Evaluation of thimerosal removal on immunogenicity of aluminum salts adjuvanted recombinant hepatitis B vaccine

Abstract

Thimerosal, which is approximately 50% mercury by weight is a preservative widely used in vaccines since the 1930's. It meets the requirements for a preservative as set forth by Pharmacopeia challenge test and has been shown to be effective against a broad spectrum of pathogens. In July 1999, the Public Health Service agencies and vaccine manufacturers agreed that thimerosal should be reduced or eliminated in vaccines as a precautionary measure but, due to the lack of appropriate alternative, it is still extensively used in multiple dose formulations of vaccines such as hepatitis-B in developing countries. In this study the effect of the removal of thimerosal in two formulations of hepatitis B vaccines containing either aluminum hydroxide or aluminum phosphate were evaluated in Balb/c mice. These formulations were administered interperitoneally and the titer of antibody was determined by ELISA technique after 28 days. The geometric mean of antibody titer (GMT), seroconversion and seroprotection rates, ED50 and relative potency of different formulations were determined. The ED50 of thimerosal-free formulations were reduced by more than 35% in both preparations. In addition, GMT of antibody titer, seroconversion and seroprotection indicated significantly higher immunogenicity for thimerosal free formulations for both aluminum phosphate and hydroxide adjuvants.

Keywords: Aluminum salts; GMT; Hepatitis B vaccine; Relative potency; Thimerosal.

Figure 1

SDS-PAGE analysis of purified HBsAg. Thegel was stained with silver nitrate. Lane 2 and.3 purified bulk of HBsAg, Lane 1. Low molecular weight calibration kit (14.4-97KDa, GE Healthcare), Lane 3.Diluted (1:2) purified HBsAg bulk.

Figure 2

Determination of residual host cell DNA using semi-quantitative PCR on agarose gel electrophoresis. Lanes1, 3, 4, 5 and 6 are the positive controls containing 10, 100, 50, 25 and 5 pgs of the host cell derived DNA respectively and lane 2 is the sample and lane 7 is the negative control lacking either the host DNA or the sample.

Figure 3

Seroconversion effect of different dilutions of hepatitis-B vaccines containing Adju-Phos (aluminum phosphate), Alhydrogel (Aluminum hydroxide) thimerosal-free or containing formulations and Engerix-B after 28 days of IP injection in Balb/c mice.

Figure 4

ED50 of different formulations containing Adju-Phos (aluminum phosphate) or Alhydrogel (Aluminumhydroxide) thimerosal-free or containing formulation and Engerix-B after 28 days of IP injection in Balb/c mice (p < 0.05).

Figure 5

Seroprotection effect of different dilutions of hepatitis-B vaccines containing dju-Phos (aluminum phosphate), Alhydrogel (Aluminum hydroxide) thimerosal-free or containing formulations and Engerix-B after 28 days of IP injection in Balb/c mice

Figure 6

Relative potency different formulations containing Adju-Phos(with and without thimerosal) or Alhydrogel (with and without thimerosal) and Engerix-B after 28 days of IP injectionin Balb/c mice.