Titration of IgG to rF1 plus rV in serum.
At screening, all individuals were below the lower limit of quantification except for one subject in the 20-μg dose group who was found to have an anti-rV titer prior to immunization, speculated to be due to a previous exposure to Yersinia enterocolitica. The immune response data for this individual has been omitted from the overall analysis. Sera from all individuals who received the placebo were below the lower limit of quantification for assay of IgG to rV and rF1, at every time point tested.
The immunized subjects produced specific antibodies against both subunits (rV and rF1) of the recombinant plague vaccine (rYP002). For each time point tested there was large variation of quantifiable antibody responses between individuals within each dose level group. Therefore, group mean ± standard error of the mean values and ranges have been derived and are presented in Tables and . Antibodies to rV were produced within two weeks of the first dose at day 1, with greater levels observed after the booster dose at day 21 (Table ). One individual in the 5-μg rYP002 group failed to respond with a titer to rV. Across the dose range studied, at least 50% of the maximum mean anti-rV titer was retained for up to 3 months following the first dose. Antibodies to rF1 were also generated within two weeks of the first dose at day 1, with greater levels observed after the booster dose at day 21 (Table ). Across the dose range studied, at least 50% of the maximum mean anti-rF1 titer was retained for up to 3 months following the first dose, although lack of sample numbers made this difficult to assess for the 20-μg group (Table ).
Anti-V serum IgG concentrationsa
Anti-F1 serum IgG concentrationsa
Relationship between dose level of vaccine and serum IgG response.
To study a possible dose-response effect, an area-under-the curve (AUC) estimate of IgG serum response to rF1 and to rV was calculated for each subject, during the period days 21 to 70, for each of the vaccine dose groups. The variation in response to both antigens between individuals within each dose group has been studied by plotting the AUC value for combined IgG to rF1 plus rV in the period days 21 to 70 (Fig. ). A one-way ANOVA was conducted to establish the effect of rYP002 dose level on the serum IgG response over time. The effect of vaccine dose on the combined IgG response to rF1 and rV was statistically significant (f = 3.26, P < 0.05); however the influence of dose on the IgG response to F1 (f = 4.22, P = 0.02) was greater than that on the IgG response to rV (f = 0.93, P = 0.45). The ANOVA indicated that the mean IgG values for rF1 alone, or for rF1 plus rV in combination, were significantly lower for the 5-μg dose group than the means for the other three dose groups. There was no significant effect of vaccine dose on the IgG response to rV.
Interdependence of dose level and IgG response to rF1 and rV in individuals across groups. Combined AUC for individuals versus vaccine dose level.
Relationship between serum IgG response and time.
To study the time trends in specific IgG response for each subject, regression analysis of the serum IgG values with time (days 28 to 91) has been carried out. A one-way ANOVA of the resulting slopes as a function of vaccine dose level showed no significant treatment effect for either rF1 (f = 1.27, P = 0.32) or rV (f = 0.92, P = 0.45). The average rate of decline in IgG titer was estimated at 2% per week for rF1 and rV.
Isotyping of serum.
For all subclasses assayed, titers generally developed after the second dose of vaccine and peaked at approximately days 28 to 35. The dominant response to rV was IgG1 followed by IgG2, although the 20-μg dose group showed a strong IgG3 response. By comparison, low levels of IgG4 were generally detected to rV, although one individual in each of the 20-μg and 40-μg groups had a prevaccination IgG4 titer (assumed nonspecific) and developed very high levels of IgG4. The data from these individuals have been omitted from the overall serological analysis. No IgG4 to rF1 was detected at any dose level. The dominant subclass to rF1 was IgG1, closely followed by IgG2 and IgG3.
To study a possible dose-response effect, the IgG1 values for each subject in the period days 21 to 91 were combined to form an AUC for IgG1 to rF1 and rV, allowing comparison between groups. A one-way ANOVA of these AUC values as a function of vaccine dose level showed no significant treatment effect of either rF1 (f = 1.82, P = 0.19) or rV (f = 0.87, P = 0.48).
Serum samples from individuals in each of the dose groups in the period days 21 to 70 of the immunization schedule were also assayed for their ability to compete with a constant concentration of the murine MAb 7.3 for binding to rV on a solid phase. The human serum displaced the mouse monoclonal antibody, to produce a titration curve for loss of binding of the mouse antibody, with increased concentration of human serum. The percentage inhibition of binding of MAb 7.3 to rV in the presence of the test and reference serum (where each were at a 1:10 dilution) was calculated for each individual at each time point and a ratio of test:reference serum derived.
Effect of dose level on the development of competing antibody.
When the development of an antibody titer which competed with MAb 7.3 for binding to rV was monitored by competitive ELISA with time, the majority of individuals in the 10-μg, 20-μg, and 40-μg rYP002 groups had competitive antibody by day 28, whereas the majority of individuals in the 5 μg group (5/6) had competitive antibody by day 70.
Effect of time on development of competing antibody.
When the total number of individuals across the immunization groups with competitive antibody to rV was monitored using the competitive ELISA assay, the following proportions had developed antibody which competed with MAb 7.3 for binding to rV: 12/20 at day 21, 18/20 at day 28, and 14/20 at day 70. A one-way ANOVA of IgG titer to rV (expressed as an AUC for rV over days 28 to 91) as a function of vaccine dose showed no significant effect of treatment (f = 0.92, P = 0.45). However, Pearson correlation of log10 IgG to rV at days 21, 28, and 70 for all individuals across all rYP002 dose groups, against the ratio of test:reference serum determined by competitive ELISA, showed a significant correlation at days 21 (r = 0.72, P < 0.001) and 28 (r = 0.82, P = <0.001) but not at day 70 (r = 0.43, P = 0.06) when serum IgG titers were declining.
The relationship between serum IgG to rV and the test:reference serum ratio for all individuals across the rYP002 dose groups at day 28 is shown (Fig. ). The one individual who had a preexisting IgG titer to rV as well as the two individuals with a preexisting IgG4 titer to rV were not included in the regression analysis, but their serum samples were assayed by competitive ELISA. The individual from the 20-μg rYP002 group with preexisting IgG titer was positive by competitive ELISA at days 8, 21, 28, and 70 (data not shown). This was not the case for the other two individuals (one from each of the 20-μg and 40-μg rYP002 groups) who were negative by competitive ELISA at days 8 and 21 but positive at days 28 and 70.
Correlation of IgG to rV with test:reference serum ratio determined by competitive ELISA for day 28 serum samples. The ratios are plotted against individual serum IgG titers to rV.
The mean AUC21-70 for IgG to rV for all individuals with a titer of competing antibody by competitive ELISA and the mean AUC for IgG to rV for all individuals without detectable competing antibody, were compared using a Student's t test. There was a significant difference between the means at day 21 (t = 3.08, P = <0.01) but not at days 28 or 70.
Estimation of the half-life of the reference serum in the mouse.
The t1/2 of the reference serum, which comprised a polyclonal macaque IgG specific for rF1 plus rV, in the mouse was estimated to be 8 days for IgG to rV and six days for IgG to rF1, with a decline to undetectable levels by 10 days (data not shown). The duration of the passive transfer assay was therefore limited to 10 days. As for the test sera, the dominant isotype in the reference serum was IgG1.
Establishment of passive transfer assay.
Pooled test sera from each of the vaccine dose groups on day 35, conferred some degree of protective immunity on recipient mice for the first 7 days postchallenge. The protection conferred was dose-related, so that it declined with dilution of the serum. Mice receiving human serum from the placebo groups did not survive and neither did untreated control mice. The assay was terminated at day 10, at which time pooled serum from the 40-μg rYP002 group was outperforming sera from the other dose groups (data not shown).
The protection provided by pooled test sera, collected from each of the rYP002 dose groups, was compared with that provided by the reference serum, either undiluted or diluted across the same range (75% to 25% in PBS). The data for day 35 serum samples from the 5 μg rYP002, 10 μg rYP002, 20 μg rYP002, and 40 μg rYP002 groups are shown in Fig. . Although sera from all the groups except the 5 μg rYP002 group conferred some partial protection at this time point, only sera from the 40 μg rYP002 group conferred protection equivalent to the reference serum (Fig. ).
FIG. 3. Survival of mice passively immunized with day 35 serum pooled from the 5-, (a), 10-, (b), 20-, (c), and 40-μg (d) dose groups prior to challenge. Values are survival rates postchallenge for up to 10 days. Negative control mice received human placebo (more ...) Determination of transferable protective immunity for individual serum samples.
The effect of time and dose level on the development of transferable protective immunity was studied by repeating the passive transfer experiments with individual (rather than pooled) sera collected from the 20 and 40 μg rYP002 dose groups at days 21, 28, and 70. Serum samples from all individuals receiving the placebo were tested and at each time point were negative in the passive transfer assay. At day 21, serum from only 4/8 vaccinated individuals tested was scored positive by passive transfer with a test:reference ratio ≥1.0; at day 28 3/8 sera from vaccinees scored positive. Passive transfer of individual sera from the 10 μg rYP002 dose group was tested only at day 70, at which time only 2/6 individuals were scored positive. At this time point, 3/5 individuals tested in the 40 μg rYP002 group were also positive by passive transfer bringing the overall total to 5/14 sera tested scoring positive by passive transfer at day 70.
Sera from the three individuals who were excluded from the analysis due to an antibody titer to rV at screening were also tested by passive transfer, in an attempt to determine whether the preexisting titer was protective. When tested at the day 21 and 28 time points, none of these sera were scored positive in the passive transfer assay. By day 70, only one of the three sera was scored positive, indicating that the preexisting IgG4 titer to rV in this individual from the 20 μg rYP002 group did not confer any cross-protection as determined by this assay.
Correlation between total IgG to rF1 plus rV and transferable protective immunity.
For all individuals across the dose groups, the specific log10 values for IgG to rF1 and to rV have been determined and a regression analysis for these values against the ratio of test:reference derived from the passive transfer assay at days 21 to 70 has been conducted. Significant correlations were obtained at day 21 for IgG to rF1 and rV (r2 = 98.6%; P < 0.001). At day 28, IgG to rV only was significantly related to the test:reference ratio (r2 = 76.8%; P < 0.03), whereas at day 70, only the IgG to rF1 was significantly related to the ratio, but the degree of correlation was weak (r = 55.7%, P < 0.01).
The difference between the mean combined AUC21-70 for IgG to rF1 plus rV for all individuals with a positive score by passive transfer and the mean combined AUC for IgG to rF1 plus rV for all individuals without a positive score by passive transfer, was compared by Student's t test but was not significant at any time point.
Prior to vaccination, leukocyte counts in peripheral blood ranged between 3.99 × 109 and 1.32 × 1010 cells/ml and for most subjects were within the accepted normal range. There was wide variation between individuals and no significant effect of vaccination could be detected.
In contrast, lymphocyte counts which comprised 22.1 to 44.6% of leukocytes, were within the normal range for all subjects prior to immunization, and fluctuated within the normal range until immediately after the second dose was administered, when they decreased. This decrease was attributed to a loss of cells to the draining lymph nodes (9
) and also occurred for the placebo group, possibly in response to the injection of alhydrogel as a mild immunostimulant. Normal levels of lymphocytes, as a percentage of leukocytes in the peripheral blood, were restored from day 35.
Flow cytometric analysis.
The response to immunization involves activation at the cellular level. Flow cytometric analysis was carried out to investigate whether immunization resulted in changes in markers of cellular activation or gross changes in cell counts and whether either of these represents an immune correlate. However, large intra- and interindividual variation existed in all treatment groups including placebo, and thus the median was used to describe group tendencies.
Within the lymphocyte population, changes in the number and percentage of CD 19+ B cells, CD3+CD4+ T helper cells and CD3+CD8+ cytotoxic T cells in peripheral blood were monitored at several time-points following immunization in comparison with preimmunization levels. Large natural interindividual variation existed prior to immunization, in all lymphocyte parameters investigated. No significant differences were observed in the number or percentages of B-cell and T-cell populations between placebo and any dose group at any time-point following immunization and in comparison with preimmunization levels.
The expression of the memory and activation markers CD45RO, CD25, and HLA-DR was investigated on CD4+ and CD8+ lymphocytes. High expression of CD45RO was determined to examine memory lymphocyte populations. CD25 and HLA-DR were examined on CD45ROhi lymphocytes and CD45RO negative cells. Again, large variation existed between individuals and no significant differences were detected in the expression of activation or memory markers between placebo and any dose group. No significant differences were found in expression of activation or memory markers between baseline and any time-point postimmunization.