Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins

To understand the differential expression of epitopes on monomeric and oligomeric forms of the envelope glycoproteins, nine human monoclonal antibodies (mAbs) were derived from the cells of human immunodeficiency virus-infected subjects by selection with soluble oligomeric gp140 (o.140). These nine mAbs and 12 human mAbs selected with V3 peptides, viral lysates, and rgp120, specific for the V2, V3, C5, CD4-binding domain (CD4bd), and gp41, were tested in a binding assay to compare the exposure of these regions on monomeric gp120 or gp41 and on o.140. None of the 21 mAbs were oligomer specific. However, mAbs to V3 and CD4bd were "oligomer sensitive," whereas mAbs to V2 and the distal epitope of C5 tended to be "monomer sensitive" (i.e., to react better with the oligomer or monomer, respectively). The majority of anti-gp41 mAbs reacted similarly with monomer and oligomer. Although the uncleaved o.140 used in this study differs from the cleaved gp120/41 oligomer found on the native virus particle, these results suggest that new epitopes are not introduced by oligomerization of viral envelope proteins, that such oligomer-specific epitopes, if they exist, are not highly immunogenic, and/or that they are not efficiently selected using soluble o.140.     

Identification of immunoglobulin recombinant elements in human immunodeficiency virus type 1 envelope gene.

Recently, it has been recognised that the amino acid motif VQL(N/V)ES is shared between the second conserved domain of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and the first framework (FW1) of human IgG heavy chain variable region (subgroup III) (VHIII). We have found that nucleotide sequence corresponding to this motif contains some recombination elements characteristic for the genes of human Ig heavy-chain variable region. The possible role of the Ig recombination elements in HIV-1 envelope gene variability, AIDS pathogenesis and vaccine design is discussed.     

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.     

Immunogenicity and ability of variable loop-deleted human immunodeficiency virus type 1 envelope glycoproteins to elicit neutralizing antibodies

It has been extremely difficult to elicit broadly cross-reactive neutralizing antibodies (Nabs) against human immunodeficiency virus type 1 (HIV-1). In this study, we compared the immunogenic properties of the wild-type and variable loop-deleted HIV-1 envelope glycoproteins. Mice were immunized with recombinant vaccinia viruses expressing either the wild-type or the variable loop-deleted (V1-2, V3, V4, and V1-3) HIV-1(DH12) gp160s. The animals were subsequently boosted with respective recombinant gp120s. All envelope constructs elicited similar levels of gp120-binding antibodies when analyzed by enzyme-linked immunosorbent assay (ELISA). However, the highest neutralizing activity was observed in sera from animals immunized with the wild-type envelope protein, followed by those immunized with DeltaV4 and DeltaV1-2. No neutralizing activity was detected in sera from animals immunized with DeltaV3 or DeltaV1-3. To identify immunogenic epitopes, ELISA was performed with overlapping 15-mer peptides that cover the entire length of gp120. For the wild-type gp120, the immunogenic epitopes mapped primarily to the variable loops V1-2 and to the conserved regions C1 and C5. When they were plotted onto known coordinates of gp120 core crystal structure, the epitopes in the conserved regions mapped predominantly to the inner domain of the protein. By immunizing with variable loop-deleted envelopes, the immune responses could be redirected to other regions of the protein. However, the newly targeted epitopes were neither on the exposed surface of the protein nor on the receptor binding regions. Interestingly, the removal of the V3 loop resulted in loss of immunoreactivity for both V3 and V1/V2 loops, suggesting structural interaction between the two regions. Our results suggest that obtaining broadly reactive Nabs may not be achieved simply by deleting the variable loops of gp120. However, the observation that the immune responses could be redirected by altering the protein composition might allow us to explore alternative strategies for modifying the antigenic properties of HIV-1 envelope glycoprotein.     

人类免疫缺陷病毒-1包膜糖蛋白gp41的表达及优化

目的 优化HIV-1本地流行株06044 gp41蛋白表达设计,为gp41免疫原的制备提供实验基础.方法 以含HIV-1 06044 gp41基因的质粒为模板,采用聚合酶链反应(PCR)扩增gp41目的基因,分别构建原核表达载体pET26b-gp41T(含546～683 aa片段)及去掉N-端(NHR)和C-端(CHR)七价重复序列中间部分loop区(582～627aa)的pET26b-gp41 T(△L),经测序确认后,转化至大肠杆菌感受态细胞BL21 (DE3)中,IPTG诱导表达.用聚丙烯酰氨凝胶电泳(SDS-PAGE)及Western blot检测并鉴定重组表达蛋白.优化了重组蛋白的诱导表达条件.结果 pET26b-gp41T和pET26b-gp4 1T (△L)重组表达质粒均能表达gp41蛋白,gp41T (△L)蛋白表达量高于gp41T;不同IPTG浓度诱导的蛋白表达量没有区别;用1 mmol/L IPTG诱导后,37℃条件下表达量最高.Western blot结果显示,gp41T (△L)与His抗体结合性能好.结论 实验获得了稳定表达HIV-1 06044 gp41的原核表达载体以及表达条件,为大量制备gp41蛋白免疫原奠定了基础.     

人免疫缺陷病毒1(HIV-1)膜蛋白基因片段在酵母中的克隆表达

目的为获得足够量的膜糖蛋白,以便于对不同ＨＩＶ分离株膜糖蛋白的结构与功能进行进一步的研究。方法从人免疫缺陷病毒１（ＨＩＶ－１）ＨＸＢ２分离株原病毒基因组的重组质粒ｐＨＸＢ２中克隆了两段膜糖蛋白基因（ＥＮＶ）片段。以酵母穿梭诱导表达质粒ｐＹＥＳ２为载体,构建了两个相应的重组表达质粒ｐＹＥＮＶ１和ｐＹＥＮＶ２;进一步利用大肠杆菌β－半乳糖苷酶基因（β－ｌａｃＺ）构建了ＨＩＶ－１膜外糖蛋白ＤＮＡ片段与β－ｌａｃＺ基因的融合表达质粒。将此３种质粒分别转化单细胞真核生物酿酒酵母ＢＪ１９９１,得到的转化子经半乳糖诱导表达后进行菌体全蛋白的ＳＤＳ－ＰＡＧＥ分析。结果克隆的基因片段在酿酒酵母中产生了分子质量为５０×１０３的特异性诱导蛋白;对含此融合表达质粒的酵母转化子半乳糖诱导后表达产物的免疫检测表明,与对照菌株相比,融合表达产物具有和ＨＩＶ－１阳性血清抗体反应的抗原性。结论可通过β－半乳糖苷酶活性的测定直接指示抗原片段的表达;为表达的膜糖蛋白片段的进一步分离纯化打下了一定基础     

Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.     

Effect of rev on the cytoplasmic localization of intron-containing human immunodeficiency virus type 1 RNA

Human immunodeficiency virus type 1 (HIV-1) proteins are expressed from both intron-containing and completely spliced RNAs. Rev, an HIV-1 regulatory protein, is necessary for the expression of intron-containing RNAs. The effect of Rev on the subcellular localization of intron-containing HIV-1 RNA was examined by in situ RNA hybridization. In the presence of Rev, intron-containing HIV-1 RNA accumulated at the nuclear membrane and within the cytoplasm of transfected cells. In the absence of Rev, intron-containing HIV-1 RNA accumulated within the nucleus. In approximately 20% of the cells transfected in the absence of Rev, intron-containing HIV-1 RNA was also found in the cytoplasm. Differences in the subcytoplasmic localization of intron-containing HIV-1 RNA in the presence and absence of Rev were not observed using in situ RNA hybridization. To determine the effect of Rev on RNA localization within the cytoplasm, an extensive fractionation protocol involving both hypotonic and detergent lysis was used. In the presence of Rev, 40.9 +/- 4.6% of the cytoplasmic intron-containing HIV-1 RNA was released by hypotonic lysis. A similar fractionation profile was seen for several other translated viral and cellular RNAs. However, in the absence of Rev, only 16.5 +/- 5.1% of the cytoplasmic intron-containing HIV-1 RNA was released on hypotonic lysis (P < 0. 005). Thus the cytoplasmic fractionation pattern of this RNA was altered in the absence of Rev.     

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.     

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.     

Purification of envelope glycoproteins of human T cell lymphotropic virus type I (HTLV-I) by affinity chromatography

The external envelope glycoprotein (gp46) and transmembrane glycoprotein (gp21) of human T-cell lymphotropic virus type I (HTLV-I) were isolated from lysates of HTLV-I-infected HUT-102 cells by affinity chromatography. Fifty ml aliquots of packed HUT-102 cells were extracted with 1% Triton X-100, and lysates were treated sequentially with an affinity column containing IgG from an HTLV-I+ human subject followed by chromatography of the bound fraction over a lentil lectin column. The identity of the purified envelope proteins was confirmed with a human monoclonal antibody (0.5 alpha) to gp46 and with rabbit antisera raised to a synthetic peptide from the C-terminus of gp21. Affinity-purified envelope glycoproteins were bound to microtiter wells and used in radioimmunoassay to detect murine and human anti-envelope antibodies to gp46 and gp21 molecules.     

A genotypic resistance assay for the detection of drug resistance in the human immunodeficiency virus type 1 envelope gene

Since it is not clear yet whether enfuvirtide resistance is restricted to gp41, it was decided to develop a genotypic assay for the detection of drug resistance in the entire human immunodeficiency virus type 1 (HIV-1) env gene. Given the increasing prevalence of HIV-1 non-B subtypes in Europe, it is important to evaluate the performance of the assay on a panel of genetically divergent samples. A panel of 1 laboratory and 10 clinical isolates from 10 patients was tested, all enfuvirtide naive and chosen according to the subtype as determined in the pol region (A, B, C, H, CRF01-AE, CRF02-AG, CRF05-DF, CRF11-cpx and U), while their env sequences belonged to subtypes A, B, C, H, A/G recombinant, B/H recombinant, CRF01-AE, CRF02-AG, CRF05-DF and CRF11-cpx. The detection limits of the gp120 and the gp41 PCRs ranged between 500 and 5000 RNA copies/ml plasma. The highest sensitivity was obtained for the laboratory strain, whereas the detection limit for all patient samples, except for the subtype C sample, was 1000 RNA copies/ml. The numerous insertions and deletions in the gp120 gene, that were often present as quasi-species, necessitated the sequencing of cloned PCR products. The gp41 gene displayed less diversity and less insertions/deletions. Especially, the heptad repeat 1 was highly conserved and none of the enfuvirtide naive samples contained any of the already known enfuvirtide resistance mutations at amino acid positions 36-45. This study demonstrates that the assay is able to genotype genetically diverse HIV-1 strains with a good sensitivity.     

Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function.

Transmembrane envelope protein (TM) residues 100, 105, and 128 of human immunodeficiency virus type 1 (HIV-1) strain HXB2 are potential sites for asparagine-linked oligosaccharide additions which are conserved among HIV-1 isolates, and all other lentivirus TM proteins. Site-specific mutants of each of the asparagine residues did not eliminate the ability of the virus to infect and replicate in CD4+ cells, but infectivity was reduced with all of these mutants, and syncytia induction was attenuated with two of these mutants. Studies of envelope expression of the mutant with the most severe defect demonstrated no significant effects on envelope protein synthesis, conformation, processing, multimerization, or release into the culture medium, suggesting that N-linked oligosaccharides are important in the specific fusion activity of TM.     

Neutralization and infectivity characteristics of envelope glycoproteins from human immunodeficiency virus type 1 infected donors whose sera exhibit broadly cross-reactive neutralizing activity

In this study, we tested the hypothesis that donors with broadly cross-reactive HIV-1 neutralizing (BCN) sera are infected with viruses encoding envelope glycoproteins (Envs) with unusual immunogenic properties. Cloned env genes were from samples of donors previously identified as having BCN antibodies (BCN donors) and from other donors not known to have such antibodies (non-BCN donors). Neutralization properties of viruses pseudotyped with BCN and non-BCN Envs were determined using BCN, non-BCN sera and broadly cross-neutralizing monoclonal antibodies (Mabs). BCN sera neutralized with higher frequency and geometric mean titers than non-BCN sera. Viruses pseudotyped with BCN Envs were mostly resistant to neutralization by anti-gp120 Mabs but tended to be more sensitive to the anti-gp41 Mabs, 2F5 and 4E10 than non-BCN Env-pseudotyped viruses. Sequence analysis of clones obtained from sequential samples of two BCN donors revealed respective 2F5 epitope mutations T662A and K665T. The K665T mutation evolved as the predominant genotype in the respective donor, consistent with an escape mutation event. The A662T mutation reduced sensitivity to 4E10, as well as 2F5 and homologous sera, consistent with neutralization escape mutation and targeting of the 2F5 epitope region by the serum. Our study suggests that viruses infecting these BCN donors encoded Envs that may have been unusually competent for induction of antibodies against the membrane proximal epitope region (MPER) of gp41, and these Envs may be useful vaccine components.     

Immunological analysis of human immunodeficiency virus type 1 envelope glycoprotein proteolytic cleavage.

Two potential cleavage sites have been identified on precursor gp 160 of human immunodeficiency virus type 1. Using antibodies directed against the C-terminus of gp 120, including the sequence between the two sites, we have shown that nonmutated viral and recombinant gp 160 are cleaved at both sites: the great majority of molecules are cleaved at site 1 (Arg-Glu-Lys-Arg), and gp41 can then associate as an oligomer; a minority of molecules are cleaved at site 2 (Lys-Ala-Lys-Arg-Arg) and the corresponding gp41 appears to present as a monomer. This could reflect two different processing pathways for gp41 biosynthesis, one of which only may result in biologically active molecules according to the literature.     

Epitopes of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins recognized by antibodies in the sera of HIV-1-infected individuals.

Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENVgp) that were recognized by antibodies. Two epitopes of HIV ENVgp, ENVP466 (amino acids 466-481) and ENVP497 (amino acids 497-509), were recognized by antibodies in the sera of most HIV-infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIV's ENVgp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections.     

Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope glycoproteins of Lassa virus

A recombinant vaccinia virus that expresses the nucleoprotein gene of Lassa virus (Josiah strain) under the control of the P7.5 promoter was constructed using the lacZ coexpression transfer vector pSC11. Southern blot analysis demonstrated that recombination of the sequences inserted within the thymidine kinase gene of the transfer vector into the HindIII J fragment of vaccina virus genomic DNA occurred properly. A 63-kDa protein identical in electrophoretic mobility to authentic Lassa nucleoprotein was observed in recombinant virus-infected cell lysates. The reactivity of vaccinia-expressed Lassa proteins to a panel of monoclonal antibodies representing multiple epitopes on each of the N, G1, and G2 proteins was determined by indirect immunofluorescence. Lassa proteins expressed in recombinant vaccinia virus-infected cells reacted in a manner indistinguishable from that of the proteins expressed in Lassa virus-infected cells, indicating that there are no significant differences between authentic and recombinant virus-expressed proteins. Vaccine efficacy trials in guinea pigs indicated that both the nucleoprotein and the envelope glycoproteins are capable of eliciting a protective immune response against a lethal dose of Lassa virus. Ninety-four percent of the animals vaccinated with V-LSN, 79% vaccinated with V-LSGPC, and 58% vaccinated with both recombinant viruses survived a Lassa virus challenge in which only 14% of unvaccinated animals and 39% of animals vaccinated with the New York Board of Health (NYBH) strain of vaccinia virus survived. The protection resulting from vaccination with the recombinant virus vaccines did not correlate with the levels of prechallenge serum antibodies, suggesting that a cell-mediated immune response is a critical component of protective immunity to Lassa fever.