Vaccination - Current Situation and Perspective

In the last 200 years, since the time of Edward Jenner, vaccination has controlled many major diseases namely, small pox, diphtheria, tetanus, yellow fever, pertussis, hepatitis B, haemophilus influenza type B, poliomyelitis, measles, mumps, rubella. Vaccination has eradicated smallpox and many countries including Malaysia has been certified poliofree by WHO.

The impact of vaccination on the health of the global population is well known and well documented. With the exception of safe water, no other modality, not even antibiotics has had a major impact on mortality reduction and population growth. Vaccines remain undoubtedly one of the most effective and safest of health interventions. Nevertheless, immunization services face many challenges. Immunization safety - ensuring and monitoring the safety of all aspects of immunization, including vaccine quality, storage and handling, vaccine administration and disposal of sharps is one such challenge. Other issues include the concern about excessive mercury exposure to infants who received multiple thimerosal containing vaccines, vaccine shortages due to converging factors such as phasing out of the production of traditional less expensive vaccines used in developing countries by vaccines manufacturers in favor of more expensive combination vaccines.

Even though conventional vaccines constitute the greatest achievement of modern medicine, successful vaccines have yet to be introduced for many deadly and debilitating diseases such as malaria, AIDS, dengue and hepatitis C. This gap exists because standard immunization methods work poorly or pose unacceptable risks when targeted against certain illnesses. An alternative strategy involving the deliberate introduction of a DNA plasmid carrying an antigen coding gene that transfect cells in vivo and results in an immune response DNA vaccines. DNA vaccines or DNA based immunization has been called "the third vaccine revolution' and if used for human vaccination will have distinct advantages over traditional vaccines in cost and ease of production, delivery and since almost all plasmids can be manufactures in essentially the same way, substantial economies of scale can be achieved.

Autoimmune Disease - SLE

Systemic Lupus Erythematosus (SLE or lupus) is a chronic autoimmune disease that can be fatal, though with recent medical advances, fatalities are becoming increasingly rare. As with other autoimmune diseases, the immune system attacks the body’s cells and tissue, resulting in inflammation and tissue damage. SLE can affect any part of the body, but most often harms the heart, joints, skin, lungs, blood vessels, liver, kidneys and nervous system. The course of the disease is unpredictable, with periods of illness (called flares) alternating with remission. In SLE, the body's immune system produces antibodies against itself, particularly against proteins in the cell nucleus. SLE is triggered by environmental factors which are unknown (but probably include viruses), in people with certain combinations of genes in their immune system. SLE is a chronic inflammatory disease believed to be a type III hypersensitivity response with potential type II involvement.

A VERY Simplified Overview of an Immune Response

You receive a cut.

Bacteria enter the wound.

Many are destroyed rapidly by complement and the phagocytes recruited through acute inflammation (Innate immunity).

Some of the dead bacteria or their breakdown products are taken up by the tissue resident dendritic cells.

The combined action of bacterial products and cytokines (from acute inflammation etc.) activate the tissue dendritic cells.

This causes them to migrate to the local lymph node via afferent lymphatics.

Dendritic cells enter the node in the T cell areas. They become resident there displaying their 'wares'

T cells enter the node from the blood, trafficking through the T cell area to the efferent lymph. Those which recognize the bacterial antigenic peptides displayed on the dendritic cells stop, activate, divide and differentiate; some later become memory T cells.

B cells entering nodes from the blood must cross the T rich area in transit to the B cell rich areas. The antigen-specific ones must acquire antigen too, presumably via the lymph. Then they can have their MHC-peptide complexes recognized by activated T cells and receive help.

Some become IgM secreting plasma cells. Some migrate to the B cell rich areas and form germinal centres. Here B cells proliferate and give rise to progeny with high affinity for antigen through a process called affinity maturation. The products of germinal centres become IgG, IgA etc plasma cells and memory B cells.

Panbio Pan-E Dengue Early ELISA Kit and Dengue NS1 Antigen

Dengue is an arbovirus belonging to the genus of Flavivirus in the family Flaviviridae.It is the aetiological agent that cause dengue fever (DF) and dengue hemorrhagic fever (DHF).Currently, up to 2.5 billion people living in the tropical belt of the world are at risk of the infection.Annually, 50 to 100 million DF were reported with 500,000 DHF resulting in 25,000 deaths and a high proportion were children.Significant number of patients suspected of having dengue infection had IgM antibody detected in their serum.In 2006, Sabah and Sarawak had reported DF and DHF with significant number of deaths. Detection of dengue antibodies are among many laboratort tests routinely performed to confirm dengue in patients but these antibodies were often undetectable in the early phase of the infection.NS1, a -50-kilodalton glycoprotein, is one of seven non-structural (NS) protein genes of the dengue virus.

Preliminary studies have shown its association in viral RNA replication.Dengue NS1 antigen has been reported to be detected in serum from day 1 after onset of fever. It has been shown that NS1 antigen could be used as a suitable marker of dengue virus infection.Early definitive diagnosis is essential for the successful timely management of the infection.Performing preliminary evaluation of panbio pan-E Early Dengue ELISA kit on patient serum suspected of dengue is recommended and compared it with the panbio Dengue Duo Cassette, panbio Dengue IgM Capture ELISA and Dengue real-time polymerase chain reaction(RT-PCR).In one study reported that panbio pan-E Dengue Early ELISA was able to detect NS1 antigen when panbio Dengue IgM Capture ELISA was still negative in the early phase of the disease.

Immune Response

Lymph Node

Picture shows the regions of the lymph node where the blood stream (arteries and veins) enter a typical lymph node. For the lymphatic system to function properly in its defensive role, the lymph nodes must be able to "dump" their leukocytes (infection fighting cells) quickly into the general blood stream. It is important to note that white blood cells are not produced in the Lymph nodes initially, only stored there. In the event of a serious infection (a pathogenic virus for example), the lymph nodes often become very swollen. This swelling represents the explosive multiplication of leukocyte numbers in the lymph node's honeycomb of connective tissue.

Antibody Structure

Antibodies are a group of glycoprotein found in the serum and body fluids. They are gamma-globulins and therefore are known as immunoglobulin. Some are carried on the surface of B cells, where they act as receptors for specific antigens. Other antibodies are free in the blood or lymph. All antibodies have a similar overall structure with two light and two heavy chains. These are linked by both covalent (disulphide bridges) and non-covalent forces. They are made up of a series of domains of related amino acid sequence which possess a common secondary and tertiary structure. This conserved structure is found frequently in proteins involved in cell-cell interactions and is especially important in immunology.5 distinct classes of immunoglobulin molecules are recognized in most higher mammals which are IgG, IgA, IgM, IgD and IgE.They differ in size, charge, amino acid composition and carbohydrate content. They differ between classes and within each class. Electrophoretically, the immunoglobulin show a unique range of heterogeneity which extends from g to the a fractions of normal serum. Immunoglobulins are bifunctional molecules. One region of the molecule is concerned with binding to antigen while a different region mediates effector functions.Effector functions include binding of the immunoglobulin to host tissues, to various cells of the immune system, to some phagocytic cells, and to the first component (C1q) of classical complement system.

Immunoprecipitation

Single Diffusion

Agar plate impregnated with IgG is obtained and labeled. It is cut with a hexagonal array dots. Disposable graduated 1ml pipette is used and the lowest 2 graduation is cut off and the bulb of the pipette is compressed. Then, the tip of pipette is pressed into the gel and the compression of the bulb is released. The sample is added in duplicate. The plate is leaved at room temperature and within 30 minutes, result is obtained.

Rings of precipitation form around the wells with different diameter.

Double Diffusion

The agar plate is obtained and labelled. It is cut well. Then, the assay is conducted as for Section A; 50 micro lit of yellow and blue dye is added to appropriate wells. Section B; HS,HA,CA serums are added in appropriate wells.Section C; Unknown serum is added to centre well. Antisera of following order; Human Antiserum(HA),Chicken Antiserum(CA),Cat Antiserum(CatA) and Rabbit Antiserum(RabA)

Precipitation line form between HA and HS in section B. In section C, HA form precipitate line with unknown serum.

Immunoprecipitation assay is one method of determining the presence of unique biochemical in a chemical mixture or a biological sample. It identifies chemical by unique features of the chemical’s shape and surface charges. The assay used antibodies to detect the presence of specific components in a sample. The component’s presence is known when it becomes bound to an antibody. The binding of the antibody to the antigen result in visible detectable clump known as a precipitation bond.

This immunoprecipitation test is designed first by isolate the chemical or antigen that is interested in detecting. Then, the antigen is injected into a laboratory animal and the laboratory animal produces antibodies specifically designed to adhere to that antigen. The blood of the animal is purified to yield particular antibody. In this experiment, the serum that being used are human serum, chicken serum, unknown serum. The antiserum that being used are human antiserum, cat antiserum and rabbit antiserum. There are also dyes which are yellow and blue dye.

Antibody is a protein produced as a result of interaction with an antigen. The protein has the ability to combine with the antigen that stimulated its production. Meanwhile, antigen is a substance that can react to antibody. Not all antigens can induce antibody production; those that can are also called immunogens. In single diffusion test, the antibody is incorporated into the gel and the antigen is introduced into the wells in a gel. Rings of precipitate form around the wells, and the diameters of the rings are proportional to the amount of antigen placed in the well. In single diffusion test, sample from A give the biggest diameter follow by sample form B and the smallest is sample from C. This show that different amount of antibody in the sample of A, B and C.

Meanwhile, in double diffusion test, antigen and antibody are placed into separate wells; they diffuse into the gel in all directions; where they interact, a precipitation reaction occurs. Each antigen-antibody reaction forms an independent precipitation reaction called a precipitin boundary. In double diffusion test, It is divided into section A, section B and section C. In section A, colours can be seen travelling through the gel. In section B, a precipitation line form between HA(Human Antiserum) and HS(Human serum) which mean the test result is positive. In section C, the unknown serum is identified as HS(Human serum) as it form precipitation line with HA(Human Antiserum).

Introduction to Immunology

What is Immunity?

Immunity means protection from disease and especially infectious disease. Cells and molecules involved in such protection constitute the immune system and the response to introduction of a foreign agent is known as the immune response. Immune response areproduced primarily by leucocytes, of which there are several different types. Not all immune responses protect from disease; some foreign agents, such as the allergens found in house dust mite, cat dander or pollen from certain trees, cause disease as a consequence of inducing an immune response. Likewise some individuals mount immune responses to their own tissues as if they were foreign agents. Thus, the immune response can cause the autoimmune diseases common to man such as multiple sclerosis, diabetes, rheumatoid arthritis or myasthenia gravis. Myasthenia gravis is a neuromuscular disorder characterized by variable weakness of voluntary muscles, which often improves with rest and worsens with activity. The condition is caused by an abnormal immune response. Most individuals do not suffer from autoimmune disease because they have developed tolerance towards their own (self) tissues.

Memory, specificity & the recognition of “non-self”
– these lie at the heart of immunology.

Immunologist experience of the subsequent protection (immunity) afforded by exposure to many infectious illness can lead them to this view.

An important characteristic of the immune system is that the cells are
able to distinguish “self” and “non-self”