Indian Journal of BiotechnologyVol 2, October 2003, pp 494-498

Recent Advances in Vaccinology

Y Udaya Bhaskara Rao*Pasteur Institute of India, Coonoor 643 103, Nilgiris, India

Received 26 August 2002; accepted /6 December 2002

Vaccination continues to be the most cost effective, safest and most powerful tool of medicine for preventingsufferings, disability and death from infectious diseases. The traditional vaccines were developed when one knewvery little about how the diseases were caused and how the immunity worked. Most of the vaccines were developedby trial and error in animal and human experiments. With the recent advances in molecular biology, immunologyand recombinant DNA technology, one can understand how the antigens are processed and presented to the immunesystem and how that affects immune response. Also the current researches are underway into the mechanism of useof pathogens to infect and cause disease. The developments in vaccinology with DNA vacinnes, transgenic plantvaccines and combination of vaccines promises an exciting area in prevention and control of infectious diseases.

Keywords: vaccination, DNA vaccines, immunization, immune response, cytotoxic T ceUs

IntroductionThe sciences of vaccinology and inmmunology

were born two centuries ago when Edward Jennerproved scientifically the prevention of small pox byinoculation of cow pox virus. This weakened form ofsmall pox was called a "Vaccine" from Vacca, theLatin word for "Cow". Subsequently, it was LouisPasteur who suggested that all the inoculations to becalled "Vaccinations" to honour Edward Jenner.Today, the most accurate term "Immunization" ispreferred instead of vaccination. Vaccinationcontinues to be the most cost effective, safest andmost powerful tool of medicine for preventingsufferings, disability and death from infectiousdiseases.

The wide spread use of vaccines over the last fewdecades has resulted in a reduction in the incidence ofmany infectious diseases in developed countries andespecially in the prevention and control of nine majorinfections, e.g. diphtheria, mumps, rubella, tetanus,poliomyelitis, measles, yellow fever, pertussisincluding the eradication of small pox (Moxon, 1990).It is now possible to control hepatitis- B virusinfection with recombinant hepatitis vaccine (Kurstak,1999).

Development of a large number of new vaccinescould greatly reduce the estimated 12 million deaths

*Tel: 0423-2238250,2231250, 2231846Fax: 0423-2238250,2231655E-mail: piicnr@md4.vsnl.net.in

in children caused by infectious diseases in each yearworldwide (CVI Forum, 1999).

History of VaccinationSome Indian Buddhists drank snake venom in the

7th century in an attempt to become immune to itseffect. They may have been inducing toxoid likeimmunity (Plotkin & Plotkin, 1999). The ancientpractice of variolation by Chinese for preventingnatural small pox by inoculating pus from small poxpatients was introduced in Europe in the earlyeighteenth century.

The first milestone in vaccination was laid byEdward Jenner in 1796 with a systematic, successfulattempt at eradication of small pox. Subsequently.Louis Pasteur accidentally observed that chickenCholera bacillus cultures left on the bench for twoweeks lost their pathogenicity (Pasteur, 1880) andretained the ability to protect birds against subsequentinfection by them lead to the discovery of the processof attenuation and the development of live vaccines.In 1885 he developed first Rabies vaccinesuccessfully, providing attenuated germs (virus) wheninjected into the body confers immunity against thedisease. Louis Pasteur is one of the pioneers in thefield of Vaccinology and it was he who coined theterm 'Vaccine' for such prophylactic preparations.Calmette and Guerin developed a stable andattenuated strain of Mycobacterium bovis againsttuberculosis, by repeated subculturing of tuberclebacillus originally isolated from a cow by Nocard in

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1902 (Calmette et al, 1924). The introduction ofinactivated vaccine in 1890 heralded a dramatic shiftin the methodology used to produce safe vaccines.Numerous bacterial and viral vaccine preparationscontaining either whole-inactivated bacteria, virus orsemi purified detoxified bacterial toxins wereintroduced during early twentieth century (Table 1).

Vaccinology: Traditional TechnologiesIn the pre-recombinant DNA era, most of the

vaccines were developed by a process of trial anderror in animal and human experiments. In tropicalcountries, vaccines are not available for manydiseases and the old conventional and traditionalapproaches to vaccination are not successful. Thereasons for failure are inability to grow the organismsand complexity of the life cycle of organism. Theprincipal aim of vaccination is to prime the immunesystem to destroy specific disease causing pathogenbefore the pathogen can multiply enough to producethe disease or symptoms. The priming can beachieved by using killed or weakened (attenuated)version of pathogen or of some piece of the pathogen(subunit). The immune system by detecting thepresence of organism in vaccine behaves as if thebody was under attack and mobilises all its defenceforces to destroy the invader. The immune systemalso leads to the creation of memory cells, whichremain on alert in future attacks with the samepathogen.

Some of the traditional vaccines (Table 2), whichcontain killed pathogens, give rise to primarilyhumoral response and do not activate killer T cells.Such responses are ineffective against manymicroorganisms that infiltrate cells. Attenuated livevaccines, usually viruses, do enter cells and makeantigens that are displayed by the inoculated cells.They thus spur attack by killer T lymphocytes as wellas by antibodies. That dual activity is essential forblocking infection by many viruses and for ensuringimmunity. Life long immunity is generally conferredby live vaccines such as measles, mumps, rubella andsmall pox vaccines.

The eradication. of small pox showed that anattenuated virus vaccine could achieve the goal andstimulated greatly to implement the followingvaccination programmes:

(a) Expanded Programme on Immunization [EPI] in1974 with the objective of providingimmunization to all children of the world againstdiphtheria, pertussis, tetanus, tuberculosis,

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Table l--Chronology of important human vaccines used

Year Disease Vaccine

1721179818851896189618971923192619271955196119631974197619811986

'Variolation' with live virus'Naturally attenuated' cow pox virusAttenuated live and inactivated virusInactivated intact bacteriaInactivated intact bacteriaInactivated intact bacteriaPartially purified formalin toxoidInactivated intact bacteriaLive attenuated BCG strainInactivated virus IPV "Salk"Live attenuated virus OPV "Sabin"Inactivated and live attenuated virus

Small poxSmall poxRabiesTyphoid feverCholeraPlagueDiphtheriaPertussisTuberculosisPolioPolioMeaslesJapaneseencephalitisRabiesHepatitis BHepatitis B

Inactivated virus

Tissue culture inactivated virusInactivated plasma derivedInactivated, Recombinant

Table 2-Traditional vaccines used for immunization of infantsand children

Inactivated

BacterialPertussisTetanus toxoidDiptheria toxoidBCG vaccine

ViralPoliomyelitis - fPV

Live attenuated

MeaslesMumpsRubellaPoliomyelitis - OPV

Poliomyelitis and Measles. The EPI programmewas introduced in our country in 1978 and thevaccine coverage against all these diseases ismore than 80%.

(b) Global eradication of poliomyelitis by the year2000, which has been revised to 2005. More than85% of the countries have achieved the goalalready.

Vaccinology: Newer TechnologiesThe recent advances in pathogenesis are primarily

due to advances in molecular biology, immunology,virology, bacteriology, recombinant DNA technology,protein biochemistry, fermentation andpolysaccharide chemistry. The scope of traditionalvaccines has been the prevention or prophylaxis ofinfectious diseases whereas the new technologieshave extended the scope to include vaccines for non-infectious diseases (fertility, cancer and autoimmunediseases) and therapeutic vaccines (cancer, allergyand some infectious diseases). The new era ofvaccinology is based on a) DNA vaccines,b) transgenic plant vaccines, c) sugar glass vaccines,

496 INDIAN J BIOTECHNOL, OCTOBER 2003

d) skin patch vaccines, e) combination of vaccinesand f) immunomodulation delivery

a) DNA VaccinesThe most recent development in vaccinology is

immunization with polynucleotides or geneticimmunization or DNA immunization, which wasdiscovered by chance when plasmids carrying a genefrom influenza virus could be used to immunize miceand protect them from a lethal viral dose (Felgner,1997). Subsequently these findings were confirmed inanimals and expanded them into the development ofDNA vaccines. The DNA vaccines induce immuneresponse to viruses, bacteria, fungi and parasite(Table 3) and response was seen in birds,chimpanzees, cows, monkeys, mice, guinea pig and inmany other animals.

The DNA vaccines are different from traditionalvaccines and usually consist of plasrnids-small ringsof double stranded DNA derived from a bacterium(like Escherichia coli) but unable to produceinfection. One or more genes are isolated from diseasecausing pathogen and inserted into plasmids anddelivered by injection or gene gun into muscle. Theforeign genes express proteins (antigen) that elicithumoral and cellular immunity. The immunologistswere excited by the ability of DNA to elicit a broadimmune response of not only humoral immunity(antibodies) but also cell mediated immunityespecially cytotoxic T cells (CTL) (Donnelly et al,1997; Lewis et al, 1997; Tang et al, 1992; Ulmer etal, 1993; Hoffmen et al, 1994). The immune responseof CTL is noticed only when live or attenuatedvaccines were inoculated. The American Academy of

Table 3--Pathogens for which DNA vaccines provokes immuneresponse in animal models

Viruses Rabies, Rota virus, Measles, Rubella, Dengue,Ebola, Simian HIV, Herpes, Influenza, Japaneseencephalitis, Cytomegalo virus, Coxsackie virus,Foot and mouth disease virus, Hanta virus,Canine distemper virus, Canine parvovirus

Bacteria Mycoplasma, Shigella, Chlamydia, Clostridiumtetani (Tetanus), Mycobacterium tuberculosis(TB), Bacillus anthracis (Anthrax), Borreliaburgdonferi (Lyme disease), Enterotoxigenic E.coli, Mycobacterium sp.

Fungi Coccidioides immitis

Parasites Leishmania, Plasmodia (Malaria), Schistosomes,Onchocerca (River blindness), Taenia crassiceps,Trypanosoma cruzi

Microbiologists wrote "The use of DNA, a non livingagent, to raise cytolytic T cells" represents a milestonein vaccinology (CVI Forum, 1999).

The naked DNA expressing the full length Cas-Br-M murine leukemia virus genome induced virusspecific CTL-mediated response when inoculated newborn mice intramuscularly (Sarzotti et al, 1997). Inanother study, a single vaccination of DNA encodingthe G of CVS rabies virus inoculated intradermally orintramuscularly elicited neutralizing antibody andprotected mice against a highly lethal dose of a streetrabies virus (Ray et al, 1997). First time ovaladministration of a PLG (poly-lactide-coglycolide)encapsulated rotavirus VPG DNA vaccinedemonstrated protection against an infection agent(Chen et at, 1998).

The DNA vaccination offers many advantages(Table 4) over traditional vaccines. However, thereare a few lingering questions on safety of thesevaccines: a) Could the DNA vaccine integrate intohost's own DNA?; b) DNA vaccines stays for a longtime in host cells and could this prolonged stimulationcause chronic inflammation or auto-immune reaction?and c) Could antibodies to a DNA vaccine recognisethe vaccine recipient's own DNA and causeautoimmune disease?

The studies in animals have shown no ill effects tilldate. About 40 DNA vaccines against 30 differentconditions are under progress and five vaccinesagainst hepatitis-B, herpes Simplex, HIV, influenzaand malaria are being used in human trials. They arein early stage in examining their immunogenicity andsafety.

b) Transgenic Plant VaccinesThey are also known as edible vaccines. The

concept of expressing the viral epitopes and subunitsof bacterial toxins in transgenic plants have beensuccessfully shown in various studies and with ediblevaccines, the plants not only manufacture the antigensbut also deliver it into the host. The foreign DNA

Table 4--Advantages of DNA vaccines

1 Safe and no risk of disease2 Broad range of immune response3 Antigen confirmation normal4 No injection site reaction5 Duration of immunity long6 Simple, cheap to produce7 Multiple vaccines can be administered simultaneously8 Neonatal immunization in presence of maternal antibody

possible9 Various delivery methods available

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inserted in the genomes of plants-notably, tobaccoand petunia (Broglie, 1984), successfully expressedthe foreign DNA and using this approach vaccineswere produced in the edible parts of plants, whichcould be eaten when inoculations were needed(Arntzen & Mason, 1997, CVI Forum, 1996). In theearly 1990's, the potential of this concept wasannounced to the world at large. During that timeWorld Health Organization has given a call fordeveloping inexpensive oral vaccines that needed norefrigeration. In 1992, Artnzen and his groupannounced the first successful report of expression ofhepatitis B surface antigen in transgenic tobacco(Hoffman et al, 1994; Kurstak, 1999; CVI Forum,1999). Subsequently, transgenic tobacco and potatoplants expressed with the genes encoding E. coli heatlabile entero toxin (LT-B) or LT-B fusion proteinwere used to immunize mice and demonstrated serumand gut mucosal anti LT-B immunoglobulins (Haq etal, 1995). The first report of human trial of an ediblevaccine, published in May 1998 (CVI Forum, 1999),showed strong immune responses in 10 of 11volunteers who were given 100 g of raw potatoengineered to express the B subunit (LT-B) ofenterotoxigenic E. coli, a major cause. of travellersdiarrhoea. Edible vaccines provided protection oflaboratory animals against challenge by rabies virus,Helicobacter pylori and the mink enteric virus(Langride, 2000). The clinical trials in humanvolunteers with potatoes expressing the hepatitis Bsurface antigen and other candidate vaccines areunder investigation (Kurstak, 2001).

Some of the foods under study are alternatively toinjectable vaccines include bananas, potatoes andtomatoes as well as rice, lettuce, wheat, com andsoyabean. The edible vaccines are safe, lackcontamination with animal pathogens, requireminimal downstream processing, easy to administerand do not require refrigeration and syringes. Theplants can be grown locally, cheaply and they act asnatural bioreactors. The results of these studies areencouraging. The day is not far off when children areimmunized by munching on foods instead of shots.

c) Sugar Glass VaccinesBruce Roser, an Australian scientist in 1990, began

to use trehalose to vaccines (CVI Forum, 1999). Thesaturated solution of trehalose, on cooling, slidessmoothly from a liquid to a viscous and ultimately toa solid glass like state called "Sugar Glass". The glassdissolves on contact with water and releases its

contents. In trehalose formulations, the vaccineappears to suffer no detectable loss of potency afterlong periods of exposure to heat or freezing. Thesugar glass immobilizes, preserves and protectsprotein and other molecules from solutions. Themeasles vaccine losses over 90% of potency after twomonths at room temperature and shows no loss ofpotency as a trehalose powder under the sameconditions. The influenza and tetanus vaccines did notshow any adverse effect on potency even after storingat below -700 C for nine months. Pertussis antigenretained its original activity for three months at 370 C.However, the application of this technology haslimited interest for vaccine producers.

d) Skin Patch VaccinesSuperficial layers of skin are the perfect gateway to

many key areas of immune system known astranscutaneous immunization. This technologyappears to target highly accessible antigen presentingcells in the skin that can be exploited for a variety ofimmune outcomes. Cholera toxin (CT) is a powerfulstimulator of the immune system and humanvolunteers have been vaccinated by skin patchmethod. The results are encouraging and the CTmixed with vaccinating antigens have shown cellularand antibody mediated responses. This technology isbeing used to develop vaccines against viral andbacterial infections.

e) Combination of VaccinesCombination of vaccines, developed and exploited

in 20th century, can be both live [Measles-Mumps-Rubella (MMR), Polio] or inactivatedlkilled subunit[Diphtheria- Tetanus-Pertussis (DTP),. Hepatitis B(HB), Haemophilus influenza type b (Hib)]. Presently,the main thrust in the development of new combinedvaccines has been DTP based multi diseasecombination for pediatric use. Combined vaccines, ifdeveloped successfully, can reduce the infectionsduring the first two years of life from 15 to 5. Thecombinations in use are combined Pneuma coccal andMeningo coccal vaccines, DTP-Hib, DTP-IPY, DTP-HB, MMR and attenuated Varicella vaccine(MMRV), hepatitis A and B, DTP-HB-Hib and DTP-HB-IPV. Till recently, the Pertussis component ofDTP vaccine has been whole cell vaccine but it isbeing replaced with acellular Pertussis vaccine indeveloped countries. The advantages of combinedvaccines are: low cost, easy delivery, less number ofvisits, and less injections for immunization.

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The disadvantages of combined vaccine arereduced immunogenicity (interference) and increasedside effects. Each combination needs to be testedextensively before licensing.

1) Immunomodulation DeliveryModulation of the immuno responses is now

possible with cytokines and new adjuvants. The use ofimmunomodulators offers the hope of developingvaccines against infections that do not stimulatestrong natural immunity. In coming years thisapproach will be used against chronic infections likehepatitis B for developing therapeutic vaccines calledPharmaccines. This approach could also be used inconditions where there is malfunction of the immunesystem like allergies and autoimmune diseases.

ConclusionThe current boom in vaccine research, supported by

immunology, molecular biology, biochemistry andrelated fields, will add to many new vaccines forimmunization of young children. In 1960, vaccinationwas available against 15 human infectious diseaseswhereas in 1998 vaccines are available against 37diseases. Presently, it is well known also how theantigens are processed and presented to the immunesystem and how that affects immune responses. Also,Rand D is underway into the mechanism ofpathogens use to infect and cause diseases. There aremore than 500 candidate vaccines in the pipeline. Theprinciples of vaccination are being extended to someform of blindness, cervical cancer, gastric ulcers,coronary artery diseases, diabetes and peanut allergy,etc. However, the experiments with HIV, TB andmalaria over the past 15 years have shown thatadvances in knowledge in some other areas are alsoimportant and the researchers are quite optimistic ofdeveloping new vaccines against.these threats.

AcknowledgementThe author is thankful to Mrs J Sunitha Raju for

typing the manuscript.

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