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Infect Immun, May 1998, p. 1968-1972, Vol. 66, No. 5
The Vaccine Testing Unit,
Received 15 September 1997/Returned for modification 12 December
1997/Accepted 2 February 1998
To evaluate a standardized inoculum of Vibrio cholerae
for volunteer challenge studies, 40 healthy adult volunteers were
challenged at three different institutions with a standard inoculum
prepared directly from vials of frozen, virulent, El Tor Inaba V. cholerae N16961, with no further incubation. Groups of 5 volunteers, with each group including 2 volunteers with blood group O,
were given a dose of 105 CFU, and 34 of the 40 volunteers developed diarrhea (mean incubation time, 28 h). Transient fevers occurred in 15 (37.5%) of the volunteers. V. cholerae was excreted by 36 of 40 volunteers. Five
additional volunteers received 104 CFU, and four developed
diarrhea but with a lower average purging rate than required for the
model. Of the 40 volunteers, 37 developed rises in their vibriocidal
and antitoxin titers similar to those in previous groups challenged
with freshly harvested bacteria. We conclude that challenge
with frozen bacteria results in a reproducible illness similar to that
induced by freshly harvested bacteria. Use of this model should
minimize differences in attack rates or severity when groups are
challenged at different times and in different institutions.
Cholera continues to be a major
public health problem in nearly all developing countries, now including
the Western hemisphere (17). It became newsworthy recently
because of its introduction into Latin America in 1991 and because of
the epidemic among refugees in Zaire, which led to an estimated 50,000 deaths during this epidemic alone (7). Cholera continues in
less dramatic fashion as an endemic disease in over 100 countries
(6). An improved vaccine(s) for cholera could provide an
important public health tool with which to control the disease
(4). The development of such a vaccine has been aided
greatly by the use of volunteer studies in which volunteers are
immunized with an experimental vaccine and then experimentally
challenged with a virulent strain while hospitalized in a facility
experienced in the management of such volunteers. By comparing the
rates and severity of illness in immunized and nonimmunized volunteers,
the efficacy of the vaccine against this standard challenge is
determined (2, 9, 10, 14-16). Experience with this model
has indicated that vaccines showing efficacy in volunteers have also
demonstrated efficacy when tested in field trials (2).
While the volunteer model is valid and useful, there are constraints
which have limited its utility. The major constraint relates to the
need for each group of volunteers to receive the same virulent
challenge, in terms of both the numbers of bacteria and the virulence
of the bacterial preparation. Currently, the bacterial challenge is
prepared by a standard operating procedure so that a consistent number
of freshly grown bacteria is given to the volunteers. However, only a
few vaccine-testing centers have the resources to develop this
challenge procedure. In addition, minor differences in growth
conditions (e.g., media, time of incubation, and temperature) could
affect the virulence of the challenge bacteria. As the need grows for
additional testing sites where such volunteer studies can be performed,
there is also a need to ensure that the challenge given to volunteers
is uniform among different volunteer units and among groups of
volunteers within the same unit challenged at different times.
The National Institutes of Health therefore undertook to prepare a
batch of frozen Vibrio cholerae (El Tor Inaba strain N16961) with a large number of aliquots so that identical vials from the lot
could be used for volunteer challenge studies. It was hypothesized that
the use of this frozen inoculum would result in consistent attack
rates, and consistent severity of illnesses among different groups of
volunteers and at different clinical sites. Furthermore, it was
hypothesized that the illness seen when frozen inocula were used would
be similar to illnesses observed when freshly harvested bacteria were
used, including a geometric mean stool output of Since the blood group of the volunteer is also an important determinant
of the severity of cholera (1, 3, 8), the model also
included two persons with blood group O among each group of five
volunteers to standardize this potentially confounding variable.
This report summarizes the clinical, microbiological, and serological
findings when the frozen inoculum was given to nine groups of
volunteers at three institutions. Past studies have used a dose of
106 CFU per dose of freshly harvested organisms; hence, the
plan was to begin with a lower dose (105 CFU), expecting to
increase in 1-log increments up to 106 or even
107 CFU per dose if needed to induce a consistent illness.
Preparation and validation of the inoculum.
The challenge
lot, containing El Tor strain N16961, was prepared as a 5-liter
fermentor batch, and over 2,300 vials with 2 ml per vial were filled,
labeled, and frozen at Study plan.
The goal of the study was to determine a dose of
bacteria which would consistently induce diarrhea in Volunteer studies.
Groups of volunteers (n = 5 per group) were recruited from the community in Baltimore and
Cincinnati for our studies by advertisements in local newspapers.
Healthy, eligible, and willing volunteers between 18 and 40 years of
age were trained, passed a written examination to document their
knowledge of cholera and the procedures of the study, signed an
informed-consent form, and underwent a series of clinical and
laboratory examinations to rule out occult illness or pregnancy. The
screening examinations included a complete blood count with
differential, chemistry panel, urinalysis, hepatitis B antigen,
hepatitis C antibody, human immunodeficiency virus antibody, blood
type, and urine human chorionic gonadotropin (for females) within 2 days of the study, and an electrocardiogram. Potential volunteers were
excluded if any of the following applied: chronic illness,
immunosuppressive condition, human immunodeficiency virus antibody
positive, hepatitis B surface antigen positive, hepatitis C antibody
positive, travel to an area of endemic cholera infection within 5 years, receipt of a cholera vaccine within 5 years, previous
participation in a research study with cholera or enterotoxigenic
Escherichia coli, pregnancy (urine human chorionic gonadotropin), antibiotic administration within 7 days of the challenge, regular use of laxatives, abnormal stool pattern,
significant abnormality in screening laboratory hematology and
chemistry tests, known allergy to tetracycline, or a significant
abnormality on EKG. Within each group of five volunteers, two had blood
group O.
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Validation of a Volunteer Model of Cholera with
Frozen Bacteria as the Challenge
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
3 liters.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C by the Salk Institute, West Point, Pa.
This strain has been used frequently as a challenge strain by the
University of Maryland Center for Vaccine Development (CVD)
(10). Sample vials were tested to validate the purity and
stability of the inoculum. The frozen preparation was agglutinated with
Vibrio O1 ("poly") antiserum (Difco Laboratories) and
with Inaba antiserum but not with Ogawa antiserum or saline. These
results were identical to those exhibited by the positive control
strain obtained from the culture collection at the CVD. Both the frozen
and control samples "keyed" to V. cholerae when the
20E strip (API Analytabs, Plainview, N.Y.) was used. A total of 90 colonies from three frozen vials were obtained from Luria agar plates
and were hybridized with a probe to detect cholera toxin-encoding genes
(10). All 90 colonies hybridized with the cholera toxin
probe. The concentration of organisms (CFU/per milliliter on Luria
agar) has been consistently 5 × 109 since the
preparation of the challenge lot in August 1995 until the present
(February 1997). The vials are stored at a National Institute of
Allergy and Infectious Diseases repository and were shipped on dry ice
to the participating study sites. The vials were kept frozen at
70°C until used at the respective sites, and the contents were
diluted in phosphate-buffered saline immediately before use. The
stability of the inocula was further documented by obtaining
quantitative counts with serial dilutions in the laboratory of the
volunteer unit. After incubation of the inoculum in 1.33% sodium
bicarbonate buffer for 1 h, there was no change in the numbers of
bacteria recovered.
80% of
volunteers. Additionally, it was important that many of the illnesses
should be moderate or severe (i.e., that the geometric mean of the
total diarrhea was at least 3 liters). For safety reasons, it was
planned to start with a dose of 105 bacteria (1 log lower
than has been used with freshly grown bacteria) and to increase the
inoculum strength if the attack rate or the severity was too low. Since
the dose of 105 did, in fact, induce illnesses according to
the criteria established, the dose was not increased, but one group was
given a lower dose (104 CFU) to determine if an even lower
dose would be sufficient to be useful in the model.
Clinical management. The volunteers were monitored for the occurrence of symptoms such as nausea, vomiting, and diarrhea and were visited at least twice daily to ascertain and manage any medical problems. Fluid intake and output was continuously monitored, with subtotals being obtained every 8 h. All stools were passed into a plastic container for weighing, inspecting, sampling, and disinfection (with sodium hypochlorite [Clorox]). The stools were graded on a 1 to 5 scale (1, firm; 2, soft; 3, runny, takes the shape of the container; 4, brown liquid; 5, rice water). The hydration of volunteers who developed diarrhea was maintained with either a glucose- or rice-based oral rehydration solution (CeraLyte, Jessup, Maryland) to match diarrhea output losses with oral rehydration solution on a 1.5:1.0 basis (one-and-a-half times as much oral rehydration solution as diarrheal stool output). When clinically indicated, intravenous Ringer's lactate was given, matching diarrhea stool volumes with an equivalent volume of rehydration fluid.
Tetracycline administration (500 mg orally every 6 h for 5 days) was started at the time the volunteers met the definition of severe cholera (>5 kg of diarrhea stool) or on day 4 at 12 noon if they had not already received it. They were discharged when they were asymptomatic, had negative stool cultures for V. cholerae on two samples, and had received a complete course of tetracycline.Definitions. A patient with cholera was defined as a volunteer who passed at least two diarrheal stools (grade 3, 4, or 5) totaling at least 200 g or who passed at least one diarrheal stool totaling 300 g within a 48-h period, associated with a stool culture positive for V. cholerae. A patient with a moderate case was one who passed 3 to 5 kg of diarrheal stool during the study, and one with a severe case was one who passed at least 5 kg during the study.
Laboratory procedures. The clinical screening laboratory tests were carried out in the clinical laboratories of the participating hospitals. The microbiology tests were carried out in the research laboratories of the respective investigators.
Stool cultures were obtained daily (up to two specimens per day) after the challenge to determine the duration of excretion of virulent V. cholerae and to measure the number of V. cholerae being excreted. A rectal swab specimen was obtained if no stool was passed. The fecal specimens were inoculated into alkaline peptone water for qualitative culture and diluted 1:10 in PBS for quantitative culture. The alkaline peptone water specimen was inoculated onto a thiosulfate-citrate-bile salts-sucrose (TCBS) plate after a 6-h incubation at 37°C. For quantitative cultures, 100 µl of stool diluted serially (10-fold dilutions) was spread on TCBS agar and incubated at 35°C and the numbers of colonies of V. cholerae were counted after an 18-h incubation. Suspected colonies were confirmed to be V. cholerae on the basis of results with oxidase reagent and agglutination with O1 antiserum. Blood samples (20 ml for serological testing and 35 ml for antibody-secreting cells assays) were obtained four times: before admission and on days 7, 10, and 14. Aliquots of serum were stored at20°C until assayed for cholera serology. To compare the serological results at two laboratories, aliquots from the first three groups from
both the Johns Hopkins University Vaccine Testing Unit (JHU-VTU) and
the CVD (120 serum samples) were sent to the National Institutes of
Health, where the sera were coded and sent back to the laboratories of
the JHU-VTU and the CVD, where they were tested for vibriocidal and
antitoxin antibodies. These coded sera were arranged so that the four
specimens from each volunteer were tested in adjacent wells, but the
order of the sera was randomized within each group of four samples.
Methods used at these laboratories have been described previously
(5, 11, 13). The methods used for vibriocidal antibody assays were similar between the two institutions, and for both the end
point was defined as the highest dilution in which there is no visible
growth. The antitoxin assay, however, is performed differently; the
JHU-VTU laboratory performs the assay with serial fivefold dilutions of
sera to reach an end point (defined as the extrapolated titer yielding
an optical density [OD] of 0.4), while the CVD laboratory determines
the OD with a single 1:50 dilution of serum. A positive response is
defined as an increase of 0.2 OD unit. Thus, the JHU-VTU laboratory
result is an end point titer whereas the CVD result is read as positive
or negative.
At the same time as the serum collections, whole blood was also
collected to determine the number of antibody-secreting cells (ASC) to
cholera toxin and V. cholerae lipopolysaccharide (LPS). This assay was carried out at the CVD (12). The results were expressed as the number of specific antibody-secreting
cells/106 peripheral blood mononuclear cells.
Comparison to past challenge studies. Records from past studies conducted at the CVD with freshly harvested V. cholerae N16961 were reviewed to compare the illnesses which occurred in the past studies with those seen in the present study, which used the same strain but with a frozen inoculum.
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RESULTS |
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Introduction
Materials & Methods
Results
Discussion
References
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Challenge dose of 105 CFU. Eight groups of volunteers (n = 5 per group) received a challenge dose of 105 CFU, including three groups at JHU-VTU, three at CVD, and two at Children's Hospital Medical Center, Cincinnati.
The clinical illnesses observed among all groups of volunteers were, in general, typical of cholera, as shown in Tables 1 and 2. Of the 40 volunteers, 34 (85%) developed diarrhea; the attack rates in the groups varied from 60 to 100%. Only one group among the eight had fewer than four illnesses. Of these illnesses, 16 (47%) were mild, 8 (24%) were moderate, and 10 (29%) were severe. As expected, the most prominent symptom of the illnesses was watery diarrhea, typical of cholera. Ten (25%) of the volunteers also vomited. The interval between ingesting the inoculum and the onset of symptoms averaged 28.4 h (standard deviation SD = 8.7 h; range = 11 to 48 h), and most symptoms started the day after challenge.
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Challenge dose of 104 CFU. At JHU-VTU, one group (n = 5) was challenged with 104 CFU to determine if this lower dose would be adequate. Four of five volunteers did develop diarrhea; however, the geometric mean stool output was 1,608 g, which is less than required for the model. Three of these volunteers also had transient fever (maximum temperature = 101.7°F), including one who did not have diarrhea. Stool cultures from each volunteer were positive.
Serological test results.
A total of 37 (92.5%) of 40 volunteers who received 105 CFU and 5 of 5 who received
104 CFU had significant rises in their vibriocidal and
antitoxin titers when prechallenge sera were compared with sera
collected on days 10 and 14 after challenge. The results of the serum
antibody assays on samples from the first 30 volunteers which were
assayed at both the JHU-VTU and the CVD under code are shown in Table 3. The volunteers included five groups
challenged with 105 CFU (n = 25) and one
group challenged with 104 CFU (n = 5). Of
40 volunteers, 37 (92.5%) developed a fourfold vibriocidal response
and a twofold immunoglobulin G (IgG) (and IgA at the JHU-VTU)
antitoxin response when the day 10 and day 14 sera were compared to the
prechallenge sera. The three volunteers who failed to develop
serological responses were ones who also had no diarrheal symptoms, but
three other asymptomatic volunteers did develop vibriocidal and
antitoxin responses. All symptomatic volunteers developed significant
vibriocidal and IgG (and IgA at the JHU-VTU) antitoxin responses. Peak
vibriocidal titers were seen on day 10, while peak antitoxin titers
were seen on day 14 (Table 3).
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study, involving eight similar groups from three vaccine-testing centers, has demonstrated that ingestion of an inoculum prepared directly from a frozen vial of V. cholerae N16961, with no further incubation, resulted in a consistent, acute, watery diarrheal illness typical of cholera. The incubation period and time course were similar to those in previous studies with freshly harvested N16961. A comparison of the results obtained in the present study (with frozen inoculum) with historical data from previous challenge studies performed at the CVD (with freshly harvested organisms) is shown in Table 5.
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The only unusual symptom with the model used in the present study was the self-limited fever beginning the day after challenge in more than 50% of the volunteers. Fevers have been seen in volunteers challenged with this strain previously, but the proportion of volunteers with fever in these groups was higher than observed previously. The cause of the fever is not known; however, it seems most likely that the fever is related directly to infection with the Vibrio strain itself rather than to an adventitious agent, since the inoculum was screened carefully for contaminants. A pyrogenic contaminant or fever due to endotoxin from killed Vibrio organisms is most unlikely since the inoculum itself was diluted by a factor of 50,000 before being given to the volunteers. Fevers also occurred in the group receiving a dose of 104 CFU (dilution factor of 500,000), suggesting that the causative agent of the fever is a viable organism.
Since the challenge organism started from a frozen state, it seemed possible that either the incubation period or the immune response would be delayed relative to those seen for challenge with freshly harvested organisms. In fact, there was no difference in the time course of either of these events. Technically, the use of the frozen challenge is easier and results in a consistent inoculum. Compared to a 2-days preparation when fresh organisms are used, the frozen inoculum can now be prepared, by a standard procedure, in about 30 min. More importantly, the inoculum given to volunteers is uniform and so the identical inoculum can be given at different times and at different centers.
Since the vibriocidal and antitoxin assays are key indicators of the immune response, it seemed important to compare the results in at least two laboratories. The titers, as reflected by the GMTs were approximately four to five times higher when measured at CVD than when measured at JHU-VTU, but there was good correlation between the rises in titer between the two laboratories and there was 100% agreement in determining which volunteers had a significant vibriocidal response. The IgG responses to antitoxin also agreed between the two laboratories, but the different methods used prohibited a comparison of the magnitude of the responses.
While this study has demonstrated that challenge with a frozen inoculum results in a consistent cholera illness, further studies are needed to investigate whether immunized volunteers will be protected against this standard challenge. If these studies are successful, this will greatly assist in the efficient evaluation of candidate cholera vaccines in the future. The results of the present study may also be relevant for other volunteer studies involving oral challenge with live bacteria, e.g., E. coli or shigellae.
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ACKNOWLEDGMENTS |
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This work was supported by the following NIH grants and contracts: NIAID contract N01-AI 45252; MO1-RR08084, General Clinical Research Centers Program; NCRR-NIH (Children's Hospital, Cincinnati); RR-00035 to the General Clinical Research Center (The Johns Hopkins Hospital, Baltimore, Maryland); and NIAID contract N01-AI 45251 (University of Maryland).
We appreciate the technical assistance of Joe Gomes, Yulvonnda Brown, Wendy Luther, Yu Lim, Lou Brunner, Jennifer Hawkins, and Jim Sherwood.
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FOOTNOTES |
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* Corresponding author. Mailing address: Johns Hopkins University Vaccine Testing Unit, 550 N. Broadway, Suite 1001, Baltimore, MD 21205. Phone: (410) 955-0053. Fax: (410) 614-9483. E-mail: dsack{at}jhsph.edu.
Editor: R. N. Moore
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Infect Immun, May 1998, p. 1968-1972, Vol. 66, No. 5
0019-9567/98/$04.00+0
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