David Yang
Monica Brown
Gayane Arutunyan
Microbiology 401
November 11, 2007
CASE STUDIES AND QUESTIONS: Human Immunodeficiency Virus (HIV)
A 28-year-old man had several complaints. He had a bad case of thrush (oral candidiasis)
and low grade fever, had serious bouts of diarrhea, had lost 20 pounds in the last year
without dieting, and, most seriously, complained of difficulty breathing. His lungs
showed bilateral infiltrate on radiographic examination, characteristic of P. carinii
pneumonia. A stool sample was positive for Giardia lamblia. He was a heroin addict and
admitted to sharing needles at a “shooting gallery”
1. What laboratory tests should be done to support and confirm a diagnosis of HIV
infection and AIDS?
Detection of HIV infection may be via a direct or an indirect assay. The most common
way to detect the HIV virus infection is via an indirect ELISA (Enzyme-Linked
Immunosorbent Assay). This is specifically done by detecting how much antibody
against HIV is present in a blood serum sample. A 96-well plate is coated with viral
protein, and the serum (which may potentially contain antibodies to the virus) is
incubated in the wells along with positive and negative controls. The serum is then
removed and the wells washed with buffer to wash off weakly adhering antibodies. A
secondary antibody (usually rodent anti-human IgG conjugated to an enzyme) is added to
the wells to attach to the human antibodies if antibodies to the virus are present. There is
again an incubation and washing period. And lastly, a substrate for the enzyme is added
to make the wells containing HIV antibodies change color.
Since false positives due to cross-reacting antibodies are possible with the ELISA, the
ELISA results are then confirmed through Western Immunoblot. In this method HIV
viral proteins are added to wells in a gel (SDS-PAGE) and separated by mass and
electrical charge through gel electrophoresis. The gel is then transferred to a
nitrocellulose blotting paper. The paper is incubated with generic protein (e.g. milk
protein) to bind any sites on the membrane that are not bound by HIV proteins. This is
called the blocking step. Patient’s serum containing potential anti-HIV antibodies is
added to the membrane. If antibodies are present they will bind to the HIV proteins.
Unbouind antibodies are washed away. A secondary antibody (usually rodent anti-human
IgG conjugated to an enzyme) is added to the blot to attach to the human antibodies if
antibodies to the virus are present. There is again an incubation and washing period. And
lastly, a colorless substrate for the enzyme is added to the membrane. If anti-HIV
antibodies were presenting the patient’s serum the enzyme will convert the substrate to a
visible colored product which is photographed.
Direct tests for the presence of the virus include an RT-PCR assay that is used to detect
the presence of virus (viral RNA) from a serum sample of the patient. This assay is most
often used to follow the viral load in HIV-infected individuals.
2. How did this man acquire the HIV infection? What are other high-risk behaviors for
HIV infection?
He most likely acquired the HIV infection from sharing needles. Other high risk
behaviors are unprotected vaginal/anal/oral sex, blood transfusions or organ transplants
(not as common these days since donated blood and organs are screened), transmission
from an infected mother to her fetus, transmission from breast milk of an infected mother
to her baby, and those who already have other sexually transmitted infections are more
susceptible to HIV. Although HIV virus has been found in saliva, there is no clinical
proof that it can be transmitted through this medium.
3. What was the immunologic basis for the increased susceptibility of this patient to
opportunistic infections?
The most important immunologic basis for the increased susceptibility of this patient to
opportunistic infections is the decrease in the number of CD4+ T cells (T helper cells).
CD4+ T cells are infected by the virus and viral infection eventually leads to the death of
the cell. The exact mechanism by which this occurs has not yet been elucidated, though a
variety of different mechanisms have been proposed.
HIV also has several proteins which act to decrease host defenses and increase viral
infectivity, one of which is the Nef protein. Nef proteins downregulate the expression of
host CD4 and MHC class I molecules, which decrease host immune defense. Nef also
induces the phosphorylation of MA proteins to increase viral infectivity. Nef proteins
also alter T cell signaling to promote viral replication. Ultimately, these effects resulting
from HIV infection decrease overall host immune defense, which leaves the patient
susceptible to opportunistic infections.
4. What precautions should have been taken in handling samples from this patient?
Healthcare personnel should assume that the patient’s blood and other bodily fluids are
potentially infectious. Therefore, precaution procedures against possible infections
should be used at all time. Protective barriers, such as gloves and goggles, should be
used when in contact with the patient’s blood and bodily fluids. Immediately after any
contact with blood or body fluids, healthcare personnel must wash hands and all other
skin surfaces. Take extra precaution with the handling and proper disposal of any sharp
instruments used on the patient.
Although the most important strategy for reducing the risk of HIV transmission is to
prevent the exposures, there must also be post-exposure plans for accidental infections.
In the case of post-exposures, the Center for Disease Control and Prevention (CDC)
recommends the postexposure prophylaxis (PEP). Antiretroviral medication, such as
Tenofovir disoproxil fumarate and emtricitabine, treatments administered to HIVinfected women during labor and delivery has shown to reduce the risk of mother-tochild transmission by approximately 50%. Antiretroviral regimens have also been shown
to be associated with an 80% reduction in risk of HIV infection among healthcare
personnel following needle sticks and other accidental exposures, when treatment is
initiated promptly.
5. Several forms of HIV vaccines are being developed. What are possible components of
an HIV vaccine? Who would be appropriate recipients of an HIV vaccine?
Some components of a vaccine can be to target the gp120, gp160 and gp41 surface
proteins of the virus, to prevent it from binding to the host cell receptor CD4. Another
possible action of the vaccine can be to target reverse transcriptase so that it won’t
synthesize new copies of the viral genome. Around 13% of people of northern European
descent have a naturally occurring deletion of 32 base pairs in the CCR5 (chemokine
receptor) gene results. This is a mutant CCR5 receptor that never reaches the surface of
their cells. Homozygotes for this mutation (1-2% Caucasians) have a resistance to HIV
infection. Therefore a vaccine can target the CCR5 receptor to cleave it to make it nonfunctional. A vaccine can also target cyclophilin A, either at the cell surface to block it
from binding to the heparin sulfate receptor or within the cytoplasm to keep it from
expanding the viral core.
There are two types of vaccines in development. Therapeutic vaccines intend to boost the
immune systems of those already infected, while a Preventive vaccine intends to generate
an immune response in an uninfected person to prevent future infection. No therapeutic
vaccines have been currently approved for use by the FDA, but they are undergoing
clinical trials to ascertain their safety and effectiveness. However they are in the very
early stage of experimentation and many years away from being available. There are
three different types of preventive vaccines: subunit, recombinant vector, and DNA.

Subunit vaccines are "component" or "protein" vaccines that contain an individual
viral components rather than the whole virus. The subunits are made through
genetic engineering and administered to induce an anti-HIV immune response.
However the response is weak and may not prevent future infections.

Recombinant vector vaccines use non-HIV viruses that don't cause disease in
humans or have been rendered unable to cause disease (attenuated). These viruses
are used as vectors to carry copies of HIV genes into human cells, in which HIV
proteins will be produced. The HIV proteins can stimulate an anti-HIV immune
response. The response may be stronger than in subunit vaccines since the
recombinant vector delivers several HIV genes into the cells. Some vectors that
are being studied for use in HIV vaccines are ALVAC (canarypox virus), MVA
(type of cowpox virus), VEE (normally a horse virus), and adenovirus-5 (human
virus; usually doesn’t cause serious disease).

DNA vaccines introduce HIV genes into the body, but don’t rely on a virus vector.
“Naked" DNA containing HIV genes is injected directly into body so that the
cells will take it up and produce HIV proteins and induce an immune response
against HIV.
Appropriate recipients would be those who have high risk behaviors such as drug addicts
who share needles and those who have unprotected sex. Countries where a significant
number of the population has people infected with HIV can also be appropriate recipients
so as to protect the uninfected members. And, health care workers that may be under
occupational risk of HIV exposure.
References
Dimmock, N.J., Easton, A.J., and K.N. Leppard. Introduction to Modern Virology, 6th
Edition. Blackwell publishing Ltd. 2007.
McQueen, Nancy. Microbiology 401 Lectures. California State University, Los Angeles.
2007
http://www.bio.davidson.edu/Courses/genomics/method/ELISA.html
http://www.bio.davidson.edu/COURSES/genomics/method/Westernblot.html
http://www.niaid.nih.gov/factsheets/hivinf.htm
http://hivinsite.ucsf.edu/InSite?page=kb-02&doc=kb-02-01-01
http://www.cdc.gov/hiv