Kinetics of the vibriocidal antibody response to live oral cholera vaccines

Kinetics of the vibriocidal antibody response to live oral cholera vaccines Steven S. Wasserman *~, Genevieve A. Losonsky t, Fernando Noriega t, Carol O. Tacket*, Elizabeth Castafieda; and Myron M. Levine *+ The best correlate o f protection aoainst cholera is the level o f serum vibriocidal antibodies, which are primarily directed aoainst the 0 antioen o1 Vibrio c h o l e r a e O1 and lyse V. c h o l e r a e in the presence of complement. We established the timin9 o[peak vibriocidal antibody response usin9 sera from safety/immunogenicity studies o f live oral cholera vaccines CVD 103-HgR, CVD 103-HoR2 and CVD 110 amon(j immunolooically naive North Americans and Colombians. The serum reciprocal vibriocidal antibody titre was consistently hioher 10 days postimmunization than on either day 7 or day 14. This study suooests that recent phase 2 studies of CVD 103-HoR may have underestimated the peak vibriocidal titre by collecting serum on days 7-8 rather than on day 10; future studies of live oral cholera vaccines should take these results into account to obtain the best measurement of peak immunolooical responses. Because of the rapid drop in vibriocidal antibody titres at about 2 weeks aj?er immunization, care must be exercised in comparing immunooenicity of d(fferent vaccine candidates, formulations, dosaoe levels and immunization schedules. Keywords: Vihrio cholerae; immunological response; kinetics

Modern vaccines to prevent cholera, whether inactivated antigens or live strains, rely on oral immunization to stimulate optimally the intestinal immune responses that are critical in mediating protection. Nevertheless, the best correlate of protection is the level of serum vibriocidal antibodies, which are primarily directed against the O antigen of Vibrio cholera O1 and which lyse V. cholerae in the presence of complement 1 6. For many years, serum vibriocidal antibody has been the standard measurement by which the immunogenicity of different vaccine candidates, formulations, dosage levels and immunization schedules has been compared 7 13 Few data have been published describing the kinetics of the serum vibriocidal antibody response following either natural infection or administration of oral cholera vaccines. Among cholera patients in Calcutta, the peak vibriocidal antibody titre occurred on day 11 (bracketed by specimens taken on days 8 and 15) 14. In North American adults who ingested wild-type V. cholerae in clinical studies, we previously reported that day 10 vibriocidal titres were higher than either day 21 or day 28 titres 15. In subjects immunized parenterally with a heat-killed streptomycin-resistant whole-cell vaccine, the vibriocidal titre was higher at 7 than at 28 days 16. *Division of Geographic Medicine, Department of Medicine, and tDivision of Infectious Diseases and Tropical Pediatrics, Department of Pediatrics, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA. ++Grupo de Microbiologia, Instituto Nacional de Salud, Bogot& Colombia. ~To whom correspondence should be addressed. (Received 8 September 1993; revised 2 December 1993; accepted 2 December 1993) 0264-410x/94/11/100o~)4 ,~' 1994Butterworth-HeinemannLtd 1000 Vaccine 1994 Volume 12 Number 11

A wealth of data is available on the vibriocidal response to live oral recombinant vaccine CVD 103-HgR. In current phase 2 and phase 3 trials, this vaccine is administered as a single dose of 5 x l0 s colony-forming units (c.f.u.) in developed countries, and as a single dose of 5 x 10 9 c.f.u, in endemic areas 12,17,18. In this report, we establish the timing of peak vibriocidal antibody response using phase 1 and phase 2 studies of live oral cholera vaccine CVD 103-HgR and phase 1 studies of two recently tested candidate vaccines, CVD 103-HgR2 and CVD 110 a9'2° METHODS Vaccines

Three attenuated recombinant V. cholerae O1 vaccine candidate strains were used in these studies. CVD 103-HgR is a genetically engineered attenuated derivative of classical Inaba V. cholerae O1 strain 569B harbouring a deletion of the A subunit of the cholera toxin gene, with a mercury-resistance gene inserted into the h/yA locus 21. CVD 103-HgR2 is identical to CVD 103-HgR, except that it underwent fewer in vitro passages during its construction 19. CVD 110 is of similar design to CVD 103-HgR, except that it is derived from E1 Tor Ogawa V. cholerae strain E7946, and it lacks a 'virulence cassette' segment of the chromosome that encodes two accessory cholera toxins: accessory cholera enterotoxin (ace) and zonula occludens toxin (zOt) 2°'2z'23. For all three vaccines, the formulation consisted of two sachets, one containing 5 × 108 c.f.u. (or 5 x 109 c.f.u, for one Colombian study) lyophilized vaccine with aspartame,

Response to live oral cholera vaccines: S.S. Wasserman et al.

and the other containing buffer (2.5 g NaHCO3 and 1.65 g ascorbic acid). A sachet of vaccine and a sachet of buffer were mixed in a cup containing 100 ml water and the suspension was given orally. Only a single oral dose was administered. Several different vaccine production lots of CVD 103-HgR are represented in these studies, but single vaccine production lots of CVD 103-HgR2 and CVD 1i0 were used. Volunteer studies Two sets of studies are included in this report. 'Preliminary studies' comprise a series of 13 phase 1 and phase 2 clinical trials of CVD 103-HgR that were conducted between 1987 and 1991 among North American adult volunteers 12'13'24'z5 (also ten additional unpublished studies). Serum specimens were obtained at baseline and, after vaccination, as follows: day 7 only (one study, n = 18); days 7 and 10 (one study, n = 14); days 7 and 14 (three studies, n = 15, 14 and 14); days 7, 14, 21 and 28 (one study, n= 14); days 7 and 21 (one study, n = 15); days 21 and 28 (one study, n=96); and days 10 and 28 (five studies, n=20, 12, 11, 10 and 7). In only one of these studies were specimens obtained on both days 7 and 10. 'Kinetics studies' refer to three vaccine studies whose serum specimen collection schedules were specifically designed, after analysis of the preliminary studies, to investigate the kinetics of the vibriocidal response. Kinetics study no. 1 included 29 Colombian subjects, aged 19 to 38 (mean 27.3) years, who received 5 x 109 c.f.u, of CVD 103-HgR. Serum specimens were obtained on days 0, 7, 10, 14 and 28. Of the 29 study participants, 27 had a complete immunological series. Kinetics study no. 2 consisted of ten North American subjects, aged 18 to 34 (mean 25.8) years, who received 5 × 10s c.f.u, of CVD 103-HgR2. Kinetics study no. 3 consisted of ten North American subjects, aged 21 to 35 (mean 25.5) years, who received 5 × l0 s c.f.u, of CVD 110. In the latter two studies, serum specimens were obtained on days 0, 7, 10, 21 and 28, from all study participants. Immunological methods Serum Inaba (studies with CVD 103-HgR and CVD 103-HgR2) or Ogawa (study with CVD 110) vibriocidal antibody was measured by the microtitre method and expressed as reciprocal titres 5. For each study, immunological assays for all time points were run at the same time in a blinded fashion using coded specimens. Vibriocidal assays on specimens from the Colombian study which were performed independently at the University of Maryland (results reported here) and at the Instituto Nacional de Salud, Bogotfi (results not reported) were strongly correlated (log-transformed data, Pearson's r >~0.88, p < 0.001).

For the individual kinetics studies, serum reciprocal vibriocidal antibody titres were transformed to natural logarithms to approximate normality better. For each study, a randomized blocks analysis of variance (ANOVA) was used to detect variation in GMTs among days postimmunization. If the overall F test indicated significant heterogeneity among sampling dates, the data were further analysed by Sid~k multiple comparisons as above. Only subjects with complete immunological series were included in these analyses. Statistical significance was assessed, for all statistical tests, as a probability ~<0.05. RESULTS Preliminary studies of the Inaba vibriocidai responses to CVD 103-HgR among North American volunteers The geometric mean vibriocidal antibody titre rose to approximately 2000 between days 10 and 14, and declined slowly thereafter (Table 1). Day 28 levels were similar to day 7 GMTs. Vibriocidal antibody titres varied significantly among sampling dates (analysis of variance, p=0.003). Antibody titres were significantly higher on days 10 and 14 than on day 7 (multiple comparisons tests, p < 0.05). Kinetics study no. 1 Inaba vibriocidal antibody GMTs in Colombian volunteers immunized with CVD 103-HgR peaked at 1640 on day 10, and dropped through to day 28 (Table 2). The day 7 GMT among these subjects was 85% of the overall GMT among preliminary studies in North Americans; thereafter, GMTs of Colombians declined more rapidly, falling to between 50 and 60% of the GMTs of North Americans on days 14 and 28. There was significant heterogeneity among GMTs for days 7 to 28 (analysis of variance, p<0.001). Multiple comparisons tests indicated two homogeneous subsets of sampling dates: days 7 and 28 constituted a low GMT group, and days 10 and 14 formed a higher GMT group. A secondary analysis of the difference between days 10 and 14 indicated that the GMT on day 10 was greater than on day 14 (paired t test, p=0.005). Kinetics study no. 2 Inaba vibriocidal antibody GMTs in North American volunteers immunized with CVD 103-HgR2 peaked at Table 1 Serum vibriocidal antibody responses of North American volunteers to a single 5 x 108 cof.u, dose of live oral cholera vaccine CVD 103-HgR. Results of 13 studies (n = 260 subjects) in which immunological measurements were made at a mean of 2.2 (range 1-4) time points postimmunization Days postimmunization with CVD 103-HgR

Statistical methods For the preliminary studies, the individual observations were the GMTs on each sampling date for each study. After transformation to natural logarithms, study GMTs for days 7 and 10 showed strong right-skewness; accordingly, prior to their analysis, GMTs were transformed to ranks, and analysed by single classification analysis of variance 26. The significant overall F test was followed by Sidfik multiple comparisons, to define homogeneous subsets of sampling dates zT.

Measure

0

Geometric mean 23 of GMTs a Loge s.d. of GMTs 0.3 Number of studies 13 Number of 260 volunteers Minimum GMT 16 Maximum GMT 46

7 565 0.6 7 104 320 1902

10

14

21

2193

1857

1065

0.6 6 74 1040 5881

0.4 4 57 1222 2970

0.3 3 125 844 1345

28 753 0.5 7 170 437 1810

aUnweighted geometric mean of study GMTs

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Response to live oral cholera vaccines: S.S. Wasserman et al. Table 2 Serum vibriocidal antibody responses in three kinetics studies: Colombian medical students who received a single 5 x 109 c.f.u. dose of live oral cholera vaccine CVD 103-HgR (27 with a complete immunological series); North American volunteers who received a single 5 x 108 c.f.u, dose of live oral cholera vaccine CVD 103-HgR2 (n= 10, Inaba vibriocidal); and North American volunteers who received a single 5 x 108 c.f.u, dose of live oral cholera vaccine CVD 110 (n= 10, Ogawa vibriocidal) Days postimmunization Measure

0

7

10

CVD 103-HgR (Colombians, Inaba vibriocidal) GMT 20 480 1 640 Ln s.d. 0.8 1.7 1.9

21 a

28

1 103 1.8

369 1.5

CVD 103-HgR2 (North Americans, Inaba vibriocidal) GMT 21.4 685.9 4 159 2 744 Ln s.d. 1.3 2.1 1.7 1.7

1 576 1.9

CVD 110 (North Americans, Ogawa vibriocidal) GMT 20 5487 19108 Ln s.d. 0.7 0.9 0.2

8914 0.9

14482 0.7

aFor Colombians, these specimens were obtained on day 14

4159 on day 10 (Table2). There was no significant difference among dates postvaccination (analysis of variance, p=0.19). However, a secondary analysis indicated that the GMT on day 10 was greater than on day 7 (paired t test, p = 0.0006). Kinetics study no. 3

Ogawa vibriocidal antibody GMTs in Colombian volunteers immunized with CVD 110 peaked at 19 108 on day 10 (Table 2). There was significant heterogeneity among GMTs for days 7 to 28 (analysis of variance, p=0.002), due to two homogeneous subsets of dates postvaccination: days 10, 21 and 28 represented a higher GMT group, which overlapped with days 7 and 28, constituting a lower GMT group. A secondary analysis of the difference between days 7 and 10 indicated that the GMT on day 10 was greater than on day 7 (paired t test, p=0.001). DISCUSSION In summary, these studies of three different live oral candidate vaccines provide evidence that peak vibriocidal antibody titres, as measured in this laboratory, occur approximately 10 days after immunization with a single dose of CVD 103-HgR. Among the kinetics studies, the GMT on day 10 is between three and six times the magnitude of the GMT on day 7, in agreement with the composite ratio obtained from the 13 preliminary studies. These results are not surprising, as the vibriocidal antibody response comprises mainly IgM antibodies, directed against the O antigenic moiety of the lipopolysaccharide. IgM responses following primary exposure occur early, starting within a few days and peaking approximately 1 week after exposure 28. It is unclear whether the kinetics of the vibriocidal response to live oral vaccines are similar to responses following wild-type challenge. Our observed peak approximately 10 days after exposure compares well with the peak of ! 1 days previously reported 14. This similarity in immunological kinetics further suggests that the magnitude of the immunological response may also be

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comparable, and that it roughly predicts the protective efficacy of these live oral vaccines. Among North Americans, the peak GMT for 155 recipients of CVD 103-HgR was 1699, approximately 57% of the peak GMT (2958) among 24 recipients of V. cholerae classical Inaba strain 569B 1s. Recent and ongoing phase 2 studies of CVD 103-HgR among adults and children in Indonesia, Peru, Chile and Costa Rica have used day 7 or day 8 titres as an estimate of peak GMT, the date of serum collection representing a decision based on logistics rather than immunological kinetics ls'29-~1 (also D. Pizarro et al., unpublished data; R. Lagos et al., unpublished data). Based on the current analyses, the peak titres reported from these phase 2 studies will underestimate the actual (unmeasured) peak GMT attained. The populations evaluated in both the preliminary and kinetics studies reported here were immunologically naive with respect to V. cholerae O1 infection. The kinetics of the vibriocidal response to either infection with V. cholerae or vaccination with a live oral cholera vaccine may differ among people who have had previous clinical or subclinical immunological experience with this pathogen or with antigens that crossreact in this assay. However, the major target populations for vaccination (travellers from developed countries to endemic areas, children living in endemic areas, inhabitants of regions experiencing new cholera outbreaks) tend to be immunologically naive, suggesting that the patterns described here will be applicable to immunization programmes or for immunization of travellers. Ultimately, the actual dates of specimen collection in phase 2 and phase 3 trials represent a trade-off between measuring the peak immunological response to vaccination and convenience in scheduling follow-up visits for study participants and staff. For example, in a recent outpatient phase 2 evaluation of CVD 103-HgR among college students, a follow-up visit 1 week after immunization was practical and helpful in maintaining compliance 12. In inpatient studies, however, this constraint is not a problem ~1'~5. Because of these trade-offs and the rapid drop in vibriocidal antibody titres 2 weeks after immunization, care must be exercised in comparing the immunogenicity of different vaccine candidates, formulations, dosage levels and immunization schedules. ACKNOWLEDGEMENTS The clinical studies described here were carried out under the sponsorship of NIH Contracts NO 1-AI-62528 and NO1-AI-15096, Swiss Serum and Vaccine Institute and Instituto Nacional de Salud, Colombia. REFERENCES 1

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