RESIDENT RESEARCH APPLICATION FORM
Project Title: Cloning and Expression of Novel P.
falciparum Vaccine Candidate Antigens
Amount Requested: $ 2,000
Resident:
Christian P. Nixon
Title:
Resident Physician 1
Affiliation:
Laboratory Medicine
Email:
Christian.Nixon@ucsfmedctr.org
Box No.
0506
Phone:
401-787-1723
Program
Director:
Enrique Terrazas
Title:
Associate Clinical Professor
Affiliation:
Laboratory Medicine
Email:
Terrazas@labmed2.ucsf.edu
Box No.
0100
Phone:
415-514-8931
Associate Professor in
Mentor:
Scott Kogan
Title:
Affiliation:
Laboratory Medicine
Email:
Scott.Kogan@ucsf.edu
Box No.
0451
Phone:
514-5190
Residence
Mentoring Relationship (mentor’s role in the project and your career development; ½ page
maximum)
This research will be performed in the laboratory of Dr. Scott Kogan. Dr. Scott
Kogan is not only a mentor in the clinical pathology residency program but will
undoubtedly prove to be a great mentor in the laboratory as well.
The differential screen detailed in the research proposal below is a project that
was recently completed during a research elective at the Center for International Health
Research in Providence RI. The project was completed in two months and has provided
ample material for study that can readily be continued here at UCSF. The methodology
employed in the screen was an extension of my graduate thesis work that was involved in
identifying novel vaccine candidate antigens for falciparum malaria. The fundamental
difference is that the prior work focused on adults, whereas the current screen was
performed with sera and epidemiologic data from 2-3 year old children from a Tanzanian
cohort. This is the patient population most affected by malaria and the one in which a
vaccine would be most beneficial.
During my fellowship year after residency, I plan on applying for a K award.
Successfully competing for and being awarded small grants such as the resident grants is
critical for maximizing the likelihood that I will be able to compete for NIH funding. In
addition, it is imperative that I not only get these grants, but that I complete the tasks
outlined in the specific aims within the budgetary restrictions and in a timely fashion, to
help set a precedent for future research activities.
Research Proposal (aims and description of pilot project or planning activities; 1 page
maximum)
Malaria is a deadly disease that can found in over 107 countries throughout the
globe. There are approximately 200-300 million clinical cases of malaria each year
resulting in 1-2 million deaths (1). African children under of the age of 5, particularly
those in sub-Saharan Africa, bear the brunt of the morbidity and mortality attributable to
this disease (2). Due to the gradual spread of drug resistant parasites and insecticide
impervious mosquitoes, an effective vaccine is urgently required to augment existing
control measures (3).
Naturally acquired resistance to falciparum malaria develops in residents of
endemic areas. This protective immunity limits parasitemia and disease, providing a
model for vaccine development. Although the antigens and specific immune responses
conferring naturally acquired resistance have not been thoroughly elucidated, antiparasite antibodies have been hypothesized to play a role in this immunity as
demonstrated by ex vivo parasite growth inhibition assays (4) and adoptive transfer
experiments (5).
We have utilized these observations in conjunction with data and sera previously
collected from a longitudinal birth study cohort in Muheza, Tanzania to perform a whole
proteome screen to identify antigens associated with resistance. Using plasma obtained
from maximally resistant and susceptible individuals from this cohort, we have
performed a differential screen of a P. falciparum 3D7 asynchronous cDNA expression
library. These two groups of sera were derived by rank ordering the 465 children in the
cohort by parasitologic data. This data was collected from weekly blood film that had
been recorded for each child from birth to three years of age. The study participants with
low levels of parasitemia were classified as resistant and those with high levels of
parasitemia were classified as susceptible. After matching for a number of variables
including age, gender, sickle trait, and entomological inoculation rate, ten individuals
each were then chosen to comprise the maximally resistant and susceptible groups.
750,000 clones were screened with these two groups of sera. Although a number of
clones were recognized by both sets of sera, only four clones were recognized by
resistant sera alone.
The identity of these four clones by DNA sequencing analysis corresponded to a
carboxy-terminal portion of merozoite surface protein # 7 (MSP-7), an unidentified
protein on the P. falciparum chromosome #10 (identified twice) and an unidentified
protein on P. falciparum chromosome # 11. These clone represent rationally identified
vaccine candidates that are recognized by host antibodies that mediate resistance to
parasitemia.
Specific Aim # 1 : Cloning of the three uniquely recognized antigens.
To investigate the correlates of protection and iso-type specific antibody
responses to the antigens identified in the differential screen throughout the entire cohort,
these antigens will first be sub-cloned into an expression vector. Ligation independent
cloning will allow for transfer of the identified antigens from a pBluescript vector into a
pET32Xa vector. This allows for protein induction with IPTG and adds a thioredoxin tag
to the N-terminus of the antigen for enhanced solubility in liquid media. In addition, this
vector also attaches a His-tag to the expressed antigen to facilitate Nickel capture in
subsequent chromatographic purification steps.
Specific Aim #2: Expression of the cloned antigens in a bacterial expression strain.
After these antigens have been cloned, they will be expressed in a suitable
bacterial expression strain such as E. Coli (BL21). Antigen expression will be optimized
through variance of induction time and temperatures, IPTG concentrations and different
culture media. Once antigen concentrations of sufficient quantity can be detected in
induced bacterial lysate, the vectors and optimized expression protocol will be returned to
the Center for International Health in Providence, RI for large scale antigen prep and
chromatographic purification.
References (up to 5)
1. Anonymous. 1997. World malaria situation in 1994. Part 1. Population at risk.
Weekly Epidemiological Record 72:269-274.
2. Breman, J.G., A. Egan, and G.T. Keusch. 2001. The intolerable burden of
malaria: a new look at the numbers. Am J Trop Med Hyg 64:iv-vii.
3. White , N.J., F. Nosten, S. Looareesuwan, W.M. Watkins, K. Marsh, R.W. Snow,
G. Kokwaro, J. Ouma, T.T. Hien, M.E. Molyneux, T.E. Taylor, C.I. Newbold,
T.K. Ruebush, 2nd, M. Danis, B.M. Greenwood, R.M. Anderson, and P. Olliaro.
1999. Averting a malaria disaster. Lancet 353:1965-1967.
4. Bouharoun-Tayoun, H., P. Attanah, A. Sabchareon, T. Chongsuphajaisiddhi, and
P. Druilhe. 1990. Antibodies that protect humans against Plasmodium falciparum
blood stages do not on their own inhibit parasite growth and invasion in vitro, but
act in cooperation with monocytes. J Exp Med 172:1633-1641.
5. Sabchareon, A., T. Burnouf, D. Ouattara, P. Attanah, H. Bouharoun-Tayoun, P.
Chantavanich, C. Foucalt, T. Chongsuphajaisiddhi, and P. Druilhe. 1991.
Parasitologic and clinical human responses to imunoglobin administration in
falciparum malaria. Am J Trop Med Hyg 45:297-308.
Timeline and Milestones (1/2 page maximum)
The cloning of the aforementioned antigens will take place during November and
December of 2008. The expression of the cloned antigens will proceed from January to
March 2009. Successful protein expression will be documented with protein gels of
uninduced and induced bacterial lysate. The size of the expressed antigens will be crossreferenced with the predicted size from the amino acid sequence. Budget permitting, the
antigens will also be prepared for protein sequencing.
Project Performance/Site Locations (1/2 page maximum)
The site location is the laboratory of Dr. Scott Kogan. Details of project and performance
of research objectives will be reported to both Dr. Scott Kogan and his laboratory
personnel.
Human Subjects
Will human subjects be used:
Yes X No
If yes, please supply the CHR date of approval
N/A
00/00/00
and approval number:
Pending
Budget ($2,000 maximum per resident)
Proposed Budget
Supplies and Expenses
Amount
2,000.00
Equipment
Travel
Human studies costs
Other: (state)
Total
2,000.00
Future Full Project Funding
Budget Details and Justification (1/2 page maximum)
The reagents that will be purchased for these experiments include the following.
Luria Broth powder, ampicillin, carbenicillin, and IPTG for culture. Glycerol stocks of
E. Coli expression strains BL21 and other variants such as BL21star if required. A
pET32Xa ligation independent cloning kit will be purchased as the expression vector for
the antigens. Primers will be ordered to amplify the inserts from the current pBluescript
vector. The PCR products will be purified with a Qiagen PCR purification kit. The
pET32Xa vectors with cloned inserts will be transformed into E.Coli DE3 cell line and
DNA isolated with a Qiagen maxi prep kit. Protein gel supplies such as APS, TEMED,
and acrylamide will be ordered. Gel Code Blue will be purchased as the protein gel
staining reagent. Protein sequencing will be ordered, budget permitting.
Letter of Support (required)
Resident’s CV (required)
Other
Have you successfully completed the Resident Research Elective (Epi 150.03) or the Summer
Research Methods Course (Epi 202)? Yes
No X
Have you applied to other funding sources for this project? Yes
No X