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J Virol. 2005 August; 79(16): 10386–10396.
PMCID: PMC1182680

Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model


The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (Δ5G). In Δ5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and Δ5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in Δ5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in Δ5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.

Primate lentiviruses, human immunodeficiency viruses (HIVs), and simian immunodeficiency viruses (SIVs) share common genetic and biological properties. As SIVmac, originally isolated from macaques in primate research centers in the United States, causes AIDS in macaques with remarkable similarities to HIV type 1 (HIV-1) infection in humans, this AIDS monkey model has been utilized to study vaccine development and the pathogenesis of HIV infection (for reviews, see references 10, 14, 17, 43, and 47).

HIV/SIV infection in the host consists of two phases, the primary infection and chronic infection. During the primary infection, extensive viral replication and dissemination of the infection occur. In chronic infection, viral replication continues for a long period, eventually leading to AIDS. Due to the host immune response against the infection, these two phases are separated by a set point at which the viral load reaches its lowest level. The viral loads of the set point and chronic infection are inversely correlated with the control of SIV/HIV infection and predict disease progression (25, 31); however, it remains unclear which host responses determine the viral loads of the set point and chronic infection. Nevertheless, virus-specific immune responses have been implicated in the host's control of the infection. Cellular immunity, such as that shown by cytotoxic T lymphocytes (CTL) and helper T cells, has been reported to correlate with the control of HIV/SIV infection (for reviews, see references 2, 24, 28, and 39). The role of the neutralizing antibody (NAb) in the control of infection and the emergence of escape mutants has also been reported previously (7, 16, 51).

Despite these immune responses against HIV/SIV infection, humans and macaques fail to contain the infection due to the virus properties. HIV/SIV infects major target cells, such as CD4+ T cells and macrophages, by binding viral envelope glycoproteins (Env) to cellular surface proteins and CD4 and chemokine receptors (CCR5, CXCR4, or others) on target cells (5, 32). Since viral entry consists of multiple steps (virion binding to these viral receptors, conformational change of Env, and fusion between the virion and the cellular membrane) and the critical parts of Env used in these steps are exposed only during each step, naturally generated antibodies are only partly effective in preventing HIV/SIV infection in their hosts (7, 8). Primary isolates can be neutralized to various degrees by HIV-infected patient serum but not by contemporaneous autologous samples. Consequently, escape mutants against preexisting NAb are selectively replicated (51). Thus, effective NAb is rarely induced in HIV/SIV infection (8, 10). This could partly explain the failure of Env-based vaccine trials against HIV-1 (8, 50).

The heavy glycosylation of Env is a unique feature of HIV/SIV that is distinctive from features of other enveloped viruses and is significantly related to their neutralization-resistant property (8, 29, 44). We therefore assumed that the insufficient immune containment of HIV/SIV might be due to heavy glycosylation in Env and that the removal of some glycans might allow the host to mount a protective immune response against the infection. Thus, we studied the influence of deglycosylation on the replication of SIVmac239 in a T-cell line and created a quintuple deglycosylation mutant of SIVmac239 (Δ5G), which has maximal removal of N-glycans at amino acid residues 79, 146, 171, 460, and 479 in Env and retains a replication capability similar to that of SIVmac239 in phytohemagglutinin-stimulated rhesus peripheral blood mononuclear cells (PBMCs) (36, 40). We then examined the infection of rhesus macaques with Δ5G; although Δ5G was replicated as extensively as SIVmac239 during the primary infection, the subsequent Δ5G infection was restricted to a level less than the detection sensitivity of a plasma viral load assay by 8 weeks postinfection (p.i.), in contrast to high chronic viral replication in SIVmac239 infection. Furthermore, an almost sterilizing immunity against SIVmac239 was induced in Δ5G-infected animals (36). Interestingly, another quintuple-deglycosylation-mutation strain with mutations at amino acid residues 146, 156, 184, 244, and 247 in Env was created (44) and was demonstrated to share common features with Δ5G in viral replication in animals and in functions as an attenuated vaccine (20). Since these two viruses share only one deglycosylation mutation and other mutations distributed differently in surface envelope protein gp120 (SU), these two studies suggest that heavily glycosylated Env determines the pathogenicity of HIV/SIV.

To dissect the mechanism for notable containment of Δ5G infection after primary infection, we hypothesized that the Env of Δ5G, a viral protein that differs from that in SIVmac239, might elicit protective immunity against SIVmac239, because deglycosylation in Env might alter antigenic properties such as B-cell and T-cell epitopes and enhance the protective immunity against SIVmac239. For this purpose, we immunized animals with Env of Δ5G (Δ5G Env) or Env of SIVmac239 (the wild type; wt Env), and examined the effect of these vaccinations against SIVmac239 infection.


Generation of SU DNA vaccines.

DNA vaccine plasmids expressing SIVmac239 SU or Δ5G SU, pJWSUmac239 and pJWSUmacΔ5G, were constructed using the expression vector pJW4303 (45). To produce secreted SU efficiently, the native signal sequence in the SIVmac239 SU gene was replaced with the human tissue plasminogen activator signal in plasmid pJW4303, and a termination codon was created at the cleavage site for SU transmembrane (TM) protein (9). An SIVmac239 SU or Δ5G SU DNA sequence was amplified with a pair of primers, SUmacA (5′-TGTGCTAGCTATGTCACAGTCTTTTATGGTGTAC-3′) and SUmacB (5′-CCAGGATCCTATTACCTCTTCACATCTGTGGGGGC-3′). The SUmacA primer consisted of nucleotides (nt) 6923 to 6955 of the SIVmac239 sequence (GenBank accession number M33262) and the boldface nucleotides, which were changed to create a NheI site; primer SUmacB consisted of nt 8412 to 8381 and the boldface nucleotides, which were changed to create a BamHI site, and the underlined nucleotides, which generated tandem termination codons. The PCR-amplified fragments were digested with NheI and BamHI and cloned into the NheI- and BamHI-digested eukaryotic expression vector pJW4303 to yield pJWSUmac239 and pJWSUmacΔ5G. These plasmids were prepared using a Plasmid Mega kit (QIAGEN, Tokyo, Japan).

Generation of Env vaccinia vaccines.

Recombinant vaccinia viruses expressing Env of SIVmac239 or Δ5G, WRvvmac239 or WRvvΔ5G, respectively, were constructed using a vaccinia virus WR strain (WRvv) as described previously (15). To excise the entire coding region of the env gene from the cloned SIV plasmid, BamHI and SmaI sites were introduced by in vitro mutagenesis at 5′- and 3′-end-flanking sites of the env gene, respectively. Primer B-6808 (5′-GAAAGAGAAGAAGGATCCCGAAAAAGG-3′) consisted of nt 6796 to 9822 and the underlined mutations of the BamHI site; S-9537 (5′-TATGAATACTCCCGGGAGAAACCC-3′) consisted of nt 9527 to 9550 and the underlined mutations of the SmaI site. DNA fragments containing the env gene of SIVmac239 or Δ5G were isolated by digesting the mutated plasmids with BamHI and SmaI and were cloned into the SmaI- and BamHI-digested vaccinia virus vector plasmid pNZ68K2. To transfer the env gene from a recombinant plasmid to WRvv, the standard homologous recombination method using CV-1 cells was performed. Env expression in the recombinant vaccinia virus was confirmed by immunoprecipitation. The function of Env was confirmed by CD4- and CCR5-dependent fusion activity. The recombinant Env-expressing vaccinia viruses obtained were propagated and titrated in CV-1cells. The two recombinant viruses were propagated with similar kinetics in CV-1 cells.

Expression of SU-expressing plasmids and Env-expressing vaccinia virus in vitro.

CV-1 cells were transfected with equal amounts of the following SU-expressing plasmids: pJWSUmac239, pJWSUmacΔ5G, or the vector pJW4303. Secreted SU metabolically labeled with 35S protein labeling mix (PerkinElmer, Boston, MA) in culture supernatant was concentrated, immunoprecipitated with plasma from SIVmac239-infected monkeys, and then analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) as described previously (40). To examine Env-expressing vaccinia viruses, CV-1 cells were infected with WRvvmac239, WRvvΔ5G, or WRvv at a multiplicity of infection of 10, metabolically labeled with 35S protein labeling mix overnight, lysed, immunoprecipitated with plasma from SIVmac239-infected monkeys, and then analyzed by SDS-PAGE as described for the expression of SU-expressing plasmids.

Animals, immunization, and challenge.

Twelve juvenile rhesus macaques from Myanmar or Laos that were seronegative for SIV, simian T-cell lymphotropic virus, B virus, and type D retroviruses were used. As the polymorphism of major histocompatibility complex (MHC) genes influenced cellular immune responses against SIV/HIV infection, MHC II haplotypes and alleles of the macaques were determined (data not shown). All animals were housed in individual cages and maintained according to the rules and guidelines for experimental animal welfare stated by the National Institute of Infectious Diseases. As shown in Fig. Fig.1,1, the 12 animals were divided into three immunization groups of four animals each: the SIVmac239 (wt)-Env immunization group (Mm0005, Mm0007, Mm0010, Mm0012), the Δ5G Env immunization group (Mm0001, Mm0002, Mm0003, Mm0009), and the vector control immunization group (Mm0004, Mm0006, Mm0008, Mm0011). All animals were inoculated with 1 mg of plasmid DNA in 1 ml of saline, one into each quadriceps femoris at 0, 4, and 8 weeks after the initial prime immunization (weeks p.p.). The boost consisted of 5 × 107 PFU of vaccinia virus in 1 ml of phosphate-buffered saline (PBS), administered in two 0.1-ml intradermal inoculations, one into the skin of each femur, and two 0.4-ml inoculations, one into each quadriceps femoris at 21 weeks p.p. All animals were challenged with 10 50% tissue culture infective doses (TCID50) of SIVmac239 intravenously at 28 weeks p.p.

FIG. 1.
Outline of immunization, challenge infection, and blood sampling. Twelve juvenile rhesus macaques were divided into three immunization groups of four animals each: the wt-Env immunization group (Mm0005, Mm0007, Mm0010, and Mm0012), the Δ5G Env ...

Viral load measurement.

To monitor SIV infection, the plasma viral load was measured by the real-time-PCR method described previously (36). Viral RNA was isolated from plasma from the infected animals using a commercial viral-RNA isolation kit (PE Applied Biosystems, Urayasu, Japan). SIV gag RNA was amplified and quantified using a commercial RNA reverse transcription (RT)-PCR kit (TaqMan EZ RT-PCR; PE Applied Biosystems) with the two gag primers, namely, the forward primer 1224F (5′-AATGCAGAGCCCCAAGAAGAC-3′), the reverse primer 1326R (5′-GGACCAAGGCCTAAAAAACCC-3′), and TaqMan probe 1272T (6-carboxyfluorescein-5′-ACCATGTTATGGCCAAATGCCCAGAC-3′-6-carboxymethylrhodamine). Purified viral RNA (10 μl) was reverse transcribed and amplified in a MicroAmp optical 96-well reaction plate (PE Applied Biosystems) according to the manufacturer's instructions and with the following thermal cycle conditions: 1 cycle of three sequential incubations (50°C for 2 min, 60°C for 30 min, and 95°C for 5 min) and then 50 cycles of amplification (95°C for 5 s, 62°C for 30 s) in a 7000 Prism sequence detection system (PE Applied Biosystems). In vitro RNA transcripts were quantified by optical density at 260 nm (OD260) measurement and branched DNA assay for SIV viral RNA (Bayer Diagnostics, Tarrytown, N.Y.). RNA equivalent to 10 to 107 copies per reaction was used as the standard for each assay. The detection sensitivity of plasma viral RNA using this method was 1,000 copies/ml.

Flow cytometry.

CD4 depletion was monitored by measuring the percentage of CD4+ T cells, memory cells (CD29 high CD4+) T cells (48) in PBMCs. PBMC samples were purified from a citrate anticoagulant containing blood using standard Ficoll-Hypaque gradient centrifugation. For flow cytometry, 2 × 105 PBMCs were reacted with fluorescein isothiocyanate or phycoerythrin-labeled antibodies (anti-human CD4, Nu-Th/I [Nichirei, Tokyo, Japan]; anti-human CD8, Leu2a [Becton Dickinson, San Jose, CA]; anti-human CD29, 4B4 [Coulter, Miami, FL]; anti-monkey CD3, FN-18 [Biosource, Camarillo, CA]; and anti-human CD20, Leu16 [Becton Dickinson, San Jose, CA]) as previously described (36, 37, 48).


Overlapping peptides were synthesized by Emory University, Microchemical Facility, Winship Cancer Center (Atlanta, GA.). All SIVmac239 viral proteins except Env, Gag, Pol, Vif, Vpr, Vpx, Tat, Rev, and Nef were covered by consecutive 20-mer peptides overlapped by 12 amino acids. Env of SIVmac239 was covered by 72 consecutive 25-mer peptides overlapped by 13 amino acids. Peptides were dissolved in PBS with 10% dimethyl sulfoxide (Sigma Chemical, St. Louis, Mo.).


Recombinant Sendai viruses (rSeV) expressing SIVmac239 Gag, SU, or Δ5G SU were used to infect herpesvirus papio-transformed B-lymphoblastoid cell lines (B-LCLs) to prepare autologous B-LCLs presenting these viral antigens. rSeV Gag expressing unprocessed SIVmac239 Gag and p55 (22, 23) and rSeV SU and rSeV/Δ5G SU expressing wt SU and Δ5G SU were constructed as described previously (52) and were also used to infect autologous B-LCLs.


A 1:100 dilution of each plasma sample in PBS (pH 7.4) containing a blocking reagent (Dainippon Seiyaku, Osaka, Japan) was assayed for SIV-specific antibody by using a standard enzyme-linked immunosorbent assay (ELISA) technique with 96-well plates precoated with SIVmac239 virion lysate. The OD492 was measured using a microplate reader (range of absorbance with linearity, 0 to 3.0; Tecan Japan, Tokyo, Japan) and utilized as a relative measurement of the antibody titer.

ELISPOT assay.

Virus-specific CD4+ T cells and CD8+ T cells in PBMCs were measured using a monkey γ-IFN ELISPOT assay kit (U-CyTech, Utrecht, The Netherlands).

Cryopreserved PBMCs were thawed and cultured overnight in R-10 medium (RPMI 1640 [Sigma] supplemented with 10% heat-inactivated, defined fetal bovine serum [HyClone, Logan, Utah], 55 μM 2-mercaptoethanol, 50 U/ml penicillin, and 50 μg/ml streptomycin). PBMCs were subjected to the depletion of CD4+ cells with magnet beads coated with anti-human CD4 Ab (Dynal ASA, Oslo, Norway) or subjected to the depletion of CD8+ cells with magnet beads coated with anti-human CD8 Ab (Miltenyi Biotec, Bergisch Gladbach, Germany). Depletion of CD4+ or CD8+ cells from PBMCs was confirmed by flow cytometry. Using this depletion method, more than 95% of CD4+ or CD8 cells were removed from PBMCs. These PBMCs were used for ELISPOT assay for virus-specific CD8+ T cells and virus-specific CD4+ T cells. Virus-specific stimulation of T cells was performed with autologous B-LCLs pulsed with pooled peptides for Pol, Vif, Vpx, Vpr, Tat, Rev, and Nef or B-LCLs infected with an rSeV for Gag, wt Env, and Δ5G Env. B-LCLs were incubated with pooled peptides corresponding to each viral protein at a final concentration of 2 μg/ml or infected with rSeV at a multiplicity of infection of 10 at 37°C overnight. Peptide-pulsed or infected B-LCLs were inactivated with long-wave UV irradiation (19) in the presence of 10 μg/ml psoralen (Sigma) for 10 min at a distance of 3.5 cm from a UV light, washed three times with R-10, and then used as stimulators in an ELISPOT assay. CD4+ or CD8+ cell-depleted PBMCs were cultured with these stimulators in an anti-γ-IFN Ab-coated ELISPOT plate (U-CyTech) overnight according to the protocol for the kit. Spots on the ELISPOT plate were imaged using an Olympus model SZX12 microscope (Olympus, Tokyo, Japan) equipped with a digital camera, PDMCIe/OL (Polaroid, Cambridge, MA), and analyzed using a personal computer with MAC SCOPE version 2.61 (Mitani Corporation, Toyama, Japan). The results were calculated as numbers of spot-forming cells (SFC) per million PBMCs after subtraction of the background.

Neutralization assay.

The original protocol of this neutralization assay was reported by Means et al. (29). Plasma that was heat inactivated at 56°C for 30 min was serially diluted and incubated with a fixed concentration of SIVmac239, Δ5G, or a macrophage-tropic SIV, 239/envMERT, at room temperature for 1 h. CEMx174/SIVLTR-SEAP cells were added to the mixture and then incubated at 37°C for 3 days. Secreted alkaline phosphatase activity in the culture supernatant was measured using a Phospha-Light System (Applied Biosystems). Chemiluminescence was detected with a Wallac Microbeta plate reader.

Statistical analysis.

Statistical analysis was based on the Mann-Whitney test and performed using GraphPad Prism 4.0 software.


Experimental design.

We adopted a DNA prime-vaccinia virus boost regimen to immunize rhesus macaques with wt Env or Δ5G Env as shown in Fig. Fig.1.1. Twelve macaques were immunized at 0, 4, and 8 weeks after the initial prime immunization (weeks p.p.) with one of three different DNA expression plasmids (n = 4): pJWSUmac239 expressing SU of SIVmac239, pJWSUΔ5G expressing SU of Δ5G, or the vector pJW4303. At 21 weeks p.p., all animals were boosted with recombinant WR vaccinia viruses expressing the respective Env proteins: vaccinia virus expressing Env of SIVmac239, vaccinia virus expressing Env of Δ5G, or vaccinia virus (Fig. (Fig.11).

Expression of SU DNA plasmids and Env vaccinia viruses in vitro and in animals.

Although Δ5G replicated similarly to wild-type SIVmac239 in animals (36), quintuple deglycosylation might affect the expression of SU in a plasmid vector and the expression of Env in the vaccinia virus vector. Thus, we examined the expression of these vaccines in CV-1 cells. SU expressions in the wild-type plasmid (pJWSUmac239) and in the deglycosylated SU plasmid (pJWSUmacΔ5G) were at similar levels (Fig. (Fig.2A).2A). The expression and processing of Env in the wild type (WRvvENVmac239) and in the deglycosylated Env mutant vaccinia virus (WRvvENVΔ5G) were also at similar levels (Fig. (Fig.2B).2B). The reduced molecular size of the proteins due to deglycosylation was confirmed by PAGE (Fig. (Fig.2).2). As the amount of secreted SU in the supernatant by DNA transfection was comparable to that of Env in the cell lysate from CV-1 cells infected with WRvvEnv, a high expression of SU was achieved in a rev-independent manner by the pJW403 expression plasmid as described previously (9).

FIG. 2.
Expression of SU and Env by SU-expressing DNA vaccines and Env-expressing vaccinia viruses. A: SU secreted in supernatant from CV-1 cells transfected with SU-expressing plasmids. Lane 1, pJW4303 vector; lane 2, pJWSUmac239; lane 3, pJWSUmacΔ5G. ...

The expression of Env vaccines in the immunized animals was indirectly estimated by Env-specific antibody responses measured by a peptide ELISA using overlapping Env peptides. Env peptide-specific Ab was detected from 11 weeks p.p. after immunization with DNA vaccines, whereas there was no significant difference in the titers and the specificity of the responses between the two vaccine groups (data not shown), suggesting similar amounts of Env expressed in animals immunized with either Env vaccine. To examine the protective effect of the Env vaccines, all animals were challenged with 10 TCID50 of SIVmac239 intravenously at 28 weeks p.p.

Cellular immune responses elicited by Env vaccines.

The DNA prime-vaccinia virus boost regimen has been used in many studies, has successfully induced a high frequency of virus-specific CD8+ T cells in macaques, and has conferred protective immunity against chimeric simian/human immunodeficiency virus (SHIV) (3, 27, 45). We therefore examined the vaccine-induced Env-specific T-cell responses by IFN-γ ELISPOT assay. Since deglycosylation in Env might change T-cell epitopes in SIVmac239, we measured the wt-SU and Δ5G SU-specific T-cell response by using autologous B-LCLs infected with recombinant Sendai viruses expressing either wt SU and/or Δ5G SU, respectively.

Although there was a tendency for more ELISPOT-positive cells to be observed by homologous SU than heterologous SU, comparable results were obtained by both assays (Fig. 3A and B). As vaccinated animals were challenged with SIVmac239, the results from the wt-SU assay were subsequently used to assess the SU-specific immune response. Immunization with the DNA vaccine induced only marginal SU-specific CD8+ T cells or CD4+ T cells at 11 weeks p.p.; however, boost immunization with recombinant WR vaccinia virus significantly increased SU-specific CD8+ T cells and CD4+ T cells in PBMCs at 26 weeks p.p. (Fig. 3A, B, and C). Notably, SIVmac239 Env (wt Env) induced twofold more SU-specific CD8 T cells (mean, 770 SFC per million PBMCs; range, 540 to 880) responding to wt SU than Δ5G Env (mean, 320; range, 110 to 400) (P = 0.029) (Fig. 3A and C). Similarly, twofold more SU-specific CD4+ T cells were observed in wt-Env vaccinees (mean, 1,260; range, 840 to 1,710) than in Δ5G Env vaccinees (mean, 680; range, 150 to 1,260) at 26 weeks p.p. (P = 0.11) (Fig. 3B and C). Thus, a twofold-greater number of both SU-specific CD4+ T cells and CD8+ T cells were induced in SIVmac239 Env vaccinees than in Δ5G Env vaccinees at 26 weeks p.p. In vector controls, only negligible SU-specific CD4+ T cells and CD8+ T cells were detected in PBMCs at 26 weeks p.p. (Fig. 3A and B).

FIG. 3.
Env-specific CD4+ T-cell and CD8+ T-cell responses in 12 macaques. A: Env-specific CD8+ T cells in PBMCs were measured by ELISPOT assay for IFN-γ in three groups. B: Env-specific CD4+ T cells in PBMCs were measured ...

Humoral immune response elicited with Env vaccines.

The anti-Env Ab titer was examined by SIVmac239 virion lysate ELISA. Anti-SIV Ab was detected in both wt-Env vaccinees and Δ5G Env vaccinees after an rVV boost (Fig. (Fig.4)4) (26 weeks p.p.). Anti-SIV Ab titers were comparable between the two vaccine groups.

FIG. 4.
Humoral immune response during immunization and after challenge infection. The OD492 was used as a relative measurement of anti-SIV ELISA antibody titer.

Next, we examined the NAb against either SIVmac239, Δ5G, or a macrophage-tropic mutant, 239env/MERT (33, 35), in the two vaccine groups. Macrophage-tropic SIVs were highly susceptible to neutralization by plasma from most SIV-infected macaques (29), whereas SIVmac239 was highly resistant to neutralization as were most clinical isolates of HIV-1 (21, 29, 30). Plasma at 26 weeks p.p. from all immunized animals failed to neutralize not only SIVmac239 but also a multiple-deglycosylation-mutation strain, Δ5G (Table (Table1);1); in contrast, these plasma specimens did neutralize 239env/MERT. Furthermore, a marked difference was observed between the two vaccine groups. The NAb titer in the wt-Env vaccine group was eightfold higher than in the Δ5G Env vaccine group (Table (Table1).1). The difference of this immune response between the two vaccine groups was significant (P = 0.029).

Neutralizing-antibody titers in the vaccinated macaques at 26 weeks p.p.

SIV replication in Env-immunized animals.

As described above, wt-Env vaccine and Δ5G Env vaccine induced different magnitudes of virus-specific cellular and humoral immunity in macaques. To examine the effect of the two vaccines, we challenged the vaccinated animals with SIVmac239. Viral loads in vector controls were mostly consistent with our previous results with SIVmac239-infected rhesus macaques (36, 48). The mean peak viral load at 2 weeks p.i. was 1.4 × 107 copies/ml, with a range of 0.5 × 107 to 2.2 × 107 copies/ml. Viral loads in chronic infection diverged into two patterns (Fig. (Fig.5A).5A). Subsequent to the set point at 20 weeks p.i., the viral loads in three animals increased more than 104 copies/ml. In contrast, viral loads in one animal (Mm0011) remained as low as 1,000 copies/ml up to 45 weeks p.i.

FIG. 5.
Plasma viral loads after SIVmac239 challenge infection. Plasma viral load was measured by real-time PCR with a detection limit of 1,000 copies/ml. A: wt-Env vaccine group; B: Δ5G Env vaccine group; C: vector controls; D: comparison of viral loads ...

Compared with the vector controls, viral loads in wt-Env vaccinees were markedly reduced (Fig. (Fig.5B).5B). Peak viral loads at 2 weeks p.i. (mean, 1 × 106 copies/ml; range, 0.8 × 106 to 1.2 × 106 copies/ml) were 1-log lower than those in the vector controls. Furthermore, viral loads decreased to as low as 1,000 copies/ml by 8 to 20 weeks p.i., remaining low until autopsy at 45 weeks p.i.

Unexpectedly, viral loads in the Δ5G Env vaccine group resembled those in vector controls (Fig. (Fig.5C).5C). Peak viral loads (mean, 2.4 × 106 copies/ml; range, 0.9× 106 to 4.2 × 106 copies/ml) were slightly lower than those in vector controls. Set points and viral loads in the chronic phase were similar to those of vector controls.

In summary, as shown by the mean viral loads in primary and chronic infection (Fig. (Fig.5D)5D) and statistical analysis (Fig. (Fig.5E),5E), the effects of vaccination differed between the wt-Env vaccine and Δ5G Env vaccine. In the effect on primary infection (up to 6 weeks p.i.), wt-Env vaccination decreased viral loads more extensively and significantly than Δ5G Env vaccination (P = 0.029 versus P = 0.057); however, in chronic infection (viral loads after 8 weeks p.i.), significant reductions in viral loads compared with those in vector controls were seen only in the wt-Env vaccine group and not the Δ5G Env vaccine group (Fig. (Fig.5E).5E). Collectively, wt-Env vaccination induced significantly effective immunity to control SIVmac239 infection, whereas Δ5G Env vaccination induced a marginal effect seen only in primary and not in chronic infection.

CD4+ T-cell subsets in PBMCs.

CD4 cell depletion is a primary manifestation indicating immune disorder in HIV/SIV infection. As CD4 depletion results from HIV/SIV infection in lymphatic tissue, it correlates with the extent of viral replication. Accordingly, viral loads were correlated mostly with CD4 depletion (Fig. (Fig.55 and and6A).6A). Despite fluctuations due to immunizations and the challenge infection, the percentage of CD4+ T cells in wt-Env-immunized animals in the chronic phase recovered to the levels at the initiation of the experiment. By contrast, in vector controls and Δ5G Env vaccinees, the percentage of CD4+ T cells decreased in the chronic phase. Among them, an extensive decrease in CD4+ T cells occurred in animals with high viral loads in the chronic phase (Mm0001, Mm0008, and Mm0009) (Fig. (Fig.55 and and6A).6A). However, in the other animals, the levels of CD4+ T cells remained as before the challenge (Mm0003, Mm0011).

FIG. 6.
CD4+ T cells in PBMCs from rhesus macaques during immunization and after the challenge infection. A: Percentage of CD4+ T cells in PBMCs; B: percentage of CD4+ CD29high T cells in PBMCs.

A subset of CD4+ CD29 high cells, approximately corresponding to memory CD4+ T cells, is useful for diagnosing a deterioration in the immune function in animals with AIDS (26, 38, 48). Although this parameter usually correlates with the percentage of CD4+ T cells, remarkable differences were noted between two Env vaccine groups after the challenge infection. First, all animals in the wt-Env vaccine group showed an increased percentage of this subset in the chronic phase (Fig. (Fig.6B).6B). Second, three of the Δ5G Env vaccinees had a marked decrease after the challenge infection (Mm0001, Mm0002 and Mm0009), whereas the remaining animal (Mm0003) showed an increased percentage of this subset. In vector controls, this subset remained in the range before the challenge infection in all animals but one (Fig. (Fig.6B6B).

Env-specific-T-cell immunity after the challenge infection.

The magnitude of Env-specific T cells after the challenge infection is assumed to be influenced not only by vaccination but also by viral replication. Namely, SU-specific T cells at 4 days p.i. and those at 12 days p.i. were likely influenced by the former and the latter respectively. The magnitudes of SU-specific CD4+ T cells and CD8+ T cells at 4 days p.i. were comparable to those before challenge at 26 weeks p.p. (Fig. 3A and B); therefore, twofold-more SU-specific CD8+ T cells and CD4+ T cells were present in wt-Env vaccinees than in Δ5G Env vaccinees up to 4 days p.i. (Fig. (Fig.3C).3C). However, this difference in the magnitudes of SU-specific CD8+ T and CD4+ T cells was not sustained at 7 and 12 days p.i. (Fig. (Fig.3C).3C). Present with robust viral replication in primary infection, SU-specific CD4+ T cells immediately decreased to an undetectable level at 12 days p.i. In contrast, SU-specific CD8+ T cells increased (Fig. 3A and B). Subsequently, SU-specific CD8+ T cells gradually decreased to very low or undetectable levels by 34 weeks p.i. (Fig. (Fig.3A).3A). Thus, vaccine-induced SU-specific CD8+ T and CD4+ T cells were sustained only for a short period of time after challenge infection in both Env vaccine groups.

SIV-specific T-cell immunity after challenge infection.

Despite an Env vaccination, robust SIV infection occurred shortly after the challenge infection (Fig. 5B and C). Consequently, SIV-specific CD8+ T cells and CD4+ T cells were elicited not only in vector controls but also in Env vaccine groups (Fig. 7A and B). To examine the effect of these SIV-specific T cells on the control of SIV infection, all animals were divided into SIV infection-controlled (controlled) and SIV infection-uncontrolled (uncontrolled) animals. Viral loads in chronic infection and the percentage of CD4+ cells in PBMCs were used to classify the animals as controlled or uncontrolled (Fig. (Fig.6A).6A). All animals in the wt-Env vaccine group, Mm00011 in vector controls, and Mm0003 in the Δ5G Env vaccine group were grouped as control animals. The remaining animals, Mm0004, Mm0006, and Mm0008 in vector controls and Mm0001, Mm0002, and Mm0009 in the Δ5G Env vaccine group were grouped as uncontrolled animals. Notably, SIV-specific CD4+ T cells as well as the percentage of CD4+ CD29H cells remained high in the chronic phase in controlled animals (Fig. (Fig.7B7B and and6B,6B, respectively).

FIG. 7.
SIV-specific CD8+ T-cell and CD4+ T-cell responses in 12 animals. A: SIV viral-protein-specific CD8+ T cells in PBMCs were measured by ELISPOT assay for IFN-γ in three groups: vector controls, wt-Env vaccine group, and ...

Although overall SIV-specific CD8+ T cells were high in Env-vaccinated controlled animals, such correlation was not seen in vector controls grouped as uncontrolled animals (Fig. (Fig.7A).7A). Therefore, to examine the relevance of virus-specific T cells to the control of SIV infection, the magnitudes of every viral-protein-specific T cell in controlled and uncontrolled animals were compared. As shown in Fig. Fig.7C,7C, Gag-specific CD8+ T cells and CD4+ T cells, and Tat/Rev-specific CD4+ T cells were induced, with statistical significance (P < 0.05), in the control animals.


The heavily glycosylated structure of Env has been considered a main cause of chronically persistent viral replication and the pathogenicity of HIV/SIV, primarily because it potentially interferes with the development of the host immune response associated with protective immune functions, such as NAb and CTL (10, 36, 44). This characteristic constitutes the primary reason for the difficulty of developing effective vaccines. We therefore examined the efficacy of a deglycosylated-Env vaccine and compared it with the wt-Env vaccine. This study showed that quintuple deglycosylation neither improved the immunogenicity of the wt-Env vaccine nor elicited NAb against SIVmac239. This was in contrast to what occurred with Δ5G infection in rhesus macaques, because the host response elicited by Δ5G infection not only contained Δ5G infection but also protected the animals from SIVmac239 challenge infection (36). This study therefore suggested that an almost sterilizing immunity against SIVmac239 induced in Δ5G-infected animals could not be explained by the immunogenicity of Δ5G Env; instead, it is likely associated with the property of Δ5G as an attenuated virus. In fact, Δ5G was more neutralization-sensitive than SIVmac239 (36). Alternatively, the immunogenic property of Env in Δ5G could not successfully be duplicated by immunization with a Δ5G Env DNA prime-vaccinia virus boost regimen. Therefore, another immunization regimen might be able to elicit the protective immune response induced by Δ5G infection.

The Env vaccine is superior to other vaccines containing other viral proteins with respect to the induction of NAb; however, both the Δ5G Env vaccine and the wt-Env vaccine could not induce detectable NAb against either SIVmac239 or Δ5G. Instead, the wt-Env vaccine induced higher NAb against macrophage-tropic SIV than the Δ5G Env vaccine. Notably, this parameter most significantly correlated with the efficacies of the two Env vaccines. As Ab neutralized the macrophage-tropic variant 239/envMERT, which has only four separate amino acid substitutions distributed in env of SVmac239 (34), it might recognize unknown epitopes conserved between SIVmac239 and 239/envMERT. On the other hand, Δ5G Env may not sufficiently present this epitope due to mutations. Regarding the role of nonneutralizing Ab for the control of SIVmac239 infection, it is assumed that, as the neutralization assay did not necessarily reflect in vivo conditions, such nonneutralizing Ab with potential virus-binding ability may interfere with SIVmac239 infection in animals. Alternatively, Ab might play a role in other effector functions, such as antibody-dependent cell-mediated cytotoxicity to eliminate the infected cells. The antibody-mediated enhancement of viral antigen processing and cross presentation is also a mechanism potentially related to the control of SIV infection in vivo (49).

Reduced immunogenicity in the Δ5G Env vaccine was also noted in cellular immunity. The levels of stimulation of antigen-specific CD8+ T cells and CD4+ T cells are MHC I and MHC II dependent, respectively. As the macaques in this study have different MHC haplotypes (data not shown), the magnitude and breadth of SIV-specific T cells should vary among the animals. Nevertheless, the magnitude of SU-specific CD8+ T cells and CD4+ T cells in PBMCs was greater in the wt-Env vaccine group than in the Δ5G Env vaccine group. Although the expression of SU by expressing plasmids and that of Env by the vaccinia virus vector elicited by either the wt-Env vaccine or Δ5G Env vaccine were indistinguishable in cultured cells (Fig. (Fig.2),2), wt-Env might persist longer than Δ5G Env in vaccinated animals. T-cell epitopes in the wt-Env vaccine might therefore be more efficiently presented on MHC molecules in antigen-presenting cells than in the Δ5G Env vaccine. Differences in glycosylation levels might also affect some processes in antigen-presenting cells associated with the presentation of T-cell epitopes in Env.

Taking all results together, Env glycosylation might affect the presentation of B-cell epitopes and T-cell epitopes required for Ab-mediated and T-cell-mediated immunities related to the control of SIV infection.

As seen in viral loads and SU-specific T cell levels after challenge infection (Fig. (Fig.33 and and5),5), the effect of vaccination was limited. That seemed related to the development of escape mutants. Therefore, distinctive cellular immune responses after the challenge infection were also implicated in the control of SIVmac239 replication. The magnitude of virus-specific CD8+ T cells did not always correlate with the suppression of viral replication as reported previously (1, 6), particularly in vector controls (Fig. (Fig.55 and and7A);7A); however, selected epitope-specific CTL responses might be associated with infection control. Gag-specific CTLs are such candidates, because a high magnitude of Gag-specific CD8+ T cells was significantly elicited in five control animals (Fig. (Fig.7C).7C). The magnitude of Gag- or Tat/Rev-specific CD4+ T cells was statistically correlated with infection control (Fig. (Fig.7C).7C). This may simply indicate a lower depletion of virus-specific CD4+ T cells in animals with lower viral loads as reported previously (11). Alternatively, these virus-specific CD4+ T cells may play an important role in protective immunity (39). Taken together, these results implicated the dominant role of selected epitope-specific CD4+ T cells and CD8+ T cells for the control of SIVmac239 infection.

The challenge virus that should be used has been an important issue in AIDS vaccine studies (8, 10, 12). Many studies have reported impressive efficacy in a pathogenic-SHIV macaque model (3, 4, 45, 46); however, pathogenic SHIVs use CXCR4 as a coreceptor, whereas the majority of clinical isolates of HIV-1 use CCR5 (13, 27). Therefore, the challenge virus for an AIDS vaccine study should be an R5 virus, such as SIV (10). Consistent with this concern, a DNA prime-modified-vaccinia virus Ankara boost regimen, inducing broad SIV-specific T-cell responses, reduced the initial viral replication but did not prevent disease progression against SIVmac239 challenge (18). Thus, vaccine studies using pathogenic SHIV should be reevaluated by using an R5 virus (10).

Matano et al. reported that a DNA prime-Sendai virus boost regimen induced the CTL-based control of SIVmac239 in rhesus macaques (27). This study demonstrated that a DNA prime-vaccinia virus WR boost regimen expressing only Env controlled the chronic infection of SIVmac239 in rhesus macaques. The relatively lower viral loads in macaques from Myanmar or Laos than in those of Indian origin might contribute to the control of SIVmac239 infection. Nevertheless, it is important that these two studies demonstrated the efficacies of the two vaccine regimens against highly pathogenic SIVmac239. In earlier studies, other R5 SIVs were used as a challenge virus for an efficacy study of vaccine candidates. An Env-based vaccine in vaccinia virus vector priming and subunit protein boosting protected cynomologous macaques against homologous SIVmne clone E11S (42). In recombinant modified vaccinia virus, Ankara viruses expressing Gag-Pol and/or Env exhibited vaccine efficacy because of reduced viremia and the increased survival of rhesus macaques infected with uncloned SIVsmE660 (41). Accordingly, the efficacy of vaccine candidates might be influenced by the experimental conditions. Thus, well-defined animal models with detailed virological, immunological, and genetic information and suitable challenge viruses are required for the evaluation of vaccine candidates and the development of an AIDS vaccine.

This study demonstrated the importance of Env as a component of the AIDS vaccine, and Env-specific CD8+ and CD4+ T cells and nonneutralizing Env-specific Ab were suggested as protective immunity components. Quintuple deglycosylation in Env reduced vaccine efficacy and Env-specific immune responses. Env may therefore be comprised of appropriate antigenic properties to elicit humoral and cellular immune responses required for protective immunity against homologous or allele-specific target SIV/HIV. These properties could be modified by the alteration of glycosylation.

In conclusion, although Env is an important immunogen for the AIDS vaccine, Env properties, including glycosylation, should be carefully considered to design vaccines specific to the targeted viruses.


We thank Kayoko Ueda for excellent technical assistance.

This work was supported by AIDS research grants from the Health Sciences Research Grants, from the Ministry of Health, Labor, and Welfare in Japan, and from the Ministry of Education, Culture, Sports, Science and Technology in Japan.


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