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.

Antiviral Properties of Antiviral Properties of Milk Proteins and Milk Proteins and

PeptidesPeptidesRAVINDER 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

• Milk proteins and peptides possess biological properties beyond their nutritional significance

• In 1987, lactoferrin (LF) -Friend leukaemia virus (FLV)

• Chemically modified milk proteins & 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

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

- NON-ENVELOPED VIRUSES:

Rotavirus

Enterovirus

Poliovirus

Adenovirus and

Feline calicivirus

Antiviral effect of proteinsAntiviral 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

HCV LF o Binds to viral envelop proteins E1 & E2

o bLF administrated orally may be the effective in combination in interferon

Alphavirus LF hLF inhibits interaction of virus with heparan sulphate receptors

Hantavirus LF o Synergistic effect with ribavirin on viral replication

o bLF administered orally may be effective in combination with interferon

HPV LF Interferes with internalisation of virus into host cells

Virus(Non-Enveloped)

Protein Comments/proposed action

Rotavirus

Lactedherin

High MW glycoprotein; bovine milk

Ig concentrate

Human lactadherin protects breast-fed infants against infection

Effective in vitro; mode of action is unclear

An in vivo effect; 100 times higher than that obtained with human milk

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

Antiviral mechanisms of LF

MechanismMechanism of actionof 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 effectiveness: The differences in amino acid sequence of

antiviral regionGlycan 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 HIV-1 entry into the target cellscells

Mediated by glycoprotein gp-120 and gp-41

CD4 -receptor and CCRS, CXCR4 – co

receptors

Fusion of viral and cellular membranes

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…

PURIFICATION OF BOVINE MILK PROTEINS ANDPURIFICATION OF BOVINE MILK PROTEINS AND PEPTIDESPEPTIDES

α-S2 Casein, bovine LFcin-B and bovine k-casein

Hydrolysis with pepsin

Cation exchange chromatography

Obtained fragments characterized by HPLC and ESI-MS

• 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…

Methods to check antiviral Methods to check antiviral propertiesproperties

• ELISA

• MTT ASSAY

• RADIOISOTOPING METHOD

1. ELISA1. ELISAAdd 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 ASSAY2. 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 METHOD3. 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 RESISTANCELACTOFERRIN 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

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

Contd…

PURIFIED MILK PROTEINS & THEIR PURIFIED MILK PROTEINS & THEIR EFFECT ON HIV-1 REPLICATIONEFFECT 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

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…

LACTOFERRIN INHIBITION OF CXCR4 & LACTOFERRIN INHIBITION OF CXCR4 & CCR5-using virusesCCR5-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

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…

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

Contd…

Antiviral properties of Antiviral properties of other milk proteinsother milk proteins

Lactadherin

Glycoprotein

Immunoglobulin (Ig)

• Lactadherin :

Viral receptor bindingsialic acid plays important role in its

antiviral action Human lactadherin protected breast-fed

infants against symptomatic rotavirus infection

• Glycoprotein : High-molecular weight fraction from

bovine milk was effective against human rotavirus in

vitro

• Milk immunoglobulin : Hyperimmunised with human rotavirus

during pregnency of cows100 times- Human milk10 times – Commercial Ig

Antiviral peptides Antiviral peptides derived from milk derived from milk

proteinsproteins

Lactoferricin

GMP

Mucin complex

Virus Peptide Comments/proposed mode of action

HSV-1 & 2 LFcin An in vitro effect;

weaker than that of LF: produces a synergistic effect with acyclovir

HCMV, FCV, Adenovirus

LFcin An iv vitro effect; weaker than that of LF

Antiviral effect of Antiviral effect of peptidespeptides

Virus

(Enveloped)

Peptide Comments

Epstein–Barr viruses

GMP Prevents morphological changes in peripheral blood lymphocytes

Virus(Non-Enveloped)

Peptide Comments

Rotavirus Mucin complex;human milk

Inhibits rotavirus replication in vitro & prevents gastroenteritis in vivo

Enhancement of Antiviral Enhancement of Antiviral activity on Chemical activity on Chemical

modificationmodification• 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 chargesAmination to increase positive charges

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…

• 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…

• 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 properties of LF but

effective against influenza virus

Contd…

ConclusionConclusion

• 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

• 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

Contd…