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Candidate polyanion microbicides inhibit HIV-1 infection and dissemination pathways in human cervical explants.

Fletcher PS, Wallace GS, Mesquita PM, Shattock RJ - Retrovirology (2006)

Bottom Line: While both compounds were virucidal for X4 HIV-1, neither was virucidal for R5 virus.Our results demonstrate that PRO 2000 is a potent inhibitor of R5 HIV-1 infection and dissemination pathways in human cervical explants.DxS, while demonstrating significant inhibition of R5 infection, was less active against DC mediated dissemination pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infection, Department of Cellular and Molecular Medicine, St George's, University of London, London, UK. pfletche@sgul.ac.uk

ABSTRACT

Background: Heterosexual intercourse remains the major route of HIV-1 transmission worldwide, with almost 5 million new infections occurring each year. Women increasingly bear a disproportionate burden of the pandemic, thus there is an urgent need to develop new strategies to reduce HIV-1 transmission that could be controlled by women themselves. The potential of topical microbicides to reduce HIV transmission across mucosal surfaces has been clearly identified, and some agents are currently under evaluation in clinical trials. Many of these "first generation" microbicides consist of polyanionic compounds designed to interfere with viral attachment. Here we have evaluated two candidate polyanion compounds in clinical trials, PRO 2000 and dextrin sulphate (DxS) to determine their safety and efficacy against in vitro HIV-1 and HSV-2 infection using cellular and tissue explant models.

Results: PRO 2000 and DxS potently inhibited infection by HIV-1 X4 and R5 isolates when present during viral exposure. However PRO 2000 required 10-fold and DxS 2000-fold more compound to block infection with R5 virus than X4. While both compounds were virucidal for X4 HIV-1, neither was virucidal for R5 virus. PRO 2000 efficiently inhibited infection of cervical explants and dissemination of virus by migratory DC. DxS was less active, able to completely inhibit cervical explant infection, but providing only partial reduction of virus dissemination by DC. PRO 2000, but not DxS, also inhibited HIV-1 binding to DC-SIGN+ cells and trans infection of co-cultured target cells. The inflammatory potential of both compounds was screened by measurement of cytokine production from cervical explants, and statistically significant increases were only observed for IL-1beta and RANTES following treatment with PRO 2000. Both compounds also demonstrated potent activity against HSV-2 infection of cervical epithelial cells.

Conclusion: Our results demonstrate that PRO 2000 is a potent inhibitor of R5 HIV-1 infection and dissemination pathways in human cervical explants. DxS, while demonstrating significant inhibition of R5 infection, was less active against DC mediated dissemination pathways. PRO 2000 has now entered human phase III efficacy trials.

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Inhibitory effect of polyanionic compounds against HIV-1 infection of T-cells. HIV-1 BaL (R5, ■, solid line) or RF (X4, □, dotted line) was immobilised onto solid phase using anti-HLA-DR antibody capture, as described in the Methods. (i) Direct virucidal activity was determined by the pre-treatment of immobilised virus for 1 hour before culture with target PM-1 cells in the absence of compound. (ii) Receptor mediated blockade activity was determined by the pre-treatment of target PM-1 cells (1 hour) prior to exposure to immobilised virus in the absence of compound. (iii) Attachment/fusion inhibition was determined by the pre-treatment of immobilised virus with test compound prior to the addition of target PM-1 cells in the presence of compound. Plates were cultured for 10 days following which viral replication was determined by reverse transcriptase measurement of culture supernatants. Compounds tested were: A) PRO 2000; and B) Dextrin sulphate. Data represent the mean ± SEM of n = 5 (PRO 2000) or 4 (Dextrin sulphate) independent experiments where each condition was tested in triplicate. Inserted figures represent the mean ± SEM concentration inhibiting 50% infection (IC50) for compounds against each virus.
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Figure 1: Inhibitory effect of polyanionic compounds against HIV-1 infection of T-cells. HIV-1 BaL (R5, ■, solid line) or RF (X4, □, dotted line) was immobilised onto solid phase using anti-HLA-DR antibody capture, as described in the Methods. (i) Direct virucidal activity was determined by the pre-treatment of immobilised virus for 1 hour before culture with target PM-1 cells in the absence of compound. (ii) Receptor mediated blockade activity was determined by the pre-treatment of target PM-1 cells (1 hour) prior to exposure to immobilised virus in the absence of compound. (iii) Attachment/fusion inhibition was determined by the pre-treatment of immobilised virus with test compound prior to the addition of target PM-1 cells in the presence of compound. Plates were cultured for 10 days following which viral replication was determined by reverse transcriptase measurement of culture supernatants. Compounds tested were: A) PRO 2000; and B) Dextrin sulphate. Data represent the mean ± SEM of n = 5 (PRO 2000) or 4 (Dextrin sulphate) independent experiments where each condition was tested in triplicate. Inserted figures represent the mean ± SEM concentration inhibiting 50% infection (IC50) for compounds against each virus.

Mentions: Direct virucidal activity was assessed by compound treatment of immobilised virus, prior to washing and culture with permissive T cells as previously described [22]. Both compounds demonstrated potent activity against the X4 isolate, with 50% inhibitory concentrations (IC50) observed at 14.8 (± 1.9) and 9.3 (± 2.1) μg/ml of PRO 2000 and DxS respectively (Figure 1Ai &1Bi). In contrast, both compounds failed to exert any effect against R5 virus, even at concentrations of 1 mg/ml (Figure 1Ai &1Bi). Receptor mediated blockade was assessed by incubating target cells with compound prior to compound removal and culture with immobilised virus; this was poor or absent for both compounds (Figure 1Aii &1Bii). Inhibition of attachment/fusion was assessed by pre-treatment of virus with test compound for 1 hour prior to culture with permissive cells in the presence of compound. Both compounds exhibited potent activity against R5 and X4 infection, although greater activity was observed against X4 than R5 virus with IC50 values of 1.9 (± 1.6) and 20.8 (± 1.5) μg/ml respectively for PRO 2000, and 0.38 (± 1.9) and 782.8 (± 2.4) μg/ml respectively for DxS (Figure 1Aiii &1Biii).


Candidate polyanion microbicides inhibit HIV-1 infection and dissemination pathways in human cervical explants.

Fletcher PS, Wallace GS, Mesquita PM, Shattock RJ - Retrovirology (2006)

Inhibitory effect of polyanionic compounds against HIV-1 infection of T-cells. HIV-1 BaL (R5, ■, solid line) or RF (X4, □, dotted line) was immobilised onto solid phase using anti-HLA-DR antibody capture, as described in the Methods. (i) Direct virucidal activity was determined by the pre-treatment of immobilised virus for 1 hour before culture with target PM-1 cells in the absence of compound. (ii) Receptor mediated blockade activity was determined by the pre-treatment of target PM-1 cells (1 hour) prior to exposure to immobilised virus in the absence of compound. (iii) Attachment/fusion inhibition was determined by the pre-treatment of immobilised virus with test compound prior to the addition of target PM-1 cells in the presence of compound. Plates were cultured for 10 days following which viral replication was determined by reverse transcriptase measurement of culture supernatants. Compounds tested were: A) PRO 2000; and B) Dextrin sulphate. Data represent the mean ± SEM of n = 5 (PRO 2000) or 4 (Dextrin sulphate) independent experiments where each condition was tested in triplicate. Inserted figures represent the mean ± SEM concentration inhibiting 50% infection (IC50) for compounds against each virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1590048&req=5

Figure 1: Inhibitory effect of polyanionic compounds against HIV-1 infection of T-cells. HIV-1 BaL (R5, ■, solid line) or RF (X4, □, dotted line) was immobilised onto solid phase using anti-HLA-DR antibody capture, as described in the Methods. (i) Direct virucidal activity was determined by the pre-treatment of immobilised virus for 1 hour before culture with target PM-1 cells in the absence of compound. (ii) Receptor mediated blockade activity was determined by the pre-treatment of target PM-1 cells (1 hour) prior to exposure to immobilised virus in the absence of compound. (iii) Attachment/fusion inhibition was determined by the pre-treatment of immobilised virus with test compound prior to the addition of target PM-1 cells in the presence of compound. Plates were cultured for 10 days following which viral replication was determined by reverse transcriptase measurement of culture supernatants. Compounds tested were: A) PRO 2000; and B) Dextrin sulphate. Data represent the mean ± SEM of n = 5 (PRO 2000) or 4 (Dextrin sulphate) independent experiments where each condition was tested in triplicate. Inserted figures represent the mean ± SEM concentration inhibiting 50% infection (IC50) for compounds against each virus.
Mentions: Direct virucidal activity was assessed by compound treatment of immobilised virus, prior to washing and culture with permissive T cells as previously described [22]. Both compounds demonstrated potent activity against the X4 isolate, with 50% inhibitory concentrations (IC50) observed at 14.8 (± 1.9) and 9.3 (± 2.1) μg/ml of PRO 2000 and DxS respectively (Figure 1Ai &1Bi). In contrast, both compounds failed to exert any effect against R5 virus, even at concentrations of 1 mg/ml (Figure 1Ai &1Bi). Receptor mediated blockade was assessed by incubating target cells with compound prior to compound removal and culture with immobilised virus; this was poor or absent for both compounds (Figure 1Aii &1Bii). Inhibition of attachment/fusion was assessed by pre-treatment of virus with test compound for 1 hour prior to culture with permissive cells in the presence of compound. Both compounds exhibited potent activity against R5 and X4 infection, although greater activity was observed against X4 than R5 virus with IC50 values of 1.9 (± 1.6) and 20.8 (± 1.5) μg/ml respectively for PRO 2000, and 0.38 (± 1.9) and 782.8 (± 2.4) μg/ml respectively for DxS (Figure 1Aiii &1Biii).

Bottom Line: While both compounds were virucidal for X4 HIV-1, neither was virucidal for R5 virus.Our results demonstrate that PRO 2000 is a potent inhibitor of R5 HIV-1 infection and dissemination pathways in human cervical explants.DxS, while demonstrating significant inhibition of R5 infection, was less active against DC mediated dissemination pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Infection, Department of Cellular and Molecular Medicine, St George's, University of London, London, UK. pfletche@sgul.ac.uk

ABSTRACT

Background: Heterosexual intercourse remains the major route of HIV-1 transmission worldwide, with almost 5 million new infections occurring each year. Women increasingly bear a disproportionate burden of the pandemic, thus there is an urgent need to develop new strategies to reduce HIV-1 transmission that could be controlled by women themselves. The potential of topical microbicides to reduce HIV transmission across mucosal surfaces has been clearly identified, and some agents are currently under evaluation in clinical trials. Many of these "first generation" microbicides consist of polyanionic compounds designed to interfere with viral attachment. Here we have evaluated two candidate polyanion compounds in clinical trials, PRO 2000 and dextrin sulphate (DxS) to determine their safety and efficacy against in vitro HIV-1 and HSV-2 infection using cellular and tissue explant models.

Results: PRO 2000 and DxS potently inhibited infection by HIV-1 X4 and R5 isolates when present during viral exposure. However PRO 2000 required 10-fold and DxS 2000-fold more compound to block infection with R5 virus than X4. While both compounds were virucidal for X4 HIV-1, neither was virucidal for R5 virus. PRO 2000 efficiently inhibited infection of cervical explants and dissemination of virus by migratory DC. DxS was less active, able to completely inhibit cervical explant infection, but providing only partial reduction of virus dissemination by DC. PRO 2000, but not DxS, also inhibited HIV-1 binding to DC-SIGN+ cells and trans infection of co-cultured target cells. The inflammatory potential of both compounds was screened by measurement of cytokine production from cervical explants, and statistically significant increases were only observed for IL-1beta and RANTES following treatment with PRO 2000. Both compounds also demonstrated potent activity against HSV-2 infection of cervical epithelial cells.

Conclusion: Our results demonstrate that PRO 2000 is a potent inhibitor of R5 HIV-1 infection and dissemination pathways in human cervical explants. DxS, while demonstrating significant inhibition of R5 infection, was less active against DC mediated dissemination pathways. PRO 2000 has now entered human phase III efficacy trials.

Show MeSH
Related in: MedlinePlus