Institution: NIH Library Sign In as Member / Individual

This Article Full Text Services Alert me to new issues of the journal Request Copyright Permission

Irradiated sporozoite immunized volunteers (n = 8) were exposed to the bites of 1,005-1,366 (mean 1,162) infected bites of gamma-irradiated (1.5 ´ 10⁴ rads) Anopheles stephensi mosquitoes infected by membrane feeding with either the NF54 strain or the corresponding 3D7 clone of P. falciparum, as described (1). Exposure was during 6-7 sessions over a period of approximately 7 months. Mock immunized volunteers (n = 4) were exposed in parallel to the bites of 1,210-1,756 (mean 1,566) gamma-irradiated uninfected A. stephensi mosquitoes. Irradiated sporozoite immunized volunteers were challenged with infectious mosquitoes to ascertain the development of protective immunity; four of the eight volunteers but none of three malaria-naïve controls challenged in parallel were protected. All volunteers were seronegative for HIV gp120 antibodies and hepatitis B virus (HBV) core antibodies as determined by standard clinical screening. HLA types were established from peripheral blood samples by using standard site-specific oligonucleotide PCR typing. Peripheral blood was obtained from study subjects by venipuncture or leukopheresis (National Institutes of Health Department of Transfusion Medicine). Peripheral blood mononuclear cells (PBMCs) were isolated utilizing Ficoll-Hypaque centrifugation and frozen and stored in liquid nitrogen by using standard methodologies prior to use.

Assays to quantitatively measure peptide binding to class I and class II MHC molecules are based on the inhibition of binding of a high affinity radiolabeled peptide to purified MHC molecules (2). To measure the capacity of peptide ligands to bind MHC molecules, 1-10 nM of radiolabeled peptide was coincubated at room temperature with 1 m M to 1 nM of purified MHC in the presence of a cocktail of protease inhibitors [EDTA, pepstatin A, NEM, 1,10-phenanthroline, PMSF, and 7-amino-1-chloro-3-tosylamido-2-heptanone (TLCK)]. For class I assays, 1 m M human b ₂-microglubulin (Scripps Laboratories, San Diego) was added and NEM was omitted. After a 48-h incubation, the percent of MHC bound radioactivity was determined by capturing MHC/peptide complexes on W6/32 (class I) or LB3.1 (DR) antibody coated Optiplates (Packard Instrument., Meriden, CT), and determining bound cpm using the TopCount (Packard Instrument) microscintillation counter. The concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Under the conditions utilized, where [label] < [MHC] and IC₅₀ ³ [MHC], the measured IC₅₀ values are reasonable approximations of the true K_d values. Each competitor peptide was tested in two to four independent experiments. As a positive control, the unlabeled version of the radiolabeled probe was also tested in parallel.

Coefficients for use in PIC algorithms represent the average relative binding associated with each of the 20 naturally occurring amino acid residues for each position, and were calculated as described (3-5). Briefly, libraries of peptides of a given length are tested their capacity to bind specific MHC molecules. A relative binding value is calculated for each peptide by dividing the IC₅₀ of the positive control for inhibition by the IC₅₀ measured for each specific peptide tested. Standardized relative binding values also allow the calculation a geometric mean, or average relative binding value (ARB), for all peptides with a particular characteristic. That is, for all i positions, the geometric mean of the ARB of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji.

A preliminary matrix algorithm (PMA) score can be calculated for each peptide in the matrix generation library by multiplying (or adding, if the ARB are expressed as log values) the ARB values corresponding to the sequence of the peptide. PMA scores can be effectively used as rank scores to select candidate peptides (5-12). Specifically, if the resulting score exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds can be chosen as a function of the degree of stringency of prediction desired. For class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, after an iterative procedure.

Next, algorithm scores are generated for each peptide in the algorithm generation set by adding the log of ARB scores corresponding to each residue/position pair in the peptide sequence. An estimated IC₅₀ value (EIC) is derived by dividing the IC₅₀ nM for an allele-specific reference peptide by the inverse log of the preliminary algorithm score divided by the number of amino acids in the peptide (i.e., EIC = IC_50ref /10^[PA/length]). The EIC values of each peptide in the generation set are then plotted against their corresponding measured IC₅₀ nM values (MIC), and constants are obtained by fitting to the polynomial function y = bx^m. Scaling of the plot is adjusted using a y-intercept adjustment (YIA) to derive linearized, or "scaled," IC₅₀ values (SIC), where YIA = (EIC/b)^p, and SIC = YIA*EIC. A correction factor (CF) representing the average ratio between SIC and MIC is calculated, and used to derive the final PIC, where PIC = SIC/CF. Finally, the optimal value of P is determined empirically by measuring the power that minimizes the standard deviation in the library between MIC and PIC values.

To estimate the performance of an algorithm, two measures commonly utilized are (i) sensitivity (SENS, the fraction of binders identified) and (ii) positive predictive value (PPV) or accuracy (the fraction of predicted peptides that actually bind). The PIC method allows selection of criteria such that either accuracy or sensitivity can be maximized. In general, lowering the PIC selection criteria lowers sensitivity but increases predictive accuracy, and vice versa. We generally utilize a PIC value of 100 nM, which was empirically selected to approximately correspond to the intersection of the SENS and PPV curves.

The number of peptide-specific IFN-γ secreting cells was determined by ex vivo IFN-γ ELISPOT (13). Samples were assayed in quadruplicate (5.0 ´ 10⁵ PBMC per well), with 100 m l of test or control peptide pools at a final concentration of 1 m g/ml each peptide. Sterile 96-well multiScreen-IP MAIP plates (Millipore, Bedford, MA) were coated overnight at 4°C with 50 m l of PBS containing 10 m g/ml anti-IFN-γ mAb (clone 1-D1K; Mabtech, Stockholm, Sweden). Wells were washed six times with RPMI medium 1640 and blocked for 1 h at room temperature with RPMI medium 1640 supplemented with 10% FCS. Then, 100 μl of input PBMCs (5.0 ´ 10⁵ PBMCs) was added in quadruplicate, together with 100 μl of test or control peptide pools at a final concentration of 1 μg/ml each peptide. Cultures were incubated for 36 h at 37°C in an atmosphere of 5% CO₂. Wells were then washed six times with PBS/0.05% Tween 20 (Sigma), and incubated for 3 h at room temperature with 100 μl of 1 μg/ml biotinylated anti-IFN-γ mAb (clone 7B6-1, Mabtech). Wells were again washed six times with PBS/0.05% Tween 20 and incubated for 1 h at room temperature with 100 μl of 1:1,000 dilution of streptavidin alkaline phosphatase (Mabtech). Wells were washed six times with PBS/0.05% Tween 20 and 3 times with PBS, and then developed with 100 μl of 1:25 diluted alkaline phosphatase substrate (Bio-Rad). The colorimetric reaction was stopped after 15 min by extensive washing in tap water, and plates were air-dried. The number of spots corresponding to IFN-γ-producing cells in wells (IFN-γ spot forming cells; SFCs) were enumerated with the Zeiss KS ELISPOT system (Carl Zeiss, Thornwood, NY) using standard parameters. Responses were expressed as the number of IFN-γ SFC per 10⁶ PBMCs and were considered positive if the magnitude of response to test peptide was >5 SFCs and if the stimulation index (SI = ratio test SFCs/ control SFCs) was greater than 2.0.

The number of peptides per pool varied between antigens depending on the number of predicted epitopes. The maximum number of peptides utilized in a pool was 50 (10 per HLA type). Preliminary experiments (data not shown) were performed to establish the optimal concentrations of peptides to be utilized in the assay, and also avoid toxicity effects (mostly related to high DMSO concentrations). Specifically, pools were created incorporating increasing numbers of different peptides (final DMSO concentration ranging from 0.01-2.0%), and IFN-γ ELISPOT and lymphoproliferation assays were carried out using rhesus monkey PBMC and mouse splenocyte preparations. The viability of the peptide-stimulated cell cultures and the magnitude of immune responses were evaluated, and no adverse effects were noted with DMSO concentrations <1.0%. In the studies reported herein, peptide stocks were prepared at an initial concentration of 20 mg/ml in 100% DMSO, then used in peptide pools at a final concentration of 1 µg/ml of pool volume (equivalent to 0.05 m l of each peptide stock per pool). As peptide pools were constructed with a maximum of 50 peptides per pool, the total peptide concentration in each pool was £ 50 μg/ml, and the total amount of DMSO was £ 2.5 μl DMSO per ml (≤0.25% DMSO).

We express our gratitude to Mara Berzins, Maria Malone, Patricia de la Vega, Thomas Richie, Judith Epstein, and other members of the NMRC Malaria Program clinical team and Susan Leitman and the staff at the National Institutes of Health Department of Transfusion Medicine for acquiring the study samples; Patricia Stuart, Stephani Stewart, and Timothy Pencille for technical assistance with peptide binding assays; Fe Baraceros, Glenna Banania, Nancy Rahardjo, Gary Brice, and Ariel Freilich for assistance for sample processing and immunological assays; Jennifer Ng and the staff of the Department of Defense Bone Marrow Donor Program for HLA typing; Peter Blair and Joao Aguair for bioinformatics assistance; and Bianca Mothe and Howard Grey for helpful discussions. The studies reported herein were conducted in accordance with U.S. Navy regulations governing the protection of human subjects in medical research, and the protocols employing human subjects were reviewed and approved by the Naval Medical Research Center’s Committee for the Protection of Human Subjects. The opinions and assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy or the Department of Defense. Work was supported by funds allocated to the Naval Medical Research and Development Center by the U.S. Army Medical Research Material Command and by funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under contract N01-AI-95362.

1. Hoffman, S. L., Goh, L. M., Luke, T. C., Schneider, I., Le, T. P., Doolan, D. L., Sacci, J., de la Vega, P., Dowler, M., Paul, C., et al. (2002) J. Infect. Dis. 185, 1155-1164.

2. Sidney, J., Southwood, S., Oseroff, C., Del Guercio, M. F., Sette, A. & Grey, H. (1998) in Current Protocols in Immunology (Wiley, New Yokr), pp. 18.3.1-18.3.19.

3. Southwood, S., Sidney, J., Kondo, A., del Guercio, M. F., Appella, E., Hoffman, S., Kubo, R. T., Chesnut, R. W., Grey, H. M. & Sette, A. (1998) J. Immunol. 160, 3363-3373.

4. Gulukota, K., Sidney, J., Sette, A. & DeLisi, C. (1997) J. Mol. Biol. 267, 1258-1267.

5. Sidney, J., Southwood, S., Mann, D. L., Fernandez-Vina, M. A., Newman, M. J. & Sette, A. (2001) Hum. Immunol. 62, 1200-1216.

6. Altfeld, M. A., Livingston, B., Reshamwala, N., Nguyen, P. T., Addo, M. M., Shea, A., Newman, M., Fikes, J., Sidney, J., Wentworth, P., et al. (2001) J. Virol. 75, 1301-1311.

7. Wilson, C. C., Palmer, B., Southwood, S., Sidney, J., Higashimoto, Y., Appella, E., Chesnut, R., Sette, A. & Livingston, B. D. (2001) J. Virol. 75, 4195-4207.

8. Doolan, D. L., Hoffman, S. L., Southwood, S., Wentworth, P. A., Sidney, J., Chesnut, R. W., Keogh, E., Appella, E., Nutman, T. B., Lal, A. A., et al.(1997) Immunity 7, 97-112.

9. Doolan, D. L., Southwood, S., Chesnut, R., Appella, E., Gomez, E., Richards, A., Higashimoto, Y. I., Maewal, A., Sidney, J., Gramzinski, R. A., et al. (2000) J. Immunol. 165, 1123-1137.

10. Bertoni, R., Sidney, J., Fowler, P., Chesnut, R. W., Chisari, F. V. & Sette, A. (1997) J Clin. Invest. 100, 503-513.

11. Threlkeld, S. C., Wentworth, P. A., Kalams, S. A., Wilkes, B. M., Ruhl, D. J., Keogh, E., Sidney, J., Southwood, S., Walker, B. D. & Sette, A. (1997) J. Immunol. 159, 1648-1657.

12. Chang, K. M., Gruener, N. H., Southwood, S., Sidney, J., Pape, G. R., Chisari, F. V. & Sette, A. (1999) J. Immunol. 162, 1156-1164.

13. Brice, G. T., Graber, N. L., Hoffman, S. L. & Doolan, D. L. (2001) J. Immunol. Methods 257, 55-69.