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
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 Interfere with virus into target cells Up regulation of killer cells Synergistic antiviral effect with cidofovir
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HCV LF Binds to viral envelop proteins E1 E2 bLF administrated orally may be the effective in combination in interferon
Alphavirus LF hLF inhibits interaction of virus with heparan sulphate receptors
Hantavirus LF Synergistic effect with ribavirin on viral replication bLF administered orally may be effective in combination with interferon
HPV LF Interferes with internalisation of virus into host cells
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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
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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
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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

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

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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
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
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Virus (Enveloped) Peptide Comments
EpsteinBarr viruses GMP Prevents morphological changes in peripheral blood lymphocytes
Virus(Non-Enveloped) Peptide Comments
Rotavirus Mucin complexhuman milk Inhibits rotavirus replication in vitro prevents gastroenteritis in vivo
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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