DNA-immunization with a V2 deleted HIV-1 envelope elicits protective antibodies in macaques.

Rhesus macaques immunized with the HIV-1 SF162DeltaV2 gp140 envelope using the DNA-prime plus protein-boost vaccination methodology, developed HIV envelope-specific T-cell lymphoproliferative responses and potent neutralizing antibodies. To evaluate the protective potential of these antibodies during acute infection, the animals were depleted of their CD8+ T lymphocytes using specific monoclonal antibodies and subsequently challenged intravenously with the pathogenic SHIV(SF162P4) isolate. As compared to non-vaccinated animals (one of which died from AIDS 16 weeks post-exposure) the vaccinated macaques had lower levels of peak viremia, rapidly cleared virus from the periphery and developed delayed seroconversion to SIV core antigens.

The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region.

Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162DeltaV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162DeltaV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162DeltaV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162DeltaV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.

DNA vaccination with the human immunodeficiency virus type 1 SF162DeltaV2 envelope elicits immune responses that offer partial protection from simian/human immunodeficiency virus infection to CD8(+) T-cell-depleted rhesus macaques.

DNA immunization of macaques with the SF162DeltaV2 envelope elicited lymphoproliferative responses and potent neutralizing antibodies. The animals were depleted of their CD8(+) T lymphocytes and then challenged intravenously with SHIV162P4. Compared to unvaccinated animals, the vaccinated macaques had lower peak viremia levels, rapidly cleared plasma virus, and showed delayed seroconversion.

Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo.

Liposomes have been proposed as a vehicle to deliver proteins to antigen-presenting cells (APC), such as dendritic cells (DC), to stimulate strong T cell-mediated immune responses. Unfortunately, because of their instability in vivo and their rapid uptake by cells of the mononuclear phagocyte system on intravenous administration, most types of conventional liposomes lack clinical applicability. In contrast, sterically stabilized liposomes (SL) have increased in vivo stability. It is shown that both immature and mature DC take up SL into neutral or mildly acidic compartments distinct from endocytic vacuoles. These DC presented SL-encapsulated protein to both CD4(+) and CD8(+) T cells in vitro. Although CD4(+) T-cell responses were comparable to those induced by soluble protein, CD8(+) T-cell proliferation was up to 300-fold stronger when DC had been pulsed with SL-encapsulated ovalbumin. DC processed SL-encapsulated antigen through a TAP-dependent mechanism. Immunization of mice with SL-encapsulated ovalbumin led to antigen presentation by DC in vivo and stimulated greater CD8(+) T-cell responses than immunization with soluble protein or with conventional or positively charged liposomes carrying ovalbumin. Therefore, the application of SL-encapsulated antigens offers a novel effective, safe vaccine approach if a combination of CD8(+) and CD4(+) T-cell responses is desired (ie, in anti-viral or anti-tumor immunity).

V2 loop glycosylation of the human immunodeficiency virus type 1 SF162 envelope facilitates interaction of this protein with CD4 and CCR5 receptors and protects the virus from neutralization by anti-V3 loop and anti-CD4 binding site antibodies.

We examined the role of asparagine-linked glycosylation of the V2 loop of the human immunodeficiency virus (HIV) SF162 envelope on viral replication potential and neutralization susceptibility. We report that the asparagines located at the amino- and carboxy-terminal sites (at positions 154 and 195, respectively), as well as within the V2 loop of the SF162 envelope (at position 186), are glycosylated during in vitro replication of this virus in human peripheral blood mononuclear cells. Our studies indicate that glycosylation of the V2 loop, in particular at its base, facilitates the interaction of the HIV envelope with the CD4 and CCR5 receptor molecules present on the surface of target cells and affects viral replication kinetics in a cell type-dependent manner. In cells expressing high numbers of receptor molecules on their surfaces, the SF162-derived V2 loop-deglycosylated mutant viruses replicate as efficiently as the parental SF162 virus, while in cells expressing small numbers of receptor molecules, the mutant viruses replicate with markedly reduced efficiency. In addition to expanding the viral tropism, V2 loop glycosylation at the three sites examined prevents neutralization by anti-CD4 binding site antibodies. In contrast, glycosylation at the amino- and carboxy-terminal sites of the V2 loop but not within the loop itself offers protection against anti-V3 loop antibodies. Thus, the epitopes masked by the sugar molecules present on the three glycosylation sites examined are not identical but overlap.

Generation and structural analysis of soluble oligomeric gp140 envelope proteins derived from neutralization-resistant and neutralization-susceptible primary HIV type 1 isolates.

We generated DNA constructs expressing soluble truncated forms of the envelope of SF162, a neutralization-resistant primary human immunodeficiency virus type 1 isolate, and SF162AV2, a neutralization-susceptible virus derived from SF162 after the deletion of 30 amino acids from the V2 loop. The constructs express the entire gp120 subunit and the extracellular region of the gp41 subunit, with either the presence ("cleaved" forms, designated gp140C) or the absence ("fused" forms, designated gp140F) of the gp120-gp41 cleavage site. Both gp140 forms derived from SF162 and SF162deltaV2 are secreted in the cell medium and are recognized by the oligomer-specific anti-gp41 MAb T4. As is the case for the corresponding virion-associated envelope molecules, the CD4-binding region is occluded within both gp140F and gp140C forms. However, structural differences exist between these two forms. The gp140F proteins are less efficiently recognized than the gp140C proteins by antibodies present in the sera of HIV-infected patients with neutralizing activities against SF162 and SF162AV2. Also, the V3 loop is more exposed on gp140F than gp140C. As is the case for intact virions, on CD4 binding both the gp140F and gp140C proteins undergo conformational changes that result in the exposure of the epitope recognized by MAb 17b, which has been implicated in coreceptor binding. In contrast, during these structural changes the exposure of specific V3 loop epitopes is not increased on either gp140C or gp140F. Taken together, our data indicate that although these gp140 forms differ structurally from the native envelope, their similarities, in particular that of gp140C, outweigh their differences.

Effect of major deletions in the V1 and V2 loops of a macrophage-tropic HIV type 1 isolate on viral envelope structure, cell entry, and replication.

Two HIV-1 envelope mutant proteins were generated by introducing deletions in the first and second hypervariable gp120 regions (V1 and V2 loops, respectively) of a macrophage-tropic primary HIV-1 isolate, SF162, to study the effect of the deleted sequences on envelope structure, viral entry, and replication potentials. The first mutant lacked 17 amino acids of the V1 loop and the latter 30 amino acids of the V2 loop. A comparison of the immunochemical structure of the wild-type and mutant monomeric and virion-associated gp120 molecules revealed that the V1 and V2 loop deletions differentially altered the structure of the V3 loop, the CD4-binding site, and epitopes within conserved regions of gp120. Regardless of differences in structure, both mutated envelope proteins supported viral replication into peripheral blood mononuclear cells to levels comparable to those of the wild-type SF162 virus. However, they decreased the viral replication potential in macrophages, even though they did not alter the coreceptor usage of the viruses. These studies support and extend previous observations that a complex structural interaction between the V1, V2, and V3 loops and elements of the CD4-binding site of gp120 controls entry of virus into cells. The present studies, however, suggest that the effect of the V1 and V2 loops in viral entry is cell dependent.

An envelope modification that renders a primary, neutralization-resistant clade B human immunodeficiency virus type 1 isolate highly susceptible to neutralization by sera from other clades.

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162DeltaV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162DeltaV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162DeltaV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.

Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off.

IgG1b12, a human monoclonal antibody (MAb) to an epitope overlapping the CD4-binding site on gp120, has broad and potent neutralizing activity against most primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess whether and how escape mutants resistant to IgG1b12 can be generated, we cultured primary HIV-1 strain JRCSF in its presence. An escape mutant emerged which was approximately 100-fold more resistant to neutralization by IgG1b12. Both virion-associated and solubilized gp120 from this variant had a reduced affinity for IgG1b12, and sequencing of its env gene showed that amino acid substitutions had occurred at three positions within gp120. Two (D164N and D182N) were located in V2, and one (P365L) was in C3. By site-directed mutagenesis, we demonstrated that the D182N and P365L mutations, but not D164N, contribute to the IgG1b12-resistant phenotype. However, the former two substitutions, individually or in combination, hinder the replication of the neutralization-resistant virus. Introduction of the D164N substitution into the P365L variant results in a nonviable virus (D164N/P365L). In contrast, addition of D164N to the D182N or D182N/P365L mutant partially restored replicative function to near wild-type levels. Furthermore, we found that all of the IgG1b12-resistant mutant viruses remained sensitive to other human MAbs, such as 2G12 and 2F5, and to the CD4-IgG molecule, except that the P365L-containing mutant was slightly resistant to CD4-IgG. These results suggest that escape from IgG1b12 neutralization is due to a local rather than a global modification of the gp120 structure. Our findings have implications for the therapeutic and prophylactic applications of antibodies for HIV-1 infection.

Binding of antibodies to virion-associated gp120 molecules of primary-like human immunodeficiency virus type 1 (HIV-1) isolates: effect on HIV-1 infection of macrophages and peripheral blood mononuclear cells.

Using immunobiochemical approaches we previously studied the conformation and surface exposure of different immunodominant regions within the oligomeric, virion-associated form of the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) (L Stamatatos and C. Cheng-Mayer (1995) J. Virol. 69, 6191-6198). These studies allowed us to determine to what extent epitopes within these immunodominant regions of the oligomeric gp120 are occluded or accessible to antibody binding on the virion surface of two primary-like (HIV-1SF162 and HIV-1SF128A) and one. T-cell-line-adapted (HIV-1SF2) isolates. Here, we investigate whether binding of anti-gp120 monoclonal antibodies (MAbs) to exposed epitopes of the immunodominant regions of oligomeric gp120 results in neutralization of HIV-1 infection and whether certain exposed sites are better targets for neutralization than others. We also test whether proposed mechanisms for antibody-mediated neutralization of T-cell-line-adapted HIV-1 isolates, e.g., antibody-mediated gp120-virion dissociation, are applicable to primary-like HIV-1 isolates. We assess the neutralization potential of anti-V2 loop, anti-V3 loop, and anti-CD4 binding site MAbs using human primary macrophages or peripheral blood mononuclear cells (PBMC) as target cells and HIV-1SF162 and HIV-1SF128A as infecting isolates. Our data indicate that: (i) not every exposed epitope of the immunodominant regions of gp120 oligomers on the virion surface is a target for neutralization; (ii) during virus-cell fusion specific exposure of previously occluded V3 loop epitopes within gp120 oligomers occurs, which may become targets for neutralization; (iii) antibody-mediated gp120-virion dissociation does not appear to be a major mechanism of neutralization for the primary-like HIV-1 isolates tested here; and (iv) infection of macrophages is more sensitive to neutralization by anti-gp120 monoclonal antibodies than PBMC. We also report that binding of one of the two anti-CD4 binding site MAbs tested mediated enhancement of macrophage cell infection in a concentration-dependent fashion, while binding of the other anti-CD4 binding site MAb resulted in efficient neutralization of infection of both macrophages and PBMC.

Macrophage tropism of human immunodeficiency virus type 1 and utilization of the CC-CKR5 coreceptor.

The recent identification of the CC-CKR5 beta chemokine receptor as a major cofactor for entry of macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1) raises the question of whether macrophage tropism is determined by utilization of this chemokine receptor. We observe that in addition to macrophage-tropic isolates of clades A, B, and E, macrophage-tropic isolates of clade F also utilize the CC-CKR5 molecule for entry. However, using single-round replication-competent reporter viruses carrying the envelope genes of T-cell line-tropic or macrophage-tropic phenotypic recombinant and mutant HIV-1 strains in infection of stable cell lines that coexpress the CD4 and chemokine receptors, we were unable to establish a strict correlation between macrophage tropism and utilization of the CC-CKR5 chemokine receptor. This latter finding suggests that a cofactor other than CC-CKR5 serves to determine entry into primary macrophages.

Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding.

We investigated the binding of conformation-dependent anti-V2, anti-V3, and anti-CD4-binding site monoclonal antibodies to monomeric and virion-associated gp120 from human immunodeficiency virus type 1 isolates displaying marked differences in cell tropism. For all viruses examined, we found that the half-maximal binding values of the anti-V2 and anti-CD4-binding site antibodies with virion-associated gp120 were higher than those with monomeric gp120, but the maximum amount of antibodies bound was diminished only for one of the anti-V2 antibodies tested. These observations suggest that upon gp120 oligomerization, the V2 loop and CD4-binding site undergo conformational changes and that particular epitopes within these domains are occluded in the oligomeric gp120. In contrast, although the overall binding patterns and half-maximal binding values of the anti-V3 loop antibodies tested were similar with monomeric and oligomeric gp120, all the V3 loop epitopes examined were less accessible to antibody binding on the virion surface. This masking of the V3 loop is more pronounced for the primary-like macrophage-tropic isolates examined. Lastly, we observe that upon soluble receptor-virion binding, specific V3 loop epitopes that differ for viruses displaying different tropisms are exposed.

Small amino acid sequence changes within the V2 domain can affect the function of a T-cell line-tropic human immunodeficiency virus type 1 envelope gp120.

Prior studies with recombinant viruses constructed in vitro showed that the V2 domain of envelope gp120, in addition to the required V3 domain, enhances the efficiency of infection of primary macrophages by HIV-1. Present structural studies on the gp120s of these recombinant viruses using three human monoclonal antibodies directed to the V3 loop indicate that the V2 domain affects cell tropism by modulating the conformation of the V3 loop. Additional mutational analyses of the V2 domain of the T-cell line-tropic virus HIV-1SF2 reveal that single amino acid sequence changes, mainly those affecting the location of potential N-linked glycosylation sites and the positive charge of this region, can also alter tropism. These amino acid substitutions in the V2 domain, however, do not appear to alter the conformation of the V3 loop. Thus, the V2 domain of gp120 can influence cell tropism through both an effect on V3 as well as via a V3-independent mechanism.

Differential regulation of cellular tropism and sensitivity to soluble CD4 neutralization by the envelope gp120 of human immunodeficiency virus type 1.

Using recombinant and mutant viruses generated between two human immunodeficiency virus type 1 isolates that display differences in cell tropism and sensitivity to soluble CD4 neutralization, we show that these two properties of the virus are regulated by different mechanisms. Whereas there is an association between V3 loop conformation and a particular cellular tropism, soluble CD4 neutralization sensitivity appears to be determined by amino acid differences in the C2 domain of the envelope gp120 that modulate the stability of gp120-gp41 association. Our findings further illustrate the importance of functional interactions among different regions of the envelope gp120 in regulating the biological phenotypes of human immunodeficiency virus and suggest that additional probing of the V3 loop with monoclonal antibodies may identify specific structural features of this loop that determine cell tropism.

Evidence that the structural conformation of envelope gp120 affects human immunodeficiency virus type 1 infectivity, host range, and syncytium-forming ability.

We investigated how amino acid changes within and outside the V3 loop of the envelope glycoprotein of human immunodeficiency virus type 1 influence the infectivity, host range, and syncytium-forming ability of the virus. Our studies show that on the genomic backgrounds of the human immunodeficiency virus type 1 strains SF2 and SF13, a reciprocal exchange of full-loop sequences does not alter the syncytium-forming ability of the viruses, indicating that a determinant(s) for this biological property maps outside the loop. However, specific amino acid substitutions, both within and outside the V3 loop, resulted in loss of infectivity, host range, and syncytium-forming potential of the virus. Furthermore, it appears that a functional interaction of the V3 loop with regions in the C2 domain of envelope gp120 plays a role in determining these biological properties. Structural studies of mutant glycoproteins show that the mutations introduced affect the proper association of gp120 with the transmembrane glycoprotein gp41. Our results suggest that mutations that alter the structure of the V3 loop can affect the overall conformation of gp120 and that, reciprocally, the structure of the V3 loop is influenced by the conformation of other regions of gp120. Since the changes in the replicative potential, host range, and fusogenic ability of the mutant viruses correlate well with the changes in gp120 conformation, as monitored by the association of gp120 with gp41, our results support a close relationship between envelope gp120 structural conformation and the biological phenotype of the virus.

Simian immunodeficiency virus (SIVmac251) membrane lipid mixing with human CD4+ and CD4- cell lines in vitro does not necessarily result in internalization of the viral core proteins and productive infection.

The cell binding site of simian immunodeficiency virus (SIV) is believed to be the CD4 molecule. Several CD4+ cell lines are, however, resistant to infection by SIVmac251 in vitro and additional cell membrane molecules have been implicated in SIVmac251 entry. We investigated the binding, envelope fusion and entry of the viral core proteins (p27) of SIVmac251 into two human CD4+ cell lines (H9 and Sup-T1) which are infectible, and one CD4+ (A3.01) and two CD4- cell lines (K562 and Raji) that are resistant to infection. The fusion of the viral and cellular membranes was monitored by a fluorescence assay for lipid mixing. Cell entry of the viral core was evaluated following virus-cell incubation and cell surface trypsinization. We found that SIVmac251 can bind to and fuse (membrane lipid mixing) in a temperature-dependent but pH-independent fashion with CD4+ and CD4- human-derived cell lines. In contrast, lipid mixing with CD4 expressing EL-4 mouse T cells or Mv-1-lu mink lung fibroblasts was absent or limited, suggesting that certain components of human cell membranes in addition to CD4 are involved in SIVmac envelope-cell fusion. Lipid mixing with the human cells was inhibited partially by soluble CD4. Anti-CD4 antibodies inhibited the lipid inter-mixing with H9, but not with Raji cells, whereas neutralizing anti-SIVmac sera inhibited fusion with both CD4+ and CD4- cells. Out of the five human cell lines tested, efficient entry of p27 and productive infection took place only with H9 and Sup-T1 cells. In these two cases, the amounts of p27 internalized during virus-cell fusion correlated with the extent of infection.

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells.

The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma (S49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4.9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5.4, different from that of the virus preincubated at low pH without target membranes.

Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: the role of CD4, serum and liposome-cell interactions.

We have reported previously the enhancement of the infectivity of human immunodeficiency virus type 1 (HIV-1) by liposomes composed of the cationic lipid N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA). To determine the mechanism by which this process occurs, we have investigated the role of CD4, serum concentration and liposome-cell interactions in the DOTMA-mediated stimulation of HIV-1 infection of A3.01 cells. Serum alone significantly inhibited the binding and infectivity of HIV-1, but DOTMA-mediated enhancement of infectivity was more pronounced in the presence of serum than in its absence. HIV-1 binding to cells was increased in the presence of DOTMA liposomes, DEAE-dextran and polybrene, all of which also enhanced infectivity to a similar extent at comparable concentrations. Fluorescence dequenching measurements indicated that DOTMA liposomes fused with HIV-1, but not with cell membranes, in the presence of serum. The enhancing effect of DOTMA liposomes on HIV-1 infectivity was CD4-dependent, and appeared to involve virus-liposome fusion and liposome binding to the cell surface. DOTMA liposomes did not mediate infection of the CD4-K562 and Raji cell lines.