Both treated and untreated tumors are eliminated by short hairpin RNA-based induction of target-specific immune responses.

RNA interference (RNAi) for cancer treatment relies on the ability to directly kill cancer cells via down-regulation of target genes, but issues of delivery and efficacy have limited clinical adoption. Furthermore, current studies using immune-deficient animal models disregard potential interactions with the adaptive immune system. It has previously been observed that certain viral antigens appear to be more rapidly presented to the immune system than normal proteins due to the production of defective ribosomal products by the virus. Given that RNAi could potentially result in the generation of truncated mRNAs, we wondered whether a similar mechanism of immune presentation of a target gene was possible. Here we show that RNAi-cleaved mRNAs can be translated into incomplete protein, and if cleavage was downstream of cytotoxic T cell epitopes, resulted in increased presentation of target protein and the generation of a tumor-protective immune response. We show that mice inoculated with tumor cells treated with such short hairpin RNAs (shRNAs) were protected from subsequent challenge with untreated tumors. However, protection was only found if shRNAs were targeted downstream of the dominant cytotoxic T cell (CTL) epitope. Our work suggests that RNAi can alter immunity to targets and shows that not all tumor cells require direct RNAi exposure for treatment to be effective in vivo, pointing the way to a new class of RNAi-based therapy.

Discordant detection of Chlamydia pneumoniae in patients with carotid artery disease using polymerase chain reaction, immunofluorescence microscopy and serological methods.

AIM: To determine the presence of Chlamydia pneumoniae in carotid plaque and peripheral blood mononuclear cells (PBMCs) using a combination of serology, direct antigen detection by immunofluorescence (IF) microscopy and polymerase chain reaction (PCR), and to compare the results obtained from each assay. METHODS: A total of 54 atherosclerotic carotid plaques were tested for the presence of Chlamydia by PCR and IF methods. Of these 54 patients with carotid artery disease (CAD), 43 were also tested for the presence of C. pneumoniae DNA in PBMCs and for Chlamydia antibodies using two methods, the Medac Chlamydien rELISA and Focus Chlamydia microimmunofluorescence (MIF) methods. RESULTS: Eighteen of the 54 (33%) carotid specimens were positive for the presence of C. pneumoniae DNA, whereas only two of 43 (5%) patients had C. pneumoniae DNA present within their PBMC fraction. Chlamydial antibodies were detected by MIF and/or rELISA in 56% (24/43) of the patients tested. None of the 43 patients was C. pneumoniae positive in all of the test specimens (plaque, PBMCs and serum). CONCLUSIONS: Chlamydia pneumoniae is commonly found in Australian patients with CAD. Serology and PCR-based detection of C. pneumoniae in PBMCs and plaque give highly discordant results.

The detection of Chlamydia pneumoniae in atherosclerotic plaques of Australian subjects.

AIM: To determine whether the common respiratory pathogen, Chlamydia pneumoniae, was associated with atherosclerotic plaques in Australian subjects. METHODS: A total of 29 coronary atherosclerotic lesions and 18 normal coronary arterial samples were tested for the presence of C. pneumoniae by PCR and immunofluorescence methods. RESULTS: Chlamydia pneumoniae was detected in 15 of the atheromatous lesions as well as in three of the normal tissues; the immunofluorescence assay was more sensitive (P=0.028) than PCR (P=0.26). CONCLUSIONS: These findings contradict previous Australian studies which did not detect C. pneumoniae in atherosclerotic plaques, thereby discounting the speculation that its absence was likely due to geographical variation. The detection of the bacterium in some of the normal tissues suggests that C. pneumoniae infection might be an initial trigger of atherosclerotic development.

The duration of Chlamydia muridarum genital tract infection and associated chronic pathological changes are reduced in IL-17 knockout mice but protection is not increased further by immunization.

IL-17 is believed to be important for protection against extracellular pathogens, where clearance is dependent on neutrophil recruitment and local activation of epithelial cell defences. However, the role of IL-17 in protection against intracellular pathogens such as Chlamydia is less clear. We have compared (i) the course of natural genital tract C. muridarum infection, (ii) the development of oviduct pathology and (iii) the development of vaccine-induced immunity against infection in wild type (WT) BALB/c and IL-17 knockout mice (IL-17-/-) to determine if IL-17-mediated immunity is implicated in the development of infection-induced pathology and/or protection. Both the magnitude and duration of genital infection was significantly reduced in IL-17-/- mice compared to BALB/c. Similarly, hydrosalpinx was also greatly reduced in IL-17-/- mice and this correlated with reduced neutrophil and macrophage infiltration of oviduct tissues. Matrix metalloproteinase (MMP) 9 and MMP2 were increased in WT oviducts compared to IL-17-/- animals at day 7 post-infection. In contrast, oviducts from IL-17-/- mice contained higher MMP9 and MMP2 at day 21. Infection also elicited higher levels of Chlamydia-neutralizing antibody in serum of IL-17-/- mice than WT mice. Following intranasal immunization with C. muridarumMajor Outer Membrane Protein (MOMP) and cholera toxin plus CpG adjuvants, significantly higher levels of chlamydial MOMP-specific IgG and IgA were found in serum and vaginal washes of IL-17-/- mice. T cell proliferation and IFNγ production by splenocytes was greater in WT animals following in vitro re-stimulation, however vaccination was only effective at reducing infection in WT, not IL-17-/- mice. Intranasal or transcutaneous immunization protected WT but not IL-17-/- mice against hydrosalpinx development. Our data show that in the absence of IL-17, the severity of C. muridarum genital infection and associated oviduct pathology are significantly attenuated, however neither infection or pathology can be reduced further by vaccination protocols that effectively protect WT mice.

Towards a Chlamydia trachomatis vaccine: how close are we?

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. The incidence of chlamydial sexually transmitted infections has increased rapidly and current antibiotic therapy has failed as an intervention strategy. The most accepted strategy for protection and/or control of chlamydial infections is a vaccine that induces both local neutralizing antibodies to prevent infections by the extracellular elementary bodies and a cell-mediated immune response to target the intracellular infection. This article will discuss the challenges in vaccine design for the prevention of chlamydial urogenital infection and/or disease, including selection of target antigens, discussion of effective delivery systems, immunization routes and adjuvants for induction of protective immunity at the targeted mucosal surface whilst minimizing severe inflammatory disease sequelae.

HBsAg-vectored vaccines simultaneously deliver CTL responses to protective epitopes from multiple viral pathogens.

We have previously demonstrated that the potent immunogenicity of hepatitis B surface antigen (HBsAg) may be exploited to deliver foreign antigens for cytotoxic T-lymphocyte (CTL) induction. Here we demonstrate that a single low-dose immunization with rHBsAg DNA is sufficient to prime for CTL responses against encoded foreign epitope and that the responses may be recalled many months after immunization. We show that simultaneous disease-protective CTL responses restricted through a diversity of MHC class I haplotypes are elicited by recombinant (r) HBsAg DNA containing multiple viral epitopes appended as a C'-terminal polyepitope or encoded individually within the HBsAg polypeptide. CTL responses delivered by rHBsAg DNA were elicited in the presence of HBsAg-directed antibody. These studies vindicate the use of HBsAg as a powerful vector to deliver CTL responses to foreign antigen and have implications for a multidisease vaccine applicable to an MHC-polymorphic population.

Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates.

The safety and efficacy of peripheral venous administration of a self-complementary adeno-associated viral vector encoding the human FIX gene (scAAV-LP1-hFIXco) was evaluated in nonhuman primates for gene therapy of hemophilia B. Peripheral vein infusion of 1x10(12) vg/kg scAAV-LP1-hFIXco pseudotyped with serotype 8 capsid, in 3 macaques, resulted in stable therapeutic expression (more than 9 months) of human FIX (hFIX) at levels (1.1+/-0.5 microg/mL, or 22% of normal) that were comparable to those achieved after direct delivery of the same vector dose into the portal circulation (1.3+/-0.3 microg/mL, or 26% of normal). Importantly, the pattern of vector biodistribution after systemic and portal vein administration of scAAV-LP1-hFIXco was almost identical. Additionally, comparable levels of gene transfer were achieved in macaques with preexisting immunity to AAV8 following peripheral vein administration of 1x10(12) vg/kg AAV5-pseudotyped scAAV-LP1-hFIXco. This confirms that alternative serotypes can circumvent preexisting naturally acquired immunity to AAV. Thus, peripheral venous administration of AAV5 and AAV8 vectors is safe and as effective at transducing the liver in nonhuman primates as direct vector administration into the portal circulation. These results should make vector administration to patients, especially those with a severe bleeding diathesis, significantly easier and safer.

Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque.

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10 % of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).

Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures.

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30- microm-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.