The document discusses using alligator serum to battle HIV/AIDS. It hypothesizes that alligator serum demonstrates antiviral activity against HIV through an active serum complement system and cationic peptides expressed by alligator leukocytes. A series of experiments are proposed to test this, including a cell-based assay to test antiviral activity and assays to analyze complement system function and cationic peptide expression. The expected results are that alligator serum will show powerful anti-HIV activity mediated by complement and cationic peptides. Limitations include the serum potentially losing effectiveness if alligators regularly encounter HIV.

1. University of Minnesota
Alligator serum and the battle against HIV
MicB 4131-Immunology
Petroula N Karagiannis
April 27th, 2006

2. Table of Contents
Justification..........................................................................................................................3
Objective/Hypothesis...........................................................................................................5
Plan Approach......................................................................................................................5
Expected Results..................................................................................................................7
Limitations ...........................................................................................................................8
References ...........................................................................................................................9
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3. Justification
HIV/AIDS is a world wide epidemic of enormous magnitude. The human
immunodeficiency virus (HIV) is thought to cause acquired immunodeficiency syndrome
(AIDS). The disease works by triggering a deficiency in cellular immune responses and a
decrease in the subpopulation of T cells that carry the CD4 marker (T helper cells). T
helper cells are responsible for an individual’s effective immune response to viral and
bacterial infection. In other words, T helper cells are part of an army living in a
individual’s body that helps protect the individual from harmful viral and bacterial
agents. The T helper cells accomplish this by attacking the disease and demolishing it.
Since, HIV/AIDS weakens the immune system the individual no longer has the capability
to fight off diseases such as cancer, heart disease, liver disease, stroke, diabetes,
tuberculosis, hepatitis and many others. As a result of the individual obtaining such
diseases due to the immunodeficiency, death subsequently follows (Goldsby RA, Kindt
TJ, Osborne BA, Kuby J., 2003).
Contraction of HIV/AIDS is thought to be linked to homosexual males,
polygamous individuals, intravenous drug users, individuals who received blood
transfusions prior to 1985 and infants born to HIV infected mothers. About 70 % of
individuals infected with the HIV in the world are from Sub-Saharan Africa, where more
than 25 million people have HIV/AIDS, including young people and children.
Worldwide there is an estimated 36 million individuals affected with the disease. HIV/
AIDS is a public health problem of primary importance that poses social, cultural and
economic problems to the patients, their immediate families and the community. Many
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4. children have been orphaned due to their parent’s death from the disease. Most deaths
from HIV/AIDS in the world are adolescent who are in their economic active life
(Goldsby RA, Kindt TJ, Osborne BA, Kuby J., 2003).
A vaccine that prevents infection to HIV and the progression to AIDS has not
been developed to this day. It has been suggested that the reason for this delay is due to
special conditions that must be applied in developing a safe and effective vaccine for the
disease. For instance, current vaccines mimic the natural state of infection from
individuals that have recovered from the disease and are immune from future attacks.
Since there are no documented recovered AIDS patients, a vaccine for HIV/AIDS is hard
to develop. Additionally, HIV has a rapid rate of mutation and therefore a particular
vaccine directed towards a specific strand of HIV would be ineffective for the each new
mutated strand of HIV. Furthermore, effective vaccines are either whole killed or live
attenuated organisms. Since killed HIV does not retain antigenicity, a live one would
have to be used. However, live vaccines are less stable than killed ones and may revert to
virulent form! This indeed raises some safety issues. Another reason for failure to
develop a safe and effective vaccine for human use is the fact that there are no suitable
animal models for researcher to work with. It is standard procedure that animal testing
for safety and efficacy of a vaccine be performed prior to human volunteer testing
(Goldsby RA, Kindt TJ, Osborne BA, Kuby J., 2003).
Consequently, the disease related to HIV/AIDS and the development of a vaccine
is an extremely important topic for the well being and survival of humanity (Goldsby RA,
Kindt TJ, Osborne BA, Kuby J., 2003).
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5. Objective/Hypothesis
The hypothesis is that alligator serum demonstrates antiviral activities against the
human immunodeficiency virus (HIV) (Merchant ME, Pallansch M, Paulman RL, Wells
JB, Nalca A, Ptak R., 2005). Furthermore, the antiviral properties of alligator serum are
due to an active serum complement system (Merchant ME, Roche CM, Thibodeaux D,
Elsey RM., 2005). In addition, alligator leukocytes express cationic peptides that are
responsible for their antiviral properties (Merchant ME, Leger N, Jerkins E, Mills K,
Pallansch MB, Paulman RL, Ptak RG., 2006). Also, the innate immunity of alligators is
much stronger and powerful then the innate immunity of humans (Merchant ME, Mills
K, Leger N, Jerkins E, Vliet KA, McDaniel N., 2005).
Plan Approach
To conduct this study, blood must be collected from wild alligators through the
spinal vein using a needle and a syringe. After approximately 3 hours, 15 ml of the blood
should be allowed to clot at room temperature. Eventually, the serum can be separated by
centrifugation and frozen at -20 degrees Celsius. To determine whether the alligator
serum is effective against HIV/AIDS, a cell based assay should be used. A cell based
assay involves infecting a human T lymphoblastoid cell line with the HIV virus. This
type of assay permits screening of the entire HIV life cycle, without requiring the use of
wild type biologically hazardous virus. Therefore, a cell based assay can be developed in
a cost-effective and rapid manner (Merchant ME, Pallansch M, Paulman RL, Wells JB,
Nalca A, Ptak R,. 2005).
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6. Also, to determine the function of alligator serum complement system of proteins
a sheep red blood cell (SRBC) hemolytic assay should be performed. In this assay,
SRBC’s mixed with buffer and undiluted alligator serum will be centrifuged.
Furthermore, the optical density of the supernatant can be measured using a
spectrophotometer and the function of alligator serum complement system will be
determined (Merchant ME, Roche CM, Thibodeaux D, Elsey RM., 2005).
In addition, to show that alligator leukocytes express cationic peptides that are
responsible for their antiviral properties, the leukocytes must be isolated and then
processed. To isolate the leukocytes whole blood should be mixed with dextran, allowing
two hours for the erythrocytes to settle. The top layer should be removed once per every
hour with the use of transfer pipettes. The top layer will contain the leukocytes, which
should be collected by centrifugation for 20 minutes. After re-suspending the cell pellet
in normal saline it should then be centrifuged once again for 20 minutes and the
leukocyte can be processed (Merchant ME, Leger N, Jerkins E, Mills K, Pallansch MB,
Paulman RL, Ptak RG., 2006).
Similarly, to support the fact that the alligator’s immune system is much more
powerful than that of humans, serum sample from alligators should be tested for
antibacterial activity against bacterial species using an antibacterial assay. To do this
blood samples should be drawn from the spinal vein using a needle and a syringe. After
transferring the blood samples to tubes, the serum should be separated and stored at -80
degrees Celsius. The alligator serum sample should then be tested in nutrient agar where
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7. zones of inhibition can be visible and manually measured (Merchant ME, Mills K, Leger
N, Jerkins E, Vliet KA, McDaniel N., 2005).
Expected Results
The expected results are indeed that the alligator serum expresses powerful and
effective anti-HIV activity. Thus, the cell based assay will indicate that the alligator
serum completely wipes out and extinguishes the HIV virus (Merchant ME, Pallansch M,
Paulman RL, Wells JB, Nalca A, Ptak R., 2005).
Additionally, the anti-HIV activity is expected to be mediated by the serum
complement system. The reason for this is that the heat treatment of the serum will
eliminate the effect of anti-viral activity. Thus, alligator serum complement, as well as
human complement activity, is sensitive to heat inactivation. Furthermore, the sheep red
blood cell (SRBC) hemolytic assay will show the ability of alligator serum to disrupt
SRCC’s in vitro. Untreated, as opposed to heat treated, alligator serum incubated with
SRBC’s at room temperature will show the maximum optical density. This finding will
verify that the antiviral properties of alligator serum are due to an active serum
complement system (Merchant ME, Roche CM, Thibodeaux D, Elsey RM., 2005).
Furthermore, the inability of heat-treatment to completely demolish the hemolytic
activity of alligator serum will indicate the presence of cationic peptides, which are heat
stable molecules. This suggests that alligator leukocytes express cationic peptides that
are responsible for their antiviral properties (Merchant ME, Leger N, Jerkins E, Mills K,
Pallansch MB, Paulman RL, Ptak RG., 2006).
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8. Moreover, the antimicrobial assay will demonstrate that the alligator’s innate
immune system is indeed much more powerful than that of humans. This is obvious from
the fact that alligators experience frequent injuries that could lead to potentially serious
infection, but rarely obtain an infection. Also, alligators live in an environment that is
rich in pathogenic microorganisms, while still obtaining the ability to heal injuries
without infection. Thus, alligators are shown to be resistant to various diseases,
compared to humans (Merchant ME, Mills K, Leger N, Jerkins E, Vliet KA, McDaniel
N., 2005).
Limitations
One of the limitations of the alligator serum against HIV is that fact that it may
prove to be ineffective, if HIV is regularly encountered by the alligator. The reason for
this is that alligators naturally present an innate immunity to viruses that they have not
contracted very often (Merchant ME, Pallansch M, Paulman RL, Wells JB, Nalca A, Ptak
R., 2005).
Similarly, a deficiency in the complement components may disrupt the antiviral
properties of the alligator serum. This will result is the inability of the alligator serum to
fight of HIV (Goldsby RA, Kindt TJ, Osborne BA, Kuby J., 2003).
Furthermore, an inability of leukocytes to migrate to the diseased area will cause a
malfunction in the alligator serum to destroy the HIV. Thus the cationic peptides will not
be expressed properly and the alligator’s serum anti-viral properties will vanish (Goldsby
RA, Kindt TJ, Osborne BA, Kuby J., 2003).
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9. Moreover, in the event that the alligator’s innate immunity fails to work properly,
it will no longer be able to provide a much stronger innate immunity than humans. For
instance, if the alligator has a deficiency in IFN-alpha, IFN-beta and natural killer (NK)
cells, the alligator will not be able to induce an anti-viral response or resistance to viral
replication (Goldsby RA, Kindt TJ, Osborne BA, Kuby J., 2003).
References
Merchant ME, Leger N, Jerkins E, Mills K, Pallansch MB, Paulman RL, Ptak RG.
(2006). “Broad spectrum antimicrobial activity of leukocyte extracts from the American
alligator (Alligator mississippiensis).” Vet Immunol Immunopathol. 110(3-4), 221-8.
Merchant ME, Mills K, Leger N, Jerkins E, Vliet KA, McDaniel N. (2005).
“Comparisons of innate activity of all known living crocodilian species.” Comp Biochem
Physiol B Biochem Mol Biol. 143(2), 133-7.
Merchant ME, Verret B, Elsey RM. (2005). “Role of divalent metal ions in serum
complement activity of the American alligator (Alligator mississippiensis).” Comp
Biochm Physiol B Biochem Mol Biol. 141(3), 289-93.
Merchant ME, Roche CM, Thibodeaux D, Elsey RM. (2005). “Identification of
alternative pathway serum complement activity in the blood of the American alligator
(Alligator mississippiensis).” Comp Biochem Physiol B Biochem Mol Biol. 141(3), 281-8.
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10. Merchant ME, Pallansch M, Paulman RL, Wells JB, Nalca A, Ptak R. (2005). “Antiviral
activity of serum from the American alligator (Alligator mississippiensis).” Antiviral Res.
66(1), 35-8.
Merchant M, Thibodeaux D, Loubser K, Elsey RM. (2004). “Amoebacidal effects of
serum from the American alligator (Alligator mississippiensis).” J Parasitol. 90(6),
1480-3.
Merchant ME, Roche C, Elsey RM, Prudhomme J. (2003). “Antibacterial properties of
serum from the American alligator (Alligator mississippiensis).” Comp Biochem Physiol
B Biochem Mol Biol. 136(3), 505-13.
Beven L, Chaloin L, Vidal P, Heitz F, Wroblewski H. (1997). “Effects on mollicutes
(wall-less bacteria) of synthetic peptides comprising a signal peptide or a membrane
fusion peptide, and a nuclear localization sequence (NLS) – a comparison with melittin.”
Biochim Biophys Acta. 1329(2), 357-69.
Goldsby RA, Kindt TJ, Osborne BA, Kuby J. (2003). Immunology. New York, NY: W.H.
Freeman and Company.
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