DNA vaccines against human immunodeficiency virus type 1

Summary: Development of a vaccine against human immunodeficiency virus type 1 (HIV‐1) is the main hope for controlling the acquired immunodeficiency syndrome pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4⁺ helper T cells, and CD8⁺ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell‐mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. This review focuses on the stimulation of HIV‐specific T cells and discusses in the context of the current ‘state‐of‐art’ of DNA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development.     

Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines

DNA immunization permits evaluation of possible antagonistic or synergistic effects between the encoded components. The protein expression capacity in vitro was related to the immunogenicity in vivo of plasmids encoding the HIV-1 regulatory genes tat rev, and nef. Neither Tat nor Rev expression was influenced by co-expression in vitro of all three proteins, while Nef expression was slightly inhibited. With the combination of genes, the T-cellular responses of mice against Rev and Nef were inhibited compared with those when single gene immunization was used. No interference was detected for the Tat T-cell response. Thus, co-immunization with certain genes may result in inhibition of specific immune responses.     

抗人免疫缺陷病毒1型gp120人源单链抗体的表达

目的研究人源抗人免疫缺陷病毒１型（ＨＩＶ－１）ｇｐ１２０单链抗体（ＳｃＦｖ）。方法以人工合成的ＨＩＶ－ｌｇｐ１２０Ｖ３环多肽为抗原，利用噬菌体抗体库技术，筛选含有抗－ｇｐ１２０ＳｃＦｖ基因的噬菌体，提取质粒，转化大肠杆菌ＨＢ２１５１，表达可溶性ＳｃＦｖ。结果经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ分析，表达产物分子量为２８ｋＤ左右，且具有ｃ－ｍｙｃ活性；ＥＬＩＳＡ和Ｄｏｔｂｌｏｔ结果表明，可溶性ＳｃＦｖ具有较好的抗原结合活性和较强的特异性；竞争性ＥＬＩＳＡ实验结果进一步证明表达产物的特异抗－ｇｐ１２０活性。结论该技术便捷有效，可大量获得人源抗ＨＩＶ抗体片段，为进一步研究抗ＨＩＶ抗体的生物活性和ＨＩＶ感染诊治打下基础     

Stability of human immunodeficiency virus type 1 proviral DNA in whole-blood samples

The Roche Amplicor HIV-1 Test requires that whole blood be processed within four days of collection. However, this requirement may be too limiting for use in international settings. Thus, we demonstrate that blood may be processed up to 10 days after collection if maintained under ambient conditions (2 to 25 degrees C).     

Primary human immunodeficiency virus type 1 infection

Primary human immunodeficiency virus type 1 (HIV-1) infection represents the initial stage of disease that immediately follows viral entry into the body. Primary infection is frequently accompanied by an acute retroviral syndrome with associated high levels of plasma HIV-1 RNA and the development of host immune responses. The identification of subjects during this period requires a high index of suspicion and an understanding of how to make the diagnosis, as standard HIV-1 antibody tests can initially be negative. Identifying these people provides a unique opportunity for early counseling to reduce further transmission, facilitates entry into care, and allows for further study of the immunopathogenesis of disease and the potential role of early antiretroviral therapy.     

Variability of human immunodeficiency virus type 1

The variability of human immunodeficiency virus type 1 (HIV-1) is very high. To date, three distinct lineages of HIVs, type 1 group M, type 1 group O and type 2 are described, suggesting at least three different zoonotic infections. HIV-1 group M is responsible for the global epidemic of AIDS. At least ten subtypes of HIV-1 group M, labelled A through J, have been discovered. Viral sequences from both the gag and the env gene, particularly a part of gp 120 referred to as the V3 region have been used to identify subtypes of HIV-1 group M. The nucleotide distance between viruses of different subtypes is on average 30% for the env gene. The various subtypes are geographically distributed throughout the world. Some of the subtypes were identified as recombinant or mosaic viruses. The existence of different subtypes of HIV-1 have major implication for vaccination. They may also influence the diagnosis of HIV infection. To date, it is unclear whether subtypes of HIV-1 differ with respect to transmissibility or pathogenicity.     

Quantification of human immunodeficiency virus type 1 proviral DNA by using TaqMan technology

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.     

Quantitation of human immunodeficiency virus type 1 DNA by competitive nested PCR assay

A quantitative competitive nested PCR assay was developed for quantifying HIV-1 proviral DNA in clinical samples. A competitor DNA was constructed from a conserved region of the HIV-1 gag gene by deleting a sequence of 18 base pairs. We quantitated HIV-1 proviral DNA copy number in clinical samples. Peripheral blood mononuclear cells (PBMCs) from 35 HIV-infected patients with a CD4 count range of 4-728 cell/mm3 were analyzed by this method. The copy numbers of HIV-1 DNA detected ranged between 518 to 67,340 copies per 10(6) CD4+ T-cells. The copy numbers correlated inversely with the CD4 counts.     

Immunoprecipitation of human immunodeficiency virus type 2 glycoproteins by sera positive for human immunodeficiency virus type 1.

Analysis by radioimmunoprecipitation of serum samples from 27 different human immunodeficiency virus type 1 (HIV-1)-infected individuals residing in Chile showed that the sera of 26% of these individuals also react with glycoprotein gp125 of HIV type 2 (HIV-2). This cross-reaction seems to reflect a qualitative difference among infected individuals, because the titer of antibodies against gp120 of HIV-1 in the cross-reacting samples did not differ significantly from that in the non-cross-reacting samples. Most of the HIV-1-seropositive sera, including many that did not react with gp125 of HIV-2, reacted with gp140, the precursor of HIV-2 glycoproteins. The observed cross-reactions allowed us to distinguish three groups of HIV-1-infected individuals: (i) those whose sera react with both gp140 and gp125, (ii) those whose sera react with gp140, and (iii) those whose sera react with neither of these glycoproteins. The possible cause and significance of these differences is under study.     

人免疫缺陷病毒1型CRF07_BC毒株准种间的重组

目的 通过研究人类免疫缺陷病毒1型（human immunodeficiency virus1，HIV-1）感染者个体内准种间的差异，探讨HIV的系统进化的发生模式。方法 从HIV-1感染者血浆中提取总RNA，通过逆转录多聚酶链反应（RT-PCR）获得HIV．1gp120全长基因，纯化后装入T载体，转化至TOP10大肠埃希菌内增殖，通过蓝白斑筛选获得阳性克隆，对所获得的目的克隆测序并分析。结果 获得同一患者的16个克隆的gp120全长基因序列，通过系统进化树分析，克隆序列均为CRF07_BC亚型，但在系统进化树上16个克隆可明显分为A、B两群，其中13个克隆属于A群，2个克隆属于B群，1个克隆（编号XPD7）位于A、B群之间，通过simplot软件的重组分析，发现XPD7克隆为A群和B群的重组株。结论 发现了我国广泛流行的HIV-1CRF07-BC毒株准种间的重组现象，准种间的重组作为HIV进化的一种有效手段将导致HIV毒株的快速进化的发生，可能更易逃脱宿主的免疫监控。     

PCR-Based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells

An assay that quantifies the amount of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells has been developed. PCR amplification of the HIV-1 DNA is performed in the presence of an internal quantitation standard, and colorimetric detection of the amplified product is performed with microwell plates. The copies of HIV-1 DNA are normalized to total genomic DNA input. The assay has an analytical sensitivity of 10 input copies per amplification reaction and a three-log detection range. In an analysis of sequential samples from patients on combination therapy, HIV-1 DNA was quantifiable for all individuals tested, including those with undetectable plasma HIV-1 RNA. In a separate study, a comparison of HIV-1 DNA levels was made with a group of long-term survivors and progressors. The mean HIV-1 DNA levels were lower in the long-term survivors than in the progressors (P, 0.04). The mean HIV-1 RNA levels were also lower, but the difference was not statistically significant (P, 0.164). A quantitative DNA assay will provide an additional tool to gain insight into the natural history of infection and the continued efficacy of potent antiretroviral therapies.     

Effects of human T-lymphotropic virus type II on human immunodeficiency virus type 1 phenotypic evolution

Summary.  Phenotypic change and broader coreceptor usage by HIV-1 have been associated with disease progression. HIV-1 coreceptor usage by primary isolates obtained from HIV-1-infected and HIV-1/HTLV-II-coinfected individuals was determined. HIV-1 was isolated from 15 of 20 HIV-1-infected and 17 of 24 HIV-1/HTLV-II-coinfected individuals. None of the isolates from either the HIV-1-infected or the coinfected group infected CCR5Δ32 PBMCs, suggesting that they all were R5-tropic. Further, both spontaneous and PHA-stimulated production of MIP-1β and RANTES were similar in HIV-1-infected and coinfected individuals. These data indicate that coinfection with HTLV-II has no effect on HIV-1 coreceptor usage or ex vivo β-chemokine production. Received December 23, 2000/Accepted May 16, 2001     

Quantitation of human immunodeficiency virus type 1 in breast milk

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.     

Molecular clock-like evolution of human immunodeficiency virus type 1

The molecular clock hypothesis states that the rate of nucleotide substitution per generation is constant across lineages. If generation times were equal across lineages, samples obtained at the same calendar time would have experienced the same number of generations since their common ancestor. However, if sequences are not derived from contemporaneous samples, differences in the number of generations may be misinterpreted as variation in substitution rates and hence may lead to false rejection of the molecular clock hypothesis. A recent study has called into doubt the validity of clock-like evolution for HIV-1, using molecular sequences derived from noncontemporaneous samples. However, after separating their within-individual data according to sampling time, we found that what appeared to be nonclock-like behavior could be attributed, in most cases, to noncontemporaneous sampling, with contributions also likely to derive from recombination. Natural selection alone did not appear to obscure the clock-like evolution of HIV-1.     

Cellular latency of human immunodeficiency virus type 1.

The infection of humans by human immunodeficiency virus type 1 is characterized by a prolonged stage of clinical quiescence. This clinically asymptomatic period may be based, in part, on the development of cell populations within the body that maintain human immunodeficiency virus type 1 in a state of latency. Recent advances in the understanding of the molecular mechanisms involved in various forms of cellular latency of human immunodeficiency virus type 1 have begun to shed light on the variable period of asymptomatic infection. The elucidation of cellular retroviral latency, in vivo, will also be critical to the design of novel therapeutic approaches with which to combat human immunodeficiency virus type 1 infections.     

Actin-binding cellular proteins inside human immunodeficiency virus type 1

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.     

A recombinant fusion protein and DNA vaccines against foot-and-mouth disease virus type Asia 1 infection in guinea pigs

On the basis of amino acid (aa) sequence of the tandem repeat 133-158-20-34-133-158 which consisted of aa 133-158 of VP1 and aa 20-34 of VP4 of Foot-and-mouth disease virus (FMDV) type Asia 1 a recombinant prokaryotic expression vector pAS1-P encoding a fusion protein and eukaryotic expression vectors pAS1-E and pAS1-EdeltaCpG-ODN representing DNA vaccines were constructed. Guinea pigs immunized with these vaccines showed both neutralizing antibody and T cell proliferation responses. FMDV challenge tests for the first time showed that the recombinant fusion protein and pAS1-E and pAS1-EdeltaCpG-ODN vaccines protected 86%, 60% and 43% of guinea pigs from FMDV type Asia1 challenge, respectively. The results also indicated that the immune response of animals treated with the vector pAS1-E containing an oligodeoxynucleotide (ODN), which consisted of immunostimulatory cytosine-phosphate-guanosine (CpG) motifs, was augmented by CpG ODN.