Antiviral Properties of Milk Proteins and Peptides
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Transcript of Antiviral Properties of Milk Proteins and Peptides
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 LFMilk
bLF & hLF block HIV-1 adsorption to target cellsInhibits 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 cellso Up regulation of killer cellso Synergistic antiviral effect with cidofovir
HCV LF o Binds to viral envelop proteins E1 & E2o 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 replicationo 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 METHODCell 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 RESISTANCE1. 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
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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…