A number of different strategies are being employed to develop a vaccine against ETEC infections including oral killed whole-cell preparations (14
) and subunit vaccines (12
). In addition, live attenuated vaccines against ETEC that are in development include those based on Shigella
) or Salmonella
) strains expressing CFA or LTB and attenuated ETEC derivatives (21
). Use of orally delivered live attenuated vaccines may be more advantageous for enteric diseases than killed or subunit vaccines as the antigens are delivered to the intestinal mucosal surface where immunity is required. Moreover, while oral killed or subunit vaccines present antigen only during their brief passage through the alimentary tract, where they are prone to digestive degradation, the aim of live attenuated vaccines is to achieve transient colonization of the host. This colonization is expected to result in expression of relevant antigens in situ, achieving a more appropriate presentation of antigens for a prolonged period to the immune system. A live attenuated vaccine based on a number of diverse natural ETEC strains may have the added advantage of presenting ETEC antigens in addition to CFA, which may add to the protection elicited.
Immune responses to the CFA are important for protection against ETEC infection (5
). Since different ETEC strains express a number of CFA, to protect against the majority of ETEC strains a vaccine will need to consist of several strains expressing the most common CFA. It was demonstrated previously that vaccine candidate PTL003, a toxin-negative derivative of ETEC strain E1392/75 with mutations introduced into the aroC
, and ompF
chromosomal genes and which expresses CFA/II, was well tolerated and immunogenic when administered to volunteers (21
One of the more common CFA is CFA/I (35
), which is expressed by ETEC strain WS-1858B. Using the same basis of attenuation as for PTL003, this strain was rendered toxin negative, and aroC
, and ompF
deletion mutations were introduced into the chromosome. Antibiotic-sensitive derivatives were identified, and the resulting strain, ACAM2010, now represents an important component of a live attenuated ETEC vaccine.
Southern hybridization analysis performed on the parent strain of ACAM2010, WS-1858B, revealed that the genes associated with virulence (CFA/I, ST, and EAST1) are all carried by the same plasmid. Likewise, loss of the antibiotic resistance determinants coincided with a visible change in mobility to only one other plasmid, suggesting that these determinants are encoded by a resistance plasmid. In WS-1858B the region between the ST and cfaA
open reading frames is only 1,876 bp. This intragenic region includes nucleotide sequences that are 94% identical to half of IS629
from Shigella sonnei
. For two other CFA/I- and ST-expressing ETEC strains, WS-4437A and WS-6117A (S. Savarino), the nucleotide sequence between the ST and cfaA
open reading frames was almost identical to that in strain WS-1858B (A. K. Turner, unpublished data). These three strains express different O:H serotypes (O128:H12; O153:H45, and O71:H−
, respectively) and so are not clonally related. This suggests that the CFA/I and ST loci initially became tightly linked by transposition and have been subsequently transferred together to a variety of different strains. Linkage of ETEC virulence factors is of clear evolutionary significance as it facilitates this simultaneous transfer. Spontaneous loss of toxin expression has been observed previously in ETEC strains (37
), and the pJCB12 approach used here was designed to permit selection for and easy isolation of the desired derivative. The estA
gene fragment in pJCB12-estA
did not incorporate a deletion but rather was used simply to insert a counterselectable marker into the locus. The loss of estA
in the ST-negative derivatives occurred by spontaneous recombination resulting in deletion of approximately 30 kb from the plasmid. Similarly, for the second copy of the astA
gene, presumed to be on the chromosome, the deletion construct was not incorporated, but rather the whole astA
gene and proximal flanking sequences were lost. The results for both estA
in WS-1858B are consistent with the hypothesis that these enterotoxins are encoded by unstable genetic loci that are prone to spontaneous recombination.
Previous phase I trials of live attenuated ETEC vaccine candidates (21
) have been carried out using vaccines formulated from freshly grown overnight cultures on solid medium, which maximizes expression of the CFA. While this provides an acceptable procedure for initial clinical proof-of-concept studies, it clearly bears little or no relation to a process by which a final vaccine product will be manufactured. An aim of the study with ACAM2010 was to compare this “fresh vaccine” approach with an alternative formulation which represents a step towards a scalable manufacturing process. Accordingly half of the subjects in this study received vaccine which had been produced by culturing ACAM2010 in a fermentor; washing and concentrating it and filling cryovials with it in a closed aseptic manner; and storing it at −80°C until use. Under these conditions no effort is made to maximize expression of CFA in vitro, relying on their expression by the live vaccine strains following their ingestion. The phase I trial described here thus represents the first administration to human volunteers of a new candidate ETEC vaccine strain expressing CFA/I as well as addressing the issue of formulation to move towards a process which can be carried out on a commercial scale.
ACAM2010 has been tested in a phase I clinical trial, with a total of 37 volunteers receiving nominal doses of 5 × 109 CFU, 19 doses prepared directly from a frozen suspension and 18 from a fresh overnight culture. Both formulations were generally well tolerated with no AEs occurring at frequencies significantly higher in vaccinees than in placebo recipients. Lower numbers of AEs were observed in recipients of the frozen than in those of the fresh formulation, although this difference was significant only for abdominal pain and the three aggregated GI symptoms (Table ). This is an encouraging result because as product development proceeds and vaccine is manufactured by large-scale fermentation for later-stage clinical trials it will be more similar to the frozen formulation than to freshly cultured organisms. The data presented here suggest that we can expect the safety and tolerability of larger-scale lots of vaccine to be similarly good. Equally encouraging is the observation that, although the frequencies of response were not significantly different, the magnitude of the immune responses to the CFA/I antigen following vaccination were significantly higher in recipients of the frozen formulation than in those ingesting the freshly grown vaccine.
These studies with ACAM2010 therefore represent an important milestone in the development of a broadly protective ETEC vaccine, providing a safe and immunogenic CFA/I-expressing strain and demonstrating that it is not necessary to maximize expression of CFA/I during growth of live vaccine strains in order to ensure optimal immunogenicity, the frozen vaccine being at least as immunogenic as vaccine freshly grown on CFA agar. The lower reactogenicity and higher immunogenicity of the frozen formulation, together with the greater ease of dose preparation and improved level of characterization possible, make this the protocol of choice for future clinical studies prior to full-scale cGMP manufacture of the final product.