Linda C. Green

Protection of rhesus macaques from vaginal infection by vaginally delivered Maraviroc, an inhibitor of HIV-1 entry via the CCR5 co-receptor

An effective vaginal microbicide could reduce human immunodeficiency virus type 1 (HIV-1) transmission to women. Among microbicide candidates in clinical development is Maraviroc (MVC), a small-molecule drug that binds the CCR5 co-receptor and impedes HIV-1 entry into cells. Delivered systemically, MVC reduces viral load in HIV-1-infected individuals, but its ability to prevent transmission is untested. We have now evaluated MVC as a vaginal microbicide with use of a stringent model that involves challenge of rhesus macaques with a high-dose of a CCR5-using virus, SHIV-162P3. Gel-formulated, prescription-grade MVC provided dose-dependent protection, half-maximally at 0.5 mM (0.25 mg/mL). The duration of protection was transient; the longer the delay between MVC application and virus challenge, the less protection (half life of approximately 4 h). As expected, MVC neither protected against challenge with a CXCR4-using virus, SHIV-KU1, nor exacerbated postinfection viremia. These findings validate MVC development as a vaginal microbicide for women and should guide clinical programs.

Topically Applied Recombinant Chemokine Analogues Fully Protect Macaques from Vaginal Simian-Human Immunodeficiency Virus Challenge

Effective strategies for preventing human immunodeficiency virus infection are urgently needed, but recent failures in key clinical trials of vaccines and microbicides highlight the need for new approaches validated in relevant animal models. Here, we show that 2 new chemokine (C-C motif) receptor 5 inhibitors, 5P12-RANTES (regulated on activation, normal T cell expressed and secreted) and 6P4-RANTES, fully protect against infection in the rhesus vaginal challenge model. These highly potent molecules, which are amenable to low-cost production, represent promising new additions to the microbicides pipeline.

Identification of rhesus macaque genital microbiota by 16S pyrosequencing shows similarities to human bacterial vaginosis: implications for use as an animal model for HIV vaginal infection.

The composition of the lower genital tract microbiota in women is believed to affect the risk of sexually acquiring HIV. Since macaque genital microbiota could similarly impact vaginal infection with SIV we identified microbiota in 11 rhesus macaques using multitag pyrosequencing of the 16S rRNA gene. The microbiota was polymicrobial with a median of nine distinct bacterial taxa per macaque (range 3-16 taxa, each constituting 1% or more of the sequences). Taxa frequently found included Peptoniphilus, Sneathia, Porphyromonas, Mobiluncus, Atopobacter, Dialister, Thioreductor, Prevotella, and Streptococcus, many of which are also frequently found in women with bacterial vaginosis. Lactobacillus sequences (mostly L. johnsonii) were found in only four macaques but were not predominant in any (median of 0% of sequences, range 0-39%). All macaques were resampled 6 months after the first time point to determine the stability of the microbiota. The microbiota remained polymicrobial with a median of 10 taxa (range 6-18). Microbial patterns remained similar for six of the macaques, changed substantially in two, and had a mixed pattern in three. Significant sialidase enzyme activity, a marker of bacteria vaginosis in women, was detected in genital fluid from 9/11 and 8/11 macaques from the first and second time points, respectively. These results show that the macaque lower genital microbiota resembled a bacteria vaginosis-type microbiota in women and suggest that the microbiota of macaques in captivity promote rather than protect against vaginal infection with SIV. These results also suggest macaques could be used as an animal model to study some aspects of bacterial vaginosis.

Tropism-independent protection of macaques against vaginal transmission of three SHIVs by the HIV-1 fusion inhibitor T-1249

We have assessed the potential of the fusion inhibitory peptide T-1249 for development as a vaginal microbicide to prevent HIV-1 sexual transmission. When formulated as a simple gel, T-1249 provided dose-dependent protection to macaques against high-dose challenge with three different SHIVs that used either CCR5 or CXCR4 for infection (the R5 virus SHIV-162P3, the X4 virus SHIV-KU1 and the R5X4 virus SHIV-89.6P), and it also protected against SIVmac251 (R5). Protection of half of the test animals was estimated by interpolation to occur at T-1249 concentrations of ≈40–130 μM, whereas complete protection was observed at 0.1–2 mM. In vitro, T-1249 had substantial breadth of activity against HIV-1 strains from multiple genetic subtypes and in a coreceptor-independent manner. Thus, at 1 μM in a peripheral blood mononuclear cell-based replication assay, T-1249 inhibited all 29 R5 viruses, all 12 X4 viruses and all 7 R5X4 viruses in the test panel, irrespective of their genetic subtype. Combining lower concentrations of T-1249 with other entry inhibitors (CMPD-167, BMS-C, or AMD3465) increased the proportion of test viruses that could be blocked. In the PhenoSense assay, T-1249 was active against 636 different HIV-1 Env-pseudotyped viruses of varying tropism and derived from clinical samples, with IC50 values typically clustered in a 10-fold range ≈10 nM. Overall, these results support the concept of using T-1249 as a component of an entry inhibitor-based combination microbicide to prevent the sexual transmission of diverse HIV-1 variants.

Sequence survey of the genome of the opportunistic microsporidian pathogen, Vittaforma corneae

Abstract The microsporidian Vittaforma corneae has been reported as a pathogen of the human stratum corneum, where it can cause keratitis, and is associated with systemic infections. In addition to this direct role as an infectious, etiologic agent of human disease, V. corneae has been used as a model organism for another microsporidian, Enterocytozoon bieneusi, a frequent and problematic pathogen of HIV-infected patients that, unlike V. corneae, is difficult to maintain and to study in vitro. Unfortunately, few molecular sequences are available for V. corneae. In this study, seventy-four genome survey sequences (GSS) were obtained from genomic DNA of spores of laboratory-cultured V. corneae. Approximately, 41 discontinuous kilobases of V. corneae were cloned and sequenced to generate these GSS. Putative identities were assigned to 44 of the V. corneae GSS based on BLASTX searches, representing 21 discrete proteins. Of these 21 deduced V. corneae proteins, only two had been reported previously from other microsporidia (until the recent report of the Encephalitozoon cuniculi genome). Two of the V. corneae proteins were of particular interest, reverse transcriptase and topoisomerase IV (parC). Since the existence of transposable elements in microsporidia is controversial, the presence of reverse transcriptase in V. corneae will contribute to resolution of this debate. The presence of topoisomerase IV was remarkable because this enzyme previously had been identified only from prokaryotes. The 74 GSS included 26.7 kilobases of unique sequences from which two statistics were generated: GC content and codon usage. The GC content of the unique GSS was 42%, lower than that of another microsporidian, E. cuniculi (48% for protein-encoding regions), and substantially higher than that predicted for a third microsporidian, Spraguea lophii (28%). A comparison using the Pearson correlation coefficient showed that codon usage in V. corneae was similar to that in the yeasts, Saccharomyces cerevisiae (r = 0.79) and Shizosaccharomyces pombe (r = 0.70), but was markedly dissimilar to E. cuniculi (r = 0.19).

The Large Intestine as a Major Reservoir for Simian Immunodeficiency Virus in Macaques with Long-Term, Nonprogressing Infection

Although patients with human immunodeficiency virus type 1 infection who are receiving antiretroviral therapy and those with long-term, nonprogressive infection (LTNPs) usually have undetectable viremia, virus persists in tissue reservoirs throughout infection. However, the distribution and magnitude of viral persistence and replication in tissues has not been adequately examined. Here, we used the simian immunodeficiency virus (SIV) macaque model to quantify and compare viral RNA and DNA in the small (jejunum) and large (colon) intestine of LTNPs. In LTNPs with chronic infection, the colon had consistently higher viral levels than did the jejunum. The colon also had higher percentages of viral target cells (memory CD4(+) CCR5(+) T cells) and proliferating memory CD4(+) T cells than did the jejunum, whereas markers of cell activation were comparable in both compartments. These data indicate that the large intestine is a major viral reservoir in LTNPs, which may be the result of persistent, latently infected cells and higher turnover of naive and central memory CD4(+) T cells in this major immunologic compartment.

Fractionation of sporogonial stages of the microsporidian Encephalitozoon cuniculi by Percoll gradients.

Microsporidia are obligate intracellular parasites that are increasingly recognized as a cause of opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabolic processes that regulate growth and replication of microsporidia. Examination of the individual stages of development will facilitate such studies and reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephalitozoon cuniculi were cultured in RK‐13 cells. The tissue supernatants containing multiple parasite stages, empty microsporidial husks and host cell debris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll®. Transmission electron microscopy and SDS‐polyacrylamide gel electrophoresis were used to compare the content and purity of each fraction. Mature spores formed a band at a density of approximately 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugation in 30% and 50% Percoll® resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporidia infecting humans.

Purification of Enterocytozoon bieneusi from Stools and Production of Specific Antibodies

ABSTRACT Enterocytozoon bieneusi is clinically the most significant of the microsporidia in humans, causing chronic diarrhea wasting and cholangitis in individuals with human immunodeficiency virus infection and AIDS. Little progress on this infection has been made because of the inability to propagate E. bieneusi in vitro and in vivo, which limits the source of parasite spores to the stools of infected human patients. Given the size and shape of the E. bieneusi spores (1.1 to 1.6 by 0.7 to 1.0 μm) and the lack of specific immune reagents, the identification and purification of large quantities of spores from feces are technically challenging. Consequently, diagnosis relies entirely on PCR, a labor-intensive approach that requires highly skilled personnel. We describe a method for the purification of E. bieneusi spores from human stools and the production of rabbit-specific antisera. Spores were purified by a combination of isopycnic Percoll gradient centrifugation and continuous sucrose gradient centrifugation. Specific polyclonal antibodies raised in mice and rabbits reacted by indirect immunofluorescence with E. bieneusi but not with Encephalitozoon spp., Candida albicans, Staphylococcus aureus, Escherichia coli, or other forms present in human stools.

Methionine Aminopeptidase 2 Expression in Microsporidia

ELIZABETH s. DIDIER,” AARON D. MARTIN,” MARY E. STOVALL,” XAVIER ALVAREZ,~ DEREK MITTLEIDER; LINDA C. GREEN: LISA C. BOWERS,” ARDETH K. PLAUCHE,” PETER J. DIDIERb and PAUL J. BRINDLEY‘ “Divsion of Microbiology and Immunology, bDivision of Comparative Pathology, Tulane National Primate Research Centee Covington, LA 70433 U.S.A. “Department cf Tropical Medicine and Hygiene, Tulane Universitr; New Orleans, LA 70112 U.S.A., and dXavier University of Lauisiana, New Orleans, LA 70125, U.S.A.