Induction of Fc-Mediated Effector Functions Against a Stabilized Inner Domain of HIV-1 gp120 Designed to Selectively Harbor the A32 Epitope Region.

Recent clinical trials and studies using nonhuman primates (NHPs) suggest that antibody-mediated protection against HIV-1 will require α-HIV envelope humoral immunity beyond direct neutralization to include Fc-receptor (FcR) mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC). There is also strong evidence indicating that the most potent ADCC response in humans is directed toward transitional non-neutralizing epitopes associated with the gp41-interactive face of gp120, particularly those within the first and second constant (C1-C2) region (A32-like epitopes). These epitopes were shown to be major targets of ADCC responses during natural infection and have been implicated in vaccine-induced protective immunity.

Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region.

Evidence supports a role of antibody-dependent cellular cytotoxicity (ADCC) toward transitional epitopes in the first and second constant (C1-C2) regions of gp120 (A32-like epitopes) in preventing HIV-1 infection and in vaccine-induced protection. Here, we describe the first successful attempt at isolating the inner domain (ID) of gp120 as an independent molecule that encapsulates the A32-like region within a minimal structural unit of the HIV-1 Env. Through structure-based design, we developed ID2, which consists of the ID expressed independently of the outer domain and stabilized in the CD4-bound conformation by an inter-layer disulfide bond.

Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1.

Unlabelled: Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes).

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate.

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018).

Chemokine receptor interactions with virus-like particles.

Virus-like particles (VLPs) presenting conformational envelope proteins on their surface represent an invaluable tool to study molecular interactions between viruses and cellular receptors/co-receptors, eliminating biological risks associated with working with live native viruses. The availability of target cells expressing specific chemokine receptors facilitates the dissection of specific interactions between human immunodeficiency virus (HIV) viral envelope proteins and these receptors in the laboratory. Here, we describe a method to evaluate HIV-VLP binding to cellular chemokine co-receptors, by carboxyfluorescein succinimidyl ester labeling and cellular uptake.

Developments in virus-like particle-based vaccines for HIV.

Virus-like particles (VLPs) hold great promise for the development of effective and affordable vaccines. VLPs, indeed, are suitable for presentation and efficient delivery to antigen-presenting cells of linear as well as conformational antigens. This will ultimately result in a crosspresentation with both MHC class I and II molecules to prime CD4(+) T-helper and CD8(+) cytotoxic T cells.

Targeting a Neutralizing Epitope of HIV Envelope Gp120 by Immune Complex Vaccine.

There are formidable challenges in developing HIV vaccines that elicit potent neutralizing antibodies against a broad array of HIV-1 isolates. The key targets for these neutralizing antibodies are the viral envelope antigens gp120 and gp41. Although broadly reactive neutralizing epitopes on gp120 and gp41 have been mapped and studied extensively, these epitopes are poorly immunogenic.

Immunogenicity of HIV virus-like particles in rhesus macaques by intranasal administration.

Female rhesus macaques were immunized with HIV virus-like particles (HIV-VLPs) or HIV DNA administered as sequential combinations of mucosal (intranasal) and systemic (intramuscular) routes, according to homologous or heterologous prime-boost schedules. The results show that in rhesus macaques only the sequential intranasal and intramuscular administration of HIV-VLPs, and not the intranasal alone, is able to elicit humoral immune response at the systemic as well as the vaginal level.

Effects of adjuvants on IgG subclasses elicited by virus-like particles.

Background: Virus-Like Particles (VLPs) represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN1826 or poly(I:C) adjuvants, has been evaluated in an animal model.

HIV-Gag VLPs presenting trimeric HIV-1 gp140 spikes constitutively expressed in stable double transfected insect cell line.

We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efficient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specific systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag-VLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins.

Generation of HIV-1 Virus-Like Particles expressing different HIV-1 glycoproteins.

Elicitation of a potent and broadly neutralizing antibody response is the main goal of an effective preventive HIV-1 vaccine. It has been shown by us and others that the expression of Env glycoproteins on the surface of particulate structures, such as Virus-Like Particles (VLPs), could be a more efficient strategy to deliver conformational epitopes to the immune system. To this aim, VLPs expressing native HIV Env gp140 or gp41 glycoproteins have been produced in insect cells using a baculovirus expression system and characterized for appropriate protein expression.

Constitutive expression of HIV-VLPs in stably transfected insect cell line for efficient delivery system.

We have previously developed HIV-1 Pr55gag-based virus-like particles (HIV-VLPs) as presentation and delivery model using a transient Baculovirus expression system. Here we describe the establishment and characterization of stably transfected insect cell line for the constitutive and reproducible production of HIV-VLPs. The persistence of HIV gag coding gene has been verified in clonal resistant insect cells and the protein expression has been confirmed by Western blot analysis.

The use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on HIV-1 envelope gp120.

The capacity of immune complexes to augment antibody (Ab) responses is well established. The enhancing effects of immune complexes have been attributed mainly to Fc-mediated adjuvant activity, while the ability of Abs to induce antigenic alterations of specific epitopes as a result of immune complex formation has been less well studied. Previously we have shown that the interaction of anti-CD4-binding site (CD4bs) Abs with HIV-1 gp120 induces conformation changes that lead to enhanced antigenicity and immunogenicity of neutralizing epitopes in the V3 loop.

Antibodies to the CD4-binding site of HIV-1 gp120 suppress gp120-specific CD4 T cell response while enhancing antibody response.

Background: The binding of Abs to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 has been shown to obstruct the processing and generation of helper epitopes from this antigen, resulting in poor presentation of various gp120 epitopes by MHC class II to CD4 T cells. However, the physiologic significance of these inhibitory anti-CD4bs Abs in vivo has remained unclear. In this study, we evaluated the immunologic effects of anti-CD4bs Abs in vivo using a murine model.

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4(+) T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells.

Identification of an N-linked glycosylation in the C4 region of HIV-1 envelope gp120 that is critical for recognition of neighboring CD4 T cell epitopes.

The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions.

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120.

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus.

Studies on the effect of Amadoriase from Aspergillus fumigatus on peptide and protein glycation in vitro.

Amadoriase I is a fructosyl amine oxidase from Aspergillus fumigatus that catalyzes the oxidation of Amadori products (APs) producing glucosone, H2O2, and the corresponding free amine. All the enzymes of this family discovered so far only deglycate small molecular weight products and are inactive toward large molecular weight substrates, such as glycated BSA or ribonuclease A. Therefore, they cannot be used to reverse protein glycation occurring in diabetes or in foods.

Glycation of lysine-containing dipeptides.

Protein glycation through Maillard reaction (MR) is a fundamental reaction both in foods and in the human body. The first step of the reaction is the formation of Amadori product (AP) that is converted into intermediate and advanced MR products during reaction development. Although the MR is not an enzymatic reaction, a certain degree of specificity in the glycation site has been observed.

Characterization of antibodies that inhibit HIV gp120 antigen processing and presentation.

Antibodies to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti-CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor-binding site or other gp120 regions. Anti-CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid-stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing.

Induction of systemic and mucosal cross-clade neutralizing antibodies in BALB/c mice immunized with human immunodeficiency virus type 1 clade A virus-like particles administered by different routes of inoculation.

We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model, based on virus-like particles (VLPs) expressing gp120 from a Ugandan HIV-1 isolate of clade A (HIV-VLP(A)s), which shows the induction of neutralizing antibodies as well as cytotoxic T lymphocytes (CTL) in BALB/c mice by intraperitoneal (i.p.) administration.

Induction of neutralizing antibodies and cytotoxic T lymphocytes in Balb/c mice immunized with virus-like particles presenting a gp120 molecule from a HIV-1 isolate of clade A.

We have recently developed a candidate HIV-1 vaccine based on virus-like particles (VLPs) expressing a gp120 from an Ugandan HIV-1 isolate of the clade A (HIV-VLP(A)s). In vivo immunogenicity experiments were performed in Balb/c mice, with an immunization schedule based on a multiple-dose regimen of HIV-VLP(A)s without adjuvants, showing a significant induction of both humoral and cellular immunity. The Env-specific cellular response was investigated in vitro, scoring for both the proliferative response of T helper cells and the cytolytic activity of cytotoxic T lymphocytes (CTLs).

Immunohistochemical expression of cyclin T1 in a case of AIDS-related cachexia.

The expression of cyclin T1 in an autoptic case of AIDS-related cachexia was investigated by immunohistochemistry. When contrasted with normal human tissues, a very similar pattern of expression was found. However, a peculiar distribution of cyclin T1 was noticed in the brown fat and in lymph nodes affected by AIDS-associated lymphadenopathy.