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. Author manuscript; available in PMC: 2010 May 15.
Published in final edited form as: J Immunol. 2009 May 15;182(10):6587–6599. doi: 10.4049/jimmunol.0900317

Adenovirus vector-induced immune responses in nonhuman primates: responses to prime boost regimens1

Nia Tatsis *,#, Marcio O Lasaro *,#, Shih-Wen Lin *,†,#, Zhi Q Xiang *, Dongming Zhou *, Lauren DiMenna *, Hua Li *, Ang Bian *, Sarah Abdulla *, Yan Li *, Wynetta Giles-Davis *, Jessica Engram ‡, Sarah J Ratcliffe §, Guido Silvestri ‡, Hildegund C Ertl *,∥, Michael R Betts
PMCID: PMC2711537  NIHMSID: NIHMS123595  PMID: 19414814

Abstract

In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with pre-existing neutralizing antibodies against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine's efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee derived Ad (AdC) vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the AdC vectors induced higher T and B cell responses than repeated immunizations with the AdHu5 vector especially in AdHu5-pre-exposed macaques.

Keywords: Vaccination, AIDS, T cells

Introduction

The quest for an efficacious vaccine to the human immunodeficiency (HIV)-1 virus continues despite a large number of theoretical and practical obstacles. Given the current inability to generate broadly reactive HIV envelope-specific neutralizing antibodies, the majority of current HIV-1 vaccine candidates focus on eliciting protective CD8+ T cell responses (1). In a recent phase IIb clinical trial, termed STEP trial, the most promising of such vaccines, an E1-deleted adenovirus (Ad) vector of the human serotype 5 (AdHu5) not only failed to protect, but instead showed a trend to render male volunteers with pre-existing neutralizing antibodies (NA) to the vaccine carrier more susceptible to infection (2). The negative result of the STEP trial has raised considerable doubts about the validity of the concept of CD8+ T cell-mediated protection against HIV-1 infection (3,4). In addition, the STEP trial has triggered intense studies aimed at identifying the mechanisms underlying the vaccine's “facilitating” effect on HIV-1 transmission linked to the presence of pre-existing anti-AdHu5 antibodies (5).

To circumvent the effects of NAs on the vaccine carrier in individuals that are infected during childhood with human Ad viruses such as AdHu5 (6), we developed E1-deleted Ad vectors from chimpanzee serotypes (AdC) (7,8). We derived these vectors from several AdC serotypes to allow for booster immunization with heterologous AdC vectors. The molecular organization and basic biology of AdC viruses are similar to that of human Ad viruses (9,10). In mice and nonhuman primates (NHPs) AdC vectors were shown to induce robust transgene product-specific T and B cell responses (7,8,11). Most importantly, NAs to AdC viruses are rarely detected in humans (12), thus these vectors may outperform AdHu5 vectors in clinical trials.

Here, we compared two AdC-HIV-1 gag vectors (AdC6 and AdC7 serotypes) in an alternating boost protocol to a dual immunization with an AdHu5-HIV-1 gag vector in rhesus macaques that had or had not been pre-exposed to AdHu5. The results show that heterologous booster immunizations with the AdC vectors induces markedly higher gag-specific T and B cell responses compared to repeated immunization with the AdHu5 vector and that responses to the AdC vectors, unlike those to the AdHu5 vector, are not impaired by pre-existing NAs to AdHu5.

Materials and Methods

Adenovirus vectors

The vaccine vectors express a codon-optimized gag of HIV-1 clade B. Ad vectors were derived from the human serotype 5 (AdHu5), and chimpanzee serotypes 6 (AdC6) or 7 (AdC7). Vectors were E1-deleted and generated from viral molecular clones by viral rescue on HEK 293, grown, purified, titrated and quality controlled as described (8)

Non-Human Primates (NHP)

Two to three year-old Chinese origin Macaca mulatta were purchased and housed at Bioqual, Inc. (Maryland, MD). All procedures involving handling and sacrifice of animals were performed according to approved protocols.

Isolation and preservation of lymphocytes

Peripheral blood mononuclear cells and lymphocytes from tissues were isolated as described. They were tested immediately after isolation by enzyme-linked immunospot (ELISpot) assays. Remaining cells were frozen in 90% FBS and 10% dimethyl sulfoxide (Sigma, St. Louis, MO) at −80°C.

Micro neutralization assay for adenovirus-specific neutralizing antibodies (NA)

NA titers were determined as described (11) on HEK 293 cells infected with Ad vectors expressing GFP.

ELISA for HIV gag antibodies

The ELISA assays were conducted on plates coated with HIV gag protein as described (13).

Synthetic peptides

HIV clade B consensus sequence Gag peptides, 15-mers overlapping by 11 amino acids, were obtained from the NIH Research and Reference Reagents Program.

ELISpot

The ELISpot assays for IFN-γ and IL-2 were conducted as described (13). Spots were counted using the C.T.L. Series 3A Analyzer and ImmunoSpot 3.2 (Cellular Technology Ltd, Cleveland, OH). The minimum spot size was set to 0.0016 mm2, and the maximum spot size was set to 0.0878 mm2. The criteria for determining positive samples included that for every 106 cells stimulated with peptides, at least 55 spots after subtraction of background spots (spots without antigenic stimulation) had to be detected. The number of spots upon peptide stimulation had to be at least 3 times higher than that seen in control wells. Data shown on graphs represent values of peptide-stimulated wells from which background values have been subtracted.

Intracellular Cytokine Staining (ICS)

Frozen cells were thawed and immediately washed with HBSS supplemented with 2 units/ml DNase I, resuspended with RPMIc and stimulated for 6 hrs with anti-CD28, anti-CD49d, and Brefeldin A (10 μg/ml each), with or without 1 μg/ml/peptide of the gag HIV-1 peptide pools at 37°C 5% CO2. After incubation, cells were stained with Violet-fluorescent reactive dye-Pacific Blue (Invitrogen, Carlsbad, CA), anti-human (h) CD14-Pacific Blue, anti-hCD16-Pacific Blue, anti-hCD8-PerCP-Cy5.5, anti-hCD95-PE-Cy5, and anti-hCD28-Texas Red (BeckmanCoulter, Fullerton, CA) for 30 min at 4°C. After fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences, San Jose, CA) for 20 min at 4°C, cells were stained with anti-hIFN-γ-APC, anti-hIL-2-PE, anti-hTNF-a-PE-Cy7 and anti-hCD3-APC-Cy7 for 30 min at 4°C. Cells were washed twice, fixed with BD Stabilizing Fixative (BD Biosciences, San Jose, CA), and then analyzed by FACS using LSRII (BD Biosciences, San Jose, CA) and DiVa software. Post-acquisition analyses were performed with FlowJo (TreeStar, Ashland, OR). Single color controls used CompBeads Anti-Mouse Ig, k (BD Biosciences, San Jose, CA). Unless otherwise noted, antibodies were purchased from BD (BD Biosciences, San Jose, CA).

Cell sorting

Cells from immunized NHPs were thawed and washed with HBSS supplemented with 2 units/ml DNase I, then stained as described for ICS. Cells were sorted by a FACSVantage SE using DiVa software (both BD Biosciences, San Jose, CA) in a BSL3 laboratory (University of Pennsylvania).

Detection of Ad vector genome

Genomic DNA was extracted as described (14). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified by real-time PCR. Samples were adjusted to equal amounts of GAPDH (106 copy) and then hexon sequences from AdHu5, AdC6 and AdC7 were amplified by PCR (30 cycles of 95°C for 40 seconds, 50°C for 40 seconds, and 72°C for 40 seconds) using primers that distinguished between the viruses. The amplicon (1 μL) from the first PCR product was used as template for a nested PCR (30 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 30 seconds). The following primers were used for first PCR: AdHu5: 5’-ATCATGCAGCTGGGAGAGTC’, 5’-ACACCTCCCAGTGGAAAGCA, AdC6: 5’-ATCGGTCTTATGTACTAC, 5’-GTCCATGGGGTCCAGCGACC, AdC7: f 5’-AGGTACAGATGACAGTAGCTC. The following primers were used for the nested PCR: AdHu5: 5’-GACTCCTAAAGTGGTATTGT-3’,5’-GTCTTGCAAATCTACAACAG-3’. AdC6: 5’-TCCCAGCTGAATGCTGTG-3’, 5’-GCCGTCCAAGGGGAAGCAAT-3’. AdC7: 5’-ACAGACCCAACTACATTGGC-3’, 5’-GATTCCACATACTGAAATACC-3’. Amplicons were checked on 1% agarose gels, in most samples no specific band could be detected after the first PCR. The amplicons (1 μl) from the first PCR product were used as templates for a second real-time PCR (40 cycles at 95°C for 5 seconds, 52°C for 10 seconds, 72°C for 12 seconds, and 83°C for 4 seconds). The amplicon was run on a 1% agarose gel and samples thatshowed a band of the expected size were viewed as being positive. Some of the band were sequenced to confirm the specificity of the reaction. For some experiments the copy numbers of hexon in each sample were quantified by normalization in comparison to GAPDH sequences as described (14).

Statistic analysis

Significance in ICS was determined by two-tailed Student t-tests and ANOVA analysis. ELISpot statistical analyses were conducted using two-tailed Student t-tests or Wilcoxin rank-sum tests, as appropriate. All analyses were conducted using SAS 9.0.

Results

Immunization regimens

Two experiments involving a total of 28 juvenile Chinese rhesus macaques (Macaca mulatta) divided in four groups of animals were conducted as part of this study. The four experimental groups each contained seven animals, of which six received an HIV-gag immunization protocol and one control animal that was vaccinated with the same vaccine vector containing an unrelated transgene (Table 1). All of the animals were pre-screened for lack of NAs to the Ad vector vaccines. Prior to the HIV-gag immunization phase, half of the macaques were immunized with 1011 vps of an AdHu5 vector expressing alpha 1 anti-trypsin (A1AT) given intratracheally. NA titers to AdHu5 were measured two weeks later from serum. Animals with titers below 1:10 were re-immunized with 1011 vps of AdHu5A1AT and thereafter seroconverted. In the first experiment, six AdHu5 pre-exposed and six non pre-exposed animals were immunized with 1011 vps of AdC6gag, then boosted at three month intervals with 1011 vps of AdC7gag, followed by 1011 vps of AdC6gag, followed by 1011 vps of AdC7gag. All vaccines were given intramuscularly (i.m.). The control animals received the same vaccine carriers expressing the rabies virus glycoprotein using the same regimen. In the second experiment, six AdHu5 pre-exposed and six non pre-exposed animals were immunized i.m. with 1011 vps of AdHu5gag vector. They were boosted 3 months later i.m. with the same dose of AdHu5gag vector. Control animals received an AdHu5 vector expressing the rabies virus glycoprotein.

Table 1.

NHP immunization regimen.

NHP ID (gender) Immunizations*
Pre-exposure†† (Ab Titera) Prime Boost 1b Boost 2c Boost 3d
2 (M) AdHu5 (1:80) AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
3 (M) AdHu5 (1:40) AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
4 (M) AdHu5 (1:80) AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
5 (F) AdHu5 (1:40) AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
6 (M) AdHu5† (<1:10) AdC6rabgp AdC7HIVgag AdC6HIVgag AdC7HIVgag
7 (M) AdHu5† (1:80) AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
8 (M) AdHu5 (1:320) AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
9 (F) - AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
10 (M) - AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
11 (F) - AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
12 (M) - AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
13 (M) - AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
14 (F) - AdC6HIVgag AdC7HIVgag AdC6HIVgag AdC7HIVgag
15 (F) - AdC6rabgp AdC7HIVgag AdC6HIVgag AdC7HIVgag
31 (M) AdHu5† (1:40) AdHu5HIVgag AdHu5HIVgag - -
32 (M) AdHu5† (1:160) AdHu5HIVgag AdHu5HIVgag - -
33 (F) AdHu5 (1:40) AdHu5HIVgag AdHu5HIVgag - -
34 (M) AdHu5 (1:80) AdHu5HIVgag AdHu5HIVgag - -
35 (M) AdHu5 (1:20) AdHu5HIVgag AdHu5HIVgag - -
36 (M) AdHu5 (1:20) AdHu5HIVgag AdHu5HIVgag - -
37 (M) AdHu5 (1:160) AdHu5rab.gp AdHu5rab.gp - -
38 (M) - AdHu5HIVgag AdHu5HIVgag - -
39 (F) - AdHu5HIVgag AdHu5HIVgag - -
41 (F) - AdHu5HIVgag AdHu5HIVgag - -
42 (F) - AdHu5HIVgag AdHu5HIVgag - -
43 (F) - AdHu5HIVgag AdHu5HIVgag - -
47 (F) - AdHu5HIVgag AdHu5HIVgag - -
54 (F) - AdHu5rab.gp AdHu5rab.gp - -
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*

All immunizations were given intramuscularly at 1011 vps of the indicated vector.

†

Due to low titers these animals received a second exposure to AdHu5 4 weeks after the first exposure.

††

Pre-exposure to AdHu5A1T1 occurred 9 weeks prior to priming.

a

Titers to AdHu5 virus were measured 2 weeks before priming.

b

16 weeks post-prime.

c

30 weeks post-prime.

d

42 weeks post-prime.

For both experiments, after each dose of vaccine, plasma and PBMC samples were collected in 2−4 week intervals. Between two to three months after the last immunization animals were euthanized and lymphocytes were isolated from tissues.

Magnitude and Kinetics of T cell Responses to HIV-1 Gag

T cell responses to gag peptide pools were tested by IFN-γ and IL-2 ELISpot assays (Figures 1A and B) as described (13). By week ten after immunization with the AdC6gag vector, IFN-γ responses were positive (>55 spots/106 PBMCs) in most animals and had reached peak frequencies. After booster immunization with AdC7gag, all animals developed detectable IFN-γ responses within four weeks and in most animals, responses peaked by week six. After the third and fourth immunizations peak frequencies (Figure 1C) remained below those obtained after the first boost. The response to the AdHu5gag vector came up more rapidly in non-pre-exposed animals, most of which developed peak frequencies of IFN-γ producing T cells within two weeks after immunization. Responses were higher in the non-pre-exposed than in the AdHu5 pre-exposed group. The second immunization with the AdHu5gag vector increased responses in some of the animals; this increase was largely transient. Statistical analyses revealed no significant differences in the IFN-γ response of non-exposed and AdHu5 pre-exposed AdC-immunized (p=0.904), whereas responses in the AdHu5gag vaccinated groups were significantly affected by pre-exposure to AdHu5 (p<0.001).

Figure 1. ELISpot for IFN-γ and IL-2 response in NHP.

Figure 1

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Animals were bled at 2 or 4 week intervals, and PBMCs were isolated and stimulated with gag peptide pools for IFN-γ (A) and IL-2 (B) ELISpot analyses. Lymphocytes were stimulated in 3 replicate wells, and spots in control wells were subtracted from experimental wells before plotting. Data from individual animals are plotted, and arrows below the x-axis denote the time of vaccination. Peak frequencies of IFN-γ (C) and IL-2 (D) ELISpots observed after each vaccination are also plotted for individual animals. White bars represent animals that had been pre-exposed to AdHu5, and black bars represent animals that had not been pre-exposed to AdHu5.

Most of the animals developed IL-2 responses after the initial AdC6gag immunization and all of them became positive after booster immunization with AdC7gag. Further booster immunizations were relatively ineffective (Figure 1D). After the first immunization with the AdHu5gag vector, only a fraction of the animals developed low frequencies of IL-2 producing T cells; these responses declined in all animals to below 55 spots/106 PBMCs by week 14 after immunization. After the boost, some animals had positive IL-2 responses, which in most disappeared by week six. Overall there was no significant difference in the IL-2 response of pre-exposed and non-pre-exposed animals in the AdC (p=0.668) and the AdHu5 (p=0.92) vaccinated animals, although the response was higher and more sustained in animals vaccinated with the AdC vectors (p=0.010). PBMCs from control animals were tested in parallel in both experiments, and responses were below 55 spots for IFN-γ or IL-2/106 PBMCs at all time points tested (data not shown).

Frequencies of IFN-γ and IL-2 producing CD8+ and CD4+ T cells were analyzed by ICS (Figures 2A-D), and were in general comparable to those obtained by ELISpot. Gag-specific CD8+ T cells (Figure 2A,B) were dominated by cells producing IFN-γ (Figure 2A) and frequencies were, at most time points tested, higher in animals immunized with the AdC vectors than the AdHu5 vector. The booster effects after repeated immunization with the AdC vectors were more pronounced than seen by ELISpot. A detectable CD4+ T cell response was elicited only in AdC immunized animals (Figure 2C,D). After the first and second immunization, CD4+ T cells produced mainly IL-2 (Figure 2D) while the third and fourth immunizations resulted in induction of CD4+ T cells producing IFN-γ in the AdC immunized groups.

Figure 2. ICS for IFN-γ and IL-2 in CD4+ and CD8+ T cells from NHP.

Figure 2

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The same NHP samples shown in Figure 1 were tested by ICS for expression of CD3 and CD8 and for production of IFN-γ and IL-2. The symbols for individual animals are the same as in Figure 1. (A) shows percentages of CD8+CD3+ cells positive for IFN-g/ all CD8+ CD3+ cells; (B) shows percentages of CD8+CD3+ cells positive for IL-2 / all CD8+ CD3+ cells; (C) shows percentages of CD8−CD3+ cells positive for IFN-g/ all CD8− CD3+ cells; (D) shows percentages of CD8−CD3+ cells positive for IL-2 / all CD8− CD3+ cells. The graphs show the sum of responses to the different peptide pools, background responses (responses without peptides) and responses of control animals (both of which were < 0.1%) were subtracted.

To test if the effectiveness of booster immunizations correlated with neutralizing antibody (NA) titers to the vaccine carrier, plasma from animals was tested before each boost for neutralizing antibodies to the vaccine carrier (Figure 3). As expected after each immunization, NA titers to the vaccine carrier increased. Unexpectedly, the primary antibody response to AdC7 was less pronounced than that to AdC6. There was no clear correlation between the increase of T cell responses following booster immunization and titers of NAs to the vaccine carrier at the time of vaccination.

Figure 3. Neutralizing antibody responses to Ad vectors.

Figure 3

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Plasma samples were tested at the indicated time points for neutralizing antibodies to the Ad vectors. The symbols for individual animals are identical to those shown in Figure 1. (A-C) show titers in plasma of animals immunized with the AdC vectors. (D) shows titers in plasma of animals immunized with the AdHu5 vector. (A) shows titers to AdC6; (B and D) show titers to AdHu5, (C) shows titers to AdC7. There was no statistical difference (two-tailed t-test) between anti AdHu5 titers prior to vaccination in the pre-exposed NHPs that were then vaccinated with AdC or AdHu5 vectors (p=0.19). There was a significant difference after the first vaccine dose between pre-exposed and non pre-exposed AdHu5 vaccinated animals (p=0.03).

Gag-Specific Antibody Responses

Antibody titers to gag were measured after each vaccine dose. The AdC6gag vector induced a gag-specific antibody response in all of the NHPs that increased after the AdC7gag boost (Figure 4). Additional booster immunizations with AdC6gag and AdC7gag did not cause any further increase. Responses in AdHu5 pre-exposed and non pre-exposed NHPs were comparable in both AdC immunized groups (p>0.05, two tailed Student t-test) at all time points tested. AdHu5gag vectors only induced a moderate antibody response to gag that did not increase after the second dose. In AdHu5 pre-exposed NHPs, the antibody response to gag presented by AdHu5 vectors was significantly lower than in non-pre-exposed NHPs (p<0.02 at all time points). After the second immunization, titers in both groups (pre-exposed and non-pre-exposed) were significantly higher in AdC vaccinated animals (p=0.005 for pre-exposed NHPs, p=0.036 for non pre-exposed NHPs).

Figure 4. Gag-specific antibody response.

Figure 4

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Plasma samples were tested at the indicated time points for binding antibodies to gag. Samples were tested in duplicates; standard deviations (not shown) were below 10% of the mean. The first booster immunization caused a significant increase in titers in the AdC groups (p=0.001, one-tailed t-test), the 2nd and 3rd booster immunizations did not cause further significant increases. In the AdHu5 groups the 1st booster immunization did not cause a significant increase of titers in pre-exposed (p=0.07, note that titers decreased) or the non pre-exposed (p=0.13) animals. The differences in titers between the pre-exposed and non-pre-exposed AdC groups were statistically not significant at any of the time points. The differences in titers between pre-exposed and non pre-exposed AdHu5 vaccinated animals were statistically significant at both time points (p=0.02 and 0.001). The differences between titers of pre-exposed or non pre-exposed animals that received AdC or AdHu5 vectors were not significantly different after the first immunization (p=0.08 for pre-exposed animals and 0.39 for non pre-exposed animals) but reached significance after the 2nd immunization (p=0.0051 for pre-exposed animals, p=0.036 for non pre-exposed animals.

Magnitude of T Cell Responses in Tissues

After euthanasia, lymphocytes were isolated from various tissues and tested for responses to gag peptides by ELISpot and ICS. Responses to gag were detectable in all AdC immunized animals in blood, spleen, and liver. For most AdC-immunized animals responses were comparable in blood and spleen, low in lymph nodes (data not shown), and as reported previously, high in liver (13). Responses in tissues were low in the AdHu5 immunized animals. Lymphocytes isolated from the intestine or the genital tract had high background activity and these results could not be interpreted (data not shown).

IL-2 responses were overall lower than IFN-g responses (Figure 5A, B). In most of the AdC vector immunized animals, IL-2 responses were correlated between blood, spleen and liver. A few animals had high IL-2 responses in iliac lymph nodes (data not shown). AdHu5 immunized animals had low IL-2 responses at the time of euthanasia.

Figure 5. ELISpot response in tissues.

Figure 5

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Between 2−3 months after the final immunization, all animals were euthanized and lymphocytes were isolated from the blood and tissues including the spleen and liver, shown here. Lymphocytes were stimulated with gag peptide pools for IFN-γ (A) and IL-2 (B) ELISpot analyses. Lymphocytes were stimulated in 3 replicate wells, and spots in control wells were subtracted from experimental wells before plotting. The vaccine regimen of each group of animals is noted at the top of each column. Data from the tissues of individual animals are plotted. White bars represent animals that had been pre-exposed to AdHu5, and black bars represent animals that had not been pre-exposed to AdHu5.

ICS analyses showed that most of the IFN-γ was derived from CD8+ T cells (Figure 6A-D), although some animals also had IFN-γ+CD4+ T cells in various compartments. IL-2 producing CD4+ and CD8+ T cells were detected at low frequencies in blood of non pre-exposed AdC immunized animals, but not in PBMC samples from the other groups. A fraction of the AdC immunized animals had IL-2 producing CD8+ or CD4+ T cells in their spleen, most had detectable frequencies of IL-2 producing CD8+ T cells in liver, and some had hepatic IL-2 producing CD4+ T cells. Conversely, IL-2 producing CD4+ or CD8+ T cells could not be detected in most AdHu5gag immunized animals.

Figure 6. ICS response in tissues.

Figure 6

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Lymphocytes isolated from blood, spleens and liver were tested by ICS for expression of CD3 and CD8 and for production of IFN-γ and IL-2. (A) shows percentages of CD8+CD3+ cells positive for IFN-γ / all CD8+ CD3+ cells; (B) shows percentages of CD8+CD3+ cells positive for IL-2 / all CD8+ CD3+ cells; (C) shows percentages of CD8−CD3+ cells positive for IFN-γ / all CD8−CD3+ cells; (D) shows percentages of CD8−CD3+ cells positive for IL-2 / all CD8−CD3+ cells. The graphs show the sum of responses to the different pools, background responses (responses without peptides) and responses of control animals (both of which were < 0.1%) were subtracted.

Neither the IFN-γ nor IL-2 responses in the individual tissues tested reached statistical significance when comparing the pre-exposed and non-pre-exposed groups of animals receiving either vector (p>0.05 for each tissue). Overall, the IFN-γ responses induced in the liver of animals vaccinated with the chimpanzee vector regimen were slightly higher than in the liver of animals vaccinated with the AdHu5 vaccine regimen (p=0.044). Also, at necropsy the IL-2 responses induced in the blood, liver and spleen of animals vaccinated with the chimpanzee vector regimen were higher than in these tissues of animals vaccinated with the AdHu5 vaccine regimen (p=0.009, p=0.002, and p=0.003, respectively).

In summary, the response in tissues was dominated by CD8+ T cells producing IFN-γ in AdC immunized animals regardless of pre-exposure to AdHu5, and these responses were more robust than in AdHu5 immunized animals. CD4+ T cell responses to the transgene product were only detected in AdC vaccinated NHPs.

Characteristics of cytokine secreting T cells from AdC immunized animals

Gag-specific T cells isolated from blood at two time points (week six and ten) after each immunization were characterized in more depth from three of the NHPs (# 3, 9, 11) that mounted high responses to AdC immunization (Figure 7A). One of the animals (#3) had been pre-exposed to AdHu5, while the other two had not. PBMCs were stained with antibodies to CD3, CD8, CD95 and CD28 to determine frequencies of effector (CD95+CD28−) and memory (CD95+CD28+) T cells (Figure 7A line graphs) to gag. Effector CD8+ T cells were more common than memory CD8+ T cells throughout the course of the immunizations in NHPs #9 and #11, while NHP #3 had slightly higher levels of memory CD8+ T cells. Memory CD8+ T cell cytokine production profiles in blood varied between the animals but were remarkably stable through the course of vaccination in each of the animals (Figure 7A pie charts). NHPs # 3 and #11 had mainly memory CD8+ T cells that produced IFN-γ or IFN-γ and TNF-α, although CD8+ T cells that produced IL-2 or all three cytokines could also be detected; NHP #9 had higher frequencies of CD8+ T cells producing TNF-α or IL-2.

Figure 7. Cytokine expression profile of gag-specific CD8+ T cells.

Figure 7

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(A) PBMC isolated from immunized NHPs at an early (week 6) and late (week 10) time point after each immunization were stimulated in vitro with gag peptide pools and production of IFN-γ, IL-2 and TNF-a were analyzed by ICS. Cytokine co-expression profiles (pie charts) were determined in memory (CD95+CD28+) CD8+ T cells for NHPs #3 (1st row), #9 (2nd row) and #11 (3rd row). IFN-γ+/IL-2+/TNF-a+, light blue; IFN-γ+/IL-2+/TNF-a−, red; IFN-γ+/IL-2−/TNF-a+, yellow; IFN-γ+/IL-2−/TNF-a−, green; IFN-γ−/IL-2+/TNF-a+, dark blue; IFN-γ−/IL-2+/TNF-a−, orange; and IFN-γ−/IL-2−/TNF-a+, brown. Percentage of cytokine-producing CD8+ T cells over total CD8+ T cells (line graphs) were determined for effector (CD95+CD28−, black squares) and memory (CD95+CD28+, black triangles), CD8+ T cells. Lymphocytes isolated from spleen (B and C) and liver (D and E) of immunized NHPs were stimulated in vitro with gag peptide pools and production of IFN-γ, IL-2 and TNF-a were analyzed by ICS. (B) and (D), cytokine co-expression profiles for effector (1st row) and memory (2nd row) CD8+ T cells were determined for NHP #3, 7, 9, 11. IFN-γ+/IL-2+/TNF-α+, light blue; IFN-γ+/IL-2+/TNF-α−, red; IFN-γ+/IL-2−/TNF-α+, yellow; IFN-γ+/IL-2−/TNF-α−, green; IFN-γ−/IL-2+/TNF-α+, dark blue; IFN-γ−/IL-2+/TNFα−, orange; and IFN-γ−/IL-2−/TNF-α+, brown. (C) and (E), Percentage of cytokine-producing CD8+ T cells over total CD8+ T cells were determined for effector (black bars) and memory (dotted grey bars) from NHPs #3, 7, 9 and 11.

Spleen (Figure 7B,C) and liver (Figure 7D,E) derived CD8+ T cells from the three NHPs described above, as well as from NHP #7, which had been pre-exposed to AdHu5, were also analyzed to determine frequencies of CD95+CD28− (effector) and CD95+CD28+ (memory) T cells (Figure 7C,E) and their cytokine production (Figure 7B,D) in response to gag. In spleens and liver frequencies of effector and memory CD8+ T cells were roughly comparable; NHPs #3 and 11 had in both compartments slightly higher frequencies of memory cells while NHP #9 had slightly higher frequencies of effector cells. Effector and memory CD8+ T cells to gag from spleens and liver of all 4 NHPs produced mainly IFN-γ TFN-α or IFN-γ and TNF-α. CD8+ T cells that produced IL-2 alone or together with IFN-γ or that produced all 3 cytokines were mainly found in the memory subset. There was no clear difference in subset distribution or the cytokine production profile between pre-exposed and non pre-exposed animals.

A similar analysis was conducted for CD3+CD8− cells (CD4+ T cells) (Figure 8). Throughout the course of the immunizations, animal #11 had mainly circulating gag-specific CD95+CD28+ memory CD4+ T cells, and comparatively lower levels of the more activated CD95+CD28− effector CD4+ T cells. In contrast, NHP #3 had comparable levels of both subsets. In NHP #9, the ratio of effector to memory CD4+ T cells fluctuated. In addition, CD4+ T cells were stained for intracellular IFN-γ, IL-2 and TNF-α (Figure 8A pie charts). Gag-specific CD4+ T cells from these three animals differed in their cytokine production. NHPs #3 had a very mixed profile with CD4+ T cells that produced two or all three cytokines being frequent. NHP #9 had a most time point a predominance of CD4+ T cells producing either TNF-α or IL-2. Most CD4+ T cells from NHP #11 produced either IL-2 or TNF-α. In all the latter two animals, CD4+ T cells that produced only one cytokine were more common than those positive for two or three cytokines.

Figure 8. Cytokine expression profile of gag-specific CD4+ T cells.

Figure 8

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The same NHP samples shown in Figure 7 were tested by ICS for CD3+ and CD8− (CD4+). The symbols and colors are the same as in Figure 7. (A) Cytokine co-expression profiles (pie charts) were determined in memory (CD95+CD28+) CD4+ T cells for NHPs #3 (1st row), #9 (2nd row) and #11 (3rd row). Percentage of cytokine-producing CD4+ T cells over total CD4+ T cells (line graphs) were determined for effector (CD95+CD28−, black squares) and memory (CD95+CD28+, black triangles) CD4+ T cells. Lymphocytes isolated from spleen (B and C) and liver (D and E) were stimulated in vitro with gag peptide pools and production of IFN-γ, IL-2 and TNF-a were analyzed by ICS. (B) and (D), Cytokine co-expression profiles for effector (1st row), and memory CD4+ T cells (2nd row) were determined for NHPs #3, 7, 9, 11. Cytokine profile production colors and symbols are the same as shown in Figure 7. (C) and (E), percentage of cytokine-producing CD4+ T cells over total CD4+ T cells were determined for effector (black bars), snd memory (grey dotted bars) CD4+ (i.e., CD8−CD3+ cells) T cells from NHPs #3, 7, 9 and 11.

Spleen (Figure 8B,C) and liver (Figure 8D,E) derived T cells from the three NHPs described above, as well as from NHP #7, which had been pre-exposed to AdHu5, were also analyzed to determine frequencies of CD95+CD28− (effector) and CD95+CD28+ (memory) T cells (Figure 7C,E) and their cytokine production (Figure 8B,D) in response to gag. In spleens NHPs #3, #7 and #9 had predominantly gag-specific memory CD4+ T cells, while NHP #11 had approximately equal numbers of memory and effector CD8 T cells. Effector CD4+ T cells produced mainly IFN-γ or IFN-γ and TNF-α. Frequencies of CD4+ T cells producing more than one cytokine were higher in memory CD4+ T subset. Frequencies of IL-2 producing memory CD4+ T cells were higher in pre-exposed than in the non pre-exposed animals.

In the liver of all of the NHPs gag-specific memory CD4+ T cells were more frequent than gag-specific effector CD4+ T cells. Effector CD4+ T cells producing only IFN-γ predominated and those producing only TNF-α or IFN-γ and TNF-α were also common. As in spleens memory CD4+T cells showed a mixed profile with substantial frequencies of CD4+ T cells producing 2 or 3 cytokines. Subset distribution and cytokine profiles in blood were poorly predictive for those in tissues.

Levels of cytokine production were analyzed from blood-derived T cells at various times after each immunization and from spleens and liver (Figure 9). In general, CD8+ (Figure 9A) or CD4+ (Figure 9B) T cells positive for two or three cytokines produced markedly higher levels of IFN-γ and/or IL-2 than those that were only positive for one cytokine.

Figure 9. Levels of cytokine production by gag-specific memory T cells producing three cytokines were higher than those producing only one cytokine.

Figure 9

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Lymphocytes isolated from liver, spleen and blood of immunized NHPs were stimulated in vitro with HIV-1 gag peptide pool. The mean fluorescence intensity (MFI) of IFN-γ (first row), IL-2 (second row) and TNF-α (third row) produced by CD28+CD95+CD8+ (A) and CD28+CD95+CD4+ (B) T cells were analyzed by ICS. Triple+ (black circles) and double+ (gray squares) cytokine producers were compared to single+ (white triangles) cytokine producers. *, triple+ cytokine producers were statistically different than single+ cytokine producers (p<0.05). When MFI axes were segmented The dotted grey line shows where the Y axis was segmented.

Biodistribution of vector sequences

Lymphocytes and tissue fragments of spleens, liver and intestine were analyzed for vector sequences using primers specific for the hexon coding sequence of the different Ad vectors. As shown in Table 2, most samples were positive for the Ad vectors used for immunization. Additional studies on sorted lymphocytes showed that vector sequences were mainly present in CD8+ T cells (Table 2). Previous mouse studies had shown that Ad vectors preferentially persist in activated CD8+ T cells responding to the Ad vector's transgene product (14). During ICS analyses of lymphocytes from AdC immunized animals, we detected a population of IFN-γ producing CD8+ T cells that was CD95low or CD95intermediate (Figure 10A), which is unexpected for antigen-induced T cells. Upon sorting of CD8+ T cells isolated from the blood of NHP #11 harvested at the time of euthanasia into CD95low, CD95 intermediate or CD95high populations, most of the vector sequences were detected by real-time PCR in the CD95low population (Figure 10B).

Table 2.

Biodistribution of Ad vectors sequences.

NHP # Spleen AdC6/AdC7 Liver AdC6/AdC7 Small Intestine AdC7
Tissue Lymphocytes Tissue Lymphocytes Lymphocytes
2 NT/NT +/+ NT/NT +/+ +
3 +/+ +/+ −/+ −/+ −
4 +/+ +/+ +/+ +/+ +
5 +/+ +/+ +/+ +/+ +
6 +/+ NT/NT +/+ NT/NT NT
7 NT/NT +/+ NT/NT +/+ NT
8 +/+ +/+ +/+ +/+ NT
9 +/+ +/+ +/+ +/+ +
10 −/+ +/+ +/+ +/+ +
11 +/+ +/+ +/+ +/+ +
12 −/+ +/+ +/+ +/+ +
13 +/+ +/− +/+ +/+ +
14 +/+ +/+ +/+ +/+ +
15 +/+ −/+ +/+ +/+ NT
NHP # AdHu5 AdHu5 AdHu5
Tissue Lymphocytes Tissue Lymphocytes Lymphocytes
2 NT + NT + NT
4 + + − + NT
6 + + + NT NT
7 + + + + +
8 + + + + +
31 + + + + −
32 + + + + +
33 + + + + +
35 + + − + −
36 − + − + +
37 + + + + NT
42 + + + + −
43 − + − + NT
47 + + + + +
54 + + + + +
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NT = Non-tested

Figure 10. Presence of Ad vector sequences in CD95 expressing CD8+ T cells.

Figure 10

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(A) IFN-γ producing CD8+ PBMCs (red) from NHP immunized with AdC were plotted with total CD8+ PBMCs (black/gray) for expression of CD95. CD8+ PBMCs were sorted into CD95low, CD95intermediate (int) and CD95high cells and (B) presence of specific AdC6 (open bars) and AdC7 (black bars) vector genome sequences, i.e. hexon, were determined by real-time PCR.

Discussion

The HIV-1 pandemic continues virtually unabated and by now is thought to have caused the death of over 22 million people. It is estimated that each day an additional 14,000 humans become infected, mainly in developing countries, which cannot afford to provide anti-retroviral therapy. For these reasons, an efficacious vaccine to HIV-1 is direly needed to stem the HIV-1 epidemic. Preventative vaccines to most viruses are designed to elicit protective neutralizing antibody (NA) responses. Unfortunately, the high diversity and structural plasticity of the HIV-1 envelope protein, its potential for extensive glycosylation and the structural changes that occur upon receptor binding have defied the development of a vaccine that elicits protective titers of broadly cross-reactive NAs to HIV-1 (15,16). Vaccine efforts have thus largely concentrated on induction of cellular immune responses to relatively conserved antigens of HIV-1 (17-19). However, the first large scale clinical trial of a CD8+ T cell inducing vaccine to HIV-1, the STEP trial, showed that an AdHu5 based HIV vaccine that induced a robust CD8+ T cell response to conserved antigens of HIV-1 was not only unable to prevent disease, but may have increased the risk of transmission in individuals with pre-existing moderate to high-titer NAs to AdHu5.

Our work has focused on candidate HIV-1 vaccines based on AdC vectors to which humans in the US and Asia lack NAs, while low titers of such antibodies are detectable in small percentages of humans residing in Central Africa (12). The rationale behind the development of these AdC based vaccines was to circumvent the reduction of vaccine antigen-specific immune responses in individuals with pre-existing NAs to human serotypes of Ad virus such as AdHu5. In the US, ∼40% of human adults carry NAs to AdHu5 while in some countries of Asia and Africa 80−90% of adults have such antibodies (12).

In this study, we tested two AdC vectors, AdC6 and AdC7 expressing gag of HIV-1, in a heterologous prime boost regimen, in comparison to an AdHu5 vector given twice in a homologous prime boost regimen. In both immunization protocols, the animals were divided into naïve and AdHu5 pre-exposed cohorts. The results of this study clearly show that the AdC heterologous prime boost regimen induces higher frequencies of gag-specific T cells in blood and tissues and higher titers of gag-binding antibodies in blood compared to a repeated immunization with the AdHu5gag vector. In addition, pre-existing immunity to AdHu5 reduced the antibody and CD8+ T cell response to gag presented by the AdHu5 vector without affecting responses to the AdC vectors. Memory CD4+ T cell responses have been implicated in previous NHP studies to play a major role in protection against challenge with simian immunodeficiency virus (20). In our study animals developed higher and more sustained CD4+ T cell responses to AdC immunization than upon immunization with AdHu5. We assume that this reflects in part the distinct biology of different Ad vectors that, as we and others reported previously, also affects activation of innate responses (21,22) which in turn influences activation of the adaptive immune system.

Studies from HIV-1 infected humans have shown that protection against disease progression is not linked to T cell frequencies but to T cell functionality, with T cells producing multiple cytokines being more protective compared to those secreting only one cytokine (23,24). The role of such subsets in vaccine-induced protection remains unknown. The AdC immunization regimen resulted in a multifaceted T cell response. In two of the three animals tested, most circulating CD8+ T cells belonged to effector T cell subsets, while in one animal memory CD8+ T cells predominated in blood. Circulating CD8+ T cells produced mainly IFN-γ or TNF-α or both. It was remarkable that the profile of cytokine production showed limited variability during the lengthy course of the experiment in individual animals although there was pronounced variability between animals. In spleens and tissues, most of the NHPs tested had both memory and effector CD8+ T cells that secreted mainly IFN-γ or TNF-α or both. IL-2 producing CD8+ T cells and CD8+ T cells that produced two or three cytokines were more common in memory CD8+ T cell subsets than in the more activated effector subset. Circulating CD4+ T cells, which belonged to the memory subset, showed a diverse cytokine profile of IFN-γ, IL-2, TNF-α or combinations thereof in only one NHP. In the other 2 NHPs the response was more restricted to T cells producing only one of the cytokines at most time point tested. In spleens and livers CD4+ T cells belonged mainly to the memory cell subset but for those from spleens of NHP #11, which also had high frequencies of effector-like CD4+ T cells. As was seen for CD8+ T cells, the effector CD4+ T cells produced mainly one cytokine, i.e., IL-2, IFN-γ or TNF-α while memory CD4+ T cells showed a more diverse profile with subsets producing one, two or three cytokines. CD4+ and CD8+ T cells from liver and spleen that were positive for two or three cytokines produced significantly higher levels of these cytokines compared to those that were positive for only one cytokine, suggesting that induction of these subsets by a vaccine may be highly desirable. Neither CD8+ nor CD4+ T cells in blood showed a clear shift in subset distribution or cytokine profiles during the course of immunization as had been shown previously in mice (25,26), nor were these parameters affected by pre-immunization to AdHu5 virus.

We previously reported that Ad vectors persist in mice and NHPs at low levels, mainly in activated CD8+ T cells directed to the Ad encoded transgene product (14). Again in this study we could detect Ad vector sequences in all of the animals in liver, spleen and intestine as well as in lymphocytes derived from these tissues. Upon analysis of T cell subsets we observed in blood and spleens three populations of CD8+ T cells producing IFN-γ that could be distinguished by levels of CD95 expression. Activated T cells express high levels of CD95, which facilitates their apoptosis (27); the presence of antigen-induced CD8+ T cells expressing only low or intermediate levels of CD95 upon Ad vector vaccination was therefore unexpected. Further analyses showed that vector sequences could mainly be detected in the CD95low population. The E3 encoded receptor internalization and degradation (RID) complex of Ad viruses can internalize CD95 and facilitate vector persistence (28,29), and we suggest that the same mechanism may in part explain persistence of E1-deleted Ad vectors. The long term persistence of the Ad genome that as we showed previously in mice (14) remains transcriptionally active presumably allows for the exceptionally sustained transgene product-specific T cell responses upon Ad vector immunization. Many of these T cells remain activated at the effector or effector memory stage rather than transitioning into central memory. Whether vaccine-induced effector-like CD8+ and CD4+ T cells are advantageous to prevent an infection with HIV-1 by providing an immediate layer of defense or detrimental by providing targets to HIV-1 remains to be investigated.

Vectors based on replication-defective AdHu5 were viewed as the most promising candidates for prevention of HIV-1 infections, as they were found to elicit potent and sustained T cells responses to their transgene product. Although their immunogenicity was attenuated in individuals with pre-existing NAs to AdHu5, early clinical trials yielded sufficiently promising results to advance an AdHu5-based HIV-1 vaccine into a phase II trial (STEP trial), designed to assess vaccine efficacy in volunteers with or without NAs to AdHu5 at high risk to contract HIV-1. The vaccine was found to lack efficacy in reducing HIV-1 acquisition or post-infection viral load, which raises the question if the concept of vaccine-induced T cell-mediated protection against HIV-1 infection is flawed. Available evidence argues that this is unlikely. Surrogate challenge models in NHPs show that the solid protection induced by attenuated simian immunodeficiency virus (SIV) against challenge with a virulent heterologous SIV is linked to T cell responses (30-33). Even more importantly, in humans control of HIV-1 infection can be mediated by CD8+ T cells (34-36). One could thus argue that the AdHu5 vaccine used in a homologous prime boost regimen failed by not inducing a potent enough immune response, especially in individuals with pre-existing NAs to the vaccine carrier, which may be overcome by heterologous prime boost regimens such as the one described here.

Although the first highly immunogenic HIV-1 vaccine based on AdHu5 vectors failed to induce protective immunity (37,38), more innovative vaccine regimens based on different vaccine platforms that allow for heterologous prime boost regimens are expected to induce even more potent and potentially functionally distinct immune responses to HIV-1 that may yet provide T cell mediated protection against HIV-1 infections of humans.

Acknowledgments

We thank the flow facilities of the Wistar Institute and University of Pennsylvania for assistance with flow cytometry and cell sorting, Mark Lewis at Bioqual for his assistance with the nonhuman primates, and Christina Cole and Colin Barth (Wistar Institute, Philadelphia, PA) for preparation of the manuscript.

Footnotes

Publisher's Disclaimer: This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.

1

This work was sponsored by NIH grant P01AI052271 to HCE, NIH grant U19AI074078 to MRB, NIH grant U19 AI074078 to GS and institutional grants to the Wistar Institute including an US National Cancer Institute Cancer Core Grant (CA10815), and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

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