tive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether ...
Proc. Natl. Acad. Sci. USA Vol. 93, pp. 3972-3977, April 1996 Medical Sciences
Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen M. JULIANA MCELRATH*, LAWRENCE COREY*tS, PHILIP D. GREENBERG*§, THOMAS J. MATTHEWSI, DAVID C. MONTEFIORI¶, LEE ROWEN*, LEROY HOOD*§II, AND JAMES I. MULLINS*t Departments of *Medicine, tLaboratory Medicine, §Immunology, lMolecular Biotechnology, and tMicrobiology, University of Washington School of Medicine, Seattle, WA 98195; and IDepartment of Surgery, Duke University Medical Center, Durham, NC 27710 Contributed by Leroy Hood, November 6, 1995 MATERIALS AND METHODS A 24-year-old HIV-1 seronegative, healthy Caucasian who
With efforts underway to develop a prevenABSTRACT tive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and
never received a smallpox vaccine and who reported involvement in a stable monogamous sexual relationship with an HIV-uninfected partner was recruited for the study. Over 4 years, the volunteer received 6 HIV-1 envelope vaccinations (5, 7, 11) as outlined in Table 1. Approximately 3 months (week 206) after the last vac-env booster, the volunteer had an increased HIV-1 antibody by ELISA and new Gag antibodies by Western blot assay (Table 1). Three months later (week 222), testing revealed a markedly reactive HIV-1 ELISA and Western blot assay. Upon questioning retrospectively, the subject disclosed that the previously reported monogamous relationship had ended and during week 203 the subject had engaged in unprotected anal intercourse with a new partner of unknown HIV-1 serostatus. Analysis of stored frozen specimens revealed no detectable HIV-1 RNA in sera from weeks 140, 164, 193, 197, and 201, but HIV-1 RNA was detected in serum from week 206 (Table 1). These data indicate that HIV-1 probably was acquired at approximately week 203, coincident with the high risk exposure, which we have designated as week 0 of infection. Individuals with early HIV-1 infection at the University of Washington Center for AIDS Primary Infection Clinic served as the nonvaccinated infected control group. Immune responses in these individuals were determined from peripheral blood mononuclear cells (PBMC) at various times following presumed or known date of seroconversion. Antibody and Cellular Immune Assays. HIV-1 antibody was measured by EIA (Genetic Systems, Seattle) and by Western blot (Epitope, Beaverton, OR). Neutralizing antibody assays were performed as described (10, 11). Titers represent the reciprocals of sera dilutions required to reduce infectious virus titer by one log or >90% in CEM cell lines (for HIV-1LAI and HIV-1MN) and in PBMC (for the first isolated autologous HIV-1). C'-ADE was measured in MT-2 cells (13) by p24 production and is expressed as the reciprocal of the last serum dilution to show enhancement (titer), the reciprocal serum dilution producing the greatest enhancement (peak), and the magnitude of enhancement at the peak dilution, given as the fold-increase in p24 production over background (power). LP responses were measured in vitro to HIV-1 antigens (2-10 gLg/ml) including psoralen-treated, UV-inactivated purified HIV-1LAI (kindly provided by S.-L Hu, Bristol-Myers/ Squibb), gpl60 (HIV-1LAI strain, kindly provided by MicroGeneSys, Meriden, CT), and Env 2-3 (HIV-lsF2 strain, kindly provided by K. Steimer, Biocine, Emeryville, CA), as described
neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.
progressive spread of human immunodeficiency virus (HIV) infection has made development of a preventive vaccine a global health priority. The immune responses that correlate with protection from HIV infection or control of disease are unknown. Presumably, a vaccine is more likely to be efficacious if it is capable of eliciting HIV-specific broad neutralizing antibodies, CD8+ cytotoxic T lymphocytes (CTL), and T helper responses (1). Several vaccine regimens have provided protection in nonhuman primates against challenge with HIV or simian immunodeficiency virus (SIV) (2-4). These findings have led to human trials to evaluate a variety of HIV-1 candidate vaccines (5-11). Since the initiation of HIV vaccine trials by the AIDS Vaccine Clinical Trails Network, some study participants have acquired HIV infection, all as a result of high risk behavior (12). This report provides a detailed analysis of HIV-1 infecThe
tion in a vaccinated volunteer from one of these studies and
compares these responses with a group of nonvaccinated individuals with early HIV infection. The volunteer received six vaccinations, three with a live recombinant vaccinia virus
containing HIV-1LAI gp160 (vac-env) and three with a baculovirus-derived HIV-lLAI recombinant envelope protein (rgpl60) (5, 7, 11). This immunization approach has shown promise in the macaque model with SIV infection (3) and has been particularly effective at inducing both HIV-specific neutralizing antibody and T-cell responses (7, 8). While it remains unclear why the regimen failed to protect this volunteer, the analysis establishes a framework with which to explore future cases of HIV infection in vaccinated volunteers participating in expanded clinical trials.
immunodeficiency virus type 1; CTL, cytotoxic T lymphocytes; SIV, simian immunodeficiency virus; vacenv, recombinant vaccinia containing HIV-1LAI gpl60; rgpl60, recombinant HIV-lLAI gpl60; PBMC, peripheral blood mononuclear cells; C'-ADE, complement-mediated antibody-dependent enhancement; LP, lymphoproliferative; S.I., stimulation index; E/T, effectorto-target ratio; CC, co-culture. tTo whom reprint requests should be addressed.
Abbreviations: HIV-1, human
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3972
Medical Sciences: McElrath et al. ^
=
8
^
^
t
Proc. Natl. Acad. Sci. USA 93 0%u
^
k
I0
4
e
E
r.- 00 Vn t" O "o oO0Ir4e en
^
Z ,N, oo0 o , ,v ,\-0C, o all 00 0
.
I
N
"sO00 P
I
0
"O
+ .m
(1996) 4 Y
e
0o
,
_._^
I
a c + + + i 111+
^0-S ++&+ ++
+ ++
°
VVV0o0
r~
C
0
000
0 0o t
CD
VVV
V V
V7 V-cVooO_ O4 jg
_
0
"
CD 'T
0
0
e
Ei
V
V
1O cO
V
v
o
o
ii
V
V
V
vv
i+i+i++
S
-o
S
a
++ 00. 0C
&
rQ Oae uVtt~- v oo V
h0
4)
C
v
v
W).e c
00
I)
ICX
o *1
'uICo
W~~~~ VYa 0c Vr cJo
g
s»^
-
wd!c
^
Ns
- 1 s^- kO 1Es^ V'
^^
cV
O
v
O~
LLc~~~~~
o\0oCW
v
V V
._
a*
0 0 eq 00
VV/ v v "',
r
elI Cu
xi
Coo rcr-
co
tn 'v
U"
i
I)
O 0Cu
t)
w)
4
'
rrl
v) v)
I)
0 c*") Cv en
t)
---
C v
v
V
>
I ll
z 01
>
I
I
I
v
VV
V
0 O
=0
00
00 ~e ~ _ ~ Cu _ II Cu
~
to
O 0
IS I
V
++
C4
00
v v v
~~~~II
\D C0
O +
u
"
II
\O 00 0N 0
NO
o o -
O
oo
o
o
T"
VV
It
ii~~~
V
v
-
I
O O A
vv
+
V
00
V
-
+
..) Cu
Ce
+ +
>-o-o-
~~1~
.i.
I
+ ++
+ + + +
t NFt.-: oo rsi »-^
14t cj
m
-
) 'O t tr
4,4)
ECCu Cu 0, 0 o >
C-_NOmo *
-o
~
~
~
" -r^ .^ ~C ~
o^o
t
.i ~
~
c~
Rt ~n
~
~
00 em Oo 0o
0
0aoooo rO-41
00 ~ ~
~
~
.
'
c: O >
N Oe
IY "00
I
0
BrI\5
t s
0"
f
C
eO0 Ot r-
I
-
-
'
Cgu8E
^^on
In 'll "IT I')
%
V
< -
(N
v v
.)
'"
0
'0%~ C" e, l ,,Nc,lc 0, c,l -eO",l > 0\ OO ci lC 0-4t O^ o rN . . . N . . . .4 \ . \ ^^mm
S.0
"
3974
Medical Sciences: McElrath et al.
(5, 7). A stimulation index (S.I.) of -3 was considered positive for the HIV-1LAI antigen and -4.0 was considered positive for gp160 and ENV 2-3 antigens. For analysis of CD8+ CTL, fresh PBMC were stimulated for two 1-week intervals with autologous HIV-1Ba-L-infected macrophages as described (14). Effectors were tested for lysis of autologous Epstein-Barr virustransformed B lymphoblastoid cell lines infected with recombinant vaccinia expressing either HIV-1LAI Env, Gag, or Pol and vaccinia wild-type control or pulsed with HIV-1LAI gp160 at varying E/T up to 50:1 in a standard 4-h chromium release
assay (7, 14). Virologic Assays. Plasma HIV-1 RNA was determined by branched DNA amplification (15). The lower level of sensitivity of the assay is 10,000 copies/ml. Infectious HIV-1 was measured by p24 production from either PBMC or plasma as described (16). The ability of the autologous viral isolate to induce syncytia was determined in MT-2 cells (17). HIV-1 env sequences were amplified using PCR directly from PBMC obtained at weeks 208 and 224, and after coculture (CC) of the recipient's PBMC drawn from week 233 with PBMC from an uninfected donor. The fragment from the first PBMC sample was generated by nested PCR (18) using primers ES7 and ES8 in the second round of PCR, and cloned into pUC19. The CC sequences were obtained after amplification of the entire envelope coding sequence which was then cloned into the mammalian vaccinia expression vectors pWR508 and pJW4303. Each sequence was determined using a fluorescence-based Applied Biosystems model 373 automated sequencer using dye-labeled terminator (PBMC samples) or with dye-labeled primers after random shearing, shotgun cloning, and sequencing of the entire plasmid including vector (CC clones) (19). RESULTS Vaccine-Induced Immune Responses. The subject experienced a primary vaccination response following the initial vac-env, and vaccinia was isolated from swabs (5) taken up to 14 days from the scarification site. Antibodies to HIV-1 gp160 were detected after the second dose of vac-env (Table 1). Four weeks after boosting with rgpl60 1 year later, neutralizing antibody was demonstrated against HIV-1LAI but not against the heterologous HIV-1MN and in a retrospective analysis, not against the autologous HIV-1 (Table 1, week 64). The vaccine regimen induced strong LP responses to HIV-1LAI and HIVlsF-2, envelope antigens, with S.I. of >1000 (Table 1). During the peak response, PBMC transferred into hu-PBL severe combined immunodeficient mice conferred protection in two of three animals following challenge with HIV-1LAI (20). Seven CD4+ clones lysed targets expressing HIV-1LAI gpl60, and three of the seven clones also recognized targets express-
ing HIV-1MN gp160 (7). After 1 year, HIV immunity declined and a third rgpl60 boost was administered at week 136. This boost resulted in increased envelope antibodies, low titer neutralizing antibodies to HIV-1LAI, but not to HIV-1MN, and an increase in lymphoproliferation to homologous HIV-1 antigens (Table 1, week 140). Another vac-env booster was administered at week 193 (Table 1), resulting in a secondary vaccinia reaction. Vaccinia was isolated from the scarification site 4 days after inoculation. However, little increase in HIV-1 immunity was noted (Table 1). HIV-specific CD8+ CTL, examined for the first time during the vaccine study, were not detected prior to, and at 1 month (weeks 193 and 197) following the third vac-env immunization (Table 1).
Clinical and Virologic Course of HIV Infection. The volunteer experienced no clinical illness following acute HIV-1 exposure, and after 2 years has remained asymptomatic. CD4 counts had fallen from a median of 780 cells/mm3 to 462 cells/mm3 when first diagnosed, and within 98 weeks of
Proc. Natl. Acad. Sci. USA 93 (1996)
infection, the counts declined to 255 cells/mm3 and remained -300 cells/mm3 through week 116 after infection. At week 206, 3 weeks after infection, 53,400 copies/ml of HIV RNA were detected in serum. Levels in plasma fell to < 10,000 copies/ml at 21 and 25 weeks after infection, and were intermittently detectable between 39,800 and 82,100 copies/ml over the next 62 weeks. Attempts to isolate HIV-1 from PBMC were unsuccessful until 30 weeks after infection, and subsequent PBMC cultures were intermittently positive (Table 1). The mean titer of HIV-1 in PBMCs, when positive, averaged
16 infectious units per million PBMCs (data not shown). All PBMC-derived HIV-1 isolates had the nonsyncytium-inducing phenotype. Only the last plasma HIV culture attempted at week 103 was positive (Table 1). HIV-1 envelope gene sequences were amplified using PCR, cloned, and sequenced from the volunteer's PBMC taken on weeks 5 and 21 after infection, and from a virus culture initiated with PBMC drawn on week 30 after infection (two clones, designated CCa and CCb). As shown in Fig. 1, for week 5 and 30 specimens, these sequences were closely related, reflecting their origin from a single infected individual and belonged to the B envelope sequence clade, as expected for infections acquired in the United States. They were also distinct from the HIV-lAI vaccine strain (represented by HXB2R in Fig. 1). Two features of note were the presence of the macrophage-tropic virus consensus sequence in the V3 loop, as expected for recently infected individuals (21-23), and the absence of positively charged amino acids at positions 11 and 28 of the loop (R and K residues underlined in Fig. 1), consistent with the observed absence of syncytium-inducing virus. Immune Responses After HIV-1 Infection. Neutralizing antibody was consistently measured in sera collected beginning 3 weeks after infection, with neutralization of HIV-1LAI detected first at a titer of 1:24 (Table 1). By contrast, sera from three patients infected within the same year and residing in a geographically similar area had low (1:20 titer) to no detectable neutralizing antibody to HIV-1LAI when examined over -1 year of infection (Fig. 2A). These results suggest that after infection, an anamnestic antibody response to the previous immunizing antigen occurred in the vaccinated individual. Neutralizing activity to HIV-1MN was detected at week 19 after infection and rose to 1:965 by week 70 after infection. Titers to HIV-1LAI increased to a lesser extent than to HIV-1MN (Table 1). Neutralizing responses to the first isolated autologous HIV-1 were not demonstrated until after 1 year of infection (Table 1, week 53), and the titers were low (1:15). In a retrospective analysis, sera obtained before infection, when
neutralizing responses to HIV-1LAI were present (week 64),
failed to neutralize the autologous HIV-1 (Table 1). Because of concern that vaccination may induce infectionenhancing antibodies which may, upon HIV-1 exposure, increase the susceptibility to infection or severity of disease (24), we retrospectively analyzed stored sera for the presence of C'-ADE. No enhancing antibodies were detected in any of the sera tested at six time points prior to the acquisition of HIV-1 (Table 1). C'-ADE were detected at week 3 after infection at an endpoint reciprocal dilution titer of 405 and a maximum power of enhancement of 11.2, which occurred at the lowest serum dilution tested (1:45) (Table 1). Such antibodies persisted but generally at lower levels when measured throughout the subsequent 70 weeks of infection (Table 1). By contrast, the power of C'-ADE was either low (
