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Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of “DNA prime plus protein boost”

WANG, Zheng; WANG, Shi-xia; LIU, Si-yang; BAO, Zuo-yi; ZHUANG, Dao-min; LI, Lin; ZHANG, Chun-hua; ZHANG, Lu; LI, Jing-yun; LU, Shan

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doi: 10.3760/cma.j.issn.0366-6999.2009.19.028
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In the last 20 years techniques to develop an effective vaccine to contain the HIV pandemic have been a main goal that scientists have pursued. From the initial attenuated or inactivated vaccines to the widely adopted viral vector-based vaccines, different strategies have been developed to inhibit HIV infection, but none of them have achieved an ideal protective activity.1

The gag-induced cell mediated immunity (CMI) in the rhesus monkey could protect it against the SHIV89.6 chimeric virus infection.2,3 These studies clearly achieved some protection by reducing the long-term viral setting point and avoiding the decline of CD4+ T cell counts. The STEP trial using an adenovirus Ad5 virus vector based HIV vaccine developed by Merck, once considered as a promising vaccine to induce the CMI,4,5 but it suffered a complete failure in the phase IIb clinical study. It is indicated that the CMI alone is not sufficient and it is likely that a sterilizing immunity is needed which demands an early and complete blocking of viral infection of the target cells with a high titer of anti-viral neutralizing antibodies.

Despite the effort of the last two decades there has been little progress toward the goal of generating broadly neutralizing antibody responses against HIV-1 by traditional immunization methods. The diversity of HIV-1 and frequent mutations make it difficult to design a vaccine based on the Env character of a sole isolate.6 Fusion intermediates, oligomeric proteins, and newly adopted consensus Env sequence vaccines were applied to discriminate the cross-clade neutralizing epitopes, yet antigens produced by these strategies have not been able to elicit broadly neutralizing antibodies against HIV-1 isolates of different genetic backgrounds.7-10

How to formulate a panel of vaccines containing multiple effective Env antigens is still to be resolved. A main problem was shown by the inability of the Env protein alone to elicit neutralizing antibodies in the human trial conducted by VaxGen a few years ago.11 One of the challenges to develop multiple recombinant Env proteins is screening, selection and production of highly immunogenic glycosylated proteins. DNA immunization has special advantage in the delivering polyvalent vaccines. DNA production is relatively standard, and mixing of different plasmids does not generate unnecessary inter-molecular interactions at the DNA level. But the limited quantity of DNA vaccine in vivo causes weaker immunogenicity in larger animals and human subjects.12 Protein boost in combination with DNA prime may overcome this problem.

It was reported that recombinant Env protein alone displays poor immunogenicity in both animal experiments and human subjects.13 However, it is an effective method to boost the response in animals that have received DNA primes, as the recombinant protein raises the antibody titer and broadens the neutralization breadth. DNA prime plus protein boost could increase the neutralizing antibody response and an isolate, JR-FL, that was resistant to neutralization, could be neutralized successfully by this approach.14 The HIV Env DNA vaccines from a T-cell line adapted strain (TCLA) were able to elicit neutralizing antibodies against HIV-1 Env and the antibody titer was improved after a TCLA HIV-1 Env protein boost.15,16

HIV-1 infections in China are mainly caused by clades CRF07_BC, CRF08_BC, Thai-B and CRF01_AE.17,18 This study intended to use gp120 from primary isolates as the immunogen, and to adopt the strategy of DNA prime plus protein boost to discover whether randomly selected Env immunogens of primary isolates can elicit broadly neutralizing antibody responses and make a solid basis for vaccine research against circulating HIV-1 isolates in China.


HIV-1 primary isolates andgp120genes

Five HIV-1 primary strains were isolated by co-culturing the peripheral blood mononuclear cells (PBMCs) of infected individuals and healthy volunteers. Isolates GX48, GX79 came from the Guangxi Autonomous Region; HN24 was isolated from an infected individual in Henan Province; NX22 from the Ninxia Autonomous Region, and GS22 from Gansu Province. All of these individuals were infected by intravenous drug use or commercial blood donation. Gp120 was first amplified by PCR using LA DNA polymerase (Takara) with the plus strand primer (Env-1: 5′-gaaagagcagaagacagtggcaatg-3′) and the minus strand primer (Env-2: 5′-tccagtcccccct-tttctyttaaaaag-3′), then a second PCR with primers (Ugp120: 5′-tgtgggtcacwgtctaytatggggtac-3′, Dgp120: 5′-tgcgccatagtgcttcctgctgctc-3′) was applied to achieve the whole gp120 region. Amplified gp120 genes were ligated into the T-easy vector (Takara) for cloning and sequencing.

Phylogenetic analysis

Deduced Env amino acid sequences were initially aligned using the soft Bioedit. Sequence gaps and ambiguous areas within the alignment were excluded from all comparisons. Pair wise evolutionary distances were estimated using Kimura's two-parameter method to correct for superimposed substitutions. A phylogenetic tree was constructed by the neighbor-joining method with the soft MEGA 3.0, and the reliability of branching orders was assessed by bootstrap analysis using 1000 replicates.

DNA vaccine constructs

The DNA vaccine inserts coding for the above 5 clones were first PCR amplified by using Pfu polymerase (Strategene) and subcloned into the DNA vaccine vector pJW4303. A pair of consensus PCR primers was used to amplify these 6 different gp120 genes; the plus strand primer was gp120-p-f1 (5′-cttgtgggtcacagtctattat ggggtacc-3′) and the minus strand primer was gp120-p-b1 (5′-ggtcggatccttactccaccactcttctctttgcc-3′). The HIV-1 gp120 inserts were fused in frame with a tissue plasminogen activator (tPA) leader sequence. The DNA vaccine plasmids grown in E. coli (HB101 strain) were prepared by using the Maxi-prep purification kit (Qiagen) after their expression was confirmed by Western blotting analysis using transiently expressed gp120 from 293T cells.

Immunization protocol

Female New Zealand white (NZW) rabbits aged 6-8 weeks old (0-2 kg of body weight) were purchased from Millbrook Farm (Amherst, MA) and housed in the animal facility managed by the Department of Animal Medicine at the University of Massachusetts Medical School in accordance with Institutional Animal Care and Use Committee (IACUC) approved protocol. Groups of rabbits first received four DNA immunizations at weeks 0, 2, 4, 8 by Bio-Rad Helios gene gun. The gp120 DNA vaccine plasmids or negative control vector pJW4303 were coated onto a 1.0 μm gold bead at a ratio of 2 μg of DNA per mg of gold. Each gene gun shot delivered 1 μg of DNA and a total of 36 non-overlapping shots were delivered to each rabbit on shaved abdominal skin at each immunization. The animals then received two recombinant gp120 protein boosts at weeks 12, 16.

In vitroexpression of gp120 antigens by DNA vaccines

The expression of primary gp120 antigens from DNA vaccine plasmids was applied in transiently transfected 293T cells. The 293T cells were grown in DMEM (Invitrogen, USA) with 10% heat-inactivated fetal bovine serum. Each of the 100 mm tissue culture dishes (5×106 cells) was transfected with 20 μg of individual gp120 DNA vaccine plasmids by calcium phosphate precipitation. The supernatants were removed after 12 hours and 10 ml fresh DMEM (FBS free) was supplied. The supernatants containing gp120 protein were harvested 72 hours post-transfection for enzyme-linked immunosorbent assay (ELISA) or Western blotting analysis.

Measurement of antibody levels by ELISA

Rabbit sera were tested for gp120-specific IgG titer by ELISA. Ninety-six-well microtiter plates (Corning, USA) were first incubated with ConA (5 μg per well in 100 μl of PBS, pH 7.2) for 1 hour then coated with 100 μl of gp120 antigens (1 μg/ml) from transiently transfected 293T cell supernatants. After wells were blocked with 200 μl/well of blocking buffer (4% milk-whey in PBS) overnight, 100 μl of serially diluted rabbit sera were added to duplicate wells and incubated for 1 hour. Following the antibody incubation, 100 μl of biotinylated anti-rabbit IgG at 1:1000 dilution was added to each well and incubated for 1 hour, horseradish-peroxiadase (HRP)-conjugated streptavidin at 1:2000 dilution was then added (100 μl/well) and incubate for 1 hour. Between each step, the plates were washed 5 times with washing buffer (PBS at pH 7.2 with 0.1% triton X-100). Finally, 100 μl/well of fresh TMB substrate (Sigma, USA) was added and incubated for 3.5 minutes. The reaction was stopped by adding 50 μl of 2 mol/L H2SO4. The absorption was measured at the optical density (OD) of 450 nm. The end titer was 2-fold higher than the OD450 value of the negative control wells with the normal rabbit sera.

Western blotting analysis

The cell lysates and supernatants from transiently transfected 293T cells were subjected to SDS-PAGE and blotted onto PVDF membrane. Blocking was done with 0.1% I-Block (Tropix, Bedford, USA). The membrane was incubated with immunized rabbit sera at 1:1000 dilution for 1 hour and subsequently reacted with AP-conjugated goat anti-rabbit IgG at 1:5000 dilution for 30 minutes. This rabbit had been immunized by polyvalent vaccines against HIV-1 A, B, C, D, E, G Env. Membranes were washed with blocking buffer after each step. Western-light substrate was then applied to the membrane for 5 minutes. Once the membrane was dried, X-ray films were exposed to the membrane and developed by a Kodak processor.

Neutralization assays

Neutralization assay was performed using a single round of infection on Tzm-bl cells. Briefly, sera were heat inactivated at 56°C for half an hour for the Tzm-bl neutralization assay that was performed in 96-well plates as described previously. Pre-bleeding rabbit sera were used as the negative control. Viruses (200×50% tissue culture infectious doses (TCID50)) were added to serial dilutions of each plasma in a total of 100 μl for 1 hour at 37°C. Each well received 100 μl of Tzm-bl cells resuspended in media at 1×105 cells/ml. Forty-eight hours later cells were lysed for ten minutes directly on the neutralization plate using 100 μl of Promega Luciferase Assay Substrate and immediately analyzed for luciferase activity on the Luciferase reader (Perkin Elmer, USA). All samples were tested in triplicate, all values were calculated with respect to the formula: (prebleeding serum-postimmunization serum)%/(prebleeding serum-cell only).

Statistical analysis

Paired t test method was applied to calculate the difference of antibody titers of rabbit sera between post-DNA and post-protein. All analyses were performed using a two-tailed P value and P values less than 0.05 were considered statistically significant.


Phylogenetic analysis of selectedgp120regions

Sequences for the gp120 genes had been deposited in the GenBank under accession numbers as follows: AAN83918 (HN24), EU908737 (GS22), EU908738 (GX48), EU908739 (GX79), and EU908740 (NX22). HIV gp120 sequences of different clades isolated in China were downloaded as referees from GenBank, gp120 of GX48, GX79, NX22, GS22 and HN24 were clustered with these representative sequences for mapping a phylogenetic tree. As depicted in Figure 1, circulating isolates in China were divided into clades of CRF07_BC, CRF08_BC, CRF01_AE and Thai-B, in particular, GX48, GX79 were classified as CRF01_AE; NX22, GS22 belonged to CRF07_BC; and HN24 was determined as Thai-B strain. In conclusion, selected isolates were good representatives of circulating HIV-1 strains in China.

Figure 1.
Figure 1.:
Phylogenetic analysis of Env sequences from circulating isolates in China. The newly characterized sequences are indicated by ellipse. Horizontal branch lengths are drawn to scale (the scale bar represents 0.02 amino acid substitution per site), but vertical separation is for clarity only. Values at nodes indicate the percentage of bootstraps in which the cluster to the right was found. The phylogenetic tree was rooted with subtype D Env sequences (NDK, 94UG114).

Construction of DNA vaccines and their expressionin vitro

The gp120 wild type signal peptide was substituted for the tissue plasminogen activator (tPA) leader after gp120 insert was cloned into vector pJW4303. A previous study showed that this substitution can improve the expression efficacy efficiently in vitro.19

To certify the DNA vaccine expression in vitro, 5×106 293T cells in 10-cm plates were transfected with 20 μg of plasmids. Cell lysates and supernatants were collected for Western blotting, rabbit anti-gp120 serum was applied as the primary antibody and exposure time was 3 minutes. As shown in Figure 2, smear-like gp120 bands detected in both cell lysates and supernatants indicated the DNA vaccine constructs could work well.

Figure 2.
Figure 2.:
Western blotting analysis of gp120 glycoprotein expression in the cell lysates and supernatants from DNA vaccines transiently transfected 293T cells. “L” indicates the cell lysate; “S” indicates the supernatant.

Immunization schedules of DNA plus protein formulation

Immunogenicity of constructed HIV gp120 DNA vaccines was studied as either monovalent, 3-valent (containing GX48, GX79, NX22) or 4-valent (GX48, GX79, NX22 and HN24 were included) combinations in NZW rabbits. In the DNA priming period, each rabbit received four DNA primings at weeks 0, 2, 4, 8. The total amount of DNA at each immunization was fixed at 36 μg, but the dose for each gp120 DNA vaccine varies depending on the number of DNA vaccines included in the polyvalent formulations. In the 3-valent group, the dose for each DNA plasmid was 12 μg, while in the 4-valent group, the dose was 9 μg each. The protein boosting phase consisted of two intradermal or hyperdermal immunizations of recombinant gp120 of isolate 92US715 mixed with incomplete freund adjuvant (IFA) at weeks 12 and 16, the dose of protein boost at each immunization was fixed at a total amount of 50 μg per immunization. The control group received 4 injections of the empty pJW4303 DNA vector followed by two recombinant gp120 protein boosts as described above.

Anti-homologous gp120 IgG responses in rabbit sera after DNA prime and protein boost

DNA vaccines expressed glycoprotein gp120 were used as capture antigens to detect the homologous gp120-induced specific IgG titers by ELISA, the amount of coated antigen per well was fixed as 100 ng. The dose of antigen for assaying rabbit sera from animals that received polyvalent vaccine immunizations varied depending on the number of DNA vaccines included in the polyvalent formulations. Rabbit sera were diluted as 1:1000. As shown in Figure 3A, antibody titers rose as the immunization time increased. After 4 DNA primes, antibody titers against Env reached a plateau while the boost with recombinant gp120 protein generated anti-Env antibody responses higher than the DNA prime alone (P <0.01). The anti-gp120 antibody titers did not improve significantly with the second protein boost, suggesting that two protein immunizations were sufficient to boost the anti-gp120 antibody titers to the peak levels in these animals. Animals primed with gp120 DNA and boosted with gp120 protein generated anti-Env antibody responses higher than which primed with empty vectors and boosted with protein. The peak titer of control group was approximately 1 log value lower than which generated by the Env DNA prime plus protein boost (Figure 3B).

Figure 3.
Figure 3.:
Anti-gp120 IgG responses after DNA prime plus protein boost. A: Temporal development of gp120-specific antibody titers after DNA prime (open arrow) and protein boost (black arrow) immunizations. B: Anti-gp120 IgG titers at the end of 2nd protein boost.

Neutralizing antibody responses in rabbit sera after DNA prime and protein boost

Neutralizing antibody responses of rabbit sera were analyzed using a single-round infection against pseudovirus SF162. Neutralization results are shown in Figure 4. Except for R579, all of the rabbit sera displayed remarkable differences between post-DNA and post-protein immunization (P <0.01). In R587, the rabbit sera seemed to increase the infectivity of the pseudovirus SF162 post-DNA injection, yet after protein boost, neutralizing activities were improved. The rabbits that received a single protein boost displayed weaker neutralizing activities against pseudovirus SF162. The reciprocal serum dilution at the half maximal inhibitory concentration (IC50) was 40. This indicates that DNA prime plus protein boost was an effective method to increase the neutralizing antibody titer response.

Figure 4.
Figure 4.:
NAb titers against SF162 after DNA prime and protein boost.

Neutralizing activities of immunized rabbit sera

Different pseudoviruses and lived viruses were included in the evaluation system (Table). In response to the chimeric virus SF162-HN24 (gp120 region of SF162 had been substituted by the gp120 of isolate HN24), most of the rabbit sera displayed weak neutralizing activities against the chimeric pseudovirus except for R586 that was primed by DNA gp120 of GS22 and R587, primed by DNA gp120 of HN24. Two rabbit sera that received the polyvalent DNA vaccine immunization also could neutralize the chimeric pseudovirus very well.

Neutralizing antibody titers against various HIV-1 viruses in rabbit sera

No sera could neutralize the infectious clone pCNHN24 derived virus20 (an infectious clone constructed from the isolate HN24). The T cell line adapted strain, HIV-1 IIIB isolate which was grown in the T-lymphoid cells for many passages, was sensitive to neutralizing antibodies. Rabbit sera R581, R586, R588 all displayed neutralizing activities against HIV-1 IIIB isolate.


Neutralizing antibody is very important in the HIV vaccine development. Passive immunization also confirms its function in the non-human primate model. However, it has been demonstrated that broadly neutralizing antibodies exist in some HIV infected individuals, yet the emergence of these antibodies was always one step behind the mutated virus.21

A successful vaccine should not only induce the CD8+ T cell response, but also can induce neutralizing antibodies. The function of neutralizing antibodies is to block the binding of virus to target cells, or to activate complement dependent cytotoxicity and antibody dependent cell mediated cytotoxicity.21 But limited progress has been achieved in designing an effective vaccine to induce broadly neutralizing antibodies; a result of the poor immunogenicity of HIV Env protein. During some early human clinical experiments, high levels of antibodies failed to be elicited by the recombinant Env vaccines. Data from the Phase III vaccine experiments suggested that, although the volunteers received 7 recombinant Env immunizations,22 the antibody response was still lower than the desired goal. The poor immunogenicity of Env is still unclear although it is suggested that there is a correlation with high glycosylation and the sophisticated oligomeric structure of Env.23

DNA immunization technology has been applied in the CMI and it is reported that DNA immunization alone can elicit strong antibody responses in small animals and non-human primate animal models. DNA vaccines have the same advantages as the attenuated vaccine, so endogenous antigens can be efficiently presented by major histocompatibility complex (MHC)-I and MHC-II molecules. MHC presentation is the key step to mediate CD8+ and CD4+ T cell responses and the newly produced intracellular antigens can be folded into the native conformation and receive the necessary post-translation modifications such as glycosylation. Env antigens can be conveniently designed and modified to enhance immunogenicity. In particular, it has been reported that Env DNA prime plus protein boost could improve the neutralizing antibody response, although a TCLA Env was applied in these experiments. With the strategy of DNA prime plus protein boost, Lu et al14 had elicited neutralizing antibodies against an isolate of JR-FL which was resistant to neutralization.

DNA vaccine and recombinant protein vaccine can elicit antigen-specific responses with different mechanisms. A DNA vaccine is effective for inducing CMI and CD4+ T cell responses while antigen-specific B cell responses can be induced from DNA vaccine. Recombinant protein can further stimulate antigen-specific memory B cells to differentiate into the plasma cells. This is also the key step to produce a high titer of antigen-specific antibody,12 so the DNA prime plus protein boost can complement each other to conquer each weakness.

In our experiments, using DNA or protein alone can produce neutralizing antibodies against pseudovirus SF162. However, in the animals which received Env DNA primes plus protein boosts, these neutralizing antibody responses were strengthened to increase the neutralizing antibody titers. In the rabbits that received 4 DNA primes and 2 protein boosts, gp120-specific antibody titers rose to the highest level, and the immunization results were better than DNA or protein alone. Above all, neutralizing antibody titers were also remarkably improved. It is indicated that DNA prime plus protein boost is an effective method to improve the specific target-binding antibody titers and neutralizing antibody response. In the neutralization experiments 2 pseudoviruses and 2 laboratorial strains were used. Sera from all the rabbits could neutralize the pseudovirus SF162. To the chimeric pseudoviruses SF162-HN24, R586, R587, R588, and R589 displayed their neutralizing activities, but their IC50 values were lower compared to the laboratorial strain HIV-1 IIIB; only rabbit sera R581, R586 and R588 could neutralize it. None of the sera could neutralize the infectious clone derived virus CNHN24.

Sera from rabbit R582, which received recombinant Env immunization alone, could also neutralize SF162 pseudovirus, yet the serum dilution at IC50 level was lower than the sera from a rabbit that received DNA prime plus protein boost. While it could not neutralize the other viruses, the gp120-specific antibody titer in its serum was 1 log lower than the rabbit that received the DNA prime plus protein boost. From the result, it seems that sera from a rabbit that received polyvalent DNA immunizations display broader neutralizing activities than those that received monovalent DNA immunization. These results suggest immunization with polyvalent vaccines is an effective method to broaden the neutralization breadth. In this pilot study, the Env antigens were randomly selected so not every Env was able to induce high Nab responses. Future studies should expand on our current report to screen for additional Env antigens from viruses circulating in China to identify those that can elicit strong Nab and use these to formulate truly effective polyvalent Env vaccines to elicit broadly neutralizing antibody responses. In this current study, the Env DNA vaccine did not use codon optimized sequences which can further improve the levels of antibody response. In addition, the protein boost only used one primary HIV-1 Env which does not match the DNA prime Env antigen. This may further reduce the quality of the Nab response. Producing recombinant proteins matching the DNA prime should further enhance the strength and breadth of anti-Env antibody responses.14


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human immunodeficiency virus type 1; DNA vaccine; DNA prime; protein boost; neutralizing antibody

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