2.0 Review of literature

Transcription

1 Review of literature Donald Emmeluth (2004) elucidated that, in the early 16 th century, Thomas Willis was the first person who accurately described typhoid fever based on clinical observations and that it is different from typhus fever. Later, In 1880 Karl was credited with isolating the bacterium responsible for typhoid fever and since then diagnosis of typhoid fever became easier. Further in the year 2005, Christopher described typhoid fever as a major endemic threat caused by Salmonella typhi, a gram negative bacterium belonging to the family of Enterobacreiacae, transmitted between the individuals by fecal-oral route via contaminated food and water and is therefore common where sanitary conditions are inadequate, particularly where the access to clean water is limited. Disease burden was estimated to be around 6-33 million cases annually, resulting in 216,000 deaths in endemic areas. The prevalence of typhoid disease was more in the developing countries like Asia, Africa and Latin America. In 1994, 26,55,000 cases were reported from Africa with 1,30,000 deaths; whereas Asia reported 1,33,10,000 cases with 4,40,000 deaths. The mean incidence of typhoid fever in developing countries was estimated between America to 150 cases per million populations per year in Latin 1000 cases per million populations per year in some Asian countries as reported by Bir Singh, (2001).

2 16 Sinha et al. (1999) conducted a study to estimate the incidence of typhoid fever in pre-school children to define the optimum age of immunisation and the choice of vaccines for Public-Health programmes in developing countries. This was done in Kalkaji, Delhi in 1995 between November 1, 1995, and October 31, 1996, checking around 8172 residents of 1820 households. Blood samples were obtained from febrile patients, and those who tested positive for Salmonella typhi were treated with ciprofloxacin. Around 63 culture positive typhoid fever cases were detected. Of these, 28 (44%) were in children aged under 5 years. Suman et al. (2008) identified that typhoid fever is still continuing as a major endemic disease all over the world. Hospital based studies and outbreak reports from India indicate that enteric fever is a major public health problem in this country, with Salmonella enterica serovar Typhi (S. typhi) the most common etiologic agent. The incidence of the disease was reported mostly in the areas with poor sanitation and inadequate supply of safe drinking water. 2.1 Salmonella typhi: Brenner et al. (2000) described and classified Salmonella typhi as motile, aerobic, non-spore-forming, flagellated, gram negative bacilli which belong to the family of Enterobacteriaceae that includes the genus of Shigella, Escherichia and the Vibrio. The genus of

3 17 Salmonella contains two different species, S. enterica and S. bongori. Particularly, the agents that cause enteric fever are therefore Salmonella enterica subspecies enterica serovar Typhi and serovars Paratyphi A, B and C. Any serovar or a sero type can be defined as a strain that have a unique surface molecule which is responsible for the production of specific antibody. Each serotype has subtle chemical differences in their antigenic portion. Khan et al. (2008) explained that humans and domestic animals are susceptible to ingested causative organisms through contaminated food and water. The ingested typhoid bacilli infect small intestine, rapidly penetrate the mucosal epithelium and arrive at lamina propia. Once the bacterium reaches the lamina propia in the host, the bacilli elicit an influx of antigen presenting cells, mostly macrophages that ingest the organisms but unable to kill them. 2.2 Multi-drug resistance: Many antibiotics were in use to treat typhoid fever before the avalibilty of modern typhoid vaccines. From 's chloramphenicol was the drug of choice widely used to treat typhoid fever. But unfortunately in 1987 multi drug resistant strains (MDR) of Salmonella typhi were identified. These MDR Strains were resistant to several antibiotics like chloramphenicol, ampicillin, amoxicillin, and co-trimoxazole. The first documented MDR strains outbreak was

4 18 reported in Malaysia in 1984 (Sajjad, 2005). Since then, MDR strains spread rapidly throughout Southeast Asia and China resulting as endemic regions. The reason for the occurrence of multidrug resistance to organisms is due to misuse, abuse and over use of the antibiotics. The other reason is due to the incompletion of the medication which results in killing of least resistant bacterium, leaving the most resistant one still viable. (Donald Emmeluth, 2004). The studies of Khan et al. (2008) revealed that resistance to bacterium towards antibiotics is due to plasmids present in the bacterium. Changing resistance pattern in S. typhi was also observed at Kolkata, India. 2.3 Typhoid vaccines: Typhoid fever is a systemic infection caused by S.typhi and S.paratyphi serovars, where the disease is exclusively spread by fecaloral route, poor hygiene, contaminated food and water sources. Non Typhoidal salmonellae (NTS) infection is commonly observed in HIV infected individuals and hence treating such immunocompromised patients has become a major problem. It was estimated that million cases reported every year resulting 216,000 deaths which mostly include preschool children and young adults. Vaccines against typhoid fever offered substantial protection against the disease. Initially the killed-phenol preserved vaccine whole cell vaccine was in

5 19 use though it was highly reactogenic. Later oral live attenuated Ty21a vaccine was developed in Switzerland by chemically mutating a wild type Ty2 strain lacking both the functional galactose-epimerase gene and Vi antigen. Later findings proved that Virulence (Vi) capsular polysaccharide of S.typhi is immunogenic and vaccine preparations were prepared by Dr.John Robbins at NIH and licensed to Sanofi- Pasteur. The efficacy of Vi vaccine was proved in randomized trials, exclusively in Nepal proved 75% protection against 20 months active surveillance. (WHO Diarrhoeal Diseases, html#Introduction). Ivanoff et al. (1994) described the emergence of vaccination due to the outbreak of MDR strains. The first generation vaccine against typhoid fever was inactivated whole cell (WC) vaccine which is parenterally administered. The circulating, secretory and cellmediated immune response is stronger overall after natural infection than after vaccination, and includes both prominent serum and cellmediated components. Parenteral, killed whole-cell vaccines elicit a serum response equal to a natural infection, but not a comparable cell-mediated response. With parenteral whole-cell vaccine, elicitation of serum H antibodies and sometime Vi antibodies correlates with protection, whereas 'O' antibodies do not.

6 20 Kossaczka et al. (1999) explained about orally administered attenuated S. typhi strain Ty21a vaccine and its limitations; this vaccine requires at least three doses and had a low rate of efficacy in an area with a high incidence rate of typhoid fever. Murphy et al. (1991) explained about oral S.typhi Ty21a vaccine which is made of attenuated bacteria that is delivered orally. Ty2la engenders levels of protection from typhoid fever that are similar to those of parenteral vaccines. Later, Acharya et al. (1987) investigated and revealed that Virulence (Vi) polysaccharide of S. typhi as the protective antigen against typhoid fever. The efficacy of Vi polysaccharide vaccine was greater than the inactivated whole cell and oral S. typhi 21a vaccines. In two randomized double-blinded vaccine-controlled clinical trials conducted in Nepal and the Republic of South Africa, it was evident that one injection of Vi induced about 70% efficacy in children 5 years old and above age. Now commercially Vi polysaccharide vaccine is in use for vaccination. 2.4 Limitations and Drawbacks of typhoid vaccines: Fraser et al. (2009) described the available typhoid vaccines and its limitations. The whole-cell S. typhi vaccine was introduced in 1896, its efficacy was established in 1960 in controlled trials carried out in Yugoslavia, the Soviet Union, Poland and Guyana. This review

7 21 concluded that two doses of this vaccine resulted in 73% efficacy over three years. Therefore, the inactivated whole cell typhoid vaccine is considered unsuitable for use as a public health vaccine and, although licensed, it is no longer available for use. Ivanoff et al. (1994) explained about whole-cell (WC) vaccines. According to him the method of killing cells impacts the immunological aspects. With parenteral, acetone or heat phenol inactivated vaccines the 'O' antibodies are of IgM type [LPS (lipopolysaccharide antigen) is T-independent], while the 'H' antibody response is initially IgM and then becomes IgG. The WC vaccines were in use till 1980 led to decrease in the incidence of typhoid fever and are credited with largely controlling the disease. However, the usage of this vaccine was discontinued because of their side effects (Denise et al., 2007). Tacket et al. (1990) proposed that attenuated S. typhi strain Ty21a is an alternate vaccine that has been efficacious in field trials conducted in United States. Ty2la was made by nonspecific mutagenesis with nitrosoguanidine and the exact mechanism of its attenuation is unknown. This was in use for a long time until the introduction of polysaccharide vaccines.

8 22 Later, McKenna et al. (1995) described the oral typhoid vaccine based on S. typhi strain Ty2la; it was the first live attenuated enteric vaccine to be licensed in many industrialized countries, primarily for the immunization of travellers to typhoid endemic areas. The advantages of this vaccine were: (i) it could be administered orally; (ii) it did not produce severe reactions in the vaccine; (iii) it promoted both local and systemic antibody and cellular immune responses; and (iv) it could be used to deliver antigens from other pathogens to the immune system. The Ty2la strain was derived from the virulent S. typhi strain Ty2 by successive rounds of NTG mutagenesis followed by screening for a stable mutation in the gale gene, which encodes UDP galactose 4-epimerase. Fraser et al. (2009) investigated the efficacy of oral typhoid vaccine which was licensed in 56 countries in Africa, Asia, Europe, and South America and in United States. A theoretical problem associated with the Ty21a vaccine is that it may revert to virulence; however, such hypothetical effects have never been documented in any of the multiple, large field trials conducted. According to one trial of 20,543 participants, this vaccine provided statistically significant protection in each of the first three years (First group: 35%, 95% CI 8% to 54%; Second group: 58%, 95% CI 40% to 71%; Third group: 46%, 95% CI 6% to 72%; CI-Confidence Interval) and the cumulative efficacy for 2.5 to 3 years was 48% (95% CI 34% to 58%).

9 23 Extensive studies of (Santander et al., 2008) revealed that the capsular polysaccharide, commonly known as the virulence (Vi) antigen is the immunogenic component that can raise efficient immunogenicity in humans. The Vi capsular polysaccharide is a protective antigen with majority of antibody response directed towards the O-acetyl groups. S. typhi produces the capsular polysaccharide which is an important virulence determinant during the infection. Vi polysaccharide is a linear homopolymer made up of α-1, 4-linked N-acetylgalctosamiuronate (GalNAcA), with 60-70% of monomeric units O-acetylated at C3 position. Yang et al. (2001) elucidated from study data the protection levels of Vi polysaccharide vaccine. He explained that Vi capsular polysaccharide vaccine can confer around 69% protection against culture-confirmed typhoid fever. This level of protection is similar to that observed for Vi vaccine produced in industrialized countries and tested in both Nepal and South Africa and is also similar to the level of protection observed in the earlier field trial of locally produced Vi vaccine in Jiangsu Province demonstrated in China. Later, Sur et al. (2009) summarized a clinical trial data carried out in the regions of Nepal, South Africa and China. The Vi polysaccharide vaccine was found to be 72% efficacious for the age group 17 to 21 months and 55% efficacious for a three-year period.

10 24 This polysaccharide vaccine is recommended for adults and children of 2 years and older. This vaccine is not recommended to the infants and children below two years of age because of their poor immunogenicity, and hence this vaccine is not included in the routine immunization schedule for children. 2.5 Immunology of bacterial polysaccharides: Weintraub (2003) described the antigenic properties of carbohydrate antigens. The cell wall of many pathogenic bacteria contained carbohydrates in the form of capsular polysaccharides or lipopolysaccharides. These molecules are the virulent factors in causing disease in the infected persons. Bacterial polysaccharides are considered as poor immunogens due to T-lymphocyte independency, and hence characterised by lack of T-lymphocyte memory, isotype restriction and delayed ontogeny. Due to these immunological attributes of bacterial polysaccharides, children below 2 years of age and elderly respond poorly to polysaccharide antigens. Secondly, the wide structural heterogeneity among the polysaccharides within and between species is also a problem. Lin et al. (2001) explained the Vi capsular polysaccharide of S. typhi is as an essential factor and a protective antigen in inducing anti-vi antibodies in young children and adults, but poorly immunogenic in children below 2 years of age. To overcome the

11 25 limitations of the age-related and T-cell independent immunogenicity of the vaccine, the same strategy that was used for the Haemophilus influenzae type b polysaccharide conjugate vaccine was followed; Vi was bound to a nontoxic recombinant protein that is antigenically identical to Pseudomonas aeruginosa exotoxin A (repa) under controlled conditions. Ada et al. (2003) described that pathogenic bacteria contain polysaccharide material in their cell wall which acts as protective antigen to the bacterium and as immunogenic component in the vaccine preparations against the invasive bacterial disease. Polysaccharides induce mainly IgM production, and are only moderately protective in adults and ineffective in young children. H. influenzae type b conjugated vaccine elicited, sufficiently high (>95%) seroconversion rate to suggest that this disease might, in time, be globally controlled in this way. The results of immunization with conjugate vaccines to Streptococcus pneumoniae, and Neisseria meningiditis are also very encouraging. However, Christopher Jones (2005) explained the limitations of bacterial polysaccharides as vaccines, which resulted poor immunogenicity in infants (0-2 years) due to T-Cell independent nature of the polysaccharides. Introduction of glycoconjugate vaccines, in which a cell surface carbohydrate from a micro-organism is covalently

12 26 attached to an appropriate carrier protein are proven to generate an effective immune response in children below 2 years of age. Three such vaccines, against H. influenzae type b, N. meningitidis Group-C and seven serotypes of Streptococcus pneumoniae, have already been licensed and many others are in development. Kalka-Moll et al. (2002) clearly explained the properties of pathogenic bacteria that produce polysaccharides usually in the form of capsule that coats bacterial cell surface and thereby help the bacterium escape from the phagocytosis attack. Hence, these polysaccharides act as protective antigen for the bacterium. Polysaccharides of pathogenic extracellular bacteria commonly have negatively charged groups or no charged groups at all. These molecules have been considered classic T cell-independent antigens that do not elicit cell-mediated immune responses, but rather elicit humoral immunity comprising of low affinity IgM and restricted classes of IgG Abs. Robbins et al. (1999) described that bacterial polysaccharides are immunogenic and efficient in inducing humoral immune responses. But these polysaccharides have limitations failing to induce immunogenicity in young children and re-injection of most does not induce a booster response. Several factors affect the immunogenicity of polysaccharide, probably because of their relatively

13 27 low molar ratio (Mr) which do not induce serum antibodies at any age and are considered as haptens. Another factor taken into consideration is that epitopes of polysaccharides are repeated regularly throughout the molecule and age-related antibody responsiveness towards bacterial polysaccharides. Later the immunological attributes were explained by (Latz et al., 2004) that bacterial polysaccharide antigens remain as T-Cell independent antigens resulting in lack of immunological memory and are poorly immunogenic in children less than 24 months of age. The fact is that, polysaccharide antigens are not processed by antigen presenting cells (APC's); the immunogenicity of glycoconjugate vaccine compared with bacterial polysaccharides is high due to the processing of attached carrier protein by the APC and presentation of carrier protein-derived peptides by MHC-II molecules to the T-Helper cells, followed by induction of cytokine production. The cytokines produced stimulate polysaccharide (PS)-specific B cells for production of PS-specific antibodies. Shriner et al. (2010) elucidated the antigenic differences of bacteria polysaccharides. Basically, carbohydrate antigens are classified as two types: firstly bacterial Lipo-Polysaccharides (LPS), a TI type 1 or (TI-1) antigens, and TI-2 Antigens generally referred as capsular polysaccharides. However, these are characterized by the

14 28 presence of repetitive epitopes that can efficiently cross-link B Cell Receptors (BCR's) and induce activation of Ag-specific B cells. Capsular polysaccharides on a variety of bacterial pathogens, such as S. Pneumoniae, N. meningitidis and Bacillus anthrasis, are classic examples of TI-2 antigens. TI-2 antigens are capable of inducing robust antibody responses in adults; however, they are poorly immunogenic in young children. Adam Finn (2004) explained that purified polysaccharide antigens that are used in vaccine preparations against many pathogenic bacteria are able to induce substantial protective serum antibody responses when used as vaccines. However, for most of these antigens, this is not the case in young children. Since they are not peptides they cannot be processed and presented by MHC class II antigens, so that the recruitment of T cell help by this route for B cell function is not expected. Stephen et al. (2010) proposed that usage of polysaccharides as immunogenic antigens are important components of microbial pathogens and for many vaccines. However, research concerning the activation of the adaptive immune system by polysaccharides gained interest only recently. Traditionally, polysaccharides were considered to be T cell-independent antigens that did not directly activate T cells or induce protective immune responses.

15 29 The polymeric nature of polysaccharides results in cross-linking of several IgM receptors at the surface of B-cells. This linking provides required signals for the production of anti-polysaccharide antibodies without the recruitment of T-cells, hence with no memory effect. The lack of booster responses and the inability of infants to respond to non-conjugated polysaccharide antigens are two very important limitations for the use of polysaccharides as vaccines in infants. The presentation and processing of different polysaccharide antigens cover several different overwhelming mechanisms that are still poorly understood (Icart et al., 2008). The B-cell organization of the immune system can efficiently recognize carbohydrate antigens either as carbohydrates, glycoproteins or glycolipids. However, the recognition of carbohydrates by T lymphocytes or T cells could be possible by the attachment of peptides to convert as T cell dependent carbohydrate antigens. The ability of carbohydrate to influence T cell recognition of antigen has important consequences for a wide range of immune responses as well as the current strategies for mapping T cell determinants (Carbone and Gleeson, 1997). Deck et al. (1999) explained bacterial carbohydrates as set of molecules that can be recognized by T cells. However, to elicit such

16 30 responses, as with the haptens, the carbohydrate needs to be linked to a peptide that binds to an MHC molecule. This is because many carbohydrates do not bind directly to MHC molecules, so the T cell epitope cannot be formed. The T-cell anti-carbohydrate response elicited by glycopeptides could be of importance in the response to viral or tumor antigens. Indeed, many eukaryotic and viral proteins carry covalently linked carbohydrates. The cell wall of the pathogenic bacteria consists of polysaccharide coatings which act as protective antigen against the attack of the immune system. The immunologic advantage of this coating is evasion of phagocyte killing, as the coating blocks complement binding and opsonisation. This can be overcome by C-reactive protein (CRP) binding and the production of antibodies against the polysaccharide. This immune response confers protection against disease. However, especially the young, but also the elderly, have a weak immunological response to these encapsulated bacteria due to the thymus independent (TI) nature of these bacteria (Klouwenberg and Louis Bont, 2008). Goldblatt (1998) clearly described the immunological attributes of carbohydrate antigens that are traditionally referred as T-Cell independent antigens which mount poor antibody response in infants and young children. These unique properties of carbohydrate antigens

17 31 limits their use as vaccine in children less than 24 months of age resulting dominance of IgM, failure to induce memory following immunisation, an absence of affinity maturation following immunisation and poor immunogenicity in infants, the elderly and the immunocompromised. The T-cell independent properties of carbohydrate antigens are altered by coupling an immunogenic protein carrier rendering the carbohydrate moiety of such vaccines immunogenic, even in the very young. This approach was evident in the success of the Hib conjugate vaccines, the first conjugate vaccine licensed clinically for human use. Bacteria that cause meningitis include Haemophilus influenzae, Streptococcus pneumoniae and Neisseria meningitidis, which are the three important bacteria causing invasive diseases in children. In contrast to the first generation of the polysaccharide vaccines, the newly developed polysaccharide-protein conjugate vaccines produce a T-dependent response and result in the development of an immunological memory, inducing protection even in children less than 2 years old (Laval et al., 2003). 2.6 General information of polysaccharide conjugate vaccines: In 1929, Avery and Goebel first showed that covalent binding of haptens to immunogenic proteins make the carbohydrate antigens immunogenic in even children <2 years of age. Chemical coupling of

18 32 polysaccharide-protein antigens makes polysaccharides to acquire T-cell-dependent (TD) characteristics, with increased levels of PS-specific antibodies with increased affinity and a switch from IgM to IgG upon repeated immunizations. Generally native polysaccharide vaccines are immunogenic and provide protection in adults but do not induce memory responses. This happens because polysaccharide antigens are T-cell-independent antigens and do not elicit antibodies in infants and young children. Hence, conjugating chemically modified poly-saccharides to proteins make the combined entity T-cell dependent and immunogenic at an early age (Jakobsen and Jonsdottir, 2003). Introduction of Hib conjugate vaccine resulted in the virtual elimination of invasive Hib disease over past 15 years. The polysaccharides of pathogenic bacteria of S. typhi, H. influenzae type b, S. pneumoniae and N. meningitidis provide both an opportunity and a challenge for vaccine prevention of life-threatening bacterial infections in childhood. Purified capsular polysaccharide vaccines are not satisfactory in early childhood where the burden of disease is highest. The concept of protein-polysaccharide conjugation has provided a solution to the problems of polysaccharide immunogenicity in childhood by recruiting T-cells to the immune response resulting in secondary immune response in children <2 years of age (Kelly et al., 2004).

19 33 Hilleman et al. (1984) described that initially Hib disease was most prevalent all around the world causing endemic bacterial meningitis in infants and has also been identified as the leading cause of acquired mental retardation in United States. The capsular polysaccharide commonly known as Poly-ribosyl ribtol phosphate (PRP) was identified as virulent factor and hence usage of purified capsular polysaccharide in vaccine preparations is recommended. There is much evidence that upon vaccination with purified polysaccharides specific serum antibodies were produced in children and adults. However, such polysaccharides vaccines failed to elicit sufficient antibody response in young infants below 15 months of age. Jessouroun et al. (2007) stated that introduction of polysaccharide-protein conjugate vaccine against Hib disease showed remarkable evidences against pathogenic bacteria causing invasive disease. Initially in 1980's Robbin and his colleagues utilized biotechnological process of chemically attaching carbohydrates to protein carriers, a concept developed 50 years earlier. The primary objective of conjugating polysaccharides to immunogenic proteins is to elicit T-cell dependent responses against attached saccharide molecule. Attachment of protein to a saccharide yields a number of T-cell epitopes, which interact with CD4 Helper T-cells contributing antibody responses to the attached polysaccharide.

20 34 Frasch (2009) proposed polysaccharide-protein conjugation methods that can be employed with different strategy based on the size, molar ratio (Mr), epitopic nature etc. To produce a conjugate vaccine, the purified PS must first be chemically modified to generate reactive groups that can link to the protein. The common methods used for activation of polysaccharide are by periodate oxidation and cyanylation. Determination of the molecular size of the polysaccharide before and after conjugation results in efficient conjugation. The two important critical quality control tests employed after conjugation and purification are the PS to protein ratio and the percent nonconjugated saccharide (Free saccharide). Lindberg (1999) explained that covalent attachment of purified polysaccharides to immunogenic carrier proteins creates glycolconjugates which are T-cell dependent antigens resulting in secondary immune responses. Purified capsular polysaccharides elicit T-independent antibody responses without a memory function, priming poor antibody responses in infants where much of invasive H. influenzae type b (Hib) and pneumococcal infection is seen. Granoff et al. (1994) described that encapsulated bacterium like Haemophilus influenzae type b, S. pnuemoniae, N. meningiditis can cause invasive bacterial meningitis in children and adults.

21 35 As bacterial polysaccharides are poorly immunogenic in infants, polysaccharide vaccines failed to be included in routine immunization schedule. However, Avery and Goebel reported that the immunogenicity of a polysaccharide antigen could be greatly enhanced by coupling it to an immunogenic carrier protein. This was evident with introduction H. influenzae type b (Hib) conjugate vaccines in the United States and several other countries, and immunization with these protein conjugated vaccines has been remarkably successful in decreasing the incidence of Hib disease. The licensed Hib conjugate vaccines currently are HIBTITER, referred to herein as HbOC (Lederle-Praxis Biologicals), consists of polyribosylribitol phosphate (PRP) oligomers conjugated to CRM197 carrier protein, ActHib, referred to here in as PRP-T (Pasteur Merieux, Lyon,France) and PedVAXHib, referred to herein as PRP-OMP (Merck and company Inc., West point, Pa).In India, Hib vaccines are manufactured by Bharat Biotech International Ltd., Shantha Biotechnics Ltd., Sereum Inistuite and Biological E Limited. Attachment of chemically modified polysaccharides to proteins converts polysaccharide as T-cell-dependent (TD) character. It is evident that polysaccharide-protein conjugate vaccines are able to induce antipolysaccharide antibodies in 2 to 3 months old children. (Breukels et al., 1999) investigated cellular interactions which determine the magnitude and nature of this B-lymphocyte response.

22 36 An in vitro culture system to determine the induction of an antipolysaccharide response in healthy adult volunteers who were vaccinated with the H. influenzae type b polysaccharide (PRP)-Tetanus toxoid (TT) conjugate vaccine was studied. Kuberan et al. (2000) described that infant less than 2 years of age possess immature immune systems and, hence they are more susceptible to invasive bacterial infections. For example, the capsular polysaccharide of Group 'B' meningococci is linked with homopolymer of N-acetyl neuraminic acid. Immunizations with such homopolymer polysaccharides results in poor immunogenicity due to immunotolerance. Such antigens are falsely recognized as Self by immune system since a similar polysaccharide is a component of a glycoprotein, called as neural cell adhesion molecule (NCAM). Segal and Pollard (2004) described that vaccines prepared with bacterial polysaccharides against H. influenzae type b, S. pneumoniae and N. meningitidis were first introduced decades ago. But they generate T-independent immune responses, do not induce immunological memory and poorly immunogenic in infants, making them unsuitable for implementation in universal infant immunization programmes. These considerations led to development of proteinpolysaccharide conjugate vaccines against Hib during the 1980's generating polysaccharides by eliciting T-lymphocyte dependent

23 37 immune-responses, immunoglobulin class-switching and immunological memory. 2.7 Bacterial polysaccharide conjugates: Protein antigens are capable of inducing T-cell immune responses, the reason being that the major histocompatibility complex II (MHC-II) present on the antigen presenting cells are capable of recognizing only protein antigens. T-cells are incapable of responding to the antigen, unless the antigen is presented by MHC-II complex. This immunological response leaves polysaccharides as poor immunogens, as only MHC-I complexes are capable of recognizing the polysaccharide antigens. Several factors affect the immunological properties of the polysaccharides which make them poor immunogens (Robbins et al., 1999). Schneerson et al. (2009) explained the method of preparing immuno-conjugates by linking the size excluded polysaccharide or hapten to an immunogenic carrier protein. Polysaccharides are linked with spacers like dihydrazide and linked to the carrier via hydrazone linkage. Poly-γ-glutamic acid (γpga) is extracted from culture supernatant of B. anthracis and B. pumilus and conjugated to BSA, repa and recombinant protective antigen (rpa) with B. anthracis γdpga and B. pumilus γdlpga. Immunogenicity of these conjugates

24 38 was studied in mice and serums IgG anti γdlpga antibodies were demonstrated. Hemberger et al. (2009) invented a method for coupling of proteins to derivizated polysaccharides. The terminal aldehyde group of polysaccharides forms chemical reaction with the functional group of protein with covalent linkages. The polysaccharide-protein conjugates chemically prepared may be used for the prophylaxis for human in vaccine preparation steps. Several polysaccharide and proteins were selectively used in the experiment viz. conjugation of oxidized high molecular weight HES (ox-hes-130 kda) to human serum albumin (HSA), coupling (ox-hes-130 kda) to myoglobin (Mb), coupling (ox-hes-130 kda) to superoxide dismutase (SOD), coupling of (ox-hes-130 kda) to streptokinase (SK), Human interleukin-1(il-2), TNF α (Tumor necrosis factor α ), Glucagon-like peptide (GLP-1), HSA and Glucagon. Loibner et al. (2005) proposed a method of preparing polysaccharide protein conjugates by treating the carbohydrate moieties with a protein containing at least one free amino group carrier, wherein polysaccharides are hydrolysed to create vicinal aldehyde groups, treated with base-instable proteins and then coupled to polysaccharides linkage under controlled conditions.

25 39 Lees et al. (2001) explained the methods for removing free protein in solutions containing polysaccharide-protein conjugates. The process is simplified which includes a solid phase and restricted access media material. The free or the low molecular weight proteins bind to the media material while the conjugate can be collected as purified fractions. McMaster (2000) described different methods of purification steps involved in preparation of polysaccharide-protein conjugate vaccines. He explained the simple, efficient scalable method of purification of conjugates by ultrafiltration techniques in presence of ammonium sulphate. In this experiment adipic acid dihydrazide linked Neisseria meningitidis group C polysaccharide is ultra-filtered against 50% saturated ammonium sulphate using a 30,000 Da MWCO membrane, and removal of free polysaccharide content was determined. Lees (1997) detailed the methods of preparing polysaccharideprotein conjugates, primarily the modification of polysaccharides by cynalating reagents. In this method, carbohydrate is firstly activated with CDAP and then covalently linked to the protein molecules. Porro (1994) reported the methods for producing oligosaccharide conjugate against serotypes of Streptococcus pneumoniae. In preparing

26 40 a oligosaccharide conjugate, 4-12 carbon moieties were linked to the protein in the presence of carbodiimide condensation. Chemically modified oligosaccharide residues were coupled to carrier proteins via lysine derivatives. Jennings et al. (1982) proposed the methods of producing immunogenic polysaccharide-protein conjugates against meningococcal serotypes A, B and C. Meningococcal polysaccharides were modified to obtain aldehyde groups by controlled oxidation of vicinal hydroxyls and then coupled to carrier proteins via reductive amination. Helting (1981) reported that membrane proteins isolated from N.meningitidis group B were immunogenic when used as vaccine component. The bacterial cells were treated with different concentrations of Sodium deoxycholate and purified protein fractions were used in vaccine preparations. Kuo (1980) explained the process for producing polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of H. influenzae type b. The process described the isolation and purification of antigenic PRP using ethanol, cetrimide and hydroxylapatite. It elicited high antibody responses and showed intrinsic bactericidal activity when tested for serum bactericidal assays.

27 41 Hagopian et al. (1978) described the methods of isolation and purification of high molecular weight Meningococcal group C polysaccharides. Generally, polysaccharides are extracted using cetavlon and calcium chloride treatment, phenol extractions, ultra centrifugations and ethanol precipitations. The process described the liquid medium for cultivation of bacterium, stages of inoculum development, fermentation process and purification of meningococcal antigen as an immunogenic component in final vaccine preparations. Rienstra et al. (1991) detailed a process for removal of endotoxin from polyribosyl ribitol phosphate, a capsular polysaccharide of H. influenzae type b. Generally gram negative bacterium releases large amount of endotoxins upon fermentation; several purification procedures help in limiting the endotoxin concentration in purified polysaccharide bulks or in the vaccine lots. The present study detailed the purification steps that include selective ethanol fractionations which limited endotoxin levels in Hib polysaccharide. Aunins et al. (2000) elucidated the principles of vaccine production which includes upstream process (Bacterial/Viral cultivation), purification methods and formulations. Concerning bacterial vaccine, S. typhi Ty21 and BCG are purified by continuous flow centrifugation or cross-flow membrane filtration. Vaccines against

28 42 whooping cough are obtained using B. pertussis cells, and in the case of antigens filamentous haemagglutinin (FHA) and pertussis toxin (PT) are released in the fermentation cell supernatant. Diphtheria vaccine is manufactured by isolating 58 kda toxin and converted into toxoid with formaldehyde and purified. The crude diphtheria toxoid is concentrated by ultrafiltration and separated from host cell impurities by ammonium sulphate precipitation. In case of capsular polysaccharide vaccines Meningococcus, Pneumococcus and H. influenzae type b are purified using cetavlon extractions and selective alcohol precipitations and purified polysaccharides are conjugated to proteins as effective vaccines in eliminating paediatric bacterial infections. Paz et al. (2003) explained the production of capsular polysaccharide from N. meningitidis group C in batch mode fermentation process. The study described different media compositions that increase capsular polysaccharide production. Frantz, modified Frantz and Catlin 6 media were used to grow the bacterium and fermentation studies were carried out and polysaccharide production obtained using three experimental media were compared. Michel et al. (1999) reported the methods for preparing group B Streptococcal vaccine. The polysaccharide of group Streptococcus B is

29 43 conjugated to Streptococcus C protein antigen which induced protective antibodies against Streptococcus B serotytpe. Antibody responses to conjugate vaccines are highly specific and long-lasting, particularly in young children. The T-Lymphocyte dependency of carbohydrate-protein molecules are evident in inducing protective immune responses in rats when immunized transcutaneously with Hib-CRM197 vaccine when combined with cholera toxin or mutants of heat liable enterotoxin of Escherichia coli as adjuvants (Mawas et al., 2004). Jin et al. (2003) studied the immunogenicity of group A meningococcal polysaccharide conjugated to bovine serum albumin and assessed the immune responses in mice. In the experiment adipic acid dihydrazide (ADH) was used as a linker and bound to cyanogenactivated meningococcal group A polysaccharide (GAMP); in another experiment ADH was linked to BSA in the presence of EDC and then bound to activated GAMP. It was observed that immunogenicity elicited by GAMP conjugates were improved by activating the polysaccharide with CDAP resulting high molecular weight conjugate. Barrett et al. (1983) described the ELISA methods to determine Vi antibodies against polysaccharide antigens in human serum. The antibody titres obtained by ELISA testing was compared with

30 44 haemagglutination assay (HA) which is generally used to quantify Vi antibodies. In the present ELISA testing method, Alkaline phosphatase-conjugated goat anti-human IgG was used to identify specific anti-vi IgG antibodies as Haemagglutination tests are more specific determining only IgM antibodies. Shafer et al. (2000) explained the use of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in activating bacterial polysaccharides prior to coupling with a protein. This application is majorly used in preparation of conjugate vaccines against disease H. influenzae type b, N. meningitidis and S. pneumoniae. Experiments were carried out to determine the selective proteins that could be coupled to CDAP activated polysaccharides. It was observed that proteins that are derivatized with hydrazides can be coupled to CDAP activated polysaccharides at lower or slightly acidic ph. The immunogenicity of hydrazide-treated protein coupled with CDAP polysaccharides elicited adequate immune responses when injected in mice. Determining molecular sizes of polysaccharides and polysaccharide-protein conjugates of bacterial polysaccharides is an important aspect in designing conjugate vaccines. The assessment of physico-chemical characteristics of polysaccharide-protein conjugate plays important role in eliciting specific immune responses. Analysis

31 45 of molecular size for Hib polysaccharide conjugated to tetanus toxoid was determined by high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column (Parisi and Hunolstein, 1999). Lee et al. (2009) detailed the methods of preparation and characterization of group A meningococcal polysaccharide vaccine and its immune responses in mice and humans. Men-A polysaccharides are conjugated to tetanus toxoid by reductive amination, injected in mice and tested for ELISA and intrinsic bactericidal activity using a complement source. The manufacturing process of Men-A PS TT conjugate vaccine was reproducible and scalable and hence vaccine can be manufactured and used for immunization in endemic regions particularly in sub-saharan Africa. Pawlowski et al. (2000) explained the steps in preparation of pneumococcal polysaccharide protein conjugate and tested in laboratory animals for immune responses. The production of 14 and 23 valent pneumococcal conjugate vaccines was developed by different conjugation mechanisms. The methods of size fragmentation of polysaccharides and coupling techniques were briefly discussed for preparation of PS14TT and PS23FTT conjugate vaccines and immunogenicity of the vaccine was assessed in mice and rabbits.

32 46 Anderson (1985) described the immunogenicity of H. influenzae type b oligosaccharides conjugated to CRM197 of Diphtheria toxin. Conjugates of Hib-CRM197 were injected into rabbits, and it was observed the conjugates elicited strong antibody responses to anti - H. influenzae type b capsular polysaccharide and a group of conjugates elicited antibody responses specific to diphtheria toxin. Masignani et al. (2010) reported that lipo-proteins isolated from H. influenzae type b containing different amino acid sequences can be used as immunogenic components in vaccine preparations against bacterial meningitidis. Virlogeux et al. (1996) explained the genes responsible for Vi polysaccharide expression S. typhi. He proposed that the regions viaa and viab chromosomal loci control the expression of the antigen. The viab region plays important role in bio-synthesis of Vi antigen and translocation of the polysaccharide to the cell surface. Makela (2003) discussed the breakthrough achieved by the use of conjugate vaccine throughout the world in eliminating infectious disease. He explained the success of Hib vaccine immunization in infants which reduced the incidence of the disease in parts of Europe, the Americas and Australia. The role of capsule is clearly explained in that the existence of the bacterium in the host depends upon their

33 47 polysaccharide capsule; absence of capsules in a bacterium can get it killed by phagocytosis. As capsular antigen-based vaccines are incapable of eliciting immune responses in young children, experience with Hib vaccines made us to understand the properties and function of conjugate vaccines in young children. The conversion of T-cell independent into T-cell dependent is the critical attribute in the conjugation mechanism which makes the polysaccharide-protein conjugates more capable in evoking efficient immune responses in all age groups. Merritt et al. (2001) discussed the production scale up of polyribosylribitolphosphate (PRP), a capsular polysaccharide of H. influenzae type b, from 1.5 L to 500 L of fermentation process. The strain used in study was Eagan and CY medium was optimized for seed and production medium with required hemin and NAD concentrations. Fed-batch experiments were carried out to increase PRP yield with different substrates like yeast extract and glucose fedbatch experiments and dextrose and concentrated yeast extract solutions; the effect of sterilization time on medium components were also studied. Results of the study concluded that PRP expression obtained was 1.3 g/l. Reeves et al. (1996) commonly explained the synthesis of bacterial polysaccharides and their gene nomenclature. Most species

34 48 of Salmonella, Shigella and E. coli secrete polysaccharides and therefore act as prominent antigens in raising immune responses. Bacteria generally contain lipooligosaccharides (LOS) as well as antigenic extracellular polysaccharides. Lipopolysaccharides are abundantly present in the gram negative bacterium. He proposed that LPS constitutes three regions which comprise of: Lipid A component, oligosaccharide and the O-antigen. Rose et al. (2005) cited the importance of pneumococcal conjugate vaccine for use in infants and immunocompromised patients. It was noticed that immunization with pneumococcal polysaccharide vaccine (PPV) in infants and immunocompromised patients elicited limited immunogenicity. With regard to this, they carried out the trial to determine whether immunization with pneumococcal conjugate vaccine (PCV) would elicit specific immune responses to the individuals immunized previously with pneumococcal polysaccharide vaccine (PPV). The results of study revealed generation of a rapid memory response in patients after priming with pneumococcal conjugate vaccine (PCV). Establishing quality control methods for polysaccharide conjugate vaccines gained interest in production process of bacterial vaccines. Ion exchange chromatography is used for determination of oligosaccharide chain length of polysaccharides derived from

35 49 H. influenzae type b and N. meningitidis sero groups A and C respectively. Carrier proteins (CRM197) are used to couple activated polysaccharides and the characterization of polysaccharide-protein conjugates, accurate size determination and degree of polymerization are considered to important aspects in designing glycoprotein vaccines (Ravenscroft, 1999). Recently synthetic conjugate vaccines gained interest against Haemophilus influenzea type b disease. Preparations of synthetic conjugate constitute at least one antigenic determinant with corresponding amino acid. In contrast PRP used in the study was chimeric T-B form which is lipidated that can be covalently linked to the carrier protein (Chong et al., 1997). Cabrera et al. (2006) explained novel polysaccharide protein coupling methods for obtaining conjugate vaccines against bacterial diseases like S. typhi, N. meningitidis and Vibrio cholerae. The immuno-genicity of these conjugates were studied in mice and the anti-igg titers were measured. Polysaccharides were subjected to base hydrolysis and linked to tetanus toxoid via carbodiimide-mediated condensation. Conjugates were passed through Sepharose CL-4B column and fractions corresponding to specific Kav were collected, pooled and injected to mice; IgG antibody titers were estimated using

36 50 ELISA. This approach is applicable for preparation of polysaccharide conjugates against bacterial disease. Halsey et al. (1983) studied protective efficacy of Hib vaccines in animal models upon administration of bacterial challenge. Vaccine components containing PRP antigens combined with Diphtheria toxoid, Tetanus toxoid, Pertussis vaccine (DTP) were injected to infants followed by bacterial challenge. Serum anti-prp antibodies were measured before challenge administration. Vaccination of pertussis or DTP alone did not protect from Hib bacteremia. When PRP was injected alone or combined with pertussis antigens it was noticed that the degree of protection levels were too low. Detecting serum antibodies by immunological methods upon vaccination has become more sensitive. The quantitative measurement of serum antibodies to PRP antigens by ELISA has become feasible for assaying large number of serum samples in clinical trial studies. In the present study a competitive ELISA method was described to quantify antibodies produced upon vaccination with Hib capsular polysaccharides, so called as PRP vaccines. This method was able to detect low titer of antibodies and described as conventional non-competitive method. This method was able to display a good correlation with radio antigen binding assay (RABA). (Mariani et al., 1998).

37 Vi polysaccharide-protein conjugates vaccines: With all the above evidences, a new design was developed to optimize Vi polysaccharide-protein conjugates aiming that it could be an effective approach for childhood immunisation particularly in the infants below two years of age. Szu et al. (1991) clearly explained the relation between the structure and immunological properties of Vi capsular polysaccharide in eliciting specific protective immune responses. Vi capsular polysaccharide linear homopolymer of poly-alpha(l-4)galnacp variably O-acetylated at the C-3 position, carboxyl reduction, O-deacetylation, and acid hydrolysis studies indicated that when Vi is completely subjected to O-deacetylation it eliminated the immunogenicity. This aspect showed that immunogenicity was related to degree of O-acetylation. However, a partial O-deacetylation showed increased immunogenicity profiles. It was noticed that antigenicity of acid treated and carboxyl-reduced Vi was greater for both native and O-deacetylated Vi polysaccharide. The use of antibiotics for the treatment of typhoid fever is a challenge due to the outbreak of antibiotic resistance strains. This prospect promoted interest in vaccine development against typhoid fever. Earlier inactivated whole cell vaccines were available followed by live attenuated oral vaccines. Later, the development of purified