The fimbrial CFAs were identified as potential targets of ETEC vaccines in the 1970s (13
). Initial attempts at oral vaccination with purified CFAs were thwarted by gastric enzymes which reduced the antigenicity of CFAs (7
). Microencapsulation and administration of CFAs via intestinal tubes prevented degradation in the stomach and resulted in 30% protection against challenge (22
Difficulties with production and delivery of purified CFAs stimulated interest in killed vaccines and live attenuated vaccines. A formalin-inactivated whole-cell vaccine produced by SBL Vaccin, Stockholm, Sweden, contains a variety of strains with many of the common CFAs and the B subunit of cholera toxin (CT), which is structurally similar to, and gives cross-protection against, ETEC LT. This vaccine was immunogenic in several adult and pediatric populations (1
). In a study of 685 U.S. citizens traveling to Mexico and Guatemala, the vaccine protected against moderate to severe ETEC illness (protective efficacy of 77%, P
= 0.04) but not against mild ETEC diarrhea (D. A. Sack, J. Shimko, O. Torres, A. Karnell, I. Nyquist, B. Gustafsson, A. L. Bourgeois, O. Newton, and A. Svennerholm, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. G-1217D, 2002). A second larger study of travelers to Latin America corroborated these results. (A. L Bourgeois, J. Halpern, B. Gustafasson, et al., Abstr. 45th Annual ICACC, abstr. G-408, 2005). Unfortunately, when the vaccine was field-tested in 314 Egyptian children aged 6 to 18 months, the protective efficacy was only 20% (S. J. Savarino, R. Abu-Elyazeed, M. Rao, R. Frenck, I. Abdel-Messih, E. Hall, S. Putnam, H. El-Mohamady, T. Wierzba, K. Kamal, P. Moyer, B. Morsy B, A. Svennerholm, Y. Lee, and J. Clemens, Abstr. 6th Annu. Conf. Vaccine Res. Natl. Found. Infect. Dis., abstr. 02-A-43-NFID, 2003). Since this vaccine contains whole cells and CT, it is difficult to assess the role of the CFAs in protecting travelers from moderate to severe ETEC illness.
Another method of delivering CFAs to the small intestine is via live, attenuated bacteria. Attenuated Salmonella
strains have been genetically engineered to express ETEC CFAs, often with the B subunit or a mutant form of LT or CT (2
). The immunogenicity of these strains has been demonstrated in animal models. In addition, live, attenuated ETEC strains have been studied in human trials. Administration of a toxin-positive ETEC strain protected against subsequent rechallenge with the same strain (13
). Furthermore, administration of a toxin-negative strain, E1392/75-2A, protected against challenge with a toxin-positive strain homologous only for CFAs (12
The current randomized, double-blind trial assessed the safety and immunogenicity of two attenuated ETEC strains, PTL-002 and PTL-003, which were derived from strain E1392/75-2A. The aroC
gene was deleted to form both strains. In addition, ompC
were deleted to generate PTL-003; and ompR
, part of a two-component regulatory system, which regulates the expression of ompC
, and other genes, was deleted to generate PTL-002 (24
). The ompR
mutation of PTL-002 is, therefore, more pleiotropic than the ompC
mutations of PTL003 and probably more attenuating.
As suggested by these attenuation strategies, PTL-003 caused more sustained colonization than PTL-002. PTL-003 was isolated more than twice as frequently as PTL-002. The duration of fecal excretion by the two vaccine constructs was somewhat longer than reported for other live attenuated enteric vaccine candidates, e.g., CVD908-htrA (23
). In the present study the streptomycin-resistant marker carried by the vaccine strains made the detection of organisms in stool samples very sensitive and specific. Since quantitative cultures were not done, the level of shedding was not determined.
In previous studies with the parent strain and a pilot study with PTL-002 and PTL-003, diarrhea occurred in 15 to 20% of subjects (21
). Administration of these strains in sodium bicarbonate may have caused some loose stools. In the present study the vaccine strains were administered in CeraVacx, a rice-based oral rehydration solution. Given in this buffer, the vaccine strains were well tolerated and were associated with mild diarrhea in only 2 of 40 subjects. CeraVacx was chosen as the buffer for the current study based on a comparison of three buffers for delivery of Peru-15, a live oral cholera vaccine. Vibriocidal antibody titers were higher when Peru-15 was given in CeraVacx compared to a standard bicarbonate-ascorbic acid buffer or Alka-Seltzer (16
). In the previous PTL-002/003 pilot study five subjects ingested PTL-002 and six ingested PTL-003 as a single dose of 5 × 109
CFU in bicarbonate buffer. None of these subjects developed IgA or IgG serum anti-CFA/II responses by ELISA (D. A. Sack, unpublished results); however, all had significant increases in IgA titers in the ALS assay (24
). In the present study, which utilized the same immunologic assays performed in the same laboratory as the pilot study, a slightly lower dose (2 × 109
CFU) of PTL-002 or PTL-003 given in CeraVacx resulted in similar mucosal responses but higher serum antibody responses to CFA/II measured by ELISA. Even though two doses of vaccine were given to 21 of the 40 subjects in the current study, serologic responses were similar for those in the two-dose and those in the one-dose groups. Further studies are needed to determine whether carbohydrate-based buffers will improve the tolerability and immunogenicity of live vaccines.
The more persistent excretion of PTL-003 correlates with its greater immunogenicity compared to PTL-002. For neither strain was a second dose on day 10 clearly beneficial in eliciting more serologic responses or greater increases in titer. There were, however, several new mucosal antibody responders noted in the ASC and ALS assays after the second dose. A larger study would be necessary to determine whether a second dose is beneficial.
PTL-003 induced IgA-ASC and serum IgA responses comparable to those elicited by two other prototype vaccines: a whole-cell, formalin-inactivated vaccine comprising five ETEC strains and CTB (1
) and a CFA/II vaccine encapsulated in biodegradable polymer microspheres (22
). Anti-CFA/II IgA-ASC response rates were 90% for PTL-003 compared to 76% (anti-CS1 or CS3 IgA-ASCs) and 50% for the other two vaccine candidates, respectively. In addition, immune responses induced by PTL-003 were similar to those observed after challenge with 109
CFU of E24377A, a virulent CS1+
ETEC strain (unpublished data). PTL-003 compared to E24377A elicited similar IgA-ASC response rates (90% versus 90%) and serum IgA response rates to CFA/II measured by standard colorimetric ELISA (35% versus 30%). In the TRF assay response rates to CS3 were similar (65% versus 55%), but response rates to CS1 were lower for PTL-003 compared to E245377A (55% versus 100%).
The evaluation of the comparative immunogenicity of the PTL-002 and PTL-003 vaccine constructs in this trial provided an opportunity for the first time to directly compare ALS and TRF to the more standard ASC and ELISAs for their relative abilities to quantify mucosal and serum antibody responses to ETEC antigens. The proportion of ALS responders was slightly lower than those detected by the ASC assay for both the IgA and IgG isotypes (Table ). This discrepancy may have resulted from the shorter, 48-h incubation time used for the ALS assay in the present study. In subsequent studies longer ALS incubation times of 72 or 96 h produced better concordance between these two tests. Additional studies comparing TRF with the colorimetric ELISA corroborated results in this report, indicating that TRF is more sensitive for detecting anti-CFA responses after infection or immunization with ETEC strains (2a
One limitation of this trial is the small number of subjects in each group. Although the size of the trial was adequate to demonstrate that PTL-003 was more immunogenic than PTL-002, it was not large enough to determine whether a second dose of vaccine was beneficial. In addition, the trial provided no information on the durability of the immune response beyond 1 month. Since immune correlates of protection against ETEC illness have not been determined, we do not know if the strong CFA response elicited by PTL-003 would protect against a virulent ETEC strain. However, based on the greater immunogenicity of the PTL-003 construct, this strain has been selected for further development as a prototype ETEC vaccine. A “proof of concept” challenge study is planned in which volunteers will be vaccinated with the PTL-003 construct and subsequently challenged with a fully virulent ETEC strain homologous for CFAs.