Transferring Adaptive Immunity Research Proposal Kurt

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Transferring Adaptive ImmunityKurt Whittemore

The ability to transfer acquired immunity (adaptive immunity) from one individual toanother would save countless lives. This concept has been around for quite some time, andis termed adoptive immunity. However, there are some problems with current adoptiveimmunity techniques. Immune cells transferred from one individual to another are often notrecognized by the patient's immune system and will be attacked. Alternatively, the foreignimmune cells may attack the patient. These problems can be overcome by geneticallyengineering the memory B cells from a patient so that they possess the same antibody codingregion as the memory B cells from another individual. These cells would be recognized bythe patient's immune system, and these cells would also be able to fend off the samediseases that the immune B cells could.

Table of Contents

Research Plan pg. 2Specific Aims pg. 2

Significance pg. 3Innovation pg. 4Approach pg. 4

Bibliography and References Cited pg. 11

Budget pg. 13

Biographical Sketch pg. 14

Facilities and Other Resources pg. 16


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Research PlanSignificance

Currently the only widely used way to prevent illness caused by pathogens that are notrecognized by the innate immune system is to vaccinate against that pathogen. Thesevaccines expose the adaptive immune system to targets which appear similar to pathogens,and an immune response can be mounted against the real pathogen when presented.

Vaccines are very effective, but they can also have side effects. Some patients are often soreafter a vaccine, or may even have more severe symptoms. Different patients also havedifferent variable (V), joining (J), and constant (C) regions, which means that some individualsmight be able to mount an immune response against a particular pathogen better thananother individual. While vaccines have definitely proved their worth, they should not be theonly way of conferring immunity.

InnovationThe hypothesis being tested is that it is possible to transfer the acquired adaptive

immunity from one organism to another. One major component of adaptive immunity is thestructure of the antibodies produced which bind to certain pathogens. Since the structure ofthe antibodies produced against pathogens encountered earlier in life is encoded within the

genome of memory B cells, it is possible to transfer this code to the memory B cells ofanother organism. These modified B cells would be returned to the non-immune organism,which would then have the ability to produce antibodies against the pathogen upon exposure.

Specific Aims

Aim 1: Determine whether memory B cells modified with the antibody coding regionfrom the memory B cells of another mouse exposed to influenza are able to conferimmunity to the original nave mouse. The antibody coding regions from memory B cellsisolated from a mouse that has been vaccinated against influenza will be transferred into theantibody coding region of memory B cells isolated from a nave mouse that has never beenexposed to the virus. These modified B cells will be transferred back into the nave mouse,which will then be challenged with a lethal strain of influenza.

Aim 2: Determine whether B cells genetically modified in this way exhibit expectedbehavior in vitro. Memory B cells modified in the same manner as the B cells used totransfer immunity between mice will be tested for certain properties. We hypothesize thatthese B cells will behave in a similar manner as normal B cells. The properties tested willinclude the number of possible cell divisions to determine the Hayflick limit, the ability toproduce antibodies which bind to the expected antigen, and the intactness of the genome.

Through the set of experiments outlined in this proposal, it should be possible todetermine whether it is feasible to transfer adaptive immunity from one organism to another.The idea of adoptive immunity is not new. However, the methods outlined here may provide away to avoid the problems of graft versus host disease and host versus graft problems thatcan arise with traditional adoptive immunity techniques. Even if the technique outlined heresuccessfully transfers immunity from one host to another, however, such results do notguarantee that the modified B cells are safe. Several other experiments will be performed totest whether the genetically modified B cells exhibit normal behavior.

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Research Strategy

Significance

Currently the main method for conferring immunity on an individual is to perform avaccine. A vaccine exposes the immune system to a weakened version of a pathogen or a

component of the pathogen so that it can learn to recognize the pathogen at a later time whena response is truly needed. Although vaccines have been tremendously effective over thelast several decades, they still have several shortcomings. The immune system of someindividuals can react very harshly to certain vaccines causing stress for the host. Sometimesthe pathogen may not have been weakened enough and even cause the host to acquire thedisease. The number of vaccine adverse events, both those caused by vaccines and thoseassociated with vaccines, is approximately 11,000 per year, which is more than the number ofvaccine preventable childhood disease cases combined ([1]). Many adverse events areminor inconveniences such as a fever. However, there can also be much more seriouscomplications. Some examples include acute encephalopathy after whole cell pertussisvaccine , GuillainBarre syndrome (GBS) after swine influenza vaccine , and vaccine-

associated paralytic polio (VAPP) ([1]). Subunit vaccines offer one way to make vaccinessafer, but there may be even better ways of providing immunity to an individual.The concept of adoptive immunity involves transferring the immunity possessed by one

individual to another without arousing a potentially harmful immune response from theindividual. The basic idea is to take cells or antibodies often contained in the blood from anindividual immune to a certain disease and transfuse it into an individual that is not immune tothat disease. While this technique is not widely used, it has been instituted as a treatment inmany cases. For example, one research group has shown promise in conferring hepatitis Bimmunity by transplanting a liver from a patient who has been vaccinated against hepatits B([2]). There are also many other forms of adoptive immunity techniques that have been tried.For example, one group was able to immunize mice against a certain pathogen. They then

re-engineered the antibodies produced by these mice to contain human constant antibodyregions. Such engineering would allow these mouse antibodies to be used in a human whilepreventing the human immune system from reacting adversely to the mouse antibody ([3]).

There are some major barriers to overcome before adoptive immunity techniquesbecome widely used. One barrier is that the host may recognize the foreign immune cells asthe foreign cells that they are and attack them. In this case, no immunity has beentransferred and the treatment is unsuccessful. In perhaps an even more serious situation,graft versus host disease can develop. The foreign immune cells recognize the patient's bodyas foreign and begin attacking the host. In order to attain a safer method of adoptiveimmunity, some novel approaches will need to be developed.

One novel approach involves genetically engineering the B cells of a patient rather

than transferring cells or antibodies from another individual. Genetically engineering this DNAcan be considered a type of gene therapy and will face many of the same challenges. Themain problem will be to avoid non-specific integration of the gene into the genome. If thegene integrates non-specifically, gene networks can be interrupted. Interruption of genes inthis manner often leads to cancer. For example, gene therapy trials aimed at treating severecombined immunodeficiency resulted in the development of cancer in the patients ([4]).Overcoming some of the problems associated with gene therapy will be critical to the

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development of a new method of transferring immunity.

Innovation

In order to gain the benefits of adoptive immunity and eliminate the disadvantages, anovel approach is suggested. This approach is to genetically engineer B cells from the

patient to contain the antibody coding region of an immune individual. The non-immunepatient would start with a normal immune system, but would lack previous exposure with thepathogen. Using this method, the patient would not recognize the B cells as foreign sincethese B cells would have originated from the patient originally. The B cells should alsotransfer immunity to the patient since these B cells produce the same antibodies that wereproduced in the body of the immune individual. Genetically engineering B cells in this mannerwould not have been very feasible at an earlier time. However, the molecular biology toolsneeded to accomplish this goal are now in place.

One of the key techniques which will allow this strategy to become a reality is thetechnique of using zinc finger proteins to integrate DNA into a specific location of the genomeof a cell. Specifically, integrase deficient lentiviruses will deliver the DNA along with zincfinger nucleases capable of inserting the antibody coding region into a specific location in thegenome ([5]). This technique has already been used with some success to genetically modifyhuman stem cells. Researchers were able to obtain a high rate of gene addition into cells upto 50%. Combining such techniques with adoptive immunity approaches should providemedical professionals with new innovative ways of preventing rather than treating illness.

Approach

The basic approach we will explore with this research involves challenging mice withinfluenza. The antibody coding region from the memory B cells of these mice will then betransferred into the genome of memory B cells from nave mice which have not been exposedto the virus. These mice will then be challenged with the pathogen to test whether the micehave developed an immunity to influenza as expected.

Regardless of whether or not the genetically modified B cells are able to conferimmunity on the nave mice, it is important to test the behavior of these cells in vitro. Forexample, if the antibody coding region is inserted non-specifically into the genome, the B cellsmay still produce anti-influenza antibody and confer immunity. However, after a period oftime, they may also become cancerous and cause severe problems for the mice. If themodified B cells do not confer immunity, it is still important to test their behavior in vitro as itmay reveal why they did not behave as expected. Such information may also suggest how toimprove the design of these B cells.

Aim 1: Determine whether memory B cells modified with the antibody coding regionfrom the memory B cells of another mouse exposed to influenza are able to conferimmunity to the original mouse.

Strategy

In order to transfer immunity from one mouse to another several steps must be taken.First the memory B cells must be isolated from a mouse that has immunity to a particular

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disease. For the purposes of this proposal, influenza will be the model disease. The antibodycoding region found in the memory B cells of this mouse will then be cloned. This fragment ofDNA will be used to replace the antibody coding region in memory B cells of a mouse that hasnever been exposed to influenza (Figure 1). This mouse will be challenged with influenza,and the survival rates and viral titers will be determined. The expectation is that the survivalrates and viral titers of the mice receiving this treatment will be much improved compared to a

control group.

Figure 1: Illustration of the concept of transferring the acquired adaptive immunity from onemouse to another. The antibody coding region from the memory B cells of a mouse that hasbeen exposed to influenza is cloned into the corresponding region in the genome of the non-immune mouse.

Methodology

These experiments will make use of three groups of five mice. One group of mice willbe vaccinated against the HK/483 strain of the influenza virus, which is a very lethal strain ofthe virus. Vaccination with the influenza virus produces both a cellular as well as a humoralresponse in the host ([6]) so there should be no trouble acquiring memory B cells specific tothe virus. Another group will not be vaccinated against the virus, but will receive geneticallymodified versions of their own B cells. These B cells will be modified to contain the antibody

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coding regions from the vaccinated mice. The third group of mice will act as a control and willnot receive a vaccine or genetically modified B cells. At the end of the experiment, all threegroups of mice will be challenged with the influenza virus and survival rates will bedetermined (Figure 2).

Figure 2: Experimental setup. Five mice will be immunized with the influenza vaccine. Thenon-immune group will not be immunized, but will receive genetically engineered B cells fromthe immune group. The control group will not receive a vaccine or genetically engineered Bcells.

Memory B cells will be isolated from the mice using fluorescence activated cell sorting(FACS). Blood sera will first be collected from the mice. Then the cells in the sera will beseparated using FACS which is a type of flow cytometry capable of separating cells based onfluorescence. In this case, memory B cells will fluoresce after they are bound to an antibodywhich binds the memory B cell marker CD27 ([7]). Note that no attempt will be made toisolate B cells which are specific for the vaccinated antigen. While this approach would beideal, such a procedure would be more time consuming and complicated. For our purposes,it will be sufficient to isolate memory B cells specific for many different antigens and transfertheir genes into the memory B cells of the patient animals receiving treatment. Therefore, the

animals receiving treatment will receive immunity not only to influenza, but to any diseasewhich the donor mice may have been exposed to. This beneficial artifact of the method couldprove detrimental if the donor mice have any memory B cells which produce antibody to a selfantigen or a non-self antigen that is present in the natural environment of the patient mice. Inthis situation, the patient mice would develop an autoimmune disease. However, the donormice chosen for this experiment will not be autoimmune disease models so this should not bea problem. Once these memory B cells are isolated, the antibody coding region from the

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immune mice can be cloned.The DNA that codes for the antibodies in the immune mice will be cloned into the B

cells of the non-immune mice. There are actually two antibody coding regions that must becloned since there is one region for the heavy chain on chromosome 14 and one region forthe lambda light chain on chromosome 22 ([8])([9]). These regions of DNA will be clonedusing a PCR technique with universal antibody primers. Although antibodies have variable

regions with different DNA sequences in each cell, there are conserved sequences which canbe used to amplify the whole rearranged antibody coding region. The universal primers usedin these experiments were developed by Dattamajumdar et al ([10]). After the PCR reaction,the DNA fragments will be sent to a sequencing lab to identify the sequence of the antibodyas well as confirm that the correct antibody coding region was cloned.

This cloned fragment will then be inserted into the genome of memory B cells from thenon-immune mice which are mice that have not been exposed to the influenza virus. This willbe accomplished using an integrase deficient lentivirus with zinc finger nucleases. These zincfinger nucleases will be specific for the antibody coding region of the genome. The antibodycoding regions obtained from the PCR with the universal antibody primers will also bedesigned in such a way that they will be homologous with the part of the genome where theantibody coding region of the genome is located. These features will allow the zinc fingernucleases to integrate the cloned antibody coding region into the genome in a specificmanner, avoiding the possibility of non-specific, gene interrupting, cancer causing geneintegrations.

Once the B cells from the non-immune mice have been genetically modified, they willbe injected back into the non-immune mice from which they came. At this point, all of themice will be challenged with the lethal HK/483 influenza virus intranasally. All three groupswill be monitored and a qualitative assessment of their health will be recorded. After 14 days,two mice from each group will be euthanized and the lung tissue which the influenza virusinfects will be collected. This lung tissue will be used to quantitatively measure the amount ofvirus in the tissue. A standard ELISA experiment will be used with an anti-Tern/SouthAfrica/61 (H5N3) antibody which recognizes both surface glycoproteins and internal proteinsof the virus ([11]). We expect that the vaccinated group of mice and the geneticallyengineered B cell group of mice will have high titers against the virus, whereas the controlgroup will have very low titers.

In addition to the viral titer, the anti-influenza antibody titer of each group will also bemeasured. Serum will be collected periodically from mice in each group to measure animmune response over time. This antibody containing serum will then be used in an ELISAwith plates coated with the HK/483 influenza virus. The anti-influenza antibody titer of eachgroup can then be determined and compared to the expected results. The expected resultsare that the vaccinated mice will have the highest titer and the control group will have thelowest titer.

There are several problems which may be encountered during these experiments.However, alternative methods can be used to circumvent any major problems. If it turns outto be too difficult to successfully transfer DNA using the integrase deficient lentivirus zincfinger approach, then a traditional mammalian plasmid could be used instead. Zinc fingertechnology is relatively new, and we may find that successfully integrating the gene into thecorrect location of the genome may not be possible. While using a mammalian plasmid is notideal for long term treatments, such a strategy should suffice for the purposes of this specificaim. The reason using a mammalian plasmid would not be ideal for long term treatments is

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that plasmid DNA is not as stable as DNA in the genome and can degrade over time. If thereare problems isolating B cells using the CD27 marker, then some of the other markersidentified by Kuppers can be used ([12]). When assessing the viral titers, they may be too lowto detect. In this circumstance, other detection methods such as the ConSense techniquecan be used which takes advantage of confocal microscopy to determine extremely low titers.Although these methods are good backup strategies to have in mind, it is expected that the

original plans should serve well.

Aim 2: Determine whether B cells genetically modified in this way exhibit expectedbehavior in vitro.

Strategy

The strategy for testing the viability of genetically engineered B cells in vitro involvesmeasuring a few specific traits. The B cells used will be obtained using the same integrasedeficient lentivirus zinc finger technique outlined in specific aim 1. If the zinc finger approachdoes not seem to work, then the B cells will be transformed using a mammalian plasmidcontaining the antibody coding regions. Such an alternative strategy will be necessary if aPCR experiment to check whether the antibody coding region from the influenza exposedmice has been inserted into the genome of the B cells in the nave mice returns a negativeresult. Once the modified B cells are created by either approach, there are three traits thatwill be measured. The first measure will be to determine the number of times the cells candivide. The second measure will be their antibody titer. The last measure will be to verify thatthe DNA is intact, and verify that the gene has not been inserted into multiple locations in thegenome.

Methodology

Several different methods will be used to measure traits associated with a normal Bcell. The Hayflick limit of a cell is the number of times the cell is capable of dividing beforereaching cellular senescence. The Hayflick limit of these cells will be determined usingtraditional cell culture techniques of the B cells. The Hayflick limit of a cell is reached oncethe ends of chromosomes called telomeres which shorten with each cell division reach acritically short length. Although, B cells express telomerase when proliferating to lengthentelomeres, this telomerase expression is not sufficient to maintain telomere length. Thetelomeres of memory B cells specifically shorten at a rate of 40 bp/year in vivo ([13]). In cellculture, B cells will reach the end of their proliferative capacity much more rapidly. If the Bcells have become cancerous, they will divide indefinitely. However, if there are no problemswith the genetic alterations undertaken by our research group, then the number of celldivisions should be within the range of cell divisions exhibited by a control group of normal Bcells.

The antibody titer of the B cells will be determined by performing a traditional ELISA inwhich the surface of the ELISA plate is coated with influenza virus. The B cell solution will beapplied to the ELISA plate, and then incubated with anti-IgG secondary detection antibodyconjugated to a detection enzyme such as horse radish peroxidaxe (HRP). In order for the Bcells to begin proliferate and differentiate into short lived plasma cells capable of antibodyproduction, the B cells must be incubated with T cells in addition to the antigen ([14]). Only

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some T cells will be specific for the antigen. In our experiment, we will collect a wide range ofT cells possessing different specificities using the same FACS method used to isolate the Bcells. These T cells will then be incubated with the memory B cells, and some of them willactivate them resulting in antibody production. While it is possible that the antibodiesproduced against the influenza were not of the IgG isotype, this isotype will be checked first.If no significant titer is found above background detection levels, then other isotypes will be

checked. Memory B cells can in fact produce antibodies of several different isotypes asdemonstrated by Wu et al. ([15]). The titer of anti-influenza antibody from these geneticallyengineered B cells is expected to be much higher than the titer of the same B cell solutionchecked against a negative control protein.

In order to assess whether the DNA is intact, several techniques will be used. Thechromosome number of the cells will be measured using standard cytogenetic techniques toacquire a karyogram. If the antibody coding region inserted into the genome non-specifically,then the chromosomes may become unstable and rearrangements could occur, which wouldresult in abnormally sized chromosomes. The location of the integrated genes will also bechecked. The method used to check this will be similar to the method used to produce cDNAfrom mRNA ([16]). Primers which bind to the antibody coding sequence will be used alongwith random hexamer primers. Some of these random hexamer primers will land on locationsin the genome downstream from one of the antibody coding region. The resulting PCRproduct will contain the sequence of the antibody coding region in addition to some genomicsequence which can be used to identify the location of the insertion site of the antibodycoding region. Ideally, the gene will only have integrated in the intended antibody codingregion location.

All of these procedures are well established so there should not be any major hurdlesand the information gained will be valuable.

Figure 3: Timeline of Experiments.

Summary

After these experiments have been completed, two major questions will have beenanswered. The first question is whether it is possible to transfer acquired immunity from onemouse to another using genetically engineered B cells. The second question that will havebeen answered is whether or not these B cells appear normal, and if it seems likely that theycould be used for long term treatments. It should be possible to address these two questionswithin a time frame of two or three years as illustrated in the figure below. In the first year and

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AimsMonths

0-6 6-12 12-18 18-24 24-30 30-36

Mice Vaccination

Isolate B cells using FACS

Transform B cells

Specific Aim 1Challenge mice with virus

Analyze results of challenge

Specific Aim 2

Specific Aims1 and 2

Genetically Engineer Viruswith Zinc fingers

Transfer B cells into navemice

Characterize B cells: Hayflicklimit, antibody titer, karyogram


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a half, the genetically engineered B cells will be created. These B cells can then be used forthe two different specific aims. Testing the transfer of immunity and the characterization of theB cells can be performed in parallel during the last year and a half of research. If ourresearch is successful, many diseases may be cured by preventing patients from becoming illto begin with.

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Bibliography and References Cited

[1] R.T. Chen, Vaccine risks: real, perceived and unknown, Vaccine, vol. 17 Suppl 3, Oct. 1999,

pp. S41-46.

[2] A. Schumann, M. Lindemann, C. Valentin-Gamazo, M. Lu, A. Elmaagacli, U. Dahmen, D. Knop,

C.E. Broelsch, H. Grosse-Wilde, M. Roggendorf, and M. Fiedler, Adoptive immune transfer of

hepatitis B virus specific immunity from immunized living liver donors to liver recipients,Transplantation, vol. 87, Jan. 2009, pp. 103-111.

[3] B.G. Sahagan, A genetically engineered murine/human chimeric antibody retains specificity forhuman tumor-associated antigen,Journal of Immunology (Baltimore, Md.: 1950), vol. 145, Nov.

1990, p. 3150.

[4] S. Hacein-Bey-Abina, C. Von Kalle, M. Schmidt, M.P. McCormack, N. Wulffraat, P. Leboulch, A.Lim, C.S. Osborne, R. Pawliuk, E. Morillon, R. Sorensen, A. Forster, P. Fraser, J.I. Cohen, G. de

Saint Basile, I. Alexander, U. Wintergerst, T. Frebourg, A. Aurias, D. Stoppa-Lyonnet, S. Romana,

I. Radford-Weiss, F. Gross, F. Valensi, E. Delabesse, E. Macintyre, F. Sigaux, J. Soulier, L.E.Leiva, M. Wissler, C. Prinz, T.H. Rabbitts, F. Le Deist, A. Fischer, and M. Cavazzana-Calvo,

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1,

Science (New York, N.Y.), vol. 302, Oct. 2003, pp. 415-419.[5] Y. Okada, Y. Ueshin, H. Hasuwa, K. Takumi, M. Okabe, and M. Ikawa, Targeted gene

modification in mouse ES cells using integrase-defective lentiviral vectors, Genesis (New York,

N.Y.: 2000), vol. 47, Apr. 2009, pp. 217-223.

[6] L. Danziger-Isakov, L. Cherkassky, H. Siegel, M. McManamon, K. Kramer, M. Budev, D.Sawinski, J.J. Augustine, D.E. Hricik, R. Fairchild, P.S. Heeger, and E.D. Poggio, Effects of

Influenza Immunization on Humoral and Cellular Alloreactivity in Humans, Transplantation,

Feb. 2010.[7] K. Agematsu, S. Hokibara, H. Nagumo, and A. Komiyama, CD27: a memory B-cell marker,

Immunology Today , vol. 21, May. 2000, pp. 204-206.

[8] I.M. Tomlinson, G.P. Cook, G. Walter, N.P. Carter, H. Riethman, L. Buluwela, T.H. Rabbitts, and

G. Winter, A complete map of the human immunoglobulin VH locus,Annals of the New YorkAcademy of Sciences, vol. 764, Sep. 1995, pp. 43-46.

[9] J. Erikson, J. Martinis, and C.M. Croce, Assignment of the genes for human lambda

immunoglobulin chains to chromosome 22,Nature, vol. 294, Nov. 1981, pp. 173-175.[10] A.K. Dattamajumdar, D.P. Jacobson, L.E. Hood, and G.E. Osman, Rapid cloning of any

rearranged mouse immunoglobulin variable genes,Immunogenetics, vol. 43, 1996, pp. 141-151.

[11] X. Lu, T.M. Tumpey, T. Morken, S.R. Zaki, N.J. Cox, and J.M. Katz, A mouse model for theevaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans,

Journal of Virology, vol. 73, Jul. 1999, pp. 5903-5911.

[12] R. Kppers, Human memory B cells: memory B cells of a special kind,Immunology and CellBiology, vol. 86, Dec. 2008, pp. 635-636.

[13] N.H. Son, B. Joyce, A. Hieatt, F.J. Chrest, J. Yanovski, and N. Weng, Stable telomere length andtelomerase expression from nave to memory B-lymphocyte differentiation, Mechanisms ofAgeing and Development, vol. 124, Apr. 2003, pp. 427-432.

[14] A.F. Ochsenbein, D.D. Pinschewer, S. Sierro, E. Horvath, H. Hengartner, and R.M. Zinkernagel,

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of

long-lived memory B cells to short-lived plasma cells independent of secondary lymphoidorgans,Proceedings of the National Academy of Sciences of the United States of America , vol.

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97, Nov. 2000, pp. 13263-13268.

[15] C.J. Wu, J.T. Karttunen, D.H. Chin, D. Sen, and W. Gilbert, Murine memory B cells are multi-

isotype expressors,Immunology, vol. 72, Jan. 1991, pp. 48-55.[16] A. Sthlberg, J. Hkansson, X. Xian, H. Semb, and M. Kubista, Properties of the reverse

transcription reaction in mRNA quantification, Clinical Chemistry, vol. 50, Mar. 2004, pp. 509-

515.

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Budget

13

Year 1Personnel

Name Position Months devoted to ProjRole Salary and fringe bene

TBD Post doc 12 Animal work 55000

TBD Graduate Student 12 Work with post doc on an 40000

TBD Graduate Student 12 Molecular Biology 40000


Equipment

SuppliesItem Cost

Typical Lab Supplies 24000

Mice 2000

Year 1 Total 201000

Year 2Personnel

Name Position Months devoted to ProjRole Salary and fringe beneTBD Post doc 12 Animal work 55000



Equipment

SuppliesItem Cost


Mice 2000

Year 2 Total 161000

Year 3

PersonnelName Position Months devoted to ProjRole Salary and fringe bene

TBD Post doc 12 Animal work 55000



Equipment

SuppliesItem Cost


Mice 2000

Year 3 Total 161000

No major equipment needs tobe purchased




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BIOGRAPHICAL SKETCHProvide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME

Kurt WhittemorePOSITION TITLE

Researcher

eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training andresidency training if applicable.)

INSTITUTION AND LOCATIONDEGREE

(if applicable)MM/YY FIELD OF STUDY

Southern Utah University located in Cedar City UT BS 05/08/10 ChemistryBiodesign Institute located in Phoenix AZ Ph.D. Current Biological Design

NOTE: The Biographical Sketch may not exceed four pages. Follow the formats and instructions

below.

A. Personal Statement

Briefly describe why your experience and qualifications make you particularly well-suited for your role

(e.g., PD/PI, mentor, participating faculty) in the project that is the subject of the application.My experience as a Biological Design Ph.D. student with a background in Chemistry has

prepared me well to lead this research effort. During my studies I have taken many classes which have

given me a strong foundation in biochemistry and immunology. I have also performed research in theCenter for Innovations in Medicine (CIM) in the Biodesign Institute where I had the opportunity to

collaborate with many outstanding researchers who are experts in fields varying from computer

programming to animal work. As I pursue my goals of transferring adaptive immunity for this research

proposal, I will be able to obtain guidance from these former collaborators.

B. Positions and Honors

List in chronological order previous positions, concluding with the present position. List any honors.Include present membership on any Federal Government public advisory committee.

Southern Utah University Water Lab Analyst 08/2005-05/2008

Graduated Summa Cum Laude from Southern Utah University (2008)

College of Science Outstanding Scholar at Southern Utah University (2008)Gold medal at iGEM competition (2007)

C. Selected Peer-reviewed PublicationsNIH encourages applicants to limit the list of selected peer-reviewed publications or

manuscripts in press to no more than 15. Do not include manuscripts submitted or in

preparation. The individual may choose to include selected publications based onrecency, importance to the field, and/or relevance to the proposed research. When citing

articles that fall under the Public Access Policy, were authored or co-authored by the

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applicant and arose from NIH support, provide the NIH Manuscript Submissionreference number (e.g., NIHMS97531) or the PubMed Central (PMC) reference number

(e.g., PMCID234567) for each article. If the PMCID is not yet available because the

Journal submits articles directly to PMC on behalf of their authors, indicate "PMCJournal - In Process." A list of these Journals is posted at:

http://publicaccess.nih.gov/submit_process_journals.htm. Citations that are not coveredby the Public Access Policy, but are publicly available in a free, online format mayinclude URLs or PMCID numbers along with the full reference (note that copies of

publicly available publications are not accepted as appendix material.)I have no publications at this time.

D. Research Support

List both selected ongoing and completed research projects for the past three years (Federal or non-

Federally-supported).Begin with the projects that are most relevant to the research proposed in theapplication. Briefly indicate the overall goals of the projects and responsibilities of the key person

identified on the Biographical Sketch. Do not include number of person months or direct costs.This is my first grant. However, I have contributed to many ongoing research projects

over the past year. I devoted time to these various research projects as I rotated throughdifferent labs during the first year of my Ph.D. In Neal Woodbury's lab I investigated thefeasibility of developing a polymer which could slow down the diffusion of molecules on aglass surface. Such a polymer could be useful for experiments which use peptide arrays,which are similar to DNA microarrays. In Stephen Johnston's lab, I performed work with thesepeptide arrays directly to investigate the effects of hybridizing a peptide to the peptide arraysinstead of the traditional animal sera which had been hybridized to the arrays before. InBruce Rittmann's lab, I performed some protein production and mass spectrometry work toinvestigate the glycosylation patterns of an enzyme known as fructosyl lysine 3 kinase. Such

information could have important implications for aging. In Brenda Hogue's lab, I producedmutant forms of a coronavirus protein to investigate a hypothesis about which part of theprotein was important for interacting with other coronavirus proteins. All of these projectswere part of much larger projects which were funded well.

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http://publicaccess.nih.gov/submit_process_journals.htmhttp://publicaccess.nih.gov/submit_process_journals.htm


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Facilities and Other Resources

Animal Facility

The Biodesign Institute has a well managed animal facility in the basement. Theanimals used by all of the different labs are centralized, and regular staff takes care of feeding

the animals. This staff also checks on their health. The operation of this facility will allow usto focus on our experiments rather than on the care of the animal.

Office Area

For each lab in the Biodesign Institute, there is an office area across the hall from a labarea. Researchers can study or perform data analysis in this area. Each desk is wellequipped with computers with standard specifications for computers released in the last 2-3years.

Lab Area

The lab is already equipped with the standard equipment we will need for the projectsuch as centrifuges, incubators, gel boxes, etc. Any equipment that we find that we don'thave can be easily obtained by collaborating with one of the many well stocked labs in theBiodesign Institute.

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Documents

Transferring Adaptive Immunity Research Proposal Kurt