Antiviral Properties of Milk Proteins and Peptides: A Comprehensive Overview, Slides of Public Health

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Antiviral Properties of Milk Proteins and Peptides docsity.com Introduction • Milk proteins and peptides possess biological properties beyond their nutritional significance • In 1987, lactoferrin (LF) -Friend leukaemia virus (FLV) • Chemically modified milk proteins & peptides docsity.com ACTION OF LF-ENVELOPED VIRUSES: Human immunodeficiency virus (HIV) Human cytomegalovirus (CMV) Herpes simplex virus type 1 and 2 (HSV) Hepatitis B, C and G viruses Human papillomavirus (HPV) and Alphavirus docsity.com - NON-ENVELOPED VIRUSES: Rotavirus Enterovirus Poliovirus Adenovirus and Feline calicivirus docsity.com Antiviral effect of proteins Virus (Enveloped) Protein Comments/proposed action HIV LF Milk bLF & hLF block HIV-1 adsorption to target cells Inhibits binding of HIV-1 to CD4 receptor HSV-1 & 2 LF bLF & hLF bind to virus particles & Synergistic effect with acyclovir HCMV LF o Interfere with virus into target cells o Up regulation of killer cells o Synergistic antiviral effect with cidofovir docsity.com PV LF Binds to target cells Human influenza virus LF Inhibits haem agglutination by the virus FCV LF bLF interfers with adsorption of virus to target cells Adenovirus LF bLF & hLF compete with virus for common membrane receptors docsity.com Antiviral mechanisms of LF A a aa oy oi y aes wa ttt Hal ® Mechanism of action • First, LF appears to interact with the receptors on the cell surface, such as glycosaminoglycans which are the binding sites for many viruses • Second, LF binds directly to viral particles and inhibits viral adsorption to target cells docsity.com  Interaction between the V3 loop and heparan sulfate adhere virus to the cell surface Positively charged compounds (AMD3100 and ALX40-4C) block HIV-1 replication, interact with negatively charged CXCR4 coreceptors Contd… docsity.com PURIFICATION OF BOVINE MILK PROTEINS AND PEPTIDES α-S2 Casein, bovine LFcin-B and bovine k-casein Hydrolysis with pepsin Cation exchange chromatography Obtained fragments characterized by HPLC and ESI-MS docsity.com • Βovine β-casein and bovine β lactoglobulin are modified by maleic acid (Ikura et al., 1984) • Bovine as2-casein is modified with 3- hydroxyphthalic anhydride • The degree of modification checked with ortho- phtaldialdehyde (Berkhout et al., 1997) Contd… docsity.com 2. MTT ASSAY MT2 T cell line infected with HIV-1 LA1 - increased concentration of milk proteins After 5 days post-infection Living cells convert the MTT {3-(4,5- dimethylethiazole-2-ly)-2,5-diphenyltetrazolium bromide) Blue product (formazine) docsity.com 3. RADIOISOTOPING METHOD Cell culture vessel (Nunclon 24-well plate) Nonspecific protein-inhibitors Add sup T1 cells in a complete medium (RPMI) Radioactively labelled 125 I-bLF & incubate plates at 4 ºc to 37 ºc for 1 hour Amount of radioactvity recovered was determined by GAMMA COUNTER docsity.com LACTOFERRIN RESISTANCE 1. HIV-1 LA1 isolate cultured in the presence of 10µM bLF 2. Cell free virus is passaged on to uninfected supT1 cells 3. Observe the massive syncytia formation in culture 4. Virus sample is taken after several days docsity.com Positively charged peptides nisin and lactoferricin  10 µM - moderately inhibit HIV-1  100 µM - complete inhibition but cytotoxicity is observed  bLF significantly inhibits at 0.1-1.0 µM conc Human LF- both native protein and recombinant protein moderately act as inhibitors at 3.1 µM conc Contd… docsity.com LACTOFERRIN INHIBITION OF CXCR4 & CCR5-using viruses Lactoferrin has both positively & negatively charged domains at physiological pH That will interfere with the virus –coreceptor interaction These HIV-1 used to infect U87CD4 cell line that was transfected either CXCR4/CCR5 docsity.com  bLF is a superior anti-HIV-1 compound compared to human LF and murine LF either of their native or recombinant proteins  bLF is 69% and 64% identical to hLF and mLF respectively Bovine Plasma and milk proteins are abundantly available These industrial proteins are produced at a large scale, through simple chemical modifications Contd… docsity.com • Lactadherin : Viral receptor binding sialic acid plays important role in its antiviral action Human lactadherin protected breast-fed infants against symptomatic rotavirus infection docsity.com • Glycoprotein : High-molecular weight fraction from bovine milk  was effective against human rotavirus in vitro • Milk immunoglobulin :  Hyperimmunised with human rotavirus during pregnency of cows 100 times- Human milk 10 times – Commercial Ig docsity.com Antiviral peptides derived from milk proteins  Lactoferricin  GMP  Mucin complex docsity.com Enhancement of Antiviral activity on Chemical modification • Chemical modifications lead to changes in the charges on milk proteins which can enhance their antiviral properties (Swart, Harmsen, et al., 1999; Waarts et al., 2005) • Two main approaches: Acylation to increase negative charges Amination to increase positive charges docsity.com  Succinylated and aconitylated LF has stronger anti-HIV-1 effects (2-4 times more active than native LF) (Swart, Harmsen, et al.,1999) • Several other proteins - b-Lg, a-La and HSA, also has an enhanced effect against HIV-1 and HIV-2 (Jiang, Lin, Strick, Li, & Neurath, 1996) • Additional negative charges were introduced through modifications of lysine residues Contd… docsity.com • b-Lg modified with 3- hydroxyphthaloyl acid (3HP) interfered with the infection by HIV-1, HSV-1 & 2, and HCMV (Berkhout et al., 2002; Swart, Kuipers, et al., 1996) • It was also found that 3HP-a-La and 3HP-as2-casein were also effective against HIV-1 Contd… docsity.com