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Complications with the current smallpox vaccine and the threat of a new smallpox epidemic have dramatically increased efforts to identify new antiviral compounds against orthopoxviruses. CpG oligodeoxynucleotides were investigated here for their ability to confer protection against vaccinia virus (VACV) in a model of orthopoxvirus infection in pre- and post-exposure settings. Intranasal delivery of the B-class CpG 7909, prior to intranasal challenge with VACV, previously resulted in complete protection in a Balb/C mouse model of infection. Immunological analysis found that pre-stimulation with CpG resulted in the local release of pro-inflammatory cytokines such as IFN-a, IFN-y, TNF-a and IL-6 along with chemptactic messengers such as CCL2. In addition, activated innate effector cells such as macrophages, neutrophils, and dendritic cells were identified in high numbers in the lungs of CpG treated mice prior to infection. This heightened immune response persisted throughout early VACV infection in CpG-treated animals compared to that observed in un-stimulated control mice. Mice treated with CpG-B 7909 also initiated a second wave of immune activation late in infection indicative of a more rapidly formed adaptive response. Treatment and infection of B-cell Knock-out (KO) mice and neutrophil ablated mice suggested neither B-cells nor neutrophils alone were crucial in conferring CpGmediated protection against VACV. The success of CpG-B 7909 as a prophylactic against VACV led to its investigation as a post-exposure therapeutic. Intranasal treatment with CpG-B

Epidemiological studies in rural Thailand have demonstrated that leptospirosis is a major cause of acute febrile illness in this setting. Superimposed on this was a sustained outbreak in northeast Thailand between 1999-2003, the basis for which was unknown. Outbreaks elsewhere in the world are commonly associated with the presence of a dominant, region-specific serovar of Leptospira, and it was postulated here that the Thai outbreak was due to the emergence of a single biologically successful clone. An inherent weakness in defining the population genetic structure of Leptospira is that the commonly used serovar is not a unit of genetic identity. Pulsed-field gel electrophoresis (PFGE) has been described for Leptospira but is an imperfect tool since a number of variable strains appear identical by banding pattern. In response to this, a multilocu~ sequence typing (MLST) was developed for Leptospira interrogans (the most common species associated with human leptospirosis), and this was used to investigate the Thai outbreak. MLST data were also compared with PFGE and serovar typing. MLST was successfully applied to clinical and rodent isolates cultured from Thailand and reference isolates cultured from a more diverse geographical region. MLST demonstrated that the majority ofhuman infection (68%) in Thailand was caused by a single clone ofL. interrogans serovar Autumnalis, termed sequence type (ST) 34. This clone was also found in the rodent population, making a link between maintenance and accidental hosts. STs were compared with those of Leptospira drawn from the reference collection. This indicated a much more diverse population genetic structure for the reference collection. Unlike serovar and PFGE, MLST could differentiate between species and identify genetically related clones of L. interrogans and the highly related species L. kirschneri. MLST of L. interrogans represents a useful new tool for the genetic investigation of this important pathogen.

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The balance between latent and productive human immunodeficiency virus (HIV) infection is the result of a complex interplay of viral and host factors. In the first part of this study I investigated a group of 6 HIV+ haemophilic long term nonprogressor~ (LTNP) for maintenance of functional defects of their HIV-I nefgene. Our results showed that Nef alleles from these nonprogressor hemophiliacs are defective in enhancing virus replication and CD4 down-regulation. This was not predictive of disease outcome as 3 out of the 6 individuals studied progressed later to full HIV disease. The primary viral isolates from these the 3 'late progressors' showed an extension of gpl20 Env dependent entry co-receptor use from CCR5 only to CCR5/CXCR4, a known correlate of disease progression, while the non I f progressors maintained monotropic CCR5 use. In addition to viral genes, several pro-inflammatory cytokines upregulated in HIV infection influence HIV latency and replication via activation of cellular transcription factors. Peripheral blood mononuclear cells from HIV+ individuals frequently show a constitutive activation of the cytokine-related Janus kinase/Signal Transducer and Activator of Transcription (JAKISTAT) pathway, and particularly of a C-terminally truncated isoform of STAT5 (STAT5 ). I investigated whether STAT5 and STAT5~ could affect HIV transcription and replication. STAT5 induced HIV transcription driven by the long terminal repeats (LTR). Chromatin immunoprecipitation and related molecular assays showed that STAT5~ actually bound to the HIV LTR and suppressed viral transcription and expression. Thus, a key finding from the present thesis is that the constitutiveIy activated STAT5~ present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression. Thus, both viral factors, such as the evolution of gp120 Env coreceptor use towards CXCR4 in individuals maintaining functional nef mutations, and host transcription factors, including STAT5/STAT58, regulate HIV- I replication in vitro and, likely, its pathogenicity in infected individuals.

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