• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content
Comparison of Systemic and Mucosal Delivery of 2 Canarypox ...
 

Comparison of Systemic and Mucosal Delivery of 2 Canarypox ...

on

  • 1,104 views

 

Statistics

Views

Total Views
1,104
Views on SlideShare
1,104
Embed Views
0

Actions

Likes
0
Downloads
0
Comments
0

0 Embeds 0

No embeds

Accessibility

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

    Comparison of Systemic and Mucosal Delivery of 2 Canarypox ... Comparison of Systemic and Mucosal Delivery of 2 Canarypox ... Document Transcript

    • MAJOR ARTICLE Comparison of Systemic and Mucosal Delivery of 2 Canarypox Virus Vaccines Expressing either HIV-1 Genes or the Gene for Rabies Virus G Protein Peter F. Wright,1 Jiri Mestecky,2 M. Juliana McElrath,4 Michael C. Keefer,5 Geoffrey J. Gorse,6 Paul A. Goepfert,3 Zina Moldoveanu,2 David Schwartz,7 Paul W. Spearman,1 Raphaelle El Habib,11 Michele D. Spring,1 Yuwei Zhu,1 Carol Smith,8 Jorge Flores,9 Kent J. Weinhold,10 and the National Institutes of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group 1 Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee; 2Mucosal Immunology Laboratory, 3Department of Medicine, University of Alabama at Birmingham, Birmingham; 4University of Washington School of Medicine, Seattle; 5University of Rochester School of Medicine and Dentistry, Rochester, New York; 6Saint Louis Department of Veterans Affairs Medical Center and Saint Louis University School of Medicine, St. Louis, Missouri; 7Johns Hopkins University School of Medicine, Baltimore, and 8EMMES Corporation, Rockville, and 9Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland; 10Central Immunology Laboratory, Duke University Medical Center, Durham, North Carolina; 11Pasteur Aventis, Marcy l’Etoile, France Background. Since the primary routes of human immunodeficiency type 1 (HIV-1) infection are across mucosal barriers, a randomized trial of canarypox virus–based vectors was conducted in 84 individuals, with delivery of vaccine by mucosal routes, and was accompanied by a detailed analysis of humoral, cellular, and mucosal immune responses. Methods. Over the course of 6 months, HIV-1–specific (vCP 205) and rabies (vCP 65) canarypox virus vectors were delivered systemically and/or mucosally into the nose, mouth, vagina, or rectum in a 4-dose schedule, followed by 2 doses of HIV-1 MN recombinant glycoprotein (rgp) 120 or subunit rabies vaccine administered by the intra- muscular route. Results. Administration of vaccine and collection of samples were well tolerated. Serum IgG HIV-1–specific antibodies to rgp120 were rarely seen after either systemic or mucosal delivery of canarypox virus vaccine. In contrast, serum IgG rabies and canarypox antibodies were detected in all individuals after systemic, but rarely after mucosal, delivery of vaccine. Suggestions of mucosal recognition of HIV-1 antigen included a cytotoxic T lymphocyte response in 4 of 8 individuals after administration of vaccine by the intrarectal route and a limited immunoglobulin A response at the same site. Conclusions. Each of the routes of vaccine administration was feasible in the context of a phase 1 study with motivated individuals. However, with the doses and routes of administration used, canarypox virus was not an effective mucosal immunogen. The current criteria for the development of an effective acquired disease are often neglected in consideration of AIDS vaccine include demonstration of cytotoxic T protective immunity [3]. Results of animal models have lymphocyte (CTL) responses and induction of cross- suggested that mucosal immunity may be important in reacting neutralizing antibodies against HIV-1 isolates the prevention of infection [4, 5, 6]. At present, with [1, 2], although true correlates of protection are not 1 exception [7], all attempts at immunization against well established. The roles that mucosal antibodies and HIV-1 in humans have been made with systemically local CTLs may play in what is usually a mucosally delivered vaccines. HIV-1–specific IgA antibodies have been detected in secretions obtained from highly exposed but seroneg- Received 22 May 2003; accepted 18 September 2003; electronically published ative women [8, 9, 10]. These antibodies have been 15 March 2004. suggested as a correlate of protection against infection Reprints or correspondence: Dr. Peter Wright, Vanderbilt University Medical Center, Section of Infectious Diseases, Rm. D-7219 MCN, Nashville, TN 37232 [11]. Local CTL responses can be detected in in vitro– (peter.wright@vanderbilt.edu). expanded T cells from cervical scrapings of HIV-1–in- The Journal of Infectious Diseases 2004; 189:1221–31 2004 by the Infectious Diseases Society of America. All rights reserved. fected women [12], and systemic CTLs have been de- 0022-1899/2004/18906-0013$15.00 tected in an HIV-1–exposed but uninfected group [8]. Mucosal HIV-1 Vaccine • JID 2004:189 (1 April) • 1221
    • However, other reports have demonstrated diminished IgA re- mucosal routes of delivery included 4 doses of vCP vector ad- sponses in HIV-1 infection and have suggested that IgA may ministered by the oral, intranasal (inl), intravaginal, or intrarectal not recognize the gp120 portion of the HIV-1 envelope [13, route, followed by 2 intramuscular (im) injections of subunit 14]. It has recently been shown that HIV-1–specific IgA re- vaccine. Two study groups received a vCP vector by either the sponses are absent or are difficult to measure in HIV-1–infected inl/im or intrarectal/im combined route, followed by subunit patients [15]. vaccine. In one group, im vector vaccinations alone were followed With other viral pathogens, optimal induction of mucosal by inl vector vaccination. The dose of vCP205 (Pasteur Aventis) immunity has been achieved by direct mucosal delivery of live was 106.7 TCID50, and the dose of vCP65 (Pasteur Aventis) was attenuated vaccines [16, 17]. Experience with respiratory vi- 106.3 TCID50. ruses has convincingly demonstrated that local IgA antibody The inl vaccine was administered by use of a spray device produced by topical delivery of vaccine can be a correlate of (Becton-Dickinson) that delivers a large-particle aerosol to the immunity [18]. To address the induction of immunity to HIV- upper airway. This device was successfully used in large-scale 1 by mucosal delivery of vaccine, a phase 1 safety and immu- trials of influenza virus vaccine [16]. The oral vaccine was nogenicity trial was conducted by use of live recombinant canary- gargled for 15 s before being swallowed. The intrarectal vaccine pox HIV-1 (ALVAC-HIV-1; vCP205) and rabies (ALVACRG; was administered by use of a 3-mL plastic syringe pipette vCP65) vectors delivered mucosally, followed by booster injec- (Fisher Scientific Supply). The intravaginal vaccine was ad- tions of VaxGen MN recombinant glycoprotein (rgp) 120 or ministered by use of the same type of pipette as above into the licensed rabies vaccine, by systemic delivery, in healthy adult posterior fornix of the vagina, after which the woman remained volunteers. The study was designed to establish the safety of the supine with knees bent for 15 min. canarypox preparations delivered mucosally and to determine The timing of vaccine administration is shown in table 2. The whether delivery of vaccine by these routes stimulated or primed subunit products (Imovax diploid rabies vaccine [Pasteur Mer- for mucosal or systemic humoral and cell-mediated responses. ieux Connaught] and AIDSVAX MN rgp120 [VaxGen]) were originally scheduled to be administered at 9 and 15 months after initiation of the vaccination series, but there was a delay in the SUBJECTS AND METHODS supply of MN rgp120 vaccine. The actual time of administration varied between 84 and 322 days after initiation of the vacci- Populations studied. A randomized, blinded, multicenter nation series, with a median interval of 140 days between the phase 1 safety and immunogenicity study was performed by the National Institute of Allergy and Infectious Diseases AIDS end of the primary vaccination and the first booster injection. Vaccine Evaluation Group (AVEG), a predecessor to the current The second booster injection was administered as planned, 6 HIV Vaccine Trials Network. The demographic characteristics months after the first booster injection. Randomization and of the population recruited are shown in table 1. Approval for data and study management were performed by the EMMES this study was obtained from each of the investigator’s insti- Corporation (Rockville, MD). tutional review boards. Exclusion criteria were similar to those Vaccine constructs. vCP205 is a recombinant canarypox described in other phase 1 studies conducted by the AVEG and virus capable of entering mammalian cells, expressing its genes, included any immunodeficiency or chronic illness, behavior and producing noninfectious viruslike particles. vCP205 ex- that placed the individual at higher risk of HIV-1 infection, or presses the gag gene portion of the Gag p55-polyprotein of the poor compliance with the protocol [19]. All women were tested HIV-1 LAI strain, the protease gene expressing the p15 protein for pregnancy and counseled to not become pregnant during of the LAI strain and the rgp120 of the HIV-1 MN strain, with the study. Women with abnormal results of a pelvic exami- an anchoring transmembrane region of gp41 of the LAI strain. nation or individuals with gastrointestinal symptoms or intra- The vCP65 virus expresses the G protein of rabies virus under rectal bleeding were excluded. Individuals with a history of the control of the vaccinia virus H6 promotor. vCP65 has been rabies vaccination were excluded, as were those who had re- shown to be safe in a variety of species and to induce antibody cently received blood products, experimental agents, or vac- and protection in a dose-dependent fashion [20–22]. The sub- cinations. Individuals were selected to be at low risk of ac- unit MN rgp120 (VaxGen) was produced in mammalian CHO quiring HIV-1 and were counseled at each clinic visit about cells and administered at a dose of 300 mg/mL in alum, and engaging in high-risk sexual behavior. the inactivated rabies vaccine Imovax, which was prepared in Study design. The 84 individuals (18–52 years old) in the human diploid cells, was administered at the standard potency study were divided into 7 study groups of 12 individuals, of of 2.5 IU of rabies antigen. whom 8 received the vCP205 construct and 4 received the Clinical safety. Individuals were observed for 30 min after vCP65 construct (table 2). The individuals were blinded to the each vaccination. Individuals recorded their temperatures the product they received but not to the route of delivery. The evening of vaccination and were questioned about or were ex- 1222 • JID 2004:189 (1 April) • Wright et al.
    • Table 1. Demographic characteristics and follow-up of individuals enrolled in the present study. Rabies vaccine HIV vaccine recipients recipients Total Variable (n p 28) (n p 56) (n p 84) Sex Female 13 25 38 Male 15 31 46 Race White, non-Hispanic 25 47 72 Black, non-Hispanic 2 6 8 Hispanic/Latino 0 2 2 Native American/Alaskan Native 1 0 1 Asian/Pacific Islander 0 1 1 Age, median (range), years 31 (18–52) 34 (18–50) 32 (18–52) No. of vaccinations (time) 1 (month 0) 28 56 84 2 (month 1) 28 56 84 3 (month 3) 27 (96%) 56 83 (99%) 4 (month 6) 27 (96%) 52 (93%) 79 (94%) 5 (month 9)—booster injection 18 (64%) 44 (79%) 62 (74%) 6 (month 15)—booster injection 15 (54%) 40 (71%) 55 (65%) NOTE. Data are no. of individuals, unless otherwise noted. amined for symptoms. Particular attention was paid to symptoms ing assays to the V3 loop of MN rgp120 produced in CHO related to the route of vaccine administration. Hematologic, he- cells, as described in the standard AVEG protocols performed patic, and renal functions were monitored during the trial. at the Central Immunology Laboratory (Duke University, Dur- Samples were obtained from the first 4 individuals who re- ham, NC) [24]. The data in the present study will focus on ceived vaccine by each route, at 4–8 h and at 24 h after primary the immune response 2 weeks after the primary ALVAC series vaccination, to detect any residual virus as evidence of virus and after the first and second booster injections. replication. Virus isolation was attempted in primary chick ELISPOT assays were performed at the Mucosal Immunology embryo fibroblast cells. No canarypox virus was recovered from Laboratory (University of Alabama at Birmingham, Birming- 17 nasal, 11 oral, 9 vaginal, and 30 rectal samples obtained at ham, AL) on heparinized peripheral blood samples obtained 1 either 4–8 h or 24 h after administration of vaccine. week after the completion of the primary series and after the Growth of canarypox virus on human mucosal epithelial initial booster injection. IgG or IgA antibody–secreting cells cells. To confirm growth and protein expression of canarypox were identified by use of the Chiron HIV-1 SF-2 rgp120 pro- virus, a green fluorescent protein (GFP) insert in a canarypox duced in CHO cells, as a coating antigen [25]. Detection of 13 vector was examined on primary human adenoid epithelial spots/106 peripheral blood mononuclear cells was considered cells, in a model described elsewhere [23]. The GFP construct to be positive, since none of the 38 control subjects was found was a gift from James Tartaglia (Virogenetics, Troy, NY). The to have levels above this value. canarypox GFP construct expressed its encoded GFP protein, CTL assays were performed at the Central Immunology Lab- with a peak intensity of fluorescence seen at 48 h, and the oratory, according to a standard protocol [26], by use of vac- number of fluorescent cells was proportional to the input virus, cinia vectors expressing epitopes of HIV-1 envelope, Gag, and without spread to adjacent cells. As predicted, canarypox virus protease, to stimulate the effector cells. Interleukin (IL)–7 and could initiate infection and express encoded proteins in human IL-2 were added during the 14-day stimulation. The target cells mucosal epithelium but was defective in production of infec- were Epstein Barr Virus–transformed B lymphoblastoid cell tious virus. lines established from each individual. The cells were labeled Systemic immunogenicity. Serum was collected for an- with 51Cr and infected with vaccinia vectors. At 24 h, the effector tibody responses after each dose of vaccine and finally at 9 cells were added for a 6-h 51Cr release assay. Background val- months after the last dose of vaccine. HIV-1 antibody responses ues against vaccinia were reduced by cold target competition, were measured by neutralization of the MN virus and by bind- by use of vaccinia virus (vP1170)–infected targets in a 30-fold Mucosal HIV-1 Vaccine • JID 2004:189 (1 April) • 1223
    • Table 2. Schema for mucosal delivery of canarypox virus developed for influenza virus [27]. The samples were not cor- vaccines. rected for total IgA and IgG. b Statistical analysis. The x2 or Fisher’s exact test was used Months 0, 1, 3, and 6 Months 9 and 15 to compare data between the different routes. P ! .05 was con- Route of Route of sidered to be statistically significant. The data analyses were Groupa Product administration Product administration performed by use of the statistical packages SPSS (version 11.0; A A or RG im A or RG in SPSS). B A or RG po MN or IVX im C A or RG inl MN or IVX im D A or RG ir MN or IVX im RESULTS E A or RG ivag MN or IVX im F A or RG inl/im MN or IVX im Follow-up. Of the 84 individuals enrolled in the trial, 5 did G A or RG ir/im MN or IVX im not complete the initial series of vaccinations with the vCP NOTE. A, ALVAC-HIV vCP205 (106.7 TCID50); im, intramuscular; inl, in- virus vectors: 2 individuals were lost to follow-up, 1 refused to tranasal; ir, intrarectal; ivag, intravaginal; IVX, Imovax diploid cell rabies vac- continue, 1 underwent a hysterectomy, and 1 became pregnant. cine; MN, VaxGen MN recombinant glycoprotein (rgp) 120 (300 mg/mL); po, oral; RG, ALVAC-RG (vCP65) (106.3 TCID50). Fourteen additional individuals did not complete the delayed a Each group included 12 individuals: 8 vaccine recipients and 4 control subunit booster injections. No individual withdrew because of individuals. Control individuals received ALVAC-RG instead of ALVAC-vCP205 the route of vaccine administration or because of mucosal sam- and Imovax instead of MN rgp120. b The first of the booster injections was to be administered at month 9; plings to measure immune responses. because of a delay in vaccine availability, the vaccination was administered Vaccine safety. The vaccines were well tolerated, with few as soon as vaccine was available. The final vaccinations were administered 6 months later (see Subjects and Methods). local and systemic complaints. No temperatures 139 C were recorded during the trial. The only severe symptoms noted after vaccination with vCP 205 were 1 episode of malaise after ad- excess. A positive result was scored as 110% release above back- ministration by the oral route and 1 local reaction with ery- ground values. The responses were scored as positive only if thema and induration 25 cm2 after administration by the they occurred on 11 assay. Assays were performed at effector combined inl/im route. The number of individuals reporting :target ratios of 1:25 and 1:50. moderate to severe reactions was somewhat greater for the Rabies virus antibody–binding assays were performed by use combined inl/im and intrarectal/im routes (6/16) of vCP 205 of the Sanofi rabies antibody kit (Pasteur Diagnostics), with a than for a single route (4/40) of systemic or mucosal delivery cutoff of 0.5 ELISA units/mL. The level of IgA antibody to (P p .02; table 3). rabies virus antigens in serum samples with detectable IgG Adverse events reported during the study included ankle pain, antibody was defined by use of a kinetic ELISA previously abdominal pain secondary to cholelithiasis, persistent lower back developed for influenza virus [27]. IgA and IgG antibodies to pain leading to anterior spinal fusion, and severe left temporal canarypox virus were measured by use of a modification of the headaches attributed to migraines. These complaints were all same kinetic ELISA, with purified canarypox virus supplied by considered to be unrelated to vaccination. One woman who Virogenetics. The cutoff for all kinetic ELISAs was 15 mOD/ had received vaccine by the intravaginal route complained of min, a measure of the maximum rate of change in optical den- irregular menses, underwent hysterectomy, and was found on sity over time. pathologic testing to have adenomatous hyperplasia, mild cer- Mucosal immunogenicity. Samples were obtained accord- vicitis, and leiomyomas. An intensive review of all reported ing to protocols developed at the Mucosal Immunology Lab- menstrual irregularities, reported for 8 of the 38 women in the oratory [28]. Emphasis was placed on specimens obtained from trial, showed no association with the intravaginal route of ad- the site of vaccine administration and on samples obtained af- ministration. All laboratory abnormalities were transient with ter completion of the primary canarypox series and after the no trends in lymphocyte populations or renal, hematologic, or first and second subunit booster injections. An initial blinded hepatic profiles. test of samples for HIV-1 antibodies was performed at the Mu- HIV-1 humoral responses. Total Ig HIV-1–specific anti- cosal Immunology Laboratory by use of chemiluminscence- body responses to MN rgp120, measured by ELISA, were rarely enhanced Western blots [29]. Of 1800 Western blots run, only seen after the primary series of vCP205 delivered either by a a few samples had 1 total heavy and light chain Ig bands to combined (mucosal/systemic) route or a mucosal route (figure HIV proteins, and none could be confirmed by use of IgG- or 1). After a single subunit booster injection of MN rgp120 vac- IgA-specific reagents. Mucosal antibodies of the IgA and IgG cine, 10 of the 14 individuals who had received vaccine by a classes to rabies virus antigen, HIV-1, and canarypox virus were combined route of delivery had responses, compared with only all performed by use of variations of a kinetic ELISA originally 1 of the 32 individuals who had received vCP205 vaccine by 1224 • JID 2004:189 (1 April) • Wright et al.
    • Table 3. Moderate or severe systemic reactions to administration of HIV-1 and rabies ca- narypox vectors. vCP 205 (HIV-1) vCP 65 (rabies), Mucosal delivery Systemic delivery Combined delivery delivery by all routes Symptom (n p 32) (n p 8) (n p 16) (n p 28) Malaise 3 (9) 0 4 (25) 1 (4) Myalgia 0 0 2 (13) 1 (4) Headache 1 (3) 0 2 (13) 3 (11) Nausea 0 0 1 (6) 2 (7) Overall 4 (13) 0 6 (38) 6 (21) NOTE. Data are no. (%) of individuals. mucosal delivery (P ! .001). Thus, systemically, but not muco- with specific 51Cr release 110% were seen in 10 (19%) of 53 sally, delivered vaccine led to priming for an HIV-1 response individuals receiving the vCP205 construct and in 1 (4%) of 25 after a single dose of subunit vaccine. With the second dose of individuals receiving the vCP65 construct (P p .09). All bulk subunit vaccine, most (28/45) individuals, regardless of the ini- CTL responses were mediated by CD8 T cells, as demonstrated tial route of delivery, had an immune response. The group of by depletion of CD8 T cells. The frequency of CTL responses individuals who received vaccine by systemic delivery (table 2, by route of administration of did not differ significantly: 1 of 6 group A) had no response to the initial im vCP205 vaccination occurred after systemic delivery, 3 of 15 occurred after delivery or to subsequent inl vCP205 vaccination. by the combined routes, and 6 of 32 occurred after mucosal de- HIV-1–specific serum antibody responses in the IgA isotype livery. However, it was of interest that 4 of 8 individuals who followed a pattern similar to that of the total Ig ELISA, with received vCP205 vaccine by the intrarectal route had a demonstra- no responses seen in individuals after the primary vCP205 vac- ble CTL response. cinations. Responses after the first booster injection were more Rabies systemic responses. Serum rabies antibody re- common (P ! .001) after vCP205 vaccination by combined route sponses to vCP65 were measured after the primary series of of delivery (10/12) than after vaccination by mucosal delivery vaccinations. The rabies response to vCP65 was greater than (2/22). To investigate whether a subunit vaccine alone could the HIV response to vCP205. All tested individuals who had induce an IgA response, 5 individuals who had received 3 doses received vCP65 by systemic or combined delivery had a serum of vaccine and participated in earlier AVEG trials were tested. IgG (figure 3A) and IgA (figure 3B) antibody response. Re- All had easily measurable IgG responses, and 2 of the 5 individ- sponses were detected as early as the second dose of vaccine. uals had HIV-1–specific IgA responses. Significantly fewer (2/14) individuals who had received vCP65 Neutralizing antibodies to the MN strain were not seen after by mucosal delivery had rabies IgG antibody response (P ! primary vCP205 vaccinations (figure 2). With the first booster .001). One of these had preexisting antibody, presumably as a injection of MN vaccine, there was evidence that the vaccination result of prior, unreported rabies vaccination. After a single by the combined route of delivery had primed the humoral booster injection of the Imovax rabies vaccine, 6 of 12 indi- response: 10 of 12 individuals responded, compared with 2 of viduals who received vCP65 by mucosal delivery had a serum 22 individuals who had received initial vaccination by mucosal IgG response. This is an expected response rate to a single dose delivery (P ! .001). of Imovax and is not evidence of priming [30]. However, all When IgA and IgG antibody–secreting cells were examined by 7 individuals who received vCP65 by mucosal delivery had a ELISPOT 1 week after individuals had received the last vCP205 high IgA rabies virus serum antibody response after the first vaccination, 0 of 52 had IgA and 2 of 52 had IgG HIV-1–specific booster injection of Imovax by systemic delivery, suggesting antibody–secreting cells in their peripheral blood samples. One that mucosal priming had occurred (figure 3B). week after the second booster injection of MN rgp120, IgG anti- Canarypox virus systemic responses. Canarypox virus– body–secreting cells were frequently seen (i.e., in 31 of 39 in- specific IgG and IgA antibodies were measured at the comple- dividuals). IgA antibody–secreting cells were seen in 8 of 39 tion of the primary vaccinations in 34 individuals. The data individuals. With a different measure of humoral immunity, MN for vCP65 and vCP205 are combined. IgG immune responses rgp120 vaccination by systemic delivery enhanced the very lim- in 16 individuals who received vaccine by either systemic or ited HIV-1–specific humoral response to primary vCP205 vac- combined delivery were measured; all individuals responded cination. That response was mainly in the IgG isotype. (figure 4A). Limited IgG antibody responses to mucosal delivery HIV-1 cellular responses. HIV-1–specific CTL responses of vaccine were seen (i.e., in 5 of 18 individuals). Serum IgA Mucosal HIV-1 Vaccine • JID 2004:189 (1 April) • 1225
    • Figure 1. Serum immune response, as measured by total immunoglobulin specific to HIV-1 MN recombinant glycoprotein (rgp) 120, in individuals receiving vCP205 or intramuscular (im) subunit MN rgp120 vaccine. Individuals receiving vaccine by mucosal delivery received vCP205 by 1 of the 4 mucosal routes (intranasal [inl], oral, intravaginal, or intrarectal). Individuals receiving vaccine by combined (mucosal/systemic) delivery received vCP205 by either the inl/intramuscular or the intrarectal/im route. responses were seen in 6 of 16 individuals who received vaccine examined for canarypox virus–specific antibodies. We did not by either systemic or combined delivery and in 3 of 18 indi- differentiate between individuals who received vCP65 and those viduals who received vaccine by mucosal delivery (figure 4B). who received vCP205. After the primary vaccinations, IgG ca- IgA responses were detected in 5 of 9 individuals who received narypox antibodies were seen in only 6 of 52 individuals test- intrarectal vaccine. ed; 5 of the positive responses were in individuals who had HIV-1 mucosal responses. No mucosal antibody responses received vaccine administered by the intravaginal route. This, to HIV-1 were seen after the initial vCP205 vaccinations by again, suggested a local IgG response at this site, since intra- mucosal delivery (0/19) or by combined delivery (0/10), when vaginal canarypox vaccine had not induced a systemic response. measured at the site of vaccine administration. Even after boost- Thirteen of 52 individuals tested had a mucosal IgA response, ing with subunit vaccine, only 2 vaginal and 2 nasal samples and 8 of the responders were among the 11 tested who received were positive for HIV-1–specific IgG. A rectal sample from 1 vaccine administered by the intrarectal route, suggesting that individual was found to be positive for IgA. a local IgA mucosal response was stimulated by vaccine deliv- Rabies mucosal responses. A limited number of samples, ered by this route. chosen to match the route of administration, were examined for rabies virus–specific antibodies. IgG antibodies to rabies DISCUSSION were seen at the site of vaccine administration in the 1 indi- vidual who had preexisting serum antibody. IgA antibodies were Mucosal delivery of vaccines and targeted stimulation of mu- detected in 13 samples from the site of vaccine administration. cosal immune responses is justified for infections that are ac- At the time of the first booster injection of subunit rabies quired by mucosal routes, such as HIV-1. Vaccination by the vaccine, IgG rabies antibodies were seen in only 3 cervical se- inl route with a canarypox vector expressing a canine distem- cretions. All cervical samples were obtained 5–20 days after the per hemagglutinin and fusion proteins fully protected ferrets last menstrual period, making blood contamination unlikely. from canine distemper and were as immunogenic as those with Canarypox virus mucosal responses. A limited number the same vector administered by the im route [31]. Attenuated of samples, chosen to match the route of administration, were poxviruses have been administered mucosally to animals and 1226 • JID 2004:189 (1 April) • Wright et al.
    • Figure 2. Serum neutralizing antibody to HIV-1 MN strain, in individuals receiving primary vCP205 or subunit intramuscular (im) MN recombinant glycoprotein 120 vaccine. Individuals receiving vaccine by mucosal delivery received vCP205 by 1 of the 4 mucosal routes (intranasal [inl], oral, intravaginal, or intrarectal). Individuals receiving vaccine by combined (mucosal/systemic) delivery received vCP205 by either the inl/im or the intrarectal/ im route. humans, to provide protection against variola and rabies [32], ered systemically were disappointing, they were consistent with and modified vaccinia Ankara administered by the intrarectal those seen in other recent studies of HIV-1 canarypox prod- route has been shown to induce mucosal CTLs [33]. ucts [19, 41]. There was priming for the first subunit booster In primate models, protection against infection with simian injection of subunit canarypox vaccine. With the first booster immunodeficiency virus (SIV) and the chimeric simian-human injection of gp120, a serum IgA response was detected. In pre- immunodeficiency virus (SHIV)–1 constructs has been shown vious studies, an IgA response was seen in 7 of 12 individuals to result from mucosal delivery of peptide-based vaccines after 2 doses of rgp120 subunit vaccine alone [40]. [34], vaccinia-derived vectors [35], and orally administered An effort was made in a single group of vaccine recipients to Sabin poliovirus vectors expressing overlapping peptides of determine whether canarypox virus vaccinations by systemic de- the SIV genome [36]. In the primate model, passive vacci- livery might prime for mucosal delivery of the same vaccine. nation with monoclonal antibody also protected against SHIV There was no evidence of this occurring; when measured, canary- infection [37, 38]. With modified vaccinia Ankara delivered pox virus–antibody responses continued to decline through the systemically, protection has been demonstrated with a mu- mucosal booster injections. However, because of the small num- cosal SIV challenge [6]. ber of individuals included, the present study does not defini- The delivery of canarypox vaccines by all of the mucosal tively answer the question concerning the importance of systemic routes in the present study was well tolerated and accepted. priming that precedes mucosal boosting, or vice versa. There were no safety issues posed by mucosal or systemic de- This is the first study performed in the evaluation of AIDS livery of the canarypox virus–vectored vaccines, confirming the vaccines that has systematically addressed the mucosal and sys- strong safety record of this product [39, 40]. The lack of re- temic responses to the control rabies virus antigen insert and covery of canarypox virus at mucosal sites indicates that, as to the canarypox vector itself. The canarypox virus responses would be expected, this is a replication-defective virus and that proved to be the most sensitive way of detecting a vaccine take. the original virus inoculum is not retained at the mucosal site It has suggested that, in some, but not in the majority, of in- for an extended period of time. dividuals who received vaccine by mucosal delivery, the vector Although the HIV-1–specific responses to the vaccine deliv- induced the expression of protein sufficiently to initiate an im- Mucosal HIV-1 Vaccine • JID 2004:189 (1 April) • 1227
    • Figure 3. Serum IgG (A) and IgA (B) antibody to rabies, in individuals receiving vCP65 or intramuscular (im) inactivated Imovax rabies vaccine. Individuals receiving vaccine by mucosal delivery received vCP65 by 1 of the 4 mucosal routes (intranasal [inl], oral, intravaginal, or intrarectal). Individuals receiving vaccine by combined (mucosal/systemic) delivery received vCP65 by either the inl/im or the intrarectal/im route. mune response. A hierarchy can be established of humoral im- systemic delivery. This could be a result of the protein expression munogenicity, with the vector itself being the most immuno- induced by this construct or the sensitivity of the detection assay genic, followed by the rabies insert, and finally by the HIV-1 but also may be an indication that the gp120 is a less immu- insert. The rabies construct induced more humoral immunity nogenic glycoprotein than the rabies G glycoprotein. vCP205 than did vCP205, after the primary series of vaccinations by routinely induced antibodies to the canarypox vector when ad- 1228 • JID 2004:189 (1 April) • Wright et al.
    • Figure 4. Serum IgG (A) and IgA (B) antibody to canarypox virus. Individuals receiving vaccine by mucosal delivery received vCP65 or vCP205 by 1 of the 4 mucosal routes (intranasal [inl], oral, intravaginal, or intrarectal). Individuals receiving vaccine by combined delivery (mucosal/systemic) received vCP65 or vCP205 by either the inl/intramuscular (im) or the intrarectal/im route. ministered systemically. A dose effect has been postulated for the 106.7 TCID50. Conversely, the lack of a systemic canarypox virus– canarypox product. The consistent induction of a response to antibody response by mucosal delivery suggests that a problem the vector indicates that any dose effect is not a function of a with the mucosal delivery might be a lack of initial infectivity of “nontake” of the vector at the dose used in the present study— the vector due to nonpermissiveness of the cells lining the mucosa Mucosal HIV-1 Vaccine • JID 2004:189 (1 April) • 1229
    • or innate defense mechanisms of the mucosa, including particle highly exposed, persistently seronegative female sex workers in north- ern Thailand. J Infect Dis 1999; 179:59–67. clearance by ciliary action or blockage of adherence by mucin. 11. Rowland-Jones S. Dimers are a girl’s best friend. Nat Med 1997; 11: However, when primary epithelial cells derived from adenoids 1199–200. were used as a target, a canarypox GFP was expressed, indicating 12. Musey L, Hu Y, Eckert L, et al. HIV-1–1 induces cytotoxic T lymphocytes in the cervix of infected women. J Exp Med 1997; 185:293–303. that there is no intrinsic resistance to infection with canarypox 13. Kozlowski PA, Jackson S. Serum IgA subclasses and molecular forms virus by cells from this site. in HIV-1 infection: selective increases in monomer and apparent re- Mucosal responses were much more difficult to analyze, and striction of the antibody response to IgA1 antibodies mainly directed they pose risks of overinterpretation of the limited positive at env glycoproteins. AIDS Res Hum Retroviruses 1992; 8:1773–80. 14. Belec L, Georges AJ, Steenman G, Martin P. Antibodies to human responses seen. However, 2 responses were notable and may immunodeficiency virus in vaginal secretions to heterosexual women. be significant as the field advances. In agreement with other J Infect Dis 1989; 160:385–91. studies (for review see [42]), the intrarectal route was the most 15. Wright, PF, Kozlowski PA, Rybczyk GK, et al. Detection of mucosal antibodies in HIV type 1–infected individuals. AIDS Res Hum Retro- likely to induce a local IgA response, both to the rabies and to viruses 2002; 18:1291–300. the canarypox antigens. This route also led to CTL responses 16. Belshe RB, Mendelman PM, Treanor J, et al. The efficacy of live, at- in 4 of 8 individuals, which is better than the response to vaccine tenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children. N Engl J Med 1998; 338:1405–12. delivered systemically. A cervicovaginal IgG response to the 17. Rennels MB, Glass RI, Dennehy PH, et al. Safety and efficacy of high- same antigens was seen in some of the individuals who received dose rhesus-human reassortant rotavirus vaccines—report of the na- intravaginal vaccine at a time chosen to be remote from their tional multicenter trial. Pediatrics 1996; 97:7–13. menstrual periods. 18. Belshe RB, Gruber WC, Mendelman PM, et al. Correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intra- The overall conclusion must be that very limited HIV-1– nasal influenza virus vaccine. J Infect Dis 2000; 181:1133–7. specific mucosal responses were seen with systemically or mu- 19. Belshe RB, Stevens C, Gorse GJ, et al. Safety and immunogenicity of cosally delivered vaccine. However, a template has now been a canarypox-based human immunodeficiency virus type 1 vaccine with and without gp 120: a phase 2 study in higher- and lower-risk vol- established for the mucosal delivery of HIV-1 vaccines. Other unteers. J Infect Dis 2001; 183:1343–52. vaccines, after establishment of preliminary data on immu- 20. Fries LF, Tartaglia J, Taylor J, et al. Human safety and immunogenicity nogenicity in animal models and a theoretical basis for pre- of a canarypox rabies glycoprotein recombinant vaccine: an alternative suming mucosal effectiveness, should be evaluated using pro- poxvirus vector system. Vaccine 1996; 14:428–34. 21. Taylor J, Trimarchi C, Weinberg R, et al. Efficacy studies on a canary- tocols such as the one used in the present study. pox-rabies recombinant virus. Vaccine 1991; 9:190–3. 22. Taylor J, Meignier B, Tartaglia J, et al. Biologic and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in References non-avian species. Vaccine 1995; 13:539–49. 23. Endo Y, Carroll KN, Ikizler MR, Wright PF. Growth of influenza in 1. Haynes BF, Pantaleo G, Fauci AS. Toward an understanding of the primary, differentiated epithelial cells derived from adenoids. J Virol correlates of protective immunity to HIV-1 infection. Science 1996; 1996; 70:2055–8. 271:324–8. 24. Graham BS, Matthews TJ, Belshe RB, et al. Augmentation of human 2. Graham BS, Wright PF. Candidate AIDS vaccines. N Engl J Med 1995; immunodeficiency virus type 1 neutralizing antibody by priming with 333:1331–9. gp 160 recombinant vaccinia and boosting with rgp 160 in vaccinia- 3. Vermund SH. Transmission of HIV. In: Devita V, Hellman S, Rosenberg naive adults: the NIAID AIDS Vaccine Trials Network. J Infect Dis 1993; SA, eds. AIDS. 4th ed. Philadelphia: Lippincott-Raven Press, 1997:147–65. 167:533–7. 4. Van Rompay K, Berardi C, Dillard-Telm S, et al. Passive immunization 25. Czerkinsky C, Moldoveanu Z, Mestecky J, et al. A novel two colour of newborn rhesus macaques prevents oral simian immunodeficiency ELISPOT assay: simultaneous detection of distinct types of antibody virus infection. J Infect Dis 1998; 177:1247–59. secreting cells. J Immunol Methods 1988; 115:31–7. 5. Lehner T, Wang Y, Cranage M, et al. Protective musosal immunity 26. Evans TG, Keefer MC, Weihold KJ, et al. A canarypox vaccine expressing elicited by targeted iliac lymph node immunization with a subunit SIV multiple human immunodeficiency virus type 1 genes given alone or envelope and core vaccine in macaques. Nat Med 1996; 2:767–75. with gp120 elicits broad and durable CD8+ cytotoxic T lymphocyte re- 6. Amara RR, Villinger F, Altman JD, et al. Control of a mucosal challenge sponses in seronegative volunteers. J Infect Dis 1999; 180:290–8. and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 27. Boyce TG, Gruber WG, Coleman-Dockery SD, et al. Mucosal immune 2001; 292:69–74. response to trivalent live attenuated intranasal influenza vaccine in 7. Lambert JS, Keefer M, Mulligan MJ, et al. A phase I safety and im- children. Vaccine 1999; 18:82–8. munogenicity trial of UBI(r) microparticulate monovalent HIV-1 MN 28. Jackson S, Mestecky J, Moldoveanu Z, Spearman P. Collection and oral peptide immunogen with parenteral boost in HIV-1 seronegative processing of human mucosal secretions. In: Ogra PL, Mestecky J, human subjects. Vaccine 2001; 19:3033–42. Lamm ME, Strober W, Bienenstock J, McGhee JR, eds. Mucosal im- 8. Mazzoli S, Trabattoni D, Lo Caputo S, et al. HIV-1–specific mucosal munology. San Diego: Academic Press, 1999:1567–75. and cellular immunity in HIV-1–seronegative partners of HIV-1–sero- 29. Mohamed OA, Ashley R, Goldstein A, et al. Detection of rectal anti- positive individuals. Nat Med 1997; 3:1250–7. bodies to HIV-1 by a sensitive chemiluminescent western blot im- 9. Kaul R, Trabattoni D, Bwayo J, et al. HIV-1–specific mucosal IgA in munodetection method. J Acquir Immune Defic Syndr 1994; 7:375–80. a cohort of HIV-1–resistant Kenyan sex workers. AIDS 1999; 13:23–9. 30. Cadoz M, Strady A, Meignier B, et al. Immunization with canarypox 10. Beyrer C, Artenstein A, Rugpao S, et al. Epidemiologic and biologic virus expressing rabies glycoprotein. Lancet 1992; 339:1429–32. characterization of a cohort of human immunodeficiency virus type 1 31. Welter J, Taylor J, Tartaglia J, Paoletti E, Stephensen CB. Mucosal 1230 • JID 2004:189 (1 April) • Wright et al.
    • vaccination with recombinant poxvirus vaccines protects ferrets against simian-human immunodeficiency virus infection. Nat Med 2000; 6: symptomatic CDV infection. Vaccine 1999; 17:308–18. 200–6. 32. Gherardi MM, Esteban M. Mucosal and systemic immune responses 38. Mascola JR, Steigler G, Van Cott T, et al. Protection of macaques against induced after oral delivery of vaccinia virus recombinants. Vaccine vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by pas- 1999; 17:1074–83. sive infusion of neutralizing antibodies. Nature Med 2000; 6:207–10. 33. Belyakov IM, Wyatt LS, Ahlers JD, et al. Induction of a mucosal cy- 39. Keefer MC, Wolff M, Gorse GJ, et al. Safety profile of phase I and totoxic T-lymphocyte response by intrarectal immunization with a rep- phase II preventive HIV-1 type 1 envelope vaccination: experience of lication-deficient recombinant vaccinia virus expressing human im- the NIAID AIDS Vaccine Evaluation Group. AIDS Res Hum Retro- munodeficiency virus 89.6 envelope protein. J Virol 1998; 72:8264–72. viruses 1997; 13:1163–77. 34. Belyakov I, Hel Z, Kelsall B. Mucosal AIDS vaccine reduces disease 40. Gorse GJ, Corey L, Patel GB, et al. HIV-1MN recombinant glycoprotein and viral load in gut reservoir and blood after mucosal infection of 160 vaccine induced cellular and humoral immunity boosted by HIV- macaques. Nat Med 2001; 7:1320–7. 1MN recombinant glycoprotein 120 vaccine. AIDS Res Hum Retrovi- 35. Stevceva L, Alverez X, Lackner AA. Both mucosal and systemic routes ruses 1999; 15:115–32. of immunization with the live, attenuated, NYVAC/simian immuno- deficiency virus SIVgpe result in gag-specific CD8+ T-cell responses. J 41. Gupta K, Hudgens M, Corey L, et al. Safety and immunogenicity of a Virol 2002; 76:11659–76. high-titered canarypox vaccine in combination with rgp120 in a diverse 36. Crotty S, Miller CJ, Lohman BL, et al. Protection against simian im- population of HIV-1 uninfected adults: AIDS Vaccine Evaluation Group munodeficiency virus vaginal challenge by using Sabin poliovirus vec- Protocol 022A. J Acquir Immune Defic Syndr 2002; 29:254–61. tors. J Virol 2001; 75:7435–52. 42. McGhee JR, Czerkinsky C, Mestecky J. Mucosal vaccines: an overview. 37. Baba TW, Liska V, Hofmann-Lehmann R, et al. Human neutralizing In: Ogra PL, Mestecky J, Lamm ME, Srober W, Bienstock J, McGhee JR, monoclonal antibodies of the IgG1 subtype protect against mucosal eds. Mucosal immunology. San Diego: Academic Press, 1999:741–57. Mucosal HIV-1 Vaccine • JID 2004:189 (1 April) • 1231