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Engineered

Plasmid DNA
Vaccine
For Staphylococcus aureus

Dr .M.Muruganandam

Engineered Plasmid DNA Vaccine


For Staphylococcus aureus

Dr.M.Muruganandam

Dedicated to My
Science Teacher Mr. Ramasamy

First Edition -2013


ISBN-978-9982-22-4185

Publisher and Author


Dr.M.Muruganandam,
Email- vaccine.m@gmail.com

Preface

Still Staph vaccine research is going


on. This book is help to move one step forward
towards staph vaccine Research. This is
prepared based on my lab work. I referred many
previous researchers work during this book
preparation, I thank to all of them. During my
lab work time many students assist me, I
sincerely thank to everyone and I thank to Mr.
Das

lab

Technician

for

kind

help

in

Haematological analysis. Finally I thank to Mr.


Stalin

Arockiaraj,

chairman,

SMCET

for

providing necessary lab facilities for this work.

M.Muruganandam

Contents
Biotherapy
Mutant Strain Vaccine
Mutant Strain plasmid
DNA Vaccine
Engineered plasmid
DNA Vaccine
Heat shock protein
Vaccine
Mixer Staph Vaccine
Recommendations
Bibliography

1. Biotherapy

In

2011,

Nobel

Prize

was

awarded in Medical Science for work in


Biotherapy.

It

is

otherwise

called

immunotherapy. It helps to boost up immune


system against pathogens. It is helping to control
various diseases. In Biotherapy, various methods
were employed to induce humoral and cell
mediated immunity.
Now Immuno stimulant, Vaccines,
probiotics, micronutrients, etc and various
natural products are used for Biotherapy. It is
designed to repair, stimulate and enhance the
functions of immune system. It boosts up the
activity of T cells, Natural killer cells,
Macrophages,

etc

and

humoral

mediated

immunity. This is an alternative to other


therapies.
Some beneficial bacteria present in
digestive track which is suggested that these are
constitute

non-pathogenic

members

of

indigenous intestinal micro flora. These are act


as probiotics against various pathogens .These
candidate are able to colonise the gut and act as
antagonistic against pathogens .These harmless
strains producing antibacterial substance may
reduce the use of antibiotics. These probiotics
bacteria act on complex carbohydrates and split
into simpler compounds for absorption. It is also
synthesizing vitamins B complex group, vitamin
K, etc. In the intestinal track some probiotic
bacteria increases appetite and health.

The probiotics bacteria stimulated the


immune
8

system

in

non-specific

way.

Researchers tested Lactobacillus as an adjuvant


to an oral vaccine to rotavirus in children and
they confirmed Lactobacillus preparations act as
immunomodulators .The probiotics bacteria
enhance immunity and alter the intestinal
metabolic activity.

The

vaccines

are

biological

preparations .It induce biological memory of


immune cells and it is also used to store
information regarding pathogens in immuno
memory cells. The antigenic portions of the
pathogens are used to prepare vaccines. Now
different type of vaccines were discovered .The
important vaccines are Killed vaccine, Sub unit
vaccine,

Live

attenuated

vaccine,

Peptide

vaccine, DNA vaccine, etc. Now various


vaccination
important
9

methods
methods

are
are

employed,
injection,

the

topical

application,

oral

drops,

eye

drops,

bath

vaccination, etc. The main components of


vaccine

are

antigenic

part

of

pathogens,

preservatives,/stabilisers and adjuvant .The main


role of adjuvant is boost up the functions of
immune system. Nowadays various Biological
materials are used as Bio-adjuvant.

DNA vaccines are new generation


vaccines. In Europe, countries mostly use rDNA
based vaccines. In this vaccine, antigenic part of
DNA was identified and isolated from pathogens
and companied with known vector DNA, then it
will insert into harmless microbes. It will culture
and used as vaccine. It gives long-term
immunity

compared

to

.Nowadays

cocktail

DNA

other

vaccines

vaccines

were

introduced for more than one infectious disease.


10

In our lab trials shows that naked plasmid DNA


and their digested parts acted as good vaccine.

1. Restriction digestion of plasmid DNA


during vaccine preparation.
Nutrients are mainly divided into
micro and macro nutrients. The macronutrients
are carbohydrates, proteins and lipids. Animals
require more macro nutrients for their growth
and maintenances. The micro nutrients are
vitamins and minerals .Many micronutrients are
functions as antioxidants and immunostimulants,
11

for eg vitamins C&E. They reduce stress,


especially

vitamin

reduce

stress.

The

micronutrients require very lesser concentrations


in diet. But it is essential for most of the
physiological functions. It is help to boost up
growth and health. The lesser intake or lack of
micronutrient in diet produces disease condition
for eg hypovitaminosis. Sometimes the excess
levels in diet produce diseases foreg hyper
vitaminosis. So we should identify optimum
requirements level for reduce stress and boost up
immune system.

Immune system is stimulated by


various methods that is called Biotherapy. All
these methods help to improve the functions of
immune system and reduce the stress. So if
identify optimum level of one or more methods
12

are helpful to expose maximum potential of


functions of our immune system.
Naked Plasmid DNA vaccine
Plasmid

DNA

vaccine

were

prepared from Naked Bacterial Plasmid DNA.


The Plasmid DNA are self replicating, double
stranded circular DNA molecule present in
bacteria. It is an extra chromosomal DNA
molecule .It always carries one or more gens
responsible for useful characteristics displayed
by the host. They have their own origin of
replication and they replicate independently of
the origins on the host chromosome. The size of
the plasmid ranges from 1kb to 500kb.
Most of the Plasmid DNA exists as
double strand circular DNA ,with both the
strands intact

called as covalently closed

circular DNA .If only one strand is intact then


the molecules are described as open circular
13

DNA.

Plasmids

are

used

as

vectors

in

recombinant DNA technology.

2. Serum protein profile analysis.


The extraction of plasmid DNA
protocol describes growth of bacterial cell
culture, harvesting and lysis of bacteria and
isolation of plasmid DNA .Various methods are
available

for isolation

of

plasmid

DNA,

However the convenient methods is alkaline


lysis method.
14

3. Molecular Weight determination of antibody


raised against Staphylococcus aureus.
In our lab trials, plasmid DNA was
isolated from pathogenic bacteria by alkaline
lysis method and it was purified. It is then
dissolved

in

double

distilled

water

and

administrated an intramuscular injection or


15

provided oral drops to albino rats. This plasmid


DNA integrates into the Chromosomal DNA and
produce antigenic proteins and it may induce
long term immunity. After two to three weeks
of vaccination, killed pathogens was injected
into albino rat. After one weeks of injection,
blood samples were collected and analysed
which shows elucidated antibody responses and
cellular responses. So it is concluded that this
plasmid DNA can act as immunogen.

In our lab works, plasmid DNA


Vaccine were prepared and tested in various
bacterial

pathogens

such

as

Aeromonas

hydrophila, Salmonella typhi E. coli and


Staphylococcus aureus.

In these trials, first

maximum immune response was observed in


killed vaccine and second maximum immune
16

responses were observed in plasmid DNA


Vaccine and protein vaccine.

4. Modified method of Counter current Immuno


electrophoresis for antibody analysis during
various vaccine treatments.
However compared to killed and
protein vaccines, plasmid DNA vaccine gives
long term immunity. It can be stored at room
temperature and transport is also easy. So it is
recommended to DNA vaccine preparation for
17

other bacterial infections. Furthermore in our


lab trails we have studied about mutant strain
plasmid DNA vaccine, digested plasmid DNA
vaccines, cocktail plasmid DNA vaccine from
various bacterial pathogens were prepared and
tested.

5. Modified method of Rocket Immuno


electrophorosisis for antibody analysis during
various vaccine treatments.
All these works, the naked plasmid
DNA produce good results. In our current lab
18

works proves that plasmid DNA, various protein


vaccines and killed pathogens were combined
and mixer vaccine was produced, which gives
good results because killed vaccine and protein
vaccines induce immediate immune responses,
and the

DNA vaccine produce long term

immunity. So the mixer vaccines produce good


results.

6. Antibody Titre (96 Well) for different


vaccine treatments.
Staphylococcus aureus (staph) is a
bacterial pathogen, which mainly affect the
19

Immuno suppressed or Immuno compromised


patients that means, it affect patients who have
already weak immune system. It produces
various diseases in humans.
Staph causes superficial skin lesions such
as boils, styes and furunculosis; more serious
infection

such

as

Pneumonia,

mastitis,

meningitis and urinary tract infections and deep


seated infections. It causes food poisoning by
releasing enterotoxins into food and toxic shock
syndrome by release of super antigens into the
blood stream.
Staphylococcus aureus is most offenly
spread to others by contaminated hands and skin.
Mucous membranes is usually an effective
barrier against infection, However if these
barriers are breached (e.g. skin damage due to
trauma or mucosal damage due to viral
infection.) S. aureus may gain access founder
20

lying tissues or the blood stream and cause


infection.
Traditionally,

Methicillin

Resistant

Staphylococcus aureus (MRSA) infections have


been associated with hospitalization or other
health care associated risk factors. In recent
years physicians and other health care providers
have observed an increasing number of people
with MRSA infections who lack traditional
health care associated risk factors. These
people appear to have community illnesses and
deaths caused by MRSA infection, mainly cause
death at a level higher than HIV infection.
MRSA

has

become

more

prevent as nosocomial pathogens causing severe


infections. MRSA become resistant to beta
lactams

antibiotics

especially

Methicillin,

Cefoxitin and Gentamicin. The mean incidence


of MRSA has drastically increased and become
21

worldwide

problem.

The

resistance

to

Methicillin is due to resistant genes. Recently


MRSA strains become resistant to several
different antibiotics such as penicillin, oxacillin
and erythromycin.
Coins have the possibility to be one
of the potential sites of MRSA has the
opportunity to transfer from nasal to fingers and
nails followed by the transmission of MRSA
through the hand contact with things such as
coins and others .
Still there is no good vaccine for
human use. Vaccine development research
programmes are going on various parts of the
world. Plasmid DNA vaccines were proposed
for Bacterial food borne pathogens, such as
Aeromonas

hydrophila,

Salmonella typhi, etc.


22

Escherichia

coli,

In our lab, Plasmid DNA vaccines were


prepared from naked plasmid DNA. For staph
infections many vaccines were proposed such as
mutant strain vaccine, Heat stress protein
vaccine, plasmid DNA vaccine, Engineered
plasmid DNA vaccine, combined vaccines etc., .
Combined vaccine is a combination of various
staph proteins and DNA. It produced good
results, because proteins are acts as bio adjutants.
In this book, five chapters were
discussed regarding various experiments on
staph

vaccine

development.

These

works

abstracts are as follows.


The

second

Chapter

describes

about mutant strain staph vaccine preparation. In


this work, pathogens was isolated from patients
sample in a hospital and cultured in our
laboratory.
23

After

that

mutant

strain

was

developed by using U.V treatments then


prepares killed vaccines form these mutant
strains. These vaccines were tested in albino
rats.
The maximum immune response was
observed in six minutes U.V treated mutant
strain. It is best for produce killed vaccine
against Staphylococcus aureus infections.
In the third chapter, mutant strains
plasmid DNA vaccine developments were
discussed. In this work, staphylococcus aureus
mutant strains were produced by using U.V.
radiation and plasmid DNA was isolated from
all these mutant strains. These DNA were used
as Vaccine.
The constant amount of vitamin C
was provided as adjuvant. After 15 days,
pathogens were injected to all the treatments,
including control treatments. After that blood
24

samples were taken for analysis. The maximum


immune response was observed in six minute
treated U.V. strains plasmid DNA. So it is
concluded that, this plasmid DNA is suitable for
S. aureus vaccine preparation.
In the fourth Chapter, Engineered plasmid
DNA vaccines preparation for Staphylococcus
aureus was discussed. Plasmid DNA has wide
variety of applications in vaccine research. Here
it is modified and used as vaccines. First plasmid
DNA was isolated form Staphylococcus aureus
and engineered by various restrictions enzymes.
In this work, two experiments were conducted.
In the first experiment, plasmid DNA
was isolated and digested individually by five
restriction enzymes such as ECOR-I, Hind-III,
Pst-I, Bam-I and Hae-III, then digested plasmid
DNA was used as vaccines. In the second
experiment, isolated plasmid DNA was double
25

digested by using these enzymes and used as


vaccines. Albino rats were used as test animals
in all the experiments. In the first experiment,
maximum immune response was observed in
Pst-I and Hae-III digested treatment. In the
second experiment, maximum immune response
was observed in EcoR-I+Hind-III and Hind-III +
Bam H-I digested treatments. So it is concluded
that, compared to two experiments, double
digested treatments are highly suitable for
plasmid

DNA

vaccine

preparation

of

Staphylococcus aureus.
Our studies showed that, plasmid
DNA vaccine is one of the best vaccines. So in
this attempt, try to develop a common plasmid
DNA

vaccine

for

staphylococcus

aureus,

Salmonella typhi and Escherichia coli. In this


work, four treatments were tested and one
control treatment was also tested. In the first
26

treatment, plasmid DNA was collected from


Salmonella

typhi,

Escherichia

coli

and

Staphylococcus aureus. These plasmids DNA


was

mixed

well

and

delivers

through

intramuscular injection. In the second treatment,


all the plasmid DNA were digested by Bam H-I
enzyme and mixed well then these digested
plasmid DNA was used as vaccine.
In the third treatment, all the plasmid
were digested by Pst-I enzyme and these also
mixed well and used as vaccine. In the fourth
treatment, all plasmids are double digested by
Bam H I and Pst-I enzymes and used as
vaccines.
The maximum immune response
was observed in double digested treatment
compared to other treatments. So it is concluded
that it is best for development a common vaccine
for these bacterial diseases.
27

In the fifth chapter, Heat stress


protein vaccines developments were discussed.
In this study, the pathogens were exposed two
different temperatures such as 50 0 C and 550 C
with different time intervals. All the treatments
albino rats were used as test animals. The Heat
stress proteins were isolated and used as
vaccines.
The maximum immune response
was observed in 10 minutes exposed pathogens
heat

stress

proteins

in

both

temperature

treatments. However compared to these two


treatments, 550 C treatment with 10 minutes time
exposed organisms produced heat stress proteins
induce maximum immune response, so it is
concluded that these Head stress proteins are
recommended to further staph protein vaccine
development process.
28

Optimization of a heat stress protein


is another study. In this study Staph-pathogens
were exposed to 45oC temperature for 10
minute.

It produces heat stress protein then

these were isolated and used as vaccine. Graded


levels of these protein vaccines were tested in
albino rat.

The maximum immune responses

were observed in 25gm protein vaccine


treatment.

So

this

concentration

is

recommended to further vaccine development


process.
Staphylococcus aureus produces
toxins.

In this next experiment, the toxins

proteins were isolated and inactivated then used


as vaccines. Different quantity of these toxin
vaccines were tested into the albino rat. The
maximum immune response was observed in 15
gm toxoid vaccine treatments. After this level
treatments the immune response were slowly
29

decreased. So this is recommended for further


vaccine development process.
In the sixth chapter, Mixer vaccine was
discussed. In this study, various vaccines were
prepared, such as killed vaccines, Heat stress
protein vaccine, Toxoid vaccine, plasmid DNA
vaccine and the mixer of all these vaccines were
tested for their immune responses. Albino rats
were

used

as

experimental

animal.

The

maximum immune responses were observed in


killed vaccine.
The second

maximum immune

response was observed in mixer vaccine. The


remaining vaccine treatments have low immune
responses compared to these two vaccine
treatments. The mixer vaccine is good for their
longer immunity aspects compared to other
vaccines. So it is recommended for further
vaccine preparation process.
30

Finally, based on all these works,


recommendations were pointed out at the end of
this book and all the supporting data for findings
were also provided.

31

2. Mutant Strain Vaccine

Food borne diseases are the major


problem in the worldwide. Around 250 different
foods borne diseases have been described.
Bacteria are the causative agents of two thirds of
food borne disease out breaks. Staphylococcus
aureus is a leading cause of gastroenteritis
resulting from consumption of contaminated
food. The symptoms of food borne diseases are
very widely, depending on the etiological agents.
The common symptoms of food borne disease
are Diarrhea and Vomiting.
The S.aureus causes disease
when they get inside the host, because they cant
penetrate the skin. So they are associated with
wounds, cuts needle pricks, etc., once inside the
host, they stick to host tissues and produce
32

toxins. In this study, first try to produce mutant


strain by using U.V. radiation and select best
mutant for prepare killed vaccine against
S.aureus infection.
This pathogen was collected from the
patients sample in the hospital and then
cultured. The pathogen was confirmed by
regular microbiological and Biochemical tests.
After that, the pathogen was subjected to
mutations under U.V. treatment at various time
intervals such as 0, 2, 4, 6 and 8 minutes and the
colonies were isolated and cultured separately.
All these cells are purified and formalin killed
by using 0.5% formalin at 40c during overnight
treatment than used as whole cell killed vaccine.
Samples 1-5 in the figures shows
pathogen exposure time such as 0,2,4,6 and 8
minutes respectively.
33

5
4
3
2
1
0

Fig-1: Mutant Strains Vaccines influence on


RBC Counts (millions) in Albino rat.
All the mutant strains were serially diluted
up to 10 5 dilution and inject to five sets of
animals (albino rats) including control. Vitamin
C 300 mg was given as adjuvant for each
individual. After 15 days formalin treated cells
were injected to all the animals including control
treatments. The immune responses of all the
strains

were

analyzed

on

the

basis

immunological and haematological aspects.


34

of

4000
3000
2000
1000
0

Fig-2: Mutant Strains Vaccines influence on


WBC Counts (Cells/Cu mm) in Albino rats.
Staphylococci can survive dry surfaces
with in the increasing of transmission. S. aureus
expresses many potential virulence factors such
as surface proteins, and toxins which damage
host tissues; it is inherent and acquired
resistance to antimicrobial agents.
The aim of the present work is to select
best mutant strain for prepared killed vaccine. So
if induce mutation through U.V. treatment leads
to increase efficacy at certain limit. Based on
35

immune responses, best mutant strain was


selected for preparation of best vaccine. In the
present experiment, the WBC count is increased,
as the U.V. treatment time increases, it attain
peak value in 6 minutes mutant strain.
80
60
40
20
0

Lym
pol

Fig-3: Mutant Strains Vaccines influence on


WBC Differential Counts (%) in albino rats.
The

maximum

lymphocyte

and

antibody levels were observed in 6 minutes


treatment. Based on these results, it is concluded
36

that 6 min. U.V. mutation strain is best for


Killed vaccine preparations.
20
15
10
5
0

Fig-4: M.S Vaccines influence on Hb (gm %).

30
20
10
0

Fig-5: Mutant Strains Vaccines influence on


Packed Cell Volume (%).

37

8
6
4
2
0

Fig-6: Mutant Strains Vaccines influence on


Antibody levels.

38

3. Mutant strains plasmid DNA Vaccine


Staphylococcus

aureus

is

an

antibiotic resistant pathogen. So there is an


alternative way to control its infection is vaccine
development for immuno therapy. The major
drawback of conventional approaches is that
large quantities of organism usually required to
isolate sufficient antigens for use in vaccine. The
novel approach in the development of new
vaccine is the use of proteins polysaccharides or
peptide fragments corresponding to specific
antigenic determinants of the infecting agents.
One of the novel and powerful method for
vaccine

development

is

DNA

vaccines

development which has several advantages. In


this work, efficacy of Mutant strain plasmid
DNA was tested.
39

Here

pathogen

was

collected from patients sample in a hospital and


it

was

cultured

in

our

laboratory.

For

confirmation, all routine microbiological and


Biochemical tests were done. During the
experiment, five sets of spread plate culture were
prepared and four sets were exposed to U.V.
radiation with different time intervals such as 0,
2, 4, 6 and 8 minutes.
First set was maintained as a control.
After 24 hours, the mutant strains were
observed. Then it was isolated and put into
subculture. The mutant strains plasmid DNA
was isolated by alkaline lysis method. After
isolation, it was dissolved in double distilled
water and this was used as vaccine. In this
experiment, albino rats were used as test animal.
Fifteen albino rats were purchased and put
in five groups with separate cages and
maintained the laboratory at one week for
40

acclamentation, commercial feed were provided


into all groups . First mutant strains plasmid
DNA

was

injected

(200l)

through

intramuscular injection to four sets of animals,


physiological saline was injected to control
group.
After one week same injection was
provided as booster dose. After two weeks,
formalin killed pathogens ( 10 5 ) was injected to
all the group of animals. After 24 hrs and 120
hrs

blood

samples

were

collected

for

immunological and Haematological analysis.


Antibody titre was done only in 120 hrs samples,
with the help of 96 well titre plates.
Samples

1-5

in

figures

shows

pathogen exposure time 0,2,4,6 and 8 minutes


respectively

41

6
4
2
0

Sample-I
Sample-II

`
Fig-7: Mut. Stra. Plasmid DNA Vaccines
influence on RBC count (millions) of Albino
rats.
10000
5

5000

sample-I
sample-II

Fig-8:Mut.Str.plasmid DNA Vaccines influence


on WBC count (cells/cu mm) of Albino rats.

42

80
60
40
20
0

sample-I
sample-II

Fig-9: Mutant Strains plasmid DNA Vaccines


influence on Lymphocytes (%) of Albino rats.

60

40
20

sample-I
sampe-II

Fig-10: Mutant Strains plasmid DNA Vaccines


influence on polymorphs (%) of Albino rats.

43

30
20

10

sample-I

sample-II

Fig-11: Mutant Strains plasmid DNA Vaccines


influence on Eosinophils (%) counts of Albino
rats.
This experiment is aimed to develop a
DNA

vaccine

Staphylococcus
manufactured

for

the

aureus.
more

easily

infection
They
than

can

of
be

vaccines

composed of a whole cell vaccine, sub cellular


fraction or recombination protein. The DNA is
very stable and resistant temperature extremes,
consequently

the

storage,

transport

and

distribution of DNA based vaccines are more


practical and less expensive.
44

40
30
20
10
0

sampe-I
sampe-II

Fig-12: Mutant Strains plasmid DNA Vaccines


influence on packed cell volume (%) of Albino
rats.
In this work, the mutation was
induced in S. aureus by using U.V. radiation
with different time exposure hen mutant strains
plasmid DNA was isolated and used as vaccines.
After that, primary immune response was
studied. In our previous lab works, the plasmid
DNA induces maximum humoral

responses

compared to other vaccines and it also gives


equal level of immunity of protein vaccines.
45

15

10
5

sample-I
sample-II

Fig13: Mutant Strains plasmid DNA Vaccines


influence on Haemoglobin (gm %) of Albino
rats.
According to the results, it is clear that
the treatment of 6 minutes induce higher WBC
counts of nearly 8,100 cells/cu mm. In the
second sample, the same trend was observed. In
the case of antibody production six minute
Mutant strain also produced maximum level
compared to other, treatments. The maximum
antibody production and WBC Count was
observed in plasmid DNA vaccine treatments.
46

8
6
4
2
0

Fig-14: Mutant Strains plasmid DNA Vaccines


influence on Antibody levels of Albino rats.
Our previous lab work proved that
plasmid DNA is good for induce immune
response during long term conditions. So in this
work, it is concluded that, 6 minutes U.V.
treated mutant pathogens plasmid DNA is act as
best vaccine.

47

4. Engineered plasmid DNA vaccine

Due to antibiotic resistant problem,


pathogen control and prevention is very difficult.
But there is another way to control the infection
by development of therapeutic vaccines for
immunotherapy. DNA vaccine development is
one of the novel powerful methods. It has
several advantages. In addition, DNA vaccines
are a greater interest among researchers around
the world.
In the present study the plasmid
DNA was isolated from S. aureus and digested
by single restriction enzymes and also double
digested, all these treatments are tested in albino
rat. The best treatments are recommended for
new

DNA

vaccine

Staphylococcus aureus.
48

development

against

The bacterial pathogen Staphylococcus


aureus was collected from the patients samples
from local hospital and confirm through regular
biochemical and microbial tests. The plasmid
DNA was isolated by alkaline lysis method. The
plasmid

DNA was

digested

by different

restriction enzymes and used as vaccine.


Samples 1-6 in the figures shows
various enzyme digested vaccines such as
1.control, 2.EcoR-I enzyme digested vaccine,
3.Hind-III enzyme digested vaccine 4.Pst-I
enzyme digested vaccine 5.BamH-I digested
vaccine, 6.Hae-III digested vaccine.

First treatment is undigested plasmid


DNA another five treatments were single
digested plasmid DNA which are digested by
different enzymes (Table.1). Then remaining
treatment
49

were

double

digested

using

combination of two enzymes. The digested


plasmids DNA were provided by intramuscular

6000
5000
4000
3000
2000
1000
0

Fig 15 Various enzymes digested DNA


vaccine influences on WBC counts (cells / cu
mm) of Albino rat.
injection. After one week, same dose was given
as booster dose. Then after two weeks, blood
samples were collected for analysis.

50

1
0
5

Fig 16 Various enzymes digested DNA


vaccine influences on RBC counts (millions) of
Albino rat.

60
40
20
0

1
2

3
4

Fig 17 Various enzymes digested DNA


vaccine influences on polymorph (%) of Albino
rat.

51

Table 1: Various restriction enzymes and their


restriction sites
S.No Name

Source

Recognition
Sequence

1.

EcoR-1

E. coli RY 13

AATTC 3
2.

Bsh I Bacillus sphaericus


(Hae

5GG CC 3

III)
3.

Pst-I

An E. coli strain that 5

CTGCAG

carries the cloned 3


Pst I gene from
Providencia stuartii
4.

5.

Bam H Bacillus

GATCC 3

Hind
III

anyloliquefacies H
Haemophilus
influence Rd

5....

AGCTT...3
3....TTGA
A....5

52

Staphylococcus aurous is a major


cause of hospital and community acquired
infection. It causes serious and fatal diseases.
Stills there are no proper vaccine for these
infections. The research is going on. The whole
cell killed vaccine is commonly used in many
diseases. The next step is preparation of mutant
strain whole cell killed vaccine. Our lab work
stated that the 6 minutes UV treated mutant
strain is best for preparing killed vaccine. If
increase the UV treatment more than 6 minutes
most of the potential of virulence factors may
decreased. Our lab studies proved that the
plasmid DNA alone acts as good vaccine for
Aeromonas hydrophila infection. It induces
maximum immune response.
The

potential

of

virulence

was

increased in mutant strain plasmid DNA


vaccine. The maximum antibody production and
53

WBC counts was observed in 6 minutes UV


treated mutant strains plasmid DNA treatment,
which is also act as best vaccine compared to
other plasmid DNA vaccines in Staphylococcus
aureus .

80
60
40
20
0

Fig 18 Various enzymes digested DNA


vaccine influences on Lymphocytes (%) of
Albino rat.
Next level trail is the enzyme
digested plasmid DNA role in immune response.
The single and double digested plasmid DNA
54

used as vaccine in the case of Salmonella typhi.


The maximum response was observed in double
digested plasmid DNA treatments. So that, here
various digested plasmid DNA were used as test
vaccines. In the present work, two experimental
trails were conducted. In the first experiment
five restriction enzymes were individually used.

First plasmid was isolated and


digested by these enzymes. The digested
plasmid DNA was used as vaccine. The
maximum immune response was observed in Pst
I digested treatment and Hae III digested
treatments. The second maximum immune
response was observed in BamH I digested
other treatments.
In the second experiment nine
treatments and one control treatments were
tested.
55

All the treatments contain double

digested plasmid DNA with various enzyme


combinations.

5
4
3
2
1
0

Fig 19 Various enzymes digested DNA


vaccine influences on Eosinophils (%) counts of
Albino rat.
The maximum immune response was
observed in EcoR I + Hind III digested
treatment. The second maximum immune
response was observed in Hind III + BamH I
digested treatments. So compare to single
digestion, the double digestion plasmid DNA

56

treatments are suitable for new DNA vaccine


preparation.
10
6

1
2

5
0

3
4

Fig 20 Various enzymes digested DNA


vaccine influences on Haemoglobin (gm %) rat

30
20
10
0

1
2

3
4

Fig 21 Various enzymes digested DNA


vaccine influences on packed cell volume (%) of
Albino rats.

57

10
8
6
4
2
0

Fig 22 Various enzymes digested DNA


vaccine influences on Antibody levels of Albino
rats.
Samples 1-10 in figures shows double
digested

vaccines

by

various

Restriction

Enzymes Such as 1. Control, 2 EcoR I + Pst


I, 3.EcoR I + BamH I, 4. EcoR I + HaeIII,
5. Hind III + Pst I, 6. Hind III + BamH I, 9.
Bam H I + HaeIII, 10.Eco R I + Hind III.

58

10

6000

1
2

4000
2000

0
8

4
7

5
6

Fig 23 Various enzymes digested DNA


vaccine influences on WBC counts (cells / cu
mm) of Albino rats.
80
10 60
40
9
20
0
8

1
2
3
4

5
6

Fig 24 Various enzymes digested DNA


vaccine influences on Lymphocytes (%) of
Albino rats.

59

60
10
40
9
20
0
8

1
2
3
4

5
6

Fig 25 Various enzymes digested DNA


vaccine influences on polymorph (%) of Albino
rats.

10
9

5
4
3
2
1
0

1
2
3

4
7

5
6

Fig26 Various enzymes digested DNA


vaccine influences on Eosinophils (%) of Albino
rats.

60

4
10 3
2
9
1
0
8

1
2
3
4

5
6

Fig 27 Various enzymes digested DNA


vaccine influences on RBC counts (millions) of
Albino rat.

10

10

0
8

4
7

5
6

Fig 28 Various enzymes digested DNA


vaccine influences on Haemoglobin (gm %) of
Albino rats.

61

10

30

1
2

20
10

0
8

4
7

5
6

Fig 29 Various enzymes digested DNA


vaccine influences on packed cell volume (%) of
Albino rats.

10
9

10
8
6
4
2
0

1
2
3

4
7

5
6

Fig 30 Various enzymes digested DNA


vaccine influences on Antibody levels of Albino
rats.
62

Naked Plasmid DNA Mixer Vaccine


Many bacterial pathogens act as
food borne pathogenic organisms. In these study
pathogens such as staphylococcus aureus,
salmonella typhi and Escherichia coli were
collected from patients samples at local hospital,
done the entire biochemical and biological test
for confirmation. Then prepared three separates
broth and individually inoculated. After 24
hours, plasmid DNA was separately isolated by
alkaline lysis method. In the first treatment, all
the plasmid DNA were mixed and used as
vaccine. In the second treatment, all the plasmid
DNA were digested by Bam-I restriction
enzymes. Then it was used as vaccine.
In the third treatment, plasmid DNA were
isolated individually and digested by Pst-I
restriction enzyme and these are mixed used as
63

vaccine. In the fourth treatment, plasmid DNA


was isolated from all the pathogen and double
digested by using Bam-I + Pst-I enzymes and
were mixed well and used as vaccine.

One

control treatment was used. Albino rats were


used as test animals in all the treatment.
All the vaccines were delivered
through

intramuscular

injections.

After

delivered the test vaccines, two weeks later,


blood samples were collected for analysis.
In this attempt maximum immune
response was observed in double digested
plasmid DNA treatment compared to other
treatments.
Sample 1-5 in figures shows various
plasmid DNA mixer vaccines such as 1.control
2.whole plasmid DNA mixer vaccine.3.BamH-I
digested mixer vaccine 4.Pst-I digested mixer
64

vaccine 5.Double digested Plasmid DNA mixer


vaccine.

8000
6000
4000
2000
0

Figure 31 Various enzymes digested Plasmid


DNA mixer vaccine influences on WBC counts
(cells / cu mm) of Albino rats.
100
80
60
40
20
0

65

2
poly
lym

Figure 32 Various enzymes digested DNA


vaccine influences on WBC differential counts
(%) of Albino rats.
The second maximum immune response
was observed in undigested plasmid DNA
treatment.

The Pst-I digested plasmid DNA

treatment gives better results than Bam-I


digested treatment.
6

4
2

Figure 33 Various enzymes digested Plasmid


DNA mixer vaccine influences on RBC counts
(millions) of Albino rats.

66

The RBC count was more or less same


level in undigested plasmids DNA treatment and
double digested plasmid DNA treatments. The
Pst-I and BamH-I digested plasmid DNA have
similar RBC counts. Higher level of polymorph
observed in digested plasmid DNA treatments,
compare to other treatments.
15

10
5

Figure 34 Various enzymes digested Plasmid


DNA mixer vaccine influences on Haemoglobin
(gm %) of Albino rats.
The antibody levels were higher in
single and double digested treatments.

The

control treatment has lesser antibody levels


67

compared to other treatments. In this attempt,


maximum immune response was observed in
double digested treatment.

So it is highly

suitable for cocktail plasmid DNA vaccine


preparation
10
8
6
4
2
0

Figure 35 Various enzymes digested Plasmid


DNA mixer vaccine influences on Antibody
levels of Albino rats.
.

68

5. Heat Stress Protein Vaccine

New types of vaccine for staph infection


were reported, such as mutant strain vaccine,
Heat stress protein vaccine, plasmid DNA
vaccine, engineered plasmid DNA vaccine, etc.
In this attempt, pathogens are exposed in two
different

temperatures

with

various

time

intervals then heat stress proteins were isolated


and used as vaccine. Our main objective is
identification of suitable heat stress protein for
vaccine preparation process for staphylococcus
aureus.
Staphylococcus aureus was collected from
patients in hospital and confirmed by regular
microbiological and biochemical tests. After that
it was introduced into nutrient medium for 24
hrs, the pathogens were isolated and exposed to
two different temperatures (50 and 55o C) with
69

various time duration such as 0, 10, 20, 30 and


40 minutes. During this time they releases heat
stress proteins for their stress resistant process
which were isolated by Acetone precipitation
method and these proteins were used as
vaccines. These proteins were mixed with 0.5ml
of

physiological

saline

and

injected

as

intramuscular injection to every albino rat, then


blood samples were collected and analysed by
using routine haematological tests for their
cellular immune responses.
In the present study, two experiments
were conducted. In the first experiment,
pathogens were exposed to 50oc, and then heat
stress proteins were isolated and used as vaccine.
The maximum total WBC count, polymorphic
cells, haemoglobin, packed cell volume and
RBC Counts were observed in 10 minutes
70

exposed pathogens heat stress protein treatment,


compared to all other treatments.
The total WBC count was increased
in 10 minutes treatment and slowly decreased in
remaining higher dose treatments. So compared
to all these treatments, 10 minutes exposed
pathogen producing cells induce maximum
responses .In the second treatments, Pathogens
were exposes to 50oc and their heat stress
proteins were isolated and used as vaccines.
Here maximum total WBC counts (7600 cells/cu
mm). There observed in 10 minutes treatments.
After 10 minutes treatments the total WBC
count was slowly decreased.

Samples 1-5 in figures shows pathogen


exposure time 0, 10, 20, 30, and 40 minutes
respectively.
71

8000

6000
4000

2000

Temp-50

Temp-55

Fig: 36-Hsp vaccine influence on Total WBC


counts (cells/cu mm) of albino rats.
.
4
3
2
1
0

Temp-50
Temp-55

Fig: 37-Hsp vaccine influence on Total RBC


counts (millions) of albino rat.
72

The maximum platelets count was also


observed in 10 minutes treatments. The total
RBC count, differential WBC count and packed
cell volume levels are more or less not much
changed. So 10 minutes treatment produced heat
stress proteins industries maximum immune
responses.

Compared

to

both

temperature

treatments, 55oC produce highest immune


response.
60

55

50

45

Temp-50
Temp-55

Fig:38-Hsp vaccine influence on polymorph


counts (%) of albino rat.

73

Generally,

microorganisms,

release

proteins during stress conditions for their


survival.

If

rising

the

environmental

temperature, microbes releases some heat stress


proteins. It is isolated and used as protein
vaccine against the infection. For staphylococcus
aureus still vaccines development research for
human is going on. Many researchers proposed
various types vaccines.
50
40
30
20
10
0

Temp-50
Temp-55

Fig:39-Hsp vaccine influence on Lymphocyte


counts(%) of albino rats.

74

2
1.5
1
0.5
0

Temp-50
Temp-55

Fig: 40-Hsp vaccine influence on Eosinophils


counts (%) of albino rat

10
8
6
4
2
0

Temp-50
Temp-55

Fig41. Hsp vaccine influence on Haemoglobin


(gm %) of albino rats.
75

30

20
10

Temp-50
Temp-55

Fig:42-Hsp vaccine influence on packed cell


volume (%) of albino rats.
4
3
2
1
0

Temp-50
Temp-55

Fig:43-Hsp vaccine influence on platelets


counts of albino rats.

76

In this study, pathogens were


exposed to two different temperature treatments
with four exposure timings. The maximum
immune responses were observed in 10 minutes
treatments in both temperature treatments.
However compared to 50 0 C treatment, 550 C
temperature exposure treatment produce more
immune responses, especially 10 minutes time
exposure

at

55 0 C

temperature

treatment

producing Heat stress proteins induce highest


immune responses compared to other treatments.

So it is concluded that these heat stress


proteins

are

highly

suitable

for

vaccine

preparations and also useful to new bioadjuvant preparation for staph DNA vaccines.

77

Optimization work-I

The next step work is optimization


process. Here graded level of Hsp vaccine was
provided and immune responses were studied in
albino rats. Pathogen was collected from
patients samples in hospital tests were carried
out for confirmation, and then introduced in
Nutrient medium.
Toxic proteins were isolated from
broth

culture medium by using

Acetone

precipitation method and inactivated by 0.5%


formalin at 40 C temperatures with overnight
incubation. Graded level of these toxoid proteins
were provided through intramuscular injection to
different group of albino rats .At the end of the
experiment, blood samples were collected and
analysed for their cellular immune responses.
78

Samples 1-5 in figures show Graded


level of Toxoid protein vaccine such as 1.
Control, 2. 15gm Toxoid Protein vaccine ,3.
30gm Toxoid Protein vaccine, 4. 45 gm
Toxoid Protein vaccine ,and 5. 60gmToxoid
Protein vaccine.
3.1
3
2.9
2.8

Fig-44.Graded level of Toxoid protein vaccine


influence on RBC counts (millions) in Albino
rats.
Staph toxin were isolated,
inactivated and provided

79

various quantities to

albino rats for tested their immune responses.


8000
6000
4000
2000
0

Fig-45.Graded level of Toxoid protein vaccine


influence on WBC counts (cells/cu mm) in
Albino rats
60
40
20
0

LYM
POLY
EOIS

Fig-46.Graded level of Toxoid protein vaccine


influence on WBC differential counts (%) in
Albino rats.
80

15

10
5

Fig-47.Graded level of Toxoid protein vaccine


influence on Haemoglobin (gm %) contents in
Albino rats.
If increases the level of toxoid
proteins, the immune responses was slowly
decreased. The maximum total WBC counts,
lymphocyte counts, packed cell volume and
Haemoglobin levels were observed in 15gm
toxoid protein vaccine treatments. After that,
higher concentration of toxoid protein vaccine
leads to lesser immune responses.

81

29

28
27

26

Fig-48.Graded level of Toxoid protein vaccine


influence on packed cell volume (%) in Albino
rats.
The total RBC count has more or
less equal level in all the treatments. The lesser
amount platelets counts were observed in 15gm
toxoid vaccine treatments.

The immune

response of 60gm toxoid vaccine treatment was


similar to control treatment. Protein vaccines
usually induce immune responses quickly. In
staph vaccine Heat stress protein vaccine (HSP)
was reported.
82

2.5
2
1.5
1
0.5
0

Fig-49.Graded level of Toxoid protein vaccine


influence on platelets counts of Albino rats.
. The HSP vaccine and Toxin
protein vaccine are also act as Boiadjuvents.
But there is lack of study in the optimization
process.
In this study, graded level of toxoid
protein vaccine was provided to albino rats
maximum immune response was observed in
15gm toxoid protein vaccine. If go to higher
doses leads to low immune response that is why
83

these concentrates act as saturation point for this


vaccine.

So it is concluded that this 15gm is

recommended

to

further

protein

vaccine

development process for staph infection.

Optimization-II
In this study graded level of staph Hsp
vaccine was provided to albino rats for
optimization study.

Here the pathogen was

collected from patient sample and conforms by


routine tests then they were introduced in
Nutrient medium, 24hours later, they were
collected and exposed to 450C temperature for
10 minutes. They produced heat shock protein
for their survival.
Sample 1-5 in the figures shows
Graded level of staph Hsp vaccine such as
1.Control 2. 12.5gm Heat Stress Protein
vaccine 3. 25gm Heat Stress Protein vaccine 4
84

37.5 gm Heat Stress Protein vaccine and 5.50


gm Heat Stress Protein vaccine.
4
3
2
1
0

Fig-50.Graded levels of Staph-Hsp vaccine


influence on Total RBC counts (millions) in
Albino rats.
These proteins were isolated by
Acetone precipitation method, and then these
were used as vaccine. Graded levels of these
proteins were mixed with 0.5ml of physiological
saline and injected as intramuscular injection to
different group of albino rats. After one week
blood samples were collected and analysed for
study the cellular immune responses.
85

6000

4000
2000

Fig- 51 .Graded levels of Staph-Hsp vaccine


influence on Total WBC counts (cells/cu mm)in
Albino rats.
60
40
20
0

1
2

POLY
LYM
EOIS

Fig- 52.Graded levels of Staph-Hsp vaccine


influence on WBC differentials counts(%) in
Albino rats.

86

15

10
5

Fig-53.Graded levels of Staph-Hsp vaccine


influence on Haemoglobin (gm %) content of
Albino rats.
40
30
20
10
0

Fig-54.Graded levels of Staph-Hsp vaccine


influence on packed cell volume (%) in Albino
rats.
87

2.5
2
1.5
1
0.5
0

Fig-55.Graded levels of Staph-Hsp vaccine


influence on platelets counts in Albino rats.
In this study, the maximum amount
of total WBC count, packed cell volume and
haemoglobin were observed in 25gm protein
treatment. So it is concluded that these level is
suitable for further process in staph protein
vaccine development.

88

6. Mixer staph vaccine

Staphylococcus aureus spread through


contaminated

food.

Many

vaccines

were

proposed for food borne diseases, such as


Genomic DNA vaccine for common food borne
diseases, DNA vaccine for E.coli , Engineered
DNA vaccines for Typhoid , cocktail plasmid
DNA vaccine for common food borne diseases.
For staph infection many vaccines also
proposed. Heat stress protein vaccine, Mutant
strain vaccine, plasmid DNA vaccine etc., but
there is no work for comparison for various
vaccines efficacy. So the aim of this work is
comparison of four different vaccines efficacy
and finally finds out which one is induce
maximum immune response in albino rat .In this
Study, Pathogens were collected from patients
sample in a hospital, Regular microbiological
89

and Biochemical tests were carried out for


confirmation. Then they were introduced in
nutrient medium, it was maintained in laboratory
.After that various vaccines were prepared and
tested for their efficacy. These vaccines are
killed vaccine, Heat stress protein vaccine (HSP)
or Heat shock protein vaccine, plasmid DNA
vaccine, Toxoid vaccine and mixer of all these
four vaccines. For killed vaccine preparation,
cells

were

isolated

from the broth

and

inactivated by 0.5% formalin at 40 C in


overnight incubation.
For

Heat

stress

protein

vaccine

preparation, pathogens were exposed to 50 0 C


temperature for 10 minutes then heat stress
proteins were isolated by acetone precipitation
method. During plasmid DNA preparation,
whole plasmid DNA was isolated by alkaline
lysis method and used as vaccine. For mixer
90

vaccine

preparation,

all

these

vaccines

preparations were equally mixed and used as


vaccine. All these vaccines were provided at
equal quantity by intramuscular injection to
different groups of albino rats. At the end of the
experiment, blood samples were collected and
analyzed for the cellular immune response.
Samples 1-6 in figures shows
various vaccines such as 1.Toxoid vaccine 2.
Hsp vaccine 3.Killed vaccine 4. Plasmid DNA
vaccine 5.Mixer vaccine and 6. Control.

4
3
2
1
0

1
2

3
4

Fig-56-Various staph vaccines influence on


total RBC counts (millions) in albino rats.
91

8000
6000
4000
2000
0

1
2

Fig-57-Various staph vaccines influence on total


WBC counts (cells /cu mm) in albino rats
60

40

20

LYM

POLY

3
4

Fig-58-Various staph vaccines influence on


WBC differential counts (%) in albino rats.

92

15
10
5
0

1
2

3
4

Fig-59-Various staph vaccines influence on


Haemoglobin (gm %) in albino rats.

40
30
20
10
0

Fig-60-Various staph vaccines influence on


packed cell volume (%) in albino rats
93

2
1.5
1
0.5
0

Fig-61-Various staph vaccines influence on plate


lets counts in albino rats.
In

this

study,

various

staph

vaccines were tested for their efficacy. The


maximum amount of WBC total counts,
Haemoglobin, packed cell volume and total
RBC counts were observed in killed vaccine.
The second maximum total WBC counts,
Haemoglobin, packed cell volume and total
RBC counts were observed in mixer vaccine

94

treatment. Remaining treatment has low immune


responses compared to these two treatments.
The maximum platelets counts were
observed in Heat stress protein vaccine; Plasmid
DNA vaccine and killed vaccine have same
range of platelets counts. Mixer vaccine has
third range of platelets count. In the packed cell
volume, more or less same values observed in
Toxoid vaccine and Heat stress protein vaccine.

Our previous Studies reported that various


vaccines efficacy of Aeromonas hydrophila
pathogen. They observed killed vaccine produce
maximum immune responses second maximum
immune response was observed in protein
vaccine. But they recommended plasmid DNA
vaccine, because the killed vaccine and protein
vaccines slowly lose their efficiency during long
95

duration. The DNA vaccine merged to host


DNA produce long term immunity.

In the present attempt, similar thing was


observed, but the mixer vaccine produce second
highest immune responses. The efficacy of the
mixer vaccine is better because the killed
vaccine part and protein vaccine part induce
immediate highest immune responses. The
toxoid protein vaccine and Heat stress protein
vaccines are also act as good bio-advents and
vaccine.
The plasmid DNA vaccine gives
long term immunity. So during long duration,
these mixer vaccine shows good efficacy
compared to other vaccines So the mixer vaccine
is recommended for New generation

staph

vaccine development program for human use.


96

Conclusion and Recommendation


Normally

killed

vaccines

stimulate

immune responses. But the mutant strain


killed vaccine stimulates slightly higher
immune responses.
Naked plasmid DNA act as vaccines,
similarly mutant strain naked plasmid
DNA produce slightly higher immune
responses.
The Engineered plasmid DNA also acts as
good vaccines.
Heat stress proteins are act as good
boiadjuvents and vaccines.
Especially 550 C temperature exposed
pathogens (at 10 minutes time) produced
97

Heat stress- protein gives good immune


responses.
The mixer of plasmid DNA with various
proteins such as Heat stress protein,
Toxoid protein and killed vaccines i.e.)
mixer vaccines are also act as best vaccine
against Staphylococcus aureus infections.
Because the killed vaccines and protein
vaccines stimulate immediate cellular
immune responses, the DNA vaccine has
long term viability.
So the combination of all these things is
act as good vaccine. This is also
recommended for new generation vaccine
preparations.

98

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About the Author

Dr.M.Muruganandam is
working in Einsteein Bio-Engineering
Research Foundation. He is an Editor
of African journal of Biotechnology and
International journal of Medicine and
Biomedical

Research.

He

is

also

Reviewer and Editorial board member


in Various National and International
journals.

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