The Different Types Of Vaccines

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02 Nov 2017

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The term "vaccine" was coined by Louis Pasteur to commemorate first successful immunization against small poxby Edward Jenner. The term vaccine was derived from "vacca", meaning cow, since Edward Jenner used cowpoxvirus (Vaccinia) to prevent smallpox infection(Rao 2006).T-cell memory is very important for long-lasting immunity, because T-cells control both humoral and cell mediated immunity. When the immune system recognizes a foreign antigen for the first time, an immune response is produced. When T cells are involved, immunological T-cell memory is produced. When the body encounters same antigen subsequently, a stronger immune response is produced. This is because of existing immunological memory against that antigen. Further antigenic stimulus increases the immune response. First antigenic stimulus is "priming" whereas subsequent stimuli are "booster". This is the principle of active immunization(Rao 2006).Vaccination is aimed at inducing active immunity in an individual, so that subsequent contact with the microorganism following natural infection induces strong protective immune response. The protective immunity may involve secretion of neutralizing antibodies or production of memory CTL or Th1 cells. The use of vaccines is now being extended to immunize against tumors or to block fertilization (contraceptive vaccines). Today the term ‘vaccine’ applies to all biological preparations, produced from living organisms, that enhance immunity against disease and either prevent (prophylactic vaccines) or, in some cases, treat disease (therapeutic vaccines), Vaccines are administered in liquid form, either by injection, by oral, or by intranasal routes(WHO 2012).Vaccines are composed of either the entire disease-causing microorganism or some of its components. They may be constructed in several ways.

From living organisms that have been weakened, usually from cultivation under sub-optimal conditions (also called attenuation), or from genetic modification, which has the effect of reducing their ability to cause disease(WHO 2012).

From whole organisms that have been inactivated by chemical, thermal or other means also known as Killed Vaccines.

From components of the disease-causing organism, such as specific proteins and polysaccharides, or nucleic acids(WHO 2012).

From inactivated toxins of toxin-producing bacteria.

From the linkage (conjugation) of polysaccharidesto proteins (this increases the effectiveness of polysaccharide vaccines in young children.

1.2 Properties of an Ideal Vaccine:

Provide long lasting immunity.

Should induce both humoral and cellular immunity.

Should not induce autoimmunity or hypersensitivity(Rao 2006).

Should be inexpensive to produce, easy to store and administer.

Vaccines must also be perceived to be safe.

The vaccine vial may contain relevant antigen, adjuvant (usually alum), preservatives and/or traces of protein derived from the cells in which the vaccine agent was cultured e.g. egg protein(Rao 2006).

Types Of Vaccines

Vaccines Containing Live, Attenuated Micro-organisms -These vaccines are composed of live, attenuated microorganisms that cause a limited infection in their hosts sufficient to induce an immune response, but insufficient to cause disease. To make an attenuated vaccine, the pathogen is grown in foreign host such as animals, embryonated eggs or tissue culture, under conditions that make it less virulent. The strains are altered to make it non-pathogenic, for example, by changing its tropism the pathogen can no longer grow at a site where it can cause disease. Some mutants will be selected that are capable of growing in a foreign host. For the original host this will be non-pathogenic in nature. These vaccines may be given by injection or by the oral route. A major advantage of live virus vaccines is that because they cause infection, the vaccine very closely reproduces the natural stimulus to the immune system. Examples include yellow fever, measles, rubella, and mumps. A related strain called BCG [Bacillus Calmette-Guérin] is a weakened version of the live tuberculosis vaccine which can cause tuberculosis in cows but it is not dangerous like the contagious live tuberculosis strain for example measles, mumps, and rubella ("German measles") vaccines. The Sabin oral polio vaccine (OPV) is another example.

Vaccines containing KILLED microorganisms - These pathogenic microorganisms are chemically killed or killed by heat to make it harmless. These are preparations of the normal (wild type) infectious, pathogenic microorganisms that have been rendered non-pathogenic.Examples are vaccines against flu, cholera, bubonic plague, and hepatitis A, typhoid etc. The viral vaccines contain whole virus particles which are inactivated (usually with formaldehyde) so that they cannot replicate in the host cells. They still keep some unchanged immunogenic properties which can induce an immune response for example polio vaccine IPV (Salk vaccine).

SUBUNIT - Subunit vaccines contain purified antigens instead of whole organisms. Such a preparation consists of only those antigens that elicit protective immunity. Subunit vaccines are composed of toxoids, subcellular fragments, or surface antigens. Administration of whole organism, as in case of pertussis was found unfavorable immune reactions resulting in severe side effects. The effectiveness of subunit vaccines is increased by giving them an adjuvant. Adjuvants help to slow down release of antigen thereby giving more prolonged immune stimulation. An adjuvant can increase the effectiveness of subunit vaccine. Examples are the subunit vaccine against HBV and the virus like particle vaccine against HPV. Vaccine against HBV is composed of surface proteins of virus(produced in yeast) and the virus like particle(VLP) vaccine against human papillomavirus (HPV) is composed of the viral major capsid protein.

Advantages:

They can safely be given to immunosuppressed people

They are less likely to induce side effects.

Dis-Advantages:

Antigens may not retain their native conformation, so that antibodies produced against the subunit may not recognize the same protein on the pathogen surface.

Isolated protein does not stimulate the immune system as well as a whole organism vaccine.

TOXOIDS - These are inactivated bacterial toxins which are no longer toxic but can induce a strong immunogenic response and thereby providing immunity towards a specific disease. Diseases like diphtheria and tetanus are caused not by the growth of the bacterium itself but due to the toxic proteins liberated by the causative organisms. These toxins can be denatured with certain chemicals for example, formaldehyde. These denatured toxic proteins are no longer dangerous, but continue to keep some epitopes on the molecules sothat they can induce immunogenic response. Examples of toxoid-based vaccines include tetanus and diphtheria.

CONJUGATE VACCINES:

Conjugate vaccines are primarily developed against capsulated bacteria. While the purified capsular antigen can act as subunit vaccine, they stimulate only humoral immunity. Polysaccharide antigens are T independent, theygenerate short-lived immunity. Immunity to these organisms requires opsonizing antibodies. Infants cannot mount good T-independent responses to polysaccharide antigens. By covalently linking the polysaccharides to proteincarriers, they are converted into T-dependent antigens and protective immunity is induced.Example.Haemophilus influenzae HiB polysaccharide is complexed with diphtheria toxoid. Tetramune vaccine, which combines the tetanus and diphtheria toxoids, whole-cell pertussis vaccine, and H. influenzae type bconjugate vaccine.

RECOMBINANT VACCINES:

Genetic engineering is also used to produce vaccines. In this desired antigen producing gene of a microbe is isolated and inserted in to a suitable cloning vector. Different strategies are used to produce recombinant vaccine.

Genetically modified vector (e.g., Vaccinia virus) that is expressing desired antigen is used to create a vaccine. The modified vector (e.g., yeast) is grown in a suitable medium to express desired antigen. This antigen is purified and used as a subunit vaccine. Other expression vectors include the bacteria Escherichia coli, mutant Salmonella spp. and BCG.

Introduction of a mutation by deleting a portion of DNA such that they are unlikely to revert can create an attenuated live vaccine. Attenuated live vaccines can be produced by mutation. In order to use as an attenuated vaccine, a part of DNA is deleted. This prevents its reversion to its original state.

Genomes of virulent and avirulent strains are reasserted to produce live attenuated vaccine.

Fruits bearing foreign antigens may be produced by introducing antigens coding genes in to respective plants. Experiments on such edible vaccines are still going on.

Examples:

Hepatitis B Virus (HBV) vaccine is a recombinant subunit vaccine. Hepatitis B surface antigen is produced from a gene transfected into yeast (Saccharomyces cerevisiae) cells and purified for injection.

Vaccinia virus may be engineered to express protein antigens of HIV, rabies etc. Foreign genes cloned into the viral genome are expressed on the surface of infected cells in association with class I MHC molecules. The antigen-MHC complex induces a Tc cell response.

B subunit of cholera toxin, the B subunit of heat-labile E. coli enterotoxin (LT), and one of the glycoprotein membrane antigens of the malarial parasite are being developed using this technique.

Salmonella typhimurium engineered to express antigens of Vibrio cholerae.

BacilleCalmette-Guérin vaccine strain engineered to express genes of HIV-1.

Reassortment of genomes between human and avian strains to create Influenza vaccine. Human and swine strains to create Rotavirus vaccine.

Advantages:

Vectors which are safe as well as easy to grow and store can be chosen.

Antigens which do not induce protective immunogenic response or which cause damaging responses are eliminated from the vaccine. Example Cholera toxin A can be safely removed from cholera toxin.

Disadvantages:

Locating and cloning of the gene for desired antigen and its efficient expression in the new vector increases the cost of production.

When engineered vaccinia virus is used to vaccinate, care must be taken to spare immunodeficient individuals.

DNA VACCINES - These vaccines are still in experimental stage. Like recombinant vaccines, genes for the desired antigens are located and clonedThe "gene gun" with compressed gas blows the DNA in to the muscle cells of the animals to be vaccinated. DNA-coated gold particles are used in bombarding the skin and thereby introducing DNA in to the tissues. DNA can also be introduced in to nasal tissue using nasal drops. Some muscle cells express the pathogen DNA to stimulate the immune system. DNA vaccines have induced both humoral and cellular immunity.

Advantages:

DNA is very stable, it resists extreme temperature and hence storage and transport are easy.

A DNA sequence can be changed easily in the laboratory.

The introduced DNA only encodes the proteins of interest but does not replicate.

Since protein components are absent there is no immune response against the vector itself.

Because of the way the antigen is presented, there is a cell-mediated response that may be directed against any antigen in the pathogen.

Disadvantages:

Insertional mutagenesis is caused due to potential integration of DNA into host genome.

Induction of autoimmune responses: Against introduced DNA, anti-DNA antibodies may be produced.

Induction of immunologic tolerance: The expression of the antigen in the host may lead to specific non-responsiveness to that antigen.

ANTI-IDIOTYPIC VACCINE - In an antibody the antigen binding site (paratope) is a reflection of the three-dimensional structure of part of the antigen (epitope). This unique structure in the antibody is known as the idiotype., it is considered as a mirror of the epitope in the antigen. By introducing the antibody into another animal, antibodies can be produced against the idiotype. This anti-idiotype antibody mimics part of the three dimensional structure of the antigen. This can be used as a vaccine. When the anti-idiotype antibody is introduced into a vaccinee, antibodies (antianti - idiotypeantiobodies) are formed that identify a structure similar to part of the virus and may help to neutralize the virus.

Advantage:

Antibodies against potentially significant antigen can be produced.

Dis- Advantage:

1.Only humoral immunity is produced. There is no cellular immunity and poor memory. Identification and preparation of idiotypes is labor intensive and difficult.

WORKING PRINCIPLE OF VACCINES:

Disease causing organisms have at least two distinct effects on the body:

The first effect exhibiting symptoms such as fever, nausea, vomiting, diarrhea, rash, and many others, the second effect is the effect that generally leads to eventual recovery from the infection: the disease causing organism induces an immune response in the infected host. As the response increases in strength over time, the infectious agents are slowly reduced in number until symptoms disappear and recovery is complete(WHO 2012).

Induction of immune response occurs by the disease causing organisms contain proteins called "antigens" which stimulate the immune response. The resulting immune response is multi-fold and includes the synthesis of proteins called "antibodies." These proteins bind to the disease causing organisms and lead to their eventual destruction. In addition, "memory cells" are produced in an immune response(WHO 2012). These are cells which remain in the blood stream, sometimes for the life span of the host, ready to mount a quick protective immune response against subsequent infections with the particular disease causing agent which induced their production. If such an infection were to occur, the memory cells would respond so quickly that the resulting immune response could inactivate the disease causing agents, and symptoms would be prevented.

EFFICACY

Vaccines do not guarantee complete protection from a disease. Sometimes this is because the host's immune system simply doesn't respond adequately or at all. This may be due to a lowered immunity in general (diabetes, steroid use, HIV infection) or because the host's immune system does not have a B-cell capable of generating antibodies to that antigen(BC center for disease control n.d.).

Even if the host develops antibodies, the human immune system is not perfect and in any case the immune system might still not be able to defeat the infection.

Adjuvants are typically used to boost immune response. Adjuvants are sometimes called the dirty little secret of vaccines in the scientific community, as not much is known about how adjuvants work. Most often aluminium adjuvants are used, but adjuvants like squalene are also used in some vaccines and more vaccines with squalene and phosphate adjuvants are being tested.

THE EFFICACY OF THE VACCINE IS DEPENDENT ON A NUMBER OF FACTORS:

the disease itself (for some diseases vaccination performs better than for other diseases).

the strain of vaccine (some vaccinations are for different strains of the disease)

whether one kept to the timetable for the vaccinations

some individuals are 'non-responders' to certain vaccines, meaning that they do not generate antibodies even after being vaccinated correctly(Center for Disease Control and Prevention 2008)

Other factors such as ethnicity or genetic predisposition(BC center for disease control n.d.).

When a vaccinated individual does develop the disease vaccinated against, the disease is likely to be milder than without vaccination.

BACKGROUND: STREPTOCOCCUS PNEUMONIAE

Streptococcus pneumoniae was first isolated and grown by Sternberg and Pasteur in 1881. The encapsulated gram-positive bacterium of 0.5-1.25 μm in diameter was subsequently named pneumococcus by Fraenkel in 1886(Watson D 1993).It has one of the largest public health and economic impacts of any infectious disease agents in both developing and industrialised countries. According to WHO statistics, pneumococcal infections annually cause 1 to 2 million deaths among children less than 5 years of age(J 1986).Rapidly increasing antimicrobial resistance of pneumococci, as well as the HIV–pandemic, further increases the urgency of the development of effective means to control pneumococcal infections.Vaccination appears to provide the best means of prevention against pneumococcal infections. The pneumococcal polysaccharide vaccine, which was licensed as 14–valent (i. e. containing capsular polysaccharides of 14 different pneumococcal serotypes) in 1977 and as 23-valent formulation in 1983, is effective in protecting healthy adults against invasive pneumococcal disease. Unfortunately people at greatest risk, i.e. young infants, elderly and immune-compromised individuals, are the poorest responders to the vaccine.The new conjugated pneumococcal vaccines are immunogenic already at early age due to the T-cell dependent immune response, which leads to increased production of antibodies, development of immunological memory and maturation of the immune response so that the majority of the antibodies are of high avidity.The first 7-valent pneumococcal conjugate vaccine is already licensed in Australia, Europe,

VIRULENCE FACTOR OF STREPTOCOCCUS PNEUMONIAE:

Streptococcus pneumoniae was first isolated and grown by Sternberg and Pasteur in 1881. The encapsulated gram-positive bacterium of 0.5-1.25 μm in diameter was subsequently named pneumococcus by Fraenkel in 1886(Watson D 1993).The complete genome sequence of an isolate of serotype 4 was determined in 1997. The genome consists of 2,160,837 base pairs and 2236 genes or coding regions, of which 1440 (64%) have been assigned a biological role(Tettelin H 2001).The surface of the bacteria is constructed of three morphologically different layers: plasma membrane, cell wall and capsule. The plasma membrane forms the innermost structure. The cell wall consists of teichoic acid and peptidoglycan, which anchors surface proteins and cell wall polysaccharides (PS), such as C-polysaccharide (CPS).The cell wall components, primarily CPS and lipoteichoid acid, mediate inflammation and can activate the alternative complement pathway.The outermost layer consists of a PS capsule, which varies in thickness for different pneumococcal serotypes.The capsule is the primary factor for the virulence of the bacteria.It protects pneumococcus from phagocytosis by polymorphonuclearleukocytes by shielding the inner structures of the bacterium from antibodies and by separating bound antibodies and complement from receptors on phagocytes.Pneumococci that lack the capsule are normally a-virulent in normal hosts.Antibodies to the capsular PSs are the main element in protection against pneumococcal infections.Differences in the chemical structure of capsular PSs provide the basis for classifying pneumococci into serotypes. The standard method for serotyping is so-called quellung reaction, in which a bacteria suspension is mixed with antiserum and the following distinct capsular microprecipitation reaction is observed under microscope.The original quelling method, described by Neufeld in 1902, has been simplified by using counter immuno-electrophoresis, co-agglutination or latex agglutination methods in a chessboard system, consisting of nine pooled antisera and 46 group- or type-specific antisera, or 12 pooled antisera.Two different systems of nomenclature exist for the classification of pneumococcal serotypes, namely the Danish and the American system. The Danish system is based on cross-reactions between different types so that serologically cross-reactive types are assigned to a common serogroup. This system classifies pneumococci into 46 serotypes/groups, numbered 1-48, with a total of 90 different serotypes. The American system numbers different serotypes sequentially according to the order of their discovery.

In addition to the PS capsule and cell wall components, several pneumococcal proteins and toxins have an important role in the virulence of the bacteria. The pneumococcal surface protein A (PspA), which binds to the cell wall choline, inhibits complement activation and thus phagocytosis of pneumococci.It is an important pneumococcal protein vaccine candidate as it extrudes from the surface of the capsule, is present in all clinical isolates and provides protection against carriege, sepsis and pulmonary infection in animal models.PspA is highly heterogeneous, but cross-protection against heterologus PspA has been demonstrated in mice.The choline binding protein A (CbpA), also called pneumococcal surface protein C (PspC), was the first pneumococcal surface protein adhesin described. It is important in human colonization by pneumococci, during the process from colonization to invasion, and in penetration of pneumococci across the blood-brain barrier.It also protects against carriage and bacteremia in mice. The pneumococcal surface adhesin A (PsaA) is a 37-kDa surface protein, which has an important role in ATP-binding transport system responsible for the import of manganese and zinc.In mouse model/mouse models, the antibodies to PsaA seem to be protective, especially against the carriage of pneumococci.

Pneumolysin (Ply) is a 52.8 kDa cytoplasmic toxin released by autolysis of the cell. It is able to damage a wide range of eukaryotic cells, including bronchial and alveolar epithelial cells, by rupturing the target cell’s membrane resulting in leakage of intracellular solutes and colloid-osmotic lysis of the cell.Pneumolysin stimulates the production of TNF-alpha and IL-1β by human monocytesand it may activate the classical complement pathway. It also inhibits the chemotaxis of polymorphonuclear leukocytes, thus facilitating the survival of pneumococci.Antibodies to Ply appear to reduce the fatality of pneumococcal infections in mouse models, but do not necessarily protect against the infection.The Ply has, however, been shown to enhanche protection provided by PspA or CbpA in a murine model.Pneumococci undergo a spontaneous phase variation between opaque and transparent colony morphologies. By means of this phase variation, the bacterium adapts to survive on the mucosa or to invade. The phase variation involves modification of several cell wall structures including CPS, teichoic acid, choline, and proteins.The transparent phenotype with less capsule, more choline and more adhesin CbpA possesses a better ability to colonise the nasopharynx as it binds more effectively to the glycoconjugate receptor in the oral epithelial cells and to the PAF receptor in the cytokine activated lung cells or endovascular cells than the opaque phenotype.The transparent phenotype is also more efficient in invading meninges.The opaque phenotype, on the other hand, has improved virulence and survival in the blood, as it bears less choline and more PspA. It is also more resistant to phagocytosis, as it requires 1.2 to 30-fold more immune human serum in order to achieve 50% killing of opaque phenotype pneumococci in an opsono-phagocytic assay compared to the transparent phenotype.

PNEUMOCOCCAL INFECTION:

NASOPHARYNGEAL CARRIAGE:

Pneumococci cause a wide variety of infections, ranging from common mucosal infections, such as otitis media, sinusitis and conjunctivitis, to systemic infections including septic arthritis, endocarditis, pneumonia, bacteremia and meningitis. The most common entry point for pneumococci is the nasopharynx, where it can establish symptomless colonisation or from which it can spread to the respiratory system or invade to the bloodstream.Several adhesive components of pneumococcus are important in establishing the colonisation. Among those are PsaA, PspA, neuramidase, pyryvate oxidase, PspC and other choline binding proteins.An increase in teichoid acid expression correlates with an enhanced ability of transparent phase variants of pneumococci to colonise. The expression of capsular PS is also essential for the colonisation, but even those bacteria with reduced levels of capsule can remain highly virulent. The adhesion of bacteria on respiratory mucosa requires glycoconjugate receptors bearing N-acetylgalactosamine B1-3 galactose, N-acetylgalactosamine B1-4 galactose, and siliac acid. Viral respiratory infections appear to enhance the adhesion. The process of colonisation can occur simultaneously for several pneumococcal serotypes.

EPIDEMIOLOGY OF PNEUMOCOCCAL CARRIAGE:

In general, the results obtained in different carriage studies may not be directly comparable. In community studies the nasopharyngeal samples are usually collected from healthy children whereas in some hospital-based studies the samples may be collected from children attending clinics for diseases such as acute respiratory infections and AOM, which are associated with increased carriage rates. Also, the techniques for collecting samples, culture media and storage conditions used, and the serotyping process may vary in different studies. The WHO Working Group, including representatives from different pneumococcal research groups, has described a standard methodology, which should reduce the methodological variation in different study settings.Pneumococcal carriage is most common among young children.

The carriage rate increases gradually with age and peaks during the second year of life, after which it gradually decreasesto the adult rate of 20-30%, or less. In most developing countries and also in indigenous populations in some industrialized countries, the acquisition of pneumococci occurs during the very first months of life whereas in most industrialized countries the time of first colonization with pneumococcus more commonly occurs during the second six months of life or beyond.The risk for pneumococcal carriage acquisition is increased in crowded conditions, common in poor countries with large families of low socio-economic status and inadequate housing. Similarly, the high risk for carriage acquisition and epidemics of mucosal and invasive pneumococcal infections have also been demonstrated in institutions such as military training camps, prisons, nursing homes and, importantly, in day-care centres.

Other risk factors include other young siblings in the family, lack of breastfeeding, parental smoking and respiratory infections. The age of the host, presence of anti-pneumococcal protein and PS antibodies, and the immunogenicity of the serotype in question are probably the most important factors determining the duration of the pneumococcal carriage. The carriage may be followed by appearance of type-specific anti-pneumococcal antibodies.The rate of colonization of strains with reduced susceptibility to antimicrobials varies by population and population subgroups. It correlates with recent antibiotic usage and the overall use of antibiotics in the community. Most of the resistant strains belong to the common paediatric serogroups 6, 14, 19 and 23.

RISK FACTORS

Several risk factors for pneumococcal infection have been identified. The non-immunological deficiencies include suppression of cough reflex by alcohol, opiates, or aging and the damage to the clearance mechanisms by exposure to cigarette smoke or air pollutants. Pneumococcal infections are more common in certain ethnic groups such as in American Indians, Alaskan Natives, Australian Aboriginals and in black persons. Whether this is due to genetic susceptibility or other environmental and socio-economic factors is difficult to determine. The HIV -infection increases the risk of invasive pneumococcal infections by 3 to 22 –fold. Other risk factors associated with increased risk of invasive pneumococcal infections are listed below:

Prematurity

Sickle Cell Disease

Congenital or Surgical asplenia

Immuno-compromised children

HIV infection

Chronic renal failure

ACUTE OTITIS MEDIA

Acute otitis media (AOM) is a result of complex interaction between pathogen and host responses. Epidemiologic, microbiologic and immunologic studies indicate that AOM occurs concurrently with, or immediately after a viral upper respiratory tract infection. Viral respiratory infections increase the adherence of bacterial pathogens to the nasopharyngeal mucosa and impair the function of the Eustachian tube, resulting in negative middle ear pressure and capillary leakage of fluid into the middle ear. The symptoms of middle ear infection are derived from the activation of inflammatory defence system. Clinical AOM is consequently defined as a condition where both acute ear-related symptoms and signs of middle ear fluid must be present.A bacterial pathogen can be isolated from the middle ear fluid (MEF) in two thirds of AOM cases in children. The three most common bacterial pathogens are: Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis (Multiple reference).Pneumococcus, which causes 30-50% of the culture positive episodes, is the most virulent organism based on its ability to cause inflammation and symptoms.Respiratory syncytial virus (RSV), para-influenza virus, influenza virus and human rhinovirus are the most common viral pathogens detected in the MEF samples.Nasopharyngeal acquisition of a pathogen is a major risk factor for AOM. Children who have the highest rate of nasopharyngeal colonization during the first months of life are thus at greater risk for an onset of AOM.Other recognized risk factors such as day care attendance, the presence of siblings, parental smoking, lack of breast feeding, and pacifier use are also associated with an increased risk for viral respiratory tract infections and carriage acquisition of bacterial pathogens. Therefore, the independent effect of each risk factor is difficult to estimate.

EPIDEMIOLOGY:

Acute otitis media is the most common bacterial infection in childhood with a majority of children experiencing one or more episodes of AOM during the first two years of life.The peak age-specific incidence of AOM occurs between 6 and 12 months of age.Otitis media is also the most common indication for antibiotic treatment in industrialised countries, despite its mostly self-limiting nature.Furthermore, chronic otitis media is the most common indication for surgical operation in children in the U.S.The epidemiology of AOM is not well studied in developing countries. While the incidence of AOM appears to be at least similar as in industrialized countries, several community-based studies have shown that the sequelae of AOM are more common in developing countries. The perforation of tympanic membrane has been present in 0.4% – 33.3%, otorrhoea in 0.4% – 6.1%, and mastoiditis in 0.19% – 0.74% of children.Otitis media and its complications are estimated to cause 51 000 deaths annually, of which the great majority occur in developing countries, as well as substantial hearing impairment and learning disabilities.The prevalence of significant hearing impairment among primary school aged children has been from 7% to 14.1% in studies conducted in India, Nigeria and Tanzania.

PNEUMONIA:

The pathogenesis of pneumococcal pneumonia is not fully understood. Pneumococcus gains access to lungs from the nasopharynx via aspiration. The preferred target for the adherence of the bacteria in the alveolus is the type II cell.The initial attachment to the pulmonary epithelium is directed by phosphorylcholine. The choline on teichoic acid can serve as a direct ligand to the PAF receptor, which appears on the human cell surface following an inflammatory stimulus.The choline also serves as a docking station for a family of choline binding proteins (CBPs) such as CbpA1, PspA and autolytic enzyme.The interaction of the pneumococcus with the PAF receptor in activated epithelial cells is a key step in the transition to intracellular invasion and subsequent progression of pneumonia to bacteremia.The progression of adherence to disease may also require additional pre-existing events such as viral respiratory infectionor damage caused by cigarette smoke.The progression of pneumococcal pneumonia in animal model is presented in Table 2. The increase in epithelial permeability leads to the formation of serous exudate (engorgement stage), which progresses to red hepatisation as erythrocytes leak into alveoli in the absence of leukocytes. Later, when leukocytes migrate into the consolidated area, the lung exhibits grey hepatisation or fibrinous consolidation.The severity of illness needs not correlate with the extent of pulmonary consolidation.Pneumolysin is the major toxin contributing to the pathogenesis of lung injury. It causes haemolysis, suppression of ciliary beat, activation of phospholipase, changes in the ultrastructure of type I alveolar cells and inhibition of the respiratory burst of leukocytes.Pneumolysin and pneumococcal cell wall components, such as phosphorylcholine and lipoteichoic acid, can activate the alternative complement pathway, which further contributes to oedema and fibrin depositions. Pneumolysin also induces with interaction of CD14 secretion of cytokines.Although the intense leukocyte response to pneumococcus depends also on the number of bacteria in the alveolus, the main chemokines enhancing the influx of leukocytes are PAF and C5a.Once in the alveolus, the polymorphonuclear leukocytes pin the bacteria against the alveolar wall in order to engulf them in a process called surface phagocytosis.

DIAGNOSTIC CONSIDERATION:

Pneumonia can be defined as an inflammation of lung tissue due to an infectious agent stimulating an inflammatory response that damages the lung tissue.The diagnosis of pneumonia is difficult to standardise due to an array of tests available to detect the inflammation of lungs and potential aetiology of infection. In many developing countries with limited diagnostic facilities available, the acute lower respiratory tract infections (ALRI) are managed by using simple clinical signs and symptoms as defined by the WHO in 1982.The ALRI is defined as cough or difficult breathing simultaneously with increased respiratory rate (>50 per minute in infants less than 12 months of age, and > 40 per minute in children aged 12 to 59 months). This definition has > 90% sensitivity and 50%-60% specificity in detecting those ALRI cases, which present with radiological findings suggestinga bacterial infection. the golden standard for the diagnosis of pneumonia is chest radiograph. The interpretation of radiographs is, however, often difficult as universally agreed definition for radiologically confirmed pneumonia does not exist. The WHO is currently coordinating an exercise to standardise the interpretation of chest radiographs for the diagnosis of pneumonia in children. A standardised definition would probably enhance the pneumonia disease burden studies as well as allow inter-study comparisons in efficacy of vaccines preventing pneumonia.Pneumococcal pneumonia is difficult to diagnose. The most specific methods for detecting bacteria as a causative agent for pneumonia are blood culture and transthoracic needle aspiration method. Unfortunately both methodshave their limitations. Detection of bacteria by blood culture is specific, but lacks sensitivity.Other non-specific tests for bacterial pneumonia include chest radiograph and erythrocyte sedimentation rate, white blood cell count, IL-6 and acute phase proteins such as C-reactive protein (CRP) and procalcitonin. In general, dense alveolar infiltrates suggests bacterial pneumonia whereas interstitial infiltrates are associated with viral or mixed viral-bacterial pneumonias.

EPIDEMIOLOGY:

According to WHO estimates, the ALRIs including measles associated pneumonia cause 20 – 25 % of the global estimate of 12 million annual deaths among

children less than 5 years of age [See figure 2].

Figure 2

Over 95% of these deaths occur in developing countries. Considering diagnostic difficulties, effect of age, season of the year and mixed bacterial-viral infections, most studies have concluded that pneumococcal pneumonia accounts for 20% to 40% of all community-acquired ALRI in both industrialised and developing countries. The incidence of ALRI varies widely in different countries. Community based studies using the WHO definition for ALRI in six developing countries showed incidence of 104 to 1768 new episodes per 1000 child years. An alternative approach to determine the disease burden attributable to different causative agents of ALRI is to use a vaccine study as an epidemiological probe. Mulholland et al demonstrated an efficacy of 21 % in reduction of radiologically proven ALRI in Gambia with a Hib conjugate vaccine (PRP-T). This information led to an estimation of Hib disease burden. The incidences of pneumonia and meningitis caused by Hib were 1250 and 250 per 100 000 child years, respectively. The incidence of pneumococcal pneumonia was extrapolated to be at least three times higher than that of Hib. A Hib effectiveness study with PRP-T vaccine in Chile demonstrated similar results with 22% to 26% reduction in suspected bacterial pneumonia cases. The Kaiser Permanente Northern California study with 7-valent pneumococcal conjugate vaccine (7-PncCRM) reported 20.5% effectiveness against radiologically proven pneumonia in children less than 5 years of age.

DEVELOPMENT OF PNEUMOCOCCAL CONJUGATE VACCINE:

The first clinical trial with a Pneumococcal vaccine was conducted in South Africa in 1911. The objective of that study was to prevent epidemic pneumonia in gold miners with a vaccine containing whole heat-inactivated pneumococci of one or more unknown serotypes.During the following decades the protective effect of antibodies against capsular PS was discovered.During the 1980s, the limitations in the efficacy of the PncPS vaccines, especially in the high disease burden populations, and the success of Hib conjugate vaccines, lead to the development of pneumococcal conjugate vaccines.The method for improving the immunogenicity of PSs by conjugation was developed already in the 1920s, but utilised only in the 1980s in development of Hib conjugate vaccines. The new Hib vaccines, of which the first was licensed in the U.S. in December 1987, were found safe, immunogenic and effective in eradicating the vast majority of serious Hib infections in countries where the Hib vaccine was included in the national vaccination program.The pneumococcal conjugate vaccines have been developed along the same principles as the Hib conjugates. The major difference is that the protection against Hib infection requires immunity to only one capsular PS, whereas the protection against a significant proportion of pneumococcal infections requires immunity to several of the at least 90 identified capsular PSs.This results in multivalent vaccines, in which each purified capsular PS is coupled covalently to an immunogenic carrier protein.The amount of carrier protein must be sufficient to induce T –cell memory for each PS, but still small enough to minimise the induction of anti-carrier antibodies.The excess amount of carrier protein may also impair antibody response to the TD antigen through antigen competition or carrier-mediated epitope suppression.

IMMUNOGENICITY AND SAFETY STUDIES:

The development of pneumococcal conjugate vaccines began with 1-valent formulations (Fattom A 1990). Different pneumococcal conjugate vaccine candidates have been evaluated in immunogenicity and safety studies (Wuorimaa T 2002). The main characteristics for the evaluation of immunogenicity are as follows:

1. The magnitude and persistence of antibody response following the primary series and subsequent doses of the vaccine in different age groups

2. The induction of immunologic memory and avidity maturation

3. Isotype or IgG subclass distribution

4. Functional activity of antibodies

The pneumococcal conjugate vaccines are immunogenic in infants (Dagan R 2003) (Eskola 2000) (Hsu KK 2002), toddlers and adults, and prime for immunologic memory. In infants, the elicited IgG antibodies are mainly of subclass IgG1, whereas in adults the main subclass is IgG2 (Soininen A 1999). The elicited antibodies are functional when measured by OPA and show avidity maturation. The pneumococcal conjugate vaccines induce modest mucosal immune response as demonstrated by the induction of IgA -secreting cells and appearance of salivary IgA and IgG antibodies. The vaccines are also immunogenic among those not responding to PS vaccines (Musher DM 1990), in patients with recurrent respiratory infections (Barnett ED 1999)and in HIV infected individuals, lymphoma patients (Chan CY 1996), renal transplant recipients, BMT recipients (Spoulou V 2000) and in the elderly.

Most immunogenicity studies have reported antibody responses after three doses of vaccine, and following the fourth (or booster) dose. In studies where the responses to each vaccine dose have been reported, the poorly immunogenic serotypes 6B and 23F have required 3 doses for significant increase in the GMC of antibodies, whereas the highly immunogenic serotypes 4 and 19F may require only 1 or 2 vaccine doses for significant responses (Eskola 2000). The avidity and OPA of antibodies seems to increase with the number of doses, but already one dose is enough to induce immunologic memory (Goldblatt D 1998). Both conjugate and PS vaccines have been used as a booster following the primary series with a conjugate vaccine. The PncPS vaccine with higher PS content (25 μg vs. 2-10 μg ) may induce higher GMCs than conjugate vaccines (Ahman H 2001). The avidity might be higher following a booster dose of conjugate vaccine (Anttila M 1998).

INTERFERENCE WITH OTHER ANTIGENS:

Immunogenicity studies have not revealed major problems related to simultaneous administration of pneumococcal conjugate and other paediatric vaccines (K. H. Wuorimaa T 2002). Several studies have, however, reported interactions in immunogenicity between different vaccines. Concurrent and mixed administration of conjugate vaccines with the same carrier protein appears to lower responses to pneumococcal polysaccharides (Choo S 2000). However, if the carrier proteins are different such phenomenon might not occur.

SAFETY:

The pneumococcal conjugate vaccines appear to be safe and well -tolerated among children and adults (Eskola J 1999). No significant safety problems have been reported in so far conducted safety and immunogenicity studies with over 20 000 subjects receiving various numbers of doses of different vaccine candidates (K. H. Wuorimaa T 2002) (Eskola J 1999). Local reactions such as redness, swelling, induration and pain at the site of injection have been the most common adverse reactions following the immunization. The rate of local reactions has varied between 6% and 38% in different studies, but has been lower at the pneumococcal than at the DTP-Hib vaccine site (32% to 53%). Fever above 38.5Ο C has occurred in up to 52% of children, 22 % in toddlers and 4% in adults (K. H. Wuorimaa T 2002). The increased valency of conjugate vaccines has not led to an increase in adverse reactions. No increase in the incidence or severity of local reactions has occurred when giving the subsequent doses of primary series or booster in childhood (O'Brien K 2000).

EFFICACY STUDIES:

Efficacy against Pneumococcal Carriage:

Pneumococcal conjugate vaccines are able to reduce nasopharyngeal carriage of serotypes included in the vaccine. The reduced carriage may lead to the interruption of the transmission of these serotypes in the community and thus formation of herd immunity (F. D. Dagan R 2000). As the vaccine-type serotype strains are often resistant to antimicrobials, the use of pneumococcal conjugate vaccines has the potential to substantially reduce the number of infections due to resistant pneumococci (Whitney CG 2002). The efficacy against pneumococcal carriage has so far been demonstrated only in young children with high carriage rates. As adults and older children are less likely to acquire pneumococcal carriage (K. 2001).

Efficacy of Pneumococcal Conjugate Vaccine against Acute Otitis Media:

The protection against vaccine-type pneumococcal AOM varied widely between serotypes, being lowest for 19F (25%) and highest for 6B (84%) (Palmu A 2001).

Efficacy of Pneumococcal Conjugate Vaccine against Pneumonia:

The impact of pneumococcal conjugate vaccines in prevention of pneumonia was conducted in the Kaiser Permanente Northern California study, the incidences of first pneumonia episodes were 53.4 and 55.9 per 1000 person years in vaccine and control groups, respectively (Black SB 2000).

Efficacy of Pneumococcal Conjugate Vaccine against Invasive Pneumoccocal Disease:

PncCRM vaccine is effective in reducing IPD among vaccinated children. The post-licensure surveillance has further demonstrated a significant decline in IPD incidence in children less than 5 years of age (H. J. Whitney CG 2002). The incidence of IPD caused by vaccine serotypes among children less than one year of age ranged between 51.5 and 98.2 per 100 000 person years before the immunisation started.

WHO Recommendations forProduction and Control Materials/Intermediates for Pneumococcal Conjugate Vaccines

S.Pneumonia Polysachharide

Seed Lot System:

The bacteria used for the preparation of the polysaccharides are strains of S. pneumoniae isolated from patients with otitis media. Master and working seeds from these strainswere derived and have been grown and stored in the absence of any material of animal origin.

Fermentation and Purification:

The production of the polysaccharides of the different serotypes is very similar and differs just in a few aspects of the purification process, depending on the nature of the PS:

acid or neutral PS. The content of a working seed vial is used to inoculate the fermentor; the fermentation occurs undercontrolled temperature, air-flow rate conditions and stirring, and it is stopped according to opticaldensity (OD) measurement.

At the end of the fermentation, inactivation of bacteria is achieved using phenol added to the fermentation broth.

Characterization:

Characterization analysis of polysaccharide bulks documenting identity, structure,

size, integrity and content of functional groupswere performed.

Specification:

The release of the polysaccharides are based upon the water content, alcohol content, molecular size distribution, residual protein, nucleic acid and endotoxin content.The specifications applied to most of the tests are those recommended by the applicable guidelines (WHO guideline ‘Recommendations for production and control of S. pneumoniae conjugate vaccines)

Stability:

The stability of purified S. pneumoniae polysaccharides (PS) is assessed by longterm,

real-time stability studies (up to 84 months) on three batches per polysaccharide

serotype. Batches are stored at -20°C or -70°C depending on the serotype.

Purified Diptheria Toxoid

Seed Lot System:

The strain of Corynebacterium diphtheriae that is used to manufacture purified diphtheria toxoid.The seed lot system was established using meat-free culture media.

Fermentation and Purification:

The manufacturing process of the purified diphtheria toxoid consists of 3 main stages: fermentation, detoxification and purification.The production of purified diphtheria toxoid is performed following the WHO and general GMP requirements. Purified diphtheria toxoid, complies with WHO (TRS 800), (Ph. Eur) requirements.

Characterization:

Characterisation was conducted on the purified DT bulks including SDS gel electrophoresis and other physicochemical tests were performed.

Specification:

The tests and specifications for release of the DT bulks should be complying with WHO (TRS 800) and European Pharmacopea.

PRODUCTION OF CONJUGATE VACCINE:

Protein Production

(fermentation of Host Organism, collect, wash, homogenize, recover and purify protein)

Polysaccharide production

(Fermentation of donor organism, disrupt cells, purify polysaccharide, trim by hydrolysis

Derivativization: make both components reactive (certain Chemicals

Conjugation: React Components together

Purification: Remove unwanted Compounds (using Tangential Flow Filtration for removal of impurities)

Characterization of the purified conjugate ( SEC HPLC for estimation of free carrier protein and molecular distribution of polysaccharide-protein conjugate)

Finish and fill: Concentrate, Sterile Filtration under aseptic conditions, dialysis into appropriate solute.

Techniques used in Production andCharacterization of Conjugate vaccine:

In preparation of the polysaccharide-protein conjugate vaccines, the most critical steps are the removal of the unbound polysaccharide as these polysaccharide present in the final conjugate does not contribute to the immunogenicity of the vaccine. For the removal of these impurities from the final conjugate bulk techniques like ultrafiltration, micro-filtration or tangential flow filtration are used. Characterization procedures are used to ensure the reproducibility, stability and safety of the conjugate bulk that is producedfrom the above said process. The conditions used in the conjugation process may alter the structure of the polysaccharide chain by causing the loss of labile substituents. For the estimation of molecular distribution of the protein-polysaccharide conjugate, Size Exclusion High Performance Liquid Chromatography technique is used.

SIZE EXCLUSION CHROMATOGRAPHY:

Size exclusion chromatography (SEC) is a chromatographic method in which particles are segregated on the basis of their size, or in more technical terms, their hydrodynamic volume. It is used in case of large molecules or macromolecular complexes such as proteins and industrial polymers. Both molecular weight and three dimensional shape contribute to the degree of retention. There are two types of size exclusion chromatography:

Gel Filtratiion chromatography (GFC) :

It is used when an aqueous solution is used to transport the sample through the column.

Gel Permeation chromatography (GPC):

This technique is used when an organic solvent is used as a mobile phase.  GPC should always function free of interactions to assure separation by size only. Only the entropy effects should influence the separation.

 

METHODOLOGY:

This method include good separation of large molecules from the small molecules with a minimal volume of eluent.In SEC the analyte does not interact with the surface of the stationary phases. Differences in time of elution are strictly based on the volume the analyte "sees"(ie the time the analyte cover the area of the pore). Thus, a small molecule will penetrate every corner of the pore system of the stationary phase "sees" the entire pore volume and the interparticle volume, and will elute late. In case of a very large molecule it cannot penetrate the pore system and will only "sees" only the inter-particle volume (approximately 35% of the column volume) and will elute faster. This technique is then combined with HPLC.Normal HPLC methods rely on interactions between sample and stationary phase. These interactions result from ion exchange, bio-affinity, or chirality.When this volume of mobile phase has passed through the column the free (unconjugated) protein content is determined by size exclusion high performance liquid chromatography.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Liquid chromatography is a separation technique that involves:

It allows injection of small volume of sample

A tube is packed with porous particle which act as stationary phase.

The individual components of the sample are allowed to flow along the packed tube (column) by a liquid moved by gravity.

The components of the sample are separated from one another by the column packing chemistry whichinvolves various chemical and physical interactions between the sample molecule and the packing particles. The separated components are collected at the end of the column and it is allowed to be identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the colour, or by another device that can give a quantitative measurement.

WHY HPLC?

HPLCis a separation technique which involves:

It allows injection of small volume of sample

A tube is packed with porous particle which act as stationary phase(3 to 5 micron (μm) in diameter called the stationary phase)

Individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.

These components are separatedfrom each other by the column packing that involves various chemical and physical interactions between the sample molecules and the packing particles.These separated components are detected at the end of the column by a flow-through a detector that measures their amount. An output from this detector is called a "liquid chromatogram".LC and HPLC has the same principle and they work the same way except the speed,efficiency, sensitivity and ease of operation of HPLC is vastly superior.

HOW DOES A CHROMATOGRAM LOOK?

Eg: Sample of vitamin E in organic solvent. General run time for HPLC is 10 – 30 minutes.

COMPONENTS OF HPLC:

The components of HPLC are as follows:

PUMP

The role of the pump is to force a solvent (mobile phase) through a liquidchromatograph at a specific flow rate, in milliliters per min (mL/min).

Normal flow rates in HPLC are in the 1-to 2-mL/min range.

Typical pumps are capable of reaching pressures in the range of 6000-9000 psi (400-to 600-bar).

During the chromatographic experiment, a pump can deliver a constant mobile phase flow rate (isocratic) or an increasing mobile phase flow rate (gradient).

INJECTOR

The injector is used to introduce the liquid sample into the flowing mobile phase.

Sample volumes are ranged from 5-to 20-microliters (μL).

The injector should withstand the high pressures of the liquid system.

An autosampleris an automatic version for easier loading of the sample where the system will automatically pull the sample from the tray.

COLUMN

It is the most important part of the chromatograph, as the column’s stationary phase separates the sample componentsof interest using various physical and chemical parameters.

The small particles inside the column causes the high back pressure at normal flow rates.

The pump must force the mobile phase through the column and this resistance to the flow causes a high pressure within the chromatograph.

Columns are packed with small diameter porous particles.

The most commonly used sizes are: 5-μm, 3.5-μm and 1.8-μm

Columns are packed at high-pressure sothat they are stable during use.

These porous particles in the column mostly have a chemically bonded phase on their surface that interacts with the sample molecule to separate them from one another for example, C18 is a popular bonded phase.

The process of retentionof the sample components (often called analytes) is based on the choice of column packing and the mobile phase which is used to push the sample through the packed column.

DETECTOR

The detector detect the individual molecules that elute from the column.

A detector measures the amount of those molecules so that the analyst can quantitatively analyze the sample components.

The detector provides anresults to a recorder or computer that interprets the results in liquid chromatogram(i.e., the graph of the detector response).

COMPUTER OR RECORDER:

It is called the data system. It controls the all the operations of the HPLC systems, it takes signals from the detector and calculates the retention time for the eluate and will also extrapolate the amount of sample that is present.

QUANTITATIVE ANALYSIS OF HPLC CHROMATOGRAM

The measurement of concentration of a sample. There are two main ways to interpret a chromatogram(i.e. to perform quantification):

Determination of the peak height of a chromatographic peakas measured from the baseline;

Determination of the peak area. To make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured. There is a linear relationship between the height or area and the amount of sample.

TRACE ANALYSIS

A trace compound is that compound that is of interest, but it’s concentration is very low, usually less than 1% by weight, often in parts per million.

Trace compounds are very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous;

In a chromatogram trace substances are difficult to quantify and to separate or detect;

High resolution separations and very sensitive and expensive detectors are required.

TEMPERATURE CONTROL IN HPLC:

Reproducibility

Retention time in HPLC is temperature-dependent

If temperature variation occurs, then it is difficult to assign "peaks" to specific compounds in the chromatogram and the peak areas or heights may vary producing false results.

Solubility

Certain chemical compounds may have low solubility in their mobile phase. If they are injected into the flow stream of the mobile phase they may precipitate or other difficulties may arise.

Stability

Certain chemical compounds, like biological compounds such as enzymes or proteins, may not be stable at room temperature or higher

The temperature needs to be much lower down to 4°C

DETECTOR

InHPLCThere are many principles used for detection of the compounds eluting from an HPLC column.The most commonare:

SpectroscopicDetection

Refractive IndexDetection

FluorescenceDetection

Spectroscopic Detection Ultraviolet (UV) Absorption

An ultraviolet light beam is allowed through a flow cell and a sensor measures the light passing through the cell.

If a compound from the column absorbs this light energy, it will change the amount of light energy falling on the sensor.

The resulting change in this electrical signal is amplified and recorded by a recorder or data system.

A UV spectrum is sometimes also obtained which may aid in the identification of a compound or series of compounds.

DIODE ARRAY DETECTOR

OBJECTIVE OF THE STUDY

Characterization of the Conjugate Ps-Pn Active Raw Material using Analytical Techniques.

Vaccines are prophylactic measures to control specific diseases. The development of conjugate vaccine is recently the popular choice for diseases like Invasive pneumococcal, diphtheria and for haemophilus influenza B. Characterization of the conjugated Ps-Pn Active Raw Material using various analytical techniques is an in process and finally corroborative approach to confirm set specific process (USP & DSP). Our main objective of the project is:

Estimation of concentration of protein using Bi-cinchoninic acid assay (Kit Method) after dia-filtration and concentration of the CRM.

Estimation of concentration of polysaccharide content in the final purified conjugate material using Resorcinol test/Orcinol test.

Estimation of bound Polysachharide-protein conjugate and estimation of other impurities using HPLC technique (In-Process).

Studying the in process, reaction kinetics using High Performance Liquid Chromatograpgy.

Testing of sample during Tangential Flow filtration (TFF) cycles for estimation of removal of un-conjugated materials, free protein and polysaccharide using HPLC.

Endotoxin test for estimation of endotoxin level in the final Purified conjugate material.

In the PROCESS DEVELOPMENT LAB at PANACEA BIOTEC (LTD) the following sequence of processes are followed routinely for the assessment of conjugated polysaccharide-protein active raw material.



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