Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Hum Vaccin Immunother. 2013 Oct 1; 9(10): 2095–2102.
Published online 2013 Aug 13. doi: 10.4161/hv.26009
PMCID: PMC3906393
PMID: 23941868

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)

Aaron Wallace, 1 Kim West, 1 , 2 Alan L Rothman, 2 Francis A Ennis, 2 Shan Lu, 1 ,* and Shixia Wang 1
Author information Article notes Copyright and License information Disclaimer

Abstract

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8+ T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

Keywords: HIV-1, Gag, DNA vaccine, antibody, cell mediated immune responses

Introduction

With a large number of patients infected with human immunodeficiency virus type 1 (HIV-1) worldwide, the development of prophylactic HIV-1 vaccines is a priority to curb the transmission of HIV-1 and AIDS-related death. With the recent results from the RV144 trial,1 scientists started to identify what may be the correlates of protective immunity against HIV-1 infection.2 At the same time, among most HIV-1 vaccine researchers, it is believed that a strong T-cell immune response may still be needed to complement antibody-mediated protection, especially for those who may not be fully protected from initial viral exposure from antibodies.3-5

While antibody responses are critical for antiviral immunity, there is well documented evidence in the literature that cell-mediated immune (CMI) responses are also very important in the control and containment of HIV-1 infection.6-11 Clinical evidence has shown that T cells, especially CD8+ cytotoxic T lymphocytes (CTL), exert critical control over HIV-1 viremia and slow disease progression in natural infection.8,9,12,13 Studies have also demonstrated that CMI responses induced by vaccination were correlated with partial protection against experimental simian-human immunodeficiency virus (SHIV) challenge in non-human primates.14-16

Although both structural and non-structural viral proteins of HIV-1 have been used as immunogens to elicit T-cell immune responses, HIV-1 Gag is a major target of CMI responses in vaccine strategies. Gag is expressed as a 55-kD precursor (p55-Gag) and is cleaved by a viral protease to produce distinct smaller Gag products including matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7), and other small proteins (p2 and p1). Gag proteins are the driving force behind viral assembly into particles; p55-Gag can self-assemble and form virus-like particles. The myristylation site at the N-terminus of p17 and the zinc-finger motif in p7 of Gag are important for post-translational process and viral particle assembly.17

HIV-1 Gag is one of the most conserved viral proteins that induces broad, cross-clade CTL responses detected in HIV-1-infected individuals.18-20 It was demonstrated that vaccine-elicited cellular immune responses against Gag successfully contained viral infection and mitigated disease progression in monkey challenge models using pathogenic SHIV strains.14-16,21 Later AIDS vaccine designs have incorporated Gag as a component to elicit strong and effective CMI responses.22-30

The Gag product NC protein plays a pivotal role in the viral life cycle, including encapsulating and packaging the viral genome, protecting the viral genome from nuclease digestion.31-34 It contains two well conserved zinc finger motifs (C-X2-C-X4-H-X4-C, CCHC) as the major functional domain required for the most of the above processes.31-33,35-37 Mutations of CCHC motifs lead to a complete loss of these functions in addition to viral infectivity.31,38,39

To induce effective major histocompatibility complex (MHC) class-I restricted cellular immune responses, antigens need to be effectively expressed endogenously. Usually, live attenuated vaccines can express antigens endogenously and have proven highly effective in eliciting CMI responses and protection in a simian immunodeficiency virus (SIV) monkey model.40,41 However, such an approach had generated significant safety concerns for human use.42,43 Recent DNA vaccination strategies, using vector plasmids to express antigens in vivo, mimic natural antigen presentation by viruses thereby providing a unique and effective approach for generation of both humoral and cellular immune responses.10,27,28,44-46 DNA vaccines expressing Gag have been reported and used to generate CMI responses against immunodeficiency virus infections.14, 27-29

One previous Gag-based DNA vaccine study reported that the use of a tissue plasminogen activator (tPA) leader was able to significantly increase the immunogenicity of Gag DNA vaccines. Our study also demonstrated that tPA is an effective DNA vaccine component to increase the immunogenicity of HIV-1 envelope (Env) DNA vaccines47,48 as tPA was able to increase secreted Env antigen when expressed in targeted cells. However, the purpose of these two studies is different. Induction of antibody responses was the main objective for an Env-based vaccine and a higher level of secreted antigens, in theory, will be beneficial. On the other hand, CD8+ T-cell responses would be the target for a Gag-based vaccine and should require intracellular antigen presentation. Therefore, an increased secretion of Gag may not be good for the induction of Gag-specific CD8+ T cells. To resolve this issue, the current study was organized to investigate whether a tPA leader was effective in eliciting better T-cell immune responses when compared with a wild type Gag DNA vaccine design in a mouse model. Furthermore, we constructed Gag DNA vaccines with modified zinc finger motifs. The results demonstrated that the post-translational intracellular processing pathway plays a key role in determining the type of immune response observed for a DNA vaccine. These findings will have a general impact on the design of optimal DNA vaccine inserts.

Results

Construction and in vitro expression of Gag DNA vaccines

A set of Gag DNA vaccines was constructed to study the effect of leader sequence on antigen expression and immunogenicity of Gag DNA vaccines (Fig. 1A). The first Gag DNA vaccine was constructed by subcloning the full length wild type gag gene sequence from HIV-1 NL4-3 strain into the DNA vaccine vector pJW4303. The 2nd construct encoded the same full length gag gene except a tissue plasminogen activator (tPA) leader was added to the N-terminus of the Gag protein. The 3rd and 4th Gag DNA vaccines have the same inserts as the 1st and 2nd Gag DNA vaccines except similar mutations were made in the zinc finger region, as reported previously in literature (Fig. 1B).49

An external file that holds a picture, illustration, etc. Object name is hvi-9-2095-g1.jpg

Figure 1. (A) Designs of HIV-1 Gag DNA vaccines. (1) Wt-Gag: the wild type gag p55 gene as insert without adding leader sequence; (2) tPA-Gag: the wild type gag p55 gene as insert with addition of an upstream tPA leader sequence; (3) Wt-Gag-ZnM with zinc finger mutation without the leader sequence; and (4) tPA-Gag-ZnM with zinc finger mutation with the tPA leader sequence. Various cleaved Gag protein products, MA (p17), CA(p24), NC (p7), p6, p1, and p2, as well as the zinc finger location, are indicated; (B) Alignment of wild type zinc finger and mutated zinc finger sequences are indicated; (C) western blot analysis of the Gag protein expressed in lysate (L) and supernatant (S) of transiently transfected 293T cell by various HIV-1 Gag DNA vaccines.

These Gag DNA vaccines were tested for their antigen expression by transient transfection in 293T cells. Western blot analysis examined the Gag protein in both lysate and supernatant samples from 293T cells (Fig. 1C). Several interesting patterns were observed. First, the level of overall Gag antigen expression in 293T cells was lower for DNA vaccines with the wild type gag gene insert compared with those with a tPA leader. Second, there was no detectable Gag antigen in supernatants when 293T cells were transfected with the wild type Gag DNA vaccines but inclusion of a tPA leader led to significant levels of Gag expression in supernatant. Third, the overall expression level of Gag antigen, in both supernatant and lysate, was greatly increased with the inclusion of a tPA leader. Finally, mutations in the zinc finger region affected the intracellular processing of the Gag protein leading to different molecular weight species when compared with those observed in 293T cells transfected with the Gag DNA vaccine constructs without the zinc finger mutation.

Antibody responses elicited by Gag DNA vaccines

Balb/C mice were immunized by gene gun at Weeks 0, 2, 8, and 12. Sera were collected prior to the start of first immunization and 2 weeks after each immunization. ELISA was conducted to measure Gag-specific antibody responses. Figure 2 demonstrates Gag-specific, end titration IgG titers at the peak of antibody response (2 weeks after the last immunization). The mouse group that received the tPA-Gag DNA vaccine had the highest levels of Gag-specific IgG responses, which were much higher than those elicited by the wild type Gag DNA vaccine. Mutations in the zinc finger region reduced the immunogenicity of respective Gag DNA vaccines, but the vaccine with a tPA leader (tPA-Gag.ZnM) was much more immunogenic than the one without a tPA leader (Wt-Gag.ZnM). This data confirms our previous report that the addition of a tPA leader is effective in improving the immunogenicity of HIV-1 Env DNA vaccines,47 probably due to increased secretion of antigens encoded by the DNA vaccines.

An external file that holds a picture, illustration, etc. Object name is hvi-9-2095-g2.jpg

Figure 2. Gag-specific antibody responses in mice immunized with DNA vaccines expressing various NL4-3 Gag antigen designs. Gag-specific IgG titers were measured by ELISA at 2 weeks after the 3rd DNA immunization using pooled mouse sera from each group against Gag antigen produced in tPA-Gag transfected 293T cell supernatant. Each bar represents the mean antibody titers with standard error of duplicated assays for each mouse group.

T-cell responses elicited by Gag DNA vaccines

Gag-specific T-cell responses were also measured by different approaches. First, an IFN-γ ELISPOT was conducted with splenocytes stimulated by a well established Gag peptide from the p24 protein (Gag 197–205, AMQMLKETI) (Fig. 3). The relative immune response pattern was very different from that for antibody responses. The Wt-Gag DNA vaccine had the highest levels of IFN-γ ELISPOT responses. Mutations in the zinc finger region greatly reduced T-cell responses, which is similar to what was observed for antibody responses. However, tPA groups (both tPA-Gag and tPA-Gag.ZnM) had reduced T-cell responses when compared with those without a tPA leader, just the opposite of what was observed with the same set of Gag DNA vaccines for antibody responses.

An external file that holds a picture, illustration, etc. Object name is hvi-9-2095-g3.jpg

Figure 3. Gag peptide-specific IFN-γ ELISPOT induced by various NL4-3 Gag DNA vaccines in mice against clade B p24 peptide (Gag 197-205, AMQMLKETI) and measured at one week after the 4th DNA immunization. Each bar represents the mean spots/million cells in each group of 5 mice with standard error. The statistical significant differences between different groups are indicated as ** p < 0.01 and *** p < 0.001.

To further confirm this finding, intracellular cytokine staining (ICS) was conducted to measure CD8+ T cell responses (Fig. 4). As shown in one sample of ICS results, Wt-Gag DNA elicited the highest T-cell immune responses while mutations in the zinc finger region reduced the level of response. The addition of a tPA leader led to a reduced T-cell immune response (Fig. 4A). This pattern was further confirmed when the total numbers for all the mouse groups were summarized (Fig. 4B).

An external file that holds a picture, illustration, etc. Object name is hvi-9-2095-g4.jpg

Figure 4. IFN-γ responses against clade B p24 peptide (Gag 197-205, AMQMLKETI) measured by intracellular cytokine staining (ICS) in mice immunized with various NL4-3 Gag DNA vaccines at one week after the 4th DNA immunization. (A) Representative ICS results for IFN-γ secreting CD8+ T cells. (B) Group mean of CD8+ T cell IFN-γ responses with standard error. The statistical significant differences between different groups are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

To further confirm that antigen-specific T-cell cytokines reflect the functional status of cytotoxic T cells, a traditional CTL assay was conducted (Fig. 5). The Wt-Gag DNA vaccine group had the highest levels of CTL responses (> 60%) while mutations in the zinc finger region greatly reduced CTL responses. Again, the addition of a tPA leader reduced CTL activities to both Wt-Gag and Wt-Gag.ZnM antigen designs.

An external file that holds a picture, illustration, etc. Object name is hvi-9-2095-g5.jpg

Figure 5. Clade B p24 peptide (Gag 197–205, AMQMLKETI)-specific CTL responses induced by various Gag DNA vaccines. CTL responses were measured at one week after the 4th DNA immunization at Effector:Target (E:T) ratio) of 1:25. Each bar represents the mean percentages of cell lysate in each group (5 mice per group) with standard error. The statistically significant differences between groups are as indicated *** p < 0.001.

A repeat mouse study was done by using a full length gag gene sequence from a primary HIV-1 isolate 96ZM651 (Czm).50 Wt-Gag.Czm and tPA-Gag.Czm DNA vaccine designs were produced to express a codon optimized gag gene of HIV-1 Czm but not the zinc finger mutated inserts as the first mouse study proved that such mutations would lead to lower antibody and T-cell responses.

When these two new Gag DNA vaccines were tested in Balb/C mice, Gag-specific T-cell immune responses were measured (Fig. 6). Similar to what was observed in the earlier study with NL4-3 Gag DNA vaccines, the Wt-Gag.Czm DNA vaccine was more immunogenic than the tPA-Gag.Czm DNA vaccine as measured by IFN-γ ELISPOT (Fig. 6A) and ICS for CD8+ T-cell responses (Fig. 6B). In addition, the Wt-Gag.Czm group had higher CTL activities than the tPA-Gag.Czm group (Fig. 6C).

An external file that holds a picture, illustration, etc. Object name is hvi-9-2095-g6.jpg

Figure 6. Clade C p24 peptide (Gag 197-205, AMQMLKDTI)-specific ELISPOT (A), ICS (B) and CTL (C) responses induced by codon optimized Gag DNA vaccines from HIV-1 clade C 96ZM651. T cell immune responses were measured at one week after the 4th DNA immunization. (A). Each bar represents the mean spots/million cells in each group (5 mice) with standard error. (B). Each bar represents the group mean percentages of p24 peptide-specific CD8+ IFN-γ cells in each mouse group with standard error. (C). Each curve represents the mean percentages of cell lysate in each mouse group of 5 mice with standard error at different Effector:Target (E:T) ratios. The statistically significance differences between groups are indicated * p < 0.05; ** p < 0.01; *** p < 0.001.

Discussion

In the current report, we investigated the roles of intracellular pathways of post-translational processing for the immunogenicity of antigenic proteins produced by DNA vaccines. While, in our previous report, we demonstrated that a strong leader that produces an increased amount of secreted proteins improves the immunogenicity of the HIV-1 Env antigen when delivered in the form of DNA vaccines, the same approach did not appear to be the best option for antigens aimed at eliciting CTL responses. By using two HIV-1 gag genes, from either NL4-3 or Czm, we demonstrated that a Gag DNA vaccine, with the addition of a tPA leader, led to reduced Gag-specific T-cell immune responses. At the same time, antibody responses against Gag were increased, which supports our previous observation that tPA was effective in enhancing Env-specific antibody responses.

While in this pilot study, no detailed structural differences for Gag proteins expressed by various Gag DNA vaccines were analyzed, western blot analyses, with either secreted or cell lysate samples, revealed that the inclusion of tPA greatly increased the amount of secreted Gag antigens. The relative distribution of different sizes of Gag proteins in cell lysate also changed with the addition of a tPA leader. Such findings will support the concept that a tPA leader plays a key role in determining the intracellular processing of Gag proteins

Our data contradict some previously reported results showing that the addition of a tPA leader makes a Gag DNA vaccine more immunogenic.51 One major difference between that study and the current study is the relative immunogenicity. In previously reported study, the overall immune response was low: positive Gag-specific antibody was shown only with immunoblots but not actual titer and only 50–60/million interferon-γ-secreting cells were obtained based on ELISPOT assay. In our current study, titers for anti-Gag antibody reached ~1:25 000 and the cell numbers based on ELISPOT were 400–800/million cells. This could be due to a difference in DNA immunization methods. The previous report used needle injection by intramuscular (IM) route while our study used a gene gun. When the overall immunogenicity of the DNA vaccine was low, the addition of a tPA antigen may increase overall antigen production and thus, improve immune responses. However, when overall immune responses are high, the role of the intracellular pathway may play a more important role in determining the immunogenicity of differently designed Gag DNA vaccine inserts.

In current study, we also examined the impact of mutations in the zinc finger region as such changes have been incorporated in previous studies.52,53 At least based on our Gag antigen design, mutations at these zinc finger sites significantly reduced the immunogenicity of Gag DNA vaccines, as measured by both antibody and CMI responses.

The impact of the current study goes beyond the design of optimal Gag DNA vaccines. Manipulation of the leader sequence has been a key strategy in the design of highly immunogenic DNA vaccines. Our data suggest that it is necessary to consider the type of immune responses to be induced before a decision is made on whether a tPA leader is beneficial. If the purpose is to induce high titer antibody responses, a tPA leader may be useful. On the other hand, if the purpose is to elicit T-cell immune responses, especially CD8+ T cells, then such a leader may be counterproductive.

Material and Methods

Production of Gag DNA vaccines

DNA vaccines were first constructed expressing Gag p55 from HIV-1 clade B laboratory strain NL4-3. The full length wild type gag gene insert (from HIV-1 NL4-3) without any leader sequence was PCR-amplified and subcloned individually into DNA vaccine vector pJW430354,55 at the PstI and BamHI sites. For Gag DNA vaccines with an additional tPA leader sequence, the same gag gene inserts were subcloned into pJW4303 at the NheI and BamHI sites downstream of a human tissue plasminogen activator (tPA) leader sequence. In addition, site directed mutagenesis was conducted to five G > C sequence changes as shown in Figure 1C to produce two additional Gag DNA vaccines with mutations in the zinc finger region. Each individual DNA vaccine plasmid was produced from Escherichia coli (HB101 strain) and purified with a Mega purification kit (Qiagen) for both in vitro transfection and in vivo animal immunization studies.

Another set of DNA vaccines were constructed expressing a codon optimized gag gene from HIV-1 clade C isolate 96ZM651 (Czm) (GenBank Accession# AF286224.1).50 The codon optimized gag-Czm gene was chemically synthesized by Geneart. During the codon optimization process, following cis-acting sequence motifs were avoided: internal TATA-boxes, chi-sites and ribosomal entry sites; AT-rich or GC-rich sequence stretches; ARE, INS, CRS sequence elements; cryptic splice donor and acceptor sites; and branch points. Despite such DNA level sequence changes, the final codon optimized Gag DNA sequence still produced the same Gag amino acid sequences as in the original HIV-1 isolate 96ZM651. These codon optimized gag-Czm gene was cloned into DNA vaccine vector pSW3891 with or without a tPA leader sequence in the same design NL4-3 Gag DNA vaccines (Fig. 1).

In vitro expression of Gag antigen

The expression of Gag DNA vaccine constructs was examined by transient transfection of 293T cells56 with individual Gag DNA vaccines. Transfection was done when cells were at approximately 50% confluence on 60 mm dishes by calcium phosphate co-precipitation, using 10 μg of plasmid DNA per dish. The supernatants and cell lysates were harvested 72 h after transfection. Gag antigen expression was verified by western blot.

Western blot analysis

The Gag antigen produced in supernatant and cell lysate of transiently transfected 293T cell was subjected to denaturing SDS-PAGE and blotted onto PVDF membrane (BioRad). Blocking was done with 0.1% I-Block (Tropix).

Gag-specific mouse sera were used as the detecting antibody at 1:500 dilution and incubated for 45 min. Subsequently, the membranes were washed with blocking buffer and then reacted with AP-conjugated goat anti-rabbit (Tropix) at 1:5000 dilution. After final wash, Western-light substrate was applied to the membranes for 5 min. Once the membranes were dry, Kodak films were exposed to the membrane and developed with an X-Omat processor.

DNA immunization and sample processing

Female Balb/C mice of 6–8 weeks old were purchased from Taconic Farms and housed in the animal facility managed by the Department of Animal Medicine at the University of Massachusetts Medical School in accordance with IACUC approved protocol. The animals received three DNA immunizations at Weeks 0, 4, 8, and 12 by a Bio-Rad Helios gene gun (Bio-Rad). The Gag DNA vaccine or empty vector plasmids were coated onto the 1.0-micron gold beads at 2 μg of DNA/mg of gold. Each gene gun shot delivered 1 μg of DNA and a total of 6 non-overlapping shots were delivered to each mouse at the shaved abdominal skin after animals were anesthetized. Serum samples were collected at Week 0, 2, 6, 10, and 13. The mice were sacrificed at 1 week after the last immunization. Spleens were removed and single-cell suspensions of splenocytes were prepared as previously described.57 The final splenocyte preparations were 5 × 106 cells/ml in R10 medium (RPMI1640 medium with 10% FBS, 1% Penicillin-Streptomycin).

Measurement of Gag-specific antibody responses

Mouse sera were tested for Gag-specific IgG antibody responses by enzyme-linked immunosorbent assay (ELISA), as described.58 Briefly, microtiter plates were coated with 100 μl/well of Gag antigen (1 μg/ml in PBS, pH7.2) produced from 293T cell supernatant transiently transfected with tPA-Gag DNA vaccine construct. After incubation at 4 °C overnight, the plates were washed 5 times with washing buffer (PBS at pH 7.2 with 0.1% Triton X-100) and blocked with 200 µl/well of 4% milk-whey blocking buffer for 1 h at room temperature. Subsequently, plates were incubated with 100 μl of serially diluted mouse sera for 1 h. Next, the plates were incubated with 100 μl of biotinylated anti-mouse IgG (Vector Laboratories) diluted at 1:1000 for 1 h. Then, horseradish peroxidase-conjugated streptavidin (Vector Laboratories), diluted at 1:2000, was added (100 μl/well) and incubated for 1 h. Washes, as described above, were performed between steps. After the final set of washes, 100 μl of fresh TMB substrate (Sigma) was added to each well and incubated for 3.5 min. The reaction was stopped by adding 25 μl of 2 M H2SO4, and the optical density (OD) of the plate was measured at 450 nm.

IFN-γ ELISPOT assay

ELISPOT assays were performed on fresh mouse splenocytes as previously described.57,59,60 Multiscreen Immobilon P membrane White sterile 96-well plates (Millipore) were coated with 5 μg/ml of purified rat anti-mouse IFN-γ IgG1 (clone R4-6A2) in PBS at 4 °C overnight. After the plates were washed three times with PBS, each plate was blocked by the addition of 200 μl of R10 medium per well for 2–3 h at 37 °C. HIV Gag p24 peptide (Gag 197–205, AMQMLKETI)57,61 was used to measure Gag-specific T-cell responses and the non-Gag peptide from HIV Env V3 (IGPGRAFYT) peptide was used as the negative control. The peptides (final concentration 4 μg/ml) were added to the wells with 100 μl of freshly isolated splenocytes (500 000 or 100 000 cells/well in R10 medium) in duplicate. The plates were incubated for 20–24 h overnight at 37 °C in 5% CO2. The plates were then washed, incubated with 100 μl of biotinylated rat anti-mouse IFN-γ (Rat IgG1 [clone XMG1.2, BD Biosciences]) (1 μg/ml in dilution buffer: PBS with 0.005% Tween 20 and 5% FBS), and incubated at 4 °C overnight. After additional washes, 100 μl of AP-conjugated streptavidin complex (BD Bioscience) was added to each well in the above dilution buffer for 2 h at room temperature. The plates were washed and spots representing individual IFN-γ-producing cells were detected after a 7 min color reaction using 1-STEP NBT/BCIP (PIERCE). IFN-γ spot-forming cells (SFC) were counted. The results were expressed as the number of SFC per 106 input cells. The number of peptide-specific IFN-γ-secreting T cells was calculated by subtracting the background (no-peptide) control value from the established SFC count.

Intracellular-cytokine staining for detection of Gag-specific CD8+ T-cell responses

Freshly isolated splenocytes (106 cells in 200 μl) were cultured at 37 °C for 5 h in 96-well round-bottom plates in completed R10 medium supplemented with 20 U/ml of human IL-2 and 0.4 µg/ml of Golgi-plug (BD Biosciences). Stimulatory conditions included 5 μg/ml of the given Gag p24 peptide (AMQMLKETI) or a non-relevant HIV Env V3 peptide (IGPGRAFYT) as negative control. After incubation, the following procedures were performed at 4 °C. At first, cells were washed with FACS buffer (PBS with 2% FBS and 0.01% sodium azide) and then incubated for 10 min in 100 μl of FcBlock (2.4 G2 mAb, BD Biosciences). After washes, the FITC-conjugated anti-CD8 mAb (clone 53-6.7; BD Biosciences) was used for cell surface staining for 30 min at 4 °C and then washed. Being subjected to intracellular cytokine staining, the cells were fixed and permeabilized using the Cytofix/Cytoperm kit in accordance with the manufacturer's recommendations (BD PharMingen). Then, the cells were stained using PE-conjugated rat anti-mouse IFN-γ mAb (clone XMG1.2) for 30 min at 4 °C. Flow cytometry analysis was performed using a FACScalibur (Becton-Dickinson), and data were analyzed using FlowJo software (TreeStar, Inc.).

CTL assay

For bulk cultures splenocytes (stimulators) were incubated with Gag peptides at a concentration of 5–10 μg/ml in RPMI 10% FBS, 2.5 × 10 −5 ME and 25 μ/ml rhuIL2 in T25 flasks. Bulk culture 51Cr release assays were performed on day 7 of culture. P815 cells target cells were labeled with 0.25 mCi of 51Cr for 60 min at 37 °C. Following labeling, cells were washed three times and then resuspended in R10 medium. Effector cells were then added to 2 × 103 peptide-pulsed (10 μg/ml) P815 cells in 96-well round-bottom plates at various E:T ratios. Plates were incubated for 4 h at 37 °C, supernatants were harvested (Skatron Instruments), and specific lysis was calculated as follows: [(experimental release – spontaneous release)/(maximum release – spontaneous release)] × 100(%). All assays were performed in triplicate. Spontaneous lysis was < 15% in all assays.

Statistical analysis

Statistical analysis was done using Student t-test to compare T-cell responses induced by different Gag DNA vaccine groups.

Acknowledgments

This study was supported in part by NIH grants U19 AI082676 and P01 AI082274. Authors would like to thank Dr Jill M Serrano for her careful reading and editing of the manuscript.

Glossary

Abbreviations:

CAcapsid
CMIcell-mediated immune
CTLcytotoxic T lymphocytes
ELISAenzyme-linked immunosorbent assay
EnvHIV-1 envelope
HIV-1human immunodeficiency virus type 1
ICSintracellular cytokine staining
IMintramuscular
MHCmajor histocompatibility complex
MAmatrix
NCnucleocapsid
ODoptical density
SHIVsimian-human immunodeficiency virus
SIVsimian immunodeficiency virus
SFCspot-forming cells
tPAtissue plasminogen activator
tPAtissue plasminogen activator
Wt-Gagwild type gag gene

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Footnotes

Previously published online: www.landesbioscience.com/journals/vaccines/article/26009

References

1. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, et al.MOPH-TAVEG Investigators Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med. 2009;361:2209–20. doi: 10.1056/NEJMoa0908492. [PubMed] [CrossRef] [Google Scholar]
2. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 2012;366:1275–86. doi: 10.1056/NEJMoa1113425. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
3. Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S, Peyerl FW, Krivulka GR, Beaudry K, Lifton MA, Gorgone DA, et al. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature. 2002;415:335–9. doi: 10.1038/415335a. [PubMed] [CrossRef] [Google Scholar]
4. Pantaleo G, Koup RA. Correlates of immune protection in HIV-1 infection: what we know, what we don’t know, what we should know. Nat Med. 2004;10:806–10. doi: 10.1038/nm0804-806. [PubMed] [CrossRef] [Google Scholar]
5. Barouch DH, Liu J, Peter L, Abbink P, Iampietro MJ, Cheung A, Alter G, Chung A, Dugast AS, Frahm N, et al. Characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype 26 HIV-1 Env vaccine in healthy adults (IPCAVD 001) J Infect Dis. 2013;207:248–56. doi: 10.1093/infdis/jis671. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
6. Hazuda DJ, Young SD, Guare JP, Anthony NJ, Gomez RP, Wai JS, Vacca JP, Handt L, Motzel SL, Klein HJ, et al. Integrase inhibitors and cellular immunity suppress retroviral replication in rhesus macaques. Science. 2004;305:528–32. doi: 10.1126/science.1098632. [PubMed] [CrossRef] [Google Scholar]
7. Bray M, Prasad S, Dubay JW, Hunter E, Jeang KT, Rekosh D, Hammarskjöld ML. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc Natl Acad Sci U S A. 1994;91:1256–60. doi: 10.1073/pnas.91.4.1256. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
8. Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky W, Farthing C, Ho DD. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol. 1994;68:4650–5. [PMC free article] [PubMed] [Google Scholar]
9. Pantaleo G, Demarest JF, Soudeyns H, Graziosi C, Denis F, Adelsberger JW, Borrow P, Saag MS, Shaw GM, Sekaly RP, et al. Major expansion of CD8+ T cells with a predominant V beta usage during the primary immune response to HIV. Nature. 1994;370:463–7. doi: 10.1038/370463a0. [PubMed] [CrossRef] [Google Scholar]
10. Robinson HL, Sharma S, Zhao J, Kannanganat S, Lai L, Chennareddi L, Yu T, Montefiori DC, Amara RR, Wyatt LS, et al. Immunogenicity in macaques of the clinical product for a clade B DNA/MVA HIV vaccine: elicitation of IFN-gamma, IL-2, and TNF-alpha coproducing CD4 and CD8 T cells. AIDS Res Hum Retroviruses. 2007;23:1555–62. doi: 10.1089/aid.2007.0165. [PubMed] [CrossRef] [Google Scholar]
11. Nabel GJ. Challenges and opportunities for development of an AIDS vaccine. Nature. 2001;410:1002–7. doi: 10.1038/35073500. [PubMed] [CrossRef] [Google Scholar]
12. Musey L, Hughes J, Schacker T, Shea T, Corey L, McElrath MJ. Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection. N Engl J Med. 1997;337:1267–74. doi: 10.1056/NEJM199710303371803. [PubMed] [CrossRef] [Google Scholar]
13. Musey L, Hu Y, Eckert L, Christensen M, Karchmer T, McElrath MJ. HIV-1 induces cytotoxic T lymphocytes in the cervix of infected women. J Exp Med. 1997;185:293–303. doi: 10.1084/jem.185.2.293. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
14. Amara RR, Villinger F, Altman JD, Lydy SL, O’Neil SP, Staprans SI, Montefiori DC, Xu Y, Herndon JG, Wyatt LS, et al. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science. 2001;292:69–74. doi: 10.1126/science.1058915. [PubMed] [CrossRef] [Google Scholar]
15. Shiver JW, Fu TM, Chen L, Casimiro DR, Davies ME, Evans RK, Zhang ZQ, Simon AJ, Trigona WL, Dubey SA, et al. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature. 2002;415:331–5. doi: 10.1038/415331a. [PubMed] [CrossRef] [Google Scholar]
16. Barouch DH, Santra S, Schmitz JE, Kuroda MJ, Fu TM, Wagner W, Bilska M, Craiu A, Zheng XX, Krivulka GR, et al. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science. 2000;290:486–92. doi: 10.1126/science.290.5491.486. [PubMed] [CrossRef] [Google Scholar]
17. Dou J, Wang JJ, Chen X, Li H, Ding L, Spearman P. Characterization of a myristoylated, monomeric HIV Gag protein. Virology. 2009;387:341–52. doi: 10.1016/j.virol.2009.02.037. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
18. McAdam S, Kaleebu P, Krausa P, Goulder P, French N, Collin B, Blanchard T, Whitworth J, McMichael A, Gotch F. Cross-clade recognition of p55 by cytotoxic T lymphocytes in HIV-1 infection. AIDS. 1998;12:571–9. doi: 10.1097/00002030-199806000-00005. [PubMed] [CrossRef] [Google Scholar]
19. Kulkarni V, Rosati M, Valentin A, Ganneru B, Singh AK, Yan J, Rolland M, Alicea C, Beach RK, Zhang GM, et al. HIV-1 p24(gag) derived conserved element DNA vaccine increases the breadth of immune response in mice. PLoS One. 2013;8:e60245. doi: 10.1371/journal.pone.0060245. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
20. Ladell K, Hashimoto M, Iglesias MC, Wilmann PG, McLaren JE, Gras S, Chikata T, Kuse N, Fastenackels S, Gostick E, et al. A molecular basis for the control of preimmune escape variants by HIV-specific CD8+ T cells. Immunity. 2013;38:425–36. doi: 10.1016/j.immuni.2012.11.021. [PubMed] [CrossRef] [Google Scholar]
21. Lakhashe SK, Wang W, Siddappa NB, Hemashettar G, Polacino P, Hu SL, Villinger F, Else JG, Novembre FJ, Yoon JK, et al. Vaccination against heterologous R5 clade C SHIV: prevention of infection and correlates of protection. PLoS One. 2011;6:e22010. doi: 10.1371/journal.pone.0022010. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
22. Manuel ER, Yeh WW, Seaman MS, Furr K, Lifton MA, Hulot SL, Autissier P, Letvin NL. Dominant CD8+ T-lymphocyte responses suppress expansion of vaccine-elicited subdominant T lymphocytes in rhesus monkeys challenged with pathogenic simian-human immunodeficiency virus. J Virol. 2009;83:10028–35. doi: 10.1128/JVI.01015-09. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
23. Hammonds J, Chen X, Zhang X, Lee F, Spearman P. Advances in methods for the production, purification, and characterization of HIV-1 Gag-Env pseudovirion vaccines. Vaccine. 2007;25:8036–48. doi: 10.1016/j.vaccine.2007.09.016. [PubMed] [CrossRef] [Google Scholar]
24. Koup RA, Roederer M, Lamoreaux L, Fischer J, Novik L, Nason MC, Larkin BD, Enama ME, Ledgerwood JE, Bailer RT, et al.VRC 009 Study Team. VRC 010 Study Team Priming immunization with DNA augments immunogenicity of recombinant adenoviral vectors for both HIV-1 specific antibody and T-cell responses. PLoS One. 2010;5:e9015. doi: 10.1371/journal.pone.0009015. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
25. Quinn KM, Da Costa A, Yamamoto A, Berry D, Lindsay RW, Darrah PA, Wang L, Cheng C, Kong WP, Gall JG, et al. Comparative analysis of the magnitude, quality, phenotype, and protective capacity of simian immunodeficiency virus gag-specific CD8+ T cells following human-, simian-, and chimpanzee-derived recombinant adenoviral vector immunization. J Immunol. 2013;190:2720–35. doi: 10.4049/jimmunol.1202861. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
26. Acierno PM, Schmitz JE, Gorgone DA, Sun Y, Santra S, Seaman MS, Newberg MH, Mascola JR, Nabel GJ, Panicali D, et al. Preservation of functional virus-specific memory CD8+ T lymphocytes in vaccinated, simian human immunodeficiency virus-infected rhesus monkeys. J Immunol. 2006;176:5338–45. [PubMed] [Google Scholar]
27. Cristillo AD, Wang S, Caskey MS, Unangst T, Hocker L, He L, Hudacik L, Whitney S, Keen T, Chou TH, et al. Preclinical evaluation of cellular immune responses elicited by a polyvalent DNA prime/protein boost HIV-1 vaccine. Virology. 2006;346:151–68. doi: 10.1016/j.virol.2005.10.038. [PubMed] [CrossRef] [Google Scholar]
28. Pal R, Kalyanaraman VS, Nair BC, Whitney S, Keen T, Hocker L, Hudacik L, Rose N, Mboudjeka I, Shen S, et al. Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype. Virology. 2006;348:341–53. doi: 10.1016/j.virol.2005.12.029. [PubMed] [CrossRef] [Google Scholar]
29. Wang S, Kennedy JS, West K, Montefiori DC, Coley S, Lawrence J, Shen S, Green S, Rothman AL, Ennis FA, et al. Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers. Vaccine. 2008;26:3947–57. doi: 10.1016/j.vaccine.2007.12.060. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
30. Smith JM, Amara RR, Campbell D, Xu Y, Patel M, Sharma S, Butera ST, Ellenberger DL, Yi H, Chennareddi L, et al. DNA/MVA vaccine for HIV type 1: effects of codon-optimization and the expression of aggregates or virus-like particles on the immunogenicity of the DNA prime. AIDS Res Hum Retroviruses. 2004;20:1335–47. doi: 10.1089/aid.2004.20.1335. [PubMed] [CrossRef] [Google Scholar]
31. Morellet N, Déméné H, Teilleux V, Huynh-Dinh T, de Rocquigny H, Fournié-Zaluski MC, Roques BP. Structure of the complex between the HIV-1 nucleocapsid protein NCp7 and the single-stranded pentanucleotide d(ACGCC) J Mol Biol. 1998;283:419–34. doi: 10.1006/jmbi.1998.2098. [PubMed] [CrossRef] [Google Scholar]
32. Poon DT, Wu J, Aldovini A. Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity. J Virol. 1996;70:6607–16. [PMC free article] [PubMed] [Google Scholar]
33. Freed EO. HIV-1 gag proteins: diverse functions in the virus life cycle. Virology. 1998;251:1–15. doi: 10.1006/viro.1998.9398. [PubMed] [CrossRef] [Google Scholar]
34. Chow JY, Jeffries CM, Kwan AH, Guss JM, Trewhella J. Calmodulin disrupts the structure of the HIV-1 MA protein. J Mol Biol. 2010;400:702–14. doi: 10.1016/j.jmb.2010.05.022. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
35. Wellensiek BP, Sundaravaradan V, Ramakrishnan R, Ahmad N. Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission. Retrovirology. 2006;3:21. doi: 10.1186/1742-4690-3-21. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
36. Baba S, Takahashi K, Koyanagi Y, Yamamoto N, Takaku H, Gorelick RJ, Kawai G. Role of the zinc fingers of HIV-1 nucleocapsid protein in maturation of genomic RNA. J Biochem. 2003;134:637–9. doi: 10.1093/jb/mvg200. [PubMed] [CrossRef] [Google Scholar]
37. Aduri R, Briggs KT, Gorelick RJ, Marino JP. Molecular determinants of HIV-1 NCp7 chaperone activity in maturation of the HIV-1 dimerization initiation site. Nucleic Acids Res. 2013;41:2565–80. doi: 10.1093/nar/gks1350. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
38. Ramboarina S, Druillennec S, Morellet N, Bouaziz S, Roques BP. Target specificity of human immunodeficiency virus type 1 NCp7 requires an intact conformation of its CCHC N-terminal zinc finger. J Virol. 2004;78:6682–7. doi: 10.1128/JVI.78.12.6682-6687.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
39. Mark-Danieli M, Laham N, Kenan-Eichler M, Castiel A, Melamed D, Landau M, Bouvier NM, Evans MJ, Bacharach E. Single point mutations in the zinc finger motifs of the human immunodeficiency virus type 1 nucleocapsid alter RNA binding specificities of the gag protein and enhance packaging and infectivity. J Virol. 2005;79:7756–67. doi: 10.1128/JVI.79.12.7756-7767.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
40. Daniel MD, Kirchhoff F, Czajak SC, Sehgal PK, Desrosiers RC. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science. 1992;258:1938–41. doi: 10.1126/science.1470917. [PubMed] [CrossRef] [Google Scholar]
41. Marthas ML, Miller CJ, Sutjipto S, Higgins J, Torten J, Lohman BL, Unger RE, Ramos RA, Kiyono H, McGhee JR, et al. Efficacy of live-attenuated and whole-inactivated simian immunodeficiency virus vaccines against vaginal challenge with virulent SIV. J Med Primatol. 1992;21:99–107. [PubMed] [Google Scholar]
42. Baba TW, Jeong YS, Pennick D, Bronson R, Greene MF, Ruprecht RM. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science. 1995;267:1820–5. doi: 10.1126/science.7892606. [PubMed] [CrossRef] [Google Scholar]
43. Baba TW, Liska V, Khimani AH, Ray NB, Dailey PJ, Penninck D, Bronson R, Greene MF, McClure HM, Martin LN, et al. Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nat Med. 1999;5:194–203. doi: 10.1038/8859. [PubMed] [CrossRef] [Google Scholar]
44. Lu S, Santoro JC, Fuller DH, Haynes JR, Robinson HL. Use of DNAs expressing HIV-1 Env and noninfectious HIV-1 particles to raise antibody responses in mice. Virology. 1995;209:147–54. doi: 10.1006/viro.1995.1238. [PubMed] [CrossRef] [Google Scholar]
45. Ulmer JB, Wahren B, Liu MA. DNA vaccines: recent technological and clinical advances. Discov Med. 2006;6:109–12. [PubMed] [Google Scholar]
46. Wang S, Goguen JD, Li F, Lu S. Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge. Vaccine. 2011;29:6802–9. doi: 10.1016/j.vaccine.2010.12.062. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
47. Wang S, Farfan-Arribas DJ, Shen S, Chou TH, Hirsch A, He F, Lu S. Relative contributions of codon usage, promoter efficiency and leader sequence to the antigen expression and immunogenicity of HIV-1 Env DNA vaccine. Vaccine. 2006;24:4531–40. doi: 10.1016/j.vaccine.2005.08.023. [PubMed] [CrossRef] [Google Scholar]
48. Wang S, Hackett A, Jia N, Zhang C, Zhang L, Parker C, Zhou A, Li J, Cao WC, Huang Z, et al. Polyvalent DNA vaccines expressing HA antigens of H5N1 influenza viruses with an optimized leader sequence elicit cross-protective antibody responses. PLoS One. 2011;6:e28757. doi: 10.1371/journal.pone.0028757. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
49. Berthoux L, Péchoux C, Ottmann M, Morel G, Darlix JL. Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis. J Virol. 1997;71:6973–81. [PMC free article] [PubMed] [Google Scholar]
50. Rodenburg CM, Li Y, Trask SA, Chen Y, Decker J, Robertson DL, Kalish ML, Shaw GM, Allen S, Hahn BH, et al.UNAIDS and NIAID Networks for HIV Isolation and Characterization Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents. AIDS Res Hum Retroviruses. 2001;17:161–8. doi: 10.1089/08892220150217247. [PubMed] [CrossRef] [Google Scholar]
51. Qiu JT, Liu B, Tian C, Pavlakis GN, Yu XF. Enhancement of primary and secondary cellular immune responses against human immunodeficiency virus type 1 gag by using DNA expression vectors that target Gag antigen to the secretory pathway. J Virol. 2000;74:5997–6005. doi: 10.1128/JVI.74.13.5997-6005.2000. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
52. Aldovini A, Young RA. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990;64:1920–6. [PMC free article] [PubMed] [Google Scholar]
53. Urbaneja MA, Kane BP, Johnson DG, Gorelick RJ, Henderson LE, Casas-Finet JR. Binding properties of the human immunodeficiency virus type 1 nucleocapsid protein p7 to a model RNA: elucidation of the structural determinants for function. J Mol Biol. 1999;287:59–75. doi: 10.1006/jmbi.1998.2521. [PubMed] [CrossRef] [Google Scholar]
54. Lu S, Wyatt R, Richmond JF, Mustafa F, Wang S, Weng J, Montefiori DC, Sodroski J, Robinson HL. Immunogenicity of DNA vaccines expressing human immunodeficiency virus type 1 envelope glycoprotein with and without deletions in the V1/2 and V3 regions. AIDS Res Hum Retroviruses. 1998;14:151–5. doi: 10.1089/aid.1998.14.151. [PubMed] [CrossRef] [Google Scholar]
55. Lu S, Manning S, Arthos J. Antigen engineering in DNA immunization. Methods Mol Med. 2000;29:355–74. [PubMed] [Google Scholar]
56. Pear WS, Nolan GP, Scott ML, Baltimore D. Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A. 1993;90:8392–6. doi: 10.1073/pnas.90.18.8392. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
57. Chen X, Rock MT, Hammonds J, Tartaglia J, Shintani A, Currier J, Slike B, Crowe JE, Jr., Marovich M, Spearman P. Pseudovirion particle production by live poxvirus human immunodeficiency virus vaccine vector enhances humoral and cellular immune responses. J Virol. 2005;79:5537–47. doi: 10.1128/JVI.79.9.5537-5547.2005. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
58. Wang S, Pal R, Mascola JR, Chou TH, Mboudjeka I, Shen S, Liu Q, Whitney S, Keen T, Nair BC, et al. Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein boosting approach are effective in generating neutralizing antibodies against primary human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E. Virology. 2006;350:34–47. doi: 10.1016/j.virol.2006.02.032. [PubMed] [CrossRef] [Google Scholar]
59. Shen M, Wang S, Ge G, Xing Y, Ma X, Huang Z, Lu S. Profiles of B and T cell immune responses elicited by different forms of the hepatitis B virus surface antigen. Vaccine. 2010;28:7288–96. doi: 10.1016/j.vaccine.2010.08.081. [PubMed] [CrossRef] [Google Scholar]
60. Xing Y, Huang Z, Lin Y, Li J, Chou TH, Lu S, Wang S. The ability of Hepatitis B surface antigen DNA vaccine to elicit cell-mediated immune responses, but not antibody responses, was affected by the deglysosylation of S antigen. Vaccine. 2008;26:5145–52. doi: 10.1016/j.vaccine.2008.03.072. [PubMed] [CrossRef] [Google Scholar]
61. Mata M, Travers PJ, Liu Q, Frankel FR, Paterson Y. The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D. J Immunol. 1998;161:2985–93. [PubMed] [Google Scholar]
Articles from Human Vaccines & Immunotherapeutics are provided here courtesy of Taylor & Francis