Comparison of Systemic and Mucosal Delivery of 2 Canarypox ...Document Transcript
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
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
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-
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 . 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 , 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 . Local CTL responses can be detected in in vitro–
(firstname.lastname@example.org). 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 , and systemic CTLs have been de-
0022-1899/2004/18906-0013$15.00 tected in an HIV-1–exposed but uninfected group .
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 . 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 . 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 . 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 . 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
Rabies vaccine HIV vaccine
recipients recipients Total
Variable (n p 28) (n p 56) (n p 84)
Female 13 25 38
Male 15 31 46
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) . 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 . 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 . 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 , 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 . 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
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
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
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.
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 . 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 . 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 . 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-
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 . 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
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 . 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/
humans, to provide protection against variola and rabies , 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 . 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 .
, vaccinia-derived vectors , 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 . 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 . 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.
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However, when primary epithelial cells derived from adenoids 1199–200.
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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
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studies (for review see ), 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.
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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.
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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-
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