Antiviral Properties of Milk Proteins and Peptides

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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_at_gmail.com

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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

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

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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

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• 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

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

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Antiviral effect of proteins
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Antiviral mechanisms of LF
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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

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• 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

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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

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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

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PURIFICATION OF BOVINE MILK PROTEINS AND PEPTIDES

• a-S2 Casein, bovine LFcin-B and bovine
• k-casein
• Hydrolysis with pepsin
• Cation exchange chromatography
• Obtained fragments characterized by HPLC and
  ESI-MS

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Contd

• ?ovine ß-casein and bovine ß 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)

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Methods to check antiviral properties

• ELISA
• MTT ASSAY
• RADIOISOTOPING METHOD

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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

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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-dimethylethi
  azole-2-ly)-2,5-diphenyltetrazolium bromide)
• Blue product (formazine)

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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

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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

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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

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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

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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 µM conc
• Human LF- both native protein and recombinant
  protein moderately act as inhibitors at 3.1 µM
  conc

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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

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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

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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

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Antiviral properties of other milk proteins

• Lactadherin
• Glycoprotein
• Immunoglobulin (Ig)

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

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• 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

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Antiviral peptides derived from milk proteins

• Lactoferricin
• GMP
• Mucin complex

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Antiviral effect of peptides
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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

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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

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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

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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

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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

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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