Indian Journal of Medical Sciences Medknow Publications on behalf of Indian Journal of Medical Sciences Trust ISSN: 0019-5359 EISSN: 1998-3654 Vol. 58, Num. 4, 2004, pp. 141-149

Indian Journal of Medical Science Vol. 58 No. 4, April 2004 , pp. 141-149

Comparison of the Immunogenicity and Safety of Two Different Brands of Salmonella typhi Vi Capsular Polysaccharide Vaccine

P Sabitha, M R Prabha Adhikari,* Abhijit Chowdary,** Malathi Prabhu,* Mohammad Soofi,* Meenakshi Shetty,* Asha Kamath,*** S S Lokaranjan.**** S S Bangera*****

Departments of Pharmacology, *Medicine, ***Preventive and Social Medicine Kasturba Medical College, Mangalore, India; ** Department of Gastroenterology, Institute of Post Graduate Medical Education and Research Kolkotta, India; ****Bharath Biotech International Ltd, Hyderabad, India; and *****Asian Clinical Trials Pvt Ltd, Hyderabad, India.
Correspondence:Dr. P. Sabitha, Department of Pharmacology, Kasturba Medical College, P. B. No. 53, Mangalore - 575001, India. E-mail: sabita_rao1@rediffmail.com

Accepted Date: 21-04-2004

Code Number: ms04024

ABSTRACT

Background: The recent emergence of multi-drug-resistant Salmonella strains highlights the need for better preventive measures, including vaccination. Safeand immunologic vaccines have been developed based on purified Vi polysaccharide. Objective: To compare the immune response elicited by two different brands of Salmonella Vi capsular polysaccharide vaccine (ViCPS). Setting and Design: Double blind, randomized (3:1), controlled, parallel, phase III study was conducted at two centres in India to compare the safety and immunogenicity of Typbar^TM, the investigational vaccine with an already marketed vaccine "X", in healthy subjects aged between 12 -25 years. Material and Methods: A sample size of 184 subjects was calculated. Subjects were randomly distributed in two groups, immunized with single dose of Typbar^TM or Vaccine "X". Serum samples were taken before 7 days and 4 weeks after immunization for the determination of antibodies to Vi polysaccharide, by ELISA method. Safety was assessed by physical examination, laboratory parameters before and after vaccination and by monitoring adverse events. Statistics: The geometric mean antibody titre (GMT) 4 weeks after vaccination was compared from respective pre-vaccination values by Wilcoxon signed rank test. Geometric mean of antibody levels before and after immunization and the ratio between them (Mann-Whitney test), the Seroconversion rates (Z test of proportions) and the adverse events (Fisher's exact testand Chi square test), were compared between two groups. P value < 0.05 was considered statistically significant. P values and 95% confidence intervals were estimated in two-tailed fashion. Results: 153 subjects (Typbar^TM =116 and Vaccine "X" =37) were studied. 71.6% (95% CI=63.4%-79.8%) and 75.7% (95% CI=64.9% - 89.5%) were the seroconversion rates with Typbar^TM and vaccine "X" respectively. The GMT values for Vi antibodies induced after Typbar^TM and vaccine "X" were 10.23 Typbar^TM and 13.46 mg/mL respectively and these values showed high significance when compared to their respective pre-immunization GMT values (P<0.0001) at 95% CI (-10.49 to -7.19 mg/mL for Typbar^TM and -14.69 to -8.86 mg/mL for Vaccine "X"). The induction of antibody response appeared to be slightly stronger (P=0.032) with vaccine "X" when compared to that of Typbar^TM. This is justifiable as the same group also had high pre-immunization GMT values (P=0.021). Conclusion: The immunogenicity and safety of the investigational vaccine Typbar^TM was found to be similar to that of already marketed brand of Vi CPS, Vaccine "X". The availability of a single dose of vaccine that is safe and effective enhances the prospective for control of typhoid fever.

Key Words: Salmonella typhi Vi polysaccharide vaccine, Vi-anti body, Sero conversion rate.

INTRODUCTION

Antibiotic resistance among Salmonella is a rising concern.¹ The use of an effective safe vaccine, especially in children could lessen the incidence of typhoid fever in endemic areas.² Of the three types of vaccines that are presently available in India, the whole-cell vaccine being highly reactogenic, produces significant adverse effects.³ Ty21a oral vaccine exists in two formulations, three doses of which administered is better tolerated but confers low grade protection.^4-6 Studies suggest that single injection of Vi capsular polysaccharide vaccine (ViCPS) confers protection for more than three years and may be protective even after ten years and better tolerated compared to whole cell killed vaccine.^2,7-10 Although the ViCPS has been marketed in India, efficacy data among Indian population has rarely been published. This article compares the immunogenicity (efficacy) and safety of two different brands of Vi CPS vaccine, ie; Typbar^TM, the investigational vaccine and an already marketed vaccine with brand name "X", in healthy subjects aged between 10 and 25 years.

MATERIAL AND METHODS

Double blind, randomized (3:1), controlled, parallel, phase III study was conducted at two centres (Centre I and Centre II) in India. The study protocol was submitted to the respective Institutional Review Boards and approval was obtained. Informed consent was obtained from the participants during group meetings or home / hostel / canteen visits. In case of subjects below 12 years of age, the consent form was countersigned by the parent/ guardian. The written consent document was in the language which the participant could read and understand. The study subjects consisted of healthy male / female between 10-25 years of age, without prior exposure to typhoid. Subjects with fever of any origin more than 3 days duration within one month prior to screening or on the day of screening. anti-Vi titres of > 5 mg/mL at screening, history of allergic, immunological disorders, pregnant, nursing women were excluded from the study. This study was carried out between 20-08-2002 to 30-9-2002 .

The subjects were screened at the base line visit i.e.: day -7 with medical history, physical examination, laboratory safety parameters, HIV status (ELISA), urine pregnancy test, and anti-Vi assay (ELISA) and those who fulfilled the inclusion criteria were assigned identification number. On day 0, these participants were randomized into two groups either to receive injections of Typbar^TM, the vaccine under investigation (Bharat Biotech International limited, Hyderabad) or the already marketed vaccine with brand name "X" (Green cross Vaccine Corp. Korea), in 3:1 ratio. Each vial of both vaccines containing 25mg/0.5mLof vaccine was assigned a code number by the manufacturers and was supplied to the investigators in individual packets. Coding was done such a way that ratio between Typbar^TM and vaccine "X" was maintained 3:1. On day 0, the physicians enrolled the participants, the pharmacologists assigned the participants into either of the groups by randomly picking up any packet containing the coded vial, as the study subject came to the injection room to receive the vaccine. The packet was opened and the code number written on the vial was assigned to the respective participant as the randomization number against the subject identification number and subject initials. The vaccines (dose of 25mg/0.5mL) were injected intramuscularly in left deltoid. The participants, physicians, pharmacologists, biochemist and the statistician were blinded to group assignment. Code which was kept with the manufacturers, was broken after receiving the post vaccination antibody values. The subjects were observed closely for 60 minutes after the injection. All subjects were followed up until day 30. On the 30th day, the vaccinates were subjected to the same procedure as that of day 7. Efficacy Parameter included measurement of serum anti-Vi antibody levels by ELISA done at Centre for Diarrhoeal Disease and Nutrition Research Department of Paediatrics, AIIMS, Delhi. Primary outcome measure was to compare the mean Vi - antibody levels on day 30 from that of day -7 and percentage of subjects sero converting (> 3 fold rise in the antibody levels from baseline) on day 30 following vaccination. Secondary outcome measure included Safety as assessed by : medical history, clinical examinations, laboratory investigations- Haematology, random blood sugar, liver function tests, renal function tests, and urinalysis, on days -7 and 30. Adverse events were monitored throughout the study period. The systemic adverse event following vaccination was defined as fever, headache, muscle pain, chills/rigor, sweating, nausea and diarrhoea while a local adverse event was defined as the pain, redness, swelling at the injection site. The subjects assessed and entered the intensity of solicited adverse events in the "Subject Diary" provided to them, as mild, moderate and severe, as defined in the diary.

STATISTICAL METHODS

Sample size calculation provided a power of 80% at a level of significance 0.05 which gives sample size of 162. Allowing a 15% dropout rate altogether 184 subjects were enrolled and were divided at 2 centres in India (92 each). Sample size was determined with the help of SAMPSIZE V2.0 (software developed by David Machin et al). Variables like age and weight were compared between groups using Paired t test. Levels of anti-Vi antibodies were summarized as geometric mean antibody titres (GMT). Mean of pre- as well as post- immunization antibody levels and their ratio were compared between two groups by Mann-Whitney test. Mean antibody levels on day 30 in each group were compared with their respective mean pre-immunization antibody levels by Wilcoxon signed rank test. Percentage sero converting in each group was calculated and was compared between the groups by Z test of proportions. The safety data was described as the percentage of subjects presenting with each of the local and systemic reactions and comparisons were made between the two groups using chi-square and Fisher's exact tests. P value < 0.05 was considered statistically significant. P values and 95% confidence intervals were estimated in two-tailed fashion.

RESULTS

Altogether, 184 (92 in each of the centres) subjects were enrolled into the study of which 19 were excluded as their pre-immunization antibody levels were > 5mg/mL. Eight eligible subjects refused to receive injection on day 0 and three vaccinates from Typbar^TM and one from vaccine "X" group were not traciable for the 30th day evaluation. Hence, 116 vaccinates receiving Typbar^TM (66 from Centre I and 50 from Centre II) and 37 vaccinates receiving vaccine "X" (22 from Centre I and 15 from Centre II), total of 153 subjects were available for final analyses of results. The study population consisted of medical students, canteen workers, farmers, other students. The distribution of the study population was similar across two groups .

(Table 1) The distribution of subject characteristics like age and weight were proportionate between two groups. The male to female ratio in Typbar^TM vaccine "X" groups were 3.5:1 and 4.3:1 respectively.

(Table 2) -The mean pre-immunization Vi- antibody level was found to be significantly higher in the vaccine "X" group as compared to that of Typbar^TM group (P=0.021) so also the mean post immunization Vi- antibody values (P=0.032). The mean values of antibody levels on the 30th day after immunization with both Typbar^TM and the vaccine "X" were significantly higher compared to their respective pre-immunization mean antibody levels (P<0.0001). Although, the mean fold rise in the antibody titre were higher following vaccine "X" than that of Typbar^TM, was not statistically significant (P=0.32). Sero conversion rates on the 30th day after immunization with Typbar^TM and vaccine "X" were 71.6%(95% CI=63.4% to 79.8%) and 75.7% (95% CI=64.9 to 89.5%) respectively, without any statistically significant difference between the two values (P=0.62).

The response rate to "Adverse Event Recording in Subject Diary" was 100%. (Table 3) - Local and systemic adverse events of mild intensity, could possibly be attributed to the vaccines were reported by equal proportion of subjects in both the groups. Laboratory parameters for safety assessment were within the normal range both on the day-7 and 30^th day of vaccination in both the groups.

No statistically significant difference in the percentage of subjects reporting various adverse events, between two groups by Fisher's exact test and Chi- square test

DISCUSSION

An increase in specific serum antibodies is the predominant immune response elicited by ViCPS vaccine.¹¹ In this study, the induction of antibody response were found to be highly significant (P<0.0001) following vaccination with both the brands of ViCPS vaccine as was reported by Panchanathan V et al.⁸ The seroconversion rates four weeks after vaccination with Typbar^TM and vaccine "X" were and 71.6% (95% CI=63.4%-79.8%) and 75.7% (95% CI=64.9%-89.5%) respectively and the two vaccines did not show statistically significant difference in this regard. With reference to other studies, the sero conversion rates 4 weeks after vaccination with ViCPS vaccine were 75% in Nepal,⁷ 67% in Malaysia,⁸ 81-92% in China,¹² 93 and 98% in USA.¹³ In a recent study, Klugman et al were also able to define the serological correlates of protection following vaccination with Vi in an endemic area.^9,10 One dose of ViCPS resulted in 55% fewer cases of blood-culture confirmed typhoid fever over a period of 3 years.² The protective efficacy was found to be 69% over a period of more than a year in South Western China¹² and 71-73% in People's Republic of China.¹⁴ The subjects were not followed up for annual estimation of antibody levels or to establish the protective efficacy, are the limitations of the present study. Although, the induction of immune response were found to be highly significant following vaccination with both the ViCPS brands, subjects receiving vaccine "X" showed slightly stronger response (P=0.032) in the induction of Vi- antibody levels than those who received Typbar^TM. The vaccine "X" group also had statistically significant higher mean pre-immunization Vi- antibody levels as compared to that of Typbar^TM group (P=0.021). Individuals with higher pre-immunization Vi- antibody titres in vaccine "X" group might have experienced previous sub-clinical infection with S. typhi thus generated a strong secondary response when given the vaccine, and that may explain the proportionately better immunological response seen with vaccine "X". Moreover, ineqivalence in the sample size ie; smaller sample size in vaccine "X" group might have contributed to this significantly higher pre and post immunization antibody values in this group. However, the ratio between post to pre-immunization mean antibody tires and the seroconversion rates did not show statistically significant difference between the groups. The maximum magnitude of rise in antibody levels on the 30th day following vaccination was more than 11 folds in both the groups in this study as compared to that of 9 fold rise after 6 weeks following vaccination in another study.⁸ The biochemical measures of safety were within normal range. The occurrence of adverse events were comparable between the two groups and the adverse event profile followed the similar pattern as reported in other studies.^3,8 Hence, the immunogenicity and safety of Typbar^TM, and that of vaccine "X" were found to be comparable in the present study.

In contrast to live oral vaccine, ViCPS is not contraindicated for individuals with underlying immune defects^11,13 In addition, ViCPS offers additional advantages like it is stable in ambient temperatures simplifying its use in the field, only one dose is required and it can be combined with other polysaccharide vaccines.^7,13 However, the efficacy of ViCPS has so far been studied only in endemic areas and not among travelers visiting to endemic area from non-endemic area. Long-term protection against Salmonella infection requires the antigen specific recall immunity, with the involvement of both antibodies and T cells in addition to the cytokines like TNF-a, IL-12, IFN-g. Vi vaccine can induce antibody responses, but is believed to be unable to trigger Th1-type immunity and mucosal response¹¹ that may explain the moderate efficacy of this vaccine. Like most polysaccharide vaccines, Vi vaccine does not induce either protective levels of antibodies in children < 2 years or a booster response because, this class of vaccine is T cell-independent.^10,13,15 To overcome the age-related and T-cell-independent immunogenicity of the vaccine, a newly devised conjugate of the ViCPS has been evaluated which has enhanced immunogenicity in adults and children of 5-14 years of age and also has elicited booster response in children of 2-4 years old.^16,17

The capsular polysaccharide of S typhi Vi, is both an essential virulence factor and a protective antigen. Vi expression is associated with a cluster of 10 genes known as viaB operon, encoded on a pathogenicity island known as SPI-7 and comprises of genes encoding enzymes for the biogenesis and translocation of Vi antigen.^18,19 Vi production appears to have been a relatively recent acquisition by S. enterica following mutations in three genes following colonic acid bio-synthesis. If sufficient immunological pressure is exerted by Vi vaccine it is possible that Vi antigen could be lost or that could be replaced by alternative but immunologically distinct capsular locus which could replace the SPI-7-associated viaB locus.²⁰ S enterica strains lacking viaB sequences were found to be responsible for an epidemic of multidrug resistant typhoid fever in Kolkotta, in India.²¹ S typhi lacking Vi antigen evident during slide agglutination with Vi typing antisera are frequently found²² which on its entry into the intracellular niche are protected from Vi antibody. Since Vi vaccine does not elicit cellular immunity, would be unlikely to confer protection against an intracellular replication if Vi negative strains and it seems prudent to monitor the Vi status of primary S typhi isolates and the stability of the viaB locus during Vi vaccination trials that would help to reveal the contributory role of such strains in failure of these vaccines.^20,22 The present study had its limitation in this regard.

In humans, attenuate strains Salmonella stimulate mucosal and serum antibodies and a variety of cell-mediated immune responses resulting in long-lasting immunological memory. Serum and mucosal responses are directed towards a broad spectrum of antigens including, Vi, porins, outer membrane proteins, lipo proteins, heat-shock proteins, flagella, and fimbriae. Cellular reponses are of are of Th1 type. Class Ib CD8+ T cells capable of lysing Salmonella infected target cells also appear after oral vaccination. T- cell responses are directed towards several antigens, including protein antigens, porins, flagellar epitomes, pilin, and to the Vi antigen. T cells modulate humoral responses during immunization with live attenuated Salmonella vaccines.¹¹

Live oral typhoid vaccine Ty21a is one of the safest and best tolerated of all licensed vaccines. However, the modest immunogenicity of this vaccine, which requires that three or four spaced doses be administered to confer credible protection, constitutes an important and practical shortcoming. Several engineered mutant strains of S typhi appear promising as candidate single-dose live attenuated oral vaccines because of their increased immunogenicity over Ty21a.^23-25

SUMMARY

In the present study, which was carried out as a double-blind evaluation, at two centres in India, Typbar^TM and vaccine "X", the two different brands of the Salmonella typhi Vi capsular polysaccharide vaccine demonstrated significant immune response (P<0.0001). Although, the mean antibody response elicited by vaccine "X" was significantly higher which could be attributed to the inhomogenicity in pre immunization antibody levels and the sample size between the two groups, the two brands of Vi vaccine showed equal sero conversion rates, and and adverse event profile, four weeks following vaccination. Hence, it could be concluded that the immunogenicity and the safety of Typbar^TM, the vaccine under investigation, is equal to that of Vaccine "X". This study also reaffirms the safety and efficacy of typhoid Vi polysaccharide vaccine. The availability of a single-dose vaccine that is safe and effective enhances the prospective for control of typhoid fever.

ACKNOWLEDGEMENTS

Thanks to Bharath Biotech International Ltd for financial support; Asian Clinical Trials Pvt Ltd; Hyderabad, the Contract Agency and Dr. Ramesh Kumar, Centre for Diarrhoeal Disease and Nutrition; Department of Pediatrics, AIIMS, New Delhi, India, for carrying out Typhoid ELISA Assays.

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