Heterologous priming-boosting with DNA and modified vaccinia virus Ankara expressing tryparedoxin peroxidase promotes long-term memory against Leishmania major in susceptible BALB/c Mice.

Leishmaniasis affects 12 million people, but there are no vaccines in routine clinical use. Th1 polarizing vaccines that elicit long-term protection are required to prevent disease in susceptible populations. We recently showed that heterologous priming-boosting with tryparedoxin peroxidase (TRYP) DNA followed by TRYP-modified vaccinia virus Ankara (TRYP MVA) protected susceptible BALB/c mice from Leishmania major. Here we compared treatment with TRYP DNA with treatment with TRYP DNA/TRYP MVA. We found that equivalent levels of protection during the postvaccination effector phase correlated with equivalent levels of serum immunoglobulin G2a and gamma interferon (IFN-gamma) in draining lymph nodes. In contrast, challenge infection during the memory phase revealed that there was enhanced clinical efficacy with TRYP DNA/TRYP MVA. This correlated with higher levels of effector phase splenic IFN-gamma, sustained prechallenge levels of memory phase IFN-gamma, and a more polarized post-L. major challenge Th1 response compared to the Th2/T(reg) response. Thus, TRYP DNA/TRYP MVA, but not TRYP DNA alone, provides long-term protection against murine leishmaniasis.

Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum beta-lactamases.

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla(CTX-M) genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on beta-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum beta-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla(CTX-M) genes in 21 of 28 ESBL-producing isolates.

From genome to vaccines for leishmaniasis: screening 100 novel vaccine candidates against murine Leishmania major infection.

The genomic sequence of Leishmania major provides a rich source of vaccine candidates. One hundred randomly selected amastigote-expressed genes were screened as DNA vaccines, and efficacy determined following high-dose L. major footpad challenge in BALB/c mice. Fourteen protective novel vaccine candidates were identified; seven vaccines exacerbated disease. There were no differences in the number of predicted MHC H-2d class I or II epitopes mapping to protective versus exacerbatory antigens. A proportion of both protective (7/14; 50%) and exacerbatory (4/7; 57%) proteins showed short (8- to 18-mer) 100% amino acid sequence identities to human, mouse or gut flora proteins. A high proportion of these (4/7 protective; 3/4 exacerbatory) showed full or partial overlap with RANKPEP-predicted H-2d classes I and II epitopes. Our data suggest, therefore, that there may be little difference between antigens/epitopes that drive regulatory versus effector CD4 T cell populations. The best novel protective antigen was an amastin-like gene that maps to a 17-gene tandem array on Leishmania chromosome 8 and is closely related to 37 other amastin-like genes. Two ribosomal proteins, a V-ATPase subunit, and a dynein light chain orthologue were the only other protective genes with putative functions.

Interleukin-4 (IL-4) and IL-10 collude in vaccine failure for novel exacerbatory antigens in murine Leishmania major infection.

Leishmaniasis affects 12 million people but there are no vaccines in routine use. Recently, we used DNA vaccination in a susceptible BALB/c high-dose model of infection to screen 100 novel Leishmania major genes as vaccine candidates. In addition to finding novel protective antigens, we identified several antigens that reproducibly exacerbated disease. Here we examined the immune response to two of these antigens, lmd29 and 584C, that were originally identified in an expressed sequence tag cDNA sequencing project. We show that, in addition to exacerbating disease in susceptible BALB/c mice, these antigens retain a propensity to exacerbate disease in resistant C57BL/6 mice. This ability to exacerbate disease was lost when susceptible BALB/c mice were rendered resistant by disruption of the genes encoding interleukin-4 (IL-4) alone, IL-4/IL-13, or IL-4, IL-5, IL-9, and IL-13. Failure to exacerbate disease was associated with reduced IL-5 and IL-10 production in IL-4 knockout mice. Treatment of lmd29-vaccinated mice with anti-IL-10 receptor antibody prior to challenge infection converted exacerbation in wild-type BALB/c mice into highly significant antigen-specific protection. These studies demonstrate that some highly immunogenic antigens of L. major, while having an intrinsic capacity to exacerbate disease in the context of otherwise T helper 1-promoting DNA vaccine delivery, can be rendered protective by the removal of functional IL-10.

IL-10 from regulatory T cells determines vaccine efficacy in murine Leishmania major infection.

Leishmaniasis affects 12 million people, but there are no vaccines. Immunological correlates of vaccine efficacy are unclear. Polarized Th1 vs Th2 responses in Leishmania major-infected mice suggested that a shift in balance from IL-4 to IFN-gamma was the key to vaccine success. Recently, a role for IL-10 and regulatory T cells in parasite persistence was demonstrated, prompting re-evaluation of vaccine-induced immunity. We compared DNA/modified vaccinia virus Ankara heterologous prime-boost with Leishmania homolog of the receptor for activated C kinase (LACK) or tryparedoxin peroxidase (TRYP). Both induced low IL-4 and high IFN-gamma prechallenge. Strikingly, high prechallenge CD4 T cell-derived IL-10 predicted vaccine failure using LACK, whereas low IL-10 predicted protection with TRYP. The ratio of IFN-gamma:IL-10 was thus a clear prechallenge indicator of vaccine success. Challenge infection caused further polarization to high IL-10/low IFN-gamma with LACK and low IL-10/high IFN-gamma with TRYP. Ex vivo quantitative RT-PCR and in vitro depletion and suppression experiments demonstrated that Ag-driven CD4+ CD25+ T regulatory 1-like cells were the primary source of IL-10 in LACK-vaccinated mice. Anti-IL-10R treatment in vivo demonstrated that IL-10 was functional in determining vaccine failure, rendering LACK protective in the presence of high IFN-gamma/low IL-5 responses.

AIDS-associated progressive multifocal leukoencephalopathy : current management strategies.

Progressive multifocal leukoencephalopathy (PML) is a rare, opportunistic infection of the CNS by the ubiquitous JC virus (JCV). PML is only seen in the context of severe and prolonged immunosuppression, a phenomenon now frequently encountered since the AIDS pandemic. PML is characterised by progressive lysis of oligodendrocytes with demyelination. A rapid clinical course ensues with focal neurological deficits and a median time to death of 3.5 months without treatment. Prior to highly active antiretroviral therapy (HAART), there was no effective therapy. Since the advent of HAART, the prognosis for PML has much improved; however, a significant number of patients appear unresponsive to antiretrovirals and some worsen because of the development of immune reconstitution disease. A better understanding of the biology of JCV and its interactions with host cells is leading to new anti-JCV-specific agents that await evaluation in randomised, controlled trials. Improved diagnostic tools and the possibility of immunotherapy and gene therapy are further advancing the field.

Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans.

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.