Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia.

Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.

HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression.

BACKGROUND: RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression. RESULTS: Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well. CONCLUSION: The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

Efficacy of a multigenic protein vaccine containing multimeric HIV gp160 against heterologous SHIV clade C challenges.

OBJECTIVE: To determine whether multigenic protein immunogens including native, trimeric HIV clade C (HIV-C) gp160 could cross-protect macaques against mucosal challenge with clade C (SHIV-C) mismatched for env. DESIGN: Because AIDS vaccine recipients are unlikely to encounter exactly matched HIV strains and to represent the diversity of locally circulating HIV-C strains, we selected env genes to generate the gp160 immunogen and SHIV-C from different, recently infected infants of the same clinical cohort in Zambia. In a model of postnatal HIV-C transmission, infant macaques were immunized with soluble viral proteins, including trimeric HIV1084i Env, and challenged with SHIV-1157ip; protein-only vaccination was compared with a DNA prime/protein boost strategy. METHODS: All vaccinated and control monkeys were exposed orally to low-dose, R5-tropic SHIV-1157ip encoding heterologous env. Animals with no or only transient infection were rechallenged intrarectally with a high dose of R5 SHIV-1157ipd3N4, a 'late', animal-evolved SHIV-1157ip variant. Animals were followed prospectively for immune parameters and viral RNA loads. RESULTS: Vaccination induced cross-neutralizing antibodies. Compared to controls, vaccinees had significantly lower peak viral RNA loads, and one vaccine recipient remained completely virus-free, even in lymphoid tissues. There was a trend for the protein-only vaccine to yield better protection than the combined modality approach. CONCLUSION: Protein-only immunogens induced significant protection against heterologous viruses encoding env from locally circulating viruses.

Long lasting control and lack of pathogenicity of the attenuated Rev-independent SIV in rhesus macaques.

A cohort of 22 rhesus macaques of Indian origin infected as neonates, juveniles, or adults by Rev-independent strains of SIV was monitored over several years. After the initial acute phase, virus replication was controlled and plasma virus loads were persistently below the threshold of the assay. The animals were monitored for up to 7.6 years after infection for viral loads, cellular and humoral immune responses, hematological changes, and overall health and no signs of immune dysfunction or AIDS were observed. This study represents several years of additional observation compared to the previously published results, and indicates that the Rev-independent SIV clones tested do not cause AIDS-like progressive disease within 7.6 years from infection. All the animals showed persistent humoral and cellular SIV-specific immune responses, consistent with chronic infection. Different Rev-independent SIV strains showed similar properties and lack of pathogenicity. Multicolor flow cytometric analysis demonstrated preservation of the Central Memory subset of T cells in the attenuated SIV-infected animals. This study demonstrates a potent, long-lasting control of the Rev-independent attenuated SIV in macaques independent of the age at virus exposure.

A novel small animal model for HIV-1 infection.

Lethally irradiated normal BALB/c mice, reconstituted with murine SCID bone marrow and engrafted with human PBMC (Trimera mice), were used to establish a novel murine model for HIV-1 infection. The Trimera mice were successfully infected with different clades and primary isolates of T- and M-tropic HIV-1, with the infection persisting in the animals for 4-6 wk. Rapid loss of the human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation accompanied the viral infection. All HIV-1 infected animals were able to generate both primary and secondary immune responses, including HIV specific human humoral and cellular responses. In addition to testing the efficacy of new antiviral compounds, this new murine HIV-1 model may be used for studying host-virus interactions and, most importantly, for screening and developing potential HIV-1 protective vaccines and adjuvants (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3185fje; doi:10.1096/fj.04-3185fje.).

Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection.

We have recently developed a novel small animal model for HIV-1 infection (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3184fje; doi:10.1096/fj.04-3184fje). The mice were successfully infected with HIV-1 for 4-6 wk with different clades of either T- or M-tropic isolates. HIV-1 infection was accompanied by rapid loss of human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation. HIV specific human humoral and cellular immune responses were observed in all HIV-1 infected animals. In the present study, HIV specific human immune responses, both humoral and cellular, were generated in noninfected Trimera mice, after their immunization with gp120-depleted HIV-1 antigen, presented by autologous human dendritic cells. Addition of CpG ODN to the antigen-pulsed DCs significantly enhanced (by 2- to 30-fold) the humoral and cellular HIV-1 specific immune responses. Only mice immunized with the HIV-1 immunogen and CpG were completely protected from infection with HIV-1 after challenge with high infection titers of the virus. This novel small animal model for HIV-1 infection may thus serve as an attractive platform for rapid testing of candidate HIV-1 vaccines and of adjuvants and may shorten the time needed for the development and final assessment of protective HIV-1 vaccines in human trials.

TLR9 expression is related to immune activation but is impaired in individuals with chronic immune activation.

Millions of individuals in developing countries are infected with helminths and other chronic infectious diseases, such as HIV-1, which lead to persistent immune activation and unbalanced immune state. We have suggested that the capacity of chronically immune activated individuals to protect themselves, cope with infections, and mount protective immunity following vaccination, is highly impaired. Here we examined the expression of toll-like receptor 9 (TLR9), as an essential component in the recognition of immunostimulating bacterial CpG-DNA motifs, in different subsets of human peripheral blood mononuclear cells (PBMC) obtained from chronically immune activated and non-activated individuals. TLR9 expression was correlated to immune cell activation and was upregulated following phytohemagglutinin or anti-CD3 activation. PBMC obtained from chronically immune activated individuals had a different overall pattern of TLR9 expression, including reduced upregulation of this receptor following additional immune activation, and diminished responsiveness to CpG-DNA stimulation, in comparison to non-activated individuals. These differences may partly account for the reduced capacity of chronically immune activated individuals to mount effective immune responses and strengthen the notion that the host immune background should be considered in the design and trial of potential adjuvants and vaccines.

Live attenuated Rev-independent Nef¯SIV enhances acquisition of heterologous SIVsmE660 in acutely vaccinated rhesus macaques.

BACKGROUND: Rhesus macaques (RMs) inoculated with live-attenuated Rev-Independent Nef¯ simian immunodeficiency virus (Rev-Ind Nef¯SIV) as adults or neonates controlled viremia to undetectable levels and showed no signs of immunodeficiency over 6-8 years of follow-up. We tested the capacity of this live-attenuated virus to protect RMs against pathogenic, heterologous SIVsmE660 challenges. METHODOLOGY/PRINCIPAL FINDINGS: Three groups of four RM were inoculated with Rev-Ind Nef¯SIV and compared. Group 1 was inoculated 8 years prior and again 15 months before low dose intrarectal challenges with SIVsmE660. Group 2 animals were inoculated with Rev-Ind Nef¯SIV at 15 months and Group 3 at 2 weeks prior to the SIVsmE660 challenges, respectively. Group 4 served as unvaccinated controls. All RMs underwent repeated weekly low-dose intrarectal challenges with SIVsmE660. Surprisingly, all RMs with acute live-attenuated virus infection (Group 3) became superinfected with the challenge virus, in contrast to the two other vaccine groups (Groups 1 and 2) (P=0.006 for each) and controls (Group 4) (P=0.022). Gene expression analysis showed significant upregulation of innate immune response-related chemokines and their receptors, most notably CCR5 in Group 3 animals during acute infection with Rev-Ind Nef¯SIV. CONCLUSIONS/SIGNIFICANCE: We conclude that although Rev-Ind Nef¯SIV remained apathogenic, acute replication of the vaccine strain was not protective but associated with increased acquisition of heterologous mucosal SIVsmE660 challenges.

A live attenuated Listeria monocytogenes vaccine vector expressing SIV Gag is safe and immunogenic in macaques and can be administered repeatedly.

Listeria monocytogenes (Lm) is known to induce strong cellular immune responses. We constructed a live-attenuated Lm vector, Lmdd-BdopSIVgag, which encodes SIVmac239 gag. Intragastric (i.g.) administration of 3 × 10(12) bacteria to rhesus macaques was safe and induced anti-Gag cellular but no humoral immune responses. Boosting of Gag-specific cellular responses was observed after i.g. administration of Lmdd-BdopSIVgag to previously vaccinated RM despite preexisting anti-Lm immunity shown by lymphoproliferative responses. Surprisingly, anti-Lm cellular responses were also detected in non-vaccinated controls, which may reflect the fact that Lm is a ubiquitous bacterium. The novel, live-attenuated Lmdd-BdopSIVgag may be an attractive platform for oral vaccine delivery.

Coinfection with Schistosoma mansoni reactivates viremia in rhesus macaques with chronic simian-human immunodeficiency virus clade C infection.

We tested the hypothesis that helminth parasite coinfection would intensify viremia and accelerate disease progression in monkeys chronically infected with an R5 simian-human immunodeficiency virus (SHIV) encoding a human immunodeficiency virus type 1 (HIV-1) clade C envelope. Fifteen rhesus monkeys with stable SHIV-1157ip infection were enrolled into a prospective, randomized trial. These seropositive animals had undetectable viral RNA and no signs of immunodeficiency. Seven animals served as virus-only controls; eight animals were exposed to Schistosoma mansoni cercariae. From week 5 after parasite exposure onward, coinfected animals shed eggs in their feces, developed eosinophilia, and had significantly higher mRNA expression of the T-helper type 2 cytokine interleukin-4 (P = 0.001) than animals without schistosomiasis. Compared to virus-only controls, viral replication was significantly increased in coinfected monkeys (P = 0.012), and the percentage of their CD4(+) CD29(+) memory cells decreased over time (P = 0.05). Thus, S. mansoni coinfection significantly increased viral replication and induced T-cell subset alterations in monkeys with chronic SHIV clade C infection.

Generation of Th1 immune responses to inactivated, gp120-depleted HIV-1 in mice with a dominant Th2 biased immune profile via immunostimulatory [correction of imunostimulatory] oligonucleotides--relevance to AIDS vaccines in developing countries.

Vaccination against HIV-1 of hosts with a dominant Th2 immune profile may fail to induce essential protective Th1 immune responses. By using Schistosoma-infected mice, with a pre-existent Th2 immune background, we demonstrate that oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanosine (CpG) immunostimulatory sequences co-administered with inactivated, gp120-depleted HIV-1 viral particles (HIV-1 immunogen) lead to potent Th1 anti-HIV-1 immune responses overcoming the Th2 bias. In contrast, Schistosoma-infected mice immunized with HIV-1 immunogen in incomplete Freund's adjuvant only, induced Th2 anti-HIV-1 immune responses. These findings strongly support the advisability of using CpG ODN as a Th1 inducing adjuvant when immunizing human populations with a strong pre-existent Th2 immune profile.

Adenovirus expressing a bioluminescence reporter gene and cMAGI cell assay for the detection of HIV-1.

We report a fast, highly sensitive method for detecting and testing drug resistance of M-tropic and T-tropic laboratory and primary HIV-1 isolates. cMAGI cells are infected with an adenovirus vector harboring the luciferase reporter gene controlled by HIV-1 Tat-responsive element, TAR. HIV-1 Tat production by HIV-1 chronically infected cells, or by cMAGI cells as early as two days after being acutely infected with HIV-1, is readily monitored in the presence or absence of antiviral drugs. This method is more sensitive than HIV-1 Tat dependant production of beta-galactosidase in the cMAGI cells. The fast answer, ease and sensitivity as well as the possibility of using this method in high throughput screening, makes it an very attractive tool for phenotypic detection of HIV-1 in clinical samples as well as a sensitive assay for monitoring drug resistant HIV-1 variants. This method can also be used for discovery of novel anti HIV-1 drugs.