The role of crude saliva and purified salivary mucins in the inhibition of the Human Immunodeficiency Virus type 1

Abstract

Background: Sub-Saharan Africa is the world's worst HIV-AIDS affected region. More interventions to manage this pandemic are urgently required. Transmission of the virus through an exchange of saliva is rarely known to occur. This project sought to verify statistically previous findings in our laboratory, that crude saliva from uninfected individuals together with its purified mucin components inhibited HIV-1, whilst mucins from infected saliva did not show this inhibition, in an in vitro assay.

Methods: Saliva was extracted in 4 M guanidinium hydrochloride and proteolytic inhibitors at pH 6.5, followed by the isolation of MUC5B and MUC7 by Sepharose 4B gel filtration and further purification of these mucins by density-gradient ultra-centrifugation in caesium chloride. Agarose gel electrophoresis, Western blotting and amino acid compositional analysis determined the size, purity and identity of the mucins. The inhibitory activity of crude saliva and purified MUC5B and MUC7, from HIV negative (n=20) and HIV positive (n=20) donors, was tested by their incubation with subtype C HIV-1 and subsequent infection of peripheral blood mononuclear cells (PBMCs). PCR was done on tandem repeat regions of MUC5B and MUC7 DNA to investigate whether any association existed between gene polymorphism and susceptibility to infection.

Results: There was an inter-individual variation in the amounts of MUC5B and MUC7 in saliva. In contrast to previous studies, crude saliva and purified mucins from both HIV negative and HIV positive individuals inhibited the infection of HIV-1 in an in vitro assay. DNA analysis of the tandem repeat regions of MUC5B and MUC7 revealed no difference between groups.

Conclusions: Crude saliva and its mucins, MUC5B and MUC7, from both uninfected controls and HIV positive individuals inhibited HIV-1 in an in vitro assay.

Figure 1

The gel filtration profile of crude saliva from an HIV negative donor. Crude saliva (20 ml in 6 M GuHCl, 10 mM EDTA, 1 mM PMSF pH 6.5) from an HIV negative donor was chromatographed on a Sepharose CL-4B gel filtration column (bed volume 200 ml). The column was eluted with 0.2 M NaCl: 0.02% NaN₃ at a flow rate of 60 ml/h at room temperature. An aliquot of each fraction was assayed by the PAS and Lowry methods. V₀ indicates the void volume and V_i indicates the included volume of the column. Fractions 18–34 were pooled to recover MUC5B and fractions 50–66 were pooled to recover MUC7. Fractions were dialysed against three changes of distilled water overnight at 4°C, and freeze-dried.

Figure 2

Profile of purification by caesium chloride density-gradient ultracentrifugation of an HIV positive MUC7 sample. Freeze dried material, in this example a sample of MUC7 (V₀), was dissolved in 4 M GuHCl containing 10 mM EDTA, 5 mM NEM, and 0.05% CHAPS pH 6.5, and adjusted to a density of 1.39 to 1.40 g/ml with solid caesium chloride. Density-gradient centrifugation was performed in a Beckman L45 ultra-centrifuge for 48 h at 40 000 rpm/105 000 g, at 4°C. Fractions 3 and 4 were pooled, dialysed against 3 changes of distilled water overnight at 4°C and freeze-dried.

Figure 3

Western blotting analysis (representative) of HIV negative and HIV positive salivary mucins MUC5B (a) and MUC7 (b). a. lanes: 1, MUC5B (V₀) HIV negative;2,MUC5B (V₀) HIV positive. b. lanes: 1, MUC7 (Vi) HIV negative;2, MUC7 (Vi) HIV positive. Mucin proteins were detected using rabbit anti-MUC5B polyclonal and mouse anti-MUC7 monoclonal primary antibodies respectively. Dashed-arrows indicate loading position at the top of the gel picture and further arrows indicate the migration distance of samples.

Figure 4

Gel electrophoresis of the PCR product of MUC5B tandem repeat regions showing variations in the number of tandem repeats. Gel electrophoresis of the PCR product of MUC5B tandem repeat regions showing variations in the number of tandem repeats. A 2% agarose gel was used for MUC5B DNA samples. 20ul of reaction mixture was loaded for each sample into each lane. MWM marks those lanes loaded with a DNA molecular weight marker. Ethidium bromide enabled DNA to be viewed under ultraviolet light. Lanes 1–10 are HIV negative DNA samples and 11–20 HIV positive DNA samples (a representation of samples is shown, lanes are numbered as such for clarity purposes).

Figure 5

Gel electrophoresis of the PCR product of MUC7 tandem repeat regions showing variations in the number of tandem repeats. A 1% agarose gel was used for MUC7 DNA samples. 20ul of reaction mixture was loaded for each sample into each lane. MWM marks those lanes loaded with a DNA molecular weight marker. Ethidium bromide enabled DNA to be viewed under ultraviolet light. Lanes 1–10 are HIV negative DNA samples and 11–20 HIV positive DNA samples (a representation of samples is shown, lanes are numbered as such for clarity purposes). A single band (590 bp) represents the tandem repeat fragment with six repeats indicating a donor who is homozygous for MUC7. The double band, marked with an arrow, is of the fragment with six repeats and the smaller sized band (521 bp) is the fragment with five repeats. This indicates a heterozygous genotype for the donor for MUC7.