Recent developments in vaccine design

Key words: vaccine, immune response, HIV.

1. Introduction

Ability of an organism to evade infection by micro organisms is due to immunity, which could be active or passive immunity. Immunity could either be acquired from natural source i.e. from mother or from artificial source i.e. by injection of antibodies.

Passive immunity is induced by antibodies from human or animal source while active immunity is due to inoculation of micro organism as a whole live/killed or by antigen from micro organism. Edward Jenner and Louis Pasteur laid the foundation of vaccination or induction of active immunity. Emil von Behring and Hidesaburo Kitasato were the pioneers of passive immunization, they established that Immune response evoked in one organism can be transferred to other by injecting serum (containing antibodies). Passive immunization was the only preventive measure available before the dawn of vaccination and antibiotics.

Practice of vaccination was among the many achievements of mankind and what could be more rewarding than prevention of diseases. Vaccination along with sanitation and nutrition improved public health. HIV/AIDS, pandemic swine flu and bird influenza, sever acute respiratory syndrome (SARS) and bioterrorism has made created a spotlight on vaccinology.

Vaccinology is multifaceted with medical microbiology and immunology on one hand and public health and sociology on other hand.

Why vaccines are required?

Passive immunization is quick and effective but there are certain risks associated with it.

1. Passive immunization cannot be used in individuals with deficiency in synthesis of antibody as a result of congenital or acquired B-cell defects, alone or together with other immunodeficiencies.

2. It could not be used to Exposure or likely exposure of an individual to a disease that will cause complications (e.g., a child with leukemia exposed to varicella or measles), or when time does not permit adequate protection by active immunization.

3. Because passive immunization does not activate the immune system, it generates no memory response so the protection is transient.

4. If the antibody was produced in another species, such as a horse, the recipient can mount a strong response to the isotypic determinants of the foreign antibody. This anti-isotype response can cause serious complications.

5. Complement activation and deposition of immune complex in tissue leads to type III hypersensitivity reaction.

Active immunization as compared to passive immunization provides protective immunity and immunological memory. Re-exposure results in increased immune response that eliminates pathogen and prevents disease from occurring. As the name suggests active immunity involves formation of antibodies, Th1 and Th2 type CD4+ T cell response and CD8+ T cell response.

Table 1.1. Showing estimated infant deaths per year by various pathogens

Estimated annual deaths worldwide of children under 5 years of age, by pathogen

Pathogen Deaths(per million)

Pneumococcus

1.2

Measles 1.1

Hemophilus 0.9

Rotavirus 0.8

Malaria 0.7

HIV 0.5

RSV 0.5

Pertussis 0.4

Tetanus 0.4

Tuberculosis 0.1

Adapted from Shann and Steinhoff, 1999, Lancet 354 (Suppl II):7–11

Table 1.1 shows statistics of infant mortality per year. It depicts the need of biomedical research community to accept the challenge of developing better, safer, cheaper and easy to administer forms of vaccines.

What are vaccines?

A vaccine is any biological agent or chemical entity which is able to evoke an immune response without subjecting the individual at risk of contracting the infection. The main purpose of vaccine is to prevent infection.

WHO standards for an ideal vaccine are-

1. Affordable worldwide

2. Heat stable

3. Effective after single dose

4. Applicable to number of diseases

5. Administered by a mucosal route

6. Suitable for administration early in life

2. History

1st recorded attempts to induce immunity artificially dates back to 15th century by Chinese and Turks by variolation. But the history of practice of vaccination started in 18th with the emergence of small pox disease. It was the Edward Jenner, a British general practitioner, who first inoculated fluids cow pox pustules into small pox affected individuals and laid the foundation of vaccination. Edward Jenner’s dream of eradicating small pox become apparent in 1979, when the Global Commission on Smallpox Eradication certified the global eradication of small pox.

However, Jenner had no theory which could suggest how vaccination provided immunity neither he was able to establish the microbial basis of the disease. It was Robert Koch and Louis Pasteur who established the etiologic cause of the disease and specificity of immunity was more widely understandable. It was Pasteur who found out that attenuated Pasteurella septic gave immunity to chickens from fresh virulent cultures. He only introduced the rabies immunization and for the first time immunizing preparations was given the name vaccine in the honour of Edward Jenner and his work on vaccinia virus.

Pasteur believed that micro-organisms had to be alive inorder to evoke a successful immunity but it was challenged by Robert Koch who in 1896 after the discovery of Vibrio cholera developed vaccine for cholera by killing the whole bacterium, similarly vaccines for typhoid and plague were also developed but were less effective.

In early 1920, Antidiptheria toxin antibodies were obtained from horses after the discovery of antibodies by Von Behring and Kitasato and first active immunization against Diphtheria and Tetanus started, first with toxins and later in 1930s by toxoids.

With the advancement in microbiology techniques, by 1930’s clear difference between virus and bacteria could be made and year 1935 saw early viral vaccines for yellow fever and influenza coming up. The revolution in antiviral vaccines was witnessed in 1955 with availability of Dr. Jonas Salk’s formalin treated whole virus vaccine for the protection against polio however it was Sabin who in 1961 further developed polio vaccine by using live attenuated virus and developed oral poliomyelitis vaccine.

The eradication of polio was the result of success of polio immunization and laid way for the development of other live attenuated virus vaccines. In 1963, measles vaccine was introduced by Ender which was followed by mumps vaccine in 1967 and live attenuated rubella vaccine in 1968. In 1971, combined mumps-measles-rubella vaccine was introduced and some firms have now introduced tetravalent mumps-measles-rubella-varicella vaccine and these efforts have made several European countries free of mumps, measles and rubella transmission.

1970’s saw rise of molecular biology as an important discipline in biomedicine. Soon molecular biology became boon for better understanding of immunology and hence in vaccine development.

It was in 1974 that molecular vaccine against subunit capsular polysaccharide for Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae B (Hib) were developed. Since these vaccines were less effective in young infants, it required a breakthrough for these vaccines to be widely accepted. In 1983, new vaccines were developed with conjugation of carbohydrate antigens to a protein carrier, by this conjugation effective T cell help and immunologic memory was developed. In 1981, hepatitis B virus vaccine was developed by the use of the surface antigen from human plasma carrier and represented a new degree of purity of a single protein as a vaccine. It was in 1986, that first vaccine from recombinant DNA technology was available for human use. It was derived from yeast and was initially developed for people at high risk e.g. doctors, nurses, blood-bank workers etc. Soon its significance in chronic liver disease and primary hepatocellular carcinoma was recognized and hepatitis B vaccine became first ever anticancer vaccine.

Table 2.1. Showing achievement of eminent scientists in chronological order

Year Achievement1796 Edward Jenner introduces vaccination against smallpox1880 Louis Pasteur develops attenuated fowl cholera vaccine, and soon after vaccines against anthrax

and rabies.1896 Robert Koch discovers the cholera vibrio. Killed, whole-cell bacterial vaccines against cholera,

typhoid, plague, and other bacteria follow. They are very reactogenic.1924 Bacille Calmette-Guerin (BCG) is introduced as a live attenuated tuberculosis vaccine.Late 1920s, early 1930s

Progressively better diphtheria and tetanus vaccines are introduced based on toxins and then toxoids.

1935 17D strain of live attenuated yellow fever vaccine is introduced.1945 Chick embryo allantoic fluid-derived killed influenza vaccine is developed.1949 John Enders cultivates the poliomyelitis virus in tissue culture.1955 Jonas Salk introduces the killed injectable poliomyelitis vaccine (IPV).1961 Albert Sabin develops the live oral poliomyelitis vaccine 1960-1969 Progressively improved live attenuated measles, mumps, and rubella vaccines are developed.1974-1984 Polysaccharide vaccines against encapsulated gram-positive bacteria are introduced and

progressively improved ”Meningococcus, Pneumococcus, Hemophilus.1981 Hepatitis B vaccine is licensed using surface antigen from human carrier plasma.1983 Hemophilus influenzae carbohydrate-protein conjugate is developed as an effective vaccine for

infants.1986 Yeast-derived recombinant hepatitis B vaccine is licensed. This first vaccine derived thorough

genetic engineering effectively begins a new paradigm that will dominate the next 20 years.

3. Classification of Established Vaccines

Table 3.1. Showing classes of vaccines

Example Type of vaccinePoliomyelitis (OPV Sabin), measles, mumps, rubella, rabies, varicella, vaccinia, yellow fever, rotavirus, influenza (intranasal)

Live attenuated viral

BCG for tuberculosis or leprosy, Ty21a for typhoid fever

Live attenuated bacterial

Poliomyelitis (IPV-Salk), influenza, hepatitis A, rabies

Killed whole virus

Pertussis, cholera, anthrax, plague Killed whole cell bacteriumDiphtheria, tetanus ToxoidsAcellular pertussis, subunit influenza, hepatitis B Molecular vaccine + proteinHib, Vi typhoid, meningococci, pneumococci Molecular vaccine + carbohydrateHib, meningococci, pneumococci Molecular vaccine + carbohydrate-protein

conjugateDPT, MMRV, DPT-Hib, DPT-Hib-IPV-hep B. Combination vaccines

1. Live attenuated vaccines

Practice of using live attenuated pathogen as vaccine was established by Edward Jenner. Since then many viral vaccines have come up which uses live attenuated pathogens as platform technology. Some of these viral vaccines have efficiency of more than 90% and protection provided usually lasts for years.

But it has several drawbacks such as multiplication of pathogen inside host to induce immune response. This phenomenon could be fatal in immunodeficient or immunocompromised individuals. Some times mutation in the non-virulent pathogen can lead to restoration of its virulence. E.g. polio vaccine.

2. Killed whole micro organisms based vaccines

Since these vaccines have killed organism which is in capable of replication so to maintain the immunity booster doses have to be given. Some vaccines are highly efficient and safe like Salk’s injectiable polio vaccine or hepatitis A vaccine. Other vaccines of this class are poorly efficient and give short immunity.

3. Toxoids

Pathological processes of diseases like Diptheria, tetanus are caused by exotoxin. The vaccines for diphtheria and tetanus are toxoid vaccines. These vaccines are prepared by purifying the bacterial toxin and inactivating it to form toxoid. On vaccination toxoids induce anti-toxoid antibodies in host which binds and neutralizes the exotoxin. For manufacturing toxoid vaccine, controlled detoxification has to be achieved with minimal change in epitope structure. Vaccine for enterotoxin are available but are not much successful.

4. Molecular Vaccine: Proteins

The advancement in genetic engineering made possible to obtain pure antigenic molecules which were tested in animal models for their ability to induce immune response. Some subunit vaccines such as Hib and acellular petussis were highly effective while others such as HBsAg were effective at low doses while some required powerful adjuvants.

5. Molecular Vaccines : Carbohydrate and conjugate

The capsular polysaccharides of encapsulated bacteria are powerfully immunogenic and can make good vaccines. However, they have several drawbacks.

1. Since carbohydrates are serotype specific these vaccines provide immunity against one variant of the pathogen. So to provide effective protection different carbohydrate molecules have to added to a single vaccine. Thus, the Merck pneumococcal vaccine has 23 separate components.

2. Second, these vaccines work poorly or not at all in young infants. For a disease such as meningitis, in which the greatest rate of mortality is among children younger than 1 year, this represents a serious limitation.

3. Third, being T-independent antigens, the vaccines engender suboptimal immunological memory.

6. Combination Vaccines

As more and more vaccines are being developed, question arises how many vaccines one has to take to become immunized against prevailing infections. This has increased the demand for combination vaccines e.g. DPT vaccine. MMR as a combination vaccine was highly successful combination and has motivated researcher for more such combinations and reduce the need for multiple jabs. Recently varicella was successfully combined with MMR to produce MMR-V vaccine. Taking DPT as base, scientists have added one or more of Hib, injectable killed polio (Salk) vaccine, and hepatitis B. such pentavalent and hexavalent vaccines have started finding their role in immunization programme. But pharmaceutical companies are seriously considering the possibility for heptavalent combination vaccines and Wyeth-Pfizer has come up with Prevnar, a heptavalent pneumonococcal vaccine.

Table 3.2. Showing currently available vaccines

Disease or pathogen Types of VaccineWHOLE ORGANISMBacterial cellsAnthrax

Inactivated

Cholera InactivatedPertussis InactivatedPlague InactivatedTuberculosis Live attenuated BCGTyphoid Live attenuatedViral ParticlesHepatitis A InactivatedInfluenza InactivatedMeasles Live attenuatedPolio (Sabin) Live attenuatedPolio (Salk) InactivatedRabies InactivatedRotavirus Live attenuatedRubella InactivatedVaricella zoster Live attenuatedYellow fever Live attenuatedPURIFIED MACROMOLECULESToxoidDiptheriaTetanus

Inactivated endotoxinInactivated endotoxin

Capsular polysaccharidesHaemophilus influenza type b Polysaccharide + protein carrier Neisseria meningitides PolysaccharideStreptococcus pneumoniae 23 distinct capsular polysaccharidesSurface antigenHepatitis B Recombinant surface antigen (HBsAg)

New designs for future vaccines

Most important aspect in developing new vaccines is the stimulation of immune response. Activation of immune system itself is not sufficient for protective long term immunity. It is important that which branch of immune system is stimulated humoral or cell mediated. Second important aspect is the development of immunologic memory. A vaccine that produces

primary immune response but fail to produce memory cells leaves host susceptible to attack by same organism in future. Memory cells on encountering same organism in future differentiate into plasma cells and produces antibodies.

e.g. Graph shown below antibody titer value after immunization with single dose Salk polio vaccine. Graph shows rise in serum antibody levels which after reaching peak declines gradually whereas stimulation of immunologic memory is a slow process and reaches peak after a long time.

Recombinant Vectored vaccines

Genes encoding for antigens could be introduced into attenuated virus or bacterium, which serves as vector and it replicates inside the host expressing the gene product. A number of organisms that are used as vectors are vaccinia virus, attenuated polio virus, adenovirus, BCG strain, etc.

Use of vectors in vaccinology was for the first time demonstrated by Bernard Moss and Enzo Paoletti. They demonstrated that genes for important antigens could be introduced into vaccinia virus without disturbing its replication and the organism expresses the antigens inside the host. This “Trojan Horse” concept has been applied in the development on many other vaccines and by this technique a harmless organism serves as a vector to deliver antigen genes. This type of vaccine design utilizes best features of live attenuated vaccine and precision of rational subunit vaccine.

The large size of vaccinia genome enables insertion of multiple antigen genes so that more than one disease could be covered. Unmodified vaccinia vectors is cause of serious concern in immunocompromised (ineffective T-cell system) individuals such as those suffering from HIV. In such cases issue has been resolved by introduction of an additional IL-2 gene to boost immune system. Also non replicating vectors like fowlpox and canarypox can be used in humans.

Poor response of the experimental HIV and malaria vaccine in clinical studies have led to decrease in the interest in this area. But the overwhelming success in the preclinical studies have kept researchers bound to their work and to hope for success in near future.

DNA Vaccines

DNA vaccines usually consist of plasmid vectors, derived from bacteria that contain

genes to be inserted under the control of a eukaryotic promoter, allowing protein expression in mammalian cells. Efficacy of DNA depends on the vector chosen.

A DNA vaccine comprises of plasmid DNA vector which has eukaryotic promoter, a cloning site, a polyadenylation sequence, selectable marker and n origin of replication.

This vaccination strategy has been developed in recent years with plasmid DNA encoding antigenic proteins is injected directly into the muscle of the recipient. Muscle cells take up the

DNA and the antigen is expressed, producing with both a humoral antibody response and a cell-mediated immune response.

The injected DNA is taken up and expressed by both muscle cell and by dendritic cells. DNA either gets integrated into chromosomal DNA or remains in episomal form. It is known that muscle cells express low levels of class I MHC molecules but do not express co-stimulatory molecules suggests that local dendritic cells may be crucial to the development of antigenic responses to DNA vaccines.

DNA vaccines are advantegous over others as the encoded protein is expressed in its natural form with any denaturation and modification. So the immune response is induced as soon as

antigen is expressed, they induce both humoral and cell-mediated immunity. DNA vaccines causes prolonged expression of antigen which help in building immunologic memory.

DNA vaccines have a promising future as refrigeration is not required for handling and storage of the plasmid DNA. The same plasmid vector can be custom tailored to make a variety of proteins, so that the same manufacturing techniques can be used for different DNA vaccines

pDUO: a novel vaccine design that induces CD8+ T-cell responses by direct presentation.

The pDUO is a novel vaccine design developed to provide effective vaccine for cancer. pDUO incorporates two strong viral promoters within the plasmid. Plasmids are derived from cytomegalovirus and Simian virus 40 and each of promoter drives expression of a separate vaccine-encoded antigenic peptide sequence. First promoter from CMV, a tumour-derived major histocompatibility complex (MHC) class I-binding peptide with a leader sequence; second promoter from SV40, a fragment C (FrC)-derived MHC class II-binding peptide (p30) within the invariant chain (Ii) sequence.

Following transfection in vivo, the tumour-derived peptide binds to MHC class I in the endoplasmic reticulum (ER) and is then expressed at the cell surface. In parallel, the fragment C-derived peptide, p30, binds to MHC class II together with the Ii chain. The latter is then cleaved leaving the bound p30 peptide. CD4+ T cells that are specific for p30 can then help the CD8+ T cells that are specific for the tumour peptide. This vaccine generates CD8+ T-cell responses that are helper T cell-dependent and helper T cells can only be generated following direct transfection of antigen-presenting cells: the p30 peptide binds to the class II molecule by the ER route. Therefore, the co-delivered CD8+ epitope can only generate immunity following direct transfection of antigen-presenting cells at the injection site. This contrasts with the pDOM–peptide design, which uses the cross-priming route. The route of priming appears to influence the outcome in terms of the level and quality of the CD8+ T-cell response.

Multivalent subunit Vaccines

The major drawback of synthetic peptide vaccines and recombinant protein vaccines is that they tend to be poor stimulator of immune system. They activates humoral antibody repone but less likely to induce cell mediated immune response. Multivalent subunit vaccine overcome these drawbacks it has epitopes of both B-cell and t-cell and vaccine is delivered intracellularly which can present can present multiple copies of a given peptide or a mixture of peptides to the immune system.

One of the approaches in multivalent subunit vaccines is

1. Solid matrix–antibody- antigen (SMAA) complexes: SMMA are formed by attaching monoclonal antibodies to particulate solid matrices and then saturating the antibody with the desired antigen. The resulting complexes formed are then used as vaccines. By attaching different monoclonal antibodies to the solid matrix, it is possible to bind a mixture of peptides or proteins, comprising of epitopes for both T cells and B cells.

These types of multivalent complexes lead to vigorous induction of humoral and cell-mediated responses. Their particulate nature of these multivalent complexes increases their immunogenicity by facilitating phagocytosis by phagocytic cells.

2. Second approach to develop multivalent vaccine is by the use of detergent to integrate protein antigens into protein micelles, lipid vesicles (liposomes) or immunostimuating complexes. Mixing proteins in detergent and then removing the detergent forms micelles. Liposomes containing proteins as antigens are prepared by mixing the proteins with a suspension of phospholipids under suitable conditions that favors vesicles formation. The bilayered vesicles formed have protein incorporated into the bilayer. Immunostimulating complexes (ISCOMs) are lipid carriers prepared by mixing protein with detergent and a glycoside called Quil A.

Edible Vaccines

It was in 1990 that for the first time emphasis was give on new radical approaches to vaccines for developing nations. This laid the path for the development of edible vaccine by expressing antigens in foods such as fruits which are cheap. This idea required development of transgenic plant and also several hurdles had to be overcome such as expression of genes of antigen only in edible parts of the plant, avoidance of oral tolerance, adequate dose and avoiding enzymatic destruction of vaccine,

First edible vaccine to come up was hepatitis B edible vaccine with hepatitis B surface antigen being expressed in tobacco plant it was followed by engineered potato plant expressing B

subunit of heat labile E.coli enterotoxin. The animal studies for these vaccines have shown promising results with the development of serum IgG and mucosal IgA specific for LT-B. it is also believed that plant cell wall will offer some barrier to enzymatic degradation and help antigens to stimulate immune response.

With the emergence of transgenic plants as source of edible vaccine the idea of using plants as source of pharmaceutical protein has surfaced. This idea if implemented would reduce the use of bacteria and yeast and would lower the cost of vaccines.

Transdermal Vaccines

Contact sensitization has been known since long which implies immune response to any chemical that reaches body via skin. The precise mechanism of sensitization is now known by T and B cell activation. Transdermal vaccines are based on the idea that once antigen reaches Langerhans cell in upper epidermis, it causes their activation and consequent migration to lymph node. If vaccine is administered along with adjuvant immune response is induced. Simple skin treatments, such as cleansing with alcohol or hydration of the skin, may enhance responses, and patches, gels, creams, and ointments are also under investigation.

Another novel that is being studied is transdermal administration of vaccine via patch consisting of extremely fine needles which would minimally abrade the skin but still target antigens within the patch to superficial Langerhans cells.

Prime boostIt is a recently evolved principle from experimental vaccine development. It is observed that that priming with an antigen presented in one way and followed by a boost or again exposure with the same antigen but presented in a different way, leads to an enhanced response to the antigen. This principle is called “prime-boost.” It has been used primarily for AIDS vaccines but is also being used in other aspects.

In AIDS vaccine development, DNA plasmids coding for HIV proteins is used as the prime, whereas poxviruses containing the genes for the same proteins is used as the boost.

Reverse vaccinologyReverse vaccinology term was coined by Rino Rappuoli. It indicates mining of microbial genomes, usually bacterial genomes, to find proteins that could be used to develop a vaccine. It is a multistep process which begins with sequencing of genome followed by computer aided analysis of DNA ORFs to predict surface antigens or secreted antigens. ORF of use are then inserted into a bacterium for expression of corresponding protein. The expressed protein are then tested in animals from which samples are collected to test for bactericidal activity and surface localization. If the proteins remain unaltered then it is included in the vaccine development.

Using reverse vaccinology 5 proteins from group B Neisseria meningitides were identified and combined to form a vaccine. Owing adverse reactions this vaccine is still in clinical trial. It is exhibiting a cross-reaction between capsular antigen and neural cell adhesion molecule in the brain. Whereas the technique is showing promise in developing many other vaccines such as for group B streptococci, pneumococci, Bacillus anthracis, Chlamydia pneumoniae, and Porphyromonas gingivalis.

Antibody-based Vaccines against Human Immunodeficiency VirusHuman Immunodeficiency virus has cunning ways to evade immune system. High mutation of envelope proteins, primary structures involved in causing infection, help virus to evade from antibody response. It integrates in provirus DNA into host genome and shows no external signs of viral infection making it safe from T cell attack. The virus target CD4+ T cell and delays immune response, since T cell levels fall low virus replicates at a high rate. Most licensed vaccines for AIDS are thought to act by eliciting a neutralizing antibody response against prominent antigens, which prevents virus from infecting cells. Prominent viral antigens on the surface are envelope glycoproteins gp160, exterior viral protein gp120 and transmembrane glycoprotein gp41. Initial clinical studies have shown that gp120 exists as homodimer with gp41 and remain hidden by heavily glycosylated exposed surface making it inaccessible for neutralizing antibody. Another possible target CD4 binding site is also in accessible as it is protected by V1/V2 loops. Binding of gp120 to CD4 leads to conformational change in the structure of envelope and exposes high affinity site for coreceptors CCR5 and CXCR (chemokines). After the binding of chemokines, gp41 also changes shape leading to the insertion of a hydrophobic amino-terminal fusion peptide into the target cell membrane to

mediate membrane fusion and viral entry. One area of HIV vaccine research also focuses on ways to stop these transitions in viral structures.

Before discussing about the antibody evoking vaccine strategy. It is important to discuss about the heterogeneity of the virus. There are 3 main types of HIV-1 namely M, N and O which cause separate infection. Type M could further divided into A, B, C, D, E and G subtypes. Most important subtype in Europe, and Australia is B; in South and East Asia it is C; and in Southeast Asia is E. These subtypes further undergo inter-subtype recombination. Hence for a vaccine to be successful it should need to elicit antibodies that neutralize multiple variants.

Researchers at International AIDS Vaccine Initiative reported in 2009 the isolation and characterization of a pair of entirely novel broadly neutralizing antibodies from a single HIV positive individual in Africa. The two antibodies namely, PG9 and PG16 were found to be extremely potent neutralizers of HIV, and capable of targeting a wide spectrum of HIV variants.

PG9 and PG16 have several interesting traits:• They identify vulnerability on HIV. The spot where PG9 and PG16 bind to HIV appears to be highly accessible. Which was not possible with all the previously isolated broadly neutralizing antibodies. This means scientists could use these antibodies as platform to design a vaccine that can induce similar antibodies.• These antibodies, as well as the antibodies isolated by the Vaccine Research Center, are not only broadly neutralizing but also particularly potent neutralizers of HIV. This is important because the more potent an antibody, the less of it is needed to block infection.

Many researchers are also trying to improve the quality of both the immunogens and the vectors by which vaccines are delivered. One approach aims to create vectors that are safe yet capable of replicating like any naturally occurring virus. Researchers have removed the replication capability from most vectors being tested in HIV vaccine trials today. Replicating vectors could provoke more effective immune responses against HIV than have so far been observed.

Modern designs for adjuvants

Development of better vaccines was not limited to finding novel vaccine strategies but also revolutionized the adjuvants as well. Companies have come with several new adjuvant designs such as-

1. AS02, developed by Glaxosmithkline Biologicals

2. Iscomatrix™, developed by CSL Limited

What are adjuvants?

Adjuvants refers to broad range of substances that have the potential to increase the immunogenicity of antigens when incorporated with them or co-administered. In simple terms an adjuvant is immune potentiator or could be an antigen delivery system.

A few of the approved adjuvants approaches include alum based like aluminium hydroxide and aluminium phosphate and other is emulsion based adjuvants like MF59. Biggest hurdle with developing ideal adjuvant lies in the difficulty of developing adjuvants which retain their adjuvant properties and are non reactogenic.

New Adjuvants

Type of Adjuvant ExampleOil in water MF-59Water in oil MontanideSaponins QS21Liposomes ASO1Bacterial toxin CT,LTCytokines Interleukin-12, granulocyte macrophage

colony stimulating factorTLR dsRNA ligands (TRL-3) MPL (TLR-4)

Flagellin (TLR-5)CpG (TLR-9

AS02: It is an integrated adjuvant consisting of oil-in-water emulsion, this emulsion contains two different immunostimulants -3D MPL and QS21 . Monophosphoryl Lipid A (MPL) is a modified form of lipid A, a potent immunostimulant. It takes up LPS endotoxins of gram negative bacteria. 3D-MPL is further derivative of MPL. QS21 is also an adjuvant derived from the bark of south American tree Quillaja saponaira. Of the crude mixture only saponin are immunostimlatory. They stimulate both T cell dependent as well as T cell independent antibody formation and cell mediated immune response.

Initial animal studies have shown promising results for diseases like HIV/AIDS, tuberculosis and cancer. The clinical trials for malarial vaccine with AS02 showed AS02 as a powerful adjuvant with use for other diseases in future.

ISCOMATRIX™: early immunologist were quick to realize that poorly immunogenic proteins could make good immunostimulatory proteins on giving them mild heat treatment which causes them to aggregate and form microparticles or microggregates. They observed similar property in hepatitis B virus, the proteins have the tendency of self assembly into virus like particles. These particles have immunostimulatory effect on taking this phenomemon as platform ISCOM’s or immunostimulatory complexes were developed. Immunostimulatory properties of QS21 have already been discussed above. Early studies showed associated toxicity with the saponins (QS21) which limited its use, but use of purified forms of QS21 when used in ISCOM’s resulted in considerable lower toxicity. Using this as platform technology ISCOMATRIX™ was developed. It contains saponins known as ISCOPREP. The lipids used are chemically synthesized Di-palmytoil phosphatidyl choline and cholesterol. These kinds of formulated vaccines stimulate all types f IgG isotypes antibodies and also induces a strong CD8+ T cell response.

Extensive clinical studies have been done with this adjuvants and data shows vaccine to be well tolerated and safe both in men and women. Dose response data indicates humoral immune response at all doses while cellular immune response at high doses. Currently using this adjuvant influenza trivalent split virion vaccine and hepatitis C virus vaccine are undergoing phase II studies.

Reactogenicity and adverse reactions of Vaccines

Reactogenicity or mild side effects are associated with live attenuated and inactivated vaccines. Live attenuated vaccines like small pox and measles vaccines involves replication of altered organism to stimulate an immune response, the build up of micro organism causes minor form of disease in host for which vaccine is given. Vaccines from killed micro organism causes local redness, pain and swelling at the site of injection and some mild systemic reactions such as transient fever, irritability, anorexia, headache, and general malaise. But these reactions are mild, for short duration and can be easily controlled.

Mild side effects arise because vaccines cause immune response like inflammation and cytokine stimulation. Reactogenicity limits the use of vaccines in immunocompromised patients where risk to benefit ratio is low.

Conclusion

Vaccines have potential usefulness beyond communicable diseases, such as anticancer vaccines, birth control vaccines, and vaccines aimed at lowering immune responses, such as in autoimmunity or allergy.

References

Wang, J.Y. and Roehrl, M.H. (2005).Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin. Medical Immunology. 4:4

Montefiori, D., Sattentau, Q., Flores, F., Esparza, J. and Mascola, J. (2007).Antibody-Based HIV-1 Vaccines: Recent Developments and Future Directions. PLoS Medicine. 4:12

Kuby, J. (). Immunology.

Paul, W.E. (2008).Fundamental Immunology. 6th Ed.

Rappuoli, R. and Bagnoli,F. (2011). Vaccine Design: Innovative Approaches and Novel Strategies

Rice, J., Ottensmeier, C.H. & Stevenson, F.K. (2008). DNA vaccines: precision tools for activating effective immunity against cancer. Nature Reviews Cancer 8, 108-120

Garmory, H.S., Brown, K.A. and Titball, R.W. (2003). DNA vaccines: improving the expression of antigens. Genetic Vaccines and Therapy. 1:2

International Aids Vaccine initiative.

The College of Physicians of Philadelphia. (2010).New Discovery May Advance HIV Vaccine Design.