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Antiviral Properties of Milk Proteins and Peptides

Antiviral Properties of Milk Proteins and Peptides. RAVINDER NAGPAL 1 , CHAITANYA. S 1 , MONICA PUNIYA 2 , AARTI BHARDWAJ 3 , SHALINI JAIN 4 AND HARIOM YADAV 4* 1 Dairy Microbiology, 2 Dairy Cattle Nutrition, 4 Animal Biochemistry, National Dairy Research Institute, Karnal 132001,

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Antiviral Properties of Milk Proteins and Peptides

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  1. Antiviral Properties of Milk Proteins and Peptides RAVINDER NAGPAL1, CHAITANYA. S1, MONICA PUNIYA2, AARTI BHARDWAJ3, SHALINI JAIN4 AND HARIOM YADAV4* 1Dairy Microbiology, 2Dairy Cattle Nutrition, 4Animal Biochemistry, National Dairy Research Institute, Karnal 132001, Haryana, Meerut Institute of Engineering and Technology, Meerut-250002, U.P., India. *Email: yadavhariom@gmail.com

  2. 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

  3. Proteins with Antiviral activity: • Lactoferrin (LF) • Lactadherin • Glycoprotein • Immunoglobulin (Ig)

  4. Lactoferrin (LF) • Multifunctional Iron binding glycoprotein • Released in the stomach by pepsin cleavage at acidic pH • LF - Antiviral activity against both DNA and RNA viruses

  5. 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

  6. - NON-ENVELOPED VIRUSES: • Rotavirus • Enterovirus • Poliovirus • Adenovirus and • Feline calicivirus

  7. Antiviral effect of proteins

  8. Antiviral mechanisms of LF

  9. Mechanismof 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

  10. Antiviral effectiveness: • The differences in amino acid sequence of antiviral region • Glycan chains and the number of disulphide bridges between hLF and bLF • HIV, HSV, CMV and adenovirus, recognise cell-surface proteoglycans (heparin and heparan sulphate) as receptors

  11. HIV-1 entry into the target cells • Mediated by glycoprotein gp-120 and gp-41 • CD4 -receptor and CCRS, CXCR4 – co receptors • Fusion of viral and cellular membranes

  12. Contd… • 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

  13. PURIFICATION OF BOVINE MILK PROTEINS ANDPEPTIDES α-S2 Casein, bovine LFcin-B and bovine k-casein Hydrolysis with pepsin Cation exchange chromatography Obtained fragments characterized by HPLC and ESI-MS

  14. Contd… • 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)

  15. Methods to check antiviral properties • ELISA • MTT ASSAY • RADIOISOTOPING METHOD

  16. 1. ELISA Add milk protein(1-10 µM ),before addition of HIV-1 virus sup T1 T cell line grown in RPMI medium with 10% FCS at 37 ºC in 5% co2 Virus harvested at peak production and stored at - 70 ºc Quantified in a CA-P24 antigen ELISA

  17. 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)

  18. 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

  19. LACTOFERRIN RESISTANCE • HIV-1 LA1 isolate cultured in the presence of 10µM bLF • Cell free virus is passaged on to uninfected supT1 cells • Observe the massive syncytia formation in culture • Virus sample is taken after several days

  20. Contd… 5. Tested for parallel infection with & without LF 6. Infected cells frozen at -80 ºc for subsequent DNA analysis 7. PCR amplified , Gel purified & Cloned into a cloning vector 8. Multiple clones are inserted as Bam H1 fragment into the PLA I molecular clone 9. Tested their replication capacity with and without bLF

  21. PURIFIED MILK PROTEINS & THEIR EFFECT ON HIV-1 REPLICATION • No antiviral activity with the negatively charged peptides • b-casein 1-28 • kcasein 1-10 and • CMP-A and CMP- B at 10 mM • Complete viral inhibition - chemically modified negatively charged milk protein 3HP-CN

  22. Contd… • 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 µMconc • Human LF- both native protein and recombinant protein moderately act as inhibitors at 3.1 µM conc

  23. 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

  24. Contd… • 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

  25. Contd… • provide relatively cheap antivirals for systemic or local administration • Systemic use of chemically modified milk proteins in human may face major toxicity and immunogenicity problems • except suc-HAS & 3HP-LA show low level toxicity & immunogenicity

  26. Antiviral properties of other milk proteins • Lactadherin • Glycoprotein • Immunoglobulin (Ig)

  27. Lactadherin: • Viral receptor binding • sialic acid plays important role in its antiviral action • Human lactadherin protected breast-fed infants against symptomatic rotavirus infection

  28. 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

  29. Antiviral peptides derived from milk proteins • Lactoferricin • GMP • Mucin complex

  30. Antiviral effect of peptides

  31. 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

  32. Contd… • 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

  33. Contd… • 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

  34. Contd… • 3-HP-b-Lg might be an efficacious agent for preventing vaginal transmission of genital herpes virus infections • Increasing positive net charge on LF • Amination abrogated its anti-HIV effect but increased anti- HCMV effect • Acylation abolished anti-HCMV propertiesof LF but effective against influenza virus

  35. Conclusion • Dietary Milk proteins improve the health of patients suffering from viral infections • Bovine LF showed considerable inhibitory action against most of the viruses • Results of research undertaken to date, primarily under in vitro conditions • In more recent years, in vivo effects have been reported in mouse and rat models

  36. Contd… • In the immediate future, for prevention and therapy of viral infections in animals and humans • Benefits of some of the chemical modifications observed in vitro could be explored • For Specific applications in animal and human health care

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