4.0 Results and Discussion

Transcription

1 Results and Discussion 4.1 Results of bioprocess and conjugation process: Bacterial growth and characterization: Source of strain: The strain Salmonella typhi (Ty2) was obtained from Dr. John Robbins, National Institutes of Child and Human Development, USA. The strain was well characterised biochemically and serologically and the seed strain was used to prepare the master seed lot and working seed lot Biochemical characterization of the strain S.typhi (Ty2): Gram stained culture showed the presence of minute gram negative rods. Growth on Deoxycholate Agar (DCA) confirmed the presence of S. typhi. Since this is not a selective medium, the identity of S typhi was further confirmed by the use of Selective and Differential media. Growth on Xylose Lysine Deoxy Cholate (XLD) agar produced black-centered colonies. This is a selective medium and the growth on this confirms that the organism as gram negative, as XLD inhibits the growth of gram positive organisms. Xylose present as the carbohydrate source in the medium is fermented by the organism and the presence of lysine reverts the ph to an alkaline condition; sodium thiosulfate present is the sulphur source and ferric ammonium citrate is an indicator which results in the formation of black colonies under alkaline condition.

2 95 Growth of S. typhi on BSA (Bismuth Sulphite Agar) shows black colonies with a surrounding metallic sheen. This is due to the production of hydrogen sulphide and reduction of sulphite to black ferric sulphide. Growth on Triple Sugar Iron media confirmed the organism as S. typhi as this media inhibits the growth of other micro organisms belonging to Enterobacteriaceae. Hydrogen sulphide can react with ferrous sulphate in the medium to form ferrous sulphide. This is visible as black precipitate. There is no gas formation, which is a characteristic of other Enterobacteriaceae organisms. Isolates were identified as Salmonella serovar typhi by identification of the following characteristics: glucose positive without gas formation, H2S positive on XLD Agar, and positive serology with anti-serum to Vi polysaccharide. This is similar to the results represented by Poppe et al. (1995) and Haider et al. (2007). The photographs of growth/colony characterstics observed in our laboratories on various media were taken and presented in figures 4.1 to 4.4.

3 96 Figure 4.1 Growth of S. typhi on XLD Agar as observed in our lab Figure 4.2 Growth of S. typhi on Bismuth Sulphite Agar as observed in our lab

4 97 Figure 4.3 Growth of S. typhi on Deoxycholate Citrate Agar as observed in our lab Figure 4.4 Growth of S. typhi on TSI slants The photographs of glucose utilization test and slide agglutination test carried out on the culture are shown in figure 4.5 and 4.6 respectively.

5 98 Figure 4.5 Glucose utilization test Figure 4.6 Slide Agglutination test A - Negative B - Positive

6 99 Table 4.1 Observations of Biochemical characteristics of Salmonella typhi (Ty2) S. No. Test performed Observations 1 XLD Agar Bismuth Sulphite 2 Agar Black centered colonies with H2S Production Black coloured colonies H2S production 3 Deoxycholate Citrate Black centered colonies 4 Triple Sugar Iron Indicates glucose utilization 5 Glucose utilization No gas formation 6 Slide Agglutination test Positive serology with anti-serum to Vi polysaccharide Preparation of Master and Working seed lots: The characterization of Master and Working seed lots was carried out in R&D labs of Bharat Biotech International Limited, Hyderabad. After all the tests were found satisfactory, Master and Working seed lots were prepared as per the details given in Materials and Method from and

7 100 Table 4.2 Tests done for characterization of Salmonella typhi (Ty2) Master seed lot S. No. Test performed Observations 1 Gram staining of culture Gram negative bacilli Utilization of glucose (Durham's method) Bacterial colonies on agar plates (Oxidase test) Culture of Salmonella typhi should form agglutination with antisera Utilized glucose without gas production Negative for oxidase Agglutination observed. 5 Viable cell count 8 x 10 6 CFU/mL The working seed lot was charecterised as per the Master seed lot and viable cell count was found to be 9 x 10 6 CFU/mL and further proceed with fermentation process Fermentation process: Inoculum development: One cryovial from working seed lot of S. typhi was inoculated into 10 ml Soybean Casein Digest Medium (SCDM) and incubated at 37±1ºC for 12 hours (Stage-I), transferred to two flasks each containing 50 ml SCDM at 37±1ºC for 12 hours (Stage-II) and finally transferred to four flasks each containing 500 ml SCDM and incubated at 37±1ºC for 12 hours (Stage-III). At every stage of

8 101 subculture purity was checked by gram staining and microscopy. The OD was checked at 600 nm. OD of the culture at Stage - I: 1.2 OD of the culture at Stage - II: 2.6 OD of the culture at Stage - III: 3.8 Optical density of the S. typhi culture recorded at different stages of growth varied from 1.2 to Fermentation (Batch phase and Fed batch mode): Stage III cultures with an OD600 of 3.8 was ideal for inoculating into fermentation medium. The ph was maintained at 6.90 and dissolved oxygen level was between 40 to 60%. Ammonia (50%) solution to the fermentor was connected for auto feed and ph maintained in the range of 6.9 to 7.2. A fed batch fermentation process was to increase biomass by feeding with a solution containing glucose at a range of 1 to 2 mg/ml concentration. This feeding strategy resulted in an increase in the biomass up to OD. The increased biomass translated into a greater Vi polysaccharide production which achieved a final concentration of 923 mg/l. Fed batch fermentation process for S.typhi resulted in increased Vi polysaccharide production. Similar study was carried out by International Vaccine Institute (IVI), Seoul to optimize

9 102 Vi polysaccharide production of S. typhi Ty2 under controlled conditions in a bioreactor. The entire fermentation process was monitored so that the Vi polysaccharide is released spontaneously from the bacterial cells into the culture medium during the cultivation process. The optimal ph maintained in the study was 7.2 using 10% NH4OH and dissolved oxygen concentration was maintained at 35% air saturation. Glucose level was monitored every 30 minutes, and through the fed-batch process the glucose concentration was maintained at about 1 g/l throughout the process. The components in the feed medium used were changed for different runs of fermentation during optimisation studies. In the present process the yield of Vi polysaccharide obtained in the fed batch culture was 415 mg/l. It was found that molecular size of Vi polysaccharide was consistent and did not degrade during the different phases of bacterial cultivation (Jang et al., 2008). Similar, fed batch fermentation studies carried out with Haemophilus influenzae type b for production of capsular polysaccharide (PRP polyribosylribitolphosphate) for use in conjugate vaccines gained interest. Merritt et al. (2001) proposed a fed batch fermentation process that showed a four-fold increase of PRP when compared to the batch mode fermentation process. He identified the defined medium that could increase PRP production. He studied the effect of yeast extract at concentrations ranging from 0.5 to 7.5 g/l

10 103 and glucose (20 g/l) was also used in the feed medium to increase PRP production. These experiments resulted in a maximum of 6 g dry cell weight per liter and 1.3 g/l PRP concentration. Virlogeux et al. (1995) have shown that the expression of Vi is controlled by the viaa and viab Chromosomal loci. He explained the role of the viab locus in synthesis, transport and expression of S. typhi Vi antigen. Studies suggested that phenotypic characteristics of the mutants and complementation experiments catalyzed the synthesis of Vi antigen by TviB and TviC polypeptides. TviA protein was expected not to involve in the expression of Vi polysaccharide. Vi antigen production at the bacterial cell surface was due to the VexE protein dependence. The results demonstrated that the regulatory pathway of Vi antigen expression of S. typhi is explained due to the involvement of tvia and viaa products and the role of viab locus. Arricau et al. (1998) studied RcsB-RcsC regulatory system of S. typhi in modulating the expression of invasion-promoting proteins and Vi antigen in response to osmolarity. When S. typhi was cultured in epithelial cells, he observed the secretion of Sip proteins and flagellin and Vi antigen was differentially modulated by RcsB-RcsC regulatory system. At high osmolarities Vi antigen biosynthesis was greatly reduced, whereas under low osmolarity conditions Vi polysaccharide

11 104 was maximally produced. The results obtained in the current experiments confirm these findings. Virlogeux et al. (1995) and Zhang et al. (2006) have shown that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDP-GalNAc, followed by the TviCcatalyzed epimerization at C-4 to form UDP-GalNAcA which serves as the building block for the formation of the Vi polymer. With regard to this Zhang et al. (2006) analyzed TviB activity over a range of ph from 6.0 to 9.6 and found the ph optimum at 7.2, with a large decrease in activity near ph 8.4. In the present study, during optimized fed batch fermentation, ph was controlled and maintained at 6.9 during the mid-stationary phase and this signalled the end of Vi production. This result showed that ph 6.9 was optimum for fermentation. All the fermentation parameters are recorded in a log sheet; a scanned copy of fermentation log sheet is shown in table 4.3 below.

12 105 Table 4.3 Fermentation parameter record

13 Down stream process: Fermentation cell supernatant is subjected to different steps of purification to isolate purified Vi polysaccharide. Vi polysaccharide consists of partly 3-O-acetylated repeated units of 2-acetylamino-2- deoxy-d-galactopyranuronic acid with α-(1 4) linkages. Hence the determination of O-acetyl content could be correlated to the amount of Vi polysaccharide. The final pure Vi polysaccharide fraction should contain 2 mm of O-acetyl per gram of Vi polysaccharide (WHO TRS 840). The supernatant normally contains large amount of proteins, nucleic acid and lipopolysaccharides. Filtration techniques play an important role in downstream processing in purification of bacterial polysaccharides from host cell impurities. Retention of the desired molecule from the dissolved substances is done on the basis of size; higher sized particles will be retained at the surface and those lower than the nominal weight limit (NMWL) of the membrane flow out in the permeate (Jagannathan et al., 2008). In the present study also 100 kda cut-off membrane cassettes were used at initial step of cell supernatant concentration and 300 kda cut-off membrane cassettes at final concentration step and diafiltered using WFI.

14 107 In some cases high molecular weight molecules (mostly nucleic acids) can be easily precipitated by ethanol precipitation and finally the resulting low molecular mass oligonucleotides are filtered through membrane. In a similar method, Polyribosylribotol phosphate (PRP) from capsular polysaccharides from Haemophilus influenzae type b is purified using ethanol, cetavlon and a phosphate containing adsorbent hydroxyl apatite. Kuo et al., 1980 described in the initial step of purification ethanol and sodium acetate is added to cell supernatant, followed by cetavlon treatment; nucleic acids and proteins are further removed by ethanol precipitation and final PRP concentrate is treated with hydroxyl-apatite. The results of the physico-chemical characteristics of the purified PRP in the method was molecular size of 0.44 kda, ribose sugar at 33.8%, nucleic acid at 0.3/mL and protein at 0.7/mL. When compared to the above study, we obtained the following O-acetyl contents at different steps of downstream processing, as given in the table 4.3 below. The O-acetyl content was analyzed by Hestrin method as described in the materials and methods.

15 108 Table 4.4 O-acetyl content and Vi content at each processing step Sl. No. Step Volume (L) O-acetyl content (µmoles/ml) Vi content (mg/ml) 1 Cell supernatant Concentrated supernatant Cetrimide precipitation Ethanol precipitation Concentration & Diafiltration Sterile filtration The final sterile filtered (0.22 µ) Vi-polysaccharide bulk is lyophilized in a low temperarure vacuum dryer (Lyophilizer FTS system) Results of lyophilized Vi polysaccharide bulk: The lyophilized powder was tested for serological identification by Ouchterlony method, moisture content, protein content, nucleic acids, molecular size distribution and bacterial endotoxin content. Ouchterlony test was developed by a Swedish bacteriologist Orjan Thomas Gunnarson Ouchterlony in He employed the immunodiffusion in gels, a technique that is useful for the analysis of

16 109 antigens and antibodies. Ouchterlony is a classical immunochemical technique and classified as single or double diffusion. (Bailey, 1996).In the present study purified Vi polysaccharide and corresponding homologous antisera were filled in the wells until the meniscus just disappears. The gel plate was incubated in a humidity chamber. The precipitin lines were observed by naked eye when the plate was seen against a bright light back ground. A photograph of the same is shown in figure 4.7, showing a clear precipitin arc observed. Figure 4.7 Serological identification test of Ouchterlony Several purification procedures of Vi polysaccharides were witnessed to contain low amounts proteins, nucleic acids and lipopolysaccharides. In a study where Vi polysaccharide was used to construct Vi and Di-O-acetyl pectin protein conjugates with adipic acid dihydrazide, the purified Vi polysaccharide was found to contain

17 110 2% nucleic acids, 1% protein and 1% lipopolysaccharide (Kossaczka et al., 1997). Similarly a Vi vaccine (vax-tyvi ) was tested for immunogenicity in Cuban children and teenagers; the Vi polysaccharide bulk used in the vaccine used in clinical trial contained >2µmol/mg O-acetyl groups,<10µg/mg proteins, <20 µg/mg nucleic acids, and <150 EU/µg endotoxins (Azze et al., 2003). Results of lyophilized Vi Polysaccharide bulk obtained are tabulated in the table 4.5 below. Table 4.5 Results of Lyophilized Vi polysaccharide bulk Tests Results Serological identification of Ouchterlony Clear precipitin arc was observed Moisture content 1.60% Protein Nucleic acids O-acetyl content (Hestrin) Molecular size distribution Endotoxins 2.4 mg/g of Vi polysaccharide powder 6 mg/g of Vi polysaccharide powder 2. 2 mmoles/g of Vi polysaccharide 76.4 % of polysaccharide eluted at 0.25 kda Less than 150 EU/µg of Vipolysaccharide powder The above results met all the requirements of WHO TRS 840 (1994), British pharmacopeia-2009, 2010 and 2011 and Indian

18 111 pharmacopeia-2010 standards. The requirements of WHO TRS 840 were considered as standard specifications in present study. The standard requirements of WHO are proteins 10 mg/g, nucleic acids 20 mg/g, O-acetyl content not less than 2 mmol/g of Vi polysaccharide, molecular size of 50% polysaccharide should elute before 0.25 kda, Identity by immune precipitation method and sterility test passing. According to British pharmacopeia (2009) and European pharmacopeia (2007), the lyophilized Vi polysaccharide specifications are: protein 10 mg/g, nucleic acids 10 mg/g, O-acetyl groups 2 mmol/g, Not less than 50 per cent of the polysaccharide to be found in the pool containing fractions eluted before kda 0.25, identification using a immuoprecipitation method, and bacterial endotoxoin test. These specifications are similar to the WHO TRS 840 (1994), British pharmacopeia (2009 and 2010) and Indian pharmacopeia (2010). Vi polysaccharide isolated from Citrobacter freundii (WR7011) was quantified for optical properties for use to calibrate typhoid vaccine parameters (Stone et al., 1988). The purified polysaccharide was estimated to contain less than 1% of lipopolysaccharides, proteins and nucleic acids less than 2% and molecular weight was estimated by gel filtration through sepharose CL-4B with Dextran T-2000.

19 112 When compared to the above studies the results that were obtained for our purified Vi polysaccharide bulk were 2.2 mmoles/g of O-acetyl groups, 2.4 mg/g of protein, 6 mg/g nucleic acid and less than 150 EU/µg of bacterial endotoxin Table 4.5. The molecular size distribution of Typhoid ViPs bulk is given in the Table 4.6 below. Table 4.6 Molecular size distribution of Typhoid Vi polysaccharide bulk Fractions Time (min) O-acetyl (µmol/ml) Vicontent Vol (ml) Total Viconc. (mg) Void Void Volume: 36 ml Total= 108 ml 4.86 mg Total elution volume: 142mL Distribution Coefficient= V e- V 0 V t - V = Vi content (mg) Before 0.25 kda = 62 ml 3.12 After 0.25 kda = 81 ml 1.74 Total Vi-content eluted= 4.86 = 0.25 kda Percentage of Vi polysaccharide eluted before 0.25kDa 3.12 x =76.4 %

20 113 The molecular size distribution of Vi polysaccharide was determined by using gel permeation column with Sepharose CL-4B as stationary phase. Fractions were collected after void volume (Vo) corresponding to kda 0.25 and pooled together. 76.4% of polysaccharide eluted at kda Results of conjugated Vi polysaccharide-tetanus toxoid bulk: The methods involved in conjugating polysaccharides to proteins were actually started with the invention of Hib conjugate vaccines. In 1931 it was originally shown that the immunogenicity of polysaccharides could be enhanced by conjugating with an immunogenic protein molecule. Vaccination against Haemophilus influenza type b disease reported high success rates (>95%), the same hypothesis can be used for preparation of other immunogenic conjugates against Stretococcus pneumoniae, Nesseria meningitidis and Salmonella typhi (Ada et al., 2003). The similar methodology was employed for preparing Vi polysaccharide protein conjugates. Szu et al. (1987) prepared and characterized Vi polysaccharides protein conjugates and studied their immunogenicity in laboratory animals. In this experiment Vi polysaccharides were conjugated with different proteins like bovine serum albumin, cholera toxin and diphtheria and tetanus toxoid. The

21 114 Vi polysaccharide used in the study was isolated from Citrobacter freundii. In the study Vi is thiolated using cystamine and proteins were derivatized with SPDP (N-Succinimidyl 3-(2-pyridyldithio)- propionate). Modified Vi was coupled directly in the presence of EDC. Similarly in the same experiment pneumococcal 6B was conjugated to tetanus toxoid with adipic acid dihydrazide as a linker. Later, Szu et al. (1994) reported the preliminary clinical characterization of Vi polysaccharide protein conjugate vaccines. In this experiment the polysaccharide of S. typhi Vi was bound to the B subunit of the heat-labile toxin (LT-B) of Escherichia coli or the recombinant exoprotein A (repa) of Pseudomonas aeruginosa. Due to the large molecular size of Vi which resulted in the low yields and poor solubility of Vi-TT, the molecular size of Vi of was reduced by ultrasonic radiation and bounded to several proteins. The subunit of E. coli LT-B, and recombinant exoprotein A (repa) of P.aeruginosa were with (N-Succinimidyl 3-(2-pyridyldithio)-propionate) SPDP (SPDP/LT-B molar ratio of 16:1) and to a final SPDP/rEPA molar ratio of 20:1. Vi was treated with cystamine and conjugated to derivatized SPDP proteins. Jennings et al. (1982) explained the different methods of generating active functional groups of polysaccharides by controlled oxidation. He concluded that a reactive aldehyde end can be generated by oxidation of vicinal hydroxyls and covalently bound to the free

22 115 amino group by reductive amination. In the study, meningococcal group A polysaccharide size was reduced using sodium borohydride which facilitated oxidizable terminal vicinal hydroxyl end. Meningococcal serogroups B and C were oxidized with sodium metaperiodate in the presence of ethylene glycol; the oxidized polysaccharides were directly coupled proteins. Kossaczka et al. (1997) reported the synthesis and immunological properties of Vi and di-o-acetyl pectin protein conjugates with adipic acid dihydrazide as the Linker; adipic acid dihydrazide (ADH) treated proteins of Vi and OAcP in the presence of EDC, the immunogenic protein molecules used for coupling mechanism was bovine serum albumin and recombinant exoprotein A of Pseudomonas aeruginosa (repa). To assess the immunogenicity of the Vi polysaccharide conjugates, native Vi or OAcP and protein conjugated Vi was administered to mice and guniea pigs. It was noticed that Vi conjugates elicited 5 to 25 fold increase in antibodies when compared to native Vi or OAcP alone. These Vi polysaccharideprotein conjugates were further evaluated clinically. By citing several conjugation methodologies a process was optimized in the present study to couple Vi polysaccharide protein conjugates under controlled conditions. Vi polysaccharide is subjected to hydrolysis under alkaline conditions and cyanalated, followed by attachment with a linker. This modified polysaccharide is later

23 116 coupled to the tetanus toxoid in the presence of EDC. The conjugate mixture was loaded and eluted through Sepharose CL-4B gel filtration column by which we have collected 10 frctions (F1-F10) after the void volume. The fractions were pooled and buffer exchanged with 20mM tris buffer ph 7.0. The final conjugate bulk was sterile- filtered using a 0.22 µ filter membrane and labelled as ViPs-TT conjugate bulk. When compared to the present study a similar trial was carried out by Novartis in preparing Vi conjugates with CRM197 and tetanus toxoid as carrier proteins. In their study Vi and EDC were mixed at appropriate molar ratio (EDC/Vi) of , alternatively CRM197 and TT were derivatized with treatment with ADH and EDC. Vi was conjugated to CRM197 and TT separately and the conjugation mixture was purified using Sephacryl S-1000; fractions were analysed by SDS- PAGE and those which did not contain free protein were collected (Micoli et al., 2011). In the present study, the purified ViPs-TT conjugate bulk was tested for the O-acetyl, protein, free ViPs, free protein, molecular size analysis and HPLC. Results are presented in the Table 4.7.

24 117 Table 4.7 Results of the ViPs-TT conjugate bulk Tests Results Molecular size % of polysaccharide eluted at kda 0.3 O-acetyl content Conjugate Vi content 0.85 µmoles/ml 0.25 mg/ml Free Vi Ps 7% Protein content 0.24 mg/ml Vi Ps / protein ratio 1.04 Free protein Sterility Peak was not detectable at th minute in HPLC UV (280nm) chromatogram No growth was observed The molecular size of the Vi-TT conjugate obtained in the present study was 0.3 kda; when compared the results obtained by Szu et al. (1987) in which the molecular size of the conjugate Vi-TT conjugate was <0.1 kda and that of the Vi was 0.36 kda. Szu et al. (1994) described the molecular sizes of Vi antigen and OAcPec with an explanation that there was no change in the molecular size of OAcPec upto 3-8 C for 3 months. After storage of OAcPec at 60 C for 3 months it was observed the molecular size decreased from 400 to 30 kda. However, the Vi was more stable. Depolymerisation was noticed when Vi was incubated at 2 weeks at 60 C and the molecular size shifted from kda to 500 kda after 3 months period.

25 118 Micoli et al. (2011) characterised the physico-chemical characteristics of Vi-CRM197 and Vi-TT conjugates; characterization of Vi revealed it to have 1% nucleic acids, 0.3% of proteins and an O-acetylation of level of 86% by H-NMR. Determination of total and free (unbound) Vi polysaccharide was measured by HPAEC-PAD analysis. Similarly the gel filtration profiles obtained by using Sephacryl S-1000 illustrated peaks containing protein at higher molecular weight than either unconjugated Vi or the unconjugated carrier protein. The polysaccharide protein ratios obtained in the study were for Vi-CRM197 and for Vi-TT 0.41 to 0.67 respectively. Based on these results immunogenicity studies were carried out in laboratory animals by them. The molecular size distribution of our Typhoid Vi polysaccharide conjugate bulk is given in the Table 4.8.

26 119 Table 4.8 Molecular size distribution of Typhoid ViPS-TT conjugate bulk Fractions Time O-acetyl Vicontenconc.(mg) Total Vi- Vol (ml) (min) (µmol/ml) Void Void Volume: 36 ml Total= 108 ml 4.57 mg Total elution volume: 142mL Distribution Coefficient = V e- V V t- V Vi Content (mg) Before 0.3 kda = 62 ml 3.33 After 0.3 kda = 81 ml 1.23 Total conjugate Vi-content = 4.57 = 0.3 kda Percentage of conjugate Vi polysaccharide eluted before 0.3 kda 3.33 x = %

27 120 Szu et al. (1997) reported carbohydrate protein ratios for Vi-rEPA and OAcP conjugates purified through gel filtration column using Sephacryl S-1000 using 0.15 M NaCl as eluent, the obtained ratio for Vi-rEPA was 1.17 and for OAcP-rEPA was Kossacka et al. (1999) prepared Vi-rEPA with two different linkers SPDP and ADH. The polysaccharide protein ratios obtained were 1.05 for Vi-rEPA1 and 1.11 for Vi-rEPA2, respectively. Frasch (2009) recommended the quality control testing and analytical methods used in preparation of bacterial polysaccharideprotein conjugate vaccines. He specified that determination of molecular size and percentage of free polysaccharide or proteins in the bulk conjugate were the two major analyses that should be carried out. Based on the published reports and our own finding, the following specifications for polysaccharide protein in the present study were determined: Molecular size distribution 72.86% of Vi polysaccharide conjugate was eluted at kda 0.30, O-acetyl content 0.85 µmoles/ml, conjugate Vi content 0.25 mg/ml, free Vi Ps 7%, protein content 0.24 mg/ml, Vi Ps/Protein ratio-1.04, free protein peak not detectable and sterility was found to be acceptable (Refer Table 4.7).

28 HPLC Chromatograms of Vi polysaccharide, Tetanus Toxoid and ViPs-TT Conjugate bulk at different stages are shown below: Figure 4.8 HPLC (RI) for Typhoid Vi polysaccharide bulk The above HPLC Profile of the purified Vi polysaccharide was detected by RI using HP-GPC column. The peak at minutes represents native Vi polysaccharide.

29 122 Figure 4.9 HPLC (UV) for Tetanus Toxoid bulk The above HPLC profile of the purified Tetanus toxoid by UV detector using HP-GPC column. The peak at minutes represents tetanus toxoid.

30 123 Figure 4.10 HPLC (RI) for ViPs-TT conjugate bulk The above HPLC Profile of the Vi polysaccharide-tetanus toxoid conjugate was detected by RI detector using HP-GPC column. The peak at minutes represents conjugate ViPs-TT.

31 124 Figure 4.11 HPLC (UV) for ViPs-TT conjugate bulk The above HPLC Profile of the Vi polysaccharide-tetanus toxoid conjugate was detected by UV detector using HP-GPC column. The peak at minutes represents conjugate ViPs-TT. 4.2 Results of Formulation and Filled lots: Experimental Vi conjugates were formulated with suitable excipients as test vaccine for animal toxicity, animal immunogenicity and human administration by several workers. Canh et al. (2004) prepared Vi-rEPA conjugate to study the immunogenicity when injected twice in 2-5 years old Vietnamese children. In the study the Vi conjugate was formulated using saline, sodium phosphate buffer

32 125 and thiomersal as preservative. Cui et al. (2010) prepared Vi-DT lots in saline formulated with thiomersal and injected in mice to study immunological properties of Vi-DT conjugates. Similarly Micoli et al., 2011, prepared Vi-CRM197 and tested for its immunogenicity in mice. In this study Vi conjugates were formulated with Aluminium hydroxide gel, Complete Freund s Adjuvant (CFA) and Incomplete Freund s Adjuvant (IFA). In the present study the ViPs TT conjugate bulk were formulated in physiological saline into two batches as Vi-TT/BB/01 and Vi-TT/BB/02. The formulated vaccine was filled as 0.5 ml in 2 ml vials as ViPs-TT conjugate vaccine and analysed for ph content, O-acetyl content, free ViPs content, protein content and sterility HPLC profiles of ViPs-TT conjugate test lots: The HPLC profiles of ViPS-TT conjugate vaccine batches (Vi-TT/BB/01 and Vi-TT/BB/02) are shown in the figures 4.12, 4.13, 4.14 & 4.15.

33 126 Figure 4.12 HPLC (RI) for ViPs-TT conjugate vaccine lot (Vi-TT/BB /01) The above HPLC profile of the Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT/BB/01) was detected by RI detector using HP-GPC column. The peak at minutes represents conjugate ViPs-TT conjugate.

34 127 Figure 4.13 HPLC (UV) for ViPs-TT conjugate vaccine lot (Vi-TT /BB/01) The HPLC profile of the Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT/BB/01) was detected by UV detector using HP-GPC column. The peak at minutes represents conjugate ViPs-TT conjugate.

35 128 Figure 4.14 HPLC (RI) for ViPs-TT conjugate vaccine lot (Vi-TT/ BB/02) The above HPLC profile of the Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT/BB/02) was detected by RI detector using HP-GPC column. The peak at minutes represents conjugate ViPs-TT conjugate.

36 129 Figure 4.15 HPLC (UV) for ViPs-TT conjugate vaccine lot (Vi-TT/ BB/02) The HPLC profile of the Vi polysaccharide-tetanus toxoid conjugate vaccine (Vi-TT/BB/02) was detected by UV detector using HP-GPC column The peak at minutes represents conjugate ViPs-TT conjugate. All the above HPLC profiles clearly demonstrate the conjugation efficiency of the method used to couple ViPs-TT molecules. In the first step the native Vi polysaccharide was found to have molecular weight of kda and eluted at 14.9 th minutes when injected into HP- GPC column. Size exclusion of native polysaccharide coupled to tetanus toxoid was carried out which showed a molecular weight of kda and eluted at minutes.

37 Pharmacopeal tests on the test lots: The two test batches (Vi-TT/BB/01 & Vi-TT/BB/02) were tested fully in the lab as per the test methods described in Materials and Methods. The tabulated results for the two batches are shown in the table 4.9. Table 4.9 Results of the ViPs-TT conjugate final test vaccine lots Tests Vi-TT/BB/O1 Vi-TT/BB/O2 ph O-acetyl content 0.85 µmoles/ml 0.92 µmoles/ml Vi content 30 µg /0.5mL 32 µg /0.5mL Free Vi Ps 7% 6% Sterility No growth observed No growth observed The results were found satisfactory. The batches were taken up for animal testing like immunogenicity and preclinical studies Results of mice immunogenicity test: Based on the above satisfactory results obtained the two test batches were carried out for immunogenicity study.

38 131 Immunogenicity of the Vi conjugate was tested in mice and sera estimated for anti Vi-antibodies using ELISA. Kossaczka et al. (1997) tested Vi and O-acetyl pectin conjugates in mice and guniea pigs for Vi antibody titres. 100 µl containing 2.5 µg (Vi or OAcP conjugates) of conjugate was injected subcutaneously by giving two or three doses at two weeks of intervals. Alternately, 5 µg of conjugate or polysaccharide alone on 0, 21, and 42 nd day was injected into 6 weeks old Duncan- Hartley guinea pigs. ELISA test on sera collected from immunized animals was carried out by parallel line analysis and the total Vi antibody/millilitre was elucidated. Szu et al. (1993) prepared Vi conjugates using different carrier proteins such as diphtheria, tetanus toxoids, cholera toxin (CT) and Haemophilus influenzae type b protein. Native polysaccharide and polysaccharide Vi conjugates were tested in mice as three different grades doses at two weeks of interval. 5 µg of Vi-CT conjugates adsorbed with alhydrogel as an adjuvant was used for immunization. Alternately, juvenile rhesus monkeys were immunized with 25µg of Vi-CT (lot XII) conjugate in two doses. The concentration of Vi antibodies were measured by radio immunoassay and expressed in micrograms Ab/ml. Again Szu et al., 1994 injected 2.5 µg O-acetyl pectin [Poly (1->4)-α-D-GalpA]-protein conjugate or polysaccharide alone in NIH mice at three weekly doses during three weeks period. The antibody concentrations were determined by ELISA and the geometric mean calculated.

39 132 The present study was conducted to compare the immunogenicity of ViPs-TT vaccine with native polysaccharide vaccine based on the observations of other workers. The mice study was conducted as per the details given in by ELISA method Enzyme linked immunosorbent assay (ELISA): In the present study, immunogenicity in 32 groups (each group contained 3 mice) of Vi polysaccharide and the two conjugates of ViPs-TT (Vi-TT/BB/01 and Vi-TT/BB/02) was evaluated. It was noticed that mice injected with the two formulations test vaccine lots, Vi-TT/BB/01 & Vi-TT/BB/02 showed maximum percentage of seroconversion with reference to a known positive control serum. In the formulation Vi-TT/BB/01 the average ELISA absorbance was observed as 1.31, and in the formulation Vi-TT/BB/02 the average absorbance was Native polysaccharide vaccine (TYPBAR TM ) showed a value of 0.81 and the control saline group showed a value of 0.23, which are shown in table 4.10.

40 133 Table 4.10 ELISA absorbance values in different groups of mice in response to reference and test vaccines * Absorbance values (mean followed by ± SD) given were after analysis of three replicates by one-way ANOVA. * Different superscripts in the same column were significantly different at p<0.05 level (Least Significant Difference). As observed above the immunogenicity results of the two conjugate vaccine batches were greater as compared to Vi polysaccharide typhoid vaccine (TYPBAR TM ). The results of seroconversion rates were interpreted by calculating the seroconversion cut-off value. The percent seroconversion rate of Vi polysaccharide-typhoid vaccine (TYPBAR TM ) was 62.5% and the percentage of seroconversion for Vi TT/BB/01 and Vi TT/BB/02 formulation of Vi polysaccharide Tetanus toxoid conjugate vaccine was 100%. Venkatesan et al.,

41 134 (2010). Both the test vaccine lots showed more than 4 times (sero conversion) the value in control group. This is shown as bar diagram in figure Figure 4.16 Percentage of seroconversion in mice model % % % Seroconversion 80.00% 60.00% 40.00% 20.00% 0.00% TYPBAR Vi-TT/BB/01 Vi-TT/BB/02 Vaccine Groups Seroconversion cut-off value = 4 x absorbance in control group, i.e. 4 x 0.23 = 0.92 This result showed that the Vi polysaccharide-tetanus toxoid conjugate vaccine prepared in the present study was more immunogenic than the Vi polysaccharide typhoid vaccine (TYPBAR TM ). Statistical analysis by one way ANOVA showed that between the different samples there was a significant difference at p<0.05 level. Both Vi-TT/BB/01 and Vi TT/BB/02 elicited higher levels of antibodies in mice than TYPBAR TM. Results were in accordance with

42 135 Robbins and Schneerson (1990). They reported that the significant effect of polysaccharides was to induce protective levels of serum antibodies. The conjugate vaccine can be expected to be more effective than native Vi polysaccharide alone for prevention of typhoid fever, similar observation has been made by Acharya et al.1987; Keital et al., 1994 and Klugman et al., As both the test vaccine lots (Vi-TT/BB/01 and Vi-TT/BB/02) gave similar results upto immunological level, to restrict the usage of lab animals further animal tests were carried out on one test vaccine (Vi-TT/BB/01) only. Except abnormal toxicity test which was carried out on both the test vaccines lots. However in all the tests the test vaccine was compared with control group of physiological saline used Results of challenge test in mice models: Challenge test on typhoid vaccine was carried out to prove the protective efficacy of the typhoid vaccines in field trials. Typhoid fever vaccines are assessed in field trials to determine the ability of the vaccine components to induce protection when challenged with virulent S. typhi strains by several workers in the past. Many experiments were carried out with S. typhosa strain. Approximately 1000 LD50 dose in the presence of gastric mucin was challenged and relative potency of alcohol-killed, alcohol-preserved, heat-killed, phenol-preserved vaccines was studied (Geoffrey et al., 1959).

43 136 Several potency tests on acetone killed vaccines of S. typhi Ty2 were assessed using the intra-peritoneal route of challenge. With reference to method, a laboratory test was performed by University of Maryland to assess the protection of S. typhi components when vaccinated in mice. Acetone-killed S. typhi Ty2 vaccines (2x10 8 ) with the Vi antigen-free variant O-901, or with Yersinia enterocolitica and Serratia marcescens, S. typhi and S. marcescens endotoxin and corresponding lipid A components were investigated. The present study was carried out to determine the protective efficacy of a newly designed Vi polysaccharide-tetanus toxoid conjugate vaccine, including the lowest concentrations of antigen, when challenged with live S. typhi Ty2 culture in mice models. The LD50 for S. typhi cultures were determined by Reed and Muench method and the mice immunized with experimental test vaccine were challenged with live cultures at 20 LD50 concentrations (Venkatesan et al., 2011).

44 137 Table 4.11 LD 50 determination in mice models Dilutions of S. typhi culture No. of mice per group Injected volume (Intraperitoneal) Number of live and dead animals during 15 days period of observation Cumulative live mice Cumulative death in mice Mortality ratio % of mortality Live Dead ml / ml / ml / ml / Control Group (Normal Saline) ml Calculation of LD 50 by Reed and muench Method: PD - Proportionate Distance = % of Mortality above 50% 50% x log Dilution factor % of Mortality above 50% % of Mortality below 50% Using the mentioned formula the Proportionate Distance was calculated as below: PD = ( / ) x 1 = (12.5 / 37.5) = 0.33 Accordingly 1 LD50 for S. typhi Ty2 was calculated as /0.25 ml.

45 138 Table 4.12 Determination of active mice protection on immunization with test ViPs-TT conjugate vaccine and reference ViPs vaccine after challenging with a determined dose of S. typhi culture Vaccines Dilutions/ Conc. of Ag. (µg) Survivals Deaths Cumulative survivals Cumulative deaths Mortality ratio % mortality *PD ED50 in µg Test vaccine Vi T/BB/01 Reference vaccine TYPBAR TM 1/4 (6.250) / /8 (3.125) / /16 (1.562) / /4 (6.250) / /8 (3.125) / /16 (1.562) / *PD - Proportionate Distance Proportionate Distance = (% of Mortality above 50% - 50%) (% of Mortality above 50% - % of Mortality below 50%) x log Dilution factor Test vaccine ED50: Log reciprocal of higher dilution of vaccine-proportionate distance = =0.904; Antilog of =8.016 i.e test vaccine dilution protecting 50% mice = 1/8.016 The antigen concentration at 1/8 th dilution was µg and that for 1/8.016 is 3.13 µg and hence 50% of mice were protected at the concentration of 3.13 µg of antigen when challenged with 20LD50.

46 139 Reference vaccine ED50: Log reciprocal of higher dilution of vaccineproportionate distance. = =0.94; Antilog of 0.94 = The antigen concentration at 1/8th dilution was µg and that for 1/8.99 is 3.51 µg. Hence 50% of mice were protected at the concentration 3.51 µg of antigen when challenged with 20LD Results of pre-clinical toxicity study: To assess the safety of the test vaccine components a pre-clincal toxicity study was conducted to determine any toxic substance present in the vaccine components and excipients. An acute and systemic study was carried out in mice and rabbits and sera analysed for serum chemistry, haematological values and histopathological findings. Abnormal toxicity studies were conducted in mice and guinea pigs which were observed for a week period. Preclinical development studies are carried out on bacterial vaccines to assess the quality of product which includes starting materials, manufacturing process, bacterial seed characterization, potency testing, general safety, purity and identity. With regard to the choice of dose, route of administration and formulations, they are determined by preclinical toxicology studies (Falk et al., 1998). Paoletti et al. (2008) described the manufacturing process for GBS type III Capsular polysaccharide CPS-Tetanus toxoid vaccine under cgmp conditions and evaluation of type III CPS-specific

47 140 antibody on conception of early and late-stage fetal development in rabbits. Toxicity evaluation was carried out in rabbits with an equivalent human dose and found no significant changes in body weights and in feed consumption throughout the study period. Mitruka and Rawnsley (1981) elucidated enormous data on clinical biochemical and haematological values in laboratory animals. References values for laboratory animals are important in pre-clinical investigations in understanding the mechanism of test components under in vivo conditions Abnormal toxicity test: In our study, five mice and two guinea pigs each were given the two test vaccines (Vi-TT/BB01 & Vi-TT/BB/02) as per Indian Pharmacopea-2007 and British Pharmacopea These were observed for their health and activity for 7 days after vaccine administration. No deaths or abnormal animal behaviour were observed and the animals remained healthy and active during the test period Acute toxicity test: The 0.5 µg/0.1ml dose of test vaccine (Vi-TT/BB/01) was injected to mice and 2.5 µg/0.5 ml dose injected in rabbits. They were observed for 14 days period. The results are shown in tables 4.13 and 4.14 These represent body weight observations in mice and rabbits

48 141 respectively. Food intake consumption data during 14 days of the study period in mice and rabbits are presented in tables 4.15 and 4.16 respectively. During the study period animals were observed for general appearance, behaviour, mortality and food intake. Table 4.13 Body weight (grams) observation in mice for control and test vaccine Group (I-Control II-Test) Sex Mice No. Study days I II Male Male Male Male Male Male Female Female Female Female Female Female Mean SD Male Male Male Male Male Male Female Female Female Female Female Female Mean SD

49 142 Table 4.14 Body weight (grams) observation in rabbits for control and test vaccine Group (I:Control II:Test) II I Sex Rabbit No. Study days Male Male Male Male Male Male Female Female Female Female Female Female Mean SD Male Male Male Male Male Male Female Female Female Female Female Female Mean SD

50 Daily observations: Mice and Rabbits were observed for 14 days for any signs of illness and reaction to vaccination. General appearance, behaviour mortality, food intake and bleeding reaction were observed. No abnormality was observed and all the animals were healthy at the end of 14 days period Body weight observations: The data presented in the tables show no significant effect on body weights of animals due to test vaccination. Statistical analysis of body weight gain revealed that there is no significant variation in the body weights in vaccinated group when compared with control group. The observation of animals was carried out on days 0, 3, 7, 8, 12 and 14 in both mice and rabbits. There were no premature deaths and no clinical observation of acute toxicity during the study. Body weight gain was normal and satisfactory. No abnormalities were detected. The food consumption data during the study period in mice is given in the table 4.15 and 4.16 below.

51 144 Table 4.15 Food consumption (grams) data of mice for control and test vaccine Group (I:Control II:Test) I II Day 0 Day 01 Day 02 Day 03 Day 07 Day 08 Day 13 Day 14 Mice Feed Feed Feed Feed No. Feed Feed Feed Feed left left left left added added added added over over over over

52 145 Table 4.16 Food consumption (grams) data of rabbits for control and test vaccine Group Rabbit Day 0 Day 01 Day 02 Day 03 Day 07 Day 08 Day 13 Day 14 (I:Control No. Feed Feed Feed Feed Feed Feed Feed Feed II:Test) added left added left added left added left over over over over I II

53 Food consumption details: The food consumption and left over food quantities in mice and rabbit groups were noted. It was observed that mice consumed approximately 5 g of feed per day whereas rabbits 125 g per day. There were no adverse effects of vaccination observed on feed consumption. There was no variation between test group when compared with control group Systemic toxicity test: In this study repeated dose of vaccine are given in a period of time and hence called as a repeated dose toxicity study. In this present study 0.05 µg/0.1ml of test vaccine was injected in mice and similarly 2.5 µg/0.5 ml was injected to rabbits on 0 th day. The same dose was repeatedly given on 7 th, 14th and 21 st day to animals and were observed for 28 days. Body weight observation data during the study period in mice and rabbits given the tables 4.17,4.18 and 4.19 below.

54 Body weight results in mice and rabbits: Table 4.17 Systemic toxicity study of body weight observation in control group of mice Body weights in grams Group (I:Control) Sex Mice No. Study days Male Male Male Male Male Male Male Male Male Male Mean I SD Female Female Female Female Female Female Female Female Female Female Mean SD

55 148 Table 4.18 Systemic toxicity study of body weight observation in test vaccine of mice Group (II:Test) II Body weights in grams Study days Sex Mice No Male Male Male Male Male Male Male Male Male Male Mean SD Female Female Female Female Female Female Female Female Female Female Mean SD

56 149 Table 4.19 Systemic toxicity study of body weight observation in control and test vaccine of rabbits Group (I:Control I:-Test) I II Body weights in kilograms Study days Sex Rabbit No Male Male Male Male Male Male Female Female Female Female Female Female Mean SD Male Male Male Male Male Male Female Female Female Female Female Female Mean SD

57 Daily observations: Mice and Rabbits were observed for 28 days and checked for their health status and signs of reaction to vaccination. General appearance, behaviour mortality, food intake and bleeding reaction were observed Body weight observations: The data presented in the tables 4.17 to 4.19 show no significant effect on body weights due to test vaccination. Statistical analysis of body weight gain revealed that there is no significant variation in the body weights in vaccinated group when compared with control group. The observation of animals was carried out on days 0, 7, 14, 21 and 28 in both mice and rabbits. There were no premature deaths and no clinical observation of acute toxicity during the study. Body weight gain was normal and satisfactory. No abnormalities were detected. When compared to mice, rabbits showed a considerable gain in weight in group II than in group I. However, significant variation was not observed in body weight in group I and group II. This showed that vaccine did not interfere with the normal metabolic pathway and showed no toxicity due to vaccine administration.

58 Clinical biochemistry results in mice and rabbits: Various clinical biochemistry parameters of the test vaccine were studied in mice and rabbits. They are shown in the tables: 4.20 to Table 4.20 Systemic toxicity study in mice control group serum chemistry - 0 th day Study on 0 th day serum chemistry (Group- I: Control ) Group Mice No. AST (U/L) ALT (U/L) ALP (U/L) T.P (g/dl) Blbn (mg/dl) Clstrl (mg/dl) Glucose (g/dl) Crtnn (mg/dl) ALB (g/dl) Urea (mg/dl) I Mean SD Mean SD

59 152 Table 4.21 Systemic toxicity study in mice control group serum chemistry - 0 th day Group II Continued from previous table... Study on 0 th day serum chemistry (I.U/L) ( Group- II: Test) AST ALT ALP T.P Blbn MiceNo. (U/L) (U/L) (U/L) (g/dl) (mg/dl) Clstrl Glucose Crtnn ALB (mg/dl) (g/dl) (mg/dl) (g/dl) Urea (mg/dl) Mean SD Mean SD

60 153 Table 4.22 Systemic toxicity study in mice test group serum chemistry- 28 th day Study on 28 th day serum chemistry (I.U/L) (Group-I: Control ) Group I Mice AST ALT ALP T.P Blbn Clstrl Glucose No. (U/L) (U/L) (U/L) (g/dl) (mg/dl) (mg/dl) (g/dl) Crtnn ALB Urea (mg/dl) (g/dl) (mg/dl) Mean SD Mean SD

61 154 Table 4.23 Systemic toxicity study in mice test group serum chemistry - 28 th day Continued from previous table Study on 28th day serum chemistry( Group- II: Test) Group Mice No. AST ALT ALP Blbn Clstrl Glucose Crtnn ALB Urea T.P (g/dl) (U/L) (U/L) (U/L) (mg/dl) (mg/dl) (g/dl) (mg/dl) (g/dl) (mg/dl) Mean SD II Mean SD

62 155 Table 4.24 Systemic toxicity study in rabbit control and test groups- serum chemistry - 0 th day Study on 0 th day serum chemistry (Group- I: Control and Group- II: Test) Group II I Rabbit AST Blbn Clstrl Glucose Crtnn ALT (U/L) ALP (U/L) T.P (g/dl) No. (U/L) (mg/dl) (mg/dl) (g/dl) (mg/dl) ALB Urea (g/dl) (mg/dl) Mean SD Mean SD

63 156 Table 4.25 Systemic toxicity study in rabbit control and test groups- serum chemistry - 28 th day Study on 28 th day serum chemistry (Group- I: Control and Group- II: Test) Group Rabbit No. AST (U/L) ALT (U/L) ALP (U/L) T.P (g/dl) Blbn (mg/dl) Clstrl (mg/dl) Glucose (g/dl) Crtnn (mg/dl) ALB (g/dl) Urea (mg/dl) II I Mean SD Mean SD Clinical chemistry data of mice and rabbits are presented in the above tables 4.20 to There is no significant variation in serum chemistry values in the 28-day study period. When compared with control group the values of aspartate transaminase (AST) were slightly higher in test vaccinated animals. The variation is not considered as test related due to the fact that all the values are within the biological limits.

64 : Haematological studies in mice and rabbits after 0 th day and 28 days after immunization with the test vaccine: Haematological values during the study period in mice and rabbits given the tables 4.26 to Table 4.26 Systemic toxicity study in mice control and test groups- Haematological values - 0 th day Study on 0 th day Hematological values (Group- I: Control and Group- II: Test) Group Mice No. Differential leukocyte count (10 3 /mm 3 ) WBC Ntrpl Bspl Espl Mncyt Lmpcyt Hb (g/dl) RBC Hcrit (x10 6 mm 3 ) (PCV)% MCV (µ 3 ) MCH (pg) MCHC (%) PLT (x1000) I Mean SD II Mean SD

65 158 Table 4.27 Systemic toxicity study in mice control and test groups-haematological values - 0 th day Continued from previous table Study on 0 th day Hematological values(group- I: Control and Group- II: Test) Group Mice No. Differential leukocyte count (10 3 /mm 3 ) Hb WBC Ntrpl Bspl Espl Mncyt Lmpcyt (g/dl) RBC (x10 6 mm 3 ) Hcrit (PCV) % MCV (µ 3 ) MCH (pg) MCHC PLT (%) (x1000) I Mean SD II Mean SD

66 159 Table 4.28 Systemic toxicity study in mice control and test groups- Haematological values - 28 th day Study on 28 th day Hematological values (Group- I: Control and Group- II: Test) Group Mice No. Differential Leukocyte Count (10 3 /mm 3 ) WBC Ntrpl Bspl Espl Mncyt Lmpcyt Hb (g/dl) RBC (x10 6 mm 3 ) Hcrit (PCV)% MCV (µ 3 ) MCH (pg) MCHC (%) PLT (x1000) I Mean SD II Mean SD

67 160 Table 4.29 Systemic toxicity study in mice control and test groups- Haematological values - 28 th day Continued from previous table Study on 28 th day Hematological values (Group- I: Control and Group- II: Test) Group Mice No. Differential Leukocyte Count (10 3 /mm 3 ) WBC Ntrpl Bspl Espl Mncyt Lmpcyt Hb RBC (g/dl) (x10 6 mm 3 ) Hcrit (PCV) % MCV (µ 3 ) MCH (pg) MCHC (%) PLT (x1000) I Mean SD II Mean SD

68 161 Table 4.30 Systemic toxicity study in rabbit control an test grops - Haematological values - 0 th day Study on 0 th day Hematological values (Group- I: control and Group- II: Test) Group Rabbit No. Differential Leukocyte Count (10 3 /mm 3 ) WBC Ntrpl Bspl Espl Mncyt Lmpcyt Hb (g/dl) RBC (x10 6 mm 3 ) Hcrit (PCV) % MCV (µ 3 ) MCH (pg) MCHC (%) PLT (x1000) I Mean SD II ` Mean SD

69 162 Table 4.31 Systemic toxicity study in rabbit control and test groups- Haematological values - 28 th day Study on 28 th day Hematological values (Group- I: Control and Group- II: Test) Group Rabbit No. Differential Leukocyte Count (10 3 /mm 3 ) WBC Ntrpl Bspl Espl Mncyt Lmpcyt Hb (g/dl) RBC (x10 6 mm 3 ) Hcrit (PCV) % MCV (µ 3 ) MCH (pg) MCHC (%) PLT (x1000) I Mean SD II Mean SD

70 163 Haematological values of mice and rabbits are presented in the tables 4.26 to There was no significant variation in haematological values in the study except that a slight increase of monocytes in test vaccinated mice group when compared to the control was observed. The variation was not considered as test related due to the fact that all the values are well within the biological limits. No significant variation observed in mice and rabbits Histopathological studies of mice and rabbits vaccinated with the test vaccine and compared with normal and control group: Figure 4.17 histopathology observations of mice organs (photographs) Mice lung (control) Mice lung 28 th day (test) Mice spleen (control) Mice spleen 28 th day (test)

71 164 Mice kidney(control) Mice kidney 28 th day (test) Mice brain (control) Mice brain 28 th day (test) Mice heart (control) Mice heart 28 th day (test) Figure 4.18 Histopathology observations of rabbit organs (photographs) Rabbit liver (control) Rabbit liver 28 th day (test)

72 165 Rabbit lung (control) Rabbit lung 28 th day (test) Rabbit spleen (control) Rabbit spleen 28 th day (test) Rabbit kidney (control) Rabbit kidney 28 th day (test) Rabbit brain (control) Rabbit brain 28 th day (test) Rabbit heart (control) Rabbit heart 28 th day (test)

73 Findings: Resuts revealed that no histopathological abnormalities were observed in lungs, liver, kidney, spleen, brain and heart. Results also indicated that there were no abnormalities, clinical changes, toxicity signs, no significant differences in body weights, food intake, normal ranges in haematological and clinical biochemistry values and no gross pathological changes were found when tested in control and test animals. Based on the absence of toxic effects in the systemic toxicity study of the test vaccine in mice and rabbits, it was concluded that the test vaccine was safe and capable of eliciting an immune response without any adverse effect. This satisfactory result allowed us to proceed further in carrying out the Human clinical trial with the test vaccine. 4.4 Results of human clinical trials: As the conjugate vaccine showed better immunological response as compared to native Vi polysaccharide vaccine, the test vaccine was considered to be promising and worth taking it to a logical end. Based on the clinical history and epidemiological studies carried out, it was debated how we should go ahead with the selection of age groups of the children and teenagers to be included in this study.

74 167 A final decision was taken to conduct an open label, active controlled study to evaluate the safety and immunogenicity of ViPs-TT conjugate vaccine Vs. native Vi polysaccharide vaccine in healthy teenagers (13-17 years) and children (6-12 years and 2-5 years). The study was approved by DCGI (Drugs Controller General of India) (Protocol code: BBIL/CTP/02/2008). The clinical safety evaluation based on general examination, local examination, laboratory investigation and subject evaluation of adverse events.these were monitored througout the study period. The vital sign and condition of injection site was inspected at 30 minutes, 24 hours and 48 hours. There was no serious adverse reaction noted in all the age groups. None of the enrolled subjects were withdrawn from study for vaccine related adverse reaction. Regarding studies by other workers, Vietnam carried out a safety and immunogenicity study on Vi conjugate vaccines, initially in adults and then in teenegers and 2-4 years old children. In the study the Vi polysaccharides that were conjugated to P.aeruginosa recombinant exoprotein A (repa) using two different linkers, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi-rEPA1) and adipic acid dihydrazide (ADH; Vi-rEPA2) were used. Vietnemese study was approved by NICHD and FDA and carried-out in Vietnam. In the age group years, 22 individuals were injected with one dose of

75 168 Vi-rEPA and serum samples were collected at 6 th and 26 weeks post injection year old children were injected with one dose of Vi, VirEPA1 or Vi-rEPA2 and serum samples were collected at 6 th and 26 weeks later. Similarly in the age group 2-4 years old, children were injected with one or two doses of Vi-rEPA1 or Vi-rEPA2 and sera collected after 6 weeks and followed upto 20 weeks after second injection. The anti-vi IgG antibody titers were determined by ELISA using precoated microtiter wells with C.freundii. Vaccinated sera were assayed for Vi-IgG and IgM immunoglobulins by using goat anti human IgG/IgM conjugated to alkaline phosphatase. The Vi antibody titers were expressed as ELISA units/ml (EU). Results summarized show that one single dose of Vi-rEPA2 conjugate elicited higher Vi-IgG levels in children when compared Vi-rEPA1. When the antibody titers observed in 2-4 years children, two injections of Vi-rEPA2 elicited significant level of antibodies compared with what one injection elicited in 5-14 years age group. The safety and immunogenicity of Vi-rEPA2 was further studied for its efficacy and effect of dosage in Vietnamese children (Kossaczka et al., 1999). In continuation of the above study an efficacy trial was carried out with Vi-rEPA vaccine in two to five year old children in Vietnam. The efficacy was estimated by comparing the attack rate of typhoid cases in vaccine group versus placebo group. It was reported that efficacy of Vi-rEPA conjugate vaccine was the highest at 91.5 % when compared with other typhoid vaccines. The vaccine was found to be

76 169 safe and immunogenic in two to five years age group (Lin et al., 2001). In a later study (Canh et al., 2004), the effect of dosage on immunogenicity in Vietnamese children was compared. In this study three different doses (25, 12.5 and 5 µg of Vi-rEPA) were given twice within 6 weeks interval. The results of the study recommended that 25 µg dose of Vi-rEPA conjugate would be efficient in producing strongest antibody response and secondary immune responses for at least four years. The Vietnamese Vi conjugate human clinical study proved its safety, immunogenicity and efficacy in adults, teenagers and children. In consonance with the Vietnemese findings, a human clinical trial was planned with age groups 13-17, 6-12 and 2-5 years and was conducted on typhoid conjugate vaccine to compare immunogenicities between our test (Typhoid ViPs-TT conjugate vaccine) and reference vaccine (TYPBAR TM ). The results from the clinical trials with our vaccines indicated that in the subjects between years, there was a four-fold rise in the levels of anti-vi-igg when compared to the pre-immuno titers. The dose administered to this age group was 25 µg/0.5 ml of test vaccine (ViPs-TT conjugate vaccine) as single dose. The percentage of seroconversion was 100%. The age group of year were immunized with reference vaccine (TYPBAR TM ) reported anti-vi-igg with four-fold increase and the percentage of sero-conversion was 80%. The age

77 170 group 6-12 years reported high levels of anti-vi-igg with four-fold increase when compared to the pre-immune titers. The percentage of sero-conversion was 100% with ViPs-TT conjugate vaccine. Age group 6-12 years reported anti-vi-igg with four-fold increase and the percentage of sero-conversion was 100% with reference vaccine (TYPBAR TM ). The results of ViPs-TT conjugate vaccine in the age group 2-5 years reported high levels of anti-vi-igg with four-fold increase when compared to the pre-immune titers when immunized at two doses of 25 µg and 15 µg/0.5 ml. The percentage of sero-conversion was 100% on the other hand. The results of TYPBAR TM in the age group 2-5 years reported anti-vi-igg with four-fold increase but the percentage of sero-conversion was 70% when compared to the pre-immuno titers. The two-dose administered to this age group was 15 µg/0.5 ml of the test vaccine ViPs-TT conjugate vaccine and the percentage of seroconversion was 100%. There was hardly any significant difference statistically in immune response with respect to GMT titers between single dose of ViPs-TT conjugate vaccine 25 µg/0.5 ml dosage (71.76) and two doses of 15 µg/0.5 ml dosage (88.75). The percentages of seroconversion (4-fold titer rise) in all the age groups of years, 6-12 years with (single dose) and in 2-5 years (two doses) of typhoid Vi polysaccharide tetanus toxoid conjugate test vaccine at strength of 25 µg were 100%.

78 171 The percentage of seroconversion (4-fold titer rise) in the age group of 2-5 years with two doses of typhoid Vi polysaccharide tetanus toxoid conjugate test vaccine at strength of 15 µg was 100% Immunogenicity results: Table 4.32 Age wise immunogenicity and seroconversion results Vaccine group Test vaccine (25µg) Test vaccine (15µg) Reference vaccine Age group in years (subjects) (15subjects) (1 dose) 6-12 (20 subjects) (1 dose) 2-5 (15 subjects) (2 dose) 2-5 (15 subjects) (2 dose) (10 subjects) (1 dose) 6-12 (10 subjects) (1 dose) 2-5 (10 subjects) (1 dose) GMT titers in ELISA units/ml Prevaccination Postvaccination % of Sero conversion 4 fold rise % % % % % % % The percentages of seroconversion (4-titer rise) in age groups of years, 6-12 yrs. and 2-5 yrs with a single dose of typhoid

79 172 Vi polysaccharide vaccine (TYPBAR TM ) at strength of 25 µg were 80%, 100% and 70% respectively. It is evident from the above data that GMT and percentage of seroconversion of test vaccine was higher than reference vaccine. There was a significant difference between the Test and Reference in 4-fold seroconversion rate (p-value: 0.002, Fisher s exact test). Figure 4.19 Clinical trial report on the immunogenicity of test Typhoid ViPs-TT conjugate vaccine Vs Reference Typhoid Vi Ps vaccine (25µg/0.5 ml) There is statistically slight significant difference in immune response with respect to GMT titers between single dose of 25 µg/0.5 ml

80 173 dosage (71.89) and two doses of 15 µg/0.5 ml (88.75) of the ViPs-TT conjugate vaccine. Figure 4.20 Comparison of percentate of seroconversion between the study groups The percentages of seroconversion in age groups yrs, 6-12 yrs (single dose) & in 2-5 yrs (two doses) with test vaccine at strength of 25 µg were 100%. The percentage of seroconversion rise in the age group of 2-5 years with two doses of test vaccine at strength of 15 µg was 100%.

81 174 Table 4.33 Overall immunogenicity results of test ViPs-TT conjugate vaccome amd reference vaccine groups Vaccine Group n* GMT titers in ELISA units/ml Pre- Vaccination Post- Vaccination Sero conversion 4 Fold Rise Test Vaccine (25µg) Test Vaccine (15µg) Reference Vaccine (TYPBAR) (25µg) % % % *n = No. of subjects completed the study. Figure 4.21 Comparison of seroconversion rate (4 fold) in test Vs. reference vaccine groups Geometric Mean Titers (GMT) were calculated using log transformation data and compared by both z-test and Fisher s exact