The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

Abstract

Background: The female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV) transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay.

Methods: Pregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells).

Results: The pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity.

Conclusion: Although it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions), is insufficient to cause viral inhibition or aggregation.

Figure 1

Caesium chloride density gradient purification of the pregnancy plug mucins. Samples in 4 M GuHCl were adjusted to a density of 1.39 to 1.40 g ml^-1 with solid caesium chloride. Density gradient centrifugation was performed in a Beckman L45 ultra-centrifuge for 48 h at a 105 000 g at 4°C. Mucin positive fractions (◆) at a density (▲) between 1.37–1.42 and still associated with some protein (■) (a) were pooled and prepared for the second step centrifugation (b). Finally fractions (fraction number 3, 4 and 5) were pooled, dialysed against three changes of distilled water and freeze-dried.

Figure 2

SDS-PAGE analyses of the pregnancy plug mucins. Freeze-dried pregnancy plug mucins (20 μg) before (a) and after (b) caesium chloride density gradient purification were separated on 10% SDS-PAGE and stained with Coomassie Brilliant Blue (lanes 1, 2 and 4) and PAS (lanes 3 and 5). Lane 1 is molecular weight marker in kDa.

Figure 3

Western blotting analyses of the purified pregnancy plug mucins. Lane 1, MUC1 (positive control), lane 2, salivary MUC5B (negative control), lane 4, colonic mucus (positive control), lane 5, tracheal sputum (negative control), lane 7, pseudomyxoma peritonei (positive control), lane 8, salivary MUC7 (negative control), lane 10, pseudomyxoma peritonei (positive control), lane 11, gastric mucus (negative control) and lanes 3, 6, 9 and 12 purified pregnancy plug mucins were separated by a 1% agarose gel and transferred to nitrocellulose membrane. Following overnight blocking, the membranes were incubated for 2 h with mouse anti-MUC1 monoclonal (lanes 1, 2 and 3) and rabbit anti-MUC2 (lanes 4, 5 and 6), rabbit anti-MUC5AC (lanes 7, 8 and 9) and rabbit anti-MUC5B (lanes 10, 11 and 12) polyclonal antibodies. Membranes were then incubated for 1 h with HRPO linked goat anti-mouse and goat anti-rabbit secondary antibodies and bands that interacted with the antibodies were detected by ECL detection. NB the two bands of MUC2 (lane 6) are indicated by the arrows.

Figure 4

Inhibition of HIV-1 activity by crude pregnancy plug mucus and purified pregnancy plug mucins in vitro assay. Crude pregnancy plug mucus and purified pregnancy plug mucins (0.9 mg each) were incubated with subtype D HIV-1 for 60 min and filtered through 0.45 μm pore size cellulose acetate filter. As controls HIV-1 treated with media and heat inactivated HIV-1 were used. The unfiltered samples were then incubated with CEM SS cells at a concentration of 0.5 × 10⁶cells ml^-1 for 30 min, 1 h and 3 h. After PBS wash cells were cultured and viral replication was measured by a qualitative p24 antigen assay. Letters a, b, c and d indicate the anti-HIV-1 activity of each sample in a serial tenfold dilution of 10^-1, 10^-2, 10^-3 and 10^-4 respectively. P. plug represents pregnancy plug.