staph vaccine pdf

Engineered Plasmid DNA Vaccine For Staphylococcus aureus

Dr .M.Muruganandam

2

Engineered Plasmid DNA Vaccine For Staphylococcus aureus

Dr.M.Muruganandam

3

Dedicated to My

Science Teacher Mr. Ramasamy

4

First Edition -2013

ISBN-978-9982-22-418—5

Publisher and Author

Dr.M.Muruganandam,

Email- [email protected]

5

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 researcher’s 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

6

Contents

Biotherapy

Mutant Strain Vaccine

Mutant Strain plasmid

DNA Vaccine

Engineered plasmid

DNA Vaccine

Heat shock protein

Vaccine

Mixer Staph Vaccine

Recommendations

Bibliography

7

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

8

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 system in non-specific way.

9

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 methods are employed, the

important methods are injection, topical

10

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 other vaccines

.Nowadays cocktail DNA vaccines were

introduced for more than one infectious disease.

11

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,

12

for eg vitamins C&E. They reduce stress,

especially vitamin C 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

13

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

14

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.

15

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

16

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

17

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

18

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

19

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

20

Immuno suppressed or Immuno compromised

patient’s 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

barrier’s are breached (e.g. skin damage due to

trauma or mucosal damage due to viral

infection.) S. aureus may gain access founder

21

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

22

a 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, Escherichia coli,

Salmonella typhi, etc.

23

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. After that mutant strain was

24

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 strain’s

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

25

samples were taken for analysis. The maximum

immune response was observed in six minute

treated U.V. strain’s 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

26

digested by using these enzymes and used as

vaccines. Albino rats were used as test animals

in all the experiments. In the first experiment,


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

27

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.

28

In the fifth chapter, Heat stress

protein vaccines developments were discussed.

In this study, the pathogens were exposed two

different temperatures such as 050 C and 055 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 pathogen’s

heat stress proteins in both temperature

treatments. However compared to these two

treatments, 055 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.

29

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 25µgm 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

µgm toxoid vaccine treatments. After this level

treatments the immune response were slowly

30

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.

31

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.

32

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 can’t

penetrate the skin. So they are associated with

wounds, cuts needle pricks, etc., once inside the

host, they stick to host tissues and produce

33

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

patient’s 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.

34

Fig-1: Mutant Strains Vaccines influence on

RBC Counts (millions) in Albino rat.

All the mutant strains were serially diluted

up to 510 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 of

immunological and haematological aspects.

012345

1

2

34

5

35


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

0

1000

2000

3000

40001

2

34

5

36

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.


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

0

20

40

60

801

2

34

5Lym

pol

37

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

Killed vaccine preparations.

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


Packed Cell Volume (%).

05

101520

1

2

34

5

0

10

20

301

2

34

5

38


Antibody levels.

0

2

4

6

81

2

34

5

39

3. Mutant strain’s 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. Here pathogen was

40

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

41

acclamentation, commercial feed were provided

into all groups . First mutant strain’s plasmid

DNA was injected (200µl) 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 ( 510 ) 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

42

`

Fig-7: Mut. Stra. Plasmid DNA Vaccines

influence on RBC count (millions) of Albino

rats.

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

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

0

2

4

61

2

34

5 Sample-I

Sample-II

0

5000

100001

2

34

5sample-I

sample-II

43

Fig-9: Mutant Strains plasmid DNA Vaccines

influence on Lymphocytes (%) of Albino rats.


influence on polymorphs (%) of Albino rats.

020406080

1

2

34

5 sample-I

sample-II

0

20

40

601

2

34

5sample-I

sampe-II

44


influence on Eosinophils (%) counts of Albino

rats.

This experiment is aimed to develop a

DNA vaccine for the infection of

Staphylococcus aureus. They can be

manufactured more easily than 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.

0

10

20

301

2

34

5sample-I

sample-II

45


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.

0

10

20

30

401

2

34

5sampe-I

sampe-II

46

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.

0

5

10

151

2

34

5sample-I

sample-II

47


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.

02468

1

2

34

5

48

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 development against

Staphylococcus aureus.

49

The bacterial pathogen Staphylococcus

aureus was collected from the patient’s 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 were double digested using

50

combination of two enzymes. The digested

plasmids DNA were provided by intramuscular

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.

0100020003000400050006000

1

2

3

4

5

6

51


vaccine influences on RBC counts (millions) of

Albino rat.


vaccine influences on polymorph (%) of Albino

rat.

0

1

2

31

2

3

4

5

6

0

20

40

601

2

3

4

5

6

52

Table 1: Various restriction enzymes and their

restriction sites

S.No Name Source Recognition

Sequence

1. EcoR-1 E. coli RY 13 5’ G

AATTC 3’

2. Bsh – I

(Hae –

III)

Bacillus sphaericus 5’GG CC 3’

3. Pst-I An E. coli strain that

carries the cloned

Pst – I gene from

Providencia stuartii

5’ CTGCAG

3’

4. Bam H–

1

Bacillus

anyloliquefacies H

5’ G

GATCC 3’

5. Hind –

III

Haemophilus

influence Rd

5’.... A

AGCTT...3’

3’....TTGA

A....5’

53

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

54

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 .


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

0

20

40

60

801

2

3

4

5

6

55

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. All the treatments contain double

56

digested plasmid DNA with various enzyme

combinations.


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

012345

1

2

3

4

5

6

57

treatments are suitable for new DNA vaccine

preparation.


vaccine influences on Haemoglobin (gm %) rat


vaccine influences on packed cell volume (%) of

Albino rats.

0

5

101

2

3

4

5

6

0102030

1

2

3

4

5

6

58


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.

02468

101

2

3

4

5

6

59


vaccine influences on WBC counts (cells / cu

mm) of Albino rats.


vaccine influences on Lymphocytes (%) of

Albino rats.

0

2000

4000

60001

2

3

4

5

6

7

8

9

10

020406080

1

2

3

4

5

6

7

8

9

10

60


vaccine influences on polymorph (%) of Albino

rats.

Fig26 – Various enzymes digested DNA

vaccine influences on Eosinophils (%) of Albino

rats.

0

20

40

601

2

3

4

5

6

7

8

9

10

012345

1

2

3

4

5

6

7

8

9

10

61


vaccine influences on RBC counts (millions) of

Albino rat.


vaccine influences on Haemoglobin (gm %) of

Albino rats.

01234

1

2

3

4

5

6

7

8

9

10

0

5

101

2

3

4

5

6

7

8

9

10

62


vaccine influences on packed cell volume (%) of

Albino rats.


vaccine influences on Antibody levels of Albino

rats.

0

10

20

301

2

3

4

5

6

7

8

9

10

02468

101

2

3

4

5

6

7

8

9

10

63

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

64

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

65

vaccine 5.Double digested Plasmid DNA mixer

vaccine.

Figure 31 – Various enzymes digested Plasmid

DNA mixer vaccine influences on WBC counts

(cells / cu mm) of Albino rats.

02000400060008000

1

2

34

5

020406080

1001

2

34

5poly

lym

66

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.


DNA mixer vaccine influences on RBC counts

(millions) of Albino rats.

0

2

4

61

2

34

5

67

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.


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

0

5

10

151

2

34

5

68

compared to other treatments. In this attempt,


double digested treatment. So it is highly

suitable for cocktail plasmid DNA vaccine

preparation

Figure 35– Various enzymes digested Plasmid

DNA mixer vaccine influences on Antibody

levels of Albino rats.

.

02468

101

2

34

5

69

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 o55 C) with

70

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

71

exposed pathogen’s 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.

72

Fig: 36-Hsp vaccine influence on Total WBC

counts (cells/cu mm) of albino rats.

.

Fig: 37-Hsp vaccine influence on Total RBC

counts (millions) of albino rat.

0

2000

4000

6000

80001

2

34

5Temp-50

Temp-55

0

1

2

3

41

2

34

5Temp-50

Temp-55

73

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.

Fig:38-Hsp vaccine influence on polymorph

counts (%) of albino rat.

45

50

55

601

2

34

5Temp-50

Temp-55

74

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.

Fig:39-Hsp vaccine influence on Lymphocyte

counts(%) of albino rats.

01020304050

1

2

34

5Temp-50

Temp-55

75

Fig: 40-Hsp vaccine influence on Eosinophils

counts (%) of albino rat

Fig41. Hsp vaccine influence on Haemoglobin

(gm %) of albino rats.

0

0.5

1

1.5

21

2

34

5Temp-50

Temp-55

02468

101

2

34

5Temp-50

Temp-55

76

Fig:42-Hsp vaccine influence on packed cell

volume (%) of albino rats.

Fig:43-Hsp vaccine influence on platelets

counts of albino rats.

0

10

20

301

2

34

5Temp-50

Temp-55

01234

1

2

34

5Temp-50

Temp-55

77

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 050 C treatment, 055 C

temperature exposure treatment produce more

immune responses, especially 10 minutes time

exposure at 055 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 bio-

adjuvant preparation for staph DNA vaccines.

78

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

patient’s 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.

79

Samples 1-5 in figures show Graded

level of Toxoid protein vaccine such as 1.

Control, 2. 15µgm Toxoid Protein vaccine ,3.

30µgm Toxoid Protein vaccine, 4. 45 µgm

Toxoid Protein vaccine ,and 5. 60µgmToxoid

Protein vaccine.

Fig-44.Graded level of Toxoid protein vaccine

influence on RBC counts (millions) in Albino

rats.

Staph toxin were isolated,

inactivated and provided various quantities to

2.8

2.9

3

3.11

2

34

5

80

albino rats for tested their immune responses.


influence on WBC counts (cells/cu mm) in

Albino rats


influence on WBC differential counts (%) in

Albino rats.

0

2000

4000

6000

80001

2

34

5

0

20

40

601

2

34

5 LYM

POLY

EOIS

81


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 15µgm

toxoid protein vaccine treatments. After that,

higher concentration of toxoid protein vaccine

leads to lesser immune responses.

0

5

10

151

2

34

5

82


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 15µgm

toxoid vaccine treatments. The immune

response of 60µgm 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.

26

27

28

291

2

34

5

83


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


15µgm toxoid protein vaccine. If go to higher

doses leads to low immune response that is why

00.5

11.5

22.5

1

2

34

5

84

these concentrates act as saturation point for this

vaccine. So it is concluded that this 15µgm 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. 12.5µgm Heat Stress Protein

vaccine 3. 25µgm Heat Stress Protein vaccine 4

85

37.5 µgm Heat Stress Protein vaccine and 5.50

µgm Heat Stress Protein vaccine.

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.

0

1

2

3

41

2

34

5

86

Fig- 51 .Graded levels of Staph-Hsp vaccine

influence on Total WBC counts (cells/cu mm)in

Albino rats.

Fig- 52.Graded levels of Staph-Hsp vaccine

influence on WBC differentials counts(%) in

Albino rats.

0

2000

4000

60001

2

34

5

0204060

1

2

34

5POLY

LYM

EOIS

87


influence on Haemoglobin (gm %) content of

Albino rats.


influence on packed cell volume (%) in Albino

rats.

0

5

10

151

2

34

5

010203040

1

2

34

5

88


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 25µgm protein

treatment. So it is concluded that these level is

suitable for further process in staph protein

vaccine development.

00.5

11.5

22.5

1

2

34

5

89

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 patient’s

sample in a hospital, Regular microbiological

90

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 050 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

91

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.

Fig-56-Various staph vaccines influence on

total RBC counts (millions) in albino rats.

01234

1

2

3

4

5

6

92

Fig-57-Various staph vaccines influence on total

WBC counts (cells /cu mm) in albino rats


WBC differential counts (%) in albino rats.

020004000

60008000

1

2

3

4

5

6

0

20

40

601

2

3

4

5

6

LYM

POLY

93


Haemoglobin (gm %) in albino rats.


packed cell volume (%) in albino rats

0

5

10

151

2

3

4

5

6

0

10

20

30

401

2

3

4

5

6

94

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

0

0.5

1

1.5

21

2

3

4

5

6

95

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

96

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.

97

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 055 C temperature exposed

pathogens (at 10 minutes time) produced

98

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.

99

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104

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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