Antiviral properties of milk proteins and peptides
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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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Antiviral properties of milk proteins and peptides

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: [email protected]


Introduction

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


Antiviral properties of milk proteins and peptides

  • Proteins with Antiviral

    activity:

  • Lactoferrin (LF)

  • Lactadherin

  • Glycoprotein

  • Immunoglobulin (Ig)


Lactoferrin lf

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


Antiviral properties of milk proteins and peptides

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


Antiviral properties of milk proteins and peptides

-NON-ENVELOPED VIRUSES:

  • Rotavirus

  • Enterovirus

  • Poliovirus

  • Adenovirus and

  • Feline calicivirus


Antiviral effect of proteins

Antiviral effect of proteins


Antiviral mechanisms of lf

Antiviral mechanisms of LF


Mechanism of action

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


Antiviral properties of milk proteins and peptides

  • 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


Hiv 1 entry into the target cells

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


Antiviral properties of milk proteins and peptides

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


Purification of bovine milk proteins and peptides

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


Antiviral properties of milk proteins and peptides

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)


Methods to check antiviral properties

Methods to check antiviral properties

  • ELISA

  • MTT ASSAY

  • RADIOISOTOPING METHOD


1 elisa

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


2 mtt assay

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)


3 radioisotoping method

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


Lactoferrin resistance

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


Antiviral properties of milk proteins and peptides

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


Purified milk proteins their effect on hiv 1 replication

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


Antiviral properties of milk proteins and peptides

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


Lactoferrin inhibition of cxcr4 ccr5 using viruses

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


Antiviral properties of milk proteins and peptides

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


Antiviral properties of milk proteins and peptides

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


Antiviral properties of other milk proteins

Antiviral properties of other milk proteins

  • Lactadherin

  • Glycoprotein

  • Immunoglobulin (Ig)


Antiviral properties of milk proteins and peptides

  • Lactadherin:

  • Viral receptor binding

  • sialic acid plays important role in its antiviral action

  • Human lactadherin protected breast-fed infants against symptomatic rotavirus infection


Antiviral properties of milk proteins and peptides

  • 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


Antiviral peptides derived from milk proteins

Antiviral peptides derived from milk proteins

  • Lactoferricin

  • GMP

  • Mucin complex


Antiviral properties of milk proteins and peptides

Antiviral effect of peptides


Enhancement of antiviral activity on chemical modification

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


Antiviral properties of milk proteins and peptides

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


Antiviral properties of milk proteins and peptides

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


Antiviral properties of milk proteins and peptides

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


Conclusion

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


Antiviral properties of milk proteins and peptides

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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