Coreceptor Usage of Sequential Isolates from Cynomolgus Monkeys Experimentally Infected with Simian Immunodeficiency Virus (SIVsm)

AbstractSequential isolates from eight cynomolgus monkeys experimentally infected with simian immunodeficiency virus (SIVsm, of sooty mangabey origin) were tested for coreceptor use in the human osteosarcoma indicator cell line, GHOST(3), expressing CD4 and one or another of the chemokine receptors CCR3, CCR5, CXCR4, BOB, or the orphan receptor Bonzo. The indicator cell line carries the human immunodeficiency virus type 2 long terminal repeat-driven green fluorescence protein gene that becomes activated upon infection with HIV or SIV and fluorescence can be quantitated by flow cytometric analysis. The methodological details are described in the accompanying paper (Vödrös et al., 2001, Virology 290, in press). All SIVsm inoculum viruses and reisolates used CCR5 with a high level of efficiency. CCR5 use was stable over time. BOB and Bonzo use was less efficient than CCR5 use and, in particular, late isolates obtained at the time of immunodeficiency varied greatly in their coreceptor use and often could not establish a productive infection in BOB- or Bonzo-expressing cells. Unexpectedly, early reisolates obtained 12 days postinfection could infect the entire GHOST(3) panel including the parental cells. In one case this was due to use of CXCR4, either transfected or endogenously expressed on the GHOST(3) cells. Our results demonstrate the complex coreceptor use of SIVsm isolates. Moreover, they focus attention on the initial phase of virus replication when the availability of target cells may govern the replication pattern of the virus

GM-CSF DNA: An adjuvant for higher avidity IgG, rectal IgA, and increased protection against the acute phase of a SHIV-89.6P challenge by a DNA/MVA immunodeficiency virus vaccine

AbstractSingle intradermal or intramuscular inoculations of GM-CSF DNA with the DNA prime for a simian–human immunodeficiency virus (SHIV)-89.6 vaccine, which consists of DNA priming followed by modified vaccinia Ankara (MVA) boosting, increased protection of both the blood and intestines against the acute phase of an intrarectal SHIV-89.6P challenge. GM-CSF appeared to contribute to protection by enhancing two antibody responses: the avidity maturation of anti-Env IgG in blood (p=<0.01) and the presence of long lasting anti-viral IgA in rectal secretions (p<0.01). The avidity of anti-Env IgG showed strong correlations with protection both pre and post challenge. Animals with the highest avidity anti-Env Ab had 1000-fold reductions in peak viremia over those with the lowest avidity anti-Env Ab. The enhanced IgA response was associated with the best protection, but did not achieve significance

Receptor use of primate lentiviruses

Human and simian immunodeficiency viruses (HIV and SIV) enter target cells using CD4, the primary cell surface receptor and a secondary receptor, called coreceptor. Coreceptor use of HIV can be related to other biological features of the virus and to the severity of infection as well. Animal models are important in understanding transmission, pathogenesis and immune responses of HIV infection but are essential in studying the effect of candidate drugs or vaccines. The most widely used model for HIV infection in humans is SIV infection of macaques, therefore it is important to understand and compare the biological features of the two systems. The aim of this thesis was to study the role of coreceptors in HIV and SIV pathogenesis. By the use of the GHOST(3) cell lines it was possible to set up a quantitative evaluation system. This allowed the detection of even slight changes in the coreceptor use of multicoreceptor tropic isolates obtained from individuals at different time points. Coreceptor use of HIV-1 isolates was studied in the context of mother-to-child transmission. 28 pregnant Cameroonian women and their newborn children were followed. Viruses from most of the patients (23 women) belonged to HIV-1 of genetic subtype A. Isolates predominantly used CCR5 (R5) for cell entry (26 out of 28 patients). No CXCR4 using virus could be isolated from any of the 23 patients harbouring subtype A viruses. Instead, CXCR6 (X6) use was evident in four cases; all of these isolates were R5X6 dual-tropic. Mother-tochild transmission occurred in four cases. Interestingly, prevalence of CXCR6 using viruses was higher among transmitting mothers (2/4) as compared to non-transmitting mothers (2/22). Since trophoblastic cells may express the CXCR6 receptor, we speculate that CXCR6 use by maternal viruses might be an advantage in crossing the placental barrier, thereby increasing the risk of mother to child transmission of HIV-1. Coreceptor use of SIVsm inoculum viruses and sequential isolates from experimentally infected macaques with fast or slow disease progression and known neutralizing antibody pattern was also studied. Furthermore, envelopes (Envs) were cloned from isolates and tested for their ability to mediate fusion. This allowed us to study an expanded range of receptors and to test the capacity of receptors to mediate CD4-independent fusion. Envs were multi-coreceptor tropic and fused efficiently with a number of receptors. CCR5 use was efficient and stable in all macaques, whereas the use of CXCR6, BOB/gpr15, gpr1 and Apj decreased over time. Coreceptor usage pattern of virus isolates and Env clones was similar in all monkeys. Therefore no changes were found that could be related to SIV pathogenesis. CD4-independent fusion with CCR5 was significant and revealed an interesting pattern. Envs from inoculum viruses showed distinct dependencies on CD4 for fusion via CCR5. Envs from early re-isolates maintained or increased CD4-independent use of CCR5 and displayed high variability in this regard. In contrast, Envs from late re-isolates tended to show less variation and increased CD4 dependence if derived from animals that were able to mount neutralizing antibody response to their virus. This is consistent with findings that show neutralization sensitivity of CD4-independent viruses and suggests that CD4-dependent (and neutralization resistant) viruses were selected in the macaques over time. Infection with a strictly CD4dependent virus resulted in evolution of CD4-independent Envs over time. The high level of virus replication during acute infection and the high rate of mutation during replication may result in the appearance of a highly variable virus population. Our results highlight the role of neutralizing antibodies in the control of SIV infection. Receptor tropism, availability of target cells and immune activation may shape the course of viral evolution and SIV pathogenesis

Quantitative evaluation of HIV and SIV co-receptor use with GHOST(3) cell assay.

An assay has been established for quantitative evaluation of lentivirus coreceptor use with the help of GHOST(3) cells. GHOST(3) cells were derived from the human osteosarcoma cell line, HOS, and have been engineered to stably express CD4 and one or another of the chemokine receptors CCR3, CCR5, CXCR4, CXCR6/STRL33/Bonzo, or the orphan receptor GPR15/BOB. The indicator cell line carries the HIV-2 long terminal repeat-driven green fluorescence protein (GFP) gene, which becomes activated upon infection with HIV or SIV. Viral entry is followed by Tat activation of transcription and GFP becomes expressed. Infected cells can be detected as early as 2 or 3 d after infection by simple fluorescence microscopic observation. The simplicity of the GHOST(3) cell system makes it particularly suitable for screening of a large number of isolates. In addition, the efficiency of co-receptor use can be accurately quantitated with flow cytometric analysis. Thus, the most efficiently used co-receptor of multitropic isolates can be determined. It is also possible to sensitively determine co-receptor switch of sequential isolates from the same individual

Primate models for human immunodeficiency virus infection. Evolution of receptor use during pathogenesis.

Animal models greatly facilitate understanding of transmission, pathogenesis and immune responses in HIV and SIV infection and provide models for studies on the effect of candidate drugs or vaccines. However, there are several aspects that one should consider when drawing conclusions from results obtained from animal models. First, the genetic relationship of primate lentiviruses cannot be disregarded because it is known that HIV-1 is more closely related to SIV of chimpanzee origin (SIVcpz) than to SIV from sooty mangabey (SIVsm) origin. Nevertheless, SIVsm and SIVmac are the ones most often used as model systems. Second, there are differences in the biological properties, like CXCR4 use and CD4-independent coreceptor use, of HIV and SIV. These differences might be relevant in virus transmission, pathogenesis and in evoking immune responses. Third, in vivo and in vitro selection may influence the results. Neutralizing antibodies may play a role in selection of variant viruses since neutralization sensitive, CD4-independent SIVsm variants seemed to be suppressed in animals that mounted a neutralizing antibody response. It is tempting to speculate that neutralizing antibodies shape the SIV/HIV infection by selecting variants with a more "closed" envelope conformation with consequences for both receptor binding and neutralization sensitivity. The SIV/monkey model, although it has important advantages, may not answer all questions asked about HIV-1 infection in human

Evolution of coreceptor use and CD4-independence in envelope clones derived from SIVsm-infected macaques.

AbstractCoreceptor use of HIV can evolve during infection. We previously examined coreceptor use of related SIVsm inoculum viruses and sequential reisolates from cynomolgus macaques. These viruses exhibited broad coreceptor specificities and, generally, CCR5 use remained efficient and stable, while alternative coreceptor use decreased longitudinally. Here we demonstrate that individual envelopes (Envs) from inoculum and reisolate viruses fuse via a range of coreceptors, including CCR5, CCR8, CXCR6, GPR15, GPR1, and APJ. On the whole, coreceptor use of Envs from sequential reisolates recapitulated that of reisolate viruses, thus CCR5 use remained stable while alternative coreceptor use tended to decrease over time. Rhesus CCR5, GPR15, and CXCR6 supported fusion to a similar extent as their human counterparts. Additionally, a number of Envs mediated CD4-independent fusion via CCR5 and GPR15. Envs from different inoculum viruses exhibited distinct dependencies on CD4 for fusion via CCR5, ranging from strictly CD4-dependent to efficiently CD4-independent. Early reisolates from macaques infected with CD4-independent inoculums maintained or evolved Envs with a broad range of CD4-independence. CD4-independence became less variable/efficient in late reisolates from macaques that developed neutralizing antibodies. Infection with a CD4-dependent virus resulted in evolution of CD4-independent Envs in late reisolates. While CD4 independence can potentially broaden tropism in vivo, CD4-independent viruses are particularly sensitive to neutralizing antibodies. Therefore, interplay between receptor tropism and neutralization may shape viral evolution and SIV pathogenesis

Prevention of postoperative peritoneal adhesions: Effects of lysozyme, polylysine and polyglutamate versus hyaluronic acid

Objective. Intraperitoneal adhesions are an important cause of postoperative intestinal obstruction, abdominal discomfort and infertility. In the present study we hypothesized that a combination of polypeptides with different surface properties, resulting in fine disperse low-soluble complexes, could be of benefit in the prevention of abdominal adhesions. Material and methods. Various polypeptides including lysozyme, polyglutamate, polylysine and combinations of all three were evaluated as compared to hyaluronic acid. A standard wound on the parietal peritoneum in mice was used and the evaluated agents were administered immediately postoperatively. The extent of peritoneal adhesions to the injured area was measured and expressed as a percentage of the wound length as evaluated after 7 days. Flow cytometry was performed to evaluate the effect on peritoneal macrophage survival and phagocytic function and the Pick test was used to determine peroxide production in order to estimate toxicity and potential impairment of macrophage function caused by the chemicals. Results. Significant differences were seen among the treatment groups (p< 0.001). Both polyglutamate and lysozyme, and polyglutamate together with polylysine significantly decreased adhesion formation as compared to hyaluronic acid. The polylysine - polyglutamate combination was still visible macroscopically on the peritoneal surface after 1 week, though not after 1 month. The polyglutamate - lysozyme mixture was less effective than these individual components alone. The chemicals did not show any toxic effects or altered function in macrophage cell culture. Conclusions. Lysozyme, polyglutamate and, most effectively, a polyglutamate - polylysine combination significantly decreased experimental abdominal adhesion formation. A strong mechanical connection to the wound and prolonged attendance in the surface were noted. Peritoneal phagocyte function did not seem to be influenced by the chemicals