Mary Salas

Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees.

ObjectiveA comparative study of the replication kinetics of different HIV-1 variants (including SIVcpz) was undertaken to determine which viral characteristics were associated with sustained plasma viraemia in chimpanzees. DesignPlasma samples from chimpanzees infected with six different HIV-1 clade B isolates were compared with plasma samples from SIVcpz−ant-infected chimpanzees. MethodsA pan-clade quantitative competitive reverse transcriptase–polymerase chain reaction assay was developed based on conserved primer sequences recognizing M, N and O human lentiviruses as well as different SIVcpz isolates. ResultsImportant differences between early kinetics in the human lentivirus isolates as well as compared with the chimpanzee isolate SIVcpz−ant were observed. R5-dependent non-syncytium-inducing (NSI) isolates (5016, Ba-L, SIVcpz) were found to have relatively higher viral loads than the syncytium-inducing (SI), X4-dependent primary (SF2), T cell-adapted (IIIB) or X4/R5 (Han2, DH12) SI primary isolates. ConclusionInfection of chimpanzees with NSI R5-utilizing isolates correlated with persistent viraemia (approximately 104 RNA equivalents/ml) in contrast to transient viraemia observed after infection with SI X4-utilizing isolates.

Transmission of Simian Immunodeficiency Virus SIVcpz and the Evolution of Infection in the Presence and Absence of Concurrent Human Immunodeficiency Virus Type 1 Infection in Chimpanzees

ABSTRACT Current data suggest that the human immunodeficiency virus type 1 (HIV-1) epidemic arose by transmission of simian immunodeficiency virus (SIV) SIVcpz from a subspecies of common chimpanzees (Pan troglodytes troglodytes) to humans. SIVcpz of chimpanzees is itself a molecular chimera of SIVs from two or more different monkey species, suggesting that recombination was made possible by coinfection of one individual animal with different lentiviruses. However, very little is known about SIVcpz transmission and the susceptibility to lentivirus coinfection of its natural host, the chimpanzee. Here, it is revealed that either infected plasma or peripheral blood mononuclear cells readily confer infection when exposure occurs by the intravenous or mucosal route. Importantly, the presence of preexisting HIV-1 infection did not modify the kinetics of SIVcpz infection once it was established by different routes. Although humoral responses appeared as early as 4 weeks postinfection, neutralization to SIVcpz-ANT varied markedly between animals. Analysis of the SIVcpz env sequence over time revealed the emergence of genetic viral variants and persistent SIVcpz RNA levels of between 104 and 105 copies/ml plasma regardless of the presence or absence of concurrent HIV-1 infection. These unique data provide important insight into possible routes of transmission, the kinetics of acute SIVcpz infection, and how readily coinfection with SIVcpz and other lentiviruses may be established as necessary preconditions for potential recombination.

Establishment of a neonatal rhesus macaque model to study Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) is the causative agent of human tuberculosis (TB) with an estimated 8.8 million new TB cases and 1.4 million deaths annually. Tuberculosis is the leading cause of death in AIDS patients worldwide but very little is known about early TB infection or TB/HIV co-infection in infants. A clinically relevant newborn animal model to study TB infection is urgently needed. We have successfully established an aerosol newborn/infant model in neonatal nonhuman primates (NHPs) that mimics clinical and bacteriological characteristics of Mtb infection as seen in human newborns/infants. Further, this model will allow the establishment of a TB coinfection model of pediatric AIDS. Aerosol versus intra broncho-alveolar Mtb infection was studied. Interestingly, 42 days post infection specific lesions were detected suggestive of the classic Ghon focus in human children. Concurrently, specific cellular immune responses developed 4-6 weeks after Mtb infection. Using the enzyme-linked immunospot (ELISPOT) assays, we found that IL-12 production correlated with early Mtb infection lesions seen by routine thoracic radiographs. Overall, this work represents the first example of early Mtb infection of newborn macaques. This study gives us a unique opportunity to further characterize immunopathogenesis and establish a TB/SIV co-infection model for pediatric AIDS.

Epithelial stem cells as mucosal antigen-delivering cells: A novel AIDS vaccine approach.

A key obstacle limiting development of an effective AIDS vaccine is the inability to deliver antigen for a sufficient period of time resulting in weak and transient protection. HIV transmission occurs predominantly across mucosal surfaces; therefore, an ideal vaccine strategy would be to target HIV at mucosal entry sites to prevent infection. Such a novel strategy relies on the activation of mucosal immune response via presentation of viral antigens by the mucosal epithelial cells. The use of a terminally differentiated epithelial cell promoter to drive expression of antigens leading to viral protein production in the upper layers of the epithelium is central to the success of this approach. Our results show that when administered intradermally to mice, a GFP-reporter gene under the transcriptional control of the involucrin promoter is expressed in the upper layers of the epidermis and, although transduced cells were very low in number, high and sustained anti-GFP antibody production is observed in vivo. A subsequent experiment investigates the effectiveness of GFP-tagged replication-competent SIVdeltaNef and GFP-tagged replication-deficient SIVdeltaVifdeltaNef constructs under the transcriptional control of the involucrin promoter. Optimal conditions for production of pseudotyped VSV-G viral particles destined to transduce basal epithelial stem cells at the mucosal sites of entry of SIV in our animal model were determined. Altogether, the data demonstrate the feasibility of an epithelium-based vaccine containing involucrin-driven viral antigen encoding sequences that integrate into epithelial stem cells and show long-term expression in the upper layer of the epithelium even after multiple cycle of epithelia renewal. Such epithelium-based vaccine should elicit a long-term immunity against HIV/SIV infection at the site of entry of the virus.

Neonatal macaque model to study Mycobacterium tuberculosis infection in pediatric AIDS

Tuberculosis (TB) is the leading cause of death in AIDS patients worldwide; the earliest opportunistic infection occurring in conjunction with HIV; an accelerant of HIV replication and immune system deterioration. Co-infection with Mycobacterium tuberculosis (Mtb) and HIV is an increasing global emergency. Little is known regarding the early events of Mtb infection in humans especially infants. There is an urgent need for a clinically relevant animal model that mimics TB disease in human children to better understand early immune responses, pathogenicity and disease progression in newborn/infants.

Engineering SIV-resistant macaque hematopoietic stem cells and CD4+ T cells with CCR5-specific zinc-finger nucleases

Background CCR5 is the major HIV co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Zinc finger nuclease (ZFN) technology is a class of engineered DNA-binding proteins facilitating targeted genome editing by binding to a user-specific locus and causing a double-strand break in the region of interest. As a result, the gene of interest targeted by ZFN cleavage is disrupted. We investigated the ability of a Ccr5 gene-specific ZFN to establish SIV-resistant CD4+ T cells and hematopoietic progenitor cells isolated from macaques.

Novel AIDS vaccine approach using epithelial stem cells as mucosal antigen-presenting cells

Background Because HIV transmission occurs predominantly across mucosal surfaces, the ideal vaccine strategy to prevent infection would be to target HIV at mucosal entry sites of transmission. We investigated a novel vaccine approach, which aim is to elicit long-term immunity against HIV infection at the entry site of the virus. This strategy relies on the expression of viral proteins from epithelial stem cells at the basal layer of the epithelium and using a promoter that is specific for terminally differentiated epithelial.