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Human Immunodeficiency Virus-Type 1 Replication Can Be Increased in
Peripheral Blood of Seropositive Patients After Influenza Vaccination
By William A. O’Brien, Kathie Grovit-Ferbas, Ali Namazi, Stanislava Ovcak-Derzic, He-Jing Wang, Julie Park,
Christine Yeramian, Si-Hua Mao, and Jerome A. Zack
Despite considerable evidence that cell activation enhances
human immunodeficiency virus-type 1 (HIV-1) replication in
vitro, there is very little data on the role of immune activation on in vivo HIV-1 replication. In this study, we examined
the effect of influenza vaccinationon HIV-1 replication in
the peripheral blood of 20 study subjects, and in 14 control
subjects who did not receive influenza vaccination. Blood
was obtained from each subject on three occasions during
the month before vaccination and again on three occasions
during the following month. Over the study period, there
was F i l e change in levels of proviral DNA in peripheral blood
mononuclear cells (PBMCs). However, peak PBMC viral RNA
levels after influenza vaccination were significantly in-
creased overthe mean of prevaccinationvalues. This change
was not observed to the same extent in unvaccinated controls. Therefore, this is the first report showing that HIV-1
replication can increase in temporal association with influenza vaccination. Our results suggest that continued immunologic (antigenic) stimulation may result in increased virus
load in vivo. To address the appropriateness of influenza
vaccination in HIV-infected patients, expanded studies will
be required to examine specific and generalized immune
responses to vaccination, and differences in patient response based on disease stage.
0 1995 by The American Societyof Hematology.
H
UMAN IMMUNODEFICIENCY virus-type 1 (HIVviral load, as reflected by viral DNA and RNA levels and
1) infection leads to a progressive depletion of CD4+
by infectious HIV-1
Nonetheless, factors responsilymphocytes and acquired immunodeficiency syndrome
ble for the regulation of virus expression in vivo remain
(AIDS) in a majority of HIV-infected individuals.’ Although
poorly defined.
the median time course from acute infection to AIDS is
One potential mechanism of virus induction is immune
greater than 10 years: the duration of the clinically latent
activation. Activation of CD4+ lymphocytes and mononustate is highly variable, and factors that govern clinical laclear phagocytes in vitro appears to be important for productency have not been elucidated. After acute infection, there
tion of HIV-1. Cellular transcription factors induced after
is a viremic phase with widespread distribution of v i r u ~ . ~ . ~activation of T cells can increase HIV-1
Viremia typically decreases within 2 to 3 weeks, at least in
Other studies using cultured primary T cells have shown
part because of sequestration of virus in lymph
as
that, although HIV-1 can bind and enter both quiescent and
well as development of specific anti-HIV-1 immunity.’ Alactivated cells, activation is required for completion of rethough the acute HIV-1 syndrome maybe followed by a
verse transcription, integration, andor subsequent progeny
prolonged period of clinical latency, there does not appear
virus f ~ r m a t i o n . ~Similarly,
~-*~
activation of mononuclear
to be virologic latency. High levels of circulating virus partiphagocytes, by adherence to plastic or by cytokine treatment,
cles can still be shown in the plasma of infected asymptomcan dramatically enhance virus production in vitro.28,2y
atic individuals by quantitative HIV-1 RNA polymerase
Therefore, we hypothesize that immunologic activation may
chain reaction (PCR) inplasma:
as well as in lymphoid
also increase HIV-1 replication in vivo.
tissue.6. Recently, the dynamic interaction between HIV- 1
To address this issue, we have examined the effect of
replication and CD4+ lymphocyte destruction was shown
antigenic stimulation by influenza vaccination on HIV-1 repin HIV-infected individuals after administration of potent
lication in the peripheral blood of HIV-infected patients.
Because cell-mediated immunity is important for containing
antiretroviral drugs.”.” Moreover, clinical progression and
influenza virus infection, HIV-infected patients are thought
CD4’ lymphocyte depletion are associated with increases in
to be at risk for prolonged andmore severe disease, and
annual influenza vaccination is specifically rec~mmended.~’
In this report, we show increases in HIV-1 RNA levels in
From the Veterans Affairs Medical Center, Department of Medicine, Division of Infectious Diseases, West Los Angeles, CA, and
peripheral blood mononuclear cells (PBMCs) temporally asthe Department of Medicine, Divisionof Hematology-Oncology, the
sociated with influenza vaccination.
’
University of California at Los Angeles.
Submitted November 15, 1994; accepted March 14, 1995.
Supported by Department of Veterans Affairs medical research
funds, National Institutes of Health GrantNo. AI 33259, UCLA
Institutional Training Grant No. PHS/5 T32 AI07388, and grants
from the American Foundation for AIDS Research and Genentech,
Inc,SouthSan Francisco, CA.
Address reprint requests to William A. O’Brien, MD, Divisionof
Infectious Disease, 691/11 IF, 11301 Wilshire Blvd, Los Angeles,
CA 90073.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8603-00I6$3.00/0
1082
MATERIALSANDMETHODS
Patients. Study subjects (n = 34) were HIV-l seropositive and
at an intermediate stage of HIV disease at entry to the study with
no AIDS-defining illnesses. This study was approved by the Veterans
Affairs Medical Center West Los Angeles Internal Review Board
for human subjects, and informed consent was obtained for all participants. Subjects were to have CD4+ lymphocyte counts of 200 to
500/mm3,although those having 100 to 200/mm3 or 500 to 550/mm3
at study entry were not excluded if willing to participate. Two patients (01 and 03) were studied in successive years, and are counted
twice. Study subjects were self-assigned tothe vaccinated (n =
20) or nonvaccinated control group (n = 14), because influenza
vaccination was, and still is, recommended for all HIV-infected
individuals. Patients were excluded if there was clinical or laboratory
Blood, VOI 86, N O 3 (August l),
1995:
Pp 1082-1089
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1083
INFLUENZAVACCINE-INDUCED HIV-1 REPLICATION
evidence of acute viral hepatitis, active herpes simplex virus infection, pneumonia, or other acute respiratory infection, psychosis, or
transfusion within the last 2 months. Patients receiving immunization
did not have an allergy to eggs, because vaccine antigens were
derived from influenza virus preparations grown in eggs. At each
visit, patients were specifically questioned about the presence of
fever, cough, rash, cutaneous or respiratory infection, and diarrhea.
Blood collection and processing. Blood was collected on three
occasions during the month before influenza vaccination, with the
third sample obtained on the day of vaccination. These baseline
samples were obtained at 7-to 14-day intervals, and spanned a
period of at least 3 weeks. Additional blood samples were obtained
during the first week, the second week, and at 4 to 6 weeks after
vaccination. PBMCs were purified by Ficoll/Hypaque (Pharmacia,
Uppsala, Sweden) gradient centrifugation within 3 hours after collection. For PCR analysis, lo7 cells were lysed in buffer containing
4.7 m o m urea3' and stored at -70°C. Nucleic acids were purified
simultaneously from all samples for each patient by phenolkhloroform extraction and ethanol precipitation. The remainder of PBMCs
were cryopreserved in RPM1 1640 medium, 20% fetal calf serum,
and 10% dimethylsulfoxide in the vapor phase of a liquid nitrogen
freezer. PBMCs from HIV-negative donors used for plasma infections titer analysis were obtained from Red Cross leukopaks (Los
Angeles, CA).
Quantitative PCR analysis. Samples from each patient were analyzed in batch on the same test run to avoid interassay variation.
Quantitative PCR for human P-globin sequence^"^^^^^^ was performed on samples from each patient to standardize for cell equivalents as a control both for nucleic acid recovery and for amplification
efficiency. Sample volumes were then adjusted for subsequent HIV
analysis. HIV-specific DNA in each sample was measured by quantitative PCR using "P-end-labeled long terminal repeat (LTR) R/U5
primers," followed by amplification for 30 cycles at 94°C for 1
minute and 65°Cfor 2 minutes. HIV-1 copy number was determined
by comparison with linearized, cloned HIV-1 DNA standards amplified in parallel as previously described.%,33 To measure HIV- 1 RNA
levels in PBMCs, total nucleic acids for each sample from an equivalent number of cells (standardized by cell number, and subsequently
by @-globin DNA content) were treated with RNase-free DNase I
at 37°C for 40 minutes to digest the DNA. Purified RNA from a 3
X 105 cells was amplified using rTth polymerase (Perkin ElmerCetus, Nonvalk, CT) with reverse transcription in the presence of
MnCI, and 12 pmol of the antisense gag primer LA9% at 70°C for
15 minutes, followed by 35 cycles of amplification using 6 pmol of
the y3'P-labeled sense primer LA8" and 6 pmol cold LA8 added
for a total amount of 12 pm01 per reaction, under the conditions
described above. The absence of HIV-l DNA wasshown by omitting
the reverse transcription step in a duplicate sample, and analyzing in
parallel. Cell equivalence for RNA analysis was shown by subjecting
selected samples to quantitative RT-PCR using P-actin primers.
Quantitation was achieved by comparison with amplicons generated
from serial dilutions of RNA purified from PBMCs infected in vitro
by HIV-lJR.Cs$4and subjected to reverse transcription and PCR in
parallel. The signal intensity of the amplified product was measured
from the autoradiograph by densitometry using an optical imaging
system (Ambis, San Diego, CA). Standard curves were generated
from the values obtained for 0-globin, and HIV-l DNA and RNA
standards, which were used to derive values for experimental samples after interpolation of the standard curve.
Statistics. Nonparametric analyses were usedto assess change
in viral RNA levels. The mean of the three preimmunization RNA
measurements for each patient wasdefined as the baseline RNA
level. Relative change in RNA level was defined as the difference
between the peak RNA value postimmunization and the mean baseline value, divided by the mean baseline value. The sign rank test
was used to evaluate the changes within each study group, whereas
the Wilcoxon rank sum test was used to compare the changes between the two study groups.
RESULTS
The mean C M ' lymphocyte count for all study subjects
was 301 ? 112/mm3;range, 55 to 547/mm3.Only one subject
had a CD4' lymphocyte count less than 120/mm3;this was
patient Olb on his second evaluation. The mean CD4+ lymphocyte count at entry was not significantly different for
control and vaccinated patients (337 2 112 and 2772 107,
respectively, P = .13). The racial composition of the two
study groups was similar; 40% of the study subjects were
black, 7% were Hispanic, and the remainder were white. All
of the study patients were believed to have acquired HIV-1
infection sexually, none admitted to recent intravenous drug
use. All but two of the study patients (04 and 34) were
receiving zidovudine therapy.
Patient histories and examinations at l- to 2-week intervals during the 2-month study period did not show any side
effects from vaccination, nor were there any symptoms of
acute infections in the study population. Therefore, we do not
believe overt infections with bacteria or with heterologous
viruses were important confounders during the course of
observation. This analysis is critical, because infection may
be another potential source of stimulation, and hence, viral
induction. In addition, none of the study patients reported
blood transfusions or symptoms of allergy or hay fever during the study period.
PBMCs were then analyzed for HIV-1 DNA and RNA
levels by quantitative PCR at each time point for each patient. To account for differences in nucleic acid extraction
efficiency, each sample for each patient set was standardized
for cell equivalence by using quantitative PCR for &globin
gene sequences.
HIV-1 DNA PCR results for five representative patients
are shown in Fig 1. With one exception (patient 04, data not
shown), there was no detectable increase in HIV-1 DNA
levels on a per-cell basis during the 2-month study period
in either the vaccinated or nonvaccinated control group. Of
importance, patient 0 4 , a vaccinated subject, had not been
receiving zidovudine therapy. Antiretroviral therapy with zidovudine wouldbe expected to inhibit formation ofnew
viral DNA. Patient 26, a nonvaccinated control, exhibited a
fall in PBMC HIV-1 DNA levels during the latter half of
the 2-month study period, which was roughly matched by a
fall in PBMC HIV-1 RNA levels (Fig 2). This higher HIV1 replication level at the earlier time points may reflect an
activation event that immediately preceded the study period.
HIV-1 RNA was detected in every patient and in most
(over 80%) of the individual PBMC samples. Representative
HIV-1 RT-PCR experiments from the same five patients in
Fig 1 are shown in Fig 2. In contrast with what wasobserved
for viral DNA, there was a significant relative increase in
postvaccination HIV-1 RNA levels in PBMC from the 20
patients receiving influenza vaccination (1 1.6- t 5.0-fold
increase, median 2.7, P < .002). Greater than fourfold increases were seen in 10/20 (50%)of the vaccinated subjects.
The peak HIV-1 RNA levels typically occurred at 1 or 2
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O'BRIEN ET AL
1084
HIV-I Standards
(Copy number)
i K 300 100 30 10 3
Subject
Blood Sampling
(Weeks)
-4 -2 0 +l +2 +4
B
-
22 -
23*-
28-
33-
or,
26-
e*
0
-
weeks postvaccination (in 9/10 patients showing greater than
fourfold increase), and returned to baseline at later time
points. Thus, in most patients, HIV-I RNA induction was
transient. Equivalent increases in peak PBMC RNA
levels
during the same time frame were not seen in the 14 nonvaccinated controls (2.4- ? 1.6-fold increase; median, 0.0; P =
.24), andonly 2/14 control patients (14%) had increases
greater than fourfold. These data are summarized in Table
1.
As another measure of viralinduction.
we measured
plasma infectious titer by limiting dilution culture analysis13.14 I n 15 of the 20 vaccinated subjects, and 4 of the 14
'
nonvaccinated controls. In this analysis, sequential fivefold
dilutions of patient plasma were added to IO" phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes
(PBL) in duplicate in 24-well plates, and assayed for p24
production at days 7 and 14. A fivefold or greater increase
in infectious plasma virus titer was seen in one of four nonvaccinated controls (patient 15), a patient in whom increases
in cellular HIV-I RNA were also detected. Plasma HIV-I
infectious titer increases of fivefoldor greater were also seen
in 6 of the 15 vaccinated subjects. Five of these 6 subjects
also had increases in cellular HIV-I RNA, and 1 patient (09)
in whom there was a 79-fold increase in cellular HIV-I
RNA, also had a greater than 25-fold increase in plasma
infectious virus titer. Thus, increases in plasma infectious
virus titer were consistent with results seen for cellular RNA
expression.
The peak RNA PBMC level was plotted against the mean
baseline levels for vaccinated and nonvaccinated control patients in Fig 3. Comparison of increases betweenthetwo
groups indicated a trend toward significance of the change in
vaccinated patients ( P = .089). Although there were highly
Fig1. Ouantitative HIV-1 PCR analysisofPBMC
DNA. PBMC DNA was purified and equivalent
amounts were ampliiied asdescribed in Materials
and Methods using LTR-specific primers M667 and
AA55 (140-bp product). Patients 22, 23, 28, and 33
were vaccinated (V); patient 26 was a nonvaccinated
control (C). B is the reagent controlsample subjected
to amplification in parallel for each patient.
significant changes in PBMC RNA levels after vaccination
when the mean value of baseline samples wasused as a
control for individual patients, thereweremarked
HIV-I
RNA increases at later time points in PBMCsfromtwo
nonvaccinated control patients (subjects IS and 78) that reduced the significance of vaccine-related HIV-I induction
in the overall study population. In retrospect, this type of
variation in the control subjects wouldbeexpected if immune stimulation by environmental antigens has an influence
on virus replication.
We have followed study patients longitudinally to determine the relationship between virologic response to vaccination, and clinical outcome. At more than 3 years mean follow-up, 12 of 20 vaccinated subjects have developed AIDS,
and 8 have died. We have indicated the CD4' lymphocyte
counts at baseline, and 6 months after enrollment in Table
1. Of the I O vaccinated patients who exhibited a fourfold
increase in HIV-I RNA, S had a fall in CD4' lymphocyte
number at 6 months of20% or more,andall 5 of these
subjects developed AIDS. Moreover, 3 of S vaccinated subjects with HIV-I RNA increases who did not have a decrease
in CD4 cell count have not developed AIDS. Finally, 3 of
the 10 study subjects whodid not show an HIV-I RNA
increase developed AIDS over 6 months, and 2 of these
patients had a 20% decrease in CD4 cell count. Therefore,
the pattern of virologic response to influenzavaccination
does not entirely predict outcome. 0the.r factors appear to
be involved in determining the rate of clinical progression.
DISCUSSION
These results show that HIV-I expression in PBMCs
markedly increased in one-half of patients during the 2 weeks
after influenza vaccination. This appears tobe a transient
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INFLUENZA VACCINE-INDUCED HIV-1 REPLICATION
Blood Sampling
(Weeks)
HIV-I Standards
(Relative)Subject
-4 -2
3K tK 300 (00 30 10
1085
RT-
-
0 +! +2 +4
23 V
33-
44
l
C
2 6 - M @
0
Fig 2. Quantitative HIV-1 PCR analysis of PBMC RNA. PBMC RNA
was purified after DNAse I treatment of total PBMC nucleic acid and
subjected to in vitro reverse transcription and PCR amplification as
described in Materials and Methods. RNA results are of the five patients shown in Fig l.RT indicates amplification of the most positive
PBMC sample following incubation at 4°C during the reverse-transcription step.
induction that generally does not lead to a sustained increase
in virus replication. The importance of cell activation for
HIV-I replication has been well shown in vitro. Infection of
quiescent PBLs results in an incompletely formed reverse
transcript that cannot give rise to infectious virions. Activation of cells with cytokines, lectins, or mitogens either before, or shortly after infection, results in completion of reverse transcription and infectious progeny virions.
Chronically HIV-infected cell lines havebeenused
as in
vitro models for effects of HIV-I activation. Cell clones
chronically infected with HIV-I LAV were established from
the monocytoid cell line U937 or the T-cell line CEM (U1
and ACH2 cell clones, respectively), but generally produced
low levels of HIV-I .3s.3h When these cell lines are activated
by cytokines or other agents, viral RNA and progeny virion
production is increased in association with increases in a
variety of cellular factor~.*~.''~~'
Reporter constructs have also
shown increased LTR-driven chloramphenicol acetyl transferase (CAT) expression in cell lines activated with mitogens
andor cytokines."
There have been other reports suggesting a relationship
between immune activation in vivo and induction of HIV-I
replication. Treatment of HIV-l -infected chimpanzees with
various vaccine preparations, or adjuvant alone, caused transient increases in numbers of infectious PBMCs.4" Suggestive evidence indicates that both influenza vaccination and
influenza virus infection in patients can increase numbers of
HIV-I -infected PBMCs.4'
We chose to focus our analysis on changes in PBMC
HIV-I RNA, rather than extracellular virus, because of the
immediacy of the response to stimulation. Cell activation
appears to invoke production of factors that can induce HIV1 expression, and leadto detectable increases in PBMC HIVI RNA.*4 Weaddressed extracellular virus levels using quantitative viral titers in plasma, which yielded similar results.
However, plasmaviral titer isless sensitive than cellular
HIV-I RNA assays for several reasons. First, infectious
assays measure only virus competent for replication in culture. Furthermore, plasma virus assays may be affected by
factors elaborated from CD8' lymphocytes, as well as by
antigen-antibody complexing of virions. It will be important
to confirm our observation in cells using the newer plasma
HIV-I RNA assays.
Recent studies'".'' have concluded that activation of latently infected PBMCs is not the major source of plasma
viremia. Rather, these data suggest that plasma virus originates either from recently infected cells," or via trafficking
through the lymphoreticular system."' Therefore, although
we show an increase in HIV-I RNA in circulating PBMCs,
activation may result in HIV-I induction in either the intravascular or the tissue compartments.
We do not believe that the HIV-l induction observed is
a result of the specific response to influenzaantigen, because
there is a fairly small population of influenza-specific memory cells:* Furthermore, most patients in our study are not
expected to mount a measurable specific response to vacThe HIV-I induction may be a consequence of a
generalized immune response resulting from antigen presentation by macrophages and dendritic cells. This could cause
cytokines to be produced that result inboth activation of
latently infected cells and induction of virus expression, as
well as increased replication from infection of newly activated cells.
There are several potential explanations for the transient
nature of the HIV-l induction seen in our study. First, the
period of viral induction may be limited by the duration of
minor specific T-cell responses to antigen. As influenzaspecific T-cell clones return to quiescence or the memory
state, cell factors involved in augmenting virus expression
are also down regulated.*'Alternatively, stimulation of HIVinfected, influenza-specific T cells may result in deletion
of the infected population, and a subsequent decrease in
HIV-I expression. This phenomenon has previously been
shown after in vitro antigenic stimulation of PBMCs from
HIV-infected asymptomatic individuals, butwasnot
addressed in this study:6 Finally, the fall in virus expression
may be related to immune clearance of cells acutely expressing viral antigens. Our studies cannot distinguish between
these possibilities. With this in mind, it is unclear why two
control subjects exhibited a substantial increase in viral RNA
production during the course of the study. Although we cannot document this, it is possible that subclinical infection or
other immune stimuli such as an allergic reaction could have
led to this induction of viral expression. This is consistent
with our hypothesis that other activation events in addition
to influenza vaccination can affect HIV-l replication.
However, there was no evidence in our study for increases
in HIV-I DNA load over the short term. Because most pa-
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1086
O’BRIEN ET AL
Table 1. Study Patients Characteristics and Response t o Vaccination
Patient at
No. of CD4 Cells
CDC
Entry
ImmunizationStage
Age
Previous Influenza
No. of CD4 Cells
at 6 Mos
No
No
63
240
308
286
20
Relative
RNA Change
Vaccinated
Ola
287
258
189
02
03a
04
01b
07
08
09
10
03b
21
22
23
28
29
30
31
33
56
61
Controls
12
15
26
34
47
48
75
77
78
79
80
81
83
88
49
63
42
49
256
225
263
313
50
45
57
43
65
30
37
47
51
43
46
38
31
46
41
52
39
39
37
41
59
220
259
41
351
232
111
II
II
IV c
IV B
IV c
II
II
II
IV c
287
111
IV A
111
IV c
55
4
121
348
535
365
485
474
547
250
301
283
232
321
204
352
446
235
218
111
Ill
IV
c
111
II
IV
c
111
II
II
IV c
II
111
IV c
IV A
II
IV c
IV c
IV c
II
IV B
-0.29
Yes
7.78
4.59
No
Yes
No
Yes
Yes
Yes
Yes9.15
Yes
Yes
63.15 Yes
Yes
0.17
Yes
0.44 Yes
Yes
Yes
Yes
Yes
0.20
16.79
16.15
-0.30
0.01
No
No
No
No
No
No
Yes
Yes
No
Yes
No
No
No
No
258
194
260
375
240
4
270
165
410
275
56
320
583
61
409
228
490
553
316
104
59
226
287
56
399
443
255
285
4.94
4.85
10.07
-0.60
79.04
0
-0.74
-0.14
8.96
-0.54
0.73
39.55
0.03
-0.80
0.32
1.96
-0.74
-0.18
0
0.92
0
0
Demographic and clinical data of study patients enrolled over three successive influenza vaccination seasons (1990, 1991, and 1992). CD4
cell counts are expressed per cubic millimeter; values shown are at enrollment, and at 6 months after immunization. Quantitative RT-PCR was
performed on PBMC extracts obtained on three occasions during the month before and again at 1,2, and 4 weeks postvaccination. Relative
RNA change is defined in Materials and Methods.
tients were receiving antiretroviral therapy with zidovudine,
new viral DNA formation would be inhibited, even in patients exhibiting marked increases inHIV-1 RNA expression. In addition, our assay would not reliably detect increases in HIV-1 DNA of twofold or less, which may still
be relevant for clinically important increases in proviral burden. It is also possible that vaccination increased the number
of infected cells in compartments not assayed here, such as
lymphoid tissues. Although in our study, we did not detect
increases in HIV-1 DNA over 2 months, it seems likely that
the progressive increases in viral load during the course of
HIV disease are a consequence of many such small inductions of HIV-1 replication which occur intermittently over
several years. Our study involved onlya single immune
stimulation event which may be inconsequential to a chronic
disease such as AIDS where an infected individual is expected to be exposed to numerous antigenic stimuli. Thus,
events that stimulate T cells or macrophages may result in
adverse consequences for HIV-infected patients.
In addition to immune-mediated events, another potential
cofactor in HIV-1 pathogenesis is infection with heterologous viruses. Coexpression of HIV and either herpes simplex
virus or cytomegalovirus genes in T cells, or stimulation of
HIV-infected cells by HTLV particles, can result in increases
in HIV-1 repli~ation.4”~~
This increase in HIV-1 RNA expression can be mediated by interaction of herpesvirus products with the HIV-l LTR.’”” In addition, a recent study
suggests that recurrent herpes simplex virus infection can
also lead to marked increases in HIV-1 expre~sion.~’
Furthermore, actual influenza virus infection may lead to a greater
level of HIV-1 expression than the transient nature of the
increase in viral expression observed here, because of the
prolonged nature of the infection. This relationship may hold
true for other vaccine-versus-disease combinations; how-
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1087
INFLUENZAVACCINE-INDUCED HIV-l REPLICATION
0
Fig 3. Plot of peak log R N A postvaccination versus mean log prevaccination for vaccinated IO1 and
nonvaccinated control (0)subjects.
ever, our study does not distinguish which process has the
more deleterious effect in HIV-infected individuals. Therefore, it may be necessary to balance HIV-1 induction by
influenza vaccination or other immune-based therapies, with
the HIV-1 potentiation effects of other acute infections.
However, the relative impact of these phenomena on viral
load accumulation and disease progression has not yet been
delineated, and it appears likely that progressive viral load
increases are a result of repeated episodes of immune activation and subsequent bursts of replication that can occur after
a variety of stimuli. This study does not attempt to address
the appropriateness of influenza vaccination in HIV-infected
patients, nor do we suggest that these findings should be
generalized to all vaccines. Our study does suggest that it
might be prudent to administer antiretroviral therapy to all
individuals receiving immune augmentation therapies to
minimize potential increases in viral load.
The HIV-1 inductive response to influenza vaccination
could not be used in our study to predict clinical course.
Immunologic and clinical deterioration ensued over a 3-year
period in only half of the vaccinated subjects who exhibited
a fourfold or greater increase in HIV-1 RNA expression.
Notably, patient 03, who showed vaccine-related HIV-l induction in 2 different years, has had stable CD4+ lymphocyte
counts for over 6 years, and is still without AIDS. Viral
strain differences and/or differences in host response may
account for the weak correlation between HIV-1 induction
and clinical outcome. In addition, the efficacy of vaccination,
as well as the HIV-1 response, varies in patients even at
similar stages of disease.
Our initial evaluation focused on patients at an intermediate stage of HIV-1 disease. Although there were three patients below 200 CD4+ lymphocytes/mm3 and two above
500/mm3at study entry, none of the patients had been diagnosed with AIDS. However, it is likely that patients at different stages of disease may exhibit distinct responses. At early
stages of disease (patients with over 500 CD4+ lymphocytes/
m m 3 ) , proliferative responses to antigenic stimulation tend
to be more v i g o r o ~ s . ~ These
~ . ~ ’ patients also show lower
virus
and more effective antiviral immunologic re-
1
2
3
4
Log R N A Before Immunization
(Relative Copy Number)
spon~es,5~.~’
which may prevent detectable viral induction.
However, patients at advanced stages of disease (<200
CD4+ lymphocytes/mm3)show markedly impaired proliferative responses to antigen:’”’
and have high background levels of HIV-1 replication. The virologic response to immune
stimulation at early or late stages of disease is notwell
described.
Individual genetic diferences are also likely to be important. For example, differences in response to immunization have been shown in patients based on HLA haplotype.’jO
Our studies suggest the need for expanded clinical trials to
identify subgroups of patients who might be expected to
benefit mostfrom vaccination and other immune-based therapies, as well as those in whom vaccination is not helpful
and, in fact, may be deleterious. The significance of transient
viral induction needs to be assessed using other assays and
long-term clinical observation, and inHIV-infected individuals at different stages of disease.
ACKNOWLEDGMENT
We thank DXGrace Aldrovandi for critical review of the manuscript, Thang Nguyen for8-actin RT-PCR assays, Felicitas Lorenzo
and Carole Silbar for data and sample
collection, Yun Chon for help
withthedata analysis, and Michelle Estrellado andMizue Aizeki
for manuscript preparation.
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1995 86: 1082-1089
Human immunodeficiency virus-type 1 replication can be increased
in peripheral blood of seropositive patients after influenza
vaccination
WA O'Brien, K Grovit-Ferbas, A Namazi, S Ovcak-Derzic, HJ Wang, J Park, C Yeramian, SH
Mao and JA Zack
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