Download 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
• 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)
• 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 proteins
Virus
Protein Comments/proposed action
(Enveloped)
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)
Rotavirus
Protein
Comments/proposed
action
Lactedherin Human lactadherin
protects breast-fed
infants against infection
High MW
glycoprotein; Effective in vitro; mode
bovine milk of action is unclear
Ig
concentrate
An in vivo effect; 100
times higher than that
obtained with human
milk
PV
LF
Binds to target cells
Human
LF
influenza virus
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
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
• 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
 Mediated by glycoprotein gp-120 and gp-41
 CD4 -receptor and CCRS, CXCR4 – co
receptors
 Fusion of viral and cellular membranes
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
α-S2 Casein, bovine LFcin-B and bovine
k-casein
Hydrolysis with pepsin
Cation exchange chromatography
Obtained fragments characterized by HPLC and
ESI-MS
Contd…
• Βovine β-casein and bovine β
lactoglobulin are modified by maleic acid
(Ikura et al., 1984)
• Bovine as2-casein is modified with 3hydroxyphthalic anhydride
• The degree of modification checked with orthophtaldialdehyde
(Berkhout et al., 1997)
Methods to check antiviral
properties
• ELISA
• MTT ASSAY
• RADIOISOTOPING METHOD
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
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-(4,5dimethylethiazole-2-ly)-2,5-diphenyltetrazolium
bromide)
Blue product (formazine)
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
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
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
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
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
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
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
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
 Lactadherin
Glycoprotein
Immunoglobulin (Ig)
• Lactadherin :
Viral receptor binding
sialic 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 cows
100 times- Human milk
10 times – Commercial Ig
Antiviral peptides derived
from milk proteins
 Lactoferricin
 GMP
 Mucin complex
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
Virus
(Enveloped)
Peptide
Epstein–Barr GMP
viruses
Comments
Prevents
morphological changes
in peripheral blood
lymphocytes
Comments
Virus(NonEnveloped)
Peptide
Rotavirus
Mucin
Inhibits rotavirus
complex;h replication in vitro &
uman milk prevents
gastroenteritis in vivo
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
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
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
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
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