Biomedical subjects

Julie M Decker

Publications and source records attributed to Julie M Decker.

16 recordsLinked to original sources

PubMed2006

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

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

AIDS Vaccines↗

PubMed2006

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

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

AIDS Vaccines↗

PubMed2006

Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa.

A standard panel of subtype C human immunodeficiency virus type 1 (HIV-1) Env-pseudotyped viruses was created by cloning, sequencing, and characterizing functional gp160 genes from 18 acute and early heterosexually acquired infections in South Africa and Zambia. In general, the gp120 region of these clones was shorter (most evident in V1 and V4) and less glycosylated compared to newly transmitted subtype B viruses, and it was underglycosylated but no different in length compared to chronic subtype C viruses. The gp120s also exhibited low amino acid sequence variability (12%) in V3 and high variability (39%) immediately downstream of V3, a feature shared with newly transmitted subtype B viruses and chronic viruses of both subtypes. When tested as Env-pseudotyped viruses in a luciferase reporter gene assay, all clones possessed an R5 phenotype and resembled primary isolates in their sensitivity to neutralization by HIV-1-positive plasmas. Results obtained with a multisubtype plasma panel suggested partial subtype preference in the neutralizing antibody response to infection. The clones were typical of subtype C in that all were resistant to 2G12 (associated with loss of N-glycosylation at position 295) and most were resistant to 2F5, but all were sensitive to 4E10 and many were sensitive to immunoglobulin G1b12. Finally, conserved neutralization epitopes in the CD4-induced coreceptor binding domain of gp120 were poorly accessible and were difficult to induce and stabilize with soluble CD4 on Env-pseudotyped viruses. These results illustrate key genetic and antigenic properties of subtype C HIV-1 that might impact the design and testing of candidate vaccines. A subset of these gp160 clones are suitable for use as reference reagents to facilitate standardized assessments of vaccine-elicited neutralizing antibody responses.

Gene Products, env↗

PubMed2006

Ancestral and consensus envelope immunogens for HIV-1 subtype C.

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

AIDS Vaccines↗

PubMed2006

Evidence for potent autologous neutralizing antibody titers and compact envelopes in early infection with subtype C human immunodeficiency virus type 1.

Information about neutralizing antibody responses in subtype C-infected individuals is limited, even though this viral subtype causes the majority of AIDS cases worldwide. Here we compared the course and magnitude of the autologous neutralizing antibody (NAb) response against viral envelope (Env) glycoproteins present during acute and early infection with subtypes B and C human immunodeficiency virus type 1 (HIV-1). NAb responses were evaluated in 6 subtype B-infected and 11 subtype C-infected subjects over a mean evaluation period of 25 months using a pseudovirus reporter gene assay. All subjects in the C cohort were infected through heterosexual contact, while five of the six subjects in the B cohort were infected via male-to-male contact. The kinetics and magnitude of the NAb responses varied among subjects in the B and C cohorts; however, the median 50% inhibitory concentration (IC(50) titer) reached by antibody in the plasma of subtype C-infected subjects, overall, was 3.5-fold higher than in the subtype B-infected subjects (P = 0.06). The higher titers of NAbs in the C cohort were associated with viruses having significantly shorter amino acid length (P = 0.002) in the V1 to V4 region of the surface Env glycoprotein, gp120, compared to the B cohort. Despite the potency of the autologous subtype C NAb response, it was not directed against cross-neutralizing epitopes. These data demonstrate that subtype C Envs elicit a potent yet restricted NAb response early in infection that frequently reaches IC(50) titers in excess of 1:1,000 and suggest that clade-specific differences may exist in Env immunogenicity or susceptibility to neutralization.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Antigenic conservation and immunogenicity of the HIV coreceptor binding site.

Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as important targets of the adaptive humoral immune response in natural infection and, potentially, as components of an acquired immune deficiency syndrome vaccine. However, variability in exposed epitopes and a combination of highly effective envelope-cloaking strategies have made the identification of such epitopes problematic. Here, we show that the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-induced (CD4i) antibody during natural human infection and that these antibodies bind and neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of 189 (94%) HIV-1-infected patients had CD4i antibodies that neutralized sCD4-pretreated HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and polyclonal antibodies from HIV-1-infected humans competed specifically with such monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed coreceptor binding surfaces were detected in human plasma; these viruses were neutralized directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate lentiviruses, functional constraints on receptor binding create opportunities for broad humoral immune recognition, which in turn serves to constrain the viral quasispecies.

Amino Acid Sequence↗

PubMed2005

Scorpion-toxin mimics of CD4 in complex with human immunodeficiency virus gp120 crystal structures, molecular mimicry, and neutralization breadth.

The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.

Animals↗

PubMed2005

Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies.

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

AIDS Vaccines↗

PubMed2005

Antigenicity and immunogenicity of a synthetic human immunodeficiency virus type 1 group m consensus envelope glycoprotein.

Genetic variation of human immunodeficiency virus (HIV-1) represents a major obstacle for AIDS vaccine development. To decrease the genetic distances between candidate immunogens and field virus strains, we have designed and synthesized an artificial group M consensus env gene (CON6 gene) to be equidistant from contemporary HIV-1 subtypes and recombinants. This novel envelope gene expresses a glycoprotein that binds soluble CD4, utilizes CCR5 but not CXCR4 as a coreceptor, and mediates HIV-1 entry. Key linear, conformational, and glycan-dependent monoclonal antibody epitopes are preserved in CON6, and the glycoprotein is recognized equally well by sera from individuals infected with different HIV-1 subtypes. When used as a DNA vaccine followed by a recombinant vaccinia virus boost in BALB/c mice, CON6 env gp120 and gp140CF elicited gamma interferon-producing T-cell responses that recognized epitopes within overlapping peptide pools from three HIV-1 Env proteins, CON6, MN (subtype B), and Chn19 (subtype C). Sera from guinea pigs immunized with recombinant CON6 Env gp120 and gp140CF glycoproteins weakly neutralized selected HIV-1 primary isolates. Thus, the computer-generated "consensus" env genes are capable of expressing envelope glycoproteins that retain the structural, functional, and immunogenic properties of wild-type HIV-1 envelopes.

Amino Acid Sequence↗

PubMed2004

Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission.

Heterosexual transmission accounts for the majority of human immunodeficiency virus-1 (HIV-1) infections worldwide, yet the viral properties that determine transmission fitness or outgrowth have not been elucidated. Here we show, for eight heterosexual transmission pairs, that recipient viruses were monophyletic, encoding compact, glycan-restricted envelope glycoproteins. These viruses were also uniquely sensitive to neutralization by antibody from the transmitting partner. Thus, the exposure of neutralizing epitopes, which are lost in chronic infection because of immune escape, appears to be favored in the newly infected host. This reveals characteristics of the envelope glycoprotein that influence HIV-1 transmission and may have implications for vaccine design.

AIDS Vaccines↗

PubMed2004

Determinants of human immunodeficiency virus type 1 baseline susceptibility to the fusion inhibitors enfuvirtide and T-649 reside outside the peptide interaction site.

The peptide fusion inhibitor (PFI) enfuvirtide is the first of a new class of entry inhibitors to receive FDA approval. We previously determined the susceptibility of 55 PFI-naïve-patient isolates to enfuvirtide and a second peptide inhibitor, T-649. Seven of the 55 viral isolates were insusceptible to enfuvirtide, T-649, or both inhibitors in the absence of prior exposure. To determine the molecular basis of the insusceptible phenotypes, we PCR amplified and cloned five PFI-insusceptible and one PFI-susceptible, full-length, biologically functional env genes and characterized viruses pseudotyped with the Env proteins in a single-round drug sensitivity assay. Overall, the mean 50% inhibitory concentrations of enfuvirtide and T-649 for the PFI-insusceptible Env pseudotypes were 1.4 to 1.7 log(10) and 1.2 to 1.8 log(10) greater, respectively, than those for a PFI-susceptible lab strain, NLHX; however, all of the PFI-insusceptible Env proteins conserved the sequence of a critical enfuvirtide interaction site (residues 36 to 38 of gp41, GIV) in HR-1. In contrast, multiple amino acid changes were observed C-terminal to HR-1, many of which were located in regions of HR-2 corresponding to the PFI. Nevertheless, peptides based on patient-derived HR-2 sequences were not more potent inhibitors than enfuvirtide or T-649, arguing that the basis of PFI susceptibility is not a higher-affinity, competitive HR-1/HR-2 interaction. These results demonstrate that regions of Env outside the enfuvirtide interaction site can significantly impact the PFI susceptibility of patient-derived Env, even prior to drug exposure. We hypothesize that both gp120 gene- and gp41 gene-encoded determinants that minimize the window of opportunity for PFI to bind provide a growth advantage and possibly a predisposition to resistance to this new class of drugs in vivo.

Amino Acid Sequence↗

PubMed2003

Antibody neutralization and escape by HIV-1.

Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nab resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the env gene that were unexpectedly sparse, did not map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape, and the exceptional density of HIV-1 envelope glycosylation generally, led us to postulate an evolving 'glycan shield' mechanism of neutralization escape whereby selected changes in glycan packing prevent Nab binding but not receptor binding. Direct support for this model was obtained by mutational substitution showing that Nab-selected alterations in glycosylation conferred escape from both autologous antibody and epitope-specific monoclonal antibodies. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire.

Amino Acid Sequence↗

PubMed2003

Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice.

Codon usage optimization of human immunodeficiency virus type 1 (HIV-1) structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo; however, all optimized genes reported thus far are derived from HIV-1 (group M) subtype B viruses. Here, we report the generation and biological characterization of codon usage-optimized gag, pol, env (gp160, gp140, gp120), and nef genes from a primary (nonrecombinant) HIV-1 subtype C isolate. After transfection into 293T cells, optimized subtype C genes expressed one to two orders of magnitude more protein (as determined by immunoblot densitometry) than the corresponding wild-type constructs. This effect was most pronounced for gp160, gp140, Gag, and Pol (>250-fold), but was also observed for gp120 and Nef (45- and 20-fold, respectively). Optimized gp160- and gp140-derived glycoproteins were processed, incorporated into virus particles, and mediated virus entry when expressed in trans to complement an env-minus HIV-1 provirus. Mice immunized with optimized gp140 DNA developed antibody as well as CD4+ and CD8+ T cell immune responses that were orders of magnitude greater than those of mice immunized with wild-type gp140 DNA. These data confirm and extend previous studies of codon usage optimization of HIV-1 genes to the most prevalent group M subtype. Our panel of matched optimized and wild-type subtype C genes should prove valuable for studies of protein expression and function, the generation of subtype-specific immunological reagents, and the production of DNA-based sub-unit vaccines directed against a broader spectrum of viruses.

AIDS Vaccines↗

PubMed2003

Potential role for CD63 in CCR5-mediated human immunodeficiency virus type 1 infection of macrophages.

Macrophages and CD4(+) lymphocytes are the principal target cells for human immunodeficiency virus type 1 (HIV-1) infection, but the molecular details of infection may differ between these cell types. During studies to identify cellular molecules that could be involved in macrophage infection, we observed inhibition of HIV-1 infection of macrophages by monoclonal antibody (MAb) to the tetraspan transmembrane glycoprotein CD63. Pretreatment of primary macrophages with anti-CD63 MAb, but not MAbs to other macrophage cell surface tetraspanins (CD9, CD81, and CD82), was shown to inhibit infection by several R5 and dualtropic strains, but not by X4 isolates. The block to productive infection was postfusion, as assessed by macrophage cell-cell fusion assays, but was prior to reverse transcription, as determined by quantitative PCR assay for new viral DNA formation. The inhibitory effects of anti-CD63 in primary macrophages could not be explained by changes in the levels of CD4, CCR5, or beta-chemokines. Infections of peripheral blood lymphocytes and certain cell lines were unaffected by treatment with anti-CD63, suggesting that the role of CD63 in HIV-1 infection may be specific for macrophages.

Antibodies, Monoclonal↗

PubMed2002

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells.

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 degrees C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.

Amino Acid Sequence↗

PubMed2002

Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.

The synthetic peptide T-20 (enfuvirtide) represents the first of a new class of antiretroviral compounds to demonstrate in vivo potency by targeting a step in viral entry. T-20 inhibits a conformational change in the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein (gp41) that is required for fusion between HIV-1 and target cell membranes. The initial phase I clinical trial of T-20 treatment for HIV-infected patients thus provided a unique opportunity to evaluate the emergence of resistant virus in vivo to this novel class of antiretroviral agents. All four patients who received an intermediate dose of T-20 (30 mg twice daily) had an initial decline in plasma viral load over the first 10 days but a rising trend by day 14, suggestive of selection for resistant virus. Plasma virus derived from patients enrolled in all dosage groups of the phase I T-20 trial was analyzed by population sequencing before and after treatment. While no mutations were found within a highly conserved 3-amino-acid sequence (GIV) known to be critical for fusion at baseline, after 14 days of therapy, virus from one patient in the 30-mg dose group (30-1) developed a mutation in this motif, specifically an aspartic acid (D) substitution for glycine (G) at position 36. Multiple env clones were derived from the plasma virus of all four patients in the 30-mg dosage group. Sequence analysis of 49 clones derived from the plasma of patient 30-1 on day 14 revealed that 25 clones contained the G36D mutation, while 8 contained the V38A mutation. Dual mutations involving G36D and other residues within the HR1 domain were also identified. In 5 of the 49 env clones, other mutations involving residues 32 (Q32R or Q32H) and 39 (Q39R) were found in combination with G36D. Cloned env sequences derived from the plasma virus of subject 30-3 also had single mutations in the GIV sequence (V38M and I37V) detectable following therapy with T-20. The plasma virus from subjects 30-2 and 30-4 did not contain changes within the GIV sequence. To analyze the biological resistance properties of these mutations, we developed a novel single-cycle HIV-1 entry assay using JC53BL cells which express beta-galactosidase and luciferase under control of the HIV-1 long terminal repeat. Full-length env clones were derived from the plasma virus of patients 30-1 and 30-3 and used to generate pseudotyped virus stocks. The mean 50% inhibition concentrations (IC(50)s) for mutants G36D and V38A (patient 30-1) were 2.3 microg/ml and 11.2 microg/ml, respectively, statistically significant increases of 9.1- and 45-fold, respectively, compared with those of wild-type Env. The IC(50) for the V38 M mutation (patient 30-3) was 7.6 microg/ml, an 8-fold increase compared with that of the wild type. The I37V mutation resulted in an IC(50) 3.2-fold greater than that of the wild type. Envs with double mutations (Q32R plus G36D and Q32H plus G36D) exhibited a level of resistance similar to that of G36D alone. These findings provide the first evidence for the rapid emergence of clinical resistance to a novel class of HIV-1 entry inhibitors and may be relevant to future treatment strategies involving these agents.

Amino Acid Sequence↗