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Detection of HIV-1-Specific CTLs in the Semen of HIV-Infected Individuals This information is current as of June 18, 2017. Alison J. Quayle, Wanda M. P. Coston, Alicja K. Trocha, Spyros A. Kalams, Kenneth H. Mayer and Deborah J. Anderson J Immunol 1998; 161:4406-4410; ; http://www.jimmunol.org/content/161/8/4406 Subscription Permissions Email Alerts This article cites 38 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/161/8/4406.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References Detection of HIV-1-Specific CTLs in the Semen of HIV-Infected Individuals1 Alison J. Quayle,2* Wanda M. P. Coston,* Alicja K. Trocha,† Spyros A. Kalams,† Kenneth H. Mayer,‡§ and Deborah J. Anderson* he World Health Organization estimates that .70% of HIV infections are established as a result of sexual contact (1). HIV in semen is found in both cell-associated and cell-free forms (2), and HIV variants differ from those found in blood, which suggests viral compartmentalization (3). The highest viral loads in semen are associated with peripheral blood CD4 counts of ,200/ml (4, 5), concomitant sexually transmitted infections (6), and asymptomatic genital tract inflammation (4, 5). Conversely, semen virus levels decrease with antiviral therapy and with treatment of sexually transmitted diseases (4, 6, 7).3 Since intracellular virus is most readily eliminated by CTLs, the presence of anti-HIV CTLs in urogenital mucosa would be predicted to decrease viral load in genital secretions, contain virus locally, and decrease the chance of transmission to sexual partners. Vigorous memory CTL responses against all the major HIV proteins have been demonstrated in the blood of infected individuals (8 –11). HIV-specific CTLs have also been isolated from various sites including the lymph node (12), spleen (12, 13), cerebrospinal fluid (14), and lung (15). Recently, SIV-specific CTLs were isolated from the vaginal mucosa of chronically infected macaques inoculated with SIV by the vaginal route (16), and HIV-specific CTLs were isolated from the cervical mucosa of infected women (17) indicating that, at least in the female, an antiviral response can T * Fearing Laboratory, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; †AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129; ‡Fenway Community Health Center, Boston, MA 02115; and §Memorial Hospital of Rhode Island, Pawtucket, RI 02860 and Brown University AIDS Program, Providence, RI 02903 Received for publication January 22, 1998. Accepted for publication June 5, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant AI35564 and by the Massachusetts Department of Public Health. 2 Address correspondence and reprint requests to Dr. Alison Quayle, Fearing Laboratory. Thorn 217, 75 Francis Street, Brigham and Women’s Hospital, Boston, MA 02115. E-mail address: [email protected] 3 Mayer, K. H., S. L. Boswell, R. S. Goldstein, W. Lo, C. Xu, L. Tucker, M. P. Pasquale, R. D’Aquila, and D. J. Anderson. 1997. HIV persistence in semen after adding indinavir to combination antiretroviral therapy. Submitted for publication. Copyright © 1998 by The American Association of Immunologists be generated and maintained locally in the genital tract mucosa. We have recently demonstrated that the semen of HIV-infected men contains functional T lymphocytes, and that the majority of these cells are CD81 and express a marker of cytolytic activity (18). In this study, we sought to determine whether T lymphocytes from the semen of seropositive men also had anti-HIV cytotoxic activity. Materials and Methods Patients Five seropositive men were recruited from Fenway Community Health Center and Massachusetts General Hospital to provide semen and blood samples. Informed consent was obtained from all individuals. Eligibility criteria included a previous leukocytic semen specimen (.5 3 104 viable leukocytes per ejaculate) and a CD4 count of .500/ml. The clinical data for each individual are summarized in Table I. Isolation and cloning of effector cells Semen was obtained by masturbation into sterile specimen cups containing 10 ml of RPMI 1640. Blood was collected in EDTA-treated vacutainers. Viable semen round cells (SRCs)4 and PBMCs were isolated on density gradient separation medium (Ficoll-Hypaque, Pharmacia, Piscataway, NJ and washed three times in HBSS containing 100 mg/ml gentamicin and 1 mg/ml amphotericin B before resuspension in RPMI supplemented with 2 mM L-glutamine, 50 mg/ml gentamicin, 0.25 mg/ml amphotericin B, and 10% FCS (all obtained from Life Technologies, Grand Island, NY) (cRPMI). Since the isolated SRC fraction contains a large proportion of immature germ cells, SRCs (10% of sample) and PBMCs were applied to 8-spot slides, allowed to air dry, fixed in acetone, and later immunostained with Abs to CD3 and CD8 to enumerate exactly the number of T cells (18). Using published data obtained from leukocytic HIV1 individuals, we ascribed arbitrary values to the proportion of CD3 cells in each SRC population for immediate cloning purposes and adjusted the values after staining (18). Arbitrary values were 1% for SRCs and 70% for PBMCs. Cells were seeded in 96-well plates at numbers shown previously to approximate limiting dilution (10 –300 cells/well) in 100-ml volumes containing 1.5 3 106 irradiated (5000 rad) allogeneic PBMCs/ml, 1 mg/ml PHA, and 50 U/ml IL-2 (Boehringer Mannheim, Indianapolis, IN) in cRPMI. After 2 to 3 wk, wells exhibiting growth were restimulated with irradiated PBMCs, PHA, and IL-2. 4 Abbreviations used in this paper: SRC, semen round cell; BLCL, B lymphoblastoid cell line; IEL, intraepithelial lymphocyte. 0022-1767/98/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 CTLs play an important role in controlling cell-associated HIV. Since the majority of HIV infections are acquired through sexual transmission, we investigated whether antiviral CTLs were present in the male urogenital tract using semen as a source of T cells. We were able to establish anti-HIV cytolytic lines in five of five HIV-infected men with CD4 counts of >500/ml, although cloning efficiencies were lower than with peripheral blood-derived T cells. CTLs generated from the semen of three men were analyzed in detail and showed a broadly active response, recognizing gag, env, and pol proteins. Detailed analysis of two gag-specific clones from one of the individuals demonstrated HLA class I restriction and recognition of the same p24 epitope (EQASQEVKNWMT). In summary, our results demonstrate the presence of a broad CTL response to HIV in the urogenital tract and provide a rationale for further studies of local enhancement of genital mucosal responses by anti-HIV immunization. The Journal of Immunology, 1998, 161: 4406 – 4410. The Journal of Immunology 4407 Table I. HIV-related clinical history of donors Donor CD4 Counta Viral Loadb Clinical Coursec Drugsd No. of Years Infectede Mode of Transmission 9320 9408 POGO 221L 161J 932 725 .1400 575 .1400 NDf ND 1.1 3 10c 6.0 3 10d ,400 Aysmp. Aysmp. Asymp. K.S. Aysmp. None None None None None 9 .10 17 .12 .20 Sexual intercourse Sexual intercourse Sexual intercourse Sexual intercourse Contaminated blood products Peripheral blood CD41 cell count/ml. HIV RNA copies/ml plasma. c Asymp., asymptomatic; K.S., Kaposi’s sarcoma. d HIV-related medication (antiretrovirals, protease inhibitors). e Documented positive serological test excluding POGO. POGO documented seropositive from 1996, but single high risk exposure in 1981 (personal history). f ND, not done. a b Immunostaining B lymphoblastoid cell lines (BLCLs) Before cloning, BLCLs were established by transformation of peripheral blood B cells by EBV obtained from the supernatant of the B95-8 cell line (8). Recombinant vaccinia viruses and synthetic HIV-1 peptides Recombinant vaccinia viruses constructed from the HIV IIIB isolate and expressing the following proteins were used in this study: env, gag, and pol (Vabt 408); gag (Vabt 401); p17 (Vabt 228); p24 (Vabt 286) (kind gifts of Therion Biologics, Cambridge, MA); env (PE16); and pol (CF21) (National Institutes of Health AIDS Repository, Rockville, MD). Wild-type vaccinia virus was used as a control (NYCBH, Therion Biologics). Synthetic p24 HIV peptides (12–21 aa) were synthesized as described previously (20). Lyophilized peptides were resuspended at 2 mg/ml in 10% DMSO. Cytotoxicity assay Cloned T cells were tested for HIV-specific cytolytic activity in a standard chromium release assay (8). Target cells were autologous BLCLs pulsed with HIV peptides or infected with vaccinia constructs expressing HIV proteins (14). Partially MHC-mismatched allogeneic BLCLs were also used in MHC restriction experiments. In brief, BLCLs to be vaccinia infected were incubated with 3 to 4 multiplicities of infection per cell of recombinant virus for 16 to 18 h at 37°C. Cells were washed twice, labeled with 150 mCi Na251Cr for 2 to 3 h, and washed four times. The sensitization of BLCLs with peptides was achieved by incubating cells with 10 mg/ml of peptide with the chromium. Clones and labeled BLCLs were incubated together in various ratios in 150-ml volumes in 96-well plates at 37°C for 4 to 6 h. Supernatants (100 ml) were subsequently harvested and counted in a Cobra gamma counter (Packard Instrument, Meriden, CT). The percentage of specific lysis was calculated from the following formula: 100 3 ([experimental release 2 spontaneous release]/[maximum release 2 spontaneous release]). Maximum release values were obtained by the lysis of targets with 5% Triton X-100. Assays were only evaluated if spontaneous release was ,30% of maximum release. Clones were considered positive if lysis of the targets was at least three times greater than the lysis of the control vaccinia target and if the HIV-1-specific lysis was .10% (14, 21). Results Isolation, phenotyping, and HIV-specific cytolytic activity of semen T cells Our previous studies have shown that viable CD31 cell counts in HIV1 men range from undetectable to .2.2 3 105 per ejaculate, HIV-1 env-, gag-, and pol-specific CTLs in semen To precisely define the HIV cytolytic responses in semen, lines from three individuals were expanded and subsequently tested for activity against the individual gag, pol, and env proteins (Table III). Activity against all three proteins was noted in each donor, indicating that CTLs derived from the urogenital tract have a broad-based activity to HIV. With the exception of two lines, all lines had activity against a single protein. In donor 9320, the predominant protein recognized was pol (43% of all specific activity), followed by gag (36%) and envelope (21%). In donors POGO and 161J, the predominant protein recognized by CTLs was gag (67 and 57% of specific activities, respectively). In one individual (9320), we also performed a parallel analysis on a small number of Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 T cells were enumerated by a standard indirect immunohistology technique that has been described previously in detail (18). Primary Abs recognizing CD3 and CD8 Ags (Dako, Carpinteria, CA) were used, and Ab-positive cells were visualized using an alkaline phosphatase/anti-alkaline phosphatase kit (Dako). A total of 200 cells were counted per Ab, and the number of positive cells in the semen sample was calculated from the SRC count. Clones were phenotyped using similar methodology, with Abs recognizing the following Ags: CD3, CD8, CD4, CD57 (Dako), TCRab (BF1, a kind gift of Dr. Michael Brenner, Department of Rheumatology, Brigham and Women’s Hospital), and TIA-1 (a kind gift of Dr. P. Johnson, Dana-Farber Cancer Institute, Boston, MA) (19). with a median value of 9.0 3 103 in men taking antiretroviral medication and 2.5 3 104 in men who are not on therapy (18). Since the number of cells recovered from semen is relatively small and the estimated frequency of HIV-specific T cells in asymptomatic individuals is estimated to range from 1/102 to 104 in PBMCs (11, 22, 23), we optimized the conditions of this study by recruiting men who had previously had a leukocytic semen sample (defined as .5 3 104 viable mononuclear leukocytes per ejaculate). A previous study from our laboratory indicates that this is not a major selection step, since 83% of HIV1 men with peripheral blood CD4 counts of .200/ml that are not on antiretroviral therapy are included in this category (18). As shown in Table I, the median CD4 count of the patients was 932, and the median length of infection was 12 yr. None of the individuals were on antiretroviral therapy, and all were asymptomatic, with the exception of one man who had recently developed Kaposi’s lesions. The numbers of viable round cells recovered from the semen samples used in this study ranged from 2.5 to 34.0 3 105 cells per ejaculate (Table II). Immunostaining revealed that between 1.1% and 14.3% of round cells (median 2.5%) were CD31, with CD81 lymphocytes contributing from 33 to 95% of the T cell population. Cells were successfully cloned from semen at a limiting dilution with PHA and IL-2, but cloning efficiencies were considerably lower than those derived from blood (Fig. 1). Fungal contamination was also observed in some semen-derived cultures, but these wells were not taken into account when calculating cloning efficiencies. Wells exhibiting proliferation were restimulated, and those which were successfully expanded were screened for antiHIV cytolytic activity using autologous BLCLs infected with a trivalent vaccinia vector expressing env, gag, and pol proteins. HIV-specific cytolytic activity was found in all five donors, with multiple lines isolated from each donor (Table II). 4408 HIV-SPECIFIC CTLs IN SEMEN Table II. Enumeration, phenotyping, and assessment of HIV-specific cytolytic activity of semen-derived T cells Viable Cells in Semen Semen CTL Activityd Peripheral Blood CTL Activityd Patient Round cells 3 105a CD31 cells 3 103b CD81 cells 3 103b Wells exhibiting HIVspecific lysis wells evaluablec Wells exhibiting HIVspecific lysis wells evaluablec 9320 9408 221L POGO 161J 34.0 0.25 1.60 0.8 2.6 486.2 0.3 21.6 1.8 7.5 387.6 0.1 14.7 1.7 3.9 12/46 3/23 18/66 8/43 8/16 4/90 NDe 32/109 ND ND a Total number of viable cells (mononuclear cells and immature germ cells) recovered from Ficoli density gradient separation of semen cellular fraction. b Cells were phenotyped by standard immunocytochemical staining using an alkaline phosphatase/anti-alkaline phosphatase detection system. A total of 200 cells were counted per mAb, and the numbers of cells per ejaculate expressing this Ag were calculated from the total round cell count. c Total number of wells exhibiting growth and which could be expanded by one round of restimulation. d Lines were considered to have HIV-specific cytolytic activity if lysis of autologous BLCLs infected with the trivalent vaccinia construct expressing env, gag, and pol was three times the value of BLCLs pulsed with wild-type vaccinia and .10%. e ND, not done. to determine that both of these lines were restricted by HLA B44 (Fig. 3). Phenotyping studies indicated that 100% of the cells in both lines stained positively for TCRab, CD8, and TIA-1. MHC restriction and epitope recognition of gag-specific semenderived HIV-1-specific CTLs Discussion Two of the gag-specific lines from donor POGO were examined in greater depth for fine epitope specificity, phenotype, and MHC restriction. We first used vaccinia constructs expressing the p17 and p24 regions of gag to demonstrate that both gag-specific lines recognized epitopes within the p24 protein (Fig. 2). When a series of nested 21-mer peptides covering the p24 sequence in pools of four (data not shown) were used followed by individual peptides, data indicated that both lines recognized the gag peptide representing aa 303 to 324. Analysis with shorter (12-mer) peptides indicated that both recognized the aa sequence 307 to 318. Using BLCLs partially matched at MHC class I loci, we were also able FIGURE 1. Relative cloning efficiencies of T cells derived from semen (E) and from peripheral blood (l). Results are expressed as the percentage of wells exhibiting growth after 15 to 20 days of culture with irradiated allogeneic PBMCs, IL-2, and PHA. The number of wells at each cell concentration ranged from 30 to 50. In this study, we describe a novel method for investigating CTL activity in the male urogenital tract using semen as a source of locally derived T cells. We reported previously that the semen of asymptomatic, seropositive men not taking antiretroviral therapy contains viable, functional T cells that are predominantly of the CD81 phenotype and express markers of mucosal derivation and cytolytic activity (18). Seminal T cells may originate from many sites within the male genital tract including the rete testis, epididymis, prostate, vas deferens, and urethra (24). Since the potential antiviral cytolytic activity of male urogenital tract cells has not been investigated, we optimized study conditions by recruiting individuals who had peripheral blood CD4 counts of .500/ml and a previous leukocytic ejaculate. We found substantial cytolytic activity in all five individuals examined, although cloning efficiencies were lower than in blood. This may reflect a lower functional capacity of T cells recovered from semen due to their exposure to highly immunosuppressive seminal plasma (25) or may reflect innate differences between T cells derived from a mucosal vs a peripheral location. For example, intraepithelial lymphocytes (IELs) derived from normal gastrointestinal mucosa demonstrate a considerably lower proliferative capacity than their blood counterparts (26), a phenotypically distinct profile (27), and an oligoclonal T cell repertoire (28, 29), and may include unique regulatory subsets of T cells (30). The presence of HIV-specific CTLs in a mucosal site was first demonstrated with T cells isolated from bronchoalveolar lavages (15). The first isolation of HIV- or SIV-specific CTLs from the genital tract was by Lohman et al. (16). Macaques were vaginally infected with SIV, acutely and chronically infected animals were sacrificed, and vaginal IELs were extracted, expanded, and tested for CTL activity. IELs were characteristically CD81, TCRab1, and CD21; precursor frequencies were similar to that in macaque blood; and chronically infected monkeys had greater numbers of virus-specific cells than those that were acutely infected. More recently, HIV-specific CTLs were cloned from cervical mononuclear cells that had been extracted from HIV-infected women by cervical cytobrush collection (17). Although cytobrush sampling Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 lines derived from the peripheral blood. Although pol-specific activity dominated in the semen of this individual, only one of four blood lines was pol-specific. The Journal of Immunology 4409 Table III. HIV-specific cytolytic activity of semen-derived T cell lines Specific Lysis (%)a Donor tri gag pol env 9320 60-1 60-2 60-3 60-4 60-5 60-6b 100-1 100-2 100-3b 100-4 100-5 100-6 Total 2 2 2 1 2 4 2 2 2 2 3 2 24 19 18 40 82 18 87 84 32 7 8 80 2 1 1 48 8 3 80 79 16 3 5 64 5 38 28 25 3 90 14 3 5 24 5 3 2 6 8 9 1 5 8 20 8 4 5 11 10 4 3 POGO 10.4 30.17 30.35 30.37 30.39 30.42 Total 0 0 0 9 0 0 47 23 34 30 40 36 31 1 0 26 17 15 4 0 0 24 0 0 0 1 NDc 10 ND ND ND ND 1 161J 60.1 60.2b 30.14 30.16 30.12 Total 0 0 0 0 0 56 25 32 46 38 2 57 18 11 14 4 3 22 0 0 0 1 67 54 0 4 0 2 a Lines were considered cytolytic against HIV env, gag, or pol proteins if lysis of autologous BLCLs infected with specific HIV vaccinia constructs was .10% and three times greater than lysis of BLCLs infected with wild-type vaccinia. Cytolytic clones are denoted in boldface. b CTL analysis of lines indicated activity against two or three HIV proteins. c ND, not done. can induce local bleeding, the IEL-like phenotype of these cells was suggestive of their mucosal derivation. The authors noted HIV-specific activity in the cervix of 63% of the subjects sampled; FIGURE 2. Semen-derived, gag-specific clones from donor POGO recognize the gag p24-specific epitope representing aa 307 to 318 (EQASQEVKNWMT). Autologous BLCLs were pulsed with wild-type vaccinia (control), vaccinia recombinants expressing p17 and p24 gag proteins, the peptide representing aa 303 to 324 (TLRAEQASQEVKNW MTETLLVQ), and truncations of this peptide. E:T ratios were 5:1. FIGURE 3. Semen-derived, gag-specific clones from donor POGO are restricted by HLA class I B44. Autologous (HLA class I A2, A30, B44, B51, and Bw4) BLCLs or allogeneic BLCLs matched at one locus were pulsed with the p24 gag peptide representing aa 303 to 324. E:T ratios were 5:1. Results are given as the percentage of HIV-specific cytotoxicity. Background counts (unpulsed BLCLs) were ,1% in all cases. the highest anti-HIV cervical CTL activity was associated with a peripheral blood CD4 count of .500 cells/ml. A comparison between small numbers of blood and cervix-derived gag-specific lines in one donor indicated that CTLs derived from the two sites recognized common epitopes. We did not undertake a comparison of epitope specificities in the paired lines that we derived from one of our donors, but we did note a predominance of pol specificity in semen that was not seen in the blood of this individual. Clearly, it still remains to be established whether blood and mucosal CTLs exhibit different CTL repertoires, whether these will reflect differences in TCR repertoires, where and how Ag is presented to these cells, or whether distinct HIV variants are being recognized (3). The isolation of HIV-specific cytotoxic T cells from the female and male urogenital tracts indicates that virus-specific lymphocytes are recruited to the urogenital mucosa of HIV-infected individuals. The presence of these effector cells in the genital tract may be critical in controlling local HIV infection, thus reducing the viral load in mucosal secretions and the chance of transmission to partners during sexual intercourse. A study looking at the correlation of CTL activity with viral load in semen is currently underway. Indications that a local immune response may prevent dissemination of virus come from a study which found that some seronegative women with seropositive partners and a history of multiple unprotected sexual exposures had HIV-specific secretory IgA but not IgG in their vaginal secretions (31). We presently hypothesize that an appropriately immunized individual with a strong, local, antiviral mucosal immune response should be able to eradicate or to locally contain HIV following genital transmission. If partners are concordant for MHC class I molecules, an immunized individual may also be able to specifically kill donor HIV-infected cells derived from the genital secretions of their partner (32, 33). In contrast, MHC discordance may result in a host vs donor immune response, and this allogeneic recognition may result in an alternative mechanism of eradicating virally infected cells. Whether MHC discordance between sexual partners would decrease the chance of virus transmission or concordance would increase sexual transmission, as has been recently shown in a perinatal transmission study, has yet to be investigated (34). Clearly it would be advantageous to develop an anti-HIV vaccine that would elicit HIV-specific mucosal CTLs. Although some studies indicate that immunization at a mucosal site is not a prerequisite for induction of a mucosal immune response (35, 36), there is a substantial amount of evidence to indicate that mucosal immunization strategies are more effective at generating a local Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 wt 4410 response (37). In addition, long-lived antiviral CTLs can be detected in mucosal tissues after mucosal but not systemic immunization, suggesting that the maintenance of memory cells is also dependent upon the chosen route of immunization (38). Of further note is the observation that inoculation at proximal mucosal sites appears to be more effective at inducing measurable immune responses than inoculation at distant mucosal sites. For example, recent studies have indicated the effectiveness of a vaginal or rectal immunization protocol in inducing a genital tract immune response (39 – 41). Consequently, we plan to address in our future studies whether antiviral CTL responses can be induced in the male urogenital tract by vaccination and, if so, which routes of immunization are the most effective at inducing and maintaining a local immune response. Acknowledgments References 1. Auerbach, D. M., W. W. Darrow, H. W. Jaffe, and J. W. Curran. 1984. Cluster of cases of the acquired immunodeficiency syndrome: patients linked by sexual contact. Am. J. Med. 76:487. 2. Mayer, K. H., and D. J. Anderson. 1995. Heterosexual HIV transmission. Infect. Agents Dis. 4:273. 3. Zhu, T., N. Wang, A. W. Carr, D. S. Nam, R. Moor-Jankowski, D. A. Cooper, and D. D. Ho. 1996. Genetic characterization of human immunodeficiency virus type q in the blood and genital secretions: evidence for viral compartmentalization and selection during transmission. J. Virol. 70:3098. 4. Anderson, D. J., T. R. O’Brien, J. A. Politch, A. Martinez, G. R. Seage, N. R. Padian, C. R. Horsburgh, and K. H. Mayer. 1992. 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Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women. Infect. Immun. 65: 1387. 41. Livingston, J. B., S. Lu, H. Robinson, and D. J. Anderson. 1998. DNA immunization of the female genital tract. Infect. Immun. 66:322. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 We thank the individuals who donated samples for this study. We also thank Dr. Eric Rosenberg at Massachusetts General Hospital and Robert Goldstein and Judy Erdman at Fenway Community Health Center for recruiting patients, Lidiya Shver and Lynne Tucker for excellent technical assistance, and Dr. Gail Mazzara at Therion Biologics for providing recombinant vaccinia viruses. HIV-SPECIFIC CTLs IN SEMEN