Jocelyn Fournier

Gene Expression Profiling of Host Response in Models of Acute HIV Infection

HIV infection is characterized by a host response composed of adaptive and innate immunity that partially limits viral replication; however, it ultimately fails in eradicating the virus. To model host gene expression during acute HIV infection, we infected cynomolgus macaques with the SIV/HIV-1 chimeric virus, SHIV89.6P, and profiled gene expression in peripheral blood over a 5-wk period using a high density cDNA microarray. We demonstrate that viral challenge induced a widespread suppression of genes regulating innate immunity, including the LPS receptors, CD14 and TLR4. An overexpression of 16 IFN-stimulated genes was also observed in response to infection; however, it did not correlate with control over viral titers. A statistical analysis of the dataset identified 10 genes regulating apoptosis with differential expression during the first 2 wk of infection (p < 0.004). Quantitative real-time PCR verified transcriptional increases in IFN-α-inducible genes and decreases in genes regulating innate immunity. Therefore, the persistence of high viral loads despite an extensive IFN response suggests that HIV can resist in vivo IFN treatment despite published reports of in vitro efficacy. The transcriptional suppression of genes regulating innate immunity may allow HIV to evade acute host responses and establish a chronic infection and may reduce innate host defense against opportunistic infections.

Characterization of blood-borne transmission of simian foamy virus.

BACKGROUND: Simian foamy virus (SFV) is an endemic, nonhuman primate (NHP) retrovirus that is transmitted to individuals who work with or hunt NHPs. The cross‐species transmission of simian retroviruses is believed to be the etiology of human immunodeficiency virus and human T‐lymphotropic virus infections in humans. Although SFV is not pathogenic in the native host, the shared ancestry with other simian retroviruses has brought into question the potential for acquired pathogenicity after cross‐species transmission. This study examines whether SFV also shares the traits of transmissibility through the blood supply.

A comparison of conventional microscopy, immunofluorescence microscopy and flow cytometry in the detection of Giardia lamblia cysts in beaver fecal samples

A variety of domestic and wild animals are considered to be potential sources of giardiasis in humans. As a result, numerous studies have been reported on the prevalence of Giardia lamblia infection in animals. The majority of these surveys have involved various floatation techniques followed by conventional microscopy in order to detect cysts in fecal samples. Immunofluorescence microscopy has become popular in recent years for the detection of G. lamblia cysts in both clinical and environmental samples. This technique can be automated by combining it with flow cytometry. The present study represents a direct comparison of conventional microscopy, immunofluorescence microscopy, and flow cytometry in terms of their relative efficiency in the detection of G. lamblia cysts in beaver fecal samples. As a result of viewer fatigue, or low cyst concentrations, false negatives were common with conventional microscopy, leading to low prevalence estimates. By specifically targeting the cysts, immunofluorescence microscopy provided more reliable results in a shorter time than conventional methods. When flow cytometry was used in combination with immunofluorescence, a larger number of samples could be examined in a relatively short period of time. The results obtained indicated that this technique allowed for more consistent recognition than either conventional or immunofluorescence microscopy of positive samples containing smaller numbers of cysts.

Genomic sequencing and characterization of cynomolgus macaque cytomegalovirus.

ABSTRACT Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development.

Multi-Low-Dose Mucosal Simian Immunodeficiency Virus SIVmac239 Challenge of Cynomolgus Macaques Immunized with “Hyperattenuated” SIV Constructs

ABSTRACT Hyperattenuated simian immunodeficiency virus SIVmac239-derived constructs Δ5-CMV and Δ6-CCI are an effort to render SIV incapable of, in practical terms, both reversion and recombination while maintaining the immune features of SIV as a retrovirus. Primary inoculation of cynomolgus macaques with 108 50% tissue culture infective doses (TCID50) of Δ5-CMV or Δ6-CCI induced low-level humoral and cellular responses detectable in the absence of measureable in vivo replication. The first of three DNA boosts resulted in elevated gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) responses to Gag, Pol, and Env in the Δ5-CMV vaccine group compared to the Δ6-CCI vaccine group (P = 0.001). Weekly intrarectal challenge with a low dose of SIVmac239 followed by a dose escalation was conducted until all animals became infected. The mean peak viral load of the Δ5-CMV-vaccinated animals (3.7 × 105 copies/ml) was ∼1 log unit lower than that of the control animals. More dramatically, the viral load set point of these animals was decreased by 3 log units compared to that of the controls (<50 versus 1.64 × 104 copies/ml; P < 0.0001). Seventy-five percent (6/8) of vaccine recipients controlled virus below 1,000 copies/ml for at least 6 months, with a subset controlling virus and maintaining substantial CD4 T-cell counts for close to 2 years of follow-up. The correlates of protection from SIV disease progression may lie in the rapidity and protective value of immune responses that occur early in primary SIV infection. Prior immunization with hyperattenuated SIVmac239, even if sterilizing immunity is not achieved, may allow a more advantageous host response.

Presence of circulating CTL induced by infection with wild-type or attenuated SIV and their correlation with protection from pathogenic SHIV challenge

Abstract: The aim of this study was to evaluate the role of CTLs in the protection from challenge with pathogenic SHIV in macaques vaccinated with attenuated virus. More specifically, we have analyzed the CTL response in cynomolgus macaques vaccinated/infected with the attenuated SIVmacC8 or the wild‐type SIVmacJ5 and correlated these responses to the protection from SHIV89.6P challenge. SIVmacC8‐vaccinated monkeys demonstrated a broader CTL response than the SIVmacJ5‐infected animals. Nevertheless, CTL against some proteins in SIVmacC8‐vaccinated monkeys became progressively more difficult to detect through the day of challenge. In regards to protection from superinfection with SHIV89.6P, neither the presence of circulating CTL nor the CTL precursor frequency against any of the tested proteins correlated with the outcome of the challenge when SIVmacJ5‐ and SIVmacC8‐infected animals were analyzed together. By analyzing the SIVmacC8‐vaccinated animals separately, only the protected animal had detectable CTL precursors with moderate frequencies against all three tested proteins at the day of challenge.

Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Human Varicella-Zoster Virus

ABSTRACT Varicella-zoster virus (VZV) is a member of the alphaherpesvirus family and the causative agent of chickenpox and shingles. To determine the utility of cynomolgus macaques (Macaca fascicularis) as a nonhuman primate model to evaluate VZV-based simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) vaccines, we experimentally inoculated 10 animals with the parental Oka (Oka-P) strain of VZV derived from MeWo or Telo-RF cells. VZV DNA could be detected in the lungs as late as 4 days postinfection, with replicating virus detected by shell vial culture assay in one case. Infection did not result in any overt clinical symptoms but was characterized by humoral and cell-mediated immunity in a time frame and at a magnitude similar to those observed following VZV vaccination in humans. The cell line source of VZV inoculum influenced both the magnitude and polyfunctionality of cell-mediated immunity. Animals mounted a vigorous anamnestic antibody response following a second inoculation 12 weeks later. Inoculations resulted in transient increases in CD4+ T-cell activation and proliferation, as well as a sustained increase in CD4+ T cells coexpressing CCR5 and α4β7 integrin. In contrast to previous failed attempts to successfully utilize attenuated VZV-Oka as an SIV vaccine vector in rhesus macaques due to suboptimal infectivity and cellular immunogenicity, the ability to infect cynomolgus macaques with Oka-P VZV should provide a valuable tool for evaluating VZV-vectored SIV/HIV vaccines.

Isolation and characterization of cynomolgus macaque (Macaca fascicularis) cytomegalovirus (CyCMV).

Cynomolgus macaques have been widely used as an animal model in preclinical biomedical research and are becoming more popular among HIV/SIV vaccine researchers. Here we report the isolation and characterization of a cytomegalovirus from cynomolgus macaques (CyCMV). CyCMV was isolated from a healthy captive-bred 4-year-old cynomolgus macaque of Filipino origin. The virus was identified by its characteristic growth properties in cell culture, ultrastructural morphology and sequence of viral DNA polymerase and glycoprotein B (gB). CyCMV gB shows 77% identity and 88% homology to rhesus cytomegalovirus (RhCMV) gB and 58% identity and 76% homology to human cytomegalovirus gB at the amino acid level. Phylogenetic analysis using known CMV gB protein sequences show that CyCMV is more closely related to RhCMV than to other primate CMVs. CyCMV down-regulates MHC class I expression on infected cells and we show that the colony-bred cynomolgus macaques have detectable CyCMV-specific humoral and cell-mediated immune responses.

Persistence of restricted CD4 T cell expansions in SIV-infected macaques resistant to SHIV89.6P superinfection

Attempts to evaluate the protective effect of live attenuated SIV vaccine strains have yielded variable results depending on the route of immunization, the level of attenuation, the level of divergence between the vaccine candidate and the challenge. The protective mechanisms induced by these vaccines are still not well understood. In an effort to address whether the diversity of the CD4+ T cell repertoire in cynomolgus macaques plays a role in the immunological protection following SIVmacC8 infection, we have performed a longitudinal follow-up of the CD4 repertoire by heteroduplex tracking assay in macaques mock-infected or infected with either the attenuated SIVmacC8 or its homologous SIVmacJ5 and challenged with simian-human immunodeficiency virus (SHIV89.6P). Viral load and CD4 absolute counts were determined in these animals and the presence of SHIV89.6P virus in challenged animals was evaluated by PCR and serology. In all macaques that were protected against the challenging virus, we demonstrated a reduced diversity in the CD4+ TRBV repertoire and a few dominant CD4+ T cell clones during early primary infection. In contrast, CD4 TRBV repertoire in unprotected macaques remained highly diverse. Moreover, some of the CD4 T cell clones that were expanded during primary SIV infection re-emerged after challenge suggesting their role in protection against the challenging virus. These results underline the importance of maintaining the CD4 T cell repertoire developed during acute infection and point to the restriction of the CD4 response to the vaccine as a correlate of protection.

A novel strain of cynomolgus macaque cytomegalovirus: implications for host-virus co-evolution.

BackgroundCytomegaloviruses belong to a large, ancient, genus of DNA viruses comprised of a wide array of species-specific strains that occur in diverse array of hosts.MethodsIn this study we sequenced the ~217 Kb genome of a cytomegalovirus isolated from a Mauritius cynomolgus macaque, CyCMV Mauritius, and compared it to previously sequenced cytomegaloviruses from a cynomolgus macaque of Filipino origin (CyCMV Ottawa) and two from Indian rhesus macaques (RhCMV 180.92 and RhCMV 68–1).ResultsThough more closely related to CyCMV Ottawa, CyCMV Mauritius is less genetically distant from both RhCMV strains than is CyCMV Ottawa. Several individual genes, including homologues of CMV genes RL11B, UL123, UL83b, UL84 and a homologue of mammalian COX-2, show a closer relationship between homologues of CyCMV Mauritius and the RhCMVs than between homologues of CyCMV Mauritius and CyCMV Ottawa. A broader phylogenetic analysis of 12 CMV strains from eight species recovers evolutionary relationships among viral strains that mirror those amongst the host species, further demonstrating co-evolution of host and virus.ConclusionsPhylogenetic analyses of rhesus and cynomolgus macaque CMV genome sequences demonstrate co-speciation of the virus and host.