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Abstract: Objectives: HIV is able to continuously adapt to and evade the evolving neutralizing antibody responses of the host. We investigated the ability of HIV variants to evade neutralizing antibodies in order to understand the distinct characteristics of HIV-1 Korean clade B. Methods: Three drug-naive subjects were enrolled in this study who were infected with HIV-1 Korean clade B. Neutralizations were performed using autologous plasma and pseudovirion-based assays in order to analyze and compare changes in the env gene. Results: In the early phase of infection, neutralizing activities against autologous virus variants gradually increased, which was followed by a decline in the humoral immune response against the subsequent viral escape variants. The amino acids lengths and number of potential N-linked glycosylation sites (PNGS) in HIV-1 env gene was positively correlated with neutralized antibody responses during the early stages of infection. Conclusion: This study suggests that change within the env domains over the course of infection influences reactivities to neutralized antibodies and may also have an impact on host immune responses. This is the first longitudinal study of HIV-1 humoral immunity that took place over the entire course of HIV-1 Korean clade B infection. [Copyright &y& Elsevier]

Abstract: The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) – unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery. [Copyright &y& Elsevier]

Abstract: The human immunodeficiency virus has been shown to increase its infectivity throughout the course of infection. This virus selection property has been associated with genome mutations and recombinations among virus variants, causing amino acid residue alterations in important viral proteins. In order to explore the contribution of Env signal peptide (Env-sp) to Env glycoprotein expression and its possible relationship to increased virus infectivity observed at late stages of infection, we characterized Env-sp sequences derived from twelve patients at “early” and “late” stages of HIV infection without antiretroviral therapy use. In spite of the remarkable overall similarity between both stages, we observed the deletion of a sequence of neutral and basic residues at the Env-sp amino terminus in virus from early stage specimens and the insertion of basic residues in the hydrophobic region on late-stage viral isolates. The Env-sp sequence alterations may have viral adaptive functions during HIV infection. [Copyright &y& Elsevier]

Abstract: The majority of infection by the human immunodeficiency virus (HIV-1) across the world occurs by heterosexual transmission and is likely mediated by virus present in genital secretions. In spite of this, infection is followed by clinical markers of the virus present in blood, which may not be representative of the virus involved in transmission. In fact, several studies have demonstrated that the genital tract represents a unique compartment for the virus. We assessed the relationship between immune system integrity, represented by CD4+ T cell counts, and the maintenance of viral compartmentalization between plasma and vaginal fluid virus in treatment naïve women from the Dominican Republic infected by the heterosexual transmission route. We cloned and sequenced cell free virus from plasma and genital fluid samples from six women to assess viral evolution, phylogenetic relatedness, and calculated co-receptor use for the C2V3 region of the envelope. Our analyses demonstrated plasma and vaginal fluid virus compartments remained intact only in samples from women with CD4+ T cell counts over 350cells/μl. The majority of viral forms were predicted to use the CCR5 co-receptor, although several dual tropic forms were also identified. None of the clones were found to use the CXCR4 co-receptor even though many of the patients showed severe disease. Our findings lend further support to the role of an intact immune system in maintaining compartmentalization across blood and genital quasispecies and provide a compelling rationale to specifically consider genital tract viral forms in therapeutic and vaccine research. [Copyright &y& Elsevier]

Objective: Retrovirus has been suggested as one of agents involved in the development of schizophrenia. In the present study, we examined the role of the human endogenous retrovirus W family (HERV-W) env gene in the etiopathogenesis of recent-onset schizophrenia, using molecular and epidemiological approaches. Methods: Nested RT-PCR was used to detect the messenger RNA (mRNA) of the HERV-w env gene in plasmas. Quantitative real-time polymerase chain reaction (PCR) was employed to detect the viral reverse transcriptase activity in human sera. Human U251 glioma cells were used to study the potential role of the HERV-W env gene in the etiopathogenesis of recent-onset schizophrenia. Results: We identified genes with mRNA sequences homologous to HERV-W env gene from plasmas of 42 out of 118 individuals with recent-onset schizophrenia but not from any of 106 normal persons (P < .01, t test). Quantitative real-time PCR showed a significantly increase in the reverse transcriptase activity in the sera of patients (by 35.59%) compared with controls (by 2.83%) (P < .05, t test). Overexpression of HERV-w env in human U251 glioma cells upregulated brain-derived neurotrophic factor (BDNF), an important schizophrenia-associated gene, neurotrophic tyrosine kinase receptor type 2 (NTRK2, also called TrkB), and dopamine receptor D3 and increased the phosphorylation of cyclic adenosine monophosphate response element–binding (CREB) protein. BDNF promoter reporter gene assays showed that the HERV-W env triggers BDNF production in human U251 glioma cells. Using gene knockdown, we found that CREB is required for the expression of BDNF that is regulated by env. Conclusion: Our data revealed that the transcriptional activation of HERV is associated with the development of schizophrenia in some patients and indicated that HERV-W env regulates the expression of schizophrenia-associated genes. This report is the first to elucidate the signaling pathway responsible for the upregulation of HERV-W env–triggered BDNF. Our study provides new evidence for the involvement of HERV-W in the central nervous system, which will benefit the diagnosis and treatment of the devastating schizophrenia and related disorders. [ABSTRACT FROM PUBLISHER]

Abstract: SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1–3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques. [Copyright &y& Elsevier]

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage. [ABSTRACT FROM AUTHOR]

Abstract: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation. [Copyright &y& Elsevier]

Abstract: Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection. [Copyright &y& Elsevier]

Abstract: Antibody-dependent cellular cytotoxicity (ADCC) may assist in preventing HIV or delaying disease progression. Most prior studies have analysed Env-specific ADCC responses. We hypothesized that effective ADCC-based immunity may target conserved internal viral proteins such as Pol. We analysed the ability overlapping Pol peptides to induce activation of NK cells via ADCC. We prospectively studied ADCC responses in 83 HIV+ subjects followed for 3years. Pol peptides were commonly targeted by ADCC responses in these chronically infected subjects (in 32 of the 83 subjects). However, Pol-specific ADCC responses declined over time and did not correlate with delayed HIV progression, measured by either baseline CD4 T cells, CD4 T cell loss over time, baseline viral load or the need to start antiretroviral therapy. Although Pol is frequently targeted by ADCC in HIV+ subjects, the strength or specificity of Pol-specific ADCC responses needs to be modulated to be effective in delaying HIV progression. [Copyright &y& Elsevier]

The long terminal repeat (LTR) regulates gene expression of HIV-1 by interacting with multiple host and viral factors. Cross-sectional studies in the pre-HAART era demonstrated that single nucleotide polymorphisms (SNPs) in peripheral blood-derived LTRs (a C-to-T change at position 3 of C/EBP site I (3T) and at position 5 of Sp site III (5T)) increased in frequency as disease severity increased. Additionally, the 3T variant correlated with HIV-1-associated dementia. LTR sequences derived by longitudinal sampling of peripheral blood from a single patient in the DrexelMed HIV/AIDS Genetic Analysis Cohort resulted in the detection of the 3T and 5T co-selected SNPs before the onset of neurologic impairment, demonstrating that these SNPs may be useful in predicting HIV-associated neurological complications. The relative fitness of the LTRs containing the 3T and/or 5T co-selected SNPs as they evolve in their native patient-derived LTR backbone structure demonstrated a spectrum of basal and Tat-mediated transcriptional activities using the IIIB-derived Tat and colinear Tat derived from the same molecular clone containing the 3T/5T LTR SNP. In silico predictions utilizing colinear envelope sequence suggested that the patient's virus evolved from an X4 to an R5 swarm prior to the development of neurological complications and more advanced HIV disease. These results suggest that the HIV-1 genomic swarm may evolve during the course of disease in response to selective pressures that lead to changes in prevalence of specific polymorphisms in the LTR, env, and/or tat that could predict the onset of neurological disease and result in alterations in viral function. [ABSTRACT FROM AUTHOR]

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology. [ABSTRACT FROM AUTHOR]

Abstract: The cytoplasmic tail domain (CTD) of retroviral envelope (Env) proteins has been implicated in modulating Env incorporation into viral particles. We generated a panel of murine leukemia virus (MLV) Env mutants and analyzed their ability to be recruited to human immunodeficiency virus-1 (HIV-1) assembly sites. Surprisingly, the entire CTD was dispensable for recruitment to assembly sites, but a mutation that disrupted the furin cleavage site in Env abolished recruitment. To determine if MLV Env can show selectivity for homologous assembly sites, cells were co-transfected with both HIV-1 and MLV assembly components along with each MLV Env construct and assayed for infectious particle production. MLV Env selectively formed infectious particles with the MLV components at the expense of infectious HIV-1 infectious particle production, but truncation of the CTD progressively reduced this selectivity. Collectively these data suggest that there are two separable mechanisms that govern MLV Env recruitment to viral assembly sites. [ABSTRACT FROM AUTHOR]

Abstract: While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms. [Copyright &y& Elsevier]

Abstract: The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant “core” structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD “core” do not influence specific virus–cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD “core” delayed the kinetics and reduced the efficiency of cell–cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process. [Copyright &y& Elsevier]

We used a DiscoTope 1.2 (), Epitopia () and EPCES () algorithms to map discontinuous B-cell epitopes in HIV1 gp120. The most mutable nucleotides in HIV genes are guanine (because of G to A hypermutagenesis) and cytosine (because of C to U and C to A mutations). The higher is the level of guanine and cytosine usage in third (neutral) codon positions and the lower is their level in first and second codon positions of the coding region, the more stable should be an epitope encoded by this region. We compared guanine and cytosine usage in regions coding for five predicted 3D B-cell epitopes of gp120. To make this comparison we used GenBank resource: 385 sequences of env gene obtained from ten HIV1-infected individuals were studied (). The most protected from nonsynonymous nucleotide mutations of guanine and cytosine 3D B-cell epitope is situated in the first conserved region of gp120 (it is mapped from 66th to 86th amino acid residue). We applied a test of variability to confirm this finding. Indeed, the less mutable predicted B-cell epitope is the less variable one. MEGA4 (standard PAM matrix) was used for the alignments and “VVK Consensus” algorithm () was used for the calculations. [ABSTRACT FROM AUTHOR]

Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize ~80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 Å resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue "specificity loop" on the "hammerhead" subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 Å facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets. [ABSTRACT FROM AUTHOR]

Abstract: A central, yet unresolved issue in the pathogenesis of HIV disease is the mechanism of antibody perturbation. In this study, HIV-specific memory B-cells were quantified in groups of infected subjects and compared with memory responses to other antigens and antibody titers. HIV-specific memory B-cell responses were vigorous in individuals with CD4+ T-cell counts >350/μl and weak or undetectable in subjects with CD4+ T-cell numbers <200/μl. Memory B-cell loss was permanent, because antiretroviral therapy failed to restore HIV-specific memory responses while influenza- and tetanus toxoid-specific memory B-cells remained unaffected or recovered. Antibody titers to Gag strongly correlated with memory B-cell frequencies. In contrast, Env-specific antibodies were maintained in advanced disease despite low or undetectable levels of memory B-cells. These results provide a potential mechanism by which destruction of HIV-specific CD4+ T-cells affects the humoral immune response against HIV and compromises the ability to maintain an effective antibody response. [Copyright &y& Elsevier]

Abstract: CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection. [Copyright &y& Elsevier]

Abstract: Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor–operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This system therefore could facilitate the development of Ad-based vaccine candidates that otherwise would not be feasible to generate. [Copyright &y& Elsevier]

Abstract: Human immunodeficiency virus type 1 (HIV-1) entry into host cells is mediated by the trimeric envelope glycoprotein complex (Env). Accordingly, the Env proteins are the targets for neutralizing antibodies (NAbs) and are the focus of vaccines intended to induce NAbs. Because the Env complex is labile, soluble recombinant Env (gp140) trimers require engineering to stabilize them sufficiently for use as immunogens. Trimeric forms of gp140 trimers can be created that are either cleavage-competent or cleavage-defective at the junction between the gp120 and gp41 subunits. As functional trimers are cleaved at this site, the question arises as to whether cleavage affects the antigenic structure of the Env complex in a way that is relevant to vaccine design. Here, we present a comparative analysis of the antigenicity profiles of cleaved and uncleaved gp140 trimers derived from the KNH1144 (subtype A) virus that are otherwise closely sequence-matched. While cleavage did not affect the exposure of NAb epitopes on the gp140 trimers, non-neutralizing antibodies to gp41 epitopes bound much more strongly to uncleaved trimers. Hence cleavage does alter the structure of the HIV-1 Env complex. [Copyright &y& Elsevier]

Abstract: It is unknown which HIV proteins to target by vaccination in order to generate the most effective CD8 T-cell immunity. We recently immunized SIVmac251-infected pigtail macaques with Gag peptides or a cocktail of peptides spanning all SIV proteins, including SIV Env. High-level SIV Env-specific CD8 T-cell responses were generated and 7 novel Env-specific CD8 T-cell epitopes in 10 animals were mapped. Env-specific CD8 T-cell responses were significantly inferior to Gag-specific responses, and no better than unvaccinated control animals, in the control of SIV replication and prevention of disease. Escape mutations emerged within several Env-specific CTL epitopes, suggesting at least some pressure imparted by the Env CTL responses, but this did not correlate with significantly reduced SIV replication. We conclude Env-specific CTL may not be the most effective response to induce by vaccination. [Copyright &y& Elsevier]

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope (Env) is an essential determinant of viral infectivity, tropism and spread between T cells. Lentiviral Env contain an unusually long 150 amino acid cytoplasmic tail (EnvCT), but the function of the EnvCT and many conserved domains within it remain largely uncharacterised. Here, we identified a highly conserved tryptophan motif at position 757 (W757) in the LLP-2 alpha helix of the EnvCT as a key determinant for HIV-1 replication and spread between T cells. Alanine substitution at this position potently inhibited HIV-1 cell–cell spread (the dominant mode of HIV-1 dissemination) by preventing recruitment of Env and Gag to sites of cell–cell contact, inhibiting virological synapse (VS) formation and spreading infection. Single-molecule tracking and super-resolution imaging showed that mutation of W757 dysregulates Env diffusion in the plasma membrane and increases Env mobility. Further analysis of Env function revealed that W757 is also required for Env fusion and infectivity, which together with reduced VS formation, result in a potent defect in viral spread. Notably, W757 lies within a region of the EnvCT recently shown to act as a supporting baseplate for Env. Our data support a model in which W757 plays a key role in regulating Env biology, modulating its temporal and spatial recruitment to virus assembly sites and regulating the inherent fusogenicity of the Env ectodomain, thereby supporting efficient HIV-1 replication and spread. [ABSTRACT FROM AUTHOR]

HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS. [ABSTRACT FROM AUTHOR]

Abstract: Infection of a human T-cell leukaemia cell line (HSB-2) with HHV-6 led to the induction of exosome-like-particles attached to newly formed HHV-6 enveloped particles and to amplification of a 1642 bp molecule consisting of a partial human endogenous retrovirus (HERV)-E polymerase gene and repetitive sequences. We initiated an analysis of transcriptional patterns of predicted genes from HERV-E sequences in normal and malignant haematopoietic cells. Transcription patterns of regions corresponding to gag, pol and env genes at different chromosomal loci varied among cell types tested. Several specific transcripts were only observed in malignant haematopoietic cells and transcriptional activity varied among different malignant cell types. A transcript of 7.1 kb spanning the complete gag, pol and env gene region, originating from chromosome 8p23, was identified in normal peripheral blood cells and cells of the chronic myeloid leukaemia cell line K562. Our study describes new active HERV-E sequences and new loci throughout the human genome. [Copyright &y& Elsevier]

Mechanisms of HIV-mediated CD4+ T cell loss leading to immunodeficiency are amongst the most extensively studied yet unanswered questions in HIV biology. The level of CD4+ T cell depletion in HIV infected patients far exceeds the number of infected T cells, suggesting an indirect mechanism of HIV pathogenesis termed bystander cell death. Evidence is accumulating that the HIV envelope glycoprotein (Env) is a major determinant of HIV pathogenesis and plays a critical role in bystander cell death. The complex structure and function of HIV Env makes the determination of the mechanism of Envmediated apoptosis more complex than previously thought. This review will examine the complex relationship between HIV Env phenotype, coreceptor expression and immune activation in determining HIV pathogenesis. We review data here corresponding to the role of HIV Env hemifusion activity in HIV pathogenesis and how it interplays with other AIDS associated factors such as chemokine receptor expression and immune activation. [ABSTRACT FROM AUTHOR]

The genome of replication-competent BERV γ4 provirus, which is the most abundant ERV family in the bovine genome, was characterized in detail. The BERV γ4 genome showed that BERV γ4 harbors 8576 nucleotides and has the typical 5'-long terminal repeat (LTR)-gap-pro-pol-env-LTR-3' retroviral organization with a long leader region positioned before the gag open reading frame. Multiple sequences analysis showed that the nucleotide difference between 5' and 3' LTRs was 4.2% (mean value 0.042) in average, suggesting that the provirus formed at most 13.3 million years ago. Gag separated by a stop codon from pro-pol in the same reading frame, while env resides in another reading frame lacking of a functional surface domain. According to the current bovine genome sequence assembly, the full-length BERV γ4 provirus sequences were only found in the chromosomes 1, 2, 6, 10, 15, 23, 26, 28, X, and unassigned, although the partial sequences almost evenly distributed in the entire bovine genome. This is the first detailed study describing the genome structure of BERV γ4, the most abundant ERV family present in bovine genome. Combined with our recent reports on characterization of ERVs in bovine, this study will contribute to illuminate ERVs in the cattle of which no information was previously available. [ABSTRACT FROM AUTHOR]

Abstract: To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations between increased Env net charge and lowered CD4+ T cell counts, enhanced viral fitness, reduced sensitivity to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5 HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness. [Copyright &y& Elsevier]

Summary: For the development of feline immunodeficiency virus (FIV) vaccines mostly structural proteins have been evaluated for their capacity to induce protective immunity. In the present study, subunit vaccines containing recombinant FIV accessory proteins Rev and OrfA were evaluated in cats. Cats were vaccinated repeatedly with these proteins, adjuvanted with immune stimulating complexes (ISCOMs). In addition, cats were vaccinated with bacterially expressed fragments spanning the entire FIV envelope protein, either alone or in combination with the regulatory proteins. Subsequently, the cats were challenged with a homologous FIV strain to assess the level of protective immunity achieved with the respective vaccination regimens. Although the vaccines proved to be immunogenic, vaccinated cats were not protected from infection with FIV. [Copyright &y& Elsevier]

Abstract: We have studied the relationship between disease progression and HIV-1 evolution in 24 infants classified as rapid or non-rapid progressors, during nearly the entire disease progression cycle from infection to AIDS. Specifically, we examined the temporal relationship between clinical status and changes in genetic diversity, divergence, selection and recombination at the C2V3C3 region of the env gene during a period of 3 years. Statistical analyses were performed using linear mixed models that are particularly well-suited for longitudinal studies in which repeated measures are taken from the same patients. We did not observe significant differences in genetic diversity or overall substitution rates between clinical categories. However, the nonsynonymous substitution rate per nonsynonymous site (dN) evolved differently between groups. Changes in dN explained the evolutionary slowdown of the dN/dS ratio in the rapid progressors, while in non-rapid progressors the dN/dS ratio continuously increased through time. The number of positively selected sites had limited power for predicting disease progression. Recombination rate estimates were different among groups, although not significantly in the linear mixed models analysis. They showed some power predicting clinical categories and, interestingly, they were significantly correlated with the frequency of positively selected sites. Overall, the results obtained confirm that viral adaptation in the C2V3C3 region of the env gene is related to disease progression, although the statistical characterization of such pattern seems rather difficult. [Copyright &y& Elsevier]

Abstract: The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120–gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B. [Copyright &y& Elsevier]

We recently used degenerate PCR and locus-specific PCR methods to identify the endogenous retroviruses (ERV) in the bovine genome. Using the ovine ERV classification system, the bovine ERVs (BERVs) could be classified into four families. Here, we searched the most recently released bovine genome database with the partial nucleotide sequence of the pro/pol region of the BERV β3 family. This allowed us to obtain and analyze the complete genome of BERV β3. The BERV β3 genome is 7666 nucleotides long and has the typical retroviral organization, namely, 5'-long terminal repeat (LTR)-gag-pro-pol-env-LTR-3'. The deduced open reading frames for gag, pro, pol and env of BERV β3 encode 507, 271, 879 and 603 amino acids, respectively. BERV β3 showed little amino acid similarity to other betaretroviruses. Phylogenetic analysis showed that it clusters with HERV-K. This is the first report describing the genetic structure and sequence of an entire BERV. [ABSTRACT FROM AUTHOR]

Abstract: Removal of the R peptide (residues 617–632) from the Moloney murine leukemia virus (MoMuLV) envelope protein (Env) cytoplasmic tail potentiates fusion. We examined the role of the membrane-proximal cytoplasmic domain (598–616) of the MoMuLV Env in the Env-mediated membrane fusion and incorporation. The Env truncated at 616 exhibits maximum fusogenicity in cell-to-cell fusion assay. By comparison, full tail Env (632) and the Env truncated to residue 601 mediated fusion at 40%. The Envs truncated to residues 598 or 595 are not fusogenic. Progressive cytoplasmic tail truncation correlated with decreased Env incorporation into virions. Substitution of the domain 598–616 with an amphiphilic α-helix from melittin results in maximally fusogenic Envs that efficiently incorporated into transduction competent virions. However, substitution of the domain 598–616 with random or hydrophilic sequences caused loss of the Env fusogenicity and titer while retaining incorporation. Further, a secondary structure prediction analysis of 27 unrelated Env cytoplasmic tails indicates a common (23/27) propensity for an amphiphilic α-helical domain at immediate proximity to the viral membrane. These results support the suggestion that viral fusion is enhanced by a membrane-proximal cytoplasmic amphiphilic α-helix in Env tail. The model of its action is proposed. [Copyright &y& Elsevier]

Abstract: In the Moloney murine leukemia virus (MoMuLV) envelope glycoprotein (Env) we identified a membrane-proximal cytoplasmic domain (residues 598–616) that facilitates the Env incorporation into virions and Env-mediated fusion [Rozenberg, Y., Conner, J., Aguilar-Carreno, H., Chakraborti, S., Dimiter, D.S., Anderson, W.F., 2008. Viral entry: membrane-proximal cytoplasmic domain of MoMuLV envelope tail facilitates fusion. In the same issue. (accompanying paper)]. By biophysical methods (CD, EPR) a corresponding peptide (membrane-proximal peptide, 598–616) was demonstrated to form a membrane-parallel amphiphilic α-helix in the presence of membranes. Electrophysiological studies with planar bilayers and liposomes indicate that the membrane-proximal peptide is membrane destabilizing. This peptide and the fusion peptide from the MoMuLV transmembrane (TM) ectodomain were tested for their effect on the bilayer for hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (TH). Importantly, the external fusion peptide and the internal membrane-proximal peptides of MoMuLV env exert opposite effects on membrane curvature. The fusion peptide lowers TH while the membrane proximal peptide raises it. These effects on TH correlate with the ability of these peptides to induce lipid mixing in large unilamellar vesicles composed of dioleoylphosphatidylethanolamine: dioleoylphosphatidylcholine:cholesterol (1:1:1 mol). When added externally to preformed liposomes, the N-terminal fusion peptide promotes lipid mixing while the cytoplasmic membrane-proximal peptide inhibits this effect. These finding indicate a possible mechanism by which the membrane-proximal domain in MoMuLV Env may affect the formation of membrane fusion intermediates. [Copyright &y& Elsevier]

The env gene of human immunodeficiency virus (HIV) is a functionally important gene responsible for the production of protein products (gp120 and gp41) involved in host cell recognition, binding, and entry. This occurs through a complex and, as yet, not fully understood process of protein–protein interaction and within and between protein functional communication. Exposure on the surface of active HIV virions means the gp120–gp41 complexes are subjected to intense immune system pressure and have, therefore, evolved mechanisms to avoid neutralization. Using protein-coding sequences representing all the HIV type-1 (HIV-1) group M subtypes, we have identified amino acids within the env gene whose evolution is inextricably linked over the entire HIV-1 group M epidemic. We identified 848 pairs of coevolving residues (involving 263 out of 764 amino acid sites), which represent 0.29% of all possible pairs. Of the coevolving pairs, 68% were significantly correlated by hydrophobicity, molecular weight, or both hydrophobicity and molecular weight. Subsequent grouping of coevolving pairs resulted in the identification of 290 groups of amino acid residues, with the size of these groups ranging from 2 to 10 amino acid residues. Many of these dependencies are correlated by function including CD4 binding, coreceptor binding, glycosylation, and protein–protein interaction. This analysis provides important information regarding the functional dependencies observed within all the HIV-1 group M subtypes and may assist in the identification of functional protein domains and therapeutic targets within the HIV-1 env gene. [ABSTRACT FROM PUBLISHER]

Abstract: Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag–pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice. [Copyright &y& Elsevier]

Abstract: To assess the potential of native Envelope glycoprotein (Env) trimers as neutralizing antibody vaccines, we immunized guinea pigs with three types of VLPs and soluble gp120. Particles included “SOS-VLPs” (bearing disulfide-shackled functional trimers), “UNC-VLPs” (bearing uncleaved nonfunctional Env) and “naked VLPs” (bearing no Env). The SOS-VLPs were found to have a density of about 27 native trimers per particle, approximately twice that of live inactivated HIV-1 preparations. As immunogens, UNC- and SOS-VLP rapidly elicited anti-gp120 antibodies focused on the V3 loop and the gp120 coreceptor binding site. Reactivity to the gp41 immunodominant domain was absent in SOS-VLP sera, presumably because gp120–gp41 association is stabilized, effectively covering this epitope. Gp120-immune sera reacted with the receptor binding sites of gp120 and were less focused on the V3 loop. Some Env-VLP sera neutralized primary isolates at modest titers. The measurement of neutralization was found to be affected by the cell lines used. Depending on the assay particulars, non-Env specific antibodies in VLP sera could enhance infection, or nonspecifically neutralize. However, a neutralization assay using TZM-BL cells was essentially clear of these effects. We also describe a native trimer binding assay to confirm neutralization activity in a manner that completely eliminates nonspecific effects. Overall, our data suggests that Env-VLP sera were primarily focused on nonfunctional forms of Env on VLP surfaces, possibly gp120/gp41 monomers and not the trimers. Therefore, to make progress toward a more effective VLP-based vaccine, we will need to find ways to refocus the attention of B cells on native trimers. [Copyright &y& Elsevier]

Abstract: Bovine leukemia virus (BLV) is an oncogenic virus widespread in cattle. It belongs to the genus Deltaretrovirus of the family Retroviridae along with human and simian T-lymphotropic viruses. Here we report the addition of 28 new sequences to the current literature of 16 full-length BLV envelope gene sequences. The phylogenetic clustering, genotyping, and geographic distribution of BLV env variations corresponded in most cases. Most natural variations are mapped to the surface of the proposed conformational models of BLV gp51 N-terminus and gp30 external domain, overlapping with or adjacent to immunogenic epitopes. Analyses for evidence of possible selection pressures suggest the BLV env is under stringent negative selection overall, while strong positive selection is indicated for immunogenic epitope G. Natural env deletions bounded by similar flanking sequences were observed in multiple isolates and would result in truncated signal peptides, missing gp51, and aberrant coding frames for other proteins. [Copyright &y& Elsevier]

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Abstract: Jembrana disease virus (JDV) is a lentivirus associated with an acute disease syndrome with a 20% case fatality rate in Bos javanicus (Bali cattle) in Indonesia, occurring after a short incubation period and with no recurrence of the disease after recovery. Partial regions of gag and pol and the entire env were examined for sequence variation in DNA samples from cases of Jembrana disease obtained from Bali, Sumatra and South Kalimantan in Indonesian Borneo. A high level of nucleotide conservation (97–100%) was observed in gag sequences from samples taken in Bali and Sumatra, indicating that the source of JDV in Sumatra was most likely to have originated from Bali. The pol sequences and, unexpectedly, the env sequences from Bali samples were also well conserved with low nucleotide (96–99%) and amino acid substitutions (95–99%). However, the sample from South Kalimantan (JDVKAL/01) contained more divergent sequences, particularly in env (88% identity). Phylogenetic analysis revealed that the JDVKAL/01 env sequences clustered with the sequence from the Pulukan sample (Bali) from 2001. JDV appears to be remarkably stable genetically and has undergone minor genetic changes over a period of nearly 20 years in Bali despite becoming endemic in the cattle population of the island. [Copyright &y& Elsevier]

Abstract: Heterozygosity for the CCR5 Δ32 allele is associated with delayed progression to AIDS in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an unusual HIV-1 isolate from the blood of an asymptomatic individual who was heterozygous for the CCR5 Δ32 allele and had reduced levels of CCR5 expression. The primary virus used CCR5, CXCR4, and an unusually broad range of alternative coreceptors to enter transfected cells. However, only CXCR4 and CCR5 were used to enter primary T cells and monocyte-derived macrophages, respectively. Full-length Env clones had an unusually long V1/V2 region and rare amino acid variants in the V3 and C4 regions. Mutagenesis studies and structural models suggested that Y308, D321, and to a lesser extent K442 and E444, contribute to the broad coreceptor usage of these Envs, whereas I317 is likely to be a compensatory change. Furthermore, database analysis suggests that covariation can occur at positions 308/317 and 308/321 in vivo. Y308 and D321 reduced dependence on the extracellular loop 2 (ECL2) region of CCR5, while these residues along with Y330, K442, and E444 enhanced dependence on the CCR5 N-terminus compared to clade B consensus residues at these positions. These results suggest that expanded coreceptor usage of HIV-1 can occur in some individuals without rapid progression to AIDS as a consequence of changes in the V3 region that reduce dependence on the ECL2 region of CCR5 by enhancing interactions with conserved structural elements in G-protein-coupled receptors. [Copyright &y& Elsevier]

Abstract: Rotifers of class Bdelloidea, a group of aquatic invertebrates in which males and meiosis have never been documented, are also unusual in their lack of multicopy LINE-like and gypsy-like retrotransposons, groups inhabiting the genomes of nearly all other metazoans. Bdelloids do contain numerous DNA transposons, both intact and decayed, and domesticated Penelope-like retroelements Athena, concentrated at telomeric regions. Here we describe two LTR retrotransposons, each found at low copy number in a different bdelloid species, which define a clade different from previously known clades of LTR retrotransposons. Like bdelloid DNA transposons and Athena, these elements are found preferentially in telomeric regions. Unlike bdelloid DNA transposons, many of which are decayed, the newly described elements, named Vesta and Juno, inhabiting the genomes of Philodina roseola and Adineta vaga, respectively, appear to be intact and represent recent insertions, possibly from an exogenous source. We describe the retrovirus-like structure of the new elements, containing gag, pol, and env-like open reading frames, and discuss their possible origins, transmission, and behavior in bdelloid genomes. [Copyright &y& Elsevier]

Abstract: A DNA plasmid encoding human immunodeficiency virus type 1 (HIV-1) env, nef and tat genes was used in mice in a prime-boost immunization regimen with the corresponding recombinant proteins. The genetic immunogen was delivered with a gene gun and the proteins were injected intramuscularly together with the adjuvant AS02A. Immunizations were followed by experimental challenge with pseudotyped HIV-1 subtype A or B virus. In an initial experiment in which animals were challenged four weeks after the final immunization, all single modality and prime-boost vaccinations resulted in a significant level of protection as compared to control animals. There was a trend for DNA-alone immunization yielding the highest protection. In a subsequent study, a late challenge was performed 19 weeks after the final immunization. All groups having received the DNA vaccine, either alone or in combination with adjuvanted protein, exhibited strong protection against HIV replication. The subtype-specific protection against the experimental HIV challenge was significantly stronger than the cross-protection. Cellular and humoral immune responses were assessed during immunization and after challenge, but without clear correlation to protection against HIV replication. The data suggest that either DNA or protein antigens alone provide partial protection against an HIV-1/MuLV challenge and that DNA immunization is essential for achieving very high levels of efficacy in this murine HIV-1 challenge model. While prime-boost combinations were more immunogenic than DNA alone, they did not appear to provide any further enhancement over DNA vaccine mediated efficacy. The DNA immunogen might prime low levels of CD8+ T cells responsible for virus clearance or possibly a yet unidentified mechanism of protection. [Copyright &y& Elsevier]

Abstract: Virally regulated HIV-1 particles were expressed from DNA plasmids encoding Gag, protease, reverse transcriptase, Vpu, Tat, Rev, and Env. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted. Mutations were engineered into the VLP genome to produce particles deficient in activities associated with viral reverse transcriptase, RNase H, and RNA packaging. Each plasmid efficiently secreted particles from primate cells in vitro and particles were purified from the supernatants and used as immunogens. Mice (BALB/c) were vaccinated intranasally (day 1 and weeks 3 and 6) with purified VLPs and the elicited immunity was compared to particles without Env (Gagp55), to soluble monomeric Envgp120, or to soluble trimerized Envgp140. Only mice vaccinated with VLPs had robust anti-Env cellular immunity. In contrast, all mice had high titer anti-Env serum antibody (IgG). However, VLP-vaccinated mice had antisera that detected a broader number of linear Env peptides, had anti-Env mucosal IgA and IgG, as well as higher titers of serum neutralizing antibodies. VLPs elicited high titer antibodies that recognized linear regions in V4-C5 and the ectodomain of gp41, but did not recognize V3. These lentiviral VLPs are effective mucosal immunogens that elicit broader immunity against Env determinants in both the systemic and mucosal immune compartments than soluble forms of Env. [Copyright &y& Elsevier]

Abstract: Late stage AIDS associated CCR5 tropic HIV-1 clones (R5-AIDS HIV-1) exhibit greater cytopathic effects (CPE) than earlier isolates from the same patients. In this study, envelopes from a series of three biological clones derived from the same patient were evaluated as a cytopathic determinant of R5-AIDS HIV-1 for thymocytes. In a single round of replication in thymocytes, the AIDS associated clone mediated greater initiation of reverse transcription. This enhancement was not due to broadened coreceptor tropism, as all clones studied were exclusively R5 tropic. The full-length R5-AIDS env mediated greater infectivity than R5 pre-AIDS env when used to pseudotype a reporter virus. R5-AIDS env pseudotypes were more resistant to TAK-779 and showed more rapid infection kinetics but similar resistance to a CD4 blocking mAb. We conclude that the enhanced thymic replication and CPE shown by the R5-AIDS clone is due to enhanced efficiency of Env-mediated entry via CCR5. [Copyright &y& Elsevier]