Live Attenuated Vaccine

Md. Riaz SarkerJagannath University, DhakaDepartment of MicrobiologySession: 2010-11

Vaccine designing

1Mr. Riaz Sarker

IntroductionAvaccineis a biological preparation that provides activeacquired immunityto a particulardisease. A vaccine typically contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The term vaccine was coined by Louis Pasteur.The term vaccine was derived from vacca, meaning cow the term devised byEdward Jennerto denotecowpox.The administration of vaccines is calledvaccination.Vaccines have two major effects : a)Vaccines protect individuals against disease.b)If there are sufficient immune individuals in populations, transmission of the infection is prevented . This is known as herd immunity.

Properties of an ideal vaccineProvide long lasting immunity.Should induce both humoral and cellular immunity.Effective in all subjects (the old & very young)Should not induce autoimmunity or hypersensitivity.Should be inexpensive to produce, easy to store and administer.Vaccines must also be perceived to be safe.Factors required for a successful vaccine1) EffectivenessThe vaccine would be effective against all current and future strains of the pathogen.Should give life long immunity.Must induce effective herd immunity.Must evoke protective levels of immunity rapidly.

2) SafteyThe vaccine would be 100% safe to the recipient.An ideal vaccine should not have any potential to disease or lead to any vaccine-associated diseases like allergic responses, local inflammation, fever etc. 3) Availability: Readily cultured in bulk or accessible source of subunit.4) Stability: Stable under extreme climatic conditions, preferably not requiring refrigeration.5) Dose: only one dose required. 6) Administration: Would not need to be administered by injection.7) Compatibility: Delivery of the vaccine simultaneously with other vaccines would be possible. Many vaccines today are delivered simultaneous with other vaccines (e.g., DTP and MMR).8) Cheapness: What is cheap in the West may be expensive in developing countries.

Failure to produce vaccineA) Evasion mechanism: all microbes deploy evasion mechanisms that interfere effective immune response, & for many microbes it is not clear that which immune response provide effective protection.B) Limited range: a given vaccine tends to be effective only against individual serovars of pathogen species although some species have hundreds of serovars).C) Disease isn't immunizing: for some pathogens even exposure to disease does not confer active immunity.D) Rapid evolution: development of vaccines against particularly rapidly evolving pathogens (such as HIV) is also difficult.E) Exacerbation of disease: vaccines of certain types, against certain pathogens can actually exacerbate disease when it occurs.F) Cause of disease: live vaccines retain at least some potential for causing the disease. This is especially true with regard to immuno-depressed individuals (e.g., live polio vaccine).G) Cost-benefit problems: successful vaccine delivery is not always economically justifiable.

Live Attenuated VaccineDefintion:- An attenuated vaccine is a vaccine created by reducing the virulence of a pathogen, but still keeping it viable or "live and have the ability to confer protective immune response.For example:-

Attenuated live bacterial vaccine (LBV) includes:-Mycobacterium bovis strain Bacille Calmette-Guerin (BCG)-Salmonella typhi Ty21a-Vibrio cholerae CVD 103-HgR.

Live virus vaccine includes:-- MMR(measle, mumps, rubella vaccine)-- Yellow fever vaccine-- Rabies vaccines

ConceptAttenuated vaccines consist of bacterial or viral strains, which are weakened by stable mutations that allow the bacteria or viruses to infect humans only transiently. This transient infection elicits immune responses, while the vaccine strains are designed in such a way that they will not cause the symptoms of natural infection by the wild type pathogen.

Empirical methods of designing attenuated vaccinesFor live bacterial vaccines-Serial passaging (BCG vaccine)Chemical mutagenesis (Typhoid vaccine)Genetic engineering (Choleraevaccine)For live virus vaccines-Passaging (Yallow fever vaccine)Reassortment (FluMist for Influenza)microRNA vaccine (Novel Influenza vaccine)

General PrincipleLive attenuated vaccines stimulate protective immune response when they replicate within the host.The antigens produced within the host are released into the extracellular space surrounding the infected cells and are then acquired, internalized and digested by scavenger cells that circulate within the body. These are APCs include macrophages dendritic cells and B cells, which work together to expand the immune response.

The APCs, re-circulate fragments of the digested antigen to their surface, by MHC class II molecules. This complex of foreign peptide antigen plus host MHC molecules forms part of the specific signal with which APCs along with the MHC-peptide complex trigger the action of immune cells, ( TH cells).T cells form complexes with antigen presented by MHC-II molecules through TCR and other co-stimulatory receptors( CD28/CTLA4) and become activated. This activated T cells secret mediators which act as powerful activator for immune cells such as B cells to produce antibodies and memory B cells.

A. Serial passaging (Mycobaterium bovis BCG vaccine)Traditionally, live attenuated vaccines were developed by passing the pathogens underin vitroconditions until they had lost virulence for humans.The BCG strain was attenuated by serial passages of a virulent M. bovis strain through bile salts in selective media. Duringin vitropassage, the M. bovis microbes became attenuated because of the loss of numerous gene complexes.Md. Riaz SarkerJagannath University, Dhaka

B. Chemical mutagenesis(Salmonell typhi Ty21a)S. typhi Ty21a, underwent a more targeted attenuation approach. It is sensitive to galactose and could not express a polysaccharide coat which protects the bacteria from immune responses. The vaccine is designed by chemical mutagenesis of wildtypeS. typhiusing nitrosoguanidine and screening for clones that had a phenotype, which is negative in the enzyme galactose epimerase. This enzyme results in galactose sensitivity, and also makes the bacterium unable to express the Vi-polysaccharide capsule.Thus this strain lacks both functional galactose-epimerase (galE) gene and the Vi antigen and is highly attenuated. Besides, throughthe chemical mutgenesis the strain was also mutated as amino acid auxotroph and less resistant to environmental stresses.

C. Genetic engineering(Vibrio cholerae CVD 103-HgR)The main virulence factor ofV. choleraeis the expression of the cholera toxin. CVD 103-HgR was derived from a wild typeV. cholera strain by the targeted deletion of 95% of both chromosomal copies of the ctxA gene, which encodes the toxic A subunit of the cholera toxin while keeping the expression of the nontoxic but immunogenic B subunit, leading to intermediary strain CVD 103. Subsequently, it was inserted with a mercury resistance marker into the genome to readily allow for identifying the vaccine strain and its differentiation from wild type organisms on vaccination.

1. Attenuation of live virus through passagingThe major vaccines used in man and animals have all been derived this way. eg. Yellow fever was developed by passage in mice and then chick embryos,Polioviruses were passaged in monkey kidney cells.

Viruses may be attenuated via passage of the disease-causing virus through a series of cell cultures or animal embryos, typically chick embryos or live animal. The initial virus population is applied to a foreign host. One or more of these will possess amutationthat enables it to infect the new host. These mutations will spread, as the mutations allow the virus to grow well in the new host; the result is a population that is significantly different from the initial population, and thus will not grow well in the original host when it is re-introduced. Using chick embryos as an example, the virus is grown in different embryos in a series. With each passage, the virus becomes better at replicating in chick cells, but loses its ability to replicate in human cells. A virus may be grown through upwards of 200 different embryos or cell cultures. Eventually, the attenuated virus will be unable to replicate well in human cells. Thus the virus through a non-human host produce a version of the virus that can still be recognized by the human immune system, but cannot replicate well in a human host.

When the resulting vaccine virus is given to a human, it will be unable to replicate enough to cause illness, but will still provoke an immune response that can protect against future infection.

2. Genetic reassortmentThe capacity of influenza and rotaviruses to generate new variants through reassortment has been harnessed to produce highly effective vaccines that stimulate immune responses without causing disease. The vaccines contain reassortants generated in the laboratory that exhibit desired properties, such as high titre growth or attenuation.A live-attenuated influenza vaccine called FluMis consists of two strains of influenza A virus (H3N2 and H1N1) and two strains of influenza B virus. During vaccine production, 6/2 reassortants are generated; these contain the 6 internal genome segments from the laboratory-adapted master donor strain and the 2 haemagglutinin (HA)-encoding and neuraminidase (NA)-encoding gene segments from the field isolates.

Fig. Reassortment strategy of FluMist vaccine.

3. The microRNA (miRNA)-virus vaccine strategy(Novel live attenuated Influenza vaccine)

This technique based on species-specific expression of miRNAs. Insertion of miRNA target sites into viral genome causes attenuation that can be successfully applied for vaccine development. Insertion of target sites results in a virus that can only replicate in cells lacking that miRNA (see Figure). If the miRNA is ubiquitous the virus demonstrates no replication.

Viral replication can be regulated in a tissue-specific manner by incorporating miRNA target sites into the viral genome. In cells that express the miRNA (e.g., brain, top cell), the miRNAs are processed and transported to the cytoplasm, where they mediate cleavage of viral RNA. Viral replication is restricted to cells in which the miRNA is not expressed (e.g., intestine, bottom cell). The engineered virus can therefore trigger a natural immune response in target tissues without the associated risk of dissemination and disease.

Fig. microRNA strategy for novel Influenza vaccine designing.

Advantages/DisadvantagesAdvantagesActivates all phases of theimmune system.Provides more durable immunity, boosters are required less frequently.Low costQuick immunitySome are easy administer, for instance, polio can be taken orally.Vaccines have strong beneficialnon-specific effects.

DisadvantagesSecondary mutation can cause a reversion to virulence.Can cause severe complications inimmunocompromised patients.Some can be difficult to administer due to requirement to maintain conditions (e.g.temperature).

Conjugate vaccineAccquaintanceA conjugate vaccine is a substance that is composed of a polysaccharide antigen fused to a carrier molecule. This is designed by chemically linking a sugar chain with peptide antigen.Polysaccharide antigens on the surface of the bacterial cells are covered by slime layer thus remain protected from WBCs in human blood. Besides, the immatureimmune systemsof very young people eg. babies, children often have difficulty recognizing certain polysaccharide antigens .For establishing immunity through overcoming these problems where ordinary vaccines may not be effective, conjugate vaccines become a choice of interest.

Aconjugate vaccineis created by covalently attaching a poorantigen (polysaccharide)to a strongantigen (protein)thereby eliciting a stronger immunological response to the poor antigen. However peptide/protein and protein/protein conjugates have also been developed.For example, Haemophilus influenzaetype B (Hib) VaccinePneumoccocal VaccineMeningococcal Vaccine

ComparisionPolysaccharide antigenProtein-polysacchharide conjugate antigen

T cell independentT cell dependent

Production of disproportionate IgMLack of immunological memoryShort-lived immune responseFailure to produce high affinity antibody

Strong boosterresponse up re-exposureHigh affinity antibody

Mechanism of conjugate vaccine in shortT-cell recognizes the antigen and secretes cytokines.Polysaccharide+Protein conjugate administered.Antigen expressed at their cell surface in MHC II complex.

Cytokines activate B cell that undergoes clonal expansion and differentiated into antibody producing plasma cells and memory cells.

Protein content internalized and processed by APCs.

Antibodies bind disease causing agents to make them reach to WBCs for destruction following further invasions.

Necessity of designing of conjugate vaccineSome pathogenic bacteria like Himophilus influenza, S. pneumoniae etc. are covered with a polysaccharide capsule that primarily helps protect the bacteria from phagocytosis, or uptake of the bacteria by immune cells.Polysaccharide antigens are large molecules consisting of repeating epitopes which are not processed by antigen-presenting cells (APC) but interact directly with B cells, inducing antibody synthesis in the absence of T cells called T-independent immune response.T-independent responses are insufficient for vaccine designing in several reasons. Most importantly, they fail to induce significant and sustained amounts of antibody in young children below the age of 18 months . While polysaccharides are immunogenic in older children and adults, they are dominated by IgM and IgG2, are relatively short lived and a booster response cannot be elicited on repeated exposure. This failure to induce immunological memory is also reflected in the absence of demonstrable affinity maturation .

In contrast to polysaccharides, antibody responses to protein antigens have an absolute requirement for T cells. The consequence of this T cell help are that antibody responses to protein antigens can be elicited in the very young and immunity is long lived due the generation of immunological memory. Antibody responses to protein antigens are dominated by the IgG1 and IgG3 subclasses and affinity maturation can be demonstrated over time. In this manner, a conjugate vaccine induces immunologic memory and provides long-term protective immunity.In addition to increased antibody production, the IgG antibodies elicited by a conjugate vaccine are of high avidity and are mainly IgG1; both these characteristics correlate with greater serum bactericidal activity. More remarkably it has the effect on nasopharyngeal carriageof reduced nasopharyngeal colonization, as observed with long-term use of Hib conjugate vaccines, has resulted in herd immunity among susceptible children.

Mechanism of conjugate vaccine

In a conjugate vaccine, the polysaccharide antigen (red) linked to the protein (blue) is taken up by a surface immunoglobulin on a B cell, internalized and processed. As the hydrophilic polysaccharide cannot enter the MHCII cavity, the polysaccharide itself cannot be presented to T cells by MHCII on the surface of the B cell in order to induce T cell help. Therefore a conjugate vaccine may use two alternative ways to induce T cell help:According to the current accepted theory the processed peptide epitope derived from the carrier protein is presented by MHCII on B cell surface and recognized by a peptide-specific T helper cell. or the new mechanism suggests that the peptide anchors the sugar epitope to the MHCII and allows presentation of the sugar epitope to a polysaccharide-specific helper T cell.

Through antigen recognition in MHC complex by helper T cell it become activated and releases cytokines. Cytokines activate B cell and begins the clonal expansion and differentiation of B cell. Some of the progeny B cells become antibody producing plasma cells, all secreting the same needed antibodies. These antibodies trapped the specific bacteria or viruses to WBC for their elimination from vaccinated body upon infection.

Machanism for a glycoconjugate vaccineThe carbohydrate portion of the glycoconjugate binds to the B cell receptor (BCR). Then glycoconjugate is internalized into an endosome and the carbohydrate portion is processed by ROS into saccharides composed of smaller numbers of repeating units used in construction of the vaccine. The protein portion is processed by acidic proteases into peptides. Processing of both these components generates glycanp-peptides. MHCII binding of the peptide portion of the glycanp-peptide allows the presentation of more hydrophilic carbohydrate to the receptor of CD4+T cells. This receptor ofT-helper cell recognizes and responds to glycanpin the complex. Activation of the T cell by carbohydrate-MHCII, along with co-stimulation, results in T cell production of cytokines such as IL-4 and IL-2, which in turn induces maturation of the cognate B cell to become a memory B cell, with consequent production of carbohydrate-specific IgG antibodies.

ImplementationHaemophilus influenzaeB (Hib) The first glycoconjugate vaccine for use in humans, aHaemophilus influenzaetype b (Hib) conjugate licensed in USA in 1987. Hib is a major cause of meningitis in children