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Dedicated to
Prof. James Philip
First Edition-2017
ISBN- 978-9982-22-633-2
Author
Dr.M.Muruganandam
Email: [email protected]
Publisher
Einsteein Bio-Engineering
Research Foundation,
South India.
Preface
New strains of pathogens are emerging
day by day in the various parts of the globe
due to over population explosion and
manmade pollutants. These harmful materials
created genetic changes in the organisms as a
result, it will produce new strains of
pathogens. It will also create big burdens to
our society. For control and management of
this disease burden, we need multidirectional
combined approach of action. Vaccine
development is one of the control measures
which is simply explain in this book to young
readers. For the preparation of these
manuscripts, I have referred many researchers’
work, I thank all of them.
M.Muruganandam
Content
1. Bacterial vaccine
1.1 vaccines
1.2vaccine Immunity
1.3 vaccine compounds
1.4 vaccinations
1.5 vaccines Research
2. Live and Inactivated vaccine
2.1 First generation vaccine
2.2 Inactivated vaccine
2.3 Live vaccine
2.4 Merits and Demerits
2.5 Lab Trails
3. Peptide vaccine
3.1 whole cell protein vaccines
3.2 Heat Stress Protein vaccine
3.3 Toxoid protein vaccine
3.4 Surface protein vaccine
3.5 Anti-idiotype vaccine
3.6 Synthetic peptide vaccine
4. Nucleotide vaccine
4.1 plasmid DNA
4.2 Mutant plasmid DNA
4.3 Engineered plasmid DNA
4.4 Cocktail plasmid DNA
4.5 Genomic DNA
4.6 rDNA
4.7 Nucleotide with proteins
4.8 potential Merits
5. Antigenic Efficacy of Immunogen
5.1 Bacterial vaccines
5.2 Immunogen preparation
5.3 Efficacy of Immunogen
5.4 Analysis
5.5 Best Immunogen
6. Current Researches
6.1 Immunostimulant
6.2 vitamins
6.3 Cancer vaccine
6.4 parasite vaccine
6.5 Therapeutic vaccine
6.6Adjuvants
6.7 Preservatives
6.8 Allergies
6.9 Metabolic Disorders
6.10 Birth Control
6.11 Economics of vaccine Development
6.12 vaccines Failure
6.13 Vaccinomics
Bibliography.
1. Bacterial Vaccines
1.1Vaccine
Vaccines are used to educate our
immune system to fight against various
infectious diseases. Ideal vaccines stimulate
both humoral and cell mediated immunity.
The vaccine is an antigenic preparation used
to produce active immunity to a disease in
order to prevent the effects of infection by
any natural or wild strain of the organism.
The vaccination process present in
old times but in the modern word, First
immunization was introduced by Edward
Jenner, He vaccinate against small pox.
1.2 Vaccine Immunity The Vaccines are
close resemblance of disease causing
pathogens. It contains inactivated
pathogens or part of pathogens. Vaccines
triggers the immune system’s fighting ability
and memory without exposure to the actual
disease producing germs. So we get
immunity without suffering from the disease
producing germs. The vaccination is similar
to the immunity acquired from natural
infection. After vaccination, if pathogens
entered into the body, first body feel
sickness then our immune system
completely destroy the pathogens and we
got complete recovery of the diseases.
1.3 Vaccine Components Vaccine
contains saline or sterile water as well as
dead or weak germs and other purified
components which are supplied in vaccine
functions. These components mainly
stimulate our immune system and help to
avoid microbial contamination to vaccine.
Some vaccines are prepared with antibiotics
or preservatives to avoid bacterial and
fungal growth and it also prepared with
stabilizers which help to maintain its efficacy
during storage conditions. Another
important component is adjuvant, Such as
aluminium Salts, mineral oils, many
chemical and Biological substances are used.
It makes more effectiveness of vaccine
functions. The ideal vaccine should induce
humoral and cell mediated immunity
properly.
1.4 Vaccination Nowadays many
advanced techniques are discovered in
immunization methods. Needle free or pain
free immunization is one of the important
techniques. It is highly suitable to children
and aged population. Now skin patches,
mucous vaccination methods such as eye
drops, oral drops are commonly used for
mass vaccination programs.
In our lab trials, skin ointment based
vaccines were tested against bacterial
infections. It also produces good results. Still
searching of new methods of vaccination
research is going on all over the world.
1.5Vaccine Research
Still many important disease
need effective vaccines, especially
multidrug resistant pathogens such as
typhoid, tuberculoses, malaria , filarial,
etc. currently, effective vaccines are
required to reduce the global disease
burden. Combined vaccine for more than
one diseases and therapeutic vaccine for
cancer are also need, the vaccine biology
faces lot of challenges in cancer vaccine
development, Human Parasite vaccine
research and multidrug resistant
pathogens. Still we are fighting to reduce
global disease burden and help to
develop healthier younger generation in
our globe.
2. Live and Inactivated Vaccine
2.1 First generation vaccine
In activated and live vaccines are first
generation vaccines. First inactivated vaccines
are universally used. It is slowly replaced by
new generation vaccine. The new generation
vaccines are various peptide and nucleotide
vaccines. During the preparation of inactivated
vaccine, it needs more micro organism. The
bacterial live attenuated vaccine was first
described by Louis Pasteur. He said that
pathogen in old culture lose their virulence.
Mostly it cannot produce disease. Furthermore
these less virulent pathogens are suitable for
prepare live attenuated vaccine. He has done
similar work in some bacterial pathogens.
2.2 Inactivated pathogens
In activated vaccines are previously
virulent micro-organisms that have been killed
with chemical or heat or suitable antibiotics.
During vaccine preparation, first bacterial cells
are harvested after centrifugation; cells are
washed with saline then inactivated with 0.5%
formalin maintained in 40c at overnight
incubation1. In some other cases of inactivation,
0.2% (v/v) of 40% formaldehyde with incubation
at 20c for 4 days maintained.
The inactivation is confirmed by the
absence of growth following the seeding of
triplicate plates of tryptone soyagar with0.1ml
volumes of the cell suspension and incubation
for up to seven days at room temperature 2. In
the case of virus, it is inactivated traditionally by
formalin. However the excessive treatment can
destroy immunogenicity where as insufficient
treatment can leave infections which capable of
causing diseases. Currently available inactivated
viral vaccines are influenza, polio, rabies,
hepatitis A, etc. The bacterial vaccines are
Typhoid, cholera, plague, etc and the fractional
vaccines are hepatitis B, influenza, etc.
2.3 Live Vaccines
Live attenuated vaccines are live micro-
organisms that have been cultivated under
conditions which are able to express their lesser
virulent properties. They are typically provoke
more durable immunological responses and are
preferred types for healthy adults. These
vaccines are prepared from live attenuated
strains that are almost or completely devoid of
Pathogenicity but they are capable of inducing
protective immune responses.
They multiply in the human host and
provide continuous antigenic stimulation over a
period of time. During bacterial live vaccine
preparation, cells are harvested from old
cultures because they lose virulence. But in the
viral vaccine preparation, various methods were
followed.
The first method is use of related virus
from another animal. For example, use of
cowpox virus to prevent small pox. The second
method is the virulence of the virus is often
reduced when administrated by an unnatural
route.
Another method is the passages of the
virus in an unnatural host or host cells. The
major vaccines used in man and animals have
been derived this way. After repeated passages,
the virus is administered to the natural host.
Initial passages are made in healthy animals or
in primary cell cultures. For example, polio
viruses were passage in monkey kidney cells
and measles in chick embryo fibroblasts. The
currently available live attenuated vaccines are
measles, mumps, rubella, yellow fever, BCG,
etc.
2.4. Merits and Demerits For in activation,
viruses treat properly otherwise insufficiently
treated virus can cause diseases. In this type of
preparation increased risk of allergic reactions
due to large amount of antigen involved.
For protective response occurs after
second and third dose. So it is required multiple
doses. The immune response is mostly humoral.
But the main advantages are non-infections and
non-replicating.
In the live vaccine, the main advantages
are immune responses similar to natural
infection and it is very close to infections agent
and another thing is usually effective with one
dose. The important disadvantages are
reversion to pathogenic (wild) form, may
serious or fatal reactions possible and
interference from circulating antibody stability.
2.5 Lab Trials In our lab work, three
bacterial pathogens (Staphylococcus aureus,
Salmonella typhi and Escherichia coli were
collected from patient’s sample and prepared
mutant whole cell vaccine. The mutation is
induced by using U-V-radiation. First bacterial
pathogens are cultured individually in the lab.
Five spread plates were prepared for each
pathogen, these plates are exposed to U.V.
radiation at different time duration.(0,2,4,6 and
8 minutes). Then these organisms are isolated
and introduced in broth culture. After 24 hours
inactivated vaccines were prepared in all the
treatments. After that these vaccines were
tested in Albino rats.
In the Staphylococcus aureus, the
maximum immune response was observed in 6
minute U.V. treated mutant strain. But in the
case of salmonella typhi mutant strains produce
more or less similar immune responses. In the
case of E.coli, if increase the duration of U.V.
treatment, the virulence of pathogens was
slowly decreased. In this study, it is concluded
that, the mutant strains has more virulence in
some cases and in some other cases, the
mutant strains has lesser virulence. However It
is also highly suitable for preparation of best
new inactivated and live vaccines. 3,4,5.
Reference
1) Bakopoulous.V.,D, Volpatti., L., Gusmani,m., Galeotti, A.,
Adams and G.J. Dimitriadis (2003). Vaccination trials of
seabass, Dicentrachoslabrax(L).againstphotobacterium
damsel sub sp. Piscicido, using novel vaccine mixture.
J.fishg. Diseases 26:77.90.
2) Irianto.A., P.A.W. Robertson and B.Austin (2003).oral
administration of formalin-in activated cells of
Aeromonas hydrophila A3-51-controls infection by
atypical.A. salmonella in gold fish, carassius auratus (L).
S.fish. Diseases. 26:117-120.
3) Muruganandam.M., J.E. John Soloman and
Mahesh.S.Kini. (2010). Mutant strain vaccine for Typhoid.
Env.&Eco 28(2B):1414-1415.
4) Muruganandam.m and K.N. Veerayee kanna (2010)
mutant strains vaccine for staphylococcus aureus.
J.Curr.Sci.15 (1):229-232.
5) Muruganandam.m. and J.Jana. (2016) ultra violet
radiation alters the virulence of pathogenic bacteria.-
proceedings of the state level seminar on Global
warming and climatic changes held on 19th Aug 2016.
3. Peptide Vaccine
3.1 Whole Cell Protein Vaccine
It is used as subunit vaccine. The main
advantages are, it cannot produce disease. But
it will produce good immunity. Because of most
of the antigens are proteins. It induces higher
level of immunity at very short duration.
Acetone based method is one of simple whole
protein isolation method. In staphylococcus
aureus, the whole cell proteins are provides
with nucleotides, which produces good result.
Proteins are good for induce short term
immunity. In the longer duration, it may fade
their efficacy. If combined with nucleotide, it
will produce long term immunity.1
3.2 Heat Stress Protein: This is otherwise called
Heat shock proteins. During unfavorable
conditions like heat stress, a small number of
specific genes called heat shock genes which
will be expressed for save the life of that
organisms,2 they are act as immunogen of
many pathogens including a wide variety of
bacteria. So it is ready to develop HSP Vaccines
against various diseases. Jacoby et al.,3
identified heat shock genes by a time course
analysis. They found that most genes exhibited
maximal responses at 20 minutes heat shock,
However it was declined by 30 minutes heat
shocks treatments. In staphylococcus aureus
550c heat shock with 10 minutes produced HSP
that induces a maximum immune response in
the host. Hence it is recommended that these
HSP can be used as supportive component of
staph vaccine development. 1
3.3 Toxoid Protein
These are inactivated toxic compounds
from micro-organisms. eg. Toxoid based
vaccines are tetanus and diphtheria. These toxic
compounds are normally produced illness and it
is changed to harmless materials then it is used
as immunogen in vaccine development. In our
lab trial Toxoid protein vaccine optimization In
this method, proteins were isolated by using
acetone precipitation method. The maximum
leucocytes responses were observed in 15 µg
toxoid treatment. These are individually used as
vaccine or sometimes use with other
immunogen for increase the efficacy of
vaccines.
3.4 Surface protein Pathogen surface proteins
play important role in producing immunity in
the host. The isolation of surface protein from
the pathogens is used to prepare vaccine
against that particular pathogen. There are
various methods are available to isolation of
surface proteins. Sometimes these proteins may
be combined to other cellular components and
used as vaccine. It is mainly helpful to prepare
parasite and bacterial vaccine.
3.5 Anti-idiotype vaccine
It is mostly used in virus vaccine
preparation. Because the available quantity of
specific antigen is lesser amount, at that time
usually prefer this type of vaccine preparation.
It has no pathogenic part, so it is very safe. In
this type first antigens are isolated and purified
then injected to some animals like rabbit Albino
rat etc,. After appropriate time intervals the
antiserum will be collected and used as vaccine.
It may be used in cancer vaccine development.
In our lab trial, Anti-idiotype vaccine developed
for colorectal cancer, it was also produced good
results.4
3.6 Synthetic Peptide Vaccine
The pre required for the preparation of
synthetic peptide vaccine involves the synthesis
of a specific peptide that can elicit antibodies
which reacting with coat proteins of viruses or
other micro-organisms. Synthetic peptide can
be easily synthesized where the nucleic acid
sequence of the gene coding for the relevant
protein is known. This vaccine has some
advantages and drawbacks. The main draw
backs are the vaccine preparation cost is higher
and also effective carrier system is required for
antigen presentation.
The synthetic peptide vaccine contain
single epitope sometimes due to genetic
variability, the efficacy may low ie. not effective.
The vaccines may stimulate only a humoral
response5. Sometime the synthetic peptide may
weak efficacy, at that time it will linked with
suitable strong adjuvant, then that will induce
strong immune responce6.
Reference
1) Muruganandam.M. (2013) Engineered plasmid DNA
vaccine book.p101
2) Beckmann.R.P., Mizzen, Lit., Welch,W.J.(1990).
Interaction of HSP 70 with newly synthesised proteins:
implications for protein folding and assembly. Vol.
248.PP.850-854.
3) Jacoby.T.H., Flann gan.A., Faykin,A.G., Seto.C,
Mattison and I.ota (1997). Two protein-Tyrosine
phosphateses inactive the osmatic stress response pathway
in yeast by targeting the mitogen activated protein kinase.
J.Bio and che.272:17749-17755.
4) Ayyappan.S., Johnpaul, Dhilipan and
M.Muruganandam (2010). Anti-Idiotype vaccine for
colorectal cancer-A two day National conference on
Innovations in Bio-technology and clinical Research held on
march 11th and 12th 2010.
5) www.bioinformatic center.org.
6) Rangasamy.(2003) Fish Bio-technology-book. Chap:
Biotechnology in Health management for
Aquaculture.Pub.Agrobios. India.
4. Nucleotide Vaccine
4.1 Plasmid DNA
The plasmid DNA is self replicating double
stranded circular DNA molecules present in the
cytoplasm of bacterial cell. It always carries one
or more genes responsible for useful
characteristics. They have their own origin of
replication and they replicate independently.
Most of the plasmid DNA exists as double
stranded 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 DNA. Usually
plasmids are used as vectors in recombinant
DNA technology.
In our lab trials, plasmid DNA of
bacterial pathogens such as Aeromonas
hydrophila, E.coli, Staphylococcus aureus and
salmonella typhi were isolated and used as
vaccine and we have received good results.1 2,3, 4.
4.2 Mutant’s plasmid DNA
In Staphylococcus aureus mutant strains
are produced by using U.V radiation. In the
different mutant colonies, plasmid DNA was
isolated by alkaline lysis method. Each group’s
plasmid DNA separately injected to albino rats
used as vaccine. After 15 days, inactivated
pathogens were injected to albino rats.
Maximum immune response was observed in 6
minute U.V treated mutant strains plasmid
DNA. So it is concluded that this treatment was
best for develop vaccine against staphylococcus
aureus.6
4.3 Engineered Plasmid DNA
In this work, plasmid DNA of
staphylococcus aureus was isolated then altered
by using various restriction enzymes such as
EcoR-I, Hind-III, pst-I, Bam H-I and Hae –III. In
this study, maximum immune responses are
observed in pst-I and Hae-III digested
treatments. This plasmid DNA is double
digested by the same enzymes. In this study the
length of the nucleotide fragments may be
reduced. So the immune responses will be
changed. In this trial, the maximum immune
response was observed in EcoR-I + Hind –III
digested treatment and Hind-III and Bam-H-I
digested treatments. These are highly suitable
for develop plasmid DNA vaccine for
Staphylococcus aureus.5
Fig-1&2- Vaccine preparation
4.4 Cocktail Plasmid DNA This type of
vaccine may help to prevent groups of
common diseases. In this attempt, mainly
try to control water borne diseases.
Commonly water borne diseases are
caused due to intake of contaminated
water. Sometimes the drinking water is
contaminated to the fecal contamination
by mixing of sewage water. At that time,
it contain many pathogens such as
Escherichia Coli, Staphylococcus auras,
salmonella typhi, etc. which causes fever,
dysentery, vomiting diarrhea, rapid pulse,
cramps, etc.
In this study mixture plasmid DNA, Pst-I
digested fragments, Bam H-I-digested
fragments-and double digested plasmid
DNA fragments used as test vaccine. The
maximum immune response was
observed in double digested plasmid DNA
compared to other treatments. So it is
recommended to develop cocktail
vaccine for water borne disease.6
4.5 Genomic DNA
Genomic immunization is going to be a
revolution in vaccines and it is called
genomic up. In the mycoplasma, genomic
DNA is cut into small fragments and
shooting all the genomic bites into skin
cells of the mice. Each bit of mycoplasma
protein, which produced an immune
response on each mouse. The genomic
vaccine fools the immune system, they
think, it has been infected by the real
pathogen. This vaccine produced good
results in human parasitic vaccine.
In Helminthes parasite, mycoplasma
palmonis, a library of gene fragments was
prepared by cloning the genomic DNA
into plasmid expression vector. Since
these organisms has a relatively small
genome (about 106 base pairs). The DNA-
protein-coding sequences might be
expressed to induce immunity to the
pathogen. Since only a small part of the
genetic complement of the organisms is
expressed, from only a fragment of genes
and not entire functional proteins.
Pathogenic effects would be avoided
while all the advantages of broad-based
immunity produced by a DNA vaccine
would be present protection against
M.pulmonis has been achieved after
immunization with different expression
libraries9. In our lab trial, Genomic DNA
vaccine was prepared against common
food borne diseases and tested. It also
gives better results7.
4.6. rDNA
During recombinant DNA vaccine
development, antigenic protein
producing DNA sequences was removed
and transferred to known vector DNA.
Both DNA were attached by using ligase
enzyme. After that it will transferred to
any one of the harmless microbes. This
will be injected to host and it will produce
antigenic protein continuously vaccine
research shows great promises against
parasites.
4.7 Nucleotide with proteins
Nucleotides act as good vaccine in various
bacterial pathogens. In the
Staphylococcus aureus, Nucleotide with
whole cell protein was used as vaccine.
Additionally small amount of inactivated
cells are also mixed and used as vaccine.
It produces good immunity. The
inactivated cells and whole proteins
induce immediate immunity. But during
long term, it is possible to fade. The
nucleotide may possible to integrate the
genomic DNA; it may produce long term
immunity. So the nucleotide with protein
is highly suitable to develop new vaccine
against staphylococcus aureus8.
4.8 Potential Merits
*Nucleotide vaccine preparation method
is easy.
*It is work in room temperature. So the
cooling system is not required.
*Transport is very easy because without
cooling system we can transport one
place to another place very easy.
*Economics of vaccine is suitable for
developing countries.
*It is very specific. The accuracy is high
compared to other immunogen.
*Storage system is not need.
*It is third generation vaccine. It is
advanced one.
*Functional efficacy is more and also
best.
*It produces good immunological
memory.
*It produces long term immunity.
*It gives new scope to develop human
parasitic vaccines and next generation
vaccines.
References
1.Muruganandam.M. (2007) New DNA vaccine for
Aeromonas hydrophila infection, Aqua Tech Vol 7(8)
pp79.
2.Muruganandam.M (2010). DNA vaccine for
Bacterial pathogen Escherichia coli Int.Nat.J.Bio.Tech
: 1 (2) :110-112.
3.Muruganandam.M. (2010) plasmid DNA vaccine for
staphylococcus aureus.J.Nat.Con:2(1):73-76.
4.Muruganandam.M.(2010).Engineered DNA vaccine
for Typhoid.J.Nat.Con. 22 (1):123-126.
5.Muruganandam.M.(2011).Engineered plasmid DNA
vaccine for staphylococcus aureus. Int.J.Bio-tech 2
(1):7-10.
6.Muruganandam.M.(2011) cocktail plasmid DNa
vaccine for common food borne disease.
Int.J.biotech.2(1):1-3.
7.Muruganandam.M.(2010).Genomic DNA vaccine
for common food borne diseases.
Int.Nat.J.Bio.Tech.1(20):107-109.
8.Muruganandam.M. (2012) Engineered plasmid
DNA vaccine. Book. ISBN-978-9982-22-418-5.
9.Sean Henahan (1995) Access Excellence, Genomic
vaccines. The new way to prevent disease-act 19.
Nature.
5. Antigenic Efficacy of Different Immunogen
5.1 Bacterial vaccine
Antigenic efficacy of different Bacterial
Immunogen is varying. The qualitative and
quantitative levels of these immunogen
determine the major level of efficacy of vaccine.
Generally the vaccine efficacy depends on many
internal and external factors. The important
factors are dietary composition, (Nutritional
Factors) condition of the immune System,
Amount of specific immunogen in vaccine,
selection of adjuvant in vaccine etc. In this study
Aeromonas hydrophila is used as test organism.
Aeromonas hydrophila is a motile rod
shaped bacterium, these Aeromonas are water
borne micro-organisms that have been
implicated repeatedly as the causative agents of
clinical illnesses often Serious, Ranging from
gastro intestinal and wound infections to
septicemia. Bacteraemia is the most common
pathogenic manifestation of Aeromonas in
humans its mild symptoms include fever , chills,
abdominal pain, Nausea, vomiting and diarrhea.
Vaccines as an antigenic preparation
used to produce active immunity to a disease in
order to present the effects of infection by any
natural or wild strain of the organism. Vaccines
prepare body for fighting deadly disease by
triggering of person immune system this leads
to the production of immune response that can
fight of the germs. The vaccine contains a killed
or weak end form or derivations of the
infections germ. But recent advances in
molecular biology have provided various
methods for producing vaccines. But still
research is in the process towards of finding
proper vaccine for the Aeromonas infections
disease in world wide. Now there is no vaccine
in Aeromonas hydrophila infection due to its
antigenic diversity. In this study various
immunogen are isolated and tested their
efficacy for vaccine development against
hydrophila infection.
5.2 Immunogen Preparation
Aeromonas hydrophila MTCC-646 strain
was used for this experiment. Albino rats were
used as test animals in the whole study. Albino
rats were purchased from animal husbandry
and were acclimatized to the laboratory
environment for a week. They were maintained
in cool dry place. During this study, the room
temperature ranges from 26’c-29’c. They were
fed commercially available feed and inspected
daily. In this study two experimental trials were
conducted. In the first experiment, four sets of
albino rats were used., each set contain three
rats being exposed to killed, live attenuated and
toxoid vaccine. In the second trial, Genomic
DNA, plasmid DNA and whole proteins were
used as vaccine, Saline as control in both
experiments. After 15 days pathogens were
injected and primary immune response were
studied.
In killed vaccine preparation,
approximately 1ml of nutrient broth inclusive of
Aeromonas hydrophila was centrifuged at
10,000 rpm for 10 minutes and the pellets were
collected and removed the supernatant, then
saline was added to the pellet and it was heated
in boiling water (30minutes) followed by
centrifugation at 10,000rpm for 10 minutes and
the dead cells were collected. It was then
serially diluted 10-5 with saline. For live
attenuated vaccine, the cells were isolated from
old nutrient medium culture and then these
were serially diluted to 10-5 using saline. For
toxin preparation, the viable cells were
inoculated for 24 hrs. The content was
centrifuged at 10,000rpm for 10min. The
supernatant was collected and serially diluted
by using saline.
The genomic DNA and plasmid DNA
was isolated from nutrient broth culture. For
isolation if both DNA, kit procedure (medox kit)
was followed. For whole protein vaccine
preparation, Ammonium per sulphate was used
to protein precipitation. In this method, first
1ml culture was taken in an eppen drof and it
was centrifuged at 10,000 rpm for 10 minutes.
Supernatant was discarded and about 1ml of
saline was added. It was then centrifuged at
10,000 for 15 minutes. Again the supernatant
was discarded and add 0.5.ml of Ammonium
Sulphate was added and then centrifuged. The
supernatant was discarded and 1ml of saline
was added and it was serially diluted 10-5 using
saline. For hematological analysis regular
procedure was followed. In the antibody
analysis, 96 well micro titer plate methods was
used and also counter current immuno
Electrophoresis and Rocket immuno
electrophoresis were carried out.
5.3 Efficacy of Immunogen
In the first experiment, maximum WBC
Count (9200 cells/cumm) was observed in killed
vaccine treatment. Other parameters such as
haemoglobin (12.2 gm %), RBC counts (4.06
cells/millions) and PCV (36.6%) were lesser
compared to toxoid and live attenuated vaccine.
The second higher value of WBC count (8,200
cells/cumm) was observed in live attenuated
vaccine. The control has lesser value compared
to other treatments. The maximum
Haemoglobin (13 gm%) and RBC counts (4.36
cells/millions) were observed in toxoid
treatments. The higher antibody production
was observed in killed and live attenuated
vaccine compared to toxin and control
treatments. So in the first experiment, killed
vaccine has maximum immune response
compare to others.
In the second experiment, higher value of
WBC counts, and antibody production were
observed in protein and plasmid DNA vaccine.
In counter current electrophoresis results shows
that more than one type of antibody was
produced in plasmid DNA vaccine compared to
other vaccines.
5.4 Analysis
Each part of the pathogens has various
level of immunogenic efficacy. The increased
level of some components and appropriate level
of mixer of these components determine the
immunogenic efficacy of the vaccine. In this
trial, various components of bacterial pathogen
Aeromonas hydrophila were isolated and tested
their immunogenic efficacy in albino rats. The
conventional approaches to the process of
vaccine development include the use of
inactivated whole cells and live attenuated
vaccine.
The major drawback of these two
approaches is that large quantities of organisms
are usually required to isolate sufficient antigen
for use in vaccine. They also started that ideal
vaccine should be able to induce the production
of both humoral and cell mediated immune
response. So the modern approaches of vaccine
development are best compared to old
approaches.
Another one important draw back in
killed vaccine is it cannot multiply in the host.
So the level of antigen is always same at
longtime. But live attenuated vaccine can
multiply inside the host. So the immunity may
increase and present in long time. So recently
there has been increasing interest the use of
live attenuated vaccines against bacterial
pathogens. In general, live vaccines; elicit a
stronger cell-mediated response than bacterial.
While the greater immunity provided by
attenuated organisms compared with that
provided by dead bacteria.
In the second trial, three types of
vaccines used. One is DNA another one is
plasmid DNA and third one is whole protein.
The DNA vaccine technique that is being tested
in humans involves the direct injection of
plasmid loops of DNA that contain genes for
proteins produced by the organisms being
targeted for immunity. In DNA and plasmid
vaccines there is lesser reports are available.
5.5 Best Immunogen for vaccine development
In Canada, plasmid DNA vaccines
researches are only under trial basis. But in this
study, plasmid DNA vaccine induce antibody
production against A.hydrophila than DNA and
protein vaccine, plasmid DNA vaccine is best for
humoral response. This is proved by
staphylococcus aureus, salmonella typhi and E.
coli.
In conclusion, the higher immune
response was observed in live attenuated
vaccine in the experimental trial. The live
attenuated organisms live prolong in the host.
So it may induce long term immunity. In the
second trial plasmid DNA and protein vaccine
have more immune responses. The plasmid
DNA may induce long term immunity and
protein can induce immediate immune
responses because antigens are made up of
proteins. So complained with plasmid DNA and
protein is best for new vaccine development for
Aeromonas hydrophila.
Table 1 various immunogenic treatment effects on immune
responses in Albino rats
Parameters Control Killed
vaccine
Live
vaccine
Whole
protein
RBC
count(millions)
3.9 4.5 4.25 4.38
WBC count 7500 9200 8900 9200
Polymorphic
Nuetrophill(%0
15 23 25 18
Lymphocyte(%) 85 75 72 81
Hb(gm%) 11.8 12.2 12.8 13
PCV(%) 38.25 35.75 39.75 39.75
Antibody titre 7 10 9 9
Table-2 Immune responses of Toxoid, Genomic DNA and
Plasmid DNA vaccine in Albino rats.
Parameters Control Toxoid Genomic
DNA
Plasmid
DNA
RBC
count(millions)
3.9 4.35 4.37 4.18
WBC count 7500 8800 8700 8800
Polymorphic
Nuetrophill(%0
15 12 11 20
Lymphocyte(%) 85 88 86 79
Hb(gm%) 11.8 13.1 12.8 13.1
PCV(%) 38.25 36.75 37.25 37.25
Antibody titre 7 8 8 10
References
1) Carmen Hernanz moral, Emilio Flano Del Castill o
Pilar, Lopez Fierro, Alberto Villena Cortes, Juan Anguita
Castillo, Albert Cascon Soriano, Maria Sanctez Salazar, Blanca
Zquin Peralta and German Naharro Carrasco, “Molecular
Characterization of the Aeromonas hydrophila aro A gene
and potential use of an autotrophic aro A mutant as a Live
attenuated vaccine. Infection and Immunity vol.66:pp 1813-
1821 (1998).
2) Hazen,J.C., C.B. Fliermans, R.P, Hirsh and G.W.
Esch:Prevalence and distribution of Aeromonas hydrophila in
the united states. Appl.Emiran.Micro Bio .Vol.36.PP. 731-738.
(1978).
3) Marsden, M.J., L.M. Vaughan,J.J.Foster and
C.J.Secomber,’’A live (aro.A) Aeromonas salum onicida
vaccine for furunculosis. Preferrentially stimulates T-cell
responses in rainbow trout (on corhyrnchus mykiss). Infect
immune .Vol. 64:PP:3862-3869-(1996).
4) Muruganandam.M.(2010) Short sequence vaccine
book.p118 .
6. Current Research
6.1. Immunostimulant
The immunostimulants are mainly either
elevates the non- specific defence mechanisms
or sometimes specific immune responses. The
immunostimulants has many functions such of
prevention of viral infections; enhance the
efficacy of antimicrobial substances. Resistance
to parasites and enhance the efficacy of
vaccines. During administration of
immunostimulants more care concerning the
doses which is necessary when working with
immunostimulants.
This is because high doses of an
Immunostimulant can suppress the defence
mechanism and low doses may not effective. So
optimization is important step during the
administration of immunostimulants. The
important immunostimulants are some
chemical compounds, biological substances and
nutrition factors such as vitamin E, C, etc.,
6.2. Vitamin Immunostimulants
Vitamins are organic compounds and it is
essential for growth and immunity. It is also
require for normal metabolic function. They
require only trace amount. It stimulates our
immune system just like immunostimulants. The
important vitamin immunostimulants are
vitamin C, E, A, D, etc.
Vitamin C and vitamin E are commonly
used in feed for reduce stress effects and
stimulate immune responses. Every vitamins
has many derivative compounds which
functional efficacy are also vary against various
infections, there is limited reports in different
vitamin derivate studies on their efficacy.
Because of each derivative has different range
of absorption, digestion and Bio availability.
6.3 Cancer Vaccine
There are different types of cancer
vaccines present only at research level, among
all the vaccine stem cell based cancer vaccine is
one of the vaccines. Prof, Eaton’s team done a
experiment in embryonic stem cell based cancer
vaccine in mice. The results suggest that the
exciting possibility of embryonic stem cell
vaccines might prevent cancer. At the end of
the experiment, all the mice were then exposed
to chemicals that cause lung cancer. All the un
vaccinated mice developed the disease.
There are lesser experiments trials
present in parasite vaccine research because
the molecular level mechanisms about
pathogenesis and parasite-host immune
reactions are not fully understood. Still there is
no proper vaccine for malaria. Malaria killed
thousands of persons in Africa and some parts
of Asia in every year. First we should
understanding the complex mechanism of host-
parasite pathogenesis then we will develop
vaccines. I hope it will present very near future.
The similar thing is happen in filariosis. The
filarial vaccine research is still going on.
6.5 Therapeutic vaccine
The therapeutic vaccine contains
normal vaccine components with Antimicrobial
medicine. It will useful to after the infectious
condition. Here anti microbial medicine clears
the pathogen load and remaining vaccine
components activate the immune system
against diseases and also reset immunological
memory. It mainly helps to solve many
problems such as many infectious diseases,
cancer, metabolic disorder diseases, Auto –
immune diseases, etc.
Fig 3&4 Drug Screening for Therapeutic vaccine
preparation.
6.6 Adjuvant
Adjuvant is the important part of vaccine,
the main functions of Adjuvant is boost up the
efficacy of vaccine now chemical adjuvant one
used. But there is lot of scope in biological
source of adjuvant. If we use bio-substances the
side effects are minimum and they are also
natural and eco friendly. There are lot of new
scope in marine microbes, plants and animals.
Only thing is we should screen the source one
by one. We can get lot of new adjuvant in
future.
6.7. Preservatives
The main function of the preservatives in
vaccine is avoiding microbial contamination in
the vaccine during storage. Now chemical
preservatives are used. Naturally many Bio-
Preservatives are available in natural resources.
The only thing is we should search and select
best preservatives from natural sources. Many
herbal sources, secondary metabolites from
microbes and plants gives new scope for
develop best preservatives for vaccines.
6.8 Allergies
In the allergies vaccines are developed to
specific antigens. This antigen is called allergen.
When an individual, who was previously
sensitize is exposed to that antigen again, his or
her immune system reacts to it in a damaging
manner. Now for happens harmful reaction,
vaccines are designed and experimental trials
going to reduce the harmful action of our
immune system.
6.9. Metabolic Disorder Diseases
Metabolic diseases are now one of the
targets for vaccine development. Due to
changes in food habits, working style, stresses
and life style leads to exposure of many
metabolic diseases. These are mainly effect
younger generation and working people. So we
need to develop new vaccines. Now scientist
focus on metabolic disorders, they give more
importance to hyper-cholsterolemia, which is
commonly affect young working people due to
high work pressure and also food habits.
6.10. Birth Control
Population explosion is a major threat in
developing countries. It leads to poor,
unemployed explosion, more infectious disease,
poor sanitation and hygiene. There are so many
methods are present for control the population
explosion. Prevention of pregnancy through
vaccination is one of the new approaches.
Pregnancy prevention vaccine has been an
effort in many countries. Because long duration
of immunity seen with traditional vaccines.
6.11. Vaccine Failure
Vaccines do not guarantee for complete
protection from a disease. Even after a
vaccination, there is still possibility that a
vaccinated person may get the disease
sometimes this is because the host’s immune
system simply doesn’t respond adequately at
all. This may be due to low level of immunity in
general (diabetics, steroid use, and HIV
infection) or just weak immune system, the
host’s immune system does not have a beta-cell
which capable of generating antibodies to that
antigen. Even if the hosts develops antibodies,
the human immune system is not perfect some
germs can mutate (the common cold) and in the
any case the immune system might still not be
able to defeat the infection 2
Polyvalent vaccines that contain there
attenuated viruses have been implicated in
including immune dysfunction. It is also leads to
vaccine failure. The other factors that can cause
immuno-suppression, malnutrition, concurrent
infections, not allowing enough time between
scheduled vaccinations and the uses of drugs.
Another cause of vaccine failure is
incorrect administration including splitting a
viral between individuals. However the most
common reason for vaccine failure is thought to
be the presence of maternal antibodies. This is a
passive immunity gained from the dam’s
colostrums during the first 72 hours of nursing
maternal antibody interferes more with level
vaccines than bacterial vaccines
6.12. Economics of Vaccine Development
Economics play important role in vaccine
development in developing countries. Many
diseases mostly demanding for vaccines, such as
HIV, Malaria, Tuberculoses, Typhoid and
Filariosis, in poor countries. So if the
government policy makers support the
industrial people may possible to reduce the
cost by various financial supports, exemption
from various taxes. Another way to solve this
problem is provide low cost highly efficient
technical for vaccine development.
6.13. Vaccinomics: Vaccinomics is a new
branch of bioinformatics which is the
integration of immune-genetics and immune-
genomics with system’s biology and immune
profiling. Vaccinomics is based on the use of
cutting edge, high dimensional assays and novel
bio informatics approaches help to develop next
generation vaccine and the expansion of our
capabilities in individual medicine .Vaccinomics
also help to develop new vaccines against hyper
variable viruses, with the greatest current
impact on public health.
References
1.Miranda Hiffi (2006) stem cells may lead to cancer vaccine
source. EORTC-NCI-AACR symposium of mole color targets and
cancer the practice prague, Czech Republic Nov-07.10.2016. news
release , European organization of research and treatment of
cancer.
2.www.wikipedia.com
3.www.accrs escexcellenie.org
4.Gregory.A,Poloand,Inna.G/OV4.Gregory.A,Poloand,Inna,syan
milkova, Richard , Kenedy, Iana.H. Haralesnbiaia and Robert.
M.Jcbbson (2011) Vaccinomics and a new paradigm for the
preventive vaccines against viral infections ,Omics Journal of
Integrative Biology 15 (a): 625-636.
4.Gregory.A,Poloand,Inna,syanmilkova, Richard , Kenedy,
Iana.H. Haralesnbiaia and Robert. M.Jcbbson (2011)
Vaccinomics and a new paradigm for the preventive vaccines
99against viral infections
