Resistance Is Futile

60 Journal of College Science Teaching

C A S E S T U D Y

Resistance Is Futile . . . or Is It? The Immunity System and HIV Infection by Annie Prud’homme-Généreux

A lthough the majority of people are prone to HIV infection, some individu- als remain uninfected de-

spite repeated exposure. This case study uses the results of the land- mark paper by Paxton and his col- leagues (1996) that offered the first breakthrough in understanding why some people are protected against HIV infection. The case study uses an interrupted progressive disclo- sure format, during which students make hypotheses, predict the out- come of experiments, and compare their predictions with real data. I am aware of another case study developed using Paxton’s paper (“Rediscovering Biology,” 2011). The aims, activities, and topics covered by that case are different, and I recommend that instructors review the Rediscovering Biology case, particularly the excellent mul- timedia and introductory literature that accompanies it. This case is appropriate for first-year biology students with knowledge of the im- mune system (cellular and immoral immunity) and HIV infection.

Objectives By the end of the case, students will be able to

• formulate testable hypotheses and design experiments to investigate them;

• predict the results of experiments given competing hypotheses;

• interpret data and compare to pre- dicted outcomes;

• describe cellular and humoral im- munity, HIV structure, and HIV infection; and

• debate the pros and cons of per- sonal knowledge of HIV immu- nity.

Classroom management This case was developed for a 90-minute class but can be adapted for a longer or shorter class period. Students should be well versed in humoral and cellular immunity and the mechanism of HIV infection be- fore attempting this case. For each section of this case, teams of three to four students are provided with printed handouts of the case and are directed to work with their group on solving the questions. This is always followed by a large class discussion during which the inputs of each team are shared. Use of a whiteboard on which student volunteers show their predictions during the class discus- sion is very helpful.

Following this case, students can be assigned research projects to update their knowledge and under- standing of this issue. Several lines of investigation are suggested:

• The CCR5 mutation is common among people of European de- scent but not among other popula- tions. Students explore and com- pare the proposed hypotheses that explain the evolution of this trait.

• Since Paxton’s paper was pub- lished, mutations in human genes other than CCR5, for example in CCR2, have been shown to pro- tect against HIV infection or slow down the progression of the dis- ease to AIDS. Review what some of these genes do and how they are thought to exert their protec- tive effect (e.g., see O’Brien, 2003; O’Brien & Moore, 2000; O’Brien & Nelson, 2004).

• Most of the individuals protected against HIV appear to have more responsive T

C. What is known about this mechanism, and what does it suggest for possible HIV preventions and cures?

• It is possible that the protected in- dividual in Group A is not lucky but rather has more potent anti- bodies that defend against HIV. What’s known about this mecha- nism of protection? How could this knowledge be used to prevent or cure HIV infection?

• Lahouassa and colleagues (2012) reported yet another means of protection against HIV. This mechanism involves the protein SAMHD1, which can protect macrophages and dendritic cells from HIV-1 by hydrolyzing and depleting the cell’s dNTP pool. Without dNTP, the virus can- not copy itself into DNA. This mechanism cannot protect TH cells, which have greater concen- trations of dNTPs, nor is it effec- tive against HIV-2, which makes

61Vol. 41, No. 5, 2012

an enzyme that counteracts SAM- HD1. Read this article and pro- pose how this information could be used to develop strategies in the fight against HIV.

• Students write an in-class re- sponse paper discussing the pros and cons of making genotype testing at the CCR5 locus readily available.

Students may work individually or in teams and communicate the results of their research in a class presenta- tion or in a written report.

Case study Part I: HIV and the immune system The vast majority of people are sus- ceptible to HIV infection. However, in the 1990s, several individuals no- ticed that, despite repeated exposure to the HIV virus, they remained HIV negative. This could be due to the fact that these individuals were ex- tremely lucky, or perhaps there was something different about them that made HIV infection less likely.

William Paxton and his colleagues at the Aaron Diamond AIDS Research Center in New York became inter- ested in this phenomenon of HIV protection. In this case study, you will retrace the steps and experiments that these researchers have performed to understand the mechanism underlying the protection against HIV (Paxton et al., 1996).

B e f o r e l o o k i n g a t P a x t o n ’s research, here are a few questions to help you review the biology of HIV infection and the virus’s interactions with the human immune system.

Questions 1. Which cells are targeted by the

HIV virus? By what molecular mechanism can the virus discrim-

inate between cell types? 2. Which cells fight HIV infection

and how? 3. HIV eventually causes AIDS, a

failure of the immune system to work effectively. How does HIV cause this symptom?

4. Describe the life cycle of the HIV virus.

5. Consider how HIV infects cells and reproduces. Also consider how the immune system fights off HIV infection. Humans differ by having mutations that result in slightly different proteins and im- mune function. Suggest as many hypotheses as possible to explain why some individuals might be protected against HIV infection. In other words, where and how might new viral infections be stopped? What could be differ- ent about the people who seem protected against HIV that caused viral replication to stop? Come up with at least three possibilities.

Part II: Paxton’s hypotheses about HIV-resistant individuals Paxton and his colleagues had a few hypotheses about why some of the individuals exposed to HIV were protected against this virus.

Super cytotoxic T cells hypothesis. Perhaps the reason that some indi- viduals were protected against HIV is because they had cytotoxic T cells that were better and faster at recognizing infected T helper cells. This ability allowed the immune system to rid the body of any HIV infection before the virus could replicate inside T helper cells and transform these cells into HIV factories.

Super T helper cells hypothesis. Perhaps the T helper cells of the protected individuals were different, preventing the infection and replica- tion of the virus inside the cell. There

are many steps necessary for viral infection and replication inside T helper cells, and any of them could be impeded.

Questions 1. Classify each of your proposed

hypotheses into the two catego- ries proposed by Paxton and his colleagues (note: some hypoth- eses may not fit into either cat- egory).

2. How might you test each of your hypotheses? Propose an experi- ment. What are your controls? Experimental conditions?

Part III: Predictions from Paxton’s two hypotheses Paxton and his colleagues recruited 25 volunteers who claimed to have had repeated exposure to the HIV virus and yet were not infected with HIV. He also enlisted the help of 9 individuals not exposed to the HIV virus (and who tested negative for the virus). This latter group is the control, whose response to HIV should be the same as the majority of people.

Paxton and his colleagues wanted to identify which of their two hypoth- eses might be correct. The problem with working in vivo is that it is unethical to expose individuals to HIV. In addition, in a person the immune system is complex, with multiple interactions. To isolate the action of T helper cells, cytotoxic T cells, and the HIV virus, Paxton and his colleagues worked in test tubes. Paxton isolated T helper cells and cytotoxic T cells from individuals in each group. He then performed the following experiments:

• In one tube, he mixed HIV virus and T helper cells.

• In another tube, he mixed HIV vi-

62 Journal of College Science Teaching

CASE STUDY

rus, T helper cells, and cytotoxic T cells.

He monitored the accumulation of virus in the test tube over time by measuring the amount of p24 pro- teins produced over a 14-day period.

Questions 1. Why were HIV and T helper cells

mixed in the presence and ab- sence of cytotoxic T cells?

2. Use the graphic provided in Fig- ure 1 to illustrate the results you would expect to obtain for a. a normal/control person b. a protected individual, assum-

ing that the super cytotoxic T cell hypothesis is correct

c. a protected individual, assum- ing that the super T helper cell hypothesis is correct.

Please note that each graph re- quires two lines (the two test tubes).

3. How is this experiment able to differentiate whether the mecha- nism of protection against HIV

FIGURE 1

Expected results from Paxton’s first in vitro experiment.

is through super T helper cells or through super cytotoxic T cells?

Part IV: Paxton’s results Paxton’s results are shown in Figure 2 (adapted from Figure 1 of his pa- per; Paxton, 1996). The black lines represent the results of experiments in which HIV was incubated with T helper cells, and the red lines rep- resent experiments where HIV + T helper cells + cytotoxic T cells were mixed in the test tube.

The top left graph shows the data from control individuals. The other three graphs show the results obtained from people claiming to be protected against HIV infection. Three different patterns were ob- served in this group of volunteers. The results of only one person was categorized as Group A, two people had results represented in the figure for Group B, and Group C is made up of seven people who had different responses but whose test tubes all produced less virus in the presence of TC.

Questions 1. Do cytotoxic T cells provide pro-

tection from HIV in control indi- viduals?

2. Why might the results of people claiming to be resistant to HIV infection differ? Why might their results be categorized into three groups?

3. Compare these results with what you had predicted in the previous section. a. Are the results of the controls

as you expected? b. Which of Paxton’s hypotheses

seem to be validated by the re- sults of the protected individu- als? Why?

c. What do you make of the per- son claiming to be protected in Group A?

Part V: The super T helper cell mechanism From the results of this experi- ment, it is apparent that the person in Group A has either been lucky so far or exhibits a mode of protection not anticipated by Paxton’s team. In- dividuals in Group B do not appear to be infected by the HIV virus at all (super T helper cells). The remaining protected individuals exhibit differ- ent degrees of infection with very ac- tive cytotoxic T cells to slow down the progression of new infections (super cytotoxic T cells).

Paxton’s team was particularly interested in protected subjects in Group B and in investigating the mechanism of their protection against HIV. To investigate this, they performed an experiment in which they mixed purified T helper cells from control or protected individu- als with different strains of HIV-1. The goal was to determine whether all HIV-1 strains could infect the T helper cells from protected individu-

63Vol. 41, No. 5, 2012

als. HIV-1, the most common form of the virus and the one responsible for the pandemic, can be classified into two different types:

• M-tropic (also called nonsyncitia- inducing (NSI) or R5 HIV-1) strains

• T-tropic (also called syncitia-in- ducing (SI) or X4 HIV-1) strains.

This turned out to be a very in- formative experiment. About the same time, two other papers were published that clarified some of the differences between these two strains of virus.

• M-tropic HIV-1 strains must bind

to two cell surface proteins to en- ter and infect a cell (Dragic et al., 1996): u CD4 protein u β-chemokine receptor CCR5

• Conversely, T-tropic HIV-1 strains use different proteins to enter and infect a cell (Feng, Broder, Kennedy, & Berger, 1996): u CD4 protein u α-chemokine receptor CXCR4

Armed with this information, we can look back at the experiment performed by Paxton’s team and investigate whether CD4, CCR5, CXCR4, or another protein is mutated and “different” in individuals that are protected against HIV. Here is the design of this experiment.

• In one tube: Mix HIV-1 (T-tropic strain) + T helper cells from a control person.

• In another tube: Mix HIV-1 (T-tropic strain) + T helper cells from a protected person.

• Monitor the appearance of p24 in the test tube (i.e., production of new virus) over time.

FIGURE 2

64 Journal of College Science Teaching

CASE STUDY

• In one tube: Mix HIV-1 (M-tropic strain) + T helper cells from a control person.

• In another tube: Mix HIV-1 (M-tropic strain) + T helper cells from a protected person.

• Monitor the appearance of p24 in the test tube (i.e., production of new virus) over time.

Questions 1. Let’s assume that protected in-

dividuals in Group B have an altered CD4 protein (a mutation in the CD4 gene) compared with controls that renders the pro- tein unrecognizable by gp120 on HIV. Use the graphs in Figure 3 to draw the results you expect to obtain from the previously men- tioned experiment. Remember that each graph should have two lines and review which proteins are required for infection by the two strains.

2. Let’s assume that protected indi- viduals in Group B have an al- tered CCR5 protein (a mutation in the CCR5 gene) compared with controls. Use the graphs in Figure 4 to draw the results you expect to obtain from the previously men- tioned experiment. Remember that each graph should have two lines and review which proteins are required for infection by the two strains.

3. Let’s assume that protected indi- viduals in Group B have an al- tered CXCR4 protein (a mutation in the CXCR4 gene) compared with controls. Use the graphs in Figure 5 to draw the results you expect to obtain from the previ- ously mentioned experiment. Remember that each graph should have two lines and review which proteins are required for infection by the two strains.

FIGURE 3

Results expected if controls and protected individuals differ in their CD4 protein.

FIGURE 4

Results expected if controls and protected individuals differ in their CCR5 protein.

65Vol. 41, No. 5, 2012

Part VI: Why some people are protected against HIV A summary of Paxton’s results is shown in Figure 6 (from Figure 4 of his paper). The black lines show the results using T helper cells from controls and the blue lines the results using T helper cells from protected individuals in Group B. Several different M-tropic and T-tropic strains of HIV-1 were used, all producing similar results.

Questions 1. Which of your hypothesized

graphics do the results most re- semble?

2. On the basis of this information, what is the mechanism of HIV protection in Group B?

3. Are these people protected against all forms of HIV out there? What are the implications?

4. In biology, the terms resistance and immunity have different meanings. Resistance is a preex- isting mutation in an organism that confers protection against a threat or challenge such as a virus. Resistance is used in the same manner as “antibiotic resis- tance” in bacteria. Immunity re- fers to an active response of the immune system to the challenge of a foreign particle that con- fers protection upon the organ- ism. You have investigated many forms of protections against HIV. Which of these constitute resis- tance and which of them consti- tute immunity?

Note: Detailed teaching notes and the answer key may be found at the National Center for Case Study Teaching in Science at http://sci- encecases.lib.buffalo.edu/cs/col- lection. n

FIGURE 6

FIGURE 5

Results expected if controls and protected individuals differ in their CXCR4 protein.

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References Dragic, T., Litwin, V., Allaway,

G. P., Martin, S. R., Huang, Y., Nagashima, K. A., . . . Paxton, W. A. (1996). HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature, 381(6584), 667–673.

Feng, Y., Broder, C. C., Kennedy, P. E., & Berger, E. A. (1996). HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane G protein-coupled receptor. Science, 272, 872–877.

Lahouassa, H., Daddacha, W., Hofmann, H., Ayinde, D., Logue, E.C., Dragin, L., . . . Margottin- Goguet, F. (2012). SAMHD1 restricts the replication of human

immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nature Immunology, 13, 223–228.

O’Brien, S. J. (2003). Chapter 12: Genetic guardians. In Tears of the cheetah: The genetic secrets of our animal ancestors (pp. 198–221). New York, NY: St. Martin’s Press.

O’Brien, S. J., & Moore, J. P. (2000). The effect of genetic variation in chemokines and their receptors on HIV transmission and progression to AIDS. Immunological Reviews, 177, 99–111.

O’Brien, S. J., & Nelson, G. W. (2004). Human genes that limit AIDS. Nature Genetics, 36, 565–574.

Paxton, W. A., Martin, S. R., Tse,

D., O’Brien, T. R., Skurnick, J., VanDevanter, N. L., . . . Koup, R. A. (1996). Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposures. Nature Medicine, 2, 412–417.

Rediscovering Biology. (2011). The genetics of resistance to HIV infection. Retrieved from http:// www.learner.org/courses/biology/ casestudy/hiv.html

Annie Prud’homme-Généreux (apg@ questu.ca) is Founding Professor in the Life Sciences Department at Quest Uni- versity Canada in Squamish, British Co- lumbia, Canada.

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