Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers

Vaccine 25 (2007) 510–518

Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers夽 Paul A. Goepfert a,∗ , Georgia D. Tomaras b , Helen Horton c , David Montefiori b , Guido Ferrari b , Mark Deers c , Gerald Voss d , Marguerite Koutsoukos d , Louise Pedneault d , Pierre Vandepapeliere d , M. Juliana McElrath c , Paul Spearman e,1 , Jonathan D. Fuchs f , Beryl A. Koblin g , William A. Blattner h , Sharon Frey i , Lindsey R. Baden j , Clayton Harro k , Thomas Evans l,2 , the NIAID HIV Vaccine Trials Network a

j

University of Alabama at Birmingham, 908 20th Street South, CCB 328, Birmingham, AL 35294, United States b Duke University Medical Center, Duke, NC, United States c Fred Hutchinson Cancer Research Center and University of Washington, Seattle, WA, United States d GlaxoSmithKline Biologicals, Rixensart, Belgium e Vanderbilt University School of Medicine, Nashville, TN, United States f San Francisco Department of Public Health, San Francisco, CA, United States g The New York Blood Center, New York, NY, United States h Institute of Human Virology, University of Maryland at Baltimore, United States i Saint Louis University School of Medicine, United States Division of Infectious Disease, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, United States k Center for Immunization Research, Johns Hopkins University, Baltimore, MD, United States l University of Rochester, Rochester, NY, United States Received 4 April 2006; received in revised form 7 July 2006; accepted 25 July 2006 Available online 10 August 2006

Abstract Background: Use of the recombinant proteins NefTat and gp120W61D formulated with the AS02A adjuvant system was previously shown to protect against AIDS in a rhesus macaque SHIV animal model system. Methods: Eighty-four HIV uninfected human participants were vaccinated intramuscularly at 0, 1, and 3 months and evaluated for safety. Immune responses were analyzed for the presence of vaccine-induced antibody and T lymphocyte responses. Results: The vaccines were safe and well tolerated at all doses. Nef-, Tat-, and gp120-specific binding antibodies were induced in all individuals that received the respective antigen, lasting up to 9 months after the final immunization. Antibodies able to neutralize the T-cell laboratory-adapted strain of HIV-1W61D were detected in the majority of vacinees, but did not neutralize primary isolates. Envelopespecific antibody-dependent cell cytoxicity was detected in most of the individuals receiving gp120. Robust and persistent HIV-specific lymphoproliferative responses were detected against all subunit proteins in the majority of immunized participants. As expected, HIV-specific CD8 T-cell responses were not detected. Conclusions: Despite the lack of primary isolate neutralizing antibody induction, the observed high frequency and magnitude of other immune responses warrant further work with this vaccine or vaccine components. © 2006 Elsevier Ltd. All rights reserved. Keywords: HIV vaccine; Clinical trial; Nef; Tat; gp120; AS02A adjuvant 夽 ∗ 1 2

Part of this study was previously presented at the AIDS Vaccine 2003 Conference in New York City, 19 September 2003. Corresponding author. Tel.: +1 205 975 5667; fax: +1 205 975 5718. E-mail address: [email protected] (P.A. Goepfert). Present address: Emory University School of Medicine, Atlanta, GA, United States. Present address: Novartis Institute of Biomedical Research, Cambridge, MA, United States.

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.07.050

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1. Introduction A preventative vaccine is urgently needed for the continuing global human immunodeficiency virus type 1 (HIV-1) pandemic in which at least 40 million individuals are infected worldwide (December 2005 UNAIDS/WHO estimates). Initial efforts focused on the use of recombinant monomeric envelope surface glycoprotein (gp120) as the immunogen [1–4]; however, neutralizing antibodies elicited to these products were highly strain specific [5], and humoral responses alone are not likely to protect against HIV infection [6]. An alternative approach has focused on eliciting CD8+ T lymphocyte (CTL) responses [7,8]. However, the current CTL-based vaccines do not protect against infection in animal models of HIV [9,10] and only variably protect against progression to AIDS [11]. Furthermore, the CTL-based vaccines studied to date in humans have not induced these intended responses in all recipients [12–14]. Clearly, other vaccine approaches, inducing both humoral and cellular responses, are necessary. One strategy to overcome strain-specific immunity of the current subunit protein vaccines would be to include a greater number of antigens that may be conserved among several viral strains. Such an approach may broaden the antibody, CD4 T helper lymphocyte, and CTL responses, and target a greater number of HIV-1 strains. It may also be beneficial to direct the immune responses against viral proteins that are expressed early in the replication cycle [15]. One such protein, Tat, was shown to induce apoptosis and also inhibit antigen-specific lymphoproliferation, thereby possibly contributing to viral pathogenesis. The presence of Tat-specific antibodies [16] and CTL [17] in chronically infected patients was demonstrated to correlate inversely with HIV-1 progression. Vaccines using Tat as an immunogen have also been demonstrated to protect against AIDS in macaques [18,19] although the sole use of this immunogen may not confer protection [20]. Nef, another protein expressed early in the viral life cycle, serves multiple functions and is likely to contribute to viral pathogenesis by downregulating CD4 and MHC class I on the surface of infected cells (reviewed in [21]). Nef-defective simian immunodeficiency viruses (SIV) are highly attenuated in rhesus macaques [22]. Additionally, Nef-deleted HIV-1 appeared to result in a delayed progression to AIDS in several patients [23], and Nef-specific immune responses have correlated with AIDS protection [24,25]. In view of these factors, Nef could be an important component of a multi-antigen vaccine. GlaxoSmithKline Biologicals (GSK Bio) has developed a multi-antigen subunit protein AIDS vaccine formulated in the proprietary adjuvant AS02A. The preclinical vaccine was composed of gp120W61D , a NefTat fusion protein, and SIV Nef. This product, when given to rhesus macaques, conferred solid protection against the development of AIDS after challenge with a partially heterologous SHIV [26]. This strong protection against SHIV-induced disease was only observed

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when all four subunit proteins were given in the AS02A formulation. The present study extends this multi-component concept into the first clinical evaluation of vaccine safety and the immunogenicity of gp120W61D and NefTat fusion protein (NefTat/gp120W61D ) formulated in the AS02A proprietary adjuvant system. We demonstrate that this multi-antigen subunit vaccine is safe, well tolerated, and highly immunogenic when given to healthy, HIV seronegative adults.

2. Materials and methods 2.1. Vaccine NefTat is comprised of Nef (derived from HIVLAI ) and Tat (derived from HIVBH10 ) expressed as a single fusion protein by recombinant technology in Pichia pastoris yeast cells. The recombinant gp120W61D is a truncated form of the envelope protein of HIV-1 isolate W61D, produced in a CHO cell line. Twenty micrograms of NefTat alone or combined with 5, 20, or 100 ␮g of gp120 were lyophilized and reconstituted with the adjuvant AS02A prior to injection. The AS02A adjuvant consists of 50 ␮g of QS-21 and 50 ␮g of 3-deacylated monophosphoryl lipid A (3D-MPL) in an oil-in-water emulsion. Five hundred microliters of this adjuvant preparation was used for final vaccine administration. 2.2. Study participants Male and female participants aged 18–60 at low risk of acquisition of HIV infection were recruited [1–3]. After participants provided individual institution informed consent, a history, physical examination, and general safety laboratories were performed. Standard eligibility criteria were used for enrollment into the study [12]. 2.3. Study design This study (HVTN 041) was a multicenter, double-blind, randomized trial conducted at 10 HIV Vaccine Trials Units sponsored by the National Institutes of Allergy and Infectious Diseases. All groups received one intramuscular injection in the deltoid at months 0, 1, and 3. Ten participants received 20 ␮g of NefTat formulated with AS02A adjuvant. Sixty received 20 ␮g of NefTat with AS02A combined with either 5, 20, or 100 ␮g of gp120W61D (20 participants received each dose). Fourteen participants received the AS02A adjuvant alone (placebo group). Participants self-reported systemic and local reactions on days 1 and 2 after injection. Those with reactions other than mild were seen in clinic within 48 h of reporting. The severity of a reaction or adverse event was defined as follows: (1) mild: transient or minimal symptoms; (2) moderate: notable symptoms requiring modification of activity; and (3) severe: incapacitating symptoms requiring bed rest and/or loss of work or social activities. The clinical

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and laboratory safety data were reviewed weekly during the vaccination period by a group that included physicians, nurses, statisticians, and study monitoring personnel. Data on adverse events were collected for 18 months of study [2,13,14]. Risk reduction counselling was performed at every study visit. 2.4. Immune assays 2.4.1. CTL CD8+ CTL responses were evaluated using standard antigen-specific in vitro stimulation (IVS) and 51 Cr-release assays (CRA) [27]. The HIV recombinant vaccinia viruses used for IVS did not perfectly match the vaccine proteins but were all derived from subtype B sequences. Assessments of specific CTL activity were carried out 14–18 days after antigen-specific stimulation, using autologous 51 Crlabeled target cells infected with the appropriate recombinant vaccinia constructs. Effector cell populations were determined by depletion with MAb-coated magnetic beads (Dynal, Oslo, Norway), as previously described [27]. When vaccinia-infected target cells were used, unlabeled BLCL infected with the vaccinia control vector served as cold target competitors for anti-vaccinia reactivity in the CTL assay. Percent of specific lysis and positive results were calculated according to published methods [27]. 2.4.2. IFN-γ ELISpot A set of 15 amino acid peptide overlapping by 11 residues was synthesized matching the original sequence of the Nef, Tat, and gp120 proteins included in the vaccine. The peptides were synthesized and HPLC purified (>90%) by Anaspec (Anaspec Inc., San Jose, CA, USA). The peptides representing the gp120 were divided in three pools containing 50 peptides each. The peptides representing the NefTat fusion protein were divided in two pools of 50 and 25 peptides representing the Nef and Tat protein, respectively. The IFN-␥ ELISpot kit (Becton Dickinson, San Jose, CA) was used according to the manufacturer’s directions. PBMC were thawed in R10 (RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 25 mM HEPES [N2-hydroxyethylpiperazine-N -2-ethanesulfonic acid] buffer, 50 ␮g/ml streptomycin, 50 U/ml penicillin) containing 50 U/ml Benzonase (Novagen, Madison, WI, USA), washed, and rested overnight in R10 at 37 ◦ C, 5% CO2 before assay. PBMC were plated at 2 × 105 cells per well. Peptides were added to the wells (in triplicate) at a final concentration of 1 ␮g/ml. Wells containing medium alone (in six replicate wells) and triplicate wells containing 1 ␮g/ml phytohemagglutinin (PHA)-P (Murex, distributed by Remel, Lenexa, KS) served as negative and positive controls, respectively. The next day, the plates were developed and spots counted using a CTL Analyzer and software version 2.8 (CTL Analyzers LLC, Cleveland, OH, USA). Positivity was defined as greater than 55 spot forming cells (SFC)/106 PBMC and greater than four times the individual background control.

2.4.3. ELISA A qualitative assessment of the presence of vaccine specific antibodies was performed on cryopreserved serum. Positive serum samples were further subjected to a quantitative end-point assay to determine binding antibody titers. Six serial dilutions (3–7-fold) of serum or plasma beginning at 1/100 were tested in duplicate in microtiter plates coated with either purified gp120W61D produced in CHO cells (GSK, Rixensart, Belgium), Nef or Tat (all produced in P. pastoris yeast cells, GSK, Rixensart, Belgium). For each assay, duplicate antigen-containing and non-antigen-containing wells were set up for each serum and two negative and two HIV+ positive control sera were utilized. A score (i.e., OD antigen–OD non-antigen) with the background subtracted was regarded as positive if it exceeded 0.2. The serum titration optical density values were averaged at each serum dilution and the average plotted as a function of serum dilution. Titers were defined as the reciprocal serum dilutions that yield 50% maximum binding of a standard positive control replicated on each assay. 2.4.4. Neutralizing antibodies Neutralization was measured as a function of reductions in luciferase reporter gene expression after a single round of virus infection in TZM-bl cells as previously described [28,29]. Briefly, cell free virus (200 TCID50 ) was incubated with serial dilutions of test samples in triplicate in a total volume of 150 ␮l for 1 h at 37 ◦ C in 96-well plates. Freshly trypsinized cells (10,000 cells in 100 ␮l of growth medium containing 75 ␮g/ml DEAE dextran and 2.5 ␮M indinavir) were added to each well. One set of control wells received cells + virus (virus control) and another set received cells only (background control). After a 48 h incubation, 100 ␮l of cells was transferred to 96-well black solid plates (Costar) for measurements of luminescence using Bright Glo substrate solution as described by the supplier (Promega). Neutralization titers are the dilution at which relative luminescence units (RLU) were reduced by 50% compared to virus control wells after subtraction of background RLUs. 2.4.5. Antibody-dependent cellular cytotoxicity (ADCC) Natural killer resistant (NKR) cells [30] were incubated with gp120W61D and 51 Cr for 90 min and then washed three times. PBMCs were isolated from HIV seronegative donors and incubated with target cells for 6 h at an effector to target ratio (E:T) of 33:1. Cryopreserved immune serum was tested using a dilution of 1/2500 in triplicate. The % specific lysis (SL) was determined to be positive if %SL with serum was >15% SL without serum. 2.4.6. Lymphoproliferative assay (LPA) PBMC were suspended in RPMI with 10% human AB serum and added to 96-well microtiter plates at 1 × 105 cells per well in triplicate for each antigen/mitogen. PBMC were stimulated with proteins matching the vaccine (gp120, Nef, and Tat). PHA and medium alone were used as positive and

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negative controls, respectively. The cultures were maintained at 37 ◦ C in a 5% CO2 incubator. On the sixth day, the cells were pulsed with 3 H-thymidine and harvested after 16–18 h. The geometric mean cpm of 3 H-thymidine incorporation for each set of replicates is ascertained. A stimulation index (SI) (i.e., the ratio of stimulated to medium control values) was calculated. An SI value above three was considered positive. 2.5. Statistical analysis 2.5.1. Safety For local and systemic reactogenicities, overall differences between groups were tested using the two-sided nonparametric Kruskal–Wallis Test. Overall differences between the number of participants who experienced at least one adverse event and those who experienced none were tested using the two-sided Fisher’s Exact Test. Differences were considered to be statistically significant if p ≤ 0.05. 2.5.2. Immunogenicity For categorical data, pairwise differences between vaccines were tested using the two-sided Fisher’s Exact Test, where differences were considered to be statistically significant if p ≤ 0.05. Net response rates were also computed and considered to be statistically significant if the exact 95% confidence interval did not include zero. For continuous data, pairwise differences between vaccine groups were tested using the two-sided nonparametric Wilcoxon Rank Sum Test, where differences were considered to be statistically significant if p ≤ 0.05. Within vaccine groups, nonparametric Spearman correlation coefficients between continuous variables were computed. Correlations are considered to be statistically significant if p ≤ 0.05 and the correlation coefficient ≥0.70.

3. Results 3.1. Participant accrual, demographic data, and vaccine safety Of the 84 participants enrolled, the majority of participants were non-Hispanic white (74%) males (68%) with a median age of 34 (range 19–60). All participants received their first vaccination, 83 received the second, and 79 the third. Overall, the vaccinations were well tolerated and none were discontinued because of vaccine-associated reactogenicity. The most common local symptoms were pain and tenderness (Fig. 1). Induration was observed more commonly in groups II and III (NefTat and 5 or 20 ␮g of gp120W61D , respectively) (p = 0.03). Systemic symptoms were generally mild with the exception of moderate myalgias and headache occurring in 20 and 25%, respectively. Overall, no statistically significant differences in systemic side effects were observed between groups. All vaccine-related reactogenicities were transient and had either improved or resolved within 48 h of onset.

Fig. 1. Comparison of local and systemic symptoms among the vaccine groups. Cumulative percent of local and systemic symptoms graded as mild, moderate, or severe are demonstrated for recipients receiving the different vaccine preparations (groups I–IV) and for the summation of all vaccinations (total). Data represent any local or systemic symptoms over all three vaccinations for each volunteer. Group I, NefTat alone; group II, NefTat + 5 ␮g of gp120W61D ; group III, NefTat + 20 ␮g of gp120W61D ; group IV, NefTat + 100 ␮g of gp120W61D ; group V, placebo control. All groups received AS02A adjuvant and the NefTat dose was 20 ␮g.

Grade 3 or 4 laboratory abnormalities were observed in only one participant in group II. This participant had an elevated CPK value which may have been due to creatine supplements taken prior to exercise and likely not vaccine related. There were six adverse events during the trial that were felt to be probably or definitely due to the immunizations. These included two severe local site reactions, two occurrences of malaise (one severe), a febrile illness (103.2 ◦ F), and an episode of urticaria. All of these adverse events significantly improved within 24 h of onset. Major social harms were not detected during this trial. 3.2. Immunogenicity 3.2.1. Humoral responses Two weeks after the second immunization, all vaccine recipients possessed Nef and Tat-specific antibody responses, as measured by ELISA, which were maintained in the great majority at 9 months after the final immunization (Fig. 2A–C). Nef, Tat, and gp120 antibody titers peaked 2 weeks after the final immunization in all vaccine groups (exception for group II where Tat antibody titers peaked 2 weeks after the second vaccination) and remained elevated throughout the remainder of the study. All individuals vaccinated with gp120/NefTat had antibodies that neutralized the viral strain corresponding to the gp120 contained within the vaccine (laboratory adapted strain of W61D) (Fig. 2D). These neutralizing titers peaked at 2 weeks post the third dose and were maintained at the final time point. Additionally, the magnitude of antibodies neutralizing the vaccine strain isolate correlated directly with binding antibodies (r = 0.89; p < 0.01 for groups II–IV combined at day 98). In contrast to these findings, neutralizing antibodies directed against the HIV-1W61D primary isolate and a standard panel of clade B reference strains [31] were not detected in any subject. Antibody-dependent cellular cytotoxicity (ADCC) is another mechanism whereby antibodies can neutralize virus

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P.A. Goepfert et al. / Vaccine 25 (2007) 510–518 Table 1 Antibody-dependent cellular cytotoxicity Group

Response rate (%)

Percentage of specific lysisa

p-valueb

I II III IV V

0/9 (0) 10/15 (67) 11/16 (69) 10/15 (67) 0/11 (0)

1.7 19.3 15.9 18.8 1.3

NA <0.01 <0.01 <0.01 NA

Group I = 20 ␮g NefTat; group II = 20 ␮g NefTat + 5 ␮g gp120; group III = 20 ␮g NefTat + 20 ␮g gp120; group IV = 20 ␮g NefTat + 100 ␮g gp120; group V = ASO2A adjuvant alone. a Median percent specific lysis for all subjects in the group. b Wilcoxon ranked sum compared with groups I or V.

[32]. The lysis of gp120-coated CEM.NKR is a direct manifestation of ADCC, in which cytophilic antibodies specific for gp120 direct the lysis of target cells by arming the Fc receptorbearing Natural Killer (NK) effector cells. The majority of gp120W61D recipients had detectable ADCC responses (Table 1). We also noted that binding antibody titers tended to correlate with ADCC lysis (r = 0.44; p < 0.01 for groups II–IV combined at day 98).

Fig. 2. Vaccination induced durable HIV-specific antibody responses. The antibody binding titers as measured by ELISA specific for gp120 (A), Nef (B), or Tat (C) are shown for the various time points throughout the trial. The neutralizing antibody assay was performed using the TCLA of HIV1W61D as described in the methods (D). Group I, NefTat alone; group II, NefTat + 5 ␮g of gp120W61D ; group III, NefTat + 20 ␮g of gp120W61D ; group IV, NefTat + 100 ␮g of gp120W61D . All groups received AS02A adjuvant and the NefTat dose was 20 ␮g. Arrows represent vaccination time points.

3.2.2. Cellular responses Although we did not expect a subunit protein vaccine approach to induce CTL responses, Tat is known to be able to penetrate cells [33], and there is evidence that this process does not depend on endocytosis [34]. It is therefore possible that Tat, when fused to Nef, can induce CTL responses to these components. However, standard chromium-release assay following 14 days of antigen stimulation did not reveal HIV-specific CTL responses at day 98, with the exception of Nef-specific reactivity in one subject (data not shown). Similarly, for the vaccine groups there were no statistically significant IFN-␥ ELISpot HIV-specific responses (data not shown). A lymphocyte proliferation assay was performed to further evaluate the vaccine-induced T-cell responses. HIV-specific LPA was seen in the majority of vaccine subjects at the earliest time point measured (day 98 or 2 weeks after the last vaccination), which persisted until day 364 (39 weeks post final vaccination) (Table 2). These responses were directed against all HIV proteins contained within the vaccine (Fig. 3). There also appeared to be decreased gp120W61D -specific LPA responses in group IV (p < 0.05 when compared to groups II and III at post vaccination time points) suggesting a dampening effect with a higher dose of the glycoprotein. Supernatants were taken at the end of the LPA assay and analyzed for IFN-␥ and IL-5 to determine whether there was a skewing towards a TH1 or TH2 type response. The majority of samples, obtained from vaccine recipients, contained both IFN-␥ and IL-5 (data not shown). Analysis was performed at baseline (day 0), and 2 weeks (day 98), 11 weeks (day 168), 26 weeks (day 270), and 39 weeks (day 364) following the third and final vaccination. Group V (AS02A alone) did not have detectable HIVspecific antibody responses.

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Table 2 Lymphoproliferative response rates to vaccine-specific HIV-1 proteins Day

Group

gp120 Response rate

Nef (%)a

Tat

p-valuesb

Response rate

(%)a

p-valuesb

Response rate (%)a

p-valuesb

0

I II III IV V

0/4 (0) 0/4 (0) 0/3 (0) 0/5 (0) 0/2 (0)

NA NS NS NS NA

0/4 (0) 0/4 (0) 0/3 (0) 1/5 (20) 0/2 (0)

NA NS NS NS NA

0/4 (0) 0/4 (0) 0/3 (0) 0/5 (0) 0/2 (0)

NA NS NS NS NA

98

I II III IV V

0/9 (0) 15/15 (100) 16/17 (94) 12/18 (67) 0/9 (0)

NA <0.01 <0.01 <0.01 NA

9/9 (100) 10/15 (67) 13/17 (76) 15/18 (83) 1/9 (11)

<0.01 0.01 <0.01 <0.01 NA

8/9 (89) 3/15 (20) 6/17 (35) 8/18 (44) 0/9 (0)

<0.01 NS 0.06 0.03 NA

168

I II III IV V

0/8 (0) 15/16 (94) 17/18 (94) 9/17 (53) 0/11 (0)

NA <0.01 <0.01 <0.01 NA

7/8 (88) 13/16 (81) 16/18 (89) 15/17 (88) 2/11 (18)

0.01 <0.01 <0.01 <0.01 NA

4/8 (50) 10/16 (63) 12/18 (67) 14/17 (82) 0/11 (0)

0.02 <0.01 <0.01 <0.01 NA

364

I II III IV V

0/9 (0) 16/18 (89) 17/18 (94) 10/17 (54) 0/13 (0)

NA <0.01 <0.01 <0.01 NA

8/9 (89) 16/18 (89) 14/18 (78) 13/17 (76) 1/13 (8)

<0.01 <0.01 <0.01 <0.01 NA

7/9 (78) 13/18 (72) 12/18 (67) 11/17 (65) 1/13 (8)

<0.01 <0.01 <0.01 <0.01 NA

Day 98, 168, and 364 represent time points following the third vaccination at 2, 11, and 39 weeks, respectively; group I = 20 ␮g NefTat; group II = 20 ␮g NefTat + 5 ␮g gp120; group III = 20 ␮g NefTat + 20 ␮g gp120; group IV = 20 ␮g NefTat + 100 ␮g gp120; group V = ASO2A adjuvant alone. a Positive responses were defined as an SI of >3. b Wilcoxon ranked sum compared with group V.

4. Discussion The multi-component recombinant HIV vaccine investigated in this study, NefTat/gp120W61D , was safe and well tolerated in healthy human participants when given in combination with the AS02A adjuvant. The latter finding is significant since a prior HIV vaccine study demonstrated that QS-21 (a component in AS02A) was not well tolerated in which five volunteers had vasovagal symptoms [4]. The majority of volunteers in the current trial did have local injection site symptoms that were mild-to-moderate in severity. In addition, systemic symptoms were also generally mild and transient. Neither vaccine-induced toxicity nor vasovagal symptoms due to pain were identified in this study. Furthermore, there were no side effects that could be directly attributable to the NefTat/gp120W61D antigens themselves (i.e., over adjuvant alone). One possible explanation for the better tolerability of this vaccine was the 10-fold lower dose of QS-21 used in this trial compared to the prior HIV vaccine trial [4]. The main justification for this study stems from the ability of this vaccine strategy to result in improved SHIV control after heterologous viral challenge in a rhesus macaque animal model system [26]. Because of the nature of this vaccine (i.e., subunit proteins), we did not anticipate the induction of CD8 T-cell responses but hypothesized that robust HIV-specific CD4 T-cells and antibodies would be seen. Indeed, the candidate vaccine was highly immunogenic, eliciting CD4 T-cells and binding antibodies to all vaccine components, and as

expected, CD8 T-cell responses were not induced. It should be noted that the Nef reagent used to analyze potential Nefspecific CTL was not identical to the vaccine component. As such some responses may not have been detected, however, this is unlikely to be a major reason for the lack of CTL observed in our study in view of the fact that Nef CTL are highly cross-reactive [35]. Although the antibodies were of high titer and were maintained during the entire study period, they were not capable of neutralizing primary viral isolates. Interestingly, ADCC responses were detected in the majority of vaccine recipients, and these vaccine-elicited antibodies may even have other important antiviral functions that were not explored here [36]. HIV-specific antibodies capable of ADCC were shown to inversely correlate with viral replication in both HIV and SIV macaque model systems [37,38], therefore, it is encouraging that the current vaccine elicited HIV-specific ADCC in the majority of individuals. Recent work has also demonstrated that these types of responses correlate with protection in an animal model system [39]. However, this optimism must be tempered with the fact that the ADCC assay in our study utilized monomeric gp120, and this might not reflect reactivity against native Env glycoprotein spikes on the virus surface. Nef and Tat antibody titers were of high magnitude and, the latter were able to bind different viral subtypes including A, B, C, D, and E (data not shown). The discrepant CD4 T-cell responses obtained with the LPA and IFN-␥ ELISpot could be due to the fact that

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Fig. 3. Vaccination induced HIV-specific CD4 T helper responses to all components. LPA was performed using Nef, Tat, and gp120 for stimulation of PBMC as described in methods. The average stimulation index (SI) for each group is shown at the indicated time points for PBMC stimulated with gp120 (A), Nef (B), or Tat (C) proteins. Group I, NefTat alone; group II, NefTat + 5 ␮g of gp120W61D ; group III, NefTat + 20 ␮g of gp120W61D ; group IV, NefTat + 100 ␮g of gp120W61D ; group V, placebo control. All groups received AS02A adjuvant and the NefTat dose was 20 ␮g. An SI of 3 was used as the cutoff value for a positive response (grey dashed line). Analysis was performed at baseline (day 0), and 2 weeks (day 98), 11 weeks (day 168), and 39 weeks (day 364) following the third and final vaccination. The standard deviation is represented by error bars (positive values only).

the vaccine-induced CD4 helper T-cells secreted IL-2 and not IFN-␥ and would not be detected using the IFN-␥ ELISpot assay alone [40]. The detection of durable CD4 Tcell responses induced by this vaccine was encouraging as both animal models and human studies indicate the need for CD4 helper responses in the induction and maintenance of CD8 T-cells [41–43]. Nonhuman primate HIV vaccine animal models also demonstrated a direct correlation between the frequency of virus-specific CD4 T-cells and control of viremia post infection [44–46]. Finally, studies have demonstrated the presence of robust CD4 T-cell responses and lymphoproliferative responses in long-term nonprogressors that are virtually absent in patients with progressive infection [47–49]. It seems likely that the AS02A adjuvant made a significant contribution to increasing the HIV-specific antibody titer over giving the immunogen alone. A prior trial using this adjuvant did show a benefit over alum adjuvant in terms of eliciting antibodies earlier that are longer lasting [5]. Furthermore,

both protection against malaria [50] and strong Hepatitis B immune responses [51] were previously observed when using this adjuvant system. It is also likely that the responses seen with even the low dose of gp120W61D were likely due to the AS02A adjuvant [4]. While this is the first time that a NefTat fusion protein has been evaluated as a vaccine in healthy adults, the responses seen in this trial were higher than what is observed in natural infection [52] indicating the possibility that adjuvant played a role in these responses as well. Despite the prevailing thought that an effective vaccine will consist of CD8 CTL and/or antibodies that are capable of neutralizing primary viral isolates [7,8], an immune correlate of protection for an effective HIV vaccine is unknown. With the enormous number of new HIV infections occurring daily, we cannot rely on just a few approaches, and multiple strategies for developing an effective HIV vaccine seem warranted. This clinical trial demonstrated that the NefTat/gp120W61D vaccine was safe, well tolerated, and highly immunogenic for the induction of antibodies and CD4 T helper responses. Several of these immune responses correlated with the lack of disease progression in humans or lower viral load set point in the macaque vaccine model system [16–18,24,25]. Furthermore, there is ample evidence to suggest that an effective HIV vaccine will need to induce strong CD4 T helper responses. Although improvements to the current vaccine seem indicated, especially those designed to increase neutralizing antibody responses against primary isolates [53], these findings suggest that NefTat/gp120W61D formulated with AS02A may be a useful component in a therapeutic or preventative HIV vaccine.

Acknowledgements This trial was conducted by the HIV Vaccine Trials Network and sponsored by the National Institute of Allergy and Infectious Diseases. The authors gratefully acknowledge the participation and support of many colleagues and staff at each site. We are particularly grateful for the participation of the 84 study volunteers. We would also like to acknowledge the following investigators who participated in the trial: Mark Mulligan, University of Alabama at Birmingham; Mike Keefer, University of Rochester; Donald Burke, Johns Hopkins University; Robert Belshe and Geoffrey Gorse, Saint Louis University; Connie Celum and Marnie Elizaga, University of Washington; Peter Wright, Vanderbilt University; Scott Hammer, Columbia University; Raphael Dolin, Michelle Lally, and Kenneth Mayer, Harvard University; Susan Buchbinder and Laurence Peiperl, San Francisco Department of Public Health; Farley Cleghorn, University of Maryland; and Kent Weinhold, Duke Laboratory Program. We acknowledge Theresa Shea for excellent handling of safety evaluations during the study, Marianne Hansen for her meticulous administrative support, and Geert Leroux-Roels (University of Ghent) for the cytokine analysis of cell culture supernatants.

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This work was supported by the HVTN as part of a grant from the NIAID. GlaxoSmithKline provided the vaccine products. G.V., M.K., L.P., and P.V. are either employees of GSK or were employees at the time of the study. Conflict of interest statement: None declared.

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