Effect of HIV-specific immune-based therapy
in subjects infected with HIV-1 subtype E in Thailand
Vina Churdboonchart, Ronald B. Moss*, Worachart Sirawaraporn,
Buranaj Smutharaks†, Reungpung Sutthent, Fred C. Jensen*,
Prawut Vacharak†, Janet Grimes‡, Georgia Theofan*
and Dennis J. Carlo*
Objective: To examine the effect of treatment with an inactivated, gp120-depleted,
HIV-1 immunogen (Remune) in 30 Thai subjects infected with HIV-1 subtype E.
Design: Sixty-week open-label study.
Methods: Thirty HIV-positive volunteers with CD4 cell counts ≥ 300 × 106/l were
given intramuscular injections of Remune into the triceps muscle on day 1 and then
at weeks 4, 8, 12, 24, 36, 48 and 60.
Results: Treatment with Remune was well-tolerated and augmented HIV-1-specific
immune responses. Furthermore, subjects had a significant increase in CD4 cell
count (P < 0.0001), CD4 cell percentage (P < 0.0001), CD8 cell percentage
(P < 0.0001), and body weight (P < 0.0001) compared with pretreatment levels.
Fourteen subjects with detectable viral load at day 1 showed a decrease at week 60
(P = 0.04). Retrospective Western blot analysis showed 23 subjects with increased
intensity of antibody bands and 15 patients showed development of new reactivities
to HIV proteins, especially towards p17 and p15.
Conclusion: These results indicate that HIV-specific immune-based therapeutic
approaches such as Remune should be further examined in countries with different
clades of HIV-1 and where access to antiviral drug therapies is limited.
© 1998 Lippincott-Raven Publishers
AIDS 1998, 12:1521–1527
Keywords: Asia, CD4, CD8, therapy, vaccine
Introduction
New effective drug ‘cocktails’ that target viral replication have become part of the standard of care for many
HIV-1-infected individuals in the United States and
Europe. Factors such as adherence to complicated drug
regimens, resistance, tolerability, and immune reconstitution remain practical public health issues for patients
taking combination antiviral drug therapy.
Unfortunately, access to highly active antiretroviral
therapy has been limited to the industrialized countries.
Thus, in developing countries, access to the newer
antiviral drug combinations is limited and currently no
alternative effective treatment modalities exist.
Furthermore, current epidemiological studies suggest
an abrupt increase in AIDS cases worldwide [1].
Estimates of future rates suggest that Asia may, in fact,
surpass Africa in the incidence of AIDS cases by the
year 2000 [1].
Thailand still has a high incidence of HIV infection
despite its implementation of many public health policy
initiatives to limit the spread of disease [2,3].
Nevertheless, there is a growing need to study alternative treatments because antiviral drug combinations are
not readily available. We examined the effect of an
From Mahidol University, Bangkok, Thailand, the *Immune Response Corporation, Carlsbad, California, USA, the †Remune
Trial Center, Mahidol University, Bangkok, Thailand, and ‡Harvard School of Public Health, Boston, Massachusetts, USA.
Requests for reprints to: Dr Vina Churdboonchart, Department of Pathobiology, Faculty of Science, Mahidol University, Rama
VI, Phayathai, Bangkok 10400, Thailand.
Date of receipt: 20 February 1998; revised: 29 April 1998; accepted: 6 May 1998.
© Lippincott-Raven Publishers
1521
1522
AIDS 1998, Vol 12 No 12
HIV-1-specific immune-based therapy (Remune;
Immune Response Corp., Carlsbad, California, USA)
as an approach to treating HIV infection. Remune is
composed of an HIV-1 isolate (HZ321) from serum
collected from a patient in former Zaïre in 1976, which
has been recently sequenced and classified as having
clade A envelope and clade G gag, and grown in the
Hut78 T-cell line [4].
Previous clinical studies of HIV-1-seropositive subjects
in the United States treated with a gp120-depleted,
inactivated HIV-1 in incomplete Freund’s adjuvant
(IFA; HIV-1 immunogen, Remune) revealed an augmentation of HIV-1-specific immune responses as
measured by lymphocyte proliferation [5], antibody
[5,6] delayed-type hypersensitivity [6], and production
of β-chemokines [7,8]. In previous clinical studies conducted prior to the advent of commercial plasma RNA
assays, a slower rise in the number of HIV-1 proviral
DNA copies in peripheral blood mononuclear cells
(PBMC) and a stabilization in the CD4 cell percentage
was observed in a treatment group compared with
adjuvant control subjects [5].
In this study, 30 HIV-1-infected Thai subjects received
treatment with an inactivated gp120-depleted HIV-1
virus in IFA (Remune) and were followed for
60 weeks in an open-label treatment study. Subjects
were not taking antiviral drugs during treatment, and
29 subjects had been infected with HIV-1 subtype E.
The initial safety and immunogenicity during the first 4
months of the trial has been previously reported [9].
We now report on a longer follow-up of the same
subjects.
Methods
Study design and demographics
Approval was obtained from the National AIDS
Committee, the Technical Subcommittee on AIDS
Vaccines, and the Ethical Committee (Ministry of
Public Health, Thailand). The study enrolled 30 subjects from a single center, Ramathibodi Hospital,
Bangkok, Thailand, with CD4 cell counts of at least
300 × 106/l. Treatment was administered as open label
therapy with an intramuscular injection of Remune.
Injections were administered on the first day of study
(day 1) and then at 4, 8, 12, 24, 36, 48 and
60 weeks after the initial injection.
Treatment
Remune consisted of inactivated gp120-depleted HIV1 formulated in IFA for intramuscular injection into the
triceps muscle. Inactivated HIV-1 antigen was obtained
by concentration and purification by anion exchange
chromatography from filtered supernatant fluid or a
chronically infected (HZ321) Hut78 cell line [10].
CD marker cell phenotyping
CD4 and CD8 cell percentages were obtained using a
validated protocol performed on fresh PBMC.
Immunophenotyping was performed at the BioService
Unit (BIOTEC Center, National Science and
Technology Development Agency, Thailand), using a
FACvantage flow cytometer (Becton Dickinson, San
Diego, California, USA). Prior to each operation, the
performance of the instrument was carefully checked
according to the manufacturer’s recommendation,
which included optimal alignment, spectral compensation, and electronic standardization. Normal cells and
calibration beads (CaliBrite, Becton Dickinson) were
used to minimize autofluorescence and to perform
spectral compensation, respectively. Heparinized blood
samples were collected from HIV-infected subjects,
transported to the laboratory, and immunophenotyping
was carried out within 2 h of blood collection.
Immunophenotyping was performed strictly according
to the manufacturer’s protocol using Simultest reagent
(Becton Dickinson), a monoclonal antibody reagent for
two-color enumeration of CD4/CD8 cells. Light-scattering gating was performed to identify the lymphocyte
subset. The gated cells were further defined by the
binding of the two monochrome-tagged monoclonal
antibodies.
Safety measurements
After obtaining written informed consent, enrolled
subjects were interviewed to obtain details of medical
history, including previous HIV-related diagnoses, and
physical examinations were performed. Subjects were
monitored during the trial for adverse events at each
visit. In addition, body weight and appetite of the
enrolled subjects were monitored at each visit. Safety
laboratory investigations included liver (e.g., aspartate
aminotransferase, alanine aminotransferase) and renal
function (e.g., blood urea nitrogen, creatinine) tests.
Enzyme levels in the kidney and liver were measured
and recorded before the first inoculation then periodically during the study period.
Viral load
Plasma HIV-1 RNA was assayed in blinded samples by
Advanced Bioscience Laboratories, Inc. (Kensington,
Maryland, USA) using the nucleic acid sequence-based
amplification method [11]. The lower limit of
detection of this assay was 4000 copies/ml. Samples
below the limit of the assay were assigned a value of
4000 copies/ml for the purpose of statistical analysis.
Immunogenicity
The ability of Remune to stimulate HIV-specific
immune responses was examined by Western blotting of
subject plasma before and after treatment at each study
visit. For Western blotting, nitrocellulose strips contain-
HIV-1 immunogen therapy in Thailand Churdboonchart et al.
ing the sodium dodecyl sulfate (SDS)–polyacrylamide
gel electrophoresis (PAGE)-separated HZ321 antigen
were incubated with subject plasma diluted in 10%
non-fat milk in 0.1% phosphate-buffered saline (PBS)
at a 1 : 50 dilution. Western blot for detection of antibodies to HIV-1 immunogen (Remune) in volunteer
plasma was performed at the Immunochemistry Unit
(Department of Pathobiology, Faculty of Science,
Mahidol University). The inactivated HIV-1 antigen
was supplied by The Immune Response Corporation
(Carlsbad, California, USA) and purified as described
by Prior et al. [12]. Eight per cent PAGE in presence of
4% SDS was used [13,14]. Ten microliters of inactivated HIV-1 antigen that had been previously diluted
to a 1 : 1 ratio in standard 2× sample buffer (Sigma
Chemical Co., St Louis, Missouri, USA) were separated by electrophoresis for 2 h 30 min at 40 mA. The
separated antigens were then transferred onto the surface of a nitrocellulose membrane by the Western blot
method.
Plasma from treated volunteers was diluted at 1 : 50
ratio with 10% (weight/vol) non-fat dry milk (Sanalac;
Hunt-Wesson, Fullerton, California, USA) in Trisbuffered saline (pH 7.4) using 0.05 ml plasma in 2.45 ml
buffer. The paired plasma samples obtained at baseline
and week 60 from each subject were tested retrospectively in one batch for comparison of results. After incubation for 3 h, the strips were washed four times in
0.1% PBS and incubated with goat anti-human horseradish peroxidase-conjugated anti-IgG and developed
for visualization of the serological response to HIV antigens. The results were recorded and compared between
those obtained in the pre- and post-treatment plasma
from each volunteer to see whether there was an
increase in the number of reactive HIV-specific bands
or a demonstrably greater intensity of the pre-existing
bands in week 60 plasma relative to the subject’s baseline plasma sample.
Viral subtyping
Viral subtyping was performed by the Department of
Microbiology at Siriraj Hospital (Bangkok, Thailand),
using a peptide enzyme-limited immunosorbent assay
(ELISA) [15] and heteroduplex mobility assay (HMA)
[16]. The 14-amino-acid peptides specific for Thai A
(env subtype E, TSITIGPGQVFYRT) and Thai B (env
subtype B, KSIHLGPGQAWYTT) were used with
ELISA for this study. The primers ED3/14 and
ED5/12 were used in HMA and their PCR products
were compared in 5% polyacrylamide gels. As noted
previously, 29 out of 30 subjects had subtype E virus
and one subject had subtype B virus [9].
Statistical analysis
The non-parametric Wilcoxon signed rank test was
used to test for significant changes from baseline for the
various markers. Spearman rank correlation was used to
determine strength and direction of association
between markers. All P values are two-tailed. RNA
values were natural log-transformed to normalize the
data because of the limited range of 12 000 to 200 000
copies/ml.
Results
Baseline demographics for the subjects are shown in
Table 1. Sixteen (53%) of the subjects were women
and 14 (47%) were men. The subjects’ ages ranged
from 19 to 41 years (median, 30 years). Six of the 30
subjects were on antiviral drugs (the most common
being zidovudine followed by didanosine) a few
months prior to enrollment into the study period but
had stopped taking antiviral drugs for 3 months or
more before recruitment into the study due to lack of
funds. However, discontinuing antiviral drugs was not
a requirement for this protocol. The mean CD4
cell count at baseline was 451 × 10 6 /l (range,
220–894 × 106/l) and CD4 cell percentage was 17.1%
(range, 10.9–34.4%) in these subjects.
Treatment with Remune was well-tolerated with no
serious adverse events noted. Local injection site reactions (i.e., swelling, redness, induration), which
resolved within 30 min were the predominant adverse
events reported.
Most subjects had an increased intensity of antibody
bands and also developed new reactivities to HIV proteins on Western blots (Table 2). The most common
new reactivity was to the conserved proteins of the
virus. An increase in both the number of bands and
intensity were noted in 15 cases. The remaining eight
cases demonstrated only an intensity increase mostly
due to an already existing optimum number of bands
detected by this method. Two cases revealed stable
reactivities. Five cases showed decreased intensity of
reactive bands with one case showing a loss of antibody
to at least one polypeptide and another showing more
antibodies but decreased band intensity.
Because HIV-1 is a wasting disease, we examined
changes in weight in these subjects. Weight
significantly increased from baseline (mean ± SE,
Table 1. Baseline demographics of 30 Thai HIV-1-infected subjects.
Baseline (day 1)
No. HIV-positive patients
30
Sex [n (%)]
Male
14 (47)
Female
16 (53)
Median (range) age (years)
30 (19–41)
Antiretroviral therapy (zidovudine–didanosine)
prior to study (n)
6
451 (220–894)
Mean (range) CD4 cell count (×106/l)
Mean (range) CD4 cell percentage
17.1 (10.89–34.41)
1523
1524
AIDS 1998, Vol 12 No 12
Table 2. Changes in Western blot immunoreactivities to HIV-1.
Antibodies detected
Week 16
Volunteer
No. bands indicating
specific antibodies
towards polypeptide
(baseline)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
3
5
5
6
5
8
10
2
9
9
8
5
7
5
9
10
10
10
6
10
6
2
6
2
4
26
27
28
29
30
4
5
8
7
5
Antibodies
towards viral
polypeptide*
Stable
Gain (p17)
Stable
Gain (p21)
Stable
Gain (p24)
Gain (p17, p15)
Gain (p21, p17)
Gain (p21, p15)
Gain (p15)
Gain (p39, p21, p17, p15)
Gain (p66, p51)
Gain (p21, p17, p15)
Gain (p24, p17, p15)
Gain (p55, p45, p21,
p17, p15)
Gain (p55, p21, p17)
Stable
Gain (p17, p15)
Gain (p17, p15)
Gain (p39, p21)
Week 60
Intensity of
reactive bands
Antibodies
towards viral
polypeptide*
Decrease
Loss (p51)
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Gain (p21, p17, p15)
Stable
Gain (p21, p17, p15)
Stable
Gain (p55, p24)
Gain (p21, p17, p15)
Gain (p21, p17)
Gain (p21, p17, p15)
Gain (p15)
Gain (p45, p39)
Gain (p21, p17, p15)
Gain (p66, p55, p51)
Gain (p21, p17, p15)
Increase
Gain (p55, p45, p39, p21,
p17, p15)
Gain (p55, p45, p21, p17)
Increase
Increase
Increase
Gain (p17, p15)
Gain (p39, p17, p15)
Gain (p39, p21)
Intensity of
reactive bands
Overall
change
Decrease
Decrease
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Decrease
Increase
Loss
Loss
Gain
Stable
Gain
Stable
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Gain
Loss
Gain
Increase
Decrease
Decrease
Increase
Increase
Gain
Loss
Loss (intensity)
Gain
Gain
Increase
*Polypeptide indicated in parentheses.
54.9 ± 1.3 kg) to 60 weeks (mean ± SE, 56.0 ± 1.4 kg;
P < 0.0001; Fig. 1).
We also examined changes in phenotypic markers of
CD4 and CD8 cell percentages (Fig. 2). CD4 cell
percentage and CD4 cell count significantly increased
(P < 0.0001) from baseline (mean ± SE, 17.1 ± 1.1%
and 451 ± 29.3 × 106/l, respectively) to week 60 (mean
Fig. 1. Mean body weight of the subjects significantly
increased at 60 weeks of the trial compared with baseline
value (P < 0.0001).
± SE, 23.3 ± 1.5% and 493 ± 38.3 × 10 6 /l, respectively). Similarly, CD8 cell percentage significantly
increased (P < 0.0001) from baseline (mean ± SE,
43.1 ± 1.8%) to week 60 (mean ± SE, 53.3 ± 1.7%).
Finally, we examined changes in plasma HIV-1 RNA
during the trial. No significant change in the mean
HIV-1 plasma RNA level was evident in the entire
group of subjects studied at week 60 [mean ± SE,
6.9 ± 0.2 ln copies/0.1 ml (9923 ± 12 copies/ml)]
compared with baseline values [mean ± SE, 7.0 ± 0.2 ln
copies/0.1 ml (10 966 ± 12 copies/ml)]. Fifteen of the
29 evaluable subjects had undetectable RNA
(< 4000 copies/ml) at baseline (pretreatment). Eleven
of the 15 subjects who had undetectable viral load at
baseline remained undetectable at 60 weeks. There was
no significant change in viral load for those subjects
with undetectable viral load at baseline (four out of 15)
but detectable viral load at week 60 (P > 0.05).
Analysis of subjects (n = 14) with detectable viral load
at baseline revealed a decrease (P = 0.04) in HIV-1
plasma RNA at week 60 [mean ± SE, 7.5 ± 0.3 ln
copies/0.1 ml (18 080 ± 14 copies/ml)] compared with
HIV-1 immunogen therapy in Thailand Churdboonchart et al.
(a)
(b)
Fig. 3. Mean viral load in subjects who had detectable viral
load at baseline (n = 14). Data is presented as mean
ln(RNA copies)/0.1 ml to normalize the absolute values.
There was a decrease in mean plasma RNA viral load at
week 60 compared with baseline values (P = 0.04).
Fig. 2. Mean values of (a) CD4 and (b) CD8 cell percentage
of subjects significantly increased at 60 weeks compared
with baseline values (both P < 0.0001).
pretreatment baseline [mean ± SE, 8.1 ± 0.2 ln
copies/0.1 ml (32 945 ± 12 copies/ml); Fig. 3].
Furthermore, plasma RNA weakly inversely correlated
with CD4 cell percentage (r = −0.27, P = 0.04).
Discussion
In this group of 30 Thai subjects infected with HIV-1
subtype E and not taking antiviral drug regimens, we
have observed a significant increase in CD4 cell count,
CD4 and CD8 cell percentages, and body weight after
treatment with a gp120-depleted HIV-1 immunogen
(Remune). In addition, increased HIV-1-specific
immune responses as measured by antibody to the conserved epitopes of the virus such as p17 were observed.
Finally, this regimen was well-tolerated and no serious
adverse events were observed. The safety profile after
treatment with Remune in this trial is consistent with
that observed in trials of Remune in the United States
[17]. Local transient injection site reactions were
observed shortly after immunization in three cases,
which resolved within 30 min without treatment. This
is a common side-effect of treatment in this study as
well as in the US studies. Furthermore, a Phase II trial
in Thailand and a Phase III trial in the United States are
ongoing and will provide a safety database for thousands of HIV-1-infected subjects treated with Remune.
In a previous study of Remune, healthy, predominantly
antiviral-naive HIV-1-seropositive subjects developed
increased antibody responses to the conserved HIV-1
protein p24 [18]. Similar HIV-1-specific antibody
responses to the more conserved epitopes of the virus
have also been observed in other US studies of
Remune [refs?]. In this Thai study, increased and new
responses were demonstrated to conserved proteins
such as p24, p17 and p15. Interestingly, antibody
responses to the more conserved core proteins, but not
envelope proteins, have been demonstrated by others
to indicate a better prognostic course [19]. In this
group of Thai subjects, in contrast to the previous US
studies, subjects were predominantly infected with
HIV-1 subtype E. Interestingly, in US studies of
Remune it has been observed that HIV-1-specific
immune responses could be generated to the Asian subtype E antigen as well to the common US clade B virus
[20]. Thus, an immunogen composed of the more conserved epitopes of the virus is capable of enhancing
immune responses across clades.
Body weight also increased in these subjects. This is
consistent with an observation made in an earlier US
trial with Remune [21]. Although a modest mean gain
of 1.1 kg was noted in the Thai study, weight continues to be highly prognostic for patients with HIV-1
infection and an indicator of overall quality of life [22].
Recently, agents such as growth hormone have been
approved and made available for wasting patients in the
United States [23].
Significant increases in CD4 cell count, CD4 and CD8
cell percentages were noted in this study. CD4 cell
count and CD4 cell percentage continues to be a useful
prognostic marker of disease progression, despite the
rapid emergence of other surrogate markers [24]. CD4
1525
1526
AIDS 1998, Vol 12 No 12
cell count assays have recently been validated by different methods in Thailand [25]. The increases in CD4
cell percentage observed in this study after treatment
are also consistent with a previous US trial of Remune
[18]. Although a decline in the number of CD4 cells is
well-documented and characteristic of HIV-1 infection, virus-specific immune function is not completely
conveyed by the number of CD4 lymphocytes [20].
Interestingly, the functional immune response against
the virus, as measured as the quantity of lymphocytes
proliferating in culture to conserved core HIV antigens
(p24), was recently demonstrated to be associated with
control of plasma viremia in antiviral-naive subjects
[26,27]. Although not measured in the present study,
previous studies of Remune have revealed increased
lymphocyte proliferative responses to the highly conserved p24 antigen after treatment [8,18,20].
The increases in CD8 cell percentage observed in this
study are intriguing considering the recent interest in
CD8 cell-derived HIV-suppressive factors such as the
β-chemokines [28]. CD8 cell viral suppressive factors
such as the β-chemokines (RANTES, macrophage
inflammatory proteins 1α and 1β) have been reported
to inhibit viral replication of macrophage-tropic strains
of HIV-1 in vitro [28]. High levels of HIV-1 antigeninduced β-chemokine production have been associated
with low viral load in antiviral-naive subjects in other
studies [29]. Whether the reported increases in CD8
cell percentage observed in this group of Thai subjects
represents increased functional responses was not determined in the present study. In previous studies, though,
we have observed an increase in HIV-specific CD8 cell
production of β-chemokines after treatment with
Remune [20,30]. Studies are now underway to examine chemokine production in Thai subjects treated
with Remune.
Finally, viral load in subjects with detectable levels at
baseline had a decrease in plasma RNA at week 60
compared with pretreatment values. In the entire group
of subjects, viral load did not change, due in part to the
fact that half of the subjects had undetectable plasma
RNA at baseline and most remained undetectable during the study period. Recent studies have documented
on-going viral production that is not quantified by
peripheral blood plasma RNA levels [31,32]. Thus the
inducement of HIV-1-specific immune responses may
be a useful strategy to potentially suppress viral spread
in such subjects. The clinical utility of augmenting
HIV-1-specific immune responses against the virus may
not be easily quantifiable by plasma RNA in subjects
with already undetectable plasma RNA levels, such as
those studied here. Nevertheless, the impact of treatment on other measurements of viral load, such as
proviral DNA, are being examined in cohorts of subjects receiving Remune.
In summary, in Thai HIV-1-infected subjects, significant increases in HIV-1-specific immune responses,
body weight, CD4 cell count, and CD4 and CD8 cell
percentages were observed after treatment with
Remune. Furthermore, treatment with Remune was
well tolerated. Plasma RNA was stable in these subjects
and decreased in those subjects with detectable viral
load at baseline. These results indicate that HIV-specific
immune-based therapeutic approaches to treating HIV1 infection such as Remune should be further examined
in countries with different clades of HIV-1 and where
access to antiviral drug therapy is limited. A doubleblind adjuvant controlled study in Thailand is ongoing
in order to confirm these preliminary observations.
Acknowledgements
Professor Dr A. Limsuwan, Site Clinician, Ramathibodi
Hospital, who took care of all 30 volunteers and completed the case reports forms on physical examination.
Dr D. Glidden, Harvard University, for reviewing the
case report forms. Kanjana Sirisidthi, Tusance
Choiyarirk, Maliwan Emyeam, Marietta Alegrado and
Yupa Siristonpun for technical assistance.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Pear R: New estimate doubles rate of HIV spread. The New
York Times, 26 November 1997.
Nagachinta T, Duerr A, Suriyanon V, et al.: Risk factors for HIV1 transmission from HIV-seropositive male blood donors to
their regular female partners in Northern Thailand. AIDS
1997, 11:1765–1772.
Workshop Report from the European Commission and the Joint
United Nations Programme on HIV/AIDS: HIV-1 subtypes:
implications for epidemiology, pathogenicity, vaccines and
diagnostics. AIDS 1997, 11:UNAIDS17–UNAIDS36.
Choi DJ, Dube S, Spicer TP, Slade HB, Jensen FC, Poiesz BJ:
Sequence note: HIV type 1 isolate Z321, the strain used to
make a therapeutic HIV type 1 immunogen, is intersubtype
recombinant. AIDS Res Hum Retroviruses 1997, 13:357–361.
Trauger RJ, Ferre F, Daigle AE, et al.: Effect of immunization
with inactivated gp120-depleted human immunodeficiency
virus type-1 (HIV-1) immunogen on HIV-1 immunity, viral
DNA, and percentage of CD4 cells. J Infect Dis 1994,
169:1256–1264.
Turner JL, Trauger RJ, Daigle AE, Carlo DJ: HIV-1 immunogen
induction of HIV-1-specific delayed-type hypersensitivity:
results of a double-blind, adjuvant-controlled, dose-ranging
trial. AIDS 1994, 8:1429–1435.
Moss RB, Trauger RJ, Giermakowska WK, et al.: Effect of immunization with an inactivated gp120-depleted HIV-1 immunogen on β-chemokine and cytokine production in subjects with
HIV-1 infection. J Acquir Immune Defic Syndr Hum Retrovirol
1997, 14:343–350.
Moss RB, Wallace MR, Lanza P, et al.: In vitro p24 antigen
stimulated lymphocyte proliferation and β-chemokine production after immunization with an inactivated gp120depleted HIV-1 immunogen (REMUNE T M ) in HIV-1
seropositive subjects. Clin Diagn Lab Immunol 1998,
5:308–312.
Limsuwan A, Churdboonchart V, Moss RB, et al.: Safety and
immunogenicity of REMUNE TM in HIV-infected Thai subjects.
Vaccine 1997, 16:142–149.
HIV-1 immunogen therapy in Thailand Churdboonchart et al.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Richieri SP, Bartholomew R, Aloia CR, et al.: Characterization
of highly purified inactivated HIV-1 particles isolated by
anion exchange chromatography. Vaccine 1997, 16:119–129.
Revets H, Marissens D, De Wit S, et al.: Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICORE-HIV Monitor, and
QUANTIPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma.
J Clin Microbiol 1996, 34:1058–1064.
Prior C, Bay P, Ebert B, et al.: Process development for the
manufacture of inactivated HIV-1. Pharm Tech 1995,
19:30–52.
Laemmli UK: Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970,
227:680–685.
Churdboonchart V, Bhamarapravati N, Peampramprecha S,
Sirinavin S: Antibodies against dengue viral proteins in primary and secondary dengue hemorrhagic fever. Am J Trop
Med Hyg 1991, 44:481–493.
Pau C-P, Lee-Thomas S, Auwanit W, et al.: Highly specific V3
peptide enzyme immunoassay for serotyping HIV-1 specimens from Thailand. AIDS 1993, 7:337–340.
Delwart EL, Shaper EG, Loawagie J, et al.: Genetic relationships
determined by a DNA heteroduplex mobility assay: analysis
of HIV-1 env genes. Science 1993, 262:1257–1261.
Levine AM, Groshen S, Allen J, et al.: Initial studies on active
immunization of HIV-1 infected subjects using a gp120depleted HIV-1 immunogen: long-term follow-up. J Acquir
Immune Defic Syndr 1996, 11:351–364.
Trauger RJ, Ferre F, Daigle AE, et al.: Effect of immunization
with inactivated gp120-depleted human immunodeficiency
virus type 1 (HIV-1) immunogen on HIV-1 immunity, viral
DNA, and percentage of CD4 cells. J Infect Dis 1994,
169:1256–1264.
Binley JM, Klasse PJ, Cao Y, et al.: Differential regulation of the
antibody responses to gag and env proteins of human
immunodeficiency virus type 1. Virology 1997, 71:2799–2809.
Moss RB, Giermakowska W, Lanza P, et al.: Cross-clade
immune responses after immunization with a whole-killed
gp120-depleted human immunodeficiency virus type-1
immunogen in incomplete Freund’s adjuvant (HIV-1 immunogen, REMUNETM) in human immunodeficiency virus type-1
seropositive subjects. Viral Immunol 1997, 10:221–228.
Moss RB, Ferre F, Trauger R, et al.: Observations on the control
of progression of HIV infection following immunization with
an inactivated HIV immunogen in IFA. X International
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Conference on AIDS. Yokohama, August 1994 [abstract RR-1].
Mulligan K, Tai VW, Schambelan M: Cross-sectional and longitudinal evaluation of body composition in men with HIV
infection. J Acquir Immune Defic Syndr Hum Retrovirol 1997,
15:43–48.
Schambelan M, Mulligan K, Grunfeld C, et al.: Recombinant
human growth hormone in patients with HIV-associated wasting: a randomized, placebo-controlled trial. Ann Intern Med
1996, 125:873–882.
Mildvan D, Landay A, De Gruttola V, Machado SG, Kagan J: An
approach to the validation of markers for use in AIDS clinical
trials. Clin Infect Dis 1997, 24:764–774.
Young NL, Ponglertnapakorn P, Shaffer N, et al.: Clinical field
site evaluation of the FACS count for absolute CD3+,
CD3+CD4+, and CD3+CD8+ cell count determinations in
Thailand. Clin Diagn Lab Immunol 1997, 4:783–786.
Rosenberg ES, Billingsley JM, Caliendo AM, et al.: Vigorous HIV1-specific CD4+ T cell responses associated with control of
viremia. Science 1997, 278:1447–1450.
Kalams SA, Buchbinder SP, Billingsley JM, et al.: Coordinate
control of HIV replication mediated by CD4 and CD8 cells.
Keystone Symposia: HIV Pathogenesis and Treatment. Park City
(UT), March 1998 [abstract 4048].
Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC,
Lusso P: Identification of RANTES, MIP-1α, and MIP-1β as the
major HIV-suppressive factors produced by CD8+ T cells.
Science 1995, 270:1811–1815.
Ferbas J, Plaeger S, Amini S, Hausner M, Detels R, Giorgi JV: In
vitro production of RANTES, MIPIα and MIPIβ as a correlate of
decreased HIV burden. Conference on Advances in AIDS Vaccine
Development: Ninth Annual Meeting of the National Cooperative
Vaccine Development Groups for AIDS. Bethesda, May 1997
[poster 91].
Moss RB, Trauger RJ, Giermakowska WK, et al.: Effect of immunization with an inactivated gp120-depleted HIV-1 immunogen on β-chemokine and cytokine production in subjects with
HIV-1 infection. J Acquir Immune Defic Syndr Hum Retrovirol
1997, 14:343–350.
Finzi D, Hermankova M, Pierson T, et al.: Identification of a
reservoir for HIV-1 in patients on highly active antiretroviral
therapy. Science 1997, 278:1295–1300.
Wong JK, Hezareh M, Gunthard HF, et al.: Recovery of replication-competent HIV despite prolonged suppression of plasma
viremia. Science 1997, 278:1291–1295.
1527