Cell-mediated immune responses in humans after immunization with one or two doses of oral live attenuated typhoid vaccine CVD 909

Abstract

CVD 909 is a novel live attenuated S. Typhi oral vaccine candidate derived from strain CVD 908-htrA which constitutively expresses Vi. Herein we investigated whether the genetic manipulations involved in modifying CVD 908-htrA altered its ability to induce potent T-cell immune responses (CMI) after a single dose (five subjects) and, in a separate trial, whether a second dose (eight subjects) further enhanced its immunogenicity. In these clinical trials we observed that CVD 909 immunization elicits a wide array of CMI, including cytotoxic T cells (CTL), IFN-γ, TNF-α and IL-10 (but not IL-2, IL-4 or IL-5) production, and proliferation to S. Typhi antigens. However, the administration of a second dose did not result in increases in CMI. These results suggest that the genetic manipulations to constitutively express Vi did not adversely affect the ability of CVD 909 to elicit a wide array of CMI responses. These observations add impetus for the continuing evaluation of CVD 909 as a typhoid vaccine candidate.

Introduction

Typhoid fever remains an important public health priority, particularly in developing countries. It is estimated that about 21,650,000 episodes of typhoid fever and 216,500 deaths occurred in 2000 [1]. The appearance of antibiotic resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, has added new urgency for the development of improved typhoid vaccines [2], [3].

Currently licensed typhoid vaccines in the USA include the purified Vi polysaccharide parenteral vaccine and the live oral attenuated Vi negative galE mutant, S. Typhi strain Ty21a vaccine (henceforth Ty21a vaccine). Each of these vaccines mediates protection by distinct mechanisms. The purified Vi polysaccharide vaccine is T cell independent and mediates protection by eliciting anti-Vi antibody responses [4]. In contrast, the Ty21a vaccine likely mediates protection by eliciting antibodies to S. Typhi LPS, flagella and other antigens, as well as through a wide array of T-cell immune responses (CMI) at the systemic and mucosal levels [5], [6], [7], [8], [9], [10], [11]. However, each of these vaccines has drawbacks. Vi polysaccharide is a parenteral vaccine that cannot stimulate CMI and fails to elicit responses to purified polysaccharides in children under 2 years of age [12], [13]. Concerning Ty21a, this vaccine does not elicit anti-Vi antibodies (because it does not make Vi capsule) and is modestly immunogenic, requiring three or four doses to evoke protective immunity in ∼47–96%, depending on the number of doses and formulations used and the time of surveillance [12], [14], [15].

To overcome these limitations, we and others have recently engineered new attenuated typhoid candidate vaccine strains that are as well-tolerated as Ty21a yet more immunogenic, with the goal of eliciting protective immunity with just a single oral dose [2]. One of these strains, CVD 908-htrA, has been attenuated by deletions in genes encoding enzymes required for the biosynthesis of aromatic amino acids (aroC and aroD) and htrA, a gene encoding a heat shock protein [16]. CVD 908-htrA has been shown to be well-tolerated and highly immunogenic in phases 1 and 2 clinical trials [17], [18], [19]. After a single dose, we observed a wide array of CMI responses, including the secretion of interferon (IFN)-γ and the killing of S. Typhi-infected cells (cytotoxicity), as well as, anti-S. Typhi LPS antibody responses in serum, and anti-LPS IgG and IgA antibody secreting cells (ASC) [17], [19], [20]. Disappointingly, CVD 908-htrA, which does not express Vi constitutively, did not induce an immune response against Vi. This lack of Vi responsiveness could be the result of low expression of the Vi capsule, which is likely to be down regulated in vivo once the bacteria gain their intracellular niche in the gut associated lymphoid tissue [21]. To render the Vi expression constitutive, promoter P_tviA in serovar Typhi vaccine CVD 908-htrA was replaced with the constitutive promoter P_tac, resulting in CVD 909, which expresses Vi even under high-osmolarity conditions [22]. Our hypothesis was that this might result in serum IgG and mucosal IgA Vi antibody responses in orally vaccinated subjects, thereby enhancing the protection against typhoid fever. We recently conducted a vaccine trial in which healthy subjects were orally immunized with one dose of CVD 909. This vaccine was very well-tolerated and 16 of the 20 volunteers (80%) who received 10^8–9 cfu exhibited anti-Vi ASC responses following immunization, while only 2 of the 20 volunteers who received 10^8–9 cfu generated anti-Vi IgG or IgA antibody in serum [21]. This is the first live attenuated S. Typhi vaccine candidate shown to elicit anti-Vi responses.

To explore the possibility that stronger immunity could be elicited by a second dose, a separate phase 2 vaccine trial was conducted in which volunteers were immunized with two oral doses of CVD 909, one dose on day 0 and a subsequent dose on day 14. In the present study, we investigated whether the genetic manipulations involved in modifying CVD 908-htrA to constitutively express Vi altered its ability to induce potent CMI after a single dose and whether a second dose further enhanced the immunogenicity of oral live attenuated typhoid vaccine CVD 909.