Summer Undergraduate Research Program - Fred Hutchinson ...

Cloning of HIV-1 env genes from Kenyan women to examine role of neutralizing antibodies
in breast milk mother-to-child transmission
Amber Peden, Jennifer Maroa, Julie Overbaugh
Fred Hutchinson Cancer Research Center, Seattle, WA
Abstract Methodology Results
controls
patient env
2010 Lee Hartwell Poster Award
Figure 7. PCR product of donor env.
Study Cohort: The subjects were from a randomized breastfeeding clinical trial
conducted between 1992-1998 in Nairobi, Kenya. HIV-1 infected women were
enrolled in the clinical trial at 32 weeks gestation. BM and peripheral blood samples
were collected from women at set time points for up to two years. Sixteen women
with an early BM sample were selected for the initial screen of BM Nabs.
patient env controls
Figure 8. Donor env after gel
isolation and PCR purification.
HIV-1 env cloning
Figure 3. HIV-1 genome. The env
gene, codes for vital HIV-1 coat
proteins crucial in mediating CD4
and chemokine receptor binding
and membrane fusion.
Mother-to-child transmission (MTCT) of human immunodeficiency virus type-1 (HIV-1)
can occur in utero, during delivery and during breastfeeding (BF). In resource-limited
countries BF continues to be the preferred mode of infant feeding resulting in, more
than one-third of MTCT cases. Interestingly, two thirds of the infants remain uninfected
despite continued exposure to the virus. The mechanism by which these infants are
protected remains unclear. Neutralizing antibodies (Nabs) act by blocking the initial
virus-target cell interaction and therefore are of great interest to researchers in an effort
to develop an effective HIV vaccine. Although antibodies have been detected in breast
milk (BM) there has been no detailed study that examines the presence and correlation
of levels of Nabs in breast milk with rate of BM transmission. To examine the role of
Nabs, we attempted to clone HIV-1 env genes from HIV-1 seropositive mothers in a
breastfeeding clinical trial in Nairobi, Kenya with the prospect of examining their
neutralization sensitivities in an in vitro neutralization assay.
cut a uncut a cut b
Figure 9. Screening of recombinant
plasmid shows successful insertion
of donor env.
uncut b
insert
Background
No insert
We attempted amplification of env from three different individuals. The results shown
above are from one individual which yielded a successful clone.
Challenges
Figure 1. Adults and
children living with
HIV. Approximately
70% of people living
with HIV reside in Subsaharan
Africa
(UNAIDS, 2008).
153
HIV portrays genetic diversity within and across clades, and also within individuals. This
was the major hindrance of our DNA cloning. Mutations in the env gene of donor DNA
caused primer mismatching, prohibiting proper amplification and restriction enzyme
digestion thereafter.
MTCT accounted for approximately 20% of new HIV infections in 2008 and
accounts for approximately 1,200 new child infections everyday (UNAIDS,
2008).
Figure 4. Cloning patient env genes. Nested PCR of patient DNA using specific primers allows for selective
amplification of env. Once multiple copies are produced, DNA is digested with restriction enzymes that
produce fragments with sticky ends. Plasmid DNA and donor DNA is then mixed and ligated with DNA ligase
to yield recombinant plasmid. Recombinant molecules are replicated in E.coli to give a population of clones
that are used to create pseudoviruses.
Figure 10. DNA sequences of donor, PCR primers and env region of full-length HIV-1.
Shows comparison of DNA sequences between second round PCR primers and second
round PCR product indicating a mismatch.
Formula feeding has been successful in developed countries but its
implementation in Sub-Saharan Africa has been met with several challenges –
cultural, cost, safety and increase in child mortality.
Antiretroviral drugs significantly reduce MTCT prevention however the benefit
of short course interventions against BM transmission is minimal.
Future Directions
Figure 5. Making pseudoviruses. A mammalian cell line is transfected with our donor recombinant
plasmid and a plasmid containing the HIV-1 backbone genetically void of env to produce a HIV-1
pseudovirus.
~60% of BF infants remain uninfected despite continued exposure to HIV.
Investigating how BM protects infants from HIV infection requires an
examination of its immune factors.
Re-design DNA PCR primers.
Generate more patient clones from different PCRs.
Test functionality of inserts.
Make pseudoviruses and perform neutralization assays.
Nabs have the ability to block initial virus-target cell interactions and if present
in BM, may potentially alter the risk of BF transmission.
References
env
Kuhn, Louise, et al. "HIV prevention is not enough: child survival in the context of prevention of
mother-to-child HIV transmission." Journal of the International AIDS society (2009).
Funding Source:
Continuing Umbrella of Research Experience CURE
(3 P30 CA015704-36S2/5 P30 CA015704-35S)
Second Contributor
Walmart College Success Award
Phogat, S., R. T. Wyatt and G. B. Karlsson Hedestam. "Inhibition of HIV-1 entry by antibodies:
potential viral and cellular targets." Journal of Internal Medicine (2007): 26-43.
Objective: To clone env
genes from HIV-positive
women which will be
employed in in vitro assays
to determine the presence
and potency of Nabs in BM.
UNAIDS. "2009 AIDS epidemic update." 2009.
Figure 6. Neutralization assay using TZM-bl cells as targets. Psuedoviruses are incubated with the
breastmilk sample of interest followed by addition of TZM-bl cells that have a beta-galatosidase reporter
gene under control of HIV-1 LTR. Reporter gene expression was quantified by luminescence upon lysis and
addition of substrate.
Figure 2. Antibodies can block the virustarget-cell
interaction.