1191
Increased Plasma Human Immunodeficiency Virus Type 1 Burden following
Antigenic Challenge with Pneumococcal Vaccine
Beda Brichacek,* Susan Swindells, Edward N. Janoff,
Samuel Pirruccello, and Mario Stevenson*
Departments of Pathology and Microbiology and of Internal Medicine,
University of Nebraska Medical Center, and Eppley Institute for
Research in Cancer and Allied Diseases, University of Nebraska
Medical Center, Omaha; Infectious Disease Section, Department of
Medicine, VA Medical Center, and University of Minnesota School of
Medicine, Minneapolis
Primary factors that influence virus burden during human immunodeficiency virus type 1 (HIV1) disease progression remain a fundamental issue in pathogenesis. Because pneumococcal vaccine
is routinely given to HIV-l-infected patients and replication of HIV-l within CD4 T cells is
dependent on the activation state of the cell, it was investigated whether the T cell activation that
enhances the immune response to vaccines may also enhance HIV-1 replication. Vaccination of
asymptomatic HIV-l-infected patients led to rapid and significant increases in virus burden in
some patients. The magnitude of these increases correlated significantly with the extent of the
antibody response to the vaccination. Thus, antigenic stimulation by vaccines designed to prevent
secondary infections may promote HIV-1 replication in certain patients. These findings provide a
window for examining HIV-1 pathogenesis and for determining the appropriate preventive measures
against other diseases in HIV-1-infected persons.
Disease progression in human immunodeficiency virus type
1 (HIV-I)-infected patients is closely associated with increased HIV-I activity, as measured by circulating infectious
virus titers, quantities of virus particles, and cell-associated
viral nucleic acids [1-4]. Analysis of changes in virus burden
and numbers of CD4 T lymphocytes following initiation of
antiretroviral therapy indicates that HIV-1 replication is sustained primarily by a dynamic process that drives rapid CD4
T lymphocyte turnover [5, 6]. However, the underlying factors
that influence HIV-1 replication are poorly characterized. CD4
T lymphocytes and macrophages play a central role in the
maintenance of virus burden during disease progression [7].
Replication of HIV -1 within CD4 T lymphocytes is dependent
upon the activation state of the cell [8-10], and cytokines
released by activated macrophages regulate HIV-1 replication
in vitro [11]. Thus, the activation ofT lymphocytes and macrophages that accompanies the immune response to antigens and
pathogens also may directly enhance virus burden [7, 12].
Received 3 November 1995; revised 25 June 1996.
Presented in part: 35th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, September 1995.
Written informed consent was obtained from all volunteers involved in this
study, which was approved by the human subjects committees at the universities of Nebraska and Minnesota.
Financial support: NIH (AI-30386, AI-32890 to M.S; AI-31373, DE-42600
to E.N.J.); VA Research Service (to E.N.J.).
Reprints or correspondence: Dr. Edward N. Janoff, VA Medical Center,
Infectious Disease Section (lllF), One Veterans Dr., Minneapolis, MN 55417.
*Present affiliation: Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts.
The Journal of Infectious Diseases 1996; 174:1191-9
© 1996 by The University of Chicago. All rights reserved.
0022-1899/96/7406-0007$01.00
In support of this hypothesis, increases in HIV-1 viremia
were observed in HIV-I-seropositive persons following acute
influenza infection [13] and vaccination with protein antigens,
including influenza [13 -15] and tetanus toxoid [16]. Thus,
because pneumococcal vaccine is routinely recommended [17]
and used to prevent the high rates of invasive Streptococcus
pneumoniae infections among HIV-l-infected patients [18],
we systematically analyzed HIV-1 activity following immune
stimulation with this polysaccharide vaccine.
Methods
Vaccinations. Twelve asymptomatic HIV-1-infected and 18
HIV-1- seronegative control subjects were enrolled. Baseline characteristics of vaccinees recruited from the HIV-1 clinic population
of the University of Nebraska Medical Center are shown in table
1. Volunteers received an intramuscular injection of 0.5 mL of
pneumococcal capsular polysaccharide vaccine (PNU-immune 23;
Lederle Laboratories Division, American Cyanamid, Pearl River,
NY) containing 25 J..lg of each of the 23 capsular polysaccharide
types. A second control group was selected from among asymptomatic HIV-I-infected patients, who received an intramuscular
injection of 0.5 mL of subvirion influenza vaccine (Wyeth-Ayerst,
Marietta, PA) containing 5 J..lg of hemagglutinin from each of
the 3 influenza strains (AiTexas/36/9I, A1Beijing/353/89, and B/
Panama/45/90.23). The first control group of HIV-I-seronegative
persons allowed a comparison of the vaccination response in these
persons with those in HIV-seropositive persons. The second control group comprised HIV-I-seropositive persons who did not
mount a significant response to another vaccine (influenza). This
latter group allowed for comparison of virus load changes between
individuals who did mount an immune response to vaccine challenge and those who did not. The second control group was also
Brichacek et a1.
1192
JID 1996; 174 (December)
Table 1. Clinical, immunologic, and virologic characteristics of HIV-1- seropositive asymptomatic
vaccinees.
Vaccine,
patient
Pneumococcal
POI
P02
P03
P04
P06
P07
P08
P09
PIO
P12
P13
P14
Influenza!
Fl5
F16
F17
F18
F19
F20
F22
Antiretroviral
therapy
(months)
Age, years
CD4 T
cellslJLL
38
40
29
33
39
47
34
33
37
32
31
32
320
334
435
300
500
435
170
470
340
470
350
365
None
None
None
Zidovudine
ddI (2)
Zidovudine
Zidovudine
None
None
Zidovudine
Zidovudine
None
30
34
35
35
45
21
34
300
600
500
230
310
430
120
Zidovudine
Zidovudine
Zidovudine
Zidovudine
Zidovudine
Zidovudine
None
(28)
(1)
(3)
(18)
(27)
(20)
(25)
(1)
(5)
(26)
(1)
Fold-rise" in
polysaccharide-specific
IgG
Plasma viral
RNA
(fold
change) *
Capsule
Cell wall
13.8
9.1
586.0
2.8
250.0
34.7
1.45
2.2
1.6
13.5
ND
1.0
11.6
9.6
10.7
17.4
2.7
1.3
.9
12.7
13.0
6.2
2.6
1.9
1.8
1.5
1.1
3.6
2.4
1.4
.6
1.7
3.0
1.5
1.5
1.4
1.9
1.0
1.7
1.4
1.8
0.12
0.33
NOTE. ND, not done; ddI, didanosine.
* Calculated as highest plasma viral RNA level divided by baseline (prevaccination level).
t Determined by dividing value after immunization (defined as point at which plasma viral burden was highest)
by value prior to immunization. Absolute change in capsule-specific IgG (value after immunization minus value
before immunization) was mean of 10,136 EO (range, 0-28,480).
~ Fold rise in influenza-specific IgG for this group = 1 except for patient F20, in whom no titers were detected
before or after immunization.
needed to exclude indirect effects of study design (e.g., stress of
clinic visits, natural variation in virus load) in contributing to
the changes in virus load observed following vaccination. The
demographics of HIV -1 - infected patients receiving pneumococcal
or influenza vaccines were similar (mean age, 35.4 ± 1.5 vs. 33.8
± 2.4 years; race, 92% vs. 88% Caucasian; and sex, 92% vs. 100%
male, respectively).
Vaccinated persons were examined at ~ 1, 3, 5, 9, and 12-16
weeks after vaccination for adverse reactions. At these intervals,
blood samples were drawn for analysis of viral and immune parameters. For most analyses, "pre" represents the sampling point prior
to vaccination, and "post" represents the sampling point at which
the highest virus load was detected (the point selected in related
studies [15, 16]).
Quantitation of plasma virus burden by quantitative competitive-polymerase chain reaction (QC-PCR) [19]. Aliquots ofpatient plasma (0.6-1.0 mL) were adjusted to 1.5 mL with serumfree RPMI (GIBCO BRL, Grand Island, NY), and virus particles
were pelleted (90 min, 18,000 g, 4°C). The virus pellet was resuspended in 100 p,L of serum-free RPMI containing 5 U of DNase
1 (Worthington Biochemical, Freehold, NJ) and incubated at 37°C
for 60 min. DNase-treated virus particles were again pelleted as
above, and virions were solubilized in 400 p,L of RNAzo1 (Te1Test "B," Friendswood, TX). We added 3 p,g of yeast tRNA
(Sigma, 81. Louis), and 2000 RNA copies (1000 virions) of an
HIV -1 integrase deletion mutant (MFA DIN 2) [20] as an internal
competitor template were added to doubling dilutions of viral
RNAzo1 lysate. The internal standard was distinguishable from
target sequences due to the presence of an 89-bp deletion in the
integrase coding region. The internal standard was quantitated by
negative stain electron microscopy to determine the number of
virus particles as well as by reverse transcription PCR amplification of viral RNA followed by comparison with a known copy
number dilution series generated by PCR amplification of DNA
from a full-length HIV -1 molecular clone (HIV-1 MF) [20].
Target and internal standard virion RNAs were reverse-transcribed in a 10-p,L reaction containing 44 pmol of an HIV-1 plusstrand primer specific for HIV -1 integrase (5' -C 49 17TGTCCCTGTAATAAACCC-3' (numbering according to Ratner et al. [21]).
Reverse transcription proceeded at 42°C for 17 min and was inactivated at 99°C for 6 min. Minus-strand primer (15 pmol) (5'G4 54 1CAGGAAGATGGCCAGTA-3') was added, and reverse
transcripts were amplified by 35 cycles of PCR in which each
cycle comprised a 30-s denaturation step (95°C), a 30-s annealing
JID 1996; 174 (December)
Pneumococcal Vaccine and HIV-1 Enhancement
step (58°C), and a 60-s extension step (72°C), followed by a single
7-min extension (72°C). Southern blots of PCR products were
visualized after hybridization to an HIV-1- specific oligonucleotide probe (5'-G 4 585CTGCCATTGTCAGTATG-3') and quantitated with a molecular phosphorimager (Molecular Dynamics SF,
Sunnvale, CA) by volume integration as described [22]. Viral RNA
copy number in the original plasma sample was calculated from
the plasma dilution that resulted in a signal intensity equivalent to
that obtained with the internal standard [19]. The sensitivity of
this assay was 200 copies when compared with quantitation of
viral preparations of known particle count (as determined by negative stain electron microscopy) and genomic viral RNA content
(as determined by comparison with a dilution series of viral DNA
from a full-length molecular clone). The linear range for the assay
was 102-105 viral RNA copies.
Quantitation of peripheral blood mononuclear cell (PBMC)associated proviral DNA. Patient PBMC were isolated on cell
separation tubes (Leucoprep; Becton Dickinson, San Jose, CA),
washed twice with RPMI, counted, resuspended at 107 cells/mL
in RPMI containing 40% serum and 10% DMSO, and stored at
-80°C. Total cellular DNA was isolated using a DNA extraction
kit (IsoQuick; MicroProbe, Bothell, WA) according to the manufacturer's protocol. HIV-I sequences in sequential cell lysate dilutions corresponding to 25, 10, and I X 104 cell equivalents were
amplified using HIV-I long terminal repeat (LTR) Rand gagspecific primers (LTR R, 5' -G 485GGAGCTCTCTGGCTAACT;
gag, 5'-G9 3 1GATTAACTGCGAATCGTTC-3'), which amplify
full-length and near full-length products of reverse transcription
[22, 23]. HIV-l sequences were amplified by 30 cycles of PCR
in which each cycle comprised a 30-s (95°C) denaturation, 30-s
(58°C) annealing, and 60-s (72°C) extension, followed by a final
5-min extension (72°C). Amplified products were analyzed by
Southern blot hybridization to an HIV-I LTR D5 oligonucleotide
probe (5' _G583T AACTAGAGATCCCTCAGAC-3 '), and hybridized products were visualized on a molecular phosphorimager as
outlined above. Cell equivalents in each PBMC sample were determined by PCR quantitation of a-tubulin copy number. a-tubulin
sequences [24] were amplified by 20 cycles of PCR using tubulinspecific primers (plus strand, 5' -AAGAAGTCCAAGCTGGAGTTC-3'; minus strand, 5'-GTTGGTCTGGAATTCTGTCAG-3';
probe, 5'-CAGGTTTCCACAGCTGTAG-3'). HIV-l and tubulin
copies were quantitated by comparison with HIV -I and tubulinspecific PCR products generated from a dilution series of 8E5 cells
[25] that contain I defective HIV -I provirus per cell. Product
yields from sample reactions and from the dilution series were
quantitated by volume integration [22].
Analysis of immunologic parameters. Lymphocyte subset
markers including CD3, CD4, CD8, CD25 (interleukin-2 receptor,
p55), and HLA-DR were examined individually and in combination (e.g., CD4/CD25, CD8/CD25, and CD3/CD25) by FACScan
(Becton Dickinson) analysis. Expression of cell surface activation
markers was compared using a one-way analysis of variance (Statistics Version 4.0; Analytical Software, St. Paul, MN). As previously described [26], antibodies to pneumococcal polysaccharides were quantitated by ELISA using cell wall polysaccharide
(5 ,ug/mL; Statens Seruminstitut, Copenhagen) or the 23-valent
vaccine (11.5 ,ug/mL total polysaccharide) as the capture antigen,
serial dilutions of sera (adsorbed with 50 ,ug/mL cell wall polysac-
1193
charide for capsule-specific IgG), and horseradish-peroxidase-labeled goat anti-human IgG (detector antibody). ELISA units (ED)
were calculated from a standard serum [26]. Titers of IgG reactive
with influenza antigens were determined by IFA according to the
protocol of Riggs et al. [27].
Statistics. Differences in age, CD4 T cell counts, and virus
burden between the 2 HIV -1- infected groups were compared by
unpaired Student's t test. Differences in virus burden, antibody
responses, and lymphocyte subset profiles between individuals
who did and did not mount an immune response to the vaccine
were evaluated using a heteroscedastic t test analysis. Comparisons
in viral antibody and lymphocyte parameters within each group
were made using a paired 2-sample for means t test. As in similar
studies, to obtain a normal distribution for the tested data, plasma
RNA copy numbers were transformed to a log scale prior to statistical analysis [15, 16] The K statistic [28] was used to examine the
degree of correlation between changes in plasma viral RNA load,
pneumococcal IgG levels, and lymphocyte subset activation markers. This involved tabulating the direction of change between each
pair of consecutive measurements on each patient for each of the
variables. For each pair of consecutive measurements, I represents
an increase and 0 indicates a decrease. The K statistic was conducted with weighted and unweighted samples to determine
whether correlations between multiple measurements on the same
participant affected the analysis. The results were essentially the
same if observations were weighted to compensate for withinpatient correlations.
Results
IgG responses to pneumococcal vaccine. Because symptomatic HIV-I infection may be associated with an attenuated
response to viral and bacterial antigen vaccines [29, 30], we
studied asymptomatic HIV-1-infected persons who were more
likely to respond to the immunization. Baseline levels of pneumococcal vaccine - specific IgG were similar among HIV-1infected patients and control subjects (5.0 ± 2.8 vs. 4.4 ± 0.5
ED X 10- 3/mL). After immunization, both groups showed a
rise in vaccine-specific IgG (P = .01 for each group; paired t
test), although mean convalescent levels of specific IgG were
lower among HIV-1- infected patients than among seronegative control subjects (13.4 ± 10 vs. 41.2 ± 6.7 ED x 10-3/mL;
P < .05; unpaired t test). Changes in levels of IgG reactive
with the cell wall polysaccharide of S. pneumoniae, also present
in the vaccine, were more modest than those to the capsular
polysaccharide (table 1). Overall, pneumococcal vaccine elicited a significant immune response in patients and control subjects, independent of HIV-l status.
Changes in HIV-l virus burden following pneumococcal
vaccination. We examined changes in plasma virus load in
asymptomatic HIV-l-infected individuals before and at various intervals after immunization with pneumococcal vaccine.
In addition, PBMC-associated proviral DNA copy number was
quantitated using PCR and primers that amplify full-length
Brichacek et al.
1194
Patient P02
Patient P01
CD ::::>
.w
I
s g
et ::'!
::E ::::>
tn w
:5
z
•i
T
I
I
/
o
102 -4
-1
0 1 3 5 12
. -...--...--......-......-......--r--I
10 2 ~---r----r----r----r----r----r---' 10 2 ......
-5 -1 0 1 3 5 12
-1 0 1 3 9 12
-4
Patient P04
et!i!! 105
z ...J
0: et
.... 0
104
et 0
0
10 3
103
8"~CJ
0:
104/
104
--
Patient P03
10 6 r-- - - - - - --,
10 5
105
,...!..,...
E !..
rn E
JID 1996; 174 (December)
103
.---~
7
Patient P06
10 6
..
10 5
104 .-----;--- 103
I
102 .I-r--r---.---Y--~~~ 102
.
Patient POS
•
.....
....
I
I
I
104
I
1./
-5 -1 0 1 3 4 12
----
--~
Patient P07
-5 -1 0 1 3 9 13
Patient P09
105·
-4 -1 0 1 3 5 12
Patient P12
1()6 .,.---'----------,
Figure 1. Plasma viral RNA and pneumococcus-specific IgG levels in representative HIV -I ~
seropositive vaccinees. Patients were evaluated at
recruitment and at each sampling point; none had
clinically significant lymphadenopathy, opportunistic infection, or other symptomatology throughout sampling period (patients 10 and 14 are not
shown). Absolute virion-associated genomic RNA
and antibody levels were measured by quantitative
competitive-polymerase chain reaction and ELISA,
respectively.
105
::~ .'~
"
"•
104
'"
103
\
\
-4 -1 0 1 3 6 12
102 .......---r----r----r----r----r----r---'
-4 -1 0 1 3 9 16
-4 -1 0 2 3 6 16
TIME PRE/POST VACCINATION (WEEKS)
viral cDNA (comprising complete plus- and minus-strand viral
DNA) [10, 23].
Because the majority of patients had baseline plasma viral
RNA < 104 copies/mL (table 1), we adopted a modified QCPCR protocol in order to quantitate changes in plasma viral
RNA after vaccination. The original QC-PCR method described by Piatak et al. [19] used an in vitro-transcribed RNA
as a competitor template. In the modified QC- PCR protocol,
we substituted virus particles of an HIV -1 integrase deletion
mutant [20] as the internal competitor template for QC-PCR.
Furthermore, addition of a known number of integrase mutant
HIV-1 virus particles to the patient samples prior to isolation
of target virion RNA enabled us to accommodate sample loss
and the presence of plasma components that interfere with the
efficiency of PCR. This modified procedure was quantitative
over a 3-10g range down to a sensitivity of 200 RNA copies/
mL (unpublished data).
The immune response to pneumococcal vaccine antigens
was accompanied by rapid and in some cases profound changes
in plasma virus burden (figure 1). Baseline plasma viral RNA
varied between < 103 and 2.3 X 105 molecules/mL (figure 1).
Following vaccination, significant increases in plasma viral
RNA levels between 1.6- and 586-fold over baseline were
evident in 10 of 11 patients tested (P = .02 for all patients;
table 1, figure 1). In some vaccinees (POI, P03, and P06),
plasma viral RNA load was elevated over baseline (prevaccina-
tion) levels throughout the sampling period, whereas in other
patients (P04, P07, P09, and PI2), plasma viral RNA load had
returned to baseline levels by the end of the sampling period
(figure 1). Since we did not obtain plasma samples from patients POI, P02, P03, and P06 beyond 3 months after vaccination, we were unable to determine whether plasma virus load
eventually returned to prevaccination levels. Studies using protein antigens suggest that the vaccine-associated rises in HI V1 in plasma are typically transient [14-16]. The transient increases in plasma viral RNA that were observed in some of
our patients immunized with polysaccharide antigens appeared
to parallel changes in levels of pneumococcal capsule-specific
IgG (figure 1).
Higher levels of virus burden, particularly in patients with
late-stage disease, have been associated with high levels of
PBMC-associated proviral DNA [4, 31, 32]. Similarly, several
patients had increased levels of PBMC-associated proviral
DNA (POI, P03, and P04) following vaccination (figure 2). In
general, however, PBMC-associated proviral DNA copy numbers were relatively static throughout the sampling period.
Correlation between immune activation and changes in virus
burden. A statistically significant increase in the number of
activated CD4 T cells, as determined by CD4/CD25 (P = .02)
double-positive cells, was observed following pneumococcal
vaccination (figure 2). In addition, we observed a significant
increase in a number of CD4/HLA-DR double-positive cells
Pneumococcal Vaccine and HIV-l Enhancement
lID 1996; 174 (December)
3.0
3.0
2.5
2.0
1.5
1.0
0.5
Q)
0)
0
c
ftI
.c
CD8+ Cells
CD4+ Cells
2.5
~
pre
post
2.0
1.5
1.0
0.5
0
-4
pre
post
1195
CD4+ CD25+ Cells
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
HIV-1 RNA
1000 ....- - - - - - - - - - ,
100
10
pre
post
0.1
oL...--or----_,....-----I
pre
post
0
'C
0
LL
Proviral DNA
35
;>
32
20
Pneumococcal IgG
3.5
15
3.0
2.5
6
10
2.0
4
1.5
5
2
0
CWPS IgG
4.0
1.0
0.5
pre
0
post
pre
post
0
pre
post
Figure 2. Changes in virus burden, lymphocyte subsets, and capsular polysaccharide-specific IgG following pneumococcal vaccination. For
each variable, fold change after vaccination (post) is calculated using value from sample with highest plasma HIV-l RNA level divided by
baseline (pre) value. Viral RNA load was measured as virion-associated HIV RNA in plasma and peripheral blood mononuclear cell-associated
proviral DNA; peak values are given. Immune response to pneumococcal vaccine was determined from IgG titers to 23-valent pneumococcal
capsular polysaccharide vaccine and to pneumococcal cell wall polysaccharide (CWPS). Patient symbols are same for each patient in each
panel (1, 0; 2, D; 3, <>; 4, X; 6, +; 7, ~; 8, e; 9, .; 10, . ; 12, .A.).
(median, 1.7-fold rise; range, 1-2.8; P = .02) but not CD8/
HLA-DR cells following pneumococcal vaccination. In contrast, no significant differences (P = .8) in CD4/HLA-DR double-positive cells were evident in influenza vaccinees.
In about half the patients, the number ofCD4 T cells declined
relative to baseline after pneumococcal vaccination (figure 2).
In addition, changes in virus burden were highest among patients with a postimmunization decline in CD4 T cell counts
(e.g., PO1, P02, P04, P06, and P07), whereas no substantial
reductions in CD4 T cell counts were observed in patients
with less profound changes in plasma virus load (P08, P09,
and PIO).
We examined the relationship between changes in plasma
viral RNA load, pneumococcal IgG levels, and lymphocyte
subset activation markers by comparing the direction of change
between each pair of consecutive measurements on each patient
(1 represents an increase and 0 represents a decrease). The K
statistic [28] was used to test for agreement between the pairs
of variables in 2 X 2 tables obtained from an increase or
decrease between consecutive measurements. The K statistic
for correlation between increase in plasma viral RNA and in-
creases in IgG to pneumococcal capsular polysaccharides was
0.64 (substantial), 0.42 (moderate) for IgG to cell wall polysaccharide, and 0.33 (fair) for increases in CD4/CD25 doublepositive cells. P values for these relationships, on the basis of
the null hypothesis that the K value is 0, indicated a substantial
agreement in direction of change at all points tested between
log changes in virion number and changes in IgG to pneumococcal vaccine (P = .003), in IgG to cell wall polysaccharide
(P = .02), and, to a lesser extent, in the number of CD4/
CD25 CD4 cells (P = .08) for unweighted comparisons. When
weighted to adjust for repeated measurements on the same
patient, the P values were .02 for IgG to vaccine, .09 for IgG
to cell wall, and .21 for activated CD4 T cells. Taken together,
these data demonstrate a significant correlation between the
magnitude of increased HIV -1 activity and extent of immune
activation following pneumococcal vaccination, particularly
the capsule-specific IgG response, to which the vaccine is directed.
Plasma viral RNA load changes in nonresponder vaccinees.
To exclude the possibility that the correlation between the
magnitude of the antibody response and changes in plasma viral
Brichacek et al.
1196
Patient F15
10 1
10 1
en
..J
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W
ce (,)
Z
a:CJ W
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enu.
N
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JID 1996; 174 (December)
Patient F17
",., ",""'.
...;::e!........
.....
fI
Patient F18
10 1
•
I
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....
-4 -1 0 1 3 5 12
-5 -1 0 1 5 16
-5 -1 0 1 3 5 8
10 0
10- 1
10- 1
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101
10 0
Patient F19
-..
(- '.<t
10 1
Patient F22
...
,
10 0
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• I. ':
\
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\
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10- 1
10-1
10-1
-5 -1 0 1 3 5
-5 -1 0 1 4 12
I
-w
Figure 3. Fold changes in plasma viral RNA,
influenza antibody titers, and lymphocyte activation levels in representative nonresponder vaccinees. Case descriptions of patient status together with maximum changes in plasma viral
RNA load throughout sampling period are given
in table 1. Patients were classified as nonresponders because titers of influenza-specific IgG did
not show >2-fold increase. Fold increases in viral RNA, antibody titers, and CD4/CD25 doublepositive cells were calculated relative to levels
obtained before vaccination. Thus, in absence of
immune response, plasma viral RNA load and
indicators of lymphocyte activation remained
stable over sampling period. Log annotations for
fold rises are 10- 1 = 0; 100 = 1; 10 1 = 10).
-5 -1 0 1 4 12
TIME PRE/POST VACCINATION (WEEKS)
RNA elicited following pneumococcal vaccination reflected
unrelated fluctuations in virus burden over the sampling period,
we examined viral and immune parameters in vaccinees who
showed no humoral response to vaccination. Consistent with
previous reports [33], of the 12 pneumococcal vaccinees analyzed in our study, only 1 (P08) did not show a specific response
to the pneumococcal vaccine (table 1). In contrast, the response
rate to influenza vaccine in HIV -L-seropositive persons is less
consistent, which may reflect the T cell-dependent nature of
the immune response to influenza antigens [34]. Thus, we selected 7 HIV-I-seropositive asymptomatic adults who did not
elicit a specific response to influenza vaccination, as demonstrated by a lack of increase in any of the three influenzaspecific IgG titers over baseline (table 1, figure 3). CD4 T
cell counts were comparable to those of the pneumococcal
vaccinees. However, unlike the marked changes in plasma viral
RNA burden observed in some pneumococcal vaccinees, there
were no statistically significant changes in postvaccination
plasma viral RNA levels relative to baseline activation markers
in these nonresponders (figure 3). Over the entire sampling
period (10 - 21 weeks), the largest increase in plasma viral RNA
over baseline levels was 1.9-fold for patient FI5 (table 1, figure
3). Furthermore, these vaccine nonresponders showed no statistically significant differences in PBMC-associated proviral
DNA levels or the number or state of activation of CD4 T cells
after vaccination compared with baseline values (figure 4).
Taken together, these data support the notion that the
changes in plasma viral RNA load in pneumococcal vaccine
recipients was a consequence of the immune activation that
accompanied the response to pneumococcal antigen stimulation
rather than a consequence of temporal fluctuations in virus
burden or unrelated events over the sampling period. HIV-I
replication following pneumococcal vaccination increased by
1-2 weeks after vaccination, when pneumococcal vaccinespecific responses are apparent [35, 36] and correlated with
the magnitude of the humoral response to the vaccination.
Discussion
We have shown that immunization with pneumococcal vaccine, a clinically relevant and widely used intervention, resulted
in appreciable increases in HIV-I burden in some patients.
This enhancement of viral activity was related to the extent of
vaccine-associated immune stimulation. In contrast, no appreciable increase in HIV -1 burden accompanied immunization
in patients who did not respond to influenza vaccine. Thus,
immunization may serve as a controlled model to characterize
the impact of antigenic stimulation on HIV-1 expression and
the mechanisms involved.
The relationship between immune activation and HIV -1 burden following vaccination is in strong agreement with recent
studies [14-16] in which vaccination resulted in significant
and transient increases in plasma HIV-1 RNA levels. The magnitude of the increase in HIV-1 replication correlated with the
ability of those persons to immunologically respond to vaccine
antigens. Furthermore, the fold-increase in virus burden was
often higher in persons with higher CD4 T cell counts, presumably because they were better able to immunologically respond
to the vaccines. These studies illustrate the relationship between
immune activation and viral replication and point to a mechanism whereby exogenous agents (intercurrent infections, antigenic exposure) may promote viral replication in infected
persons.
Several mechanisms may explain the increased HIV -1 replication that followed stimulation with pneumococcal antigens.
The predominant targets for HIV-I infection and replication in
JID 1996; 174 (December)
Pneumococcal Vaccine and HIV-1 Enhancement
3.0
Figure 4. Changes in virus burden and
lymphocyte subsets in nonresponder influenza antigen vaccinees. Data were collected
from 7 influenza vaccinees who did not elicit
response to vaccine «2-fold changes in influenza antibody titers) over sampling period
similar to that of pneumococcal vaccinees.
Postvaccination values were determined as
described in figure 2 legend. Each patient is
represented by same symbol in each panel
(15,0; 16,0; 17,0; 18, X; 19, +; 20, f;..;
22, .).
CD
a»
CD4+ Cells
3.0
2.5
2.5
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0
0
C
pre
post
1197
CD8+ Cells
~
pre
post
4.0
3.5
CD4+ CD25+ Cells
~--------,
3.0
2.5
2.0
~
1.5
1.0
0.5
0"---....------...-----1
pre
post
as
.c
0
't:J
~
10
Proviral DNA
1000
8
HIV-1 RNA
100
6
10
4
2
0
-4
pre
infected persons are cells of lymphoid and monocyte/macrophage lineage [7]. Productive infection of T lymphocytes is
absolutely dependent upon the activation state of the host cell
[8-10, 20], and the T cell activation that accompanied the
pneumococcal vaccinations may directly influence the permissiveness of the T cell reservoir to productive HIV -1 infection.
Proinflammatory cytokines, which can be elicited by responses
to pneumococcal cell wall polysaccharides, may stimulate activity of the provirus [11, 37]. The extent of HIV -1 production
from infected lymphocytes and macrophages may be a consequence of the production of cytokines, which accompanies
antigen processing by macrophages [38]. Thus, activation of
the immune system, whether induced by vaccine antigens or
by natural infection, may elicit inflammatory responses, production of cytokines, and activation of lymphocyte subsets and
HIV -1 replication.
Our study provides evidence in vivo for a relationship between vaccination-mediated immune stimulation and HIV1 activity. Recent studies examining changes in virus load
following administration of antiretrovirals [5, 6] together with
mathematical modeling [39] provide evidence for a dynamic
process in HIV-1 replication involving an equilibrium between HIV -1 replication and CD4 lymphocyte turnover. An
important issue in this process is whether HIV -1 replication
is limited by the availability of infectious virus or permissive
(activated) target cells. Administration of interleukin-2 to
HIV-l-seropositive persons leads to rapid increases in both
CD4 T cell counts and virus burden [40]. That observation,
together with the correlation between the extent of CD4 lymphocyte activation and HIV -1 replication observed in our
post
0.1
~
pre
post
study, support the notion that target cell availability is an
important rate-limiting step for de novo HIV-1 replication.
Such a relationship would predict that subtle changes in the
frequency of permissive target cells have a profound effect
on the level of viral activity in HIV-1- infected persons. The
influence of immune activation on permissiveness to HIV-I
infection would also predict that HIV -1- seronegative persons
with recurrent bacterial or viral infections may be at increased
risk of infection by HIV-1 if an activation episode coincides
with exposure to the virus. Indeed, Stanley et al. [16] showed
that cells from seronegative subjects after immunization with
tetanus toxoid were more permissive to HIV-1 infection in
vitro than were cells from the same donors prior to immunization.
In this study, we do not provide evidence that a single
vaccination and the potential for a subsequent viremic response has an appreciable or clinically significant impact on
HIV-1 disease progression. However, many questions arise
about the implications of our findings with pneumococcal
vaccine and those with other routine immunizations in HIVI-infected patients. What is the duration and magnitude of
these responses? Does immunization promote immunologic
deterioration and promote progression of HIV-I-associated
disease? Do patients at all stages of HIV-1 infection show
similar responses? What is the mechanism of antigen-associated viral replication? Does repeated immune stimulation
serve to sustain elevated levels of HIV -1 replication with
concomitant expansion of HIV-1 genotypic and phenotypic
diversity? Should consideration be given to coadministration
of vaccines in conjunction with antiviral agents to counter
1198
Brichacek et al.
possible effects on viral replication, particularly in HIV-1infected infants, who often receive multivaccine regimens?
Most important, are the potential adverse consequences of
immunization worse than the diseases they are designed to
prevent? Pneumococcal vaccination offers potential benefit in
reducing the very high incidence of pneumococcal infection
(bacteremia, pneumonia, meningitis) [41], particularly at earlier stages of disease. To address these critical issues, prospective longitudinal studies of the efficacy of the vaccine and of
the virologic, immunologic, and clinical sequelae of pneumococcal vaccine and disease in HIV-1- infected persons are in
progress.
In summary, antigenic challenge, whether in association with
vaccine challenge or following acute and chronic infections,
may promote CD4 T cell activation, HIV-1 replication, and
CD4 T cell dysfunction and death. These findings provide the
rationale for more expanded studies to examine the actual clinical and immunologic consequences of antigen-induced stimulation of HIV-1 replication, the duration of these effects, their
impact on viral heterogeneity, and the role of specific T cell
subsets in modulating this process.
Acknowledgments
We thank J. Schademan for patient recruitment, S. Roumpf, L.
Halverson, W. Robertson, R. Kagaruki, and J. O'Brien for excellent technical assistance, Kay Cowels for statistical analysis, H.
Gendelman for helpful discussions, and K. Bowers and Ann Emery
for manuscript preparation.
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