B/B 05 · Adaptive immunity and vaccination
A Vaccine Trial in Mice
Read antibody titers and challenge data to follow a primary response, class switching and memory.
Passage
The adaptive immune system responds to a new antigen in stages. B cells that recognize the antigen through their surface receptors can make IgM antibodies within days. With help from CD4⁺ T cells, activated B cells can switch to making IgG antibodies, which circulate for longer and bind antigen with higher affinity. Some activated B and T cells become long-lived memory cells that respond more quickly and strongly if the same antigen is met again. Many vaccines are made of purified proteins, which cause only a weak response, so they are often given together with an adjuvant, a substance that activates cells of the innate immune system.
A researcher tests a purified protein antigen, X, as a vaccine in mice. She forms four groups of ten mice. Group 1 receives a salt solution (PBS) alone. Group 2 receives antigen X alone. Group 3 receives antigen X with an adjuvant. Group 4 receives antigen X with the adjuvant, and is also given an antibody that depletes CD4⁺ T cells. All mice are injected on day 0 and again on day 28. Blood is collected on the days shown, and the amounts of antibodies against X are reported as titers, which are the highest dilutions of serum that still bind X. A higher titer means more antibody. Table 1 shows the geometric mean titers of IgM and IgG.
Table 1. Antibody titers against antigen X.
| Group | IgM, day 7 | IgG, day 14 | IgG, day 28 | IgG, day 31 (3 days after the second dose) |
|---|---|---|---|---|
| 1: PBS only | <10 | <10 | <10 | <10 |
| 2: X alone | 100 | 100 | 200 | 400 |
| 3: X + adjuvant | 400 | 800 | 1600 | 25,600 |
| 4: X + adjuvant + CD4⁺ depletion | 400 | 100 | 100 | 100 |
Titers are measured by an enzyme-linked immunosorbent assay (ELISA). Serum is diluted in two-fold steps and added to wells coated with antigen X, and bound antibody is detected with a labeled secondary antibody that recognizes either mouse IgM or mouse IgG. The titer is the highest dilution that still gives a signal above the background. Adjuvants often work by triggering pattern-recognition receptors on antigen-presenting cells, which then display antigen and send the costimulatory signals that T cells need to become fully active.
On day 42, each mouse is exposed to a pathogen that carries antigen X on its surface, and the amount of pathogen in the blood is measured two days later. In a separate experiment, serum is taken from Group 3 on day 31 and injected into naive mice, which are then exposed to the same pathogen either one day or 42 days later. Table 2 shows the results, expressed relative to the PBS group, whose pathogen level is set to 100.
Table 2. Pathogen level in the blood two days after exposure (relative to PBS).
| Mice | Exposed to pathogen | Pathogen level |
|---|---|---|
| Group 1 (PBS) | Day 42 | 100 |
| Group 2 (X alone) | Day 42 | 40 |
| Group 3 (X + adjuvant) | Day 42 | 5 |
| Group 4 (X + adjuvant, CD4⁺ depleted) | Day 42 | 60 |
| Naive mice given Group 3 serum | 1 day after the transfer | 15 |
| Naive mice given Group 3 serum | 42 days after the transfer | 90 |
The pathogen is a bacterium that carries antigen X on its surface and grows in the blood of unprotected mice. Pathogen level is determined by counting the colonies that form when a small sample of blood is spread on a plate, and the count for Group 1 is set to 100 so that the other groups can be compared with it. Mice in the serum-transfer experiment receive the same volume of serum, and their blood contains no detectable antibody against X before the transfer.
The researcher notes that the groups were randomly assigned and that the person who measured the titers did not know which group each sample came from.
Questions
Question 1 of 6
What is the main purpose of Group 1, which received only PBS?
The group received neither the adjuvant nor the antigen, so it cannot show anything about either alone.
Group 1 received no antigen, so no antigen-specific memory cells are expected.
That is Group 4.
Why the answer is what it is
A control group that is given only the vehicle shows the baseline. The titers in Group 1 are below the detection limit and the pathogen level is 100, so any change in the other groups can be attributed to the vaccine components.
Review: Immunology · Molecular & Cell Biology
Question 2 of 6
IgM appears at day 7, and IgG appears later. Which process explains the change in the type of antibody made by a B cell?
Negative selection removes self-reactive cells during development.
Somatic hypermutation changes the variable regions to improve affinity. It does not change the antibody class.
V(D)J recombination happens during B cell development in the bone marrow, before any exposure to antigen.
Why the answer is what it is
IgM is the first antibody class produced. After activation, with help from T cells, a B cell can switch to IgG by changing the heavy chain constant region while keeping the same antigen-binding variable region.
Review: Immunology · Molecular & Cell Biology
Question 3 of 6
What does the comparison between Group 2 and Group 3 show?
The titers were measured against antigen X, not against the adjuvant.
Antigen X alone raised the titers (100 to 400), but the response was weaker.
After the second dose, the titer in Group 3 rose to 25,600, far above that of Group 2 (400).
Why the answer is what it is
Group 3 had much higher titers than Group 2 at every time point (for example 1600 versus 200 on day 28) and had less pathogen (5 versus 40). The adjuvant therefore increased both the response and the protection.
Review: Immunology · Molecular & Cell Biology
Question 4 of 6
Group 4 made as much IgM as Group 3 on day 7, but little IgG. What is the best interpretation?
The adjuvant acts on innate immune cells. The difference in this experiment is explained by T cell help.
Group 4 made normal levels of IgM, so some antibody was made without CD4⁺ T cells.
Cells of one lineage do not change into the other.
Why the answer is what it is
Class switching to IgG and the formation of strong memory depend on help from CD4⁺ T cells. With those cells removed, the IgM response was similar (400), but IgG stayed low (100) and the second dose did not boost it.
Review: Immunology · Molecular & Cell Biology
Question 5 of 6
After the second dose, IgG in Group 3 rose from 1600 to 25,600 within three days. What best explains the speed of this response?
A response from naive cells takes about a week or longer, as in Table 1 after the first dose.
Complement proteins are not converted to antibodies.
The result shows a quick rise in antibody, which matches the response of memory cells, not the growth of a new population of naive B cells.
Why the answer is what it is
A secondary response is faster and larger than a primary response because memory B and T cells already exist. The rapid rise within three days is the signature of immune memory.
Review: Immunology · Molecular & Cell Biology
Question 6 of 6
Naive mice given Group 3 serum were protected one day later (15) but not 42 days later (90). What does this show?
The same pathogen was used, and the vaccinated mice remained protected on day 42.
Serum contains antibodies but not cells, so the recipients make no memory cells of their own.
Vaccinated mice made their own antibodies and memory cells, which is active immunity.
Why the answer is what it is
Antibodies in transferred serum protect the recipient immediately, but they are used up or broken down within weeks, and the recipient has no memory cells to make more. This is passive immunity. The vaccinated mice made their own antibodies and memory cells and stayed protected.
Review: Immunology · Molecular & Cell Biology
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