Immune activation and regulation in simian immunodeficiency virus-Plasmodium fragile-coinfected rhesus macaques.

Human immunodeficiency virus (HIV) is characterized by immune activation, while chronic malaria is associated with elevated interleukin-10 (IL-10) levels. How these apparently antagonizing forces interact in the coinfected host is poorly understood. Using a rhesus macaque model of simian immunodeficiency virus (SIV)-Plasmodium fragile coinfection, we evaluated how innate immune effector cells affect the balance between immune activation and regulation. In vitro Toll-like receptor (TLR) responses of peripheral blood myeloid dendritic cells (mDC) and monocytes were temporarily associated with acute parasitemic episodes and elevated plasma IL-10 levels. Prolonged infection resulted in a decline of mDC function. Monocytes maintained TLR responsiveness but, in addition to IL-12 and tumor necrosis factor alpha, also produced IL-10. Consistent with the role of spleen in the clearance of parasite-infected red blood cells, coinfected animals also had increased splenic IL-10 mRNA levels. The main cellular source of IL-10 in the spleens of coinfected animals, however, was not splenic macrophages but T cells, suggesting an impairment of adaptive immunity. In contrast to those in spleen, IL-10-positive cells in axillary lymph nodes of coinfected animals were predominantly mDC, reminiscent of the immunosuppressive phenotype of peripheral blood mDC. Concurrent with IL-10 induction, however, SIV infection promoted elevated systemic IL-12 levels. The continuously increasing ratio of plasma IL-12 to IL-10 suggested that the overall host response in SIV-P. fragile-coinfected animals was shifted toward immune activation versus immune regulation. Therefore, SIV-P. fragile coinfection might be characterized by earlier manifestation of immune dysfunction and exhaustion than that of single-pathogen infections. This could translate into increased morbidity in HIV-malaria-coinfected individuals.

Prolonged tenofovir treatment of macaques infected with K65R reverse transcriptase mutants of SIV results in the development of antiviral immune responses that control virus replication after drug withdrawal.

BACKGROUND: We reported previously that while prolonged tenofovir monotherapy of macaques infected with virulent simian immunodeficiency virus (SIV) resulted invariably in the emergence of viral mutants with reduced in vitro drug susceptibility and a K65R mutation in reverse transcriptase, some animals controlled virus replication for years. Transient CD8+ cell depletion or short-term tenofovir interruption within 1 to 5 years of treatment demonstrated that a combination of CD8+ cell-mediated immune responses and continued tenofovir therapy was required for sustained suppression of viremia. We report here follow-up data on 5 such animals that received tenofovir for 8 to 14 years. RESULTS: Although one animal had a gradual increase in viremia from 3 years onwards, the other 4 tenofovir-treated animals maintained undetectable viremia with occasional viral blips (≤ 300 RNA copies/ml plasma). When tenofovir was withdrawn after 8 to 10 years from three animals with undetectable viremia, the pattern of occasional episodes of low viremia (≤ 3600 RNA/ml plasma) continued throughout the 10-month follow-up period. These animals had low virus levels in lymphoid tissues, and evidence of multiple SIV-specific immune responses. CONCLUSION: Under certain conditions (i.e., prolonged antiviral therapy initiated early after infection; viral mutants with reduced drug susceptibility) a virus-host balance characterized by strong immunologic control of virus replication can be achieved. Although further research is needed to translate these findings into clinical applications, these observations provide hope for a functional cure of HIV infection via immunotherapeutic strategies that boost antiviral immunity and reduce the need for continuous antiretroviral therapy.

Evidence for an increased risk of transmission of simian immunodeficiency virus and malaria in a rhesus macaque coinfection model.

In sub-Saharan Africa, HIV-1 infection frequently occurs in the context of other coinfecting pathogens, most importantly, Mycobacterium tuberculosis and malaria parasites. The consequences are often devastating, resulting in enhanced morbidity and mortality. Due to the large number of confounding factors influencing pathogenesis in coinfected people, we sought to develop a nonhuman primate model of simian immunodeficiency virus (SIV)-malaria coinfection. In sub-Saharan Africa, Plasmodium falciparum is the most common malaria parasite and is responsible for most malaria-induced deaths. The simian malaria parasite Plasmodium fragile can induce clinical symptoms, including cerebral malaria in rhesus macaques, that resemble those of P. falciparum infection in humans. Thus, based on the well-characterized rhesus macaque model of SIV infection, this study reports the development of a novel rhesus macaque SIV-P. fragile coinfection model to study human HIV-P. falciparum coinfection. Using this model, we show that coinfection is associated with an increased, although transient, risk of both HIV and malaria transmission. Specifically, SIV-P. fragile coinfected macaques experienced an increase in SIV viremia that was temporarily associated with an increase in potential SIV target cells and systemic immune activation during acute parasitemia. Conversely, primary parasitemia in SIV-P. fragile coinfected animals resulted in higher gametocytemia that subsequently translated into higher oocyst development in mosquitoes. To our knowledge, this is the first animal model able to recapitulate the increased transmission risk of both HIV and malaria in coinfected humans. Therefore, this model could serve as an essential tool to elucidate distinct immunological, virological, and/or parasitological parameters underlying disease exacerbation in HIV-malaria coinfected people.

Characterization of a Mycobacterium ulcerans-like infection in a colony of African tropical clawed frogs (Xenopus tropicalis).

A nontuberculous Mycobacterium ulcerans-like organism was identified as the causative agent of an epizootic of mycobacteriosis in a colony of African tropical clawed frogs, Xenopus (Silurana) tropicalis, at the University of California, Berkeley. Diverse clinical signs of disease were observed, including lethargy, excess buoyancy, coelomic effusion, cutaneous ulcers, and granulomas. Visceral granulomas, ulcerative and granulomatous dermatitis, coelomitis, and septicemia were common findings at necropsy. Identification of M. ulcerans-like organisms was based on molecular and phenotypical characteristics. The findings of this investigation indicate that this M. ulcerans-like organism is a primary cause of morbidity and mortality in aquatic anurans and should be considered in the differential diagnosis of coelomic effusion in amphibians. Furthermore, if this Mycobacterium species ultimately is identified as M. ulcerans, X. tropicalis should be considered a potential source of this important public health pathogen.

A newly discovered mycobacterial pathogen isolated from laboratory colonies of Xenopus species with lethal infections produces a novel form of mycolactone, the Mycobacterium ulcerans macrolide toxin.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, produces a macrolide toxin, mycolactone A/B, which is thought to play a major role in virulence. A disease similar to Buruli ulcer recently appeared in United States frog colonies following importation of the West African frog, Xenopus tropicalis. The taxonomic position of the frog pathogen has not been fully elucidated, but this organism, tentatively designated Mycobacterium liflandii, is closely related to M. ulcerans and Mycobacterium marinum, and as further evidence is gathered, it will most likely be considered a subspecies of one of these species. In this paper we show that M. liflandii produces a novel plasmid-encoded mycolactone, mycolactone E. M. liflandii contains all of the genes in the mycolactone cluster with the exception of that encoding CYP140A2, a putative p450 monooxygenase. Although the core lactone structure is conserved in mycolactone E, the fatty acid side chain differs from that of mycolactone A/B in the number of hydroxyl groups and double bonds. The cytopathic phenotype of mycolactone E is identical to that of mycolactone A/B, although it is less potent. To further characterize the relationship between M. liflandii and M. ulcerans, strains were analyzed for the presence of the RD1 region genes, esxA (ESAT-6) and esxB (CFP-10). The M. ulcerans genome strain has a deletion in RD1 and lacks these genes. The results of these studies show that M. liflandii contains both esxA and esxB.

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis).

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mug ml(-1)), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15(T), has been deposited in the American Type Culture Collection as ATCC BAA-883(T) and the National Collection of Type Cultures (UK) as NCTC 13318(T).

Techniques and probes for the study of Xenopus tropicalis development.

The frog Xenopus laevis has provided significant insights into developmental and cellular processes. However, X. laevis has an allotetraploid genome precluding its use in forward genetic analysis. Genetic analysis may be applicable to Xenopus (Silurana) tropicalis, which has a diploid genome and a shorter generation time. Here, we show that many tools for the study of X. laevis development can be applied to X. tropicalis. By using the developmental staging system of Nieuwkoop and Faber, we find that X. tropicalis embryos develop at similar rates to X. laevis, although they tolerate a narrower range of temperatures. We also show that many of the analytical reagents available for X. laevis can be effectively transferred to X. tropicalis. The X. laevis protocol for whole-mount in situ hybridization to mRNA transcripts can be successfully applied to X. tropicalis without alteration. Additionally, X. laevis probes often work in X. tropicalis--alleviating the immediate need to clone the X. tropicalis orthologs before initiating developmental studies. Antibodies that react against X. laevis proteins can effectively detect the X. tropicalis protein by using established immunohistochemistry procedures. Antisense morpholino oligonucleotides (MOs) offer a new alternative to study loss of gene activity during development. We show that MOs function in X. tropicalis. Finally, X. tropicalis offers the possibility for forward genetics and genomic analysis.