To confirm if mice developed serological reactivity against TIGR4 strain, western blots were performed using mouse sera as a source of primary antibodies against TIGR4 cell lysate (Fig 6.7).
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Figure 6.7. Western blot using pre-immune and post-immune sera from vaccinated mice as source of primary antibodies against TIGR4 cell lysate. 10 µl of ladder and sample was loaded in lanes. Molecular weights of major bands are indicated with the help of arrows. Serum was used in 1/100 while secondary anti-mouse antibodies were used at 1:1000 dilution.
(A) Pre-immune Sera. Lane 1: Protein ladder.
Lane 2: Positive control using TIGR4 lysate in a previously immunized mouse. Lane 3: Protein ladder
Lane 4: Pre-immunization serum from Mouse M1 Lane 5: Protein ladder
Lane 6: Pre-immunization serum from Mouse M2 Lane 7: Protein ladder
Lane 8: Pre-immunization serum from Mouse M3 Lane 9: Protein ladder
Lane 10: Pre-immunization serum from Mouse M4 Lane 11: Protein ladder
Figure 6.7. Western blot using pre-immune and post-immune sera from vaccinated mice as source of primary antibodies against TIGR4 cell lysate. 10 µl of ladder and sample was loaded in lanes. Molecular weights of major bands are indicated with the help of arrows. Serum was used in 1/100 while secondary anti-mouse antibodies were used at 1:1000 dilution.
(B) Post immune sera. Lane 1: Protein ladder
Lane 2: Post-immunization serum from Mouse M1 Lane 3: Protein ladder
Lane 4: Post immunization serum Mouse M2 Lane 5: Protein ladder
Lane 6: Post immunization serum Mouse M3 Lane 7: Protein ladder
Lane 8: Post immunization serum Mouse M4 Lane 9: Protein ladder
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Western blot showed that antibodies had developed in mice after vaccination against TIGR4 proteins and serum after vaccination of mice was reactive against them.
6.4.1.1 Mouse serum also reacts with the type 4 capsular antigens:
Quellung reactions are an important tool to show if anticapsular antibodies had developed in vaccinated mice as they are considered important defence mechanism and help in providing protection against pneumococcal infection. Quellung reactions performed using sera from vaccinated mice were positive against TIGR4 as well as 403.
Further experiments were performed to see effects of vaccine administration through IN route, effect of vaccination on challenge with lesser number of bacteria and protection against different serotypes
6.4.2 Intraperitoneal immunization and low dose challenge:
10 MF1 mice were divided into two groups, and were given PBS or vaccinated 3x with strain 403 as described previously in Section 1.3.
Mice were challenged intraperitoneally with 5 x 103 cfu/ 200µl and were bled 6 hourly and monitored for development of clinical symptoms during the course of the experiment.
As compared to previous experiment, median survival time of both groups increased by thirty hrs. There was not statistical difference between the two groups. There was one survivor in the vaccinated group which was able to clear A
C E
the infection after reaching a relatively high level of bacteraemia but two of the vaccinated mice had to be sacrificed earlier in experiment (Fig 6.8).
Figure 6.8. Survival curve of mice challenged with 5 x 103 cfu/ 200 μl TIGR4 IP in groups 3x vaccinated IP with 5 x 106 cfu of strain 403/ 200μl PBS and 200 μl PBS only. Mice vaccinated with 403 strains survived longer than unvaccinated mice and 20% survived till the end of experiment though survival analysis did not show any statistically significant difference in survival time between the two groups. Median survival time for vaccinated mice was 72 hrs as compared to 48 hrs for control mice (GraphPad Prism 4.0, USA). All data plotted as percentage survival as a staircase line with points for all observations against hrs post infection. Five mice used in each group.
There was no statistical difference in the bacteraemia in the two groups. In vaccinated mice, after a very high level of bacterial load of 108 cfu/ml at 24
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hour time point, the count dropped back to 106 cfu/ml at 30 hrs though only 20% survived beyond 72 hrs (Fig 6.9).
Figure 6.9. Comparison of the bacterial load in mice challenged with 5 x103 cfu/ 200 μl TIGR4 IP in groups 3x vaccinated IP with 5 x 106 cfu of strain 403/ 200μl PBS and 200 μl PBS only. Graph shows no notable difference in bacterial load in two groups. Dotted line represents limit for detection.
Figure 6.10. Comparison of bacterial load during 6-36 hrs post-challenge with 5x103 cfu/ 200 μl TIGR4 IP in mice vaccinated 3x IP with 5 x 106 cfu of strain 403/ 200μl PBS and control
group given PBS only. Groups were compared with Mann-Whitney test (GraphPad Prism 4.0, USA). Circles mark individual mice. Horizontal dotted line represents the limit for detection. Horizontal bar represents the median. (A) Bacteraemia count at 6 hpi. (B) Bacteraemia count at 12 hpi. (C) Bacteraemia count at 18 hpi. (D) Bacteraemia count at 24 hpi.
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Figure 6.10. Comparison of bacterial load during 6-36 hrs post-challenge with 5x103 cfu/ 200 μl TIGR4 IP in mice vaccinated 3x IP with 5 x 106 cfu of strain 403/ 200μl PBS and control
group given PBS only. Groups were compared with Mann-Whitney test (GraphPad Prism 4.0, USA). (E) Bacteraemia count at 30 hpi. (F) Bacteraemia count at 36 hpi.
Though both groups did not show much difference in levels of attained bacterial load there was significant difference in clinical symptoms between the two groups (p= 0.0317)( (GraphPad Prism 4.0, USA). The vaccinated group lost less weight as compared to the control group though it was not statistically significant (Fig 6.11).
Figure 6.11. Comparison of weight and clinical symptoms at 30 hrs time points in mice challenged with 5 x 103 cfu/ 200 μl TIGR4 IP. Vaccinated group was 3x inoculated IP with 5 x 106 cfu of strain 403/ 200μl in PBS and control group was given PBS only. (A) Comparison of clinical scores. Unpaired t-test showed significant difference in clinical symptoms (GraphPad Prism 4.0, USA). Bars represent the mean ± SEM (B) Comparison of weight-loss. Weight changes are plotted as a box and whiskers plot with horizontal line in the box representing median.
B A
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6.4.3 Intranasal Immunization of mice:
Thirty MF1 mice were divided into two groups. The first group was vaccinated intranasally with 5 x 107 cfu/10µl under general anaesthesia, while control group was given 10 µl PBS. Intranasal booster doses were given on day 14 and 28 and sample bleeds were taken before each vaccination and on day 42.
6.4.3.1 Intranasal challenge of mice with TIGR4 shows weak trends:
Five mice were taken from each group and were challenged intranasally with 5x106 cfu TIGR4/50µl. Twenty percent of vaccinated mice survived and cleared
infection as compared to none surviving in the control group (Fig 6.12).
Figure 6.12. Survival curve of mice challenged with 5x106 cfu/ 50 μl TIGR4 IN. Vaccinated group was vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. 20% survival was seen in vaccinated group. All data plotted as percentage survival as a staircase line with points for all observations against hrs post infection. Log rank test did not show any significant difference between the two groups. The median survival time for both the groups was 54 hrs. Five mice were used in each group.
There was no significant difference in levels of bacteraemia the two groups (Fig 6.13).
Figure 6.13. Bacteraemia during 36 hrs post IN infection in mice challenged with 5x106 cfu/ 50 μl TIGR4. Vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in
PBS and control group was given 10 μl PBS only. There was no significant difference in level of bacteraemia between the two groups. Five mice were used in each group.
The first vaccinated mouse died at 45 hrs before any of control group mouse, though the last one to be culled at 72 hrs also belonged to vaccinated group. One of the mice from vaccinated group cleared infection and survived till the end of experiment.
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6.4.3.2 Intranasal challenge of mice with A66:
Five vaccinated and five control mice were challenged intranasally with bioluminescent serotype 3 strain A66.1 using 5 x 106 cfu/50 µl dose. The results did not show any significant differences between the two groups. There were no survivors in any group, though 40% mice from vaccinated group reached 96 hour time point as compared to 20% from the control group (Fig 6.14).
Figure 6.14. Survival curve of mice challenged with 5x106 cfu/ 50 μl bioluminescent strain A66.1 IN. Vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. There was no significant difference in the two groups. All data plotted as percentage survival as a staircase line with points for all observations against hrs post infection. . Five mice were used in each group.
Group analysis did not show significant differences in bacteraemia in vaccinated group as compared to control group, on excluding vaccinated mouse M5 as it
showed exceptionally high bacteraemia levels, some difference could be seen in the two groups though it did not reach statistically significant level (Fig 6.15).
Figure 6.15. Bacteraemia and thoracic photon emission in mice challenged with 5x106 cfu/ 50 μl bioluminescent strain A66.1 IN. The vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. (A) Bacteraemia from 0-48 hrs in the two groups. (B) Bacteraemia from 0-48 hrs excluding outlier M5. (C) Thoracic photon emission from 0-48 hrs. (D) Thoracic photon emission from 0-48 hrs excluding M5. Five mice used for each group.
Bacteraemia results were consistent with images when compared with Thoracic Photon Emission recorded by the IVIS imaging system (IVIS: Caliper Life Sciences, UK), which is an optical imaging system for non-invasive monitoring of disease progression without killing the animals at each stage. It records photon emission
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from diseased animal and uses this data with 3D reconstruction of these images to localise source of bioluminescence, allowing visualisation of the spread of infection within the body of living animal (Fig 6.16).
Figure 6.16. IVIS images showing photon emission in mice challenged with 5x106 cfu/ 50 μl bioluminescent strain A66.1 IN. Vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. Control mice are on left (M6-M10) and vaccinated mice on the right (M1-M5).
Figure 6.16. IVIS images showing photon emission in mice challenged with 5x106 cfu/ 50 μl bioluminescent strain A66.1 IN. Vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. Control mice are on left (M6-M10) and vaccinated mice on the right (M1-M5). (C) 12 hr time point. Infection developed in M5. (D) 18 hr time point. Progression of infection in M5.
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Figure 6.16. IVIS images showing photon emission in mice challenged with 5x106 cfu/ 50 μl bioluminescent strain A66.1 IN. Vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. Control mice are on left (M6-M10) and vaccinated mice on the right (M1-M5). (E) 24 hr time point. Infection has also appeared in M9. (F) 30 hr time point. Infection has developed in several mice in both the groups.
Figure 6.16. IVIS images showing photon emission in mice challenged with 5x106 cfu/ 50 μl bioluminescent strain A66.1 IN. Vaccinated group was 3x vaccinated IN with 5 x 107 cfu of strain 403/ 10μl in PBS and control group was given 10 μl PBS only. Control mice are on left (M6-M10) and vaccinated mice on the right (M1-M5). (G) 36 hr time point. (H) 42 hr time point. Infection can be seen particularly around the lung areas in heavily infected mice.