Maleic anhydride-modified chicken ovalbumin as an effective and inexpensive anti-HIV microbicide candidate for prevention of HIV sexual transmission

Abstract

Background: Previous studies have shown that 3-hydroxyphthalic anhydride (HP)-modified bovine milk protein, beta-lactoglobulin (beta-LG), is a promising microbicide candidate. However, concerns regarding the potential risk of prion contamination in bovine products and carcinogenic potential of phthalate derivatives were raised. Here we sought to replace bovine protein with an animal protein of non-bovine origin and substitute HP with another anhydride for the development of anti-HIV microbicide for preventing HIV sexual transmission.

Results: Maleic anhydride (ML), succinic anhydride (SU) and HP at different conditions and variable pH values were used for modification of proteins. All the anhydrate-modified globulin-like proteins showed potent anti-HIV activity, which is correlated with the percentage of modified lysine and arginine residues in the modified protein. We selected maleic anhydride-modified ovalbumin (ML-OVA) for further study because OVA is easier to obtain than beta-LG, and ML is safer than HP. Furthermore, ML-OVA exhibited broad antiviral activities against HIV-1, HIV-2, SHIV and SIV. This modified protein has no or low in vitro cytotoxicity to human T cells and vaginal epithelial cells. It is resistant to trypsin hydrolysis, possibly because the lysine and arginine residues in OVA are modified by ML. Mechanism studies suggest that ML-OVA inhibits HIV-1 entry by targeting gp120 on HIV-1 virions and also the CD4 receptor on the host cells.

Conclusion: ML-OVA is a potent HIV fusion/entry inhibitor with the potential to be developed as an effective, safe and inexpensive anti-HIV microbicide.

Figure 1

The effects of anhydride concentrations in the reaction system on the percentages of modified residues and anti-HIV-1 activity of the SU-, ML-, and HP-modified OVA. The concentration of the anhydrides used is associated with the percentages of modified lysine residues (A) and arginine residues (B) in the chemically modified OVAs and with their anti-HIV-1_IIIB activity (C) and their anti-HIV-1_BaL activity (D). Each sample was tested in triplicate, the experiment was repeated twice, and the data are presented in means ± SD.

Figure 2

The effects of pH value in the reaction system on the percentages of modified residues and anti-HIV-1 activity of SU-, ML-, and HP-modified OVA. The pH value of reaction systems is correlated with the percentages of modified lysine residues (A) and arginine residues (B) in the chemically modified OVAs and with their anti-HIV-1_IIIB activity (C) and their anti-HIV-1_BaL activity (D). Each sample was tested in triplicate, the experiment was repeated twice, and the data are presented in means ± SD.

Figure 3

ML-OVA-mediated inhibition of transmission of HIV-1_BaL from PBMCs to CEMx174 5.25M7 cells. All the samples were tested in triplicate, the experiment was repeated twice, and the data are presented in means ± SD.

Figure 4

Inhibition of chemically modified OVA on single round entry of HIV-1_NL4-3. Each sample was tested in triplicate, the experiment was repeated twice, and the data are presented in means ± SD.

Figure 5

Time-of-addition assay. Inhibition of infection by HIV-1_IIIB (A) and HIV-1_BaL (B) by ML-OVA and the control compounds when added at different intervals post-infection was tested using a time-of-addition assay. Each sample was tested in triplicate, the experiment was repeated twice, and the data are presented in means ± SD.

Figure 6

Flow cytometric analysis of binding of ML-OVA to cells expressing HIV-1 Env or CD4 molecule. (A) ML-OVA + CHO-WT cells; (B) ML-OVA + CHO-EE cells; (C) OVA + CHO-WT cells; (D) OVA + CHO-EE cells; (E) ML-OVA + HeLa-CD4-LTR-β-gal cells; (F) ML-OVA + HeLa cells; (G) OVA + HeLa-CD4-LTR-β-gal cells; and (H) OVA + HeLa cells.

Figure 7

The binding of ML-OVA to sCD4 and gp120 as assessed by ELISA. (A) Dose-dependent binding of ML-OVA to sCD4. (B) Dose-dependent binding of ML-OVA to gp120 from HIV-1_IIIB. Each sample was tested in quadruplicate, the experiment was repeated twice, and the data are presented as means ± SD.

Figure 8

Sensitivity of ML-OVA to digestion by trypsin. The remaining anti-HIV-1_IIIB activity of ML-OVA, T20 and C34 after incubation with trypsin for varying intervals of time was determined by ELISA for p24 antigen production. All the samples were tested in triplicate, the experiment was repeated twice, and data are presented in means ± SD.

Figure 9

The effect of human SF and VSF on the anti-HIV-1 activity of ML-OVA. The antiviral activities against HIV-1_IIIB (A) and HIV-1_BaL (B) in the presence or absence of SF and VSF were assessed using p24 assay as described in the Materials and Methods. The inhibitory activity was detected by an ELISA assay. Each sample was tested in triplicate, and the data are presented as means ± SD.