Dennis Hartigan-O'Connor

Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25–30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.

Developments in gene therapy for muscular dystrophy

Gene therapy for muscular dystrophy (MD) presents significant challenges, including the large amount of muscle tissue in the body, the large size of many genes defective in different muscular dystrophies, and the possibility of a host immune response against the therapeutic gene. Overcoming these challenges requires the development and delivery of suitable gene transfer vectors. Encouraging progress has been made in modifying adenovirus (Ad) vectors to reduce immune response and increase capacity. Recently developed gutted Ad vectors can deliver full‐length dystrophin cDNA expression vectors to muscle tissue. Using muscle‐specific promoters to drive dystrophin expression, a strong immune response has not been observed in mdx mice. Adeno‐associated virus (AAV) vectors can deliver small genes to muscle without provocation of a significant immune response, which should allow long‐term expression of several MD genes. AAV vectors have also been used to deliver sarcoglycan genes to entire muscle groups. These advances and others reviewed here suggest that barriers to gene therapy for MD are surmountable. Microsc. Res. Tech. 48:223–238, 2000.

Breast-fed and bottle-fed infant rhesus macaques develop distinct gut microbiotas and immune systems

Breast-feeding and bottle-feeding promote different gut microbial communities, which are associated with development of different immune systems in rhesus macaque infants. Diet and the Infant Gut Microbiota Diet has a strong influence on the microbes living in the intestine. Furthermore, microbial colonization of the gut is required for normal development of the immune system. Nonetheless, it has been unclear how profoundly diet might shape the primate immune system or how durable the influence might be. Ardeshir et al. now demonstrate that breast-fed and bottle-fed infant macaques develop markedly different immune systems, which remain different for at least 6 months after the animals begin receiving identical diets. These findings may partly explain the variation in human susceptibility to conditions with an immune basis, as well as the variable protection against certain infectious diseases. Diet has a strong influence on the intestinal microbiota in both humans and animal models. It is well established that microbial colonization is required for normal development of the immune system and that specific microbial constituents prompt the differentiation or expansion of certain immune cell subsets. Nonetheless, it has been unclear how profoundly diet might shape the primate immune system or how durable the influence might be. We show that breast-fed and bottle-fed infant rhesus macaques develop markedly different immune systems, which remain different 6 months after weaning when the animals begin receiving identical diets. In particular, breast-fed infants develop robust populations of memory T cells as well as T helper 17 (TH17) cells within the memory pool, whereas bottle-fed infants do not. These findings may partly explain the variation in human susceptibility to conditions with an immune basis, as well as the variable protection against certain infectious diseases.

Th17 cells and regulatory T cells in elite control over HIV and SIV.

Purpose of reviewWe present current findings about two subsets of CD4+ T cells that play an important part in the initial host response to infection with the HIV type 1: those producing IL-17 (Th17 cells) and those with immunosuppressive function (CD25+FoxP3+ regulatory T cells or T-reg). The role of these cells in the control of viral infection and immune activation as well as in the prevention of immune deficiency in HIV-infected elite controllers will be examined. We will also discuss the use of the simian immunodeficiency virus (SIV)-infected macaque model of AIDS to study the interplay between these cells and lentiviral infection in vivo. Recent findingsStudy of Th17 cells in humans and nonhuman primates (NHPs) has shown that depletion of these cells is associated with the dissemination of microbial products from the infected gut, increased systemic immune activation, and disease progression. Most impressively, having a smaller Th17-cell compartment has been found to predict these outcomes. T-reg have been associated with the reduced antiviral T-cell responses but not with the suppression of generalized T cell activation. Both cell subsets influence innate immune responses and, in doing so, may shape the inflammatory milieu of the host at infection. SummaryInteractions between Th17 cells, T-reg, and cells of the innate immune system influence the course of HIV and SIV infection from its earliest stages, even before the appearance of adaptive immunity. Such interactions may be pivotal for elite control over disease progression.

Suppression of SIV-specific CD4+ T cells by infant but not adult macaque regulatory T cells: implications for SIV disease progression

The impact of regulatory T cells (T reg cells) on the course of HIV and SIV disease is unknown. T reg cells could suppress protective antiviral responses and accelerate disease progression. Alternatively, these cells might block T cell activation and thereby limit viral replication as well as activation-associated immunopathology. Given the higher frequency of T reg cells known to be present during human fetal ontogeny, such influences may be most important in the context of perinatal infection. We found that infant macaques had higher fractions of CD4+CD25+CD127lowFoxP3+ T reg cells in the peripheral blood and in lymphoid tissues, and that these T reg cells showed greater in vitro suppressive activity on a per cell basis. Infant and adult macaques were infected with SIVmac251 to test the influence of the T reg cell compartment on SIV-specific immune responses. After infection with SIV, most (three out of four) infant macaques had persistently high viral loads, weak and transient SIV-specific CD4+ and CD8+ T cell responses, and rapid disease progression. T reg cells in the infant but not in the adult directly suppressed SIV-specific CD4+ T cell responses, which were detectable only after depletion of T reg cells. In the case of both the infant and the adult macaque, T reg cells were not able to directly suppress SIV-specific CD8+ T cell responses and had no apparent effect on T cell activation. In aggregate, these observations suggest that the T reg cell compartment of the infant macaque facilitates rapid disease progression, at least in part by incapacitating SIV-specific CD4+ T cell responses.

Replication of Lengthened Moloney Murine Leukemia Virus Genomes Is Impaired at Multiple Stages

ABSTRACT It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral “headful” was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19.2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses.

Helper‐dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high‐titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo.

Efficient Rescue of Gutted Adenovirus Genomes Allows Rapid Production of Concentrated Stocks Without Negative Selection

Gutted adenoviral (Ad) vectors have a greater cloning capacity and elicit less immune response than conventional Ad vectors. Unfortunately, clinical use of gutted vectors has been slowed by production difficulties, including low yield and a tendency for recombinant virus to emerge. These two problems are related, because expansion of dilute vector stocks requires selective pressure against helper virus. The ability to rescue gutted virus at high titer would lessen the requirement for selective pressure, thereby limiting the advantage afforded to undesirable recombinants. We tested gutted virus rescue from plasmids and from synthetic terminal protein (TP)-DNA complexes by transfection/infection or cotransfection with various forms of helper viral DNA. Optimal rescue required cotransfection of gutted and helper genomes with identical origins of replication. Transfection/infection, which introduces unequal origins, was 30 times less efficient than cotransfection of genomes that had been released from plasmid DNA and bore identical origins. Cotransfection of TP-linked genomes was several times more efficient than that of unlinked genomes, yielding average gutted viral titers above 10(7) transducing units (TU)/ml. In addition, we found that limited expression of Cre recombinase doubled the yield of gutted virus. Using these techniques, gutted viruses can be rescued at titers greater than 3 x 10(7) TU/ml, about 100 times higher than is usually achieved. Finally, we found that high-titer lysates could be serially passaged on Cre-negative cells without loss of titer, further reducing selective pressure. These methods allow large-scale production of gutted virus in three or four serial passages, while minimizing exposure to Cre recombinase.

The Human Fetal Immune Response to Hepatitis C Virus Exposure in Utero

BACKGROUND Although the rate of mother-to-child transmission of hepatitis C virus (HCV) is low, the effect of HCV exposure in utero on the fetal immune system is unknown. METHODS Umbilical cord blood was obtained from 7 neonates born to HCV-seropositive, HCV RNA-positive women and 8 neonates born to HCV-seronegative women. Cord blood mononuclear cells were analyzed by immunophenotyping and by intracellular cytokine staining after HCV-specific and polyclonal stimulation. Plasma was analyzed for anti-HCV immunoglobulin M (IgM), cytokine/granzyme concentrations, and indoleamine 2,3-dioxygenase (IDO) activity. RESULTS HCV-exposed neonates had significantly lower levels of regulatory T cells expressing HLA-DR, lower CD4(+) and CD8(+) T cell activation, and lower plasma levels of pro-inflammatory markers than did controls. However, CD4(+) and CD8(+) T cells from HCV-exposed neonates had higher IFN-γ production in response to polyclonal stimulation than did T cells from controls. IDO activity was similar between groups. No HCV-specific T cell responses or anti-HCV IgM were detected in any neonates. CONCLUSIONS HCV-exposed neonates showed a relative suppression of immune activation and pro-inflammatory markers, which was counterbalanced by an increased production capacity for IFN-γ. These results suggest that HCV encounters the fetal immune system in utero, and alters the balance between suppressive and pro-inflammatory responses.

Development of Cytomegalovirus (CMV) Immune Recovery Uveitis Is Associated with Th17 Cell Depletion and Poor Systemic CMV-Specific T Cell Responses

We tested whether impaired systemic immunoregulation and hyperactive immune responses are associated with an immune reconstitution inflammatory syndrome, CMV IRU. We found instead that T-regs in CMV IRU patients are functionally intact, while virus-specific immune responses and Th17 cells are compromised