Human Immunodeficiency Virus Env Research Articles

Improving the expression of recombinant soluble HIV Envelope glycoproteins using pseudo-stable transient transfection

The Envelope glycoprotein (Env) of the human immunodeficiency virus (HIV) is the target of neutralizing antibodies (NAbs). So far, HIV Env-derived immunogens have not been able to elicit broad neutralizing antibody responses against primary isolates. Identifying conditions that will permit the efficient production of different soluble HIV Env proteins will facilitate a high throughput comparative analysis of the immunogenicity of diverse Env constructs, potentially identifying Env forms that are more conducive to the elicitation of anti-HIV NAbs. Here we compared different cell types, transfection reagents, transfection conditions and different DNA expression vectors on soluble HIV Envelope expression levels. We identified optimal expression conditions and developed a protocol to streamline and maximize production of diverse HIV Env constructs. Using this optimized platform, milligram quantities of purified soluble HIV Env trimer can be routinely achieved in a rapid and cost-effective manner.

The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.

The CXCR4-Tropic Human Immunodeficiency Virus Envelope Promotes More-Efficient Gene Delivery to Resting CD4+T Cells than the Vesicular Stomatitis Virus Glycoprotein G Envelope

Current gene transfer protocols for resting CD4(+) T cells include an activation step to enhance transduction efficiency. This step is performed because it is thought that resting cells are resistant to transduction by lentiviral-based gene therapy vectors. However, activating resting cells prior to transduction alters their physiology, with foreseeable and unforeseeable negative consequences. Thus, it would be desirable to transduce resting CD4(+) T cells without activation. We recently demonstrated, contrary to the prevailing belief, that wild-type human immunodeficiency virus (HIV) integrates into resting CD4(+) T cells. Based on that finding, we investigated whether a commonly used, vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped lentiviral gene therapy vector could also integrate into resting CD4(+) T cells. To investigate this, we inoculated resting CD4(+) T cells with lentiviral particles that were pseudotyped with VSV-G or CXCR4-tropic HIV Env and assayed binding, fusion, reverse transcription, and integration. We found that the VSV-G-pseudotyped lentiviral vector failed to fuse to resting CD4(+) T cells while HIV Env-pseudotyped lentiviral vectors fused, reverse transcribed, and integrated in resting cells. Our findings suggest that HIV Env could be used effectively for the delivery of therapeutic genes to resting CD4(+) T cells and suggest that fusion may be the critical step restricting transduction of resting CD4(+) T cells by lentiviral gene therapy vectors.

Human Immunodeficiency Virus (HIV) Antigens and RNA in HIV-Seronegative Women with Cervical Intraepithelial Neoplasia

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase-deficient (Tk(-)) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk(-) rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins.

Chemokine receptors and its importance in the replication cycle of human immunodeficiency virus: clinical and therapeutic implications

Human immunodeficiency virus (HIV) infection of host cells begins with binding of viral envelope (Env) surface (SU) glycoprotein to specific receptors present at plasma cell membrane. One of these receptors is the CD4 molecule which can be found namely in T-helper lymphocytes (T-CD4+), macrophages and dendritic cells. Binding of SU glycoprotein to CD4 molecule, enables HIV to adsorb to target cell membrane and also initiates conformational changes in SU glycoprotein that enable it to bind to a second receptor (coreceptor). This coreceptor belongs to a family of plasma cell membrane molecules that acts, in vivo, as chemokine receptors. The SU glycoprotein binding to coreceptor molecule triggers further conformational changes and consequently the exposure of a hydrophobic region of viral envelope transmembrane glycoprotein (TM), named fusion peptide that ultimately leads to viral envelope fusion with target cell membrane. Nowadays, 19 of these chemokine receptors have been thus considered, in vitro, as coreceptors for HIV. Interestingly, despite this extensive range of potential coreceptors, only CCR5 and CXCR4 seem to be relevant in HIV transmission and in the pathogenesis of HIV infection. Identification of cell surface coreceptors, specific for HIV envelope SU glycoprotein, has provided an elucidative explanation for molecular mechanisms involved in viral cell tropism and pathogenesis. Furthermore, the recognition of a coreceptor-mediated HIV's entry has also provided novel viral and cellular targets for antiretroviral intervention. During the last few years, the inhibition of HIV entry has become an incontestable target for anti-HIV drug discovery. Enfuvirtide is one example of these new antiretroviral molecules. It is the only member of fusion inhibitors targeting fusion peptide region, which prevents HIV entry by blocking the TM-mediated fusion between viral envelope and plasma cell membrane. More recently, CCR5-specific antagonists have been described, including monoclonal antibodies, modified chemokines and more importantly small-molecules inhibitors, such as maraviroc and vicriviroc. These drugs prevent SU glycoprotein binding to CCR5 coreceptor, and thus inhibiting HIV entry into target cell. This review will focus on the influence of coreceptor engagement, by HIV Env glycoproteins, in viral replication cycle and the importance of targeting its coreceptor function, by specific inhibitors, as a new and promising class of antiretrovirals.

Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes

The human immunodeficiency virus 1 (HIV-1) envelope regulates the initial attachment of viral particles to target cells through its association with CD4 and either CXCR4 or CCR5. Although F-actin is required for CD4 and CXCR4 redistribution, little is known about the molecular mechanisms underlying this fundamental process in HIV infection. Using CD4(+) CXCR4(+) permissive human leukemic CEM T cells and primary lymphocytes, we have investigated whether HIV-1 Env might promote viral entry and infection by activating ERM (ezrin-radixin-moesin) proteins to regulate F-actin reorganization and CD4/CXCR4 co-clustering. The interaction of the X4-tropic protein HIV-1 gp120 with CD4 augments ezrin and moesin phosphorylation in human permissive T cells, thereby regulating ezrin-moesin activation. Moreover, the association and clustering of CD4-CXCR4 induced by HIV-1 gp120 requires moesin-mediated anchoring of actin in the plasma membrane. Suppression of moesin expression with dominant-negative N-moesin or specific moesin silencing impedes reorganization of F-actin and HIV-1 entry and infection mediated by the HIV-1 envelope protein complex. Therefore, we propose that activated moesin promotes F-actin redistribution and CD4-CXCR4 clustering and is also required for efficient X4-tropic HIV-1 infection in permissive lymphocytes.

Short Communication:Activating Stimuli Enhance Immunotoxin-Mediated Killing of HIV-Infected Macrophages

Abstract Strategies for purging persistent reservoirs in human immunodeficiency virus (HIV)-infected individuals may be enhanced by including agents that specifically kill virus-expressing cells. Anti-HIV envelope immunotoxins (ITs) represent one class of candidate molecules that could fulfill this function. We have previously utilized an anti-gp120 IT in conjunction with various stimulants to kill latently infected T cells ex vivo. Here we show that primary macrophages expressing HIV Env are relatively refractory to killing by IT when used alone. However, including stimulants such as prostratin or granulocyte-macrophage colony-stimulating factor to increase HIV gene expression in infected macrophages enhanced IT-mediated killing. Therefore, "activation-elimination" strategies similar to those proposed for purging the latent HIV reservoir may prove useful in clearing chronically infected macrophages in vivo.

Levels of N-linked Glycosylation on the V1 Loop of HIV-1 Env Proteins and their Relationship to the Antigenicity of Env from Primary Viral Isolates

A good understanding about the structure and function of the envelope glycoprotein (Env) from primary human immunodeficiency virus-1 (HIV-1) isolates is important in facilitating the development of effective neutralizing antibody responses as a component of an effective HIV-1 vaccine. In the current study, the antigenicity of a panel of diverse HIV-1 primary Env from different clades of HIV-1 Group M was analyzed using rabbit sera produced by either 3- or 9-valent gp120 DNA vaccine formulations. Both the 3- and 9-valent gp120 DNA vaccine formulations elicited HIV-1 gp120-specific antibodies in immunized rabbits. However, we observed two levels of primary envelope antigenicity to the same set of rabbit immune sera and that the level of glycosylation, particularly in the V1 loop, may contribute to such diversity. Bioinformatics analysis on the distribution and average number of the N-linked glycosylation sites in all variable regions (V1-V5) was conducted. A linear plot demonstrated that the average number of potential N-glycosylation sites in the V1 and V4 loops correlates to the size of the loop. These data provide further evidence on the complexity of primary HIV-1 Env antigens and offers new insight into the mechanisms that HIV-1 uses to escape protective immune responses.

Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes

Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

Critical Involvement of the ATM-Dependent DNA Damage Response in the Apoptotic Demise of HIV-1-Elicited Syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

Peptide Mimic of the HIV Envelope gp120–gp41 Interface

The human immunodeficiency virus envelope glycoprotein (Env) is composed of surface (gp120) and transmembrane (gp41) subunits, which are noncovalently associated on the viral surface. Human immunodeficiency virus Env mediates viral entry after undergoing a complex series of conformational changes induced by interaction with cellular CD4 and a chemokine coreceptor. These changes propagate from gp120 to gp41 via the gp120–gp41 interface, ultimately exposing gp41 and allowing it to form the trimer-of-hairpins structure that provides the driving force for membrane fusion. Key unresolved questions about the gp120–gp41 interface include the specific regions of gp41 and gp120 involved, the mechanism by which receptor and coreceptor-binding-induced conformational changes in gp120 are communicated to gp41, how trimer-of-hairpins formation is prevented in the prefusogenic gp120–gp41 complex, and, ultimately, the structure of the prefusion gp120–gp41 complex. Here, we develop a biochemical model system that mimics a key portion of the gp120–gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and C-peptide region binds primarily to the gp120 C5 region and that this interaction is incompatible with trimer-of-hairpins formation. Based on these data, we propose that in prefusogenic Env, gp120 sequesters the gp41 C-peptide region away from the N-trimer region, preventing trimer-of-hairpins formation until coreceptor binding disrupts this interface. This model system is a valuable tool for studying the gp120–gp41 complex, conformational changes induced by CD4 and coreceptor binding, and the mechanism of membrane fusion.

The First Hypervariable Region of the gp120 Env Glycoprotein Defines the Neutralizing Susceptibility of Heterologous Human Immunodeficiency Virus Type 1 Isolates to Neutralizing Antibodies Elicited by the SF162gp140 Immunogen

Current vaccine efforts to elicit cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus (HIV) focus on the engineering of soluble mimetics of the trimeric HIV Env glycoprotein (commonly termed gp140 immunogens). Such immunogens are thought to be more effective than previously tested monomeric gp120 immunogens at eliciting cross-reactive NAbs. Still, the breadth of neutralizing antibody responses elicited by gp140 immunogens is narrow. Understanding why antibodies elicited by gp140 immunogens fail to neutralize a wide range of heterologous primary HIV isolates is necessary for improving the design of such immunogens. We previously reported that antibodies elicited in macaques by SF162 Env-derived gp140 immunogens fail to neutralize several heterologous "neutralization-resistant" primary HIV type 1 isolates, such as JRFL, ADA, and YU2. Here we show that by replacing the V1 region of Env on these heterologous viruses with that of SF162, we render them highly susceptible to neutralization by the SF162gp140-elicited antibodies. We observed that viral neutralization was mediated not only by vaccine-elicited anti-V1 but also by anti-V3 antibodies and antibodies directed against as yet unidentified Env regions, depending on the heterologous Env background. Our study indicates that common neutralization epitopes are differentially exposed on diverse primary HIV isolates and that the V1 loop contributes to this differential exposure. Therefore, the antibody responses elicited by soluble gp140 immunogens will have to overcome several distinct obstacles in order to neutralize diverse primary HIV isolates.

Incorporation of High Levels of Chimeric Human Immunodeficiency Virus Envelope Glycoproteins into Virus-Like Particles

The human immunodeficiency virus (HIV) envelope (Env) protein is incorporated into HIV virions or virus-like particles (VLPs) at very low levels compared to the glycoproteins of most other enveloped viruses. To test factors that influence HIV Env particle incorporation, we generated a series of chimeric gene constructs in which the coding sequences for the signal peptide (SP), transmembrane (TM), and cytoplasmic tail (CT) domains of HIV-1 Env were replaced with those of other viral or cellular proteins individually or in combination. All constructs tested were derived from HIV type 1 (HIV-1) Con-S DeltaCFI gp145, which itself was found to be incorporated into VLPs much more efficiently than full-length Con-S Env. Substitution of the SP from the honeybee protein mellitin resulted in threefold-higher chimeric HIV-1 Env expression levels on insect cell surfaces and an increase of Env incorporation into VLPs. Substitution of the HIV TM-CT with sequences derived from the mouse mammary tumor virus (MMTV) envelope glycoprotein, influenza virus hemagglutinin, or baculovirus (BV) gp64, but not from Lassa fever virus glycoprotein, was found to enhance Env incorporation into VLPs. The highest level of Env incorporation into VLPs was observed in chimeric constructs containing the MMTV and BV gp64 TM-CT domains in which the Gag/Env molar ratios were estimated to be 4:1 and 5:1, respectively, compared to a 56:1 ratio for full-length Con-S gp160. Electron microscopy revealed that VLPs with chimeric HIV Env were similar to HIV-1 virions in morphology and size and contained a prominent layer of Env spikes on their surfaces. HIV Env specific monoclonal antibody binding results showed that chimeric Env-containing VLPs retained conserved epitopes and underwent conformational changes upon CD4 binding.

New way of regulating alternative splicing in retroviruses: the promoter makes a difference.

Alternative splicing has been recognized as a major mechanism for creating proteomic diversity from a limited number of genes. However, not all determinants regulating this process have been characterized. Using subviral human immunodeficiency virus (HIV) env constructs we observed an enhanced splicing of the RNA when expression was under control of the cytomegalovirus (CMV) promoter instead of the HIV long terminal repeat (LTR). We extended these observations to LTR- or CMV-driven murine leukemia proviruses, suggesting that retroviral LTRs are adapted to inefficient alternative splicing at most sites in order to maintain balanced gene expression.

Defining Carbohydrate Antigens as HIV Vaccine Candidates

The induction of high affinity antibodies capable of broad neutralization and protection against infection and/or disease is a major goal in the development of a vaccine for human immunodeficiency virus (HIV). Insights into the structure and function of the envelope (Env) protein of HIV-1 suggest that the virus is under strong selection pressure by the immune response leading to constant mutations in the Env protein including the N-glycosylation sites. Initially considered a shield against the immune system, the heavily glycosylated outer surface of the HIV Env protein has drawn attention lately as a legitimate target. The dense cluster of high mannose glycans and the great variety of complex glycans present epitopes that might impact on disease progression. Indeed a number of mannose binding proteins and at least one human anti-mannose antibody--2G12, are broadly neutralizing. Due to the low immunogenicity of carbohydrates, these targets on HIV are of limited value unless new powerful immunogens are found. One approach would be the molecular design of peptide carbohydrate mimotopes that can elicit neutralizing antibodies by recruiting optimal T cell help. Here we review existing data on carbohydrate interactions and HIV immunogenicity that serves as a basis for structural concepts and approaches used for vaccine design targeting HIV associated carbohydrate antigens. In particular, the value and the limitations of chemical (peptide libraries), structural and immunological information is illustrated.

Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector

Recombinant vesicular stomatitis virus (rVSV) is currently under evaluation as a human immunodeficiency virus (HIV)-1 vaccine vector. The most compelling reasons to develop rVSV as a vaccine vector include a very low seroprevalence in humans, the ability to infect and robustly express foreign antigens in a broad range of cells, and vigorous growth in continuous cell lines used for vaccine manufacture. Numerous preclinical studies with rVSV vectors expressing antigens from a variety of human pathogens have demonstrated the versatility, flexibility, and potential efficacy of the rVSV vaccine platform. When administered to nonhuman primates (NHPs), rVSV vectors expressing HIV-1 Gag and Env elicited robust HIV-1-specific cellular and humoral immune responses, and animals immunized with rVSV vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env were protected from AIDS after challenge with a pathogenic SIV/HIV recombinant. However, results from an exploratory neurovirulence study in NHPs indicated that these prototypic rVSV vectors might not be adequately attenuated for widespread use in human populations. To address this safety concern, a variety of different attenuation strategies, designed to produce a range of further attenuated rVSV vectors, are currently under investigation. Additional modifications of further attenuated rVSV vectors to upregulate expression of HIV-1 antigens and coexpress molecular adjuvants are also being developed in an effort to balance immunogenicity and attenuation.

Antibody Responses Elicited in Macaques Immunized with Human Immunodeficiency Virus Type 1 (HIV-1) SF162-Derived gp140 Envelope Immunogens: Comparison with Those Elicited during Homologous Simian/Human Immunodeficiency Virus SHIVSF162P4and Heterologous HIV-1 Infection

The antibody responses elicited in rhesus macaques immunized with soluble human immunodeficiency virus (HIV) Env gp140 proteins derived from the R5-tropic HIV-1 SF162 virus were analyzed and compared to the broadly reactive neutralizing antibody responses elicited during chronic infection of a macaque with a simian/human immunodeficiency virus (SHIV) expressing the HIV-1 SF162 Env, SHIV(SF162P4), and humans infected with heterologous HIV-1 isolates. Four gp140 immunogens were evaluated: SF162gp140, DeltaV2gp140 (lacking the crown of the V2 loop), DeltaV3gp140 (lacking the crown of the V3 loop), and DeltaV2DeltaV3gp140 (lacking both the V2 and V3 loop crowns). SF162gp140 and DeltaV2gp140 have been previously evaluated by our group in a pilot study, but here, a more comprehensive analysis of their immunogenic properties was performed. All four gp140 immunogens elicited stronger anti-gp120 than anti-gp41 antibodies and potent homologous neutralizing antibodies (NAbs) that primarily targeted the first hypervariable region (V1 loop) of gp120, although SF162gp140 also elicited anti-V3 NAbs. Heterologous NAbs were elicited by SF162gp140 and DeltaV2gp140 but were weak in potency and narrow in specificity. No heterologous NAbs were elicited by DeltaV3gp140 or DeltaV2DeltaV3gp140. In contrast, the SHIV(SF162P4)-infected macaque and HIV-infected humans generated similar titers of anti-gp120 and anti-gp41 antibodies and NAbs of significant breadth against primary HIV-1 isolates, which did not target the V1 loop. The difference in V1 loop immunogenicity between soluble gp140 and virion-associated gp160 Env proteins derived from SF162 may be the basis for the observed difference in the breadth of neutralization in sera from the immunized and infected animals studied here.

Effects of signal peptide exchange on HIV-1 glycoprotein expression and viral infectivity in mammalian cells

In certain cell systems, exchange of the human immunodeficiency virus (HIV) Env signal peptide (SP) sequence with that of heterologous SPs has been shown to increase gp120 transport and secretion. Here we demonstrate that exchange of the HIV-Env-SP with those from erythropoietin or tissue plasminogen activator in the proviral context does not increase wild-type membrane-bound Env expression or incorporation into released virions. In fact, virion infectivities were decreased. These infectivity decreases were largely due to effects on Env transport and/or function and only to a minor extent to cis effects as a result of the sequence exchanges themselves. Thus, in fact, it is not advantageous to employ heterologous SPs to achieve high-level expression of functional cell surface membrane- or virion-associated HIV-Env.

Large HIV‐specific CD8+ cytotoxic T‐lymphocyte (CTL) clones reduce their overall size but maintain high frequencies of memory CTL following highly active antiretroviral therapy

Cytotoxic T-lymphocytes (CTL) play an important role in the control of human immunodeficiency virus (HIV) and of human cytomegalovirus (HCMV) infection. Following highly active antiretroviral therapy (HAART), most studies have demonstrated a decline in the frequency of HIV-specific CTL. We analysed the effect of HAART on the size, phenotype and function of individual HIV- and HCMV-specific CTL clones, using clonotypic oligonucleotide probing specific for the T-cell receptor (TCR) beta-chain hypervariable sequence of defined immunodominant CTL clones specific for peptides of HIV or HCMV, and quantified the limiting dilution analysis frequencies of CTL precursors (CTLp) specific for the same viral peptides. We found that the clonal composition of CD8+ T cells specific for HIV gag and env epitopes was highly focused and did not change after HAART. Following HAART, there was progressive contraction of HIV-specific CD8+ clones, especially in the CD28- CD27- subpopulation--the remaining cells of contracting HIV-specific clones were predominantly CD28- CD27+ CD45RO(hi). We observed maintenance of strong functional HIV-specific CD8+ T-cell responses in limiting dilution analysis following HAART, indicating preferential loss of HIV-specific cells that have reduced cloning efficiency in vitro. Following HAART, we also observed selective expansion of HCMV-specific CD8+ clones. Most HCMV-specific CD8+ clones were predominantly CD28- CD27+/- CD45RA(hi) following HAART. In one subject, a Vbeta6.4+ clone specific for HCMV pp65 selectively expanded following HAART, without expansion of two other Vbeta6.4+ clones, indicating that individual clonotypes specific for the same peptide can show different kinetics and phenotypes in response to antiretroviral therapy.