Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins

"Centralized" (ancestral and consensus) HIV-1 envelope immunogens induce broadly cross-reactive T cell responses in laboratory animals; however, their potential to elicit cross-reactive neutralizing antibodies has not been fully explored. Here, we report the construction of a panel of consensus subtype B (ConB) envelopes and compare their biologic, antigenic, and immunogenic properties to those of two wild-type Env controls from individuals with early and acute HIV-1 infection. Glycoprotein expressed from full-length (gp160), uncleaved (gp160-UNC), truncated (gp145), and N-linked glycosylation site deleted (gp160-201N/S) versions of the ConB env gene were packaged into virions and, except for the fusion defective gp160-UNC, mediated infection via the CCR5 co-receptor. Pseudovirions containing ConB Envs were sensitive to neutralization by patient plasma and monoclonal antibodies, indicating the preservation of neutralizing epitopes found in contemporary subtype B viruses. When used as DNA vaccines in guinea pigs, ConB and wild-type env immunogens induced appreciable binding, but overall only low level neutralizing antibodies. However, all four ConB immunogens were significantly more potent than one wild-type vaccine at eliciting neutralizing antibodies against a panel of tier 1 and tier 2 viruses, and ConB gp145 and gp160 were significantly more potent than both wild-type vaccines at inducing neutralizing antibodies against tier 1 viruses. Thus, consensus subtype B env immunogens appear to be at least as good as, and in some instances better than, wild-type B env immunogens at inducing a neutralizing antibody response, and are amenable to further improvement by specific gene modifications.     

Ancestral and consensus envelope immunogens for HIV-1 subtype C

Immunogens based on "centralized" (ancestral or consensus) HIV-1 sequences minimize the genetic distance between vaccine strains and contemporary viruses and should thus elicit immune responses that recognize a broader spectrum of viral variants. However, the biologic, antigenic and immunogenic properties of such inferred gene products have to be validated experimentally. Here, we report the construction and characterization of the first full-length ancestral (AncC) and consensus (ConC) env genes of HIV-1 (group M) subtype C. The codon-usage-optimized genes expressed high levels of envelope glycoproteins that were incorporated into HIV-1 virions, mediated infection via the CCR5 co-receptor and retained neutralizing epitopes as recognized by plasma from patients with chronic HIV-1 subtype C infection. Guinea pigs immunized with AncC and ConC env DNA developed high titer binding, but no appreciable homologous or heterologous neutralizing antibodies. When tested by immunoblot analysis, sera from AncC and ConC env immunized guinea pigs recognized a greater number of primary subtype C envelope glycoproteins than sera from guinea pigs immunized with a contemporary subtype C env control. Mice immunized with AncC and ConC env DNA developed gamma interferon T cell responses that recognized overlapping peptides from the cognate ConC and a heterologous subtype C Env control. Thus, both AncC and ConC env genes expressed functional envelope glycoproteins that were immunogenic in laboratory animals and elicited humoral and cellular immune responses of comparable breadth and magnitude. These results establish the utility of centralized HIV-1 subtype C Env immunogens and warrant their continued evaluation as potential components of future AIDS vaccines.     

Subtype-specific conservation of isoleucine 309 in the envelope V3 domain is linked to immune evasion in subtype C HIV-1 infection

The V3 region of the HIV-1 envelope (Env) glycoprotein gp120 is a key functional domain yet it exhibits distinct mutational patterns across subtypes. Here an invariant residue (Ile 309) was replaced with Leu in 7 subtype C patient-derived Envs from recent infection and 4 related neutralizing antibody escape variants that emerged later. For these 11 Envs, I309L did not alter replication in primary CD4 T cells; however, replication in monocyte-derived macrophages was enhanced. Infection of cell lines with low CD4 or CCR5 revealed that I309L enhanced utilization of CD4 but did not affect the ability to use CCR5. This CD4-enhanced phenotype tracked with sensitivity to sCD4, indicating increased exposure of the CD4 binding site. The results suggest that Ile 309 preserves a V3-mediated masking function that occludes the CD4 binding site. The findings point to an immune evasion strategy in subtype C Env to protect this vulnerable immune target.     

Factors limiting the immunogenicity of HIV-1 gp120 envelope glycoproteins

Efficient immune responses to HIV-1 gene products are essential elements to the development and design of an effective vaccine. Ideally, both humoral and cellular responses will be optimally elicited. It is therefore important to elucidate any factors that might limit the immunogenicity of HIV-1 proteins that are likely to be included in an effective vaccine. Since the HIV-1 exterior envelope glycoprotein gp120 is a major target for neutralizing antibodies, it is a virtual certainty that this gene product will be a component of any vaccine that seeks to elicit neutralizing antibody responses from the host humoral immune system. We report here the testing of several HIV-1 gp120 variants derived from a primary isolate that appears deficient in eliciting immune responses at both the level of CD4+ help and consequently in the generation of high-affinity IgG antibody responses in small animals. Factors limiting an effective immune response include (a) envelope glycoprotein strain variation decreasing functional T-cell help, (b) alteration of the glycosylation patterns of gp120 by expression in different cell types, and (c) the native structure of gp120 itself, which may limit the elicitation of effective T-cell help during natural infection or during parenteral immunization in adjuvant. Such limiting factors and others should be considered in the design and testing of gp120-based immunogens in small animals and possibly in primates as well.     

Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120

In order to improve the immunogenicity of HIV-1 envelope glycoproteins, we have fused gp120 to a carrier protein, hepatitis B surface antigen (HBsAg), which is capable of spontaneous assembly into virus-like particles. The HBsAg-gp120 hybrid proteins assembled efficiently into 20-30 nm particles. The particles resemble native HBsAg particles in size and density, consistent with a lipid composition of about 25% and a gp120 content of about 100 per particle. Particulate gp120 folds in its native conformation and is biologically active, as shown by high affinity binding of CD4. The particles express conformational determinants targeted by a panel of broadly cross-reactive neutralizing antibodies, and they show tight packing of gp120. Because the particles are lipoprotein micelles, an array of gp120 on their surface closely mimics gp120 on the surface of HIV-1 virions. These gp120-rich particles can enhance the quality, as well as quantity, of antibodies elicited by a gp120 vaccine.     

HIV-1 黑龙江省分离株CHNHLJ03009包膜克隆及分析

目的 分析黑龙江省Ⅰ型人免疫缺陷病毒（human irnmunodeficiency virus type 1，HIV-1）原代分离株包膜糖蛋白的变异性及表型特征。方法从一名HIV阳性但未发病的感染者外周血单个核细胞提取DNA，采用保守引物进行HIV包膜直接克隆，进行序列分析及系统树分析。构建了一株带该包膜蛋白的伪病毒并利用表达CXCR4和CCR5的靶细胞进行了感染实验，观察该病毒包膜利用辅助受体的表型特征。结果共获得2个有功能全长env克隆，分别命名为CHNHIJ03009c34（GenBank序列号为AY905493）和CHNHIA03009c33。用全长env氨基酸序列与国内外分离株进行同源性比较分析发现，与分离株CHNHLJ03009c34的包膜同源性最高的病毒为云南的HIV-1 B’亚型分离株RIA2，同源性为91．52％。系统发育分析结果表明，该株为泰国B’亚型，与云南分离株RIA2的遗传距离最近。对包膜蛋白结构分析结果显示，分离株CHNHLJ03009c34包膜在抗原性和亲水性上与RIA2没有明显差别。感染性检测结果显示该病毒只能感染U87．CD4．CCR5细胞，不能感染U87．CD4．CX-CR4细胞。结论本研究从黑龙江省一名HIV-1阳性者克隆到HIV-1CHNHLJ03009病毒的包膜基因，该病毒属于B’亚型（泰国亚型），为R5亲嗜性毒株。该包膜基因克隆为首次报道的黑龙江分离株。     

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.     

Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves protein conformation and entry function

The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at eliciting humoral immunity. Env's extensive and heterogeneous N-linked glycosylation affects folding, binding to lectin receptors, antigenicity and immunogenicity. We characterized recombinant Env proteins and virus particles produced in mammalian cells that lack N-acetylglucosaminyltransferase I (GnTI), an enzyme necessary for the conversion of oligomannose N-glycans to complex N-glycans. Carbohydrate analyses revealed that trimeric Env produced in GnTI^−/− cells contained exclusively oligomannose N-glycans, with incompletely trimmed oligomannose glycans predominating. The folding and conformation of Env proteins was little affected by the manipulation of the glycosylation. Viruses produced in GnTI^−/− cells were infectious, indicating that the conversion to complex glycans is not necessary for Env entry function, although virus binding to the C-type lectin DC-SIGN was enhanced. Manipulating Env's N-glycosylation may be useful for structural and functional studies and for vaccine design.     

Cloning and analysis of the envelope protein clone of HIV-1, CHNHLJ03009c34 from an infected individual in Heilongjiang province

Envelopeglycoprotein(Env)ofHIV1isacom plexoftwononcovalentlyassociatedsubunits,Gp120andGp41.Gp120isanexternalsubunit thatbindsthecellularreceptorCD4andachemo kinereceptor,suchasCXCR4orCCR5.Gp41isatransmembranesubunit,responsibleforthere ceptor mediatedmembranefusion[1,2].Because ofthehighvariabilityofHIV1,theaminoacid sequenceaswellasthestructureofviralenvelope canvaryandresultinthechangeofviraltropism withtimeextensionanddiseaseprogressionafter infection.DuringtheearlyphaseofHIVinfec…     

A group M consensus envelope glycoprotein induces antibodies that neutralize subsets of subtype B and C HIV-1 primary viruses

HIV-1 subtype C is the most common HIV-1 group M subtype in Africa and many parts of Asia. However, to date HIV-1 vaccine candidate immunogens have not induced potent and broadly neutralizing antibodies against subtype C primary isolates. We have used a centralized gene strategy to address HIV-1 diversity and generated a group M consensus envelope gene with shortened consensus variable loops (CON-S) for comparative studies with wild-type (WT) Env immunogens. Our results indicate that the consensus HIV-1 group M CON-S Env elicited cross-subtype neutralizing antibodies of similar or greater breadth and titer than the WT Envs tested, indicating the utility of a centralized gene strategy. Our study also shows the feasibility of iterative improvements in Env immunogenicity by rational design of centralized genes.     

Functional properties of the HIV-1 subtype C envelope glycoprotein associated with mother-to-child transmission

Understanding the properties of viruses capable of establishing infection during perinatal transmission of HIV-1 is critical for designing effective means of limiting transmission. We previously demonstrated that the newly transmitted viruses (in infant) were more fit in growth, as imparted by their envelope glycoproteins, than those in their corresponding mothers. Here, we further characterized the viral envelope glycoproteins from six mother-infant transmission pairs and determined whether any specific envelope functions correlate with HIV-1 subtype C perinatal transmission. We found that most newly transmitted viruses were less susceptible to neutralization by their maternal plasma compared to contemporaneous maternal viruses. However, the newly transmitted variants were sensitive to neutralization by pooled heterologous plasma but in general were resistant to IgG1 b12. Neither Env processing nor incorporation efficiency was predictive of viral transmissibility. These findings provide further insight into the characteristics of perinatally transmissible HIV-1 and may have implications for intervention approaches.     

Murine retroviral vector that induces long-term expression of HIV-1 envelope protein

A retroviral vector was constructed that induces long-term expression of human immunodeficiency virus type 1 (HIV-1) rev, vpu and env genes. The vector contains the neo gene and a cytomegalovirus (CMV) immediate early promoter followed by HIV-1 sequence. When HeLa cells were infected with viral stocks derived from this vector, about 25% of the resulting G418-resistant clones expressed HIV-1 envelope protein (Env), easily detectable by Western blot analysis, metabolic labelling, and syncytium formation after co-cultivation with HeLa-CD4 cells. In most cases the level of Env expression was higher than in a T cell line (H9) chronically infected with HIV-1. Env-expressing HeLa cell lines also expressed Rev, detected by transfection with a Rev-dependent CAT gene construct, and Vpu, detected by immunoprecipitation with a Vpu-specific antiserum. The 75% of G418-resistant HeLa cell lines that did not express Env were found to contain proviruses that had undergone deletion of env sequences corresponding to a known intron; presumably these cell lines arose as a result of infection with virions derived from spliced RNAs. This vector should be useful for studying non-transient effects of HIV Env, Rev and Vpu in tissue culture, and for the production of Env- and/or Rev-expressing cell lines.     

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope.

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.     

Molecular determinants of HIV-1 subtype C coreceptor transition from R5 to R5X4

The molecular mechanism(s) underlying transition from CCR5 to CXCR4 usage of subtype C viruses remain largely unknown. We previously identified a subtype C HIV-1 infected child whose virus demonstrated CXCR4 usage along with CCR5 upon longitudinal follow-up. Here we delineated the molecular determinants of Env involved in expanded coreceptor usage. Residue changes in three positions of Env V3 domain are critical for the dual tropic phenotype. These include: substitution of arginine at position 11, MG or LG insertion between positions 13 and 14, and substitution of threonine at the position immediately downstream of the GPGQ crown. Introducing these mutations into V3 region of a heterologous R5 virus also conferred dual tropism. Molecular modeling of V3 revealed a possible structural basis for the dual tropic phenotype. Determining what defines a subtype C X4 virus will lead to a better understanding of subtype C HIV-1 pathogenesis, and will provide important information relevant to anti-retroviral therapy.     

Preparation and characterization of three monoclonal antibodies against HIV-1 p24 capsid protein

HIV-1 p24 detection provides a means to aid the early diagnosis of HIV-1 infection, track the progression of disease and assess the efficacy of antiretroviral therapy. In the present study, three monoclonal antibodies （mAbs） p3JB9, p5F1 and p6F4 against HIV-1 p24 were generated. All mAbs could detect p24 of HIV-1ⅢB, HIV-1Ada-M, HIV-174v mAbs p5F1 and p6F4 could detect HIV-1KM018, while p3JB9 could not. Three mAbs did not react with HIV-2ROD, HIV-2CBL-20 and SIVagmTYO-1. The recognized epitope of p5F1 was located on the Gag amino acid region DCKTILKALGPAATLEEMMTAC. The p5F1 was used to establish a modified sandwich ELISA with rabbit anti-p24 serum and showed good specificity and high sensitivity, which has been used to measure HIV-1 p24 antigen levels in research. Cellular ＆ Molecular Immunology.     

Inter-subunit disulfide bonds in soluble HIV-1 envelope glycoprotein trimers

Soluble forms of the trimeric human immunodeficiency virus (HIV-1) envelope glycoproteins are important tools for structural studies and in the construction of improved immunogens. We found that a substantial fraction of soluble envelope glycoprotein trimers contain inter-subunit disulfide bonds (inter-S-S bonds) that render the trimers resistant to heat and denaturing agents. These inter-S-S bonds can be reduced without disrupting the trimers by treatment with a low concentration of beta-mercaptoethanol or DTT. Antibody mapping studies suggest that the soluble HIV-1 envelope glycoprotein trimers lacking the inter-S-S bonds exhibit a conformation closer to that of the native HIV-1 envelope glycoprotein complex. However, reducing these inter-S-S bonds had only modest effects on the inefficient elicitation of neutralizing antibodies by the soluble trimers. These studies provide guidance in improving the resemblance of tractable, soluble forms of the HIV-1 envelope glycoproteins to the native virion spikes.     

Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates

We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140DeltaV2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV(SF162P4) virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140DeltaV2TV1 (subtype C DeltaV2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C DeltaV2 trimer; however, we did not observe significant binding for the b12 mAb. The molecular mass of subtype C DeltaV2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C DeltaV2 trimer binds to CD4 with an affinity comparable to o-gp140DeltaV2SF162 (subtype B DeltaV2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C DeltaV2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.     

Subtle alteration of residues including N-linked glycans in V2 loop modulate HIV-1 neutralization by PG9 and PG16 monoclonal antibodies

Recent discovery of several potent and broadly neutralizing monoclonal antibodies (MAbs) (such as PG9 and PG16) to HIV-1 provided clues on newer vaccine targets. In the present study, we found an env clone obtained from a slow progressor showing significant resistance to PG9 and PG16 MAbs in sharp contrast to other contemporaneous autologous env clones. By constructing chimeric envelopes and specific substitutions we found that both loop length and spatial orientation of glycan residues in addition to the net charge of the β sheet C region that directly binds to PG9 CDRH3 within V2 loop significantly modulated HIV-1 sensitivity to PG9 and PG16 MAbs. Similar observation were made with several other Envs which varied in length, glycan content and net charge in PG9 contacting complementary region in V2 loop. Our data indicated that subtle change within V2 loop alone modulates exposition of quaternary epitopes that are targets of PG9/PG16 MAbs.     

Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.