innovative vaccine for typhoid fever
TRANSCRIPT
Dedicated to My Best friend
Mr. NOVA,
First Edition-2017
ISBN 978-9982-22-636-3
Author
Dr.M.Muruganandam,
Email [email protected]
Publisher
Einsteein Bio-engineering Research Foundation, South India.
Preface
Multidrug Resistant Typhoid pathogen is
slowly increasing in developing countries. We need
suitable Vaccine for save millions of school children’s
life from Typhoid fever .In this work we have taken a
preliminary step towards new generation vaccine
development for Typhoid fever. My aim is to develop
new vaccine against multidrug resistant typhoid
pathogen and reduce global disease burden. I have
referred many researchers work for preparation for
this manuscript .I thanks all of them. I thank and
Honoured International Vaccine Institute, World
Health Organization and International Society of
Infectious Diseases for their Noble service.
M.Muruganandam.
Content
Chapter-1. Global Disease Burden
1.1 History
1.2 Enteric Fever
1.3 Disease Burden
1.4 Multi Drug Resistance
1.5 Prevention Method
Chapter-2. Inactivated vaccine
2.1 Vaccine
2.2 Vaccine Development Process
2.3 Inactivated Typhoid vaccine
2.4 Lab Trial
2.5 Current Status
Chapter-3 Current Vaccine
3.1 Ty21a.Live vaccine
3.2 Vi-capsular Polysaccharide vaccine
3.3 Effectiveness of Vaccine
3.4 Vaccine under Development
3.4.1 Vi-conjugate vaccine
3.4.2 Oral Live vaccine
Chapter-4 Nucleotide vaccine
4.1 Plasmid DNA
4.2 Mutant Strain
4.3 Engineered Plasmid DNA
4.4 Plasmid DNA with Protein
4.5 Lab Trails
4.5.1 Nucleotide vaccine
4.5.2 Optimization
4.6 Vision for Future Vaccine.
1. Global Disease Burden
1-1 Historical Events
*Alexander the Great (356-323 B.C) died in typhoid
fever at Babylon.17
*Salmonella typhi was observed and cultured for the
first time in the early 1880’s in Philadelphia.
*1897 English bacteriologist Almroth wright
introduces a killed (Heat – inactivated, phenol –
preserved, whole – cell) typhoid vaccine in Britain.
*1898-9 vaccination trials in the Indian Army
produced excellent results and typhoid vaccination
was adapted for the use of British troops serving in
the second Boer war (1899).18
*1909-typhoid vaccination starts in U.S.Army.
*1911-typhoid vaccination required for entire
U.S.Army and Navy.19
*The impact of typhoid vaccination in US armed
forces.
*World war I, 1917-1918
*2000 typhoid cases, 227 deaths
*42 typhoid cases per 100,000 soldiers.
*World war II, 1941-1945
*5 typhoid cases per 100,000 soldiers.
*1914-Typhoid vaccine first Licensed for the U.S
general population.
*July 16, 1952 – Heat / Phenol inactivated typhoid
Vaccine by Wyeth licensed in U.S
*1960s Acetone – inactivated whole-cell typhoid
vaccine developed.
*Dec.15, 1989- A live oral typhoid vaccine (Ty21a,
vivotif Berna by swiss serum Institute) licensed in U.S.
*No.28,1994 Typhoid Vi-Polysaccharide inactivated
injectable polysaccharide vaccine prepared. (Typhim vi
by Aventis Pasteur licensed in U.S).20
*Vi-Vaccination program for 2-5 years old in Delhi,
India.
*Mass Vaccination campaigns in several provinces and
districts in china for school children and food handlers
in mid – 1990s.
*Annual Campaigns in Vietnam for 3-10 years old
children in a limited number of high-risk districts since
1997.*Pondicherry and Fiji Vaccination after
outbreaks.*Srilanka vaccination campaigns for
internally displaced people (IDPS) and Food handlers.
Demonstration projects in Nepal and Pakistan.
*In view of continued high burden of Typhoid Fever
and increasing antibiotic resistance and given the
safety, efficacy, feasibility and affordability of two
licensed vaccines (Vi and Ty.21a), countries should
consider the programmatic use of typhoid vaccines for
controlling endemic disease.20
1.2. Enteric Fever
Typhoid Fever is caused by bacteria called
salmonella typhi. Typhoid caused a high Fever,
weakness, stomach pains, headache, loss of appetite
and sometimes rash. If it is not treated, it can kill up to
30% of people who get it.
Some people who get typhoid become
“Carriers”, who can spread the disease to others.
Generally, people get typhoid from contamination
food or water.2
The Typhoid fever causing pathogens are
salmonella enteric serovar typhi (Typhoid) or
Salmonella enteric serovar paratyphoid A,B and C
(paratyphoid) and caused enteric disease in school
aged children and younger people in developing
countries. As the results of disease, there is in an
estimated 216,000 – 600,000 deaths per year. Among
these deaths, Majority cases occur in Asia.3
Salmonella belongs to the group of entero
bacteriacae that are aerobic and Gram – negative
bacteria. salmonella typhi is transmitted via the oral-
Faecal route through contaminated food or water.
Usually it is associated with poor sanitation and
hygiene practices.1
Humans are the sole reservoir of this organism,
more amount of organism present in their gall
bladders. But it does not infect animals. Food may
become contaminated from food handlers or via the
use of contaminated water during processing. Shell
fish grown in polluted water may also accumulate the
organisms within their tissues. Water that has been
contaminated human faecal pollution is a transmission
route. It survives in Sea water up to nine days and in
sewage for weeks has been recorded. Survival in
ground water was better than in pond, stream or lake
water. The main transmission route is from
contaminated water or from human carriers.4
1.3. Disease Burden
The WHO estimate of the global typhoid disease
burden, based on a study from 1984, in an around 17
million cases and approximately 500,000-600,000
deaths per year.5, 6 In Asia 90% of typhoid – related
deaths occur.7 The typhoid fever mainly spread in
some parts of developing countries in Asia, Africa and
south America. The multi drug resistant strains of
S.typhi are increasing in incidence in Africa with 75-
82%.8 in recent years.
In 2004, highest incidence presents in south-
central Asia and south-east Asia. In 2012- Greatest
increased incidence in sub-Saharan Africa. In Asia
highest numbers present in Kolkata, Karachi, North
Jakarta, Hue, and He chi The main endemic areas are
Chile, South Africa, Nepal, Indonesia, India, Pakistan,
China and Vietnam.3
1.4. Multi Drug resistance Typhoid (MDR)
This resistance problem can be seen
simplistically as an equation with two main
components. The antibiotic or antimicrobial drug,
which inhibits susceptible organisms and selects the
resistant ones; and the genetic resistance determinant
in micro-organisms, selected, by the antimicrobial
drug .9, 10 Drug resistances emerges only when these
two components come together in an environment or
host, which can lead to a clinical problem. Selected
resistance genes and their hosts spread and propagate
under continued antimicrobial selection to amplify
and extend the problem to other hosts and other
geographic locations.
There are more than 15 classes of antibiotics
whose targets are involved essential physiological or
metabolic functions of the bacterial cell. None has
escaped a resistance mechanism.11
Millions of kilograms of antimicrobials are used
each year in the prophylaxis and treatment of people,
animals and agriculture globally. 11, 12, 13, 14
The multi-drug resistant S.typhi has spread too
many parts of the world. In the limited the ability of
treatment of typhoid fever with commonly-available
Antibiotics, increasing the costs of treatment and
raising the spectre of higher case fatality rates.
This multidrug resistance strains of S.typhi
resistance to first line antibiotics such as
chloramphenicol, ampicillin, co-trimoxozole. It is first
appeared In South Asia and Middle East and rapidly
spread to East Asia and Africa.15 Now these MDR –
S.typhi is increasing in incidence in Africa with 75-
82%.8 MDR typhoid has been associated with more
severe illness and higher rates of complications and
deaths, especially in children under two years of
age.15,16
1.6 Prevention Methods
*Safe Water
Typhoid fever is water borne disease and the
main preventive measure is to ensure the access to
safe water.
*Food Safety
Contaminated food is an important vehicle for
typhoid fever transmission.
*Sanitation
Proper sanitation contributes to reducing the risk,
of transmission of all diarrheal pathogens.
*Health education
Health education is paramount to raise public
awareness on all preventive methods.
*Vaccination
Safe and efficacious vaccines are available.
Literature Cited
1) Imrankhan.m (2015). Typhoid Fever vaccines-Asian Pacific
Vaccinology meeting. No 30-Dec-3, 2015. Inter Continental
Holiday. Inn, Bangkok, Thailand.
2) Typhoid vaccine (2014) med line plus. Drug information US
National Library of medicine, National Institute of Health.
www.nlmnih.gov
3) Background paper on vaccination against Typhoid fever using
New – generation vaccines presented at the SAGE Nov-2007
meeting-WHO.
4) Salmonella typhi (2001) prepared for ministry of Health by
ESR.Ltd.-online reference.
5) Typhoid vaccines WHO Position Paper. Wkly
Epidemial.Res.2000;75:257-264
6) The diagnosis, treatment and prevention of typhoid fever.
Geneva:WHO,2003
7) Crump J.A, Luby. S.P, minta. E.d.(2004) The global burden of
typhoid fever. Bull.WHO.(82(5):346-53.
8) Kariuki S.Revathi.G.mulodi:-S.Mwitwiria.s; Mungalo.A, Mirza
A., Hart. C.A. Characterization of multidrug resistant typhoid
out breaks in Kenya J.clin. micro.bio.2004; 42:14177-14182
9) Levy, S.B. Balancing the drug resistance equation, Trends
microbial.2.341-342(1994).
10) Levy.S.B. Gerrod Lecture. Factors impacting on the problem
of antibiotic resistance. J.Antimicrob. chemo. ther.49,25-
30(2002).
11) Levy.S.The Antibiotic paradox:How misuse of Antibiotics
Destroys their curative powers (Press Cembridge.,2002).
12) Mellon,m.Benbrook,C and Benbrook., K.L. Hogging it:
estimated of antimicrobial abuse in live stock (UCS
Publications,Cambridge,U.K.2001).
13) U.S. congress.office of Technology Assessment. Impacts of
Antibiotic Resistant Bacteria (OTA-F-629,U.S.Government
Printing office,Washington,D.C.1995).
14) Stuart.B. Levy and Bennie marshall (2004) Antibacterial
resistance world wide:Causes,Challenges and responses
Review Vol.l0, Num-12., Nature medicine
supplement.PP:S122-129.
15) Bhutta.ZA, Naqv. S.A.Razzaq.R.A., Farooqui.B.J, multi-drug
resistant typhoid in children: prevention and clinical
features.Rev.Infect.DIs.1991:13:832-36.
16) Bhutta.Z.A., Impact of Age and drug resistance on
mortalittyphoid fever.Arch.Dis.child.1996:72:214-217.
17) Oldach.D.W,Richard.RE,Borza.E.N,Benitez.RM.N.Engl.J.med.1
998.Juhll; 338(24) :1764-9(1998)
18) Cantile.N.History of the Army medical Dept.Vol.ll.Ed in burgh
and London: Church ill Livingstone,1974:230-373.
19) Grabenstein.J.D.et.al.Immunization to protect the U.S Armed
forces:current practice and prospects. Epide
mid.Rev.2006;28;3-26.http://www.immunix org/timeline.
20) Who -2008 WHO Position Paper on Typhoid fever.83-49-
60.(2008)
2. In activated Vaccine
2.1 Inactivated vaccine
Virulent bacterial pathogens have been killed by
heat or chemical or Suitable antibiotics, then it will use
as vaccine. This type of vaccine is called Inactivated
Vaccine. It cannot produce disease. But it can induce
more immune response in the host.
2.2 Vaccine Development Process
*During vaccine preparation, pathogens are
inactivated by heat. These vaccine called Heat killed
Vaccines.
*Sometimes pathogens are inactivated by chemicals
especially, Acetone, formalin, etc.
*During chemical inactivation, generally used low
concentration of chemicals with long time incubation
is important because these types of treatment help to
preserve the structure of the pathogen. These
inactivated organisms induce good immunity in the
host.
*Another type is use suitable antibiotics to kill the
organisms.
*All these inactivated cells are finally introduced in
culture medium. For check the status of live or dead, If
they completely inactivated will not grow in the
medium.
*The main drawback of this type is, it will not multiply
inside the host. So during long term immunity, it will
slowly fade their ability.
*Another drawback is during preparation of this
vaccine, large quantity of organism is required.
*However, if it injects into the host, it produce good
immunity and also induce good immunological
memory.
2.3 Inactivated Typhoid Vaccine
*English Bacteriologist Almroth Edward wright
developed an effective whole cell typhoid vaccine that
was introduced in 1896.
*It is Heat – inactivated, phenol preserved whole cell
vaccine, used in Britain.1
*The Heat – inactivated whole cell vaccine showed
protective efficiency rates that in controlled studies
ranged between 51% to 67%.2
*One dose inactivated vaccine provides protection. It
should be given at least two weeks before travel.
A booster does is needed every 2 years for people
who remain at risk.
*Some people should not use typhoid vaccine, due to
the following reason
*Should not be given to children younger than 2 years
of age.
*Anyone who has had a severe reaction to a previous
dose of this vaccine should not get another dose.
*Anyone who has a severe allergy to any component
of this vaccine should not get it.
*Anyone who is moderately or severely infected all at
the time the shot is scheduled should usually wait
until they recover before getting the vaccine.
*Serious problems from either typhoid vaccine are
very rare. Anyhow, sometimes it creates some
problems. These mild reactions are
*Fever (Up to about/Person in 100)
*Headache (Up to about /Person in 30)
*Redness or swelling at the site of the injection
(Up to about /Person in 15).3
2.4 Lab Trial
In our lab work, mutant strain whole cell
inactivated vaccine was developed and tested.
Usually, in the pathogenesis experiment, researchers
use salmonella typhimurium in mice. However in this
study, we use salmonella typhi in albino rats. Because
we are not entered the pathology, But just check the
antigenic property of immunogen.
In this experiment, salmonella typhi was
collected from patients sample into local hospital
Laboratory and then it was brought to the Lab then
routine microbiological and biochemical tests were
carried out for confirmation. After that it was put into
sub culture. In this experiment, first five spread plates
of salmonella typhi were prepared and expose to U.V
radiation at different time intervals. Such as 0,2,4,6
and 8 minutes. After radiation all the plates were
stored in 37’C for 24 hours.
Later, pathogens of all treatments
were isolated and inoculated into the broth than after
24hours; the cells were isolated and inactivated by
using 0.5% of formalin at 4’c in overnight period. Five
sets of albino rates (three for each set) were taken
and intramuscularly injected. After two week, blood
samples were collected and analyzed haematological
parameters and antibody levels were studied by using
96 well litre plate.
Some people may be protected by serum
antibodies to salmonella typhi but susceptible. People
may require cell mediated immunity. The whole cell
inactivated vaccine can induce humoral and cell
mediated immunity. So in this study normal and
mutant strain whole cell vaccine Influence on immune
responses were analyzed.
The maximum antibody response was observed
in four minute U.V treated strains. But in the WBC
count, maximum range was observed in normal strains
(control) compared to other mutant strain vaccines.
The anti body a level was slowly increased up to four
minute treatment, after that. It was decreased. The
RBC Count has not much difference in all the
treatments.4But in the case of Staphylococcus aureus,
six minutes U.V treated mutant strain produce more
immune response compared to normal and other
srains.6
Table:1. Mutant Strains of salmonella typhi effect on Immune
responses in Albino rats.
U.V Treatment (Minutes)
Parameters 0 2 4 6 8
WBC Total count(cells/ccmm)
6400 5100 5900 4200 4400
Polymorphic neutrophill (%)
78 82 82 70 85
Lymphosite(%) 22 16 18 21 25
RBC Count(millions)
3.6 3.8 3.6 3.2 3.8
Haemoglobin(gm%) 10.5 12 10.5 10 11.5
Antibody Titre well 9 10 11 9 9
2.5 Current Status
In early 1990s, due to side effects the
inactivated vaccine usage was slowly replaced by new
generation vaccine. They have been available on the
market and they found to be safe and moderately
efficacious. One is oral live attenuated vaccine Ty21a
and another one is Vi-polysaccharide vaccine.
In conclusion, due to some draw backs, the
inactivated vaccine is slowly replaced by new vaccines.
Now further research is going on to improve the
efficacy of these vaccines and also try to develop new
generation vaccines in International Vaccine Institute
and World Health Organization.
Literature Cited
1) Cantlie.N..History of the Army medical Dept.Vol.ll.Edinburgh
and London. Churchill Livingstone, 1974, 230, 373.
2) Typhoid fact sheet for T. Sunami affected populations-
www.who.int./vaccines/intermediate /typhoid .htm –
available .
3) Typhoid vaccine (2012) –vaccine information
statement.www.(dl.gov/acdnes)/vacl/typhoid/default.htm.U.
S.Dept of Health and Human services, centers for Disease
control and prevention.
4) Muruganandam.M., J.E.John Solomon and Mahesh.S(2010).
Mutant strain vaccine for Typhoid. Env&Eco. 28(2B) 1414-
1415.
5) Background paper on vaccination against Typhoid fever using
New – Generation Vaccines – Presented at the SAGE
Nov.2007 meeting-WHO.
6) Muruganandam.M and K.N Verrayee Kanna (2010) Mutant
Strain Vaccine for Staphylococcus aureus.J.Curr.Sci.15
(1):229-232.
3. Current Vaccines
Typhoid vaccines completely prevent typhoid
fever. Now there are two types of vaccines available in
market. One is live attenuated vaccine and another
one is Vi-polysaccharide vaccine. The inactivated
whole cell vaccine is previously used. Now it is
replaced by these new generation vaccines. They have
some more advantages. So now it is universally used.
3.1 Ty2la – Live Vaccine
The Ty21a is licensed for use from age six years
and older; Boosters are recommended every five
years.9 The Th21a vaccine is a live attenuated strain of
salmonella typhi Ty21a.
The vaccine is usually adiministrated orally as
enteric coated capsules and registered for use from six
years of age. It has shown a protective efficaly of 62%
for at least 7 years. A liquid formulation of the Ty2la
vaccine can be taken by children as young as two
years of age and it has proved more immunogenic
than the capsular formulation.
In a field trial in Chile among more than 36,000
vaccine provided in the age 5-19 years old, this
formulation provided 79% efficacy five years after
immunization. The Ty2la is remarkably well tolerated.
The vaccine may be given simultaneously with other
vaccines including cholera, measles and MMR. The
vaccine required stronger at 2-8 degree Celsius.10
3.2 Vi-Capsular Polysaccharide vaccine
The Vi-Capsular polysaccharide vaccine is
licensed for use from age of two years and older and
boosters are required every three months.11 The Vi-
Polysaccharide vaccine composed of purified Vi-
Polysaccharide from S.typhi. It is administrated
subcutaneously or intra muscularly as one dose of
25mg. This vaccine confers protection seven days after
injection.
Trial in Nepal involving people age 5-44 years
showed 75% protection during the 20months of active
surveillance. In a recent study in South Africa, 55%
efficacy was demonstrated three years after
immunization of children 5-16 years old. The vaccine
requires storage at 2 to 8 degree Celsius.3
3.3 Effectiveness of Vaccines
In south western china-Guangxi province, Vi-
Vaccination was provided to students during a school
based campaign vaccination early in the outbreak,
using a locally, provided Vi-Vaccine has effectiveness
of 71% nearly as much as the vaccine. Efficacy among
those whom had been vaccinated with Vi the year
before (73%). This study that the single dose Vi-
Vaccine is effective for outbreak control, as well as for
use against endemic disease.3
A mass vaccination campaign involved almost
20,000 Russian soldiers stationed in Dustanbe,
Tajikistan during the large epidemic of typhoid fever in
1996-1997, demonstrated the effectiveness of the
vaccine in reducing typhoid disease among these
individuals who were affected by inadequate food,
water and general living conditions during the
outbreak.2
Ty2la vaccine has thus for been limited to use
primarily for travellers and has not been used by
developing countries for control of endemic typhoid.
However, it is worth nothing that the large scale trials
of this vaccine done in several hundred thousand
school children. They employed classrooms as the unit
of randomization, much as would occur in a practical
school based immunization programme.
Indeed, the trial in Area Sur and Area central of
Santiago was undertaken as a post-licensure
effectiveness trial that represents a collaboration of
the ministry of Health and ministry of Education
undertaking a School - based immunization
programme that targeted – 225,000 School Children.
The controlled field trials in chile and Indonesia
found the Ty2la vaccine to be efficacious and to confer
several year- protections for 5-7 years. Moreover, as
noted earlier, ty2la was also found in the children to
be associated with herd protection of non-vaccines in
these studies. In addition, a large randomized place to
controlled study of Ty2la conducted in 6-7 years old
school children in Alexandria, Egypt showed efficacy of
96% after three years. The study was the first to show
the efficacy of a liquid formulation.4
3.4 Vaccine under Development
Typhoid vaccine development is currently going
in two main directions.
a) Vi- Conjugate vaccine and
b) Improved live oral vaccines
3.4.1 a)Vi-Conjugate Vaccine
The NIH - U.S developed a prototype Vi-
Conjugate Vaccine, using combinant exotoxin A of
Pseudomonas aeruginosa (Vi- rEPA) as a carrier
protein. The vaccine was found to be safe and to elicit
serum antibody levels in 2-4 year old Vietnamese
children that were three times higher than these
elicited by vi – in 5-14 years olds.6,7 In a phase III
double-randomized place to controlled trial among 2-5
years old in Mekong Delta of Vietnam, where typhoid
is highly endemic, a two doses regimen of the vi-rEPA
vaccine conferred 91% efficacy in preventing blood
culture-confirmed typhoid over 27 months of follow
up 6 and 89% after 46 months suggesting that the
vaccine is highly efficacious for at least four years.8
3.4.2 Oral Live – Attenuated Typhoid Vaccines
There are at least three oral live attenuated
typhoid vaccines in that are currently in stages of
clinical development. The aim has been to develop
more highly – immunogenic vaccine than Ty2la that
would provide protection after a single dose. All of
these vaccine candidates are derived from wild-type
strain Ty2, as are Ty2la and Vi.
All thus for appear to be markedly more
immunogenic than Ty21a.5 One live oral vaccine
candidate, CVD909, has been found to elicit gut-
derived antibody secreting cells that secrete Ig A anti
vi-antibodies, as well as cell-mediated responses and
antibody responses to other antigens. Now this
vaccine as well as CVD 908-htrA, Ty800 (developed by
the U.S.Firm AVANT) and ZH9 (developed by Emergent
Bio-Solutions in the U.K) have undergone safety and
immunogenicity trials.1
In Conclusion, Typhoid vaccine development
research may move mostly towards advancement of
current vaccines. It is not sufficient to control multi
drug resistant pathogens. It should need more effort
to develop next generation nucleotide vaccine. The
bacterial nucleotide vaccine trials open new scope in
the future generation vaccine development. These
research and development of new vaccines help to
control new strains of pathogens.
Literature Cited
1) WHO-2007-Background paper on vaccination against Typhoid
fever using New-Generation vaccines-present at the SAGE-
November-2007 meeting.pp:1-65.
2) Tarr.P.E., Kuppens.L;Jones., T.c, Ivoani f.B Aparin.P.G.,
Heymann.B.L., considerations regarding mass vaccination against
typhoid fever as an adjunct to sanitation and public health measures:
potential use in an epidemic in
Tajikistan.Am.J.Trop.med.Hyg.1999;61(1);163-170
3) Yang.H.H, Kilgore PE, Yang LH, park J.K, pan YF, kim.Yetal (1999) An
out break of typhoid fever, Xing -An country, people’s Republic of
china-Estimation of the field effectiveness of vi –polysaccaride
typhoid vaccine.j. Infect Dis 2001; 183; 1775-80.
4) Wahdan.M.H., Serie.C. Cerisier.Y, Sallam.S., Germanier.R., A
controlled field trial of live Salmonella typhi strain Ty2la oral vaccine
against typhoid: Three year results.J. Infect Dis.1982;145-292-96.
5)Levine.M.M., Typhoid fever vaccines. In: Plotkin As, orensten
W.A.,editors vaccines. 3rd edition.Philidelphia :WiB. Sawnders
Company : 1999, P. 781-814.
6) Berkly.J.A., Lowe.B.S,M., weang;- I, Williams.T., Bawni.E,
Mwarumba.S., Ngetsa.O., Slack mPE, Njenga.S., Hart.C.A. Maitland.K,
English.M., Marsh.K. Scott. JAh. Bacteroemia among children
admitted to a rural hospital in Kenya.N.Engl.J.med.2005;352:39-47.
7) Kossaczka.Z., Lin. Fy.Hova, Thuy.N.T.Van. Bay.p., Thanh.T.c. etal.,
Safety and immunogenicity of conjungate vaccines for typhoid fever
in adult, teenagar and 2-4 Years old children in Vietnam. Infect
Immun. 199;67:5806-10
8) MaiNL.Phan.VB,Vo.AH,Tran CT., Lin F.y., Bryla.DA, Chuc,
Schiloach.J., Robbins J.B, Schneerson.R., Szu sc.Peristant efficaly of Vi-
conjungate vaccine against typhoid fever in young children N.Eng
J.med.2003;14:1390-1391.
9) www.fda.gov/downloads/Biologicsblood vaccines/Approved
products/Ucmk2807 pdf.
10) Typhoid fact sheet for Tsunami affected populations-
www.sho.int/vaccines/intermediate/typhoid.htm.
11) Salisbury,D., Ramsamy.m., Noakes.K. Chapter 33 typhoid
(www.dn.gov.uk) Immunization against infections disease. Dept of
Health P.413. IS BN O-11-322 528-8
4. Nucleotide Vaccine
There are two types of nucleotide present in
bacterial cell. One is plasmid DNA and another one is
genomic DNA. The Plasmid DNA is extra chromosomal
DNA, present in the cytoplasm of the cell. It has small
nucleotides; it carries one or more genes which
regulate some specific functions. If Plasmid DNA is
digested by restriction enzymes, it will split into small
nucleotides fragments. These various fragments have
different levels of immunogenic properties. So these
are all helps to prepare new nucleotide vaccines
against typhoid fever.
4.1 Plasmid DNA
The plasmid DNA is self replicating double
stranded circular DNA molecules present in Bacterial
cell. It always carries one or more genes responsible
for useful characters. They have their own origin of
replication and they replicate independently. Usually
plasmid DNA used as vector for rDNA preparations. In
the extraction of plasmid DNA protocol, first described
about culture of bacterial cells then harvesting
analysis of bacterial cells. After that plasmid DNA was
isolated. During our lab trials, isolated plasmid DNA
was used as immunogenic which produce good
results. First naked plasmid DNA of Aeromonas
hydrophila was used as vaccine, tested in albino rats.1
It shows good results compared to other vaccines.
Similar trials are repeated in various bacterial
pathogens such as Escherichia Coli, Staphylococcus
aureus and salmonella typhi 2.
4.2 Mutant Strains
The mutant strain pathogens are some time
more virulent compared to normal strain. It is used to
prepare vaccines. The mutant plasmid DNA was also
acted as good vaccine. In the Staphylococcus aureus
mutant strain plasmid DNA act as good vaccines in
albino rats experimental trials, Compared to non
mutant strain pathogens. 3
4.3 Engineered plasmid DNA
The Engineered plasmid DNA nucleotide fragments
produce good immune response in the albino rat. This
is proved by various experimental trials using in
different bacterial pathogen. The single and double
restriction enzyme digested plasmid DNA fragment
produce good immune responses in various bacterial
pathogens such as Staphylococcus aureus, Salmonella
typhi and Escherichia coli.4, 5, 6. This type vaccine has
many advantages such as DNA sequence decoding is
easy and preparation process is also easy compared to
long sequence DNA. The preparation cost is also low.
However the efficacy of vaccine is high compared to
other vaccines.
4.4 Plasmid DNA with protein
In this type, plasmid DNA is compared with
immunogenic proteins and inactivated Pathogen cells.
Which are produced good immune responses because
different type of immunogen has various range of
functional efficacy in immune system. The killed
pathogen and various antigenic proteins immediately
induce immune responses and immunogenic
nucleotide possible to produce long term immunity. It
will not fade at long time. This mixture of these
components produces more immunity. This type of
vaccine experimental trials was conducted in
Staphylococcus aureus. It produces good results.6
4.5 Nucleotide Vaccine
In this study, salmonella typhi was collected
from patient’s sample and maintained in the
laboratory. First, in the salmonella typhi Genomic DNA
and plasmid DNA were isolated. In this experiment,
two trials were conducted. In the first trial, Genomic
DNA, BamH-I digested genomic DNA, Pst-I digested
DNA and double digested DNA treatments with one
control treatment were maintained. The BamH-I
enzyme recognition sequence is 5’G GAT CC3’ and
Pst-I enzyme recognition sequence is 5’ CTG CAG3”. In
all the trials albino rats were used as experimental
animals. In the second trial, another four treatments
were used, such as whole plasmid DNA, Bam H-I
digested plasmid DNA, Pst-I digested plasmid DNA and
double digested DNA vaccine treatment. After one
week of first injection, same dosage was injected as
booster dose for all the treatments. After one week of
booster dose. Blood samples were collected and
analyzed. In the first trial, maximum immune response
was observed in Bam H-I digested genomic DNA
treatment. Second highest level was observed in
double digested DNA treatments. The lymphocyte and
polymorphic Nuetrophill levels are also higher in Bam-
H-I digested treatment. The minimum immune
response was observed in Pst-I digested DNA
treatments. So the Bam H-I digested segments has
more antigenic nucleotides compared to all other
vaccine treatments. In the second trial, highest
immune response was observed in Pst-I digested
plasmid DNA treatment. The maximum antibody level
was observed in Bam H-I digested DNA treatment. The
second highest immune response was observed in
double digested plasmid DNA treatment. The
minimum immune response was observed in plasmid
DNA treatment. The control (non-vaccinated) has
lesser immune response compared to other
treatments.5
Table-1 Typhoid Genomic and Plasmid DNA effect on
immune responses in Albino rates.
Parameters Control Genomic DNA
BamH-I Digested DNA
Pst-I Digested DNA
Doubled Digested Genomic DNA
WBC Total count(cells/ccmm)
4800 5000 6200 4200 5800
Polymorphic neutrophill (%)
60 60 60 50 55
Lymphosite(%) 38 35 35 48 45
RBC Count(millions) 3.7 3.8 3.9 3.2 3.0
Haemoglobin (gm % )
12.5 11.5 12 10 9.5
Antibody Titre well 7 9 10 8 9
Parameters
Control Plasmid DNA
BamH-I Diges.DNA
Pst-I Diges.DNA
Doub.Digested Plasmid DNA
WBC Total count(cells/ccmm)
4800 6,600 7100 8100 7600
Polymorphic neutrophill (%)
60 56 48 70 68
Lymphosite(%) 38 42 50 25 30
RBC ount(millions) 3.7 3.9 4.2 3.9 4.5
Haemoglobin(gm%) 12.5 12 12.5 12 13.5
Antibody Titre well 7 8 10 7 10
In conclusion, best vaccine candidate is double
digested plasmid DNA nucleotide fragments. (Bam H-I
+ Pst –I) and Pst-I digested plasmid DNA fragments. So
it is recommended for new DNA vaccine development
for typhoid fever. These nucleotide fragments are
immunogenic and it is suitable to prepare Typhoid
vaccines.
4.6 Vaccine Optimization
Optimization trial is a important step in vaccine
research. In this study two trials were conducted. In
the first trial, graded level of whole plasmid DNA was
orally provided to albino rats. In the second trial,
double digested plasmid DNA was provided at
different quantity to various group of albino rats. In
the previous experiment, maximum immune response
was observed in double digested plasmid DNA
compared to undigested plasmid DNA. For the
confirmation of this finding, we have conducted this
experimental trial. At the end of this experiment,
blood samples were collected for analysis.
In the first trial, maximum immune responses
was observed in highest quantity treatment. The
immune response was slowly increased, when
increasing level of whole plasmid DNA. The total RBC
count, packed cell volume levels were more or less
equal in all the treatments.
In the second trial similar results was observed.
But the highest immune responses was observed in
highest quantity of digested DNA fragments
treatments. So compared to these two trials, digested
plasmid DNA produce highest immune responses. The
DNA vaccine is new generation vaccine, it has many
advantages, and for typhoid fever still there is lesser
work in DNA vaccine development and optimization
process. In this study optimization was done in
typhoid nucleotide vaccine. Compared to undigested
plasmid DNA, the double digested plasmid DNA
treatments produce higher immune responses. In our
previous studies proves the similar results. Now this
study was ones again confirm these findings.
Finally it is concluded that, if increasing the level
of DNA leads to produce higher immune response up
to the saturation level in the host. The double
digested plasmid DNA fragment produce more
immune responses compared to undigested plasmid
DNA.
Table 2 Optimization of Salmonella typhi Whole Plasmid DNA
Parameters 0 µl 3µl 6µl 12µl 24µl
WBC Total count(cells/ccmm)
5100 6200 6300 6600 7200
Polymorphic neutrophill (%)
60 63 78 76 78
Lymphosite(%) 35 38 30 33 40
RBC Count(millions)
3.7 3.4 3.8 3.9 4.0
Haemoglobin(gm%) 9 10.5 9 9.6 9.2
PCV (%) 26 28 29 29 30
Table-3 optimization of Salmonella typhi Double Digested
Plasmid DNA
Parameters 0µl 3µl 6µl 12µl 24µl
WBC Total count(cells/ccmm)
5200 6,200 6600 6800 7600
Polymorphic neutrophill (%)
58 62 68 60 58
Lymphosite(%) 35 40 30 35 40
RBC Count(millions)
1.6 2.8 3.1 3.4 3.6
Haemoglobin(gm%) 7 8.6 10 7.6 11
PCV (%) 24 26 29 28 33
Fig-1&2 Vaccine development work.
Fig-3 Experimental Animal -Albino Rat.
4.7 Vision for Future Vaccine
*Currently available vaccines are live attenuated
vaccine and Vi-Polysaccharide vaccines.
*Now vaccine development research moves only few
directions. It is not sufficient to reduce the current
disease burden in the globe. But we should move in all
the directions
*We should try to develop new peptides immunogen.
It may be heat stress protein vaccine or Anti-idiotype
Proteins, Antigenic surface protein etc.
*We should also move to nucleotide immunogen
research that is why we have studied some
preliminary studies in nucleotide vaccine. It gives new
hope and step to solve the vaccine development
program.
*In the DNA vaccine, there are many opening to
research and develop new vaccines candidate against
Typhoid pathogen.
*In our lab trials digested plasmid DNA Nucleotide
immunogen produce good results. It gives promising
hope to develop new vaccines candidate.
*In the DNA vaccine, there are many advantages, such
as preparation method is easy and could maintain at
room temperature. So transport is easy.
*The DNA vaccine is new generation vaccine. The
nucleotide with suitable protein mixer gives new
scope to develop new generation vaccine against
Typhoid fever.
*Another important area is adjuvants research.Now
There are lot of bio-adjuvants are available. So
screening and selection of correct adjuvants or
mixture of adjuvants is another important step.
*The third thing is preservative selection. There are
various Bio-preservatives available and screening to
find new preservative is another important step.
*The fourth step is Economic of vaccine preparation. It
must be suitable for developing countries. So they can
develop their own medicine.
*Now multi drug resistant pathogens (MDR)
population is slowly rising in many parts of the
globe.So we should give special attention to this
problem we should develop new vaccines against
MDR-pathogens.
*Drug screening from marine sources is another
important scope. We should try to find out new
Antibiotics against MDR-pathogens.
*In the vaccination, pain free vaccination
development is good. Because typhoid mainly affect
school children population they cannot tolerate
painful vaccination. The oral vaccine is best. Otherwise
we should find out new pain free methods.
*For mass vaccination to children and people, oral
vaccine, edible vaccine, skin –ointment vaccine, patch
vaccine, Nasal spray vaccination, etc are the highly
suitable.
*Typhoid fever affect children, their immune system is
weak and undeveloped. So during vaccine
development program we should remember these
facts. Based on this situation we should design vaccine
for this weak immunity population.
Literature cited 1. Muruganandam.M.(2007). New DNA Vaccine for Aeromonas
hydrophila infection. Aqua Tech. Aug-PP: 79.
2. Muruganandam.M.(2012) short sequence DNA Vaccine. Book-
ISBN-978-9982-22-460-4
3. Muruganandam.M (2010).Plasmid DNA vaccine for
staphylococcus auras. J.Nat.Con:22(1): 73-76.
4. Muruganandam.M.(2010) DNA vaccine for Bacterial pathogen
Escherichia Coli. Int. J.Bio.Tech: 1 (2) :110-112.
5. Muruganandam.M. (2010) Engineered DNA Vaccine for typhoid.
J.Nat. Con.22 (1):123-126.
6. Muruganandam.M. (2013). Engineered plasmid DNA vaccine.
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