CHAPTER 2: Testing mouse, macaque and clinical samples with ISI assay
2.2.1 Mouse samples
2.2.1.1 CS and TRAP
The first samples tested with the flow cytometry optimised ISI protocol were sera obtained from mice vaccinated with CS and TRAP expressing vaccines. Samples from three different studies were tested, where mice had been immunised with 107 or 108 iu AdCh63 followed at least 6 weeks later by 106 or 107 pfu of MVA. Serum samples were tested at a final concentration of 10, 5 or 2%.
Figure 2.1. BALB/c mice were immunised with 107 AdCh63 CS and 106 MVA CS 8 weeks apart and serum collected 4 weeks after MVA vaccination. Serum samples were tested at 2 and 10% serum dilution and Huh7 cells infected with P. falciparum CSP P. berghei sporozoites in the presence of serum samples (A: black dots) or naive sera (A: grey bars). Results are expressed as the % Infection blocked, taking infectivity in the absence of serum as a reference. The line at 30% (A) indicates the threshold level for positive responses. Correlation between % Infection blocked and ELISA-CS is shown at 2% (B1) and 10% (B2) serum dilution. Data (B1, B2) was analysed with: nonparametric Spearman correlation test (r); linear regression (R2; p-value from F test: is the slope significantly different than zero?).
Chimeric P. berghei parasites expressing the P. falciparum protein (CSP or TRAP) were mixed with each serum sample and added to hepatoma cell line Huh7, which had been plated approximately 8 hours prior to infection. Tests were performed in duplicate or triplicate. Cultures were incubated at 37ºC for 24-28 hours prior to sample acquisition on the flow cytometer to determine the percentage of infected cells. Results are shown in the three figures: Figure 2.1 (BALB/c - CS), Figure 2.2 (C57BL/6 and CD1 -TRAP) and Figure 2.3 (BALB/c - CS and TRAP). The percentage of infection blocked (percentage of infection avoided in the presence of serum) has been plotted for each serum dilution used. CS samples showed a positive concentration-dependent blocking response [Figure 2.1.A, Figure 2.3.A]. BALB/c mice vaccinated against CS with a higher dose of ChAd63 and MVA [Figure 2.3.A] showed higher blocking responses (82% Infection blocked, with 2% serum dilution), than those that received the lower doses of vaccine [Figure 2.1.A] (50% Infection blocked, with 2% serum dilution).
Figure 2.2. C57BL/6 and CD1 mice, CS samples. Mice were immunised with 108 AdCh63 and 106 MVA, both vectors encoding ME-TRAP and administered 7 (C57BL/6) or 8 (CD1) weeks apart. Serum was collected one week after MVA vaccination. Each serum sample was tested in triplicates and at two concentrations, 2 and 10% (final serum volume). P. falciparum TRAP P. berghei sporozoites were employed to infect Huh7 cells. The % Infection blocked has been plotted in A), where each dot (black-C57BL/6, blue-CD1) represents an individual mouse. The line at 30% indicates the threshold above which blocking is considered positive. B) The graph shows the correlation between % Infection blocked and LIPS data, data was analysed with a nonparametric Spearman correlation test (r); linear regression (R2; p-value from F test: is the slope significantly different than zero?).
C57BL/6 and CD1 mouse strains were tested for blocking capacity of antibodies after ME-TRAP vaccination. It was possible to detect positive responses at 10% dilution for CD1 mice, but not for C57BL/6 [Figure 2.2.A].
Figure 2.3 shows the blocking obtained with serum from BALB/c mice vaccinated against CS [Figure 2.3.A] or TRAP [Figure 2.3.B]. Both antigens elicited strong blocking responses, higher in the case of CS (at 2% serum dilution: CS generated 82% of blocking and TRAP, 69%).
Figure 2.3. BALB/c mice were immunised with 108 iu AdCh63 followed 8 weeks later by 107 pfu MVA, expressing either CS (A) or TRAP (B). Serum samples were taken 8 to 14 days after the last vaccination and each sample tested in duplicates and at three dilutions: 2, 5 and 10% serum. Transgenic P. berghei sporozoites (PfCS Pb in A and PfTRAP Pb in B) and serum were added to Huh7 cells. In both graphs, each dot represents an individual mouse and the blocking effect (non-specific background effect) of naive serum is shown as bars.
To determine whether the level of infection blocked is due to the amount of antigen specific antibodies, the level of % Infection blocked was compared to LIPS (Luminescence ImmunoPrecipitation System) or ELISA values. While ISI assay is a measurement of antibody functionality, LIPS and ELISA give information about antibody concentration. Comparing ISI assays with other studies may give an idea of the relative effect of antibody concentration on the blocking of sporozoite infection and allow appreciation of the different information obtained by each assay. In Figure 2.1, %Infection blocked at 2% (B1) and 10% (B2) serum dilution was plotted against ELISA values. Although samples with lower ELISA titres tend to have a smaller blocking effect, there was not a strong correlation between the two values, most likely due to the small number of samples tested. In Figure 2.2, the regression line for %Infection blocked against LIPS values had a slope significantly different than zero (linear regression), but the correlation between the two parameters was not statistically
significant (Spearman test). Unfortunately, LIPS / ELISA data was not available for samples used in Figure 2.3 to enable a correlation analysis on a larger data set.
The experiments described confirmed that, using the optimised flow cytometry based ISI assay, it is possible to detect a reduction on the rate of infection with the addition of post-vaccination serum samples. These first results emphasise the importance of the serum dilution employed in the ISI assay, the effect of the vaccination dose on the antibody responses, the influence of using a particular mouse strain on the antibody responses, and support (with the trends found between LIPS and ELISA assays and % Infection blocked) the search for possible correlations between ISI and other antibody assays in future studies.