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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Vaccine. Author manuscript; available in PMC Oct 16, 2010.
Published in final edited form as:
PMCID: PMC2955883
NIHMSID: NIHMS135149

Heterologous Prime/Boost Immunizations of Rhesus Monkeys Using Chimpanzee Adenovirus Vectors

1. Introduction

Considerable data have accrued in recent years suggesting that non-replicating adenovirus vectors are potent inducers of cell-mediated immune responses. Most work in this area has been done using adenovirus serotype 5 (Ad5) constructs [13]. Studies done initially in small laboratory animals and then in nonhuman primates indicated that recombinant Ad5 (rAd5) constructs elicit potent and durable CD8+ T lymphocyte responses [46]. These findings led to the development of rAd5-based vaccines for HIV prophylaxis. With promising early phase immunogenicity data in human volunteers, efficacy trials were initiated with these recombinant vaccine constructs.

However, as the clinical trials were being pursued, a number of problems with this vaccine strategy became apparent. First, it became clear that pre-exisiting immunity to Ad5, a common finding in both the developed and developing nations, can diminish the immunogenity of Ad5-based vaccine constructs [710]. Second, and much more troubling, an advanced phase HIV vaccine trial being pursued by a partnership between Merck Research Laboratories and the National Institutes of Health was stopped when data became available indicating that vaccinees with pre-exisiting immunity to Ad5 had an increased incidence of HIV acquisition when compared with control vaccinees [11].

A number of novel adenovirus vectors have been evaluated that might circumvent the problems associated with rAd5 constructs [12, 13]. Rare serotype human adenoviruses are being evaluated as potential vaccine vectors. While such vectors have proven less immunogenic than Ad5, they may prove useful in association with heterologous vectors in prime/boost immunization strategies. A chimeric human adenoviral vector has been created that has the biologic properties of Ad5 but the serologic profile of a rare serotype adenovirus [13]. The immunogenicity of this construct is not diminished by pre-existing anti-Ad5 antibodies in the vaccinees. Finally, replication incompetent chimpanzee adenovirus constructs are being evaluated as vaccine immunogens. Since humans are not infected by these adenoviruses, vaccine constructs created from them should escape neutralization by pre-existing anti-adenovirus antibodies [14].

We have previously shown that two chimpanzee adenovirus-based vectors, AdC7 and AdC68, elicit potent T cell and antibody responses in mice and nonhuman primates [15]. Both of these viruses are closely related to human serotype 4 adenoviruses. Similar to AdHu5 virus, AdC7 and AdC68 viruses use the coxsackie adenovirus receptor (CAR) ([16]), whereas, AdC1/C5 viruses use CD46 as its receptor ([17]). Importantly, the expression of transgene products and the immunogenicity of these constructs were not compromised by anti-Ad5 antibody. The present study was done to compare the immunogenicity in nonhuman primates of two chimpanzee adenovirus-based vectors with human Ad5. Further, we have assessed the use of heterologous replication incompetent chimpanzee adenoviruses as immunogens when used in a prime/boost vaccination regimen.

2. Materials and Methods

2.1. Generation of recombinant viruses

E1-deleted recombinant AdHu5, AdC7 and AdC68 adenoviruses expressed a codon-optimized HIV-1 clade B gp140 (provided by G. Nabel, Vaccine Research Center, NIH). Construction of these vectors has been described previously [15, 18, 19]. E1-deleted recombinant AdHu5 and E1-and E3-deleted recombinant AdC7 and AdC68 adenoviruses expressed a Gag-Pol fusion protein from SIVmac239. Construction of these vectors has been described before [20, 21]. Chimeric rAdC1/C5 vectors expressing HIV-1 gp140, SIVmac239 Gag and rabies glycoprotein were made as described by McCoy et al. [14].

2.2. Immunization and Challenge of Rhesus Monkeys

Twenty four Mamu-A*01-negative Indian-origin rhesus monkeys were housed at New England Regional Primate Research Center of Harvard Medical School, Southborough, MA. The animals were maintained in accordance with National Institutes of Health and Harvard Medical School guidelines. Monkeys were divided into four groups, each consisting of six animals. Immunizations were carried out as described in the Results section. At week 47, 18 weeks following the last boost, all monkeys were challenged with 50 50% monkey infectious doses (MID50) of SHIV-89.6P by intravenous route.

2.3. IFN–γ ELISpot assays

Multiscreen ninety-six well plates were coated overnight with 100µl per well of 5 µg/ml anti-human interferon-γ (IFN-γ) (B27; BD Pharmingen) in endotoxin-free Dulbecco’s-PBS (D-PBS). The plates were then washed three times with D-PBS containing 0.25% Tween-20, blocked for 2 h with D-PBS containing 5% FBS to remove the Tween-20, and incubated with peptide pools and 2 × 105 PBMCs in triplicate in 100 µl reaction volumes. The peptide pool spanning the entire SIVmac239 Gag and SIV Pol proteins comprised of 15 amino acid peptides overlapping by 11 amino acids and that covering the entire HIV-1 clade B Env protein comprised of 15 mers overlapping by 10 amino acids. Each peptide in a pool was present at a 1 µg/ml concentration. Following an 18 h incubation at 37°C, the plates were washed nine times with D-PBS containing 0.25% Tween-20 and once with distilled water. The plates were then incubated with 2 µg/ml biotinylated rabbit anti-human IFN-γ (Biosource) for 2 h at room temperature, washed six times with Coulter Wash (Beckman Coulter), and incubated for 2.5 h with a 1:500 dilution of streptavidin-AP (Southern Biotechnology). After five washes with Coulter Wash and one with D-PBS, the plates were developed with NBT/BCIP chromogen (Pierce), stopped by washing with tap water, air dried, and read with an ELISpot reader (Hitech Instruments) using Image-Pro Plus image-processing software (version 4.1) (Media Cybernetics, Des Moines, IA).

2.4. PBMC stimulation and intracellular cytokine staining

Purified PBMC were isolated from EDTA-anticoagulated blood and frozen in the vapor phase of liquid nitrogen. Cells were later thawed and allowed to rest for 6 h at 37°C in a 5% CO2 environment. The viability of these cells was >90%. PBMCs were then incubated at 37°C in a 5% CO2 environment for 6 h in the presence of RPMI/10% fetal calf serum alone (unstimulated), a pool of 15-mer Gag peptides (2 µg/ml of each peptide; AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Germantown, MD), or staphylococcal enterotoxin B (SEB) (5 µg/ml, Sigma-Aldrich, St. Louis, MO) as a positive control. All cultures contained Monensin (GolgiStop; BD Biosciences) as well as 1 µg/ml of anti-CD28 and anti-CD49d (BD Biosciences). The cultured cells were stained with monoclonal antibodies (mAbs) specific for cell surface molecules including CD3, CD4 and CD8. The antibodies used in this study were directly coupled to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinium chlorophyll protein-Cy5.5 (PerCP-Cy5.5), phycoerythrin-Cy7 (PE-Cy7), AmCyan, Pacific Blue®, allophycocyanin (APC), Alexa Fluor® 700 and Quantum-Dot 605. All reagents were validated and titrated using rhesus monkey PBMC. The following mAbs were used: anti-TNF-α-FITC (MAb11; BD Biosciences, San Jose, CA), anti-IFN-γ-PE-Cy7 (B27; BD Biosciences), anti-CD3-Pacific Blue® (SP34-2; BD Biosciences), anti-IL-2-APC (MQ1-17H12; BD Biosciences), anti-CD8α-Alexa Fluor® 700 (RPA-T8; BD Biosciences), and anti-CD4-Quantum-Dot 605 (unconjugated CD4 antibody was obtained from BD Bioscience, Quantum-Dot 605 was obtained from Invitrogen, Carlsbad, CA). A violet fluorescent reactive dye (ViViD; Invitrogen) was also used as a viability marker to exclude dead cells in the analysis. After fixing with Cytofix/Cytoperm solution (BD Biosciences), cells were permeabilized and stained with antibodies specific for IFN-γ, TNF-α, and IL-2. Labeled cells were fixed in 1% formaldehyde-PBS. Samples were collected on a LSR II instrument (BD Biosciences) and analyzed using FlowJo software (Tree Star). Approximately 200,000 to 1,000,000 events were collected per sample. The background level of cytokine staining varied from sample to sample, but was typically < 0.01% of the CD4+ T cells and < 0.05% of the CD8+ T cells. The only samples considered positive were those in which the percentage of cytokine-staining cells were at least twice that of the background or in which there was a distinct population of cytokine brightly positive cells.

2.5. Neutralizing Antibody Assays

Neutralizing antibodies were measured in TZM-bl cells (NIH AIDS Research and Reference Reagent Program) as described before [22] except that trypsinized cells were added (10,000 cells/well) to the virus serum dilutions and indinavir was added to inhibit progeny virions. A panel of genetically diverse HIV-1 Env-expressing pseudovirions was used in this study, some relatively neutralization sensitive (Tier 1) and others more neutralization resistant (Tier 2). The use of this panel of pseudovirions has previously been described [23]. Titers of neutralizing antibodies are the reciprocal plasma dilution at which relative luminescence units (RLU) were reduced 50% compared to virus control wells (no sample). VIG was utilized as a positive control antibody for all neutralization assays.

2.6. CD4+ T lymphocyte counts and viral RNA levels

CD4+ T-lymphocyte counts were determined by multiplying the total lymphocyte count by the percentage of CD3+ CD4+ T cells determined by monoclonal antibody staining and flow cytometric analysis. Plasma viral RNA levels were measured by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies per ml (Bayer Diagnostics).

3. Results

3.1. Vaccine trial design

Twenty four Indian-origin rhesus monkeys were distributed into three experimental and one control group, each consisting of six animals. On week 0, six monkeys in the first experimental group received priming immunizations with 1011 viral particles (vp) of rAdHu5 expressing HIV-1 clade B Env and 1011 vp of rAdHu5 expressing SIVmac239 Gag-Pol, administered intramuscularly. Six monkeys in the second experimental group were primed by the same route with 1011 vp of rAdC7 expressing HIV-1 clade B Env and 1011 vp of rAdC7 expressing SIVmac239 Gag-Pol. The third experimental group was primed with 1011 vp each of rAdC68 expressing HIV-1 clade B Env and SIVmac239 Gag-Pol. Monkeys in the control group received inoculations with 2×1011 vp of rAd5 expressing rabies virus glycoprotein. At week 29, monkeys in all three experimental groups were boosted with 1011 vp of rAdC1/C5-HIV-1 clade B Env and 1011 vp of rAdC1/C5-SIVmac239 Gag, administered by the intramuscular route. Monkeys in the control group were inoculated with 2×1011 vp of rAdC1/C5 expressing rabies virus glycoprotein.

3.2. Vaccine-elicited cellular and humoral immune responses

The cellular immune responses elicited by these vaccine constructs were assessed using pooled peptide IFN-γ ELISpot assays and intracellular cytokine assays (ICS). As shown in Fig. 1A, vaccine-elicited ELISpot responses to HIV-1 Env and SIV Pol peptides were detected in the PBMC of all experimentally vaccinated monkeys 2 weeks after the first inoculation. Monkeys that were primed with rAdHu5 vaccine constructs showed a response to SIV Gag and higher magnitude IFN-γ responses to HIV-1 clade B Env and SIV Pol antigens than the two groups of monkeys that received immunizations with the chimpanzee adenovirus vectors, rAdC7 and rAdC68. Monkeys receiving immunizations with rAdC7 and rAdC68 had comparable magnitude IFN-γ ELISpot responses to all three antigens tested. But the higher magnitude IFN-γ ELISpot responses seen in rAdHu5-primed monkeys than the other two groups of monkeys were not statistically significant (Mann-Whitney test).

Figure 1
Vaccine-elicited cellular immune responses to SIVmac239 Gag, SIVmac239 Pol and HIV-1 HXBc2 Env (clade B). PBMC were isolated at weeks 2 (Post-prime), 31 (Post-boost) and 47 (Day of challenge) post-immunization and assessed in IFN-γ ELISpot assays ...

At week 29, monkeys in all three experimental groups were boosted with 1011 vp of rAdC1/C5-clade B Env and 1011 vp of rAdC1/C5-SIV Gag. rAdC1/C5 was chosen as a boosting immunogen because AdC1/C5 uses CD46 as receptor and not CAR, which is used by Ad5, AdC7 and AdC68. We reasoned that the use of a genetically disparate viral vector might prove a better boosting immunogen. PBMC IFN-γ ELISpot responses to SIV Gag, SIV Pol and HIV-1 clade B Env peptides were assessed in all 3 groups of animals at week 31, 2 weeks following the boost. Monkeys primed with rAdHu5 had a 4-fold increase in their IFN-γ ELISpot responses to SIV Gag protein, whereas responses to HIV-1 Env were of same magnitudes as observed following the priming immunization (Fig. 1B). Similarly, monkeys primed with either rAdC7 or rAdC68 showed almost 10-fold increases in their IFN-γ ELISpot responses to SIV Gag and a marginal increase in their responses to the HIV-1 Env protein. As SIV Pol was not included in the construct used in the boost, none of these groups of monkeys showed any increase in their responses to SIV Pol (Fig. 1B). Total IFN-γ ELISpot responses in the rAdHu5-primed group were significantly higher than those in the rAdC68-primed group (p=0.03). The difference in magnitude of responses between rAdHu5-primed monkeys and the rAdC7-primed monkeys did not achieve statistical significance (Mann-Whitney test).

These IFN-γ ELISpot responses declined slowly in all groups of experimentally immunized monkeys, and as shown in Fig. 1C, by week 47, eighteen weeks following the last boost, there was a 1.5-2-fold decrease from the peak responses in the total spot forming cell responses. Monkeys in the rAdHu5-primed group had less of a decline in their total ELISpot responses than the groups primed with chimpanzee adenovirus vectors (Fig. 1C) and had significantly higher responses than the rAdC68-primed group (p=0.026) but not significantly different than rAdC7-primed group.

As a pooled peptide ELISpot assay only assesses the secretion of IFN-γ by peptide-stimulated T lymphocytes, we sought to assess secretion of other cytokines by these lymphocytes using a flow cytometry-based intracellular cytokine assay. Two weeks following the boost, peripheral blood lymphocytes from the monkeys were exposed to overlapping peptides spanning the SIV Gag protein, and the fractions of CD4+ or CD8+ T cells producing IFN-γ, IL-2 and TNF-α were determined by intracellular cytokine staining. No statistically significant differences were noted in the frequencies of cytokine-producing CD4+ T lymphocytes between the three groups of vaccinated monkeys (Fig. 2, upper panel). As shown in Fig. 2 (lower panel), the two groups of monkeys that were primed with recombinant chimpanzee adenovirus vectors showed very comparable frequencies of cytokine-producing CD8+ T lymphocytes. However, a trend toward higher frequencies of IFN-γ- and TNF-α-producing CD8+ lymphocytes was seen in monkeys that were primed with rAdHu5 (Fig. 2, lower panel) but these differences did not achieve statistical significance. Due to limitations in lymphocytes available for performing the cytokine analysis, we chose to assess only the SIV Gag-specific responses.

Figure 2
IFN-γ, IL-2 and TNF-α secretion by CD4+ (A) and CD8+ (B) T lymphocytes were measured by intracellular cytokine staining following stimulation of PBMC with peptide pools spanning the SIV Gag protein two weeks following the boost. The bars ...

To assess the vaccine-elicited humoral immune responses, serum samples from the vaccinated and control monkeys were analyzed for HIV-1 Env neutralizing antibodies. No HIV-1 Env-specific neutralization above background was detected in sera of the vaccinated monkeys at the time of peak vaccine-elicited immunity (data not shown).

3.3. Cellular and humoral immune responses following viral challenge

Eighteen weeks after the final immunization all monkeys were challenged with 50 MID50 of cell-free SHIV-89.6P by the intravenous route. Since virus-specific cellular immune responses post-challenge contribute to the early control of viremia, we monitored the relative magnitudes of the virus-specific cellular immune responses in these infected monkeys. Two weeks post-challenge, the magnitudes of the total IFN-γ ELISpot responses were slightly higher in the group of monkeys primed with rAdC68. The rAdHu5- and rAdC7-primed groups had comparable total ELISpot responses (Fig. 3A). The control animals had much lower ELISpot responses. Therefore, although the vaccine-elicited pre-challenge peak and plateau immune responses were greater in the group of monkeys primed with rAdHu5 than in the groups receiving immunizations with the recombinant chimpanzee adenovirus vectors, the post-challenge peak secondary responses were comparable in magnitude in all 3 groups of experimentally vaccinated animals.

Figure 3
PBMC IFN-γ ELISpot responses in the vaccinated and control monkeys following viral challenge. Freshly isolated PBMC were assessed for IFN-γ ELISpot responses after in vitro exposure to peptide pools spanning the SIVmac239 Gag, SIVmac239 ...

IFN-γ ELISpot responses were also assessed at the end of the acute phase of infection, at week 4 following viral challenge. As shown in Fig. 3B, rAdHu5- and rAdC68-primed groups of monkeys had a marginal contraction in their total ELISpot responses compared with those measured at the peak of viremia. In contrast, the rAdC7-primed monkeys had no contraction in their ELISpot responses from those measured at 2 weeks post-challenge. Twenty four weeks later, at week 28 following challenge, there was little further contraction in the ELISpot responses in these cohorts of monkeys (Fig. 3C). The monkeys in all three vaccinated groups had comparable magnitudes spot forming cell responses at this time.

Emergence of neutralizing antibody response against the challenge virus was monitored in all groups of monkeys following infection (Fig. 4). At four weeks post-challenge, 13 of 18 experimentally vaccinated monkeys had detectable ID50 NAb titers against SHIV-89.6P, and all 24 animals developed a detectable response within six weeks of infection. No significant differences were seen between the vaccinated and control groups of monkeys in either the kinetics or the magnitude of ID50 neutralization titers. On week 10 following challenge, all vaccinated and control monkeys showed moderate titer of neutralizing antibody responses specific for 89.6P envelope. To assess the breadth of the neutralizing antibody responses, sera from all animals were tested against a heterologous panel of clade B viruses. As shown in Fig. 5 no significant differences were observed following challenge between vaccine groups in the evolution of neutralizing antibodies against the challenge virus. It is interesting to note that although no significant differences were observed between vaccine groups against the majority of viruses in the panel of heterologous clade B viruses, the rAdHu5-rAdC1/C7 group showed a significantly higher neutralization titer against SF162.LS than the other two groups (p= 0.026, Mann-Whitney test).

Figure 4
Post-challenge NAb responses against SHIV-89.6P. Serum samples were obtained from vaccinated and control monkeys on the day of SHIV-89.6P challenge and subsequent time points following infection. Serial dilutions of samples were tested for NAb activity ...
Figure 5
Tier 1 and Tier 2 virus neutralization. Plasma samples were obtained from vaccinated monkeys six weeks following SHIV-89.6P challenge. Serial dilutions of samples were tested for NAb activity against a panel of Tier 1 and Tier 2 HIV-1 pseudoviruses. Data ...

3.4. Plasma viral RNA levels and CD4 T cell counts

Viral replication in the SHIV-89.6P challenged monkeys was assessed by quantitating plasma viral RNA levels. Log copies of plasma viral RNA per milliliter of plasma for each animal in each of the four groups are shown and the median values for each group of animals for each sampling time are shown in the solid line in each (Fig 6). The median values of the peak plasma viral RNA levels in each of the 3 groups of experimentally vaccinated monkeys were 6.5 (rAdHu5-rAdC1/C5), 6.0 (rAdC7-rAdC1/C5), and 7.0 (rAdC68-rAdC1/C5) log copies of viral RNA per milliliter of plasma. Median viral load of the monkeys in all three experimentally vaccinated groups was significantly lower than that of the control group (median 7.65 log copies) [p= 0.01 (rAdHu5-rAdC1/C5), 0.0002 (rAdC7-rAdC1/C5), 0.015 (rAdC68-rAdC1/C5), Mann-Whitney test]. Comparisons between the three experimental groups did not reveal significant differences in their peak viral loads.

Figure 6
Plasma viral RNA levels following intravenous challenge with SHIV-89.6P. These values were determined by an ultrasensitive branched DNA amplification assay with a detection limit of 125 copies/ml. Peak viremia values represent plasma viral RNA levels ...

The plasma viral RNA levels in each of the experimentally vaccinated and control monkeys were also assessed following challenge during the post-acute period, defined for each monkey as the median value of six determinations performed on specimens obtained between days 35 and 70 following challenge. The median plasma viral RNA levels during this post-acute period in each of the 3 groups of experimentally vaccinated monkeys were 3.7 (rAdHu5-rAdC1/C5), 3.16 (rAdC7-rAdC1/C5), and 4.13 (rAdC68-rAdC1/C5) and that of the control group was 4.8 log copies of viral RNA per milliliter of plasma. Monkeys in two of the experimentally vaccinated groups had significantly lower plasma viremia than the control vaccinees [p= 0.01 (rAdHu5-rAdC1/C5), p = 0.004 (rAdC7-rAdC1/C5), Mann-Whitney test] (Fig 6, Post-acute period panel). The plasma viral RNA levels in the rAdC68-primed monkeys and the control monkeys were not significantly different during this period. No significant differences in plasma viral RNA levels were observed between the groups of vaccinated monkeys during the post-acute infection period.

The rAdHu5-rAdC1/C5- and rAdC7-rAdC1/C5- vaccinated monkeys had lower long-term set point plasma viral RNA levels than the control monkeys. The set point plasma viral RNA values used for evaluation of each monkey were the median of eight data points obtained between days 84 and 224 post-challenge. Using these set point values, the six control monkeys had a median value of 4.5 log copies/ml. The median plasma viral RNA levels at set point in the experimentally vaccinated monkeys were 2.85 (rAdHu5-rAdC1/C5), 2.5 (rAdC7-rAdC1/C5), and 3.86 (rAdC68-rAdC1/C5) log copies/ml (Fig 5, Long-term set point panel). Therefore, at set point, the two groups of vaccinated animals (rAdHu5-rAdC1/C5 and rAdC7-rAdC1/C5) had 1.5–1.8 logs lower plasma viral RNA levels than the control animals [p= 0.015 (rAdHu5-rAdC1/C5), p = 0.009 (rAdC7-rAdC1/C5), Mann-Whitney test]. The AdC68-primed monkeys did not have statistically significant differences in their long-term set point plasma viral RNA levels when compared with the control animals.

Peripheral blood CD4+ T lymphocyte counts were also measured in all the monkeys to assess the vaccine-mediated clinical protection against the pathogenic SHIV-89.6P challenge. All six control monkeys had a greater loss of peripheral blood CD4+ T lymphocytes than the experimentally vaccinated animals by day 14 after challenge (Fig. 7, Peak panel). During the post-acute period (days 35 through 70 following challenge), all six control monkeys showed a profound loss of peripheral blood CD4+ T lymphocytes. In contrast, all experimentally vaccinated animals had a transient decline in peripheral blood CD4+ T lymphocytes detected by day 70 following challenge, but this cell loss substantially reversed in 17 of the 18 monkeys.

Figure 7
Peripheral blood CD4+ T lymphocytes following SHIV-89.6P challenge. The values were determined by multiplying the percentage of CD3+ T lymphocytes that were CD4+ by the total lymphocyte counts. Peak (top panel) represents the CD4+ T lymphocyte counts ...

To quantitate the CD4+ T lymphocyte counts of the monkeys at the time of long-term set-point plasma viremia, the median peripheral blood CD4+ T lymphocyte count was determined between days 84 and 224 post-challenge for each animal. Using these values, the control monkeys had a median CD4+ T lymphocyte count of 171 and the experimentally vaccinated animals had median CD4+ T lymphocyte counts of 596 (rAdHu5-rAdC1/C5), 672 (rAdC7-rAdC1/C5), and 407 (rAdC68-rAdC1/C5) [p= 0.04 (rAdHu5-rAdC1/C5), p = 0.015 (rAdC7-rAdC1/C5), Mann-Whitney test]. The median CD4+ T lymphocyte count for the rAdC68-primed monkeys was not significantly different than that of the control group of monkeys. Therefore, the CD4+ T lymphocyte counts in these monkeys reflected their plasma viral RNA levels post-challenge.

4. Discussion

Accumulating data are indicating that the use of heterologous recombinant adenoviruses in a prime/boost vaccination regimen can generate high frequency cellular immune responses in rhesus monkeys. This has been shown with the sequential administration of distinct serotypes of recombinant chimpanzee adenoviruses [14, 18] as well as the sequential administration of rare serotype- and adenovirus serotype 5-based vaccines [13]. Rare serotype rAd vectors like rAd26 and rAd35 have been used as potent heterologous rAd prime-boost vaccination strategy [24]. More recently it has been shown by Liu et al [25] that heterologous rAd26/rAd5 vaccination elicited more potent cellular immune responses compared to homologous rAd5/rAd5 vaccination and afforded partial control following a SIVmac239 challenge. The present study builds on those previous observations, providing further evidence for the utility of such an approach for the vaccine elicitation of cellular immune responses.

This study shows the magnitude of vaccine-elicited cellular immune responses induced by recombinant chimpanzee adenovirus vectors in comparison with the responses elicited by rAd5-based vectors. The present experiments demonstrate that the highest frequency T cell responses after a priming immunization were elicited in monkeys that received rAdHu5 immunogens. Priming with either of the two chimpanzee adenovirus constructs, rAdC7 and rAdC68, elicited less potent T cell responses. Following the heterologous boost with rAdC1/C5, the T cell responses in these two groups of recombinant chimpanzee adenovirus primed monkeys remained lower in magnitude than the rAdHu5-primed group of monkeys. Recent studies have shown that heterologous prime-boost regimens that include rAd5 and either a rare serotype rAd vector or a nonhuman rAd vector were highly immunogenic [12, 25]. The rAdHu5 vector used in this study was E1-deleted, whereas the chimpanzee adenoviral vectors used were both E1 and E3 deleted. Although the E3 region encodes polypeptides that are nonessential for viral growth, its deletion may affect vector performance. However, both E1- and E3-deleted rAd vectors have been successfully used by other laboratories to elicit potent T cell responses [24].

Two weeks following the pathogenic SHIV-89.6P challenge, the rAdC68-rAdC1/C7 group of monkeys developed larger magnitude anamnestic T cell responses than the rAdHu5-rAdC1/C7 or rAdC7-rAdC1/C7 groups of monkeys. Although anamnestic responses contribute to the early containment of viremia, this group of monkeys failed to control SHIV-89.6P replication and had the highest plasma virus RNA levels at all timepoints post-challenge. This persistent intense viremia likely stimulated the high frequency T cell response that we detected post-challenge. The magnitude of the pre-challenge vaccine-elicited T cell responses was associated with the control of viremia after challenge. Both the rAdHu5-rAdC1/C7 and rAdC7-rAdC1/C7 groups had higher pre-challenge T cell responses and lower viremia than the rAdC68-rAdC1/C7 group. These findings are consistent with the well-documented contribution of pre-challenge T cell responses to the control of viremia after virus challenge [26, 27].

The present study was done using SHIV immunogens to facilitate the assessment of the breadth of neutralizing antibodies generated through immunization with these vectors. We have previously shown that immunization with a plasmid DNA prime/rAd5 boost regimen generates a sufficiently potent neutralizing antibody response in monkeys to allow an assessment of the breadth of the vaccine-elicited neutralizing antibody responses [28, 29]. However, in this study vaccine-elicited neutralizing antibody responses were less potent when tested against a heterologous panel of clade B viruses.

Although we recognized the limitations of the data that would be generated, we performed a SHIV-89.6P challenge in these vaccinated monkeys. Most transmitted HIV-1 isolates are CCR5-tropic, and infection by these viruses causes a selective depletion of CD4+CCR5+ memory T lymphocytes. SHIV-89.6P acts in vivo like a CXCR4-tropic virus and selectively depletes naïve CD4+ T lymphocytes [30]. Moreover, vaccinated monkeys that are infected with SHIV-89.6P can control viral replication such that plasma viral RNA levels are undetectable, and these animals can survive for prolonged periods of time following challenge [31]. When that dramatic control of HIV-1 replication was not observed in vaccinees in the STEP trial, the utility of the SHIV-89.6P/rhesus monkey model was called into serious question [32].

Nevertheless, since vaccinated monkeys were available for further study, we challenged them with SHIV-89.6P. Potentially useful data were generated in this phase of the study. Although the magnitude of the vaccine-elicited virus-specific cellular immune responses differed in these groups of vaccinated monkeys, the magnitude of the cell-mediated immune responses in the first weeks following challenge were comparable. Since it is these cellular immune responses that we presume are responsible for the early containment of viral replication, this observation suggests that the differences in the magnitudes of the vaccine-elicited immune responses may not be critically important for control of an AIDS virus challenge. In fact, the control of SHIV-89.6P replication in the rAdHu5-rAdC1/C5 immunized and the rAdC7- rAdC1/C5 immunized monkeys was comparable.

Human Ad5 remains the gold standard for a vector that elicits cellular immune responses. Extensive experience with Ad5-based vectors in mice, monkeys and humans suggests that these vectors generate higher frequency and more durable cellular immune responses than vectors created using other serotype human adenoviruses. Further, these responses have proven more robust than those elicited by other live vector systems. However, in light of the data generated in the human clinical trials with Ad5-based immunogens, it may not be possible in the future to use Ad5-based vaccines in humans [33, 34].

The present study provides further evidence for the potential utility of nonhuman primate adenoviruses as vaccine vectors for generating effective AIDS virus-specific cellular immune responses. With evidence for an untoward effect of Ad5-based vectors on HIV-1 acquisition, as well as the finding that pre-existing immunity to Ad5 blunts the immunogenicity of these vectors, chimpanzee adenoviruses provide potentially powerful alternative vectors for use in generating cellular immune responses.

Acknowledgments

This work was supported by NIH grant P01-AI52271 and Harvard University Center for AIDS Research (CFAR) program grant NIH AI-060354.

Footnotes

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References

1. Barouch DH, Nabel GJ. Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Hum Gene Ther. 2005;16(2):149–156. [PubMed]
2. Shiver JW, Emini EA. Recent advances in the development of HIV-1 vaccines using replication-incompetent adenovirus vectors. Annu Rev Med. 2004;55:355–372. [PubMed]
3. Tatsis N, Ertl HC. Adenoviruses as vaccine vectors. Mol Ther. 2004;10(4):616–629. [PubMed]
4. Santra S, Seaman MS, Xu L, Barouch DH, Lord CI, Lifton MA, et al. Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates. J Virol. 2005;79(10):6516–6522. [PMC free article] [PubMed]
5. Sumida SM, Truitt DM, Kishko MG, Arthur JC, Jackson SS, Gorgone DA, et al. Neutralizing antibodies and CD8+ T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors. J Virol. 2004;78(6):2666–2673. [PMC free article] [PubMed]
6. Shiver JW, Fu TM, Chen L, Casimiro DR, Davies ME, Evans RK, et al. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature. 2002;415(6869):331–335. [PubMed]
7. Kostense S, Koudstaal W, Sprangers M, Weverling GJ, Penders G, Helmus N, et al. Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector. AIDS. 2004;18(8):1213–1216. [PubMed]
8. Sumida SM, Truitt DM, Lemckert AA, Vogels R, Custers JH, Addo MM, et al. Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein. J Immunol. 2005;174(11):7179–7185. [PubMed]
9. Vogels R, Zuijdgeest D, van Rijnsoever R, Hartkoorn E, Damen I, de Bethune MP, et al. Replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol. 2003;77(15):8263–8271. [PMC free article] [PubMed]
10. Barouch DH, Pau MG, Custers JH, Koudstaal W, Kostense S, Havenga MJ, et al. Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity. J Immunol. 2004;172(10):6290–6297. [PubMed]
11. Sekaly RP. The failed HIV Merck vaccine study: a step back or a launching point for future vaccine development? J Exp Med. 2008;205(1):7–12. [PMC free article] [PubMed]
12. Nanda A, Lynch DM, Goudsmit J, Lemckert AA, Ewald BA, Sumida SM, et al. Immunogenicity of recombinant fiber-chimeric adenovirus serotype 35 vector-based vaccines in mice and rhesus monkeys. J Virol. 2005;79(22):14161–14168. [PMC free article] [PubMed]
13. Roberts DM, Nanda A, Havenga MJ, Abbink P, Lynch DM, Ewald BA, et al. Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity. Nature. 2006;441(7090):239–243. [PubMed]
14. McCoy K, Tatsis N, Korioth-Schmitz B, Lasaro MO, Hensley SE, Lin SW, et al. Effect of preexisting immunity to adenovirus human serotype 5 antigens on the immune responses of nonhuman primates to vaccine regimens based on human- or chimpanzee-derived adenovirus vectors. J Virol. 2007;81(12):6594–6604. [PMC free article] [PubMed]
15. Reyes-Sandoval A, Fitzgerald JC, Grant R, Roy S, Xiang ZQ, Li Y, et al. Human immunodeficiency virus type 1-specific immune responses in primates upon sequential immunization with adenoviral vaccine carriers of human and simian serotypes. J Virol. 2004;78(14):7392–7399. [PMC free article] [PubMed]
16. Cohen CJ, Xiang ZQ, Gao GP, Ertl HC, Wilson JM, Bergelson JM. Chimpanzee adenovirus CV-68 adapted as a gene delivery vector interacts with the coxsackievirus and adenovirus receptor. J Gen Virol. 2002;83(Pt 1):151–155. [PubMed]
17. Tatsis N, Blejer A, Lasaro MO, Hensley SE, Cun A, Tesema L, et al. A CD46-binding chimpanzee adenovirus vector as a vaccine carrier. Mol Ther. 2007;15(3):608–617. [PubMed]
18. Pinto AR, Fitzgerald JC, Giles-Davis W, Gao GP, Wilson JM, Ertl HC. Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers. J Immunol. 2003;171(12):6774–6779. [PubMed]
19. Fitzgerald JC, Gao GP, Reyes-Sandoval A, Pavlakis GN, Xiang ZQ, Wlazlo AP, et al. A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag. J Immunol. 2003;170(3):1416–1422. [PubMed]
20. Tatsis N, Tesema L, Robinson ER, Giles-Davis W, McCoy K, Gao GP, et al. Chimpanzee-origin adenovirus vectors as vaccine carriers. Gene Ther. 2006;13(5):421–429. [PubMed]
21. Xiang Z, Gao G, Reyes-Sandoval A, Cohen CJ, Li Y, Bergelson JM, et al. Novel, chimpanzee serotype 68-based adenoviral vaccine carrier for induction of antibodies to a transgene product. J Virol. 2002;76(6):2667–2675. [PMC free article] [PubMed]
22. Montefiori DC. Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays. In: Coligan AM, Margulies DH, Shevach EM, Strober W, Coico R, editors. Current Protocols in Immunology. New York: John Wiley & Sons; 2004. pp. 12.1.1–12.1.5.
23. Mascola JR, D'Souza P, Gilbert P, Hahn BH, Haigwood NL, Morris L, et al. Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. J Virol. 2005;79(16):10103–10107. [PMC free article] [PubMed]
24. Liu J, Ewald BA, Lynch DM, Denholtz M, Abbink P, Lemckert AA, et al. Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous prime-boost regimens in rhesus monkeys. J Virol. 2008;82(10):4844–4852. [PMC free article] [PubMed]
25. Liu J, O'Brien KL, Lynch DM, Simmons NL, La Porte A, Riggs AM, et al. Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature. 2009;457(7225):87–91. [PMC free article] [PubMed]
26. Seth A, Ourmanov I, Schmitz JE, Kuroda MJ, Lifton MA, Nickerson CE, et al. Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge. J Virol. 2000;74(6):2502–2509. [PMC free article] [PubMed]
27. Barouch DH, Santra S, Kuroda MJ, Schmitz JE, Plishka R, Buckler-White A, et al. Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination. J Virol. 2001;75(11):5151–5158. [PMC free article] [PubMed]
28. Mascola JR, Sambor A, Beaudry K, Santra S, Welcher B, Louder MK, et al. Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficiency virus type 1 proteins. J Virol. 2005;79(2):771–779. [PMC free article] [PubMed]
29. Seaman MS, Xu L, Beaudry K, Martin KL, Beddall MH, Miura A, et al. Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys. J Virol. 2005;79(5):2956–2963. [PMC free article] [PubMed]
30. Nishimura Y, Igarashi T, Donau OK, Buckler-White A, Buckler C, Lafont BA, et al. Highly pathogenic SHIVs and SIVs target different CD4+ T cell subsets in rhesus monkeys, explaining their divergent clinical courses. Proc Natl Acad Sci U S A. 2004;101(33):12324–12329. [PubMed]
31. Barouch DH, Santra S, Schmitz JE, Kuroda MJ, Fu TM, Wagner W, et al. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science. 2000;290(5491):486–492. [PubMed]
32. Morgan C, Marthas M, Miller C, Duerr A, Cheng-Mayer C, Desrosiers R, et al. The use of nonhuman primate models in HIV vaccine development. PLoS Med. 2008;5(8):e173. [PMC free article] [PubMed]
33. Buchbinder SP, Mehrotra DV, Duerr A, Fitzgerald DW, Mogg R, Li D, et al. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet. 2008;372(9653):1881–1893. [PMC free article] [PubMed]
34. McElrath MJ, De Rosa SC, Moodie Z, Dubey S, Kierstead L, Janes H, et al. HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis. Lancet. 2008;372(9653):1894–1905. [PMC free article] [PubMed]