Table 3.
Immunization Protocol for Evaluating Humoral and Cell-Mediated Immune Responses to Biotinylated Antigens in Mice (n = 5 for Each Group)a
| groups | day 0 | day 12 | day 21 | day 24 | |
|---|---|---|---|---|---|
| 1 | control | PBS | PBS | spleen (T cell) | IFN-γ assay |
| serum (humoral) | |||||
| 2 | B-pVHX-6 | 500 ng | 500 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| 3 | B-pVHX-6 | 500 ng | 500 ng | spleen (T cell) | IFN-γ assay |
| anti-CD40 mAb | 25 μg | 0 | serum (humoral) | ||
| 4 | B-pVHX-6 | 500 ng | 500 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| anti-CD40 mAb | 25 μg | 0 | |||
| 5 | B-OVA | 200 ng | 200 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| anti-CD40 mAb | 25 μg | 0 | |||
| 6 | B-MUC-1 | 200 ng | 200 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| anti-CD40 mAb | 25 μg | 0 | |||
| 7 | B-GM3 | 1 μg | 1 μg | spleen (T cell) | IFN-γ assay |
| B-GM2 | 1 μg | 1 μg | serum (humoral) | ||
| bfFp | 20 μg | 0 | |||
| anti-CD40 mAb | 25 μg | 0 | |||
| groups | day 0 | day 12 | day 21 | day 24 | |
| 8 | control | PBS | PBS | spleen (T cell) | IFN-γ assay |
| serum (humoral) | |||||
| 9 | B-pEBOV GP1,2 | 500 ng | 500 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| anti-CD40 mAb | 25 μg | 0 | |||
| 10 | B-pSARS-CoV spike | 500 ng | 500 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| anti-CD40 mAb | 25 μg | 0 | |||
| 11 | B-pSARS-CoV membrane | 500 ng | 500 ng | spleen (T cell) | IFN-γ assay |
| bfFp | 20 μg | 0 | serum (humoral) | ||
| anti-CD40 mAb | 25 μg | 0 | |||
| groups | day 0 | day 12 | day 21 | day 24 | |
| 12 | control | PBS | PBS | spleen (T cell) | IFN-γ assay |
| serum (humoral) | |||||
| 13 | B-OVA | 200 ng | 200 ng | spleen (T cell) | IFN-γ assay |
| B-EBOV GP1 | 200 ng | 200 ng | serum (humoral) | ||
| B-SARS-CoV spike RBD | 200 ng | 200 ng | |||
| B-MUC-1 | 200 ng | 200 ng | |||
| B-anthrax PA | 200 ng | 200 ng | |||
| anti-CD40 mAb | 25 μg | 0 | |||
| core-streptavidin | 10 μg | 0 | |||
| 14 | B-OVA | 200 ng | 200 ng | spleen (T cell) | IFN-γ assay |
| B-EBOV GP1 | 200 ng | 200 ng | serum (humoral) | ||
| B-SARS-CoV spike RBD | 200 ng | 200 ng | |||
| B-MUC-1 | 200 ng | 200 ng | |||
| B-anthrax PA | 200 ng | 200 ng | |||
| bfFp | 20 μg | 0 | |||
| 15 | B-OVA | 200 ng | 200 ng | spleen (T cell) | IFN-γ assay |
| B-EBOV GP1 | 200 ng | 200 ng | serum (humoral) | ||
| B-SARS-CoV spike RBD | 200 ng | 200 ng | |||
| B-MUC-1 | 200 ng | 200 ng | |||
| B-anthrax PA | 200 ng | 200 ng | |||
| bfFp | 20 μg | 0 | |||
| anti-CD40 mAb | 25 μg | 0 | |||
The amounts of antigens, antibodies and bfFp are either in μg or ng per mouse. All mice were injected subcutaneously near the inguinal lymph node. Three separate experiments were conducted in 15 groups of mice and each experiment had its own control group. Groups 1, 8 and 12 were the control groups. The first experiment demonstrated the versatility of the bfFp based delivery of protein, peptide, glycolipids and DNA to DC in generating immune responses (Group 2–7 mice). The second experiment was designed to confirm the DNA delivery strategy of targeting varies infectious disease viral DNA to DC (Groups 9–11). Multivalent immune responses against a mixture of antigens (proteins and peptide) were studied in the third set of experiments (Groups 13–15). All mice were boosted with the same concentrations of antigen(s) in PBS 12 days following primary immunization. The mice were sacrificed after 9 days of boost.