Demonstration of Feasibility with Influenza Virus-Specific Responsest

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1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 1994, p X/94/$ Copyright C 1994, American Society for Microbiology Vol. 1, No. 5 Analysis of Human Antiviral Cytotoxic T-Lymphocyte Responses for Vaccine Trials Using Cryopreserved Mononuclear Leukocytes: Demonstration of Feasibility with Influena Virus-Specific Responsest NAYEF EL-DAHER,t JOAN E. NICHOLS, AND NORBERT J. ROBERTS, JR.* Infectious Diseases Unit, Department of Medicine, University of Rochester School of Medicine, Rochester, New York Received 27 December 1993/Returned for modification 10 February 1994/Accepted 13 May 1994 The feasibility of measuring virus-specific human cytotoxic T-lymphocyte (CTL) activity by using cryopreserved mononuclear leukocytes to support clinical vaccine trials was addressed. Autologous fresh and cryopreserved cells from the same sample of peripheral blood were used as sources of CTL precursors and were tested for influena virus-specific activity. The data indicated that virus-specific CTL activity could be measured by using cryopreserved cells; this could also be done in assays that are designed to characterie the responsible effector cell population. Immuniation has been recognied to be an important component of strategies for the effective prevention or control of viral infections, whether they are directed toward longencountered pathogens, such as influena virus, or toward newer but aggressive pathogens, such as the human immunodeficiency virus type 1 (HIV-1). The benefits, for a vaccine evaluation program, of being able to use cryopreserved cells to measure the induction of cellular immunity by the candidate vaccine, especially the induction of virus-specific cytotoxic T-lymphocyte (CTL) responses, are many. First, for example, responses to various components of the regimen, or the time of first appearance and/or the length of persistence of the responses, can be assessed effectively with greater control of day-to-day experimental variation. Second, when volunteer entry must be immediate or rapid, both pre- and postvaccination responses to the immunogen can be assessed. Volunteer participation does not have to be delayed for several weeks until an Epstein-Barr virus (EBV)-transformed lymphocyte cell line (EBV-LCL) can be established for use as the target cell in CTL assays. Third, the best or most broadly informative CTL assay system or the most appropriate vectors for producing target cells for the assays may not be available at the point when the clinical studies for the evaluation of candidate vaccine preparations must be undertaken. The current studies were undertaken to determine whether virus-specific CTL responses could be assayed effectively by using cryopreserved human mononuclear leukocytes (MNLs). The influena virus-specific responses of cryopreserved MNLs were compared with those of autologous fresh MNLs from the same sample of peripheral blood that did not undergo the freeing and thawing procedure. * Corresponding author. Mailing address: Infectious Diseases Unit, Box 689, University of Rochester Medical Center, Rochester, NY Phone: (716) Fax: (716) t The results of this study were from work performed in part with support from the AIDS Clinical Vaccine Trials Network, National Institute of Allergy and Infectious Diseases, Bethesda, Md. t Present address: Jordan University of Science and Technology, Amman, Jordan. 487 MATERUILS AND METHODS Cell sources, preparation, and culture conditions. MNLs were obtained from the peripheral blood of healthy adult male and female volunteers by Ficoll-Hypaque sedimentation (3). All donors had been typed previously for both human leukocyte antigen (HLA) class I and class II antigens by established serological techniques for class I antigens and by using panels of homoygous typing cells for the class II D-region antigens (23). All experiments used concomitant assays of autologous MNL preparations. For each CTL donor and for appropriately HLA-mismatched donors, target cell lines were produced by EBV-induced transformation of MNLs by standard methods to provide B-lymphocyte cell lines (EBV-LCL) (13, 22). The MNLs were cultured at 37 C in medium 199 with 10% heat-inactivated fetal calf serum except during the 1-h exposures to infectious or inactivated influena virus, when they were suspended in serum-free medium (16, 17). Cryopreservation and recovery of MNLs. MNLs were cryopreserved and recovered by previously described techniques (5, 8, 14). In brief, the cells were washed and diluted to i07 cells per ml in RPMI 1640 containing 40% autologous plasma and were cooled to 4 C. The cell suspension was then mixed 1:1 with cooled 15% dimethyl sulfoxide, and the mixture was dispensed in 1.8- to 2.0-ml aliquots into plastic vials (Nunc, Roskilde, Denmark). The vials were cooled in a controlledrate freeing apparatus (Cryomed, New Baltimore, Mich.), at a rate of 1 C/min to -40 C and then at 5 C/min to -80 C. They were then removed and placed in a liquid nitrogen storage chamber. For recovery, vials were removed from liquid nitrogen storage and the cell suspensions were thawed rapidly in a 37 C water bath. As the last ice crystal melted, the vials were placed on ice for 2 min. The cell suspension was then diluted slowly at room temperature with increasing amounts of RPMI 1640 containing 40% autologous plasma. The viable MNL concentration was adjusted to that appropriate for the assay to be performed. These cryopreservation and recovery methods were refined by members of our unit (14) and have been used for assays of other cellular immune responses (8, 14). The detailed procedures, which have been adopted by the AIDS Clinical Vaccine Trials Network of the National Institute of

2 488 EL-DAHER ET AL. Allergy and Infectious Diseases, are available from the corresponding author. Assays of CTL activity. Assays of influena virus-specific CTL activity were performed by using minor modifications of previously described techniques (5, 11, 13). In brief, fresh or cryopreserved MNLs were stimulated in vitro with influena virus or heat (56 C, 2 h)-inactivated influena virus by using the standard protocol in which 10% of the effector cell population is exposed to the virus and then added back to the remaining effector cells. The cells were exposed to influena virus A/AA/ Marton/43 HlNl, A/Scotland/840/74 H3N2, or B/Singapore/ 3/64 at a multiplicity of infection of 3 for 1 h at 37 C in serum-free medium (16, 17). In a series of experiments, fresh and cryopreserved virus-infected stimulator cells were used to make concomitant crossover measurements of the stimulation of fresh or cryopreserved effector cells (CTLs) as described in the Results. In all experiments, the cells were then cultured for 7 days to generate CTLs (effector cells), which were collected and counted by using trypan blue to determine viability. Target cells were prepared by infecting phytohemagglutininstimulated MNLs or cells of the EBV-LCL with influena virus. On the day of assay, 106 target cells were infected by exposure to infectious virus at a multiplicity of infection of 3, were labeled with 150,uCi of 51Cr for 90 min, and, at 4 h after infection (including the radiolabeling period), were washed and counted by using trypan blue to determine percent viable cells. The effector cells (CTLs) and target cells (5 x 103 MNL or EBV-LCL target cells per well) were cocultured for 4 to 6 h, commonly at an effector cell/target cell ratio of 100:1. In a series of experiments, the effector cells were added to the target cells at various ratios, and the percent lysis is given in the Results for each of the different combinations of cells. All assays were performed in triplicate, and the results are expressed as the mean percentages of lysis or of influena virus-specific lysis (termed specific lysis in Results). Spontaneous release was <15% and was commonly <10%. Maximum release was induced by adding 10% Triton X-100. The influena virus-specific lysis was calculated according to the following formulas: R1, for virus-infected cells, [(measured release-spontaneous release)/(maximum release-spontaneous release)] x 100%; R2, for sham-infected cells, [(measured release -spontaneous release)/(maximum release - spontaneous release)] x 100%. Specific lysis is R1 - R2. In a subset of experiments, effector cells were identified by depletion of subsets of potential effector cells, before addition to assay wells, by using immunomagnetic beads (Dynabeads; Dynal, Inc., Great Neck, N.Y.) (5). In these experiments, effector cells were sham depleted by using anti-murine immunoglobulin (anti-mlg) beads (control for subpopulation depletion) or were depleted by using anti-cd4 or anti-cd8 beads. Depletion of.98% of the specified cells was confirmed by immunofluorescence labeling and flow cytometric analysis of the cell populations (5). HLA restriction of virus-specific CTL activity was further established by use of HLA-mismatched target cells in assays. In another series of experiments, micro-ctl assays, designed to require fewer cells, were conducted in parallel with the standard assays. For such micro-ctl assays, 103 EBV-LCL target cells were added to each well along with effector cells at the appropriate ratios. Statistical comparisons of CTL responses. The responses of the CTLs were compared by using the SYSTAT program with Student's t test. U0 30- LU O U.20 - w 0L U,) w cc 10- we T T 0- Cells: Fresh Cryo- Fresh preserved Virus: Infectious -F- Cryopreserved Inactivated FIG. 1. Influena virus-specific lysis of infected autologous MNLs. Fresh or cryopreserved MNLs were stimulated with infectious or inactivated influena virus and were assessed for their CTL activities at an effector/target ratio of 100:1. Results represent the means ± standard errors of six experiments. RESULTS Influena virus-specific CTL responses of fresh and cryopreserved MNL. Influena virus-specific CTL activity was exhibited by fresh and cryopreserved effector MNLs. Virusspecific lysis, at an effector/target ratio of 100:1, could be demonstrated by using either virus-infected MNLs (Fig. 1) or virus-infected EBV-LCL cells (Fig. 2) as target cells. In the first series of experiments, infectious virus was used to stimulate effector cells. This is the common method for in vitro activation of precursor CTLs for the measurement of influena virus-specific CTL activity. Virus-specific lysis of infected MNL target cells and infected EBV-LCL target cells by fresh MNLs (effector cells) ranged from 17 to 42% and 16 to 36%, Cl) >-J i-l wcn 20' 0. CO) w cc wl a. Cells: Fresh Cryo- Fresh preserved Virus: Infectious CLIN. DIAGN. LAB. IMMUNOL. Cryopreserved 1 Inactivated FIG. 2. Influena virus-specific lysis of infected autologous cells of the EBV-LCL. Fresh or cryopreserved MNLs were stimulated with infectious or inactivated influena virus and were assessed for their CTL activities at an effector cell/target cell ratio of 100:1. Results represent the means ± standard errors of four experiments.

3 VOL. 1, 1994 CYTOTOXIC RESPONSES OF CRYOPRESERVED LEUKOCYTES 489 1UU B 40-2CO /E:T FIG. 3. Variation in concordance of CTL activity between fresh and cryopreserved MNLs assayed by using cells obtained from a single donor on 2 different days (A and B, respectively). Graphs show percent lysis of sham-infected (open symbols) and infected (closed symbols) autologous EBV-LCL target cells by fresh (triangles) or cryopreserved (squares) MNL effector cells that were stimulated by using infectious influena virus and assessed for CTL activity at various effector cell/target cell (E:T) ratios. respectively. Specific lysis ranged from 14 to 24% for MNL target cells and 12 to 28% for EBV-LCL target cells when cryopreserved MNLs were used as effector cells. Although less specific lysis was observed overall by using cryopreserved CTLs against either infected MNLs or infected cells of the EBV-LCL (n = 10; P = 0.007), the difference was more marked when cells of the EBV-LCL were used as target cells (n = 4; P = for fresh versus cryopreserved CTLs) than in assays in which MNLs were used as target cells (n = 4; P =0.087). Nonetheless, the existence of distinct CTL activity could clearly be demonstrated by using the cryopreserved CTLs and either MNLs or cells of the EBV-LCL as target cells (Fig. 1 and 2), and could be examined further to identify CTL populations within the total pool of effector MNLs (see below). The concomitant assays of fresh and cryopreserved MNLs for CIL activity revealed fully equivalent lysis of target cells in approximately 60% of the individual experiments. In the other experiments, greater CTL activity was exhibited by the fresh cells. Individual donor's cells were studied on more than one occasion to determine whether this finding was related to the cell donor. In each case in which a donor's cells were studied in more than one experiment, both virtually equivalent and different (but still easily delineated) CGL activity patterns were observed. Results of a representative pair of experiments with fresh and cryopreserved cells from a single donor obtained from two separate experiments are shown in Fig. 3 to illustrate this observation. Fewer cells might reasonably be used in assays for CGL activity specific for a virus (or viral vaccine) that can be expected to induce a relatively high CTL precursor frequency. This would further support the use of cryopreserved leukocytes for such immunologic measurements. This possibility was demonstrated in several experiments by using additional concomitant micro-ctl assays that required one-fifth the standard number of cells in individual assay wells. Figure 4 shows the results of the micro-ctl assays that correspond to the routine assays depicted in Fig. 3A. The reproducibility of assays that were conducted on different days with cells cryopreserved from a single blood donation could be illustrated even with the micro-ctl assay. This assay would be more susceptible to variation because of the lower number of specific CTLs that might be added to individual On & wells. Even with the micro-cgl assay, however, repetitive measurements of activity by using aliquots of cells that were cryopreserved at a single time showed reproducible CGL activity. The results of two different sets of repeated assays are shown in Fig. 5. CTL activity stimulated by infectious versus inactivated influena virus. CGL activity was also easily demonstrated when the fresh or cryopreserved MNL effector cells were stimulated by using inactivated influena virus and tested against infected target MNLs (Fig. 1). Virus-specific lysis was almost as intense as that demonstrated concomitantly by using infectious virus to stimulate the CTLs (P = and for fresh and cryopreserved CTLs, respectively, in a comparison of the methods of stimulation). This observation made it less /E:T FIG. 4. Micro-CGL assays of lysis by fresh and cryopreserved MNLs. The graph shows the lysis of sham-infected (open symbols) and infected (closed symbols) autologous EBV-LCL target cells by fresh (triangles) or cryopreserved (squares) effector MNLs that were stimulated by using infectious influena virus and assessed for their CTL activities at various effector cell/target cell (E:T) ratios. Data are from micro-ctl assays performed in parallel (with the same cell populations) with the standard CTL assays that are depicted in Fig. 3A.

4 490 EL-DAHER ET AL. CLIN. DIAGN. LAB. IMMUNOL. ra P.-O ra >4 04 E-4 w u B o-i 1/E:T FIG. 5. Reproducibility of micro-ctl assays of lysis by cryopreserved MNLs by using cells from two different donors (A and B, respectively). Graphs show lysis of sham-infected (open symbols) and infected (closed symbols) autologous EBV-LCL target cells by cryopreserved effector MNLs that were stimulated by using infectious influena virus and assessed for CTL activity at various effector cell/target cell (E:T) ratios. Each graph depicts repeated assays for CTL activity on different days (triangles and squares, respectively) by using cryopreserved cells that were obtained from an individual donor at a single point in time. likely that the greater CTL activity exhibited by infectious virus-stimulated fresh MNLs relative to that exhibited by cryopreserved MNLs (with virus-infected MNLs used as the target cells; see Fig. 1) was due mainly to the differential synthesis of influena virus target antigens by the fresh and cryopreserved MNLs (10% of the effector cell population) that were exposed to the infectious virus and used to stimulate CTLs in vitro. The results of crossover experiments, in which fresh or cryopreserved effector MNLs (i.e., CTLs) were stimulated in vitro with fresh or cryopreserved influena virusinfected cells (Table 1; no significant differences were observed between the combinations of cells), also suggested that the lower levels of response of cryopreserved MNLs boosted in vitro with infectious influena virus could not be related solely to the synthesis and expression of viral antigens by cryopreserved cells during the boosting of CTL activity. With in vitro stimulation with inactivated virus, there was a trend toward a greater CTL activity of fresh MNLs versus that of cryopreserved MNLs against infected MNLs as target cells (P = 0.069; see Fig. 1), but there was no difference in CTL activity against the infected cells of the EBV-LCL as target cells (P = 0.613; see Fig. 2). Identificaticn of the phenotype(s) of fresh and cryopreserved virus-specific CTLs. Additional studies were performed by using fresh and cryopreserved MNLs stimulated with infec- TABLE 1. Comparison of fresh versus cryopreserved MNLs as stimulators of CTL activity' Responder Stimulator MNL Specific lysis MNL (% [mean ± SE]) Fresh Fresh Fresh Cryopreserved 27 ± 8 Cryopreserved Fresh 23 ± 5 Cryopreserved Cryopreserved 27 ± 8 " The mean ± standard error percent virus-specific lysis is shown for each stimulator-responder MNL combination added to autologous EBV-LCL target cells at an effector cell/target cell ratio of 100:1. Results are from four experiments, each with the indicated combinations of cells assayed. tious virus in order to determine whether the anti-virus CTL activity was mediated by CD8+ and/or CD4+ T lymphocytes. Effector CTL activity against autologous EBV-LCL target cells was measured by using MNLs directly and after subpopulation depletion by using immunomagnetic anti-cd8 or anti-cd4 beads or was measured after sham depletion (with anti-mlg beads). Activity was also measured against HILA class I- and class II-mismatched EBV-LCL target cells. The virus-specific lysis of autologous target cells was abolished by depletion of CD8+ cells, and HLA-mismatched target cells were not lysed (Table 2). In addition, autologous cells infected with another influena A virus, but not those infected with an influena B virus, were lysed (data not shown). Thus, CD8+ CTL activity was clearly demonstrable with cryopreserved MNLs. DISCUSSION The current studies measured the potential preservation and assessment of CTL activity that could be achieved with cryopreservation of human MNLs that were collected to measure TABLE 2. Effector cell phenotype(s) for influena virus-specific CTL activity" Mean % lysis by CTL EBV-LCL target Treatment effector cells cell Fresh Cryopreserved Autologous None Anti-CD Anti-CD8 0 0 Anti-mlg 20 ND HLA mismatched None 0 0 Anti-CD4 0 0 Anti-CD8 0 0 Anti-mlg 0 ND apercent virus-specific lysis is given for each effector cell/target cell (100:1 ratio) combination. Spontaneous release was <15% for all target cells. ND, not done.

5 VOL. 1, 1994 CYTOTOXIC RESPONSES OF CRYOPRESERVED LEUKOCYTES 491 virus-specific immunological responses during clinical vaccine or antiviral drug trials that enroll healthy human subjects. The data suggest that virus-specific CD8+ CTL activity is retained and can be assayed through such procedures, and the methods should allow more accurate longitudinal assessments of antiviral immune responses as well as the application of subsequently designed in vitro immunological assays to support clinical vaccine or antiviral drug trials. The results suggest that qualitative rather than firmly quantitative comparisons of CTL activity can readily and consistently be made between fresh and cryopreserved cells obtained at the same time or between cells obtained at different times whether they are assayed fresh or after cryopreservation. Such results are consistent with the recognied variation in other cellular immune responses, such as the mitogen-induced lymphocyte proliferative responses of normal fresh MNLs obtained from a single donor at different points in time (15). Nonetheless, the presence (or absence) of specific antiviral CTL precursors as well as characteristics of the response, such as the mediation of the lysis or the epitope repertoire of the response by the cells, can be delineated by using cryopreserved cells that are obtained at various points during and after the administration of a viral vaccine regimen. Our volunteer donors were naturally infected with influena virus in the past and were not recently vaccinated. They were anticipated to have a relatively low influena virus-specific precursor CTL frequency in the peripheral blood that they donated. Their MNLs were therefore expected to require in vitro stimulation by well-established methods (1, 2, 6, 10, 11, 19, 21) to generate CTL activity. Furthermore, without selection to provide donors whose cells had a known high level of responsiveness in the assay, the assays might have required a commonly used high effector cell/target cell ratio to detect virus-specific, HLA-restricted CTL activity. Most of the data presented in the Results are for assays with a 100:1 effector cell/target cell ratio, but influena virus-specific CTL activity could be detected at lower ratios when sufficient numbers of cells were available to perform assays with a full range of effector cell/target cell ratios (Fig. 3 to 5). In the current studies, CD8+ CTL activity could be stimulated in vitro by using inactivated whole influena virus. Similar observations have been made in studies of HIV-1 gpl60- specific CTL responses in which a live virus vaccine preparation was administered in vivo and stimulation of CTL activity was accomplished in vitro by using psoralen-uv-inactivated HIV-1 (5). Such results are supported by other observations of HLA class I-restricted responses. Thus, class I-restricted T-cell responses have been described after stimulation with other complex, noninfectious antigens that might be delivered to the cytosol. For example, antigens presented in acid-sensitive liposomes have been shown to generate class I-restricted CTL activity (4). Recently, others have reported that high doses of formalin-inactivated influena whole virus vaccine could induce virus-specific MHC class I-restricted CD8+ CTL responses in mice (9). One could reasonably speculate that the uptake of inactivated virus by the antigen-presenting cells (monocytes-macrophages) in our MNL cultures resulted in virion penetration to the cytosol in at least a portion of the cells, with presentation to CD8+ T lymphocytes in a class I-restricted fashion. Since the subjects were exposed initially to a live virus via natural infections, secondary stimulation in vitro could result in clonal expansion and activation of CD8+ CTL precursors with specificity for the virus. Others have used cryopreserved human MNLs in assays conducted to examine and characterie influena virus-specific HLA-restricted CTL activity; cells from strong CTL responders were used very effectively (without comparisons with the responses of fresh cells) (1, 2, 10, 19). In studies of responses specific for another virus in our laboratory, T lymphocytemediated HIV-1 gpl60-specific cellular immune responses were demonstrated and shown to persist for at least 15 months after last receipt of vaccine by using MNLs from subjects who were initially immunied with a recombinant vaccinia virus containing gp160 and were subsequently boosted with a purified recombinant baculovirus-produced gpl60 (5). The cellular immune responses that were exhibited included gpl60-specific lymphocyte proliferation, the production of gamma interferon, and the functional gpl60-specific cytotoxic activity of CD8+ T lymphocytes (CTLs). Notably, all of the vaccine-induced gpl60-specific responses could be assessed by using cryopreserved MNLs. In those trials of HIV-1-derived candidate envelope vaccines (5), cryopreserved cells were commonly used in CTL assays because the desired assay could not be performed at the time that the volunteer provided the sample of peripheral blood. Limited numbers of MNLs were available, and parallel assays of fresh and cryopreserved cells for their gpl60-specific CTL activities could not generally be performed. In the few instances in which enough cells were available to perform comparison assays, virtually equivalent CTL activities were observed by using fresh and cryopreserved cells (Sa). The current data extend such observations by providing comparisons of virus-specific CTL responsiveness between autologous fresh and cryopreserved MNLs and support the use of cryopreserved MNLs in assays that are performed to assess immunogenicity in virus vaccine trials. Additional studies support the use of cryopreserved human MNLs for immunological assays during clinical vaccine or antiviral drug trials. Cryopreserved human MNLs have been shown to produce herpes simplex virus-induced immune-specific interferon titers not significantly different from the titers produced by fresh MNLs (14), and the gpl60-specific lymphocyte proliferation and production of gamma interferon have been demonstrated by using cryopreserved cells from volunteers who received candidate HIV-1 envelope-derived vaccine preparations (5). In a limited series of experiments, cryopreserved versus fresh MNLs have been assessed for their CTL activities in mixed leukocyte cultures with phytohemagglutinin (PHA)-stimulated target cells (20); the cryopreserved cells worked as well in the cytotoxic killing of (mitomycin-treated) stimulating cells. Others have reported highly reproducible lymphocyte cytotoxicity activity on a per lymphocyte basis, but with a definite decrement in the percentage recovery, by using assays that measured natural killer cell activity against standard target cells (12). Also in potential support of clinical vaccine field trials, other investigators have described a low-weight, low-level N2-consuming, controlled-gradient freeing apparatus suitable for the cryopreservation of MNLs under field conditions (7). The apparatus was used to show equivalent fresh and cryopreserved mitogen (PHA) and antigen (tetanus toxoid)-induced lymphoproliferative responses. The relative proportions of subsets of T cells and B cells were comparable, but the numbers of CD14+ cells decreased after freeing. It is not clear why somewhat different results were obtained by using infected PHA-stimulated MNLs versus cells of the EBV-LCL as target cells for fresh and cryopreserved CTLs induced by inactivated influena virus. It is important to recognie, however, that these are not equivalent as infected targets. In that regard, other investigators have used influena virus-specific CTLs to demonstrate differences between infected EBV-LCL cells and infected PHA-stimulated (non- EBV-transformed) lymphoblasts as target cells. Cold target

6 492 EL-DAHER ET AL. inhibition experiments showed that the lysis of infected nontransformed lymphoblasts was completely blocked by both infected cold cells of the EBV-LCL and cold lymphoblasts, whereas the lysis of EBV-LCL cells was completely inhibited by cold infected EBV-LCL cells but was only partially inhibited by cold infected lymphoblasts (18). Those data indicated that there are a subset of determinants on EBV-LCL cells that can be recognied by influena virus-specific CTLs but that are absent from nontransformed virus-infected lymphoblasts (18). Nonetheless, EBV-LCL target cells facilitate such immunological assays for vaccine trials, especially when a large number of assays must be done at any one time, and EBV-LCL target cells may provide more standardied targets than PHA-stimulated infected MNLs between assays. The overall observations of previous studies and the current studies suggest that cryopreserved human MNLs can be used effectively to assess the induction of virus-specific CTL activity in studies that support clinical trials of candidate viral vaccines. ACKNOWLEDGMENTS This work was supported by a cooperative agreement (UO1-AI ) with the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and by the Jordan University of Science and Technology (N.E.). REFERENCES 1. Biddison, W. E., and S. Shaw Differences in HLA antigen recognition by human influena virus-immune cytotoxic T cells. J. Immunol. 122: Biddison, W. E., F. E. Ward, G. M. Shearer, and S. Shaw The self determinants recognied by human virus-immune T cells can be distinguished from the serologically defined HLA antigens. J. Immunol. 124: Boyum, A Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21(Suppl. 97): Collins, D. S., K. Findlay, and C. V. 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