. Antibody-based immunoadhesins provided high serum neutralizing titers against the original SIV strain. CD4-based immunoadhesins provided a wider spectrum of neutralization against different SIV strains in comparison to antibody-based therapeutics and had the potential to protect against high viral challenging doses. Although the albumin-antibody fusion immunoadhesin provided strong and prolonged protection of the immunized animals against SIV challenge, the albumin-CD4 fusion altered the specificity and decreased the overall protection effectiveness of the immunoadhesin in comparison to the antibody-based molecules. Albumin-based immunoadhesins increase in vivo longevity of the immune protection; however, they present challenges likely linked to the induction of anti-immunoadhesin antibodies.
RESUMO
BACKGROUND: A preventive vaccine for HIV is a crucial public health need; adeno-associated virus (AAV)-mediated antibody gene delivery could be an alternative to immunisation to induce sustained expression of neutralising antibodies to prevent HIV. We assessed safety and tolerability of rAAV1-PG9DP, a recombinant AAV1 vector encoding the gene for PG9, a broadly neutralising antibody against HIV. METHODS: This first-in-human, proof-of-concept, double-blind, phase 1, randomised, placebo-controlled, dose-escalation trial was done at one clinical research centre in the UK. Healthy men aged 18-45 years without HIV infection were randomly assigned to receive intramuscular injection with rAAV1-PG9DP or placebo in the deltoid or quadriceps in one of four dose-escalating cohorts (group A, 4â×â1012 vector genomes; group B, 4â×â1013 vector genomes; group C, 8â×â1013 vector genomes; and group D, 1·2â×â1014 vector genomes). Volunteers were followed up for 48 weeks. The primary objective was to assess safety and tolerability. A secondary objective was to assess PG9 expression in serum and related HIV neutralisation activity. All volunteers were included in primary and safety analyses. The trial is complete and is registered with ClinicalTrials.gov, number NCT01937455. FINDINGS: Between Jan 30, 2014, and Feb 28, 2017, 111 volunteers were screened for eligibility. 21 volunteers were eligible and provided consent, and all 21 completed 48 weeks of follow-up. Reactogenicity was generally mild or moderate and resolved without intervention. No probably or definitely related adverse events or serious adverse events were recorded. We detected PG9 by HIV neutralisation in the serum of four volunteers, and by RT-PCR in muscle biopsy samples from four volunteers. We did not detect PG9 by ELISA in serum. PG9 anti-drug antibody was present in ten volunteers in the higher dose groups. Both anti-AAV1 antibodies and AAV1-specific T-cell responses were detected. INTERPRETATION: Future studies should explore higher doses of AAV, alternative AAV serotypes and gene expression cassettes, or other broadly neutralising HIV antibodies. FUNDING: International AIDS Vaccine Initiative, United States Agency for International Development, Bill & Melinda Gates Foundation, US National Institutes of Health.
Assuntos
Anticorpos Neutralizantes/sangue , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , Adolescente , Adulto , Anticorpos Neutralizantes/genética , Método Duplo-Cego , Seguimentos , Terapia Genética/efeitos adversos , Anticorpos Anti-HIV/genética , Voluntários Saudáveis , Humanos , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Placebos/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Reino Unido , Adulto JovemRESUMO
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.
Assuntos
Dependovirus/genética , Terapia Genética , Músculo Esquelético/metabolismo , Plasmídeos/genética , Sequência de Bases/genética , Dependovirus/metabolismo , Vetores Genéticos/genética , Genoma , Humanos , Plasmídeos/metabolismoRESUMO
This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.
Assuntos
Adenoviridae/genética , Expressão Gênica , Vetores Genéticos , Anticorpos Anti-HIV/imunologia , HIV/imunologia , Imunização Passiva/métodos , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/genética , Haplorrinos , Humanos , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transdução GenéticaRESUMO
PURPOSE OF REVIEW: In 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 FINDINGS: Large 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. SUMMARY: Vector-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.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Dependovirus/genética , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Transferência de Genes , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/genética , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Haplorrinos , CamundongosRESUMO
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.
Assuntos
Anticorpos Antivirais/imunologia , Vetores Genéticos/imunologia , Haplorrinos/imunologia , Vacinas contra a SAIDS/uso terapêutico , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Antivirais/metabolismo , Imunoadesinas CD4/imunologia , Técnicas de Transferência de Genes/veterinária , Haplorrinos/genética , Imunoterapia/métodos , Imunoterapia/veterinária , Modelos Biológicos , Testes de Neutralização , Vacinas contra a SAIDS/genética , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Resultado do TratamentoRESUMO
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.
Assuntos
Dependovirus/genética , Cromossomos Humanos Par 1 , DNA Viral/análise , Dependovirus/fisiologia , Células HeLa , Humanos , Pulmão/virologia , Tonsila Palatina/virologia , Reação em Cadeia da Polimerase , Baço/virologia , Sequências Repetidas Terminais/genética , Integração Viral/fisiologiaRESUMO
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 (approximately 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.
Assuntos
Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Infecções por Parvoviridae/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Criança , DNA Viral/química , Dependovirus/isolamento & purificação , Heparina/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Dependovirus/genética , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Vacinas Sintéticas/imunologia , Replicação Viral , Animais , Dependovirus/imunologia , Vetores Genéticos , Antígenos de Histocompatibilidade Classe I/genética , Macaca mulatta , Testes de Neutralização , Vacinas de DNA/imunologiaRESUMO
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.