Th2 polarization in peripheral blood mononuclear cells from human immunodeficiency virus (HIV)-infected subjects, as activated by HIV virus-like particles.

We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.

Baculovirus-derived human immunodeficiency virus type 1 virus-like particles activate dendritic cells and induce ex vivo T-cell responses.

We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model based on HIV-1 Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A (HIV-VLP(A)s). The HIV-VLP(A)s show the induction in BALB/c mice of systemic and mucosal neutralizing antibodies as well as cytotoxic T lymphocytes, by intraperitoneal as well as intranasal administration. In the present article, the effects of the baculovirus-expressed HIV-VLPs on human immature monocyte-derived dendritic cells (MDDCs) have been evaluated. The HIV-VLPs efficiently induce maturation and activation of MDDCs and are incorporated into MDDCs preferentially via an actin-dependent macropinocytosis and endocytosis. The HIV-VLP-activated MDDCs show enhanced Th1- and Th2-specific cytokine production, and the effects of HIV-VLPs on MDDCs are not mediated through Toll-like receptors 2 and 4 (TLR2 and -4) signaling. Finally, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. Our results on the interaction and processing of baculovirus HIV-VLPs by MDDCs give an insight into the mechanisms underlying the immune response induced by HIV-VLP(A)s in vivo.

Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1.

Conformationally constrained HIV-1 Env and gp120 immunogens induce broadly cross-reactive neutralizing antibodies. Thus, it is now feasible to rationally design an HIV-1 vaccine that affords protection through humoral mechanisms. This paper reviews our progress toward the development of an oral bacterial vaccine vector that is capable of delivering an HIV-1 DNA vaccine to host lymphoid tissues and inducing broadly neutralizing antibodies to HIV-1 in the mucosal and systemic immune compartments.

Immunogenicity of DNA vaccines that direct the coincident expression of the 120 kDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin.

Passive antibody studies unequivocally demonstrate that sterilizing immunity against lentiviruses is obtainable through humoral mechanisms. In this regard, DNA vaccines represent an inexpensive alternative to subunit vaccine for mass vaccination programs designed to induce such responses to human immunodeficiency virus type I (HIV-1). At present, however, this vaccine modality has proven relatively ineffective at inducing humoral responses. In this report, we describe the immunogenicity of DNA vaccines that direct the coincident expression of the cholera toxin catalytic domain (CTA1) with that of the human immunodeficiency virus type I gp120 through genes either encoded in individual plasmids or in a single dicistronic plasmid. In BALB/cJ mice, coincident expression of CTA1 in either a separate plasmid or in the dicistronic plasmid in the DNA vaccines induced serum IgG responses to gp120 that were at least 1000-fold greater, and remained elevated longer than, the analogous responses in mice vaccinated with a DNA vaccine that expressed gp120 alone. In addition, mice vaccinated with CTA1 and gp120 produced significantly more gp120-specific IFN-gamma ELISPOTs than mice vaccinated with the gp120 DNA vaccine. Combined, these data show that the adjuvant properties of cholera toxin can be harnessed in DNA vaccine modalities.

Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector.

CD8(+) T-cell responses provide beneficial antiviral immunity against human immunodeficiency virus 1 (HIV-1). In this study, we show that intragastric vaccination with a Salmonella HIV-1 Env DNA vaccine vector generates Env-specific CD8(+) T-cells, both in mucosal and systemic lymphoid tissue. By contrast, intramuscular vaccination with the Env DNA vaccine alone only induced systemic CD8(+) T-cells. To our knowledge, this is the first report showing both mucosal and systemic CD8(+) T-cell responses following vaccination with a Salmonella vaccine vector. These data suggest that this mode of HIV-1 DNA vaccine delivery will be advantageous over parenterally administered HIV-1 DNA vaccines.

Antigenic properties of the human immunodeficiency virus envelope during cell-cell fusion.

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4( degrees )C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.

Perforin-low memory CD8+ cells are the predominant T cells in normal humans that synthesize the beta -chemokine macrophage inflammatory protein-1beta.

The synthesis of antiviral beta-chemokines has joined cytolysis as a potential mechanism for the control of HIV-1 infection by CD8(+) T cells. Recent evidence suggests that these two effector functions can diverge in some individuals infected with HIV-1; however, little is known about the CD8(+) T cell subsets in normal individuals that synthesize antiviral beta-chemokines. In this report, we have used mutliparameter flow cytometry to characterize the T cell subsets that secrete the antiviral beta-chemokine macrophage inflammatory protein (MIP)-1beta. These studies have shown: (i) CD8(+) cells are the predominant T cell subset that synthesizes MIP-1beta; (ii) MIP-1beta and IFN-gamma are synthesized congruently in most CD8(+) T cells; however, significant numbers of these cells synthesize only one of these effector molecules; (iii) approximately 60% of the CD8(+) T cells that synthesize MIP-1beta lack perforin; (iv) MIP-1beta is synthesized with approximately equal frequency by CD28(+) and CD28(-) subpopulations of CD8(+) T cells; (v) MIP-1beta is synthesized by three distinct CD8(+) T cell subsets defined by the expression of CD45R0 and CD62L; and (vi) MIP-1beta is not synthesized in short-term cultures of naive CD8(+) T cells. These results demonstrate substantial subset heterogeneity of MIP-1beta synthesis among CD8(+) T cells and suggest that these subsets should be evaluated as correlates of protective immunity against HIV-1.

Stimulation of HIV gp120-specific cytolytic T lymphocyte responses in vitro and in vivo using a detoxified pertussis toxin vector.

CD8+ cytolytic T lymphocytes (CTL) are almost certainly an important component of a potentially protective immune response to HIV. To test the ability of pertussis toxin (PT) to deliver an HIV-derived major histocompatibility complex (MHC) class I peptide for CTL stimulation, we constructed a fusion of the gp120 P18-I10 CTL epitope with a genetically detoxified derivative of PT (PT9K/129G) and assayed this fusion for its ability to stimulate a gp120-specific CTL response in vitro and in vivo. Antigen-presenting cells incubated with this fusion protein were lysed by P18-I10-specific CTL in vitro and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A but was not inhibited by proteasome inhibitors, possibly because PT undergoes retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum and delivers epitopes directly to nascent class I molecules. Mice immunized intraperitoneally with a single dose of the fusion protein without adjuvant raised a strong gp120-specific CTL response in the spleen. This CTL response was dependent on (1) the dose of fusion administered, (2) the fusion of the epitope with the toxin (since coadministration of peptide and toxin gave no response), and (3) the activity of CD8+ cells. These data demonstrate that this detoxified derivative to PT, which is already a component of a licensed vaccine for humans, could represent a useful vaccine vector molecule for stimulation of HIV-specific CTL responses.

Choroid plexus carcinoma presenting as an intraparenchymal mass.

A 6-year-old girl with a history of a nondisplaced skull fracture diagnosed with computerized tomography (CT) scanning 3 years previously presented with a 6-week history of headaches and decreased use of her right side. On admission CT scans, a large cystic mass was identified in the left frontal lobe region of the brain. A connection between the mass and the ventricular system was not seen on radiological examination or during surgery. Gross-total resection of the mass was achieved. The histological and immunohistochemical findings in the resected tissue confirmed a diagnosis of choroid plexus carcinoma (ChPC). This is the first reported case of a ChPC arising in an extraventricular location not associated with the choroid plexus.

Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex.

The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.

Recent advances with recombinant bacterial vaccine vectors.

Bacille Calmette-Guerin (BCG), Listeria monocytogenes, Salmonellae and Shigellae have shown promise as vaccine vectors in experimental animal models. Although disappointing results in humans and non-human primates stalled the development of this vaccination strategy, interest in this approach was reinvigorated recently by the development of bacterial DNA-vaccine-vectors. The purpose of this review is to highlight the strengths and weaknesses of bacterial vaccine vectors, and to discuss the future prospects of these vaccine delivery systems.

Soluble complexes of regulated upon activation, normal T cells expressed and secreted (RANTES) and glycosaminoglycans suppress HIV-1 infection but do not induce Ca(2+) signaling.

Chemokines comprise a family of low-molecular-weight proteins that elicit a variety of biological responses including chemotaxis, intracellular Ca(2+) mobilization, and activation of tyrosine kinase signaling cascades. A subset of chemokines, including regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta, also suppress infection by HIV-1. All of these activities are contingent on interactions between chemokines and cognate seven-transmembrane spanning, G protein-coupled receptors. However, these activities are strongly inhibited by glycanase treatment of receptor-expressing cells, indicating an additional dependence on surface glycosaminoglycans (GAG). To further investigate this dependence, we examined whether soluble GAG could reconstitute the biological activities of RANTES on glycanase-treated cells. Complexes formed between RANTES and a number of soluble GAG failed to induce intracellular Ca(2+) mobilization on either glycanase-treated or untreated peripheral blood mononuclear cells and were unable to stimulate chemotaxis. In contrast, the same complexes demonstrated suppressive activity against macrophage tropic HIV-1. Complexes composed of (125)I-labeled RANTES demonstrated saturable binding to glycanase-treated peripheral blood mononuclear cells, and such binding could be reversed partially by an anti-CCR5 antibody. These results suggest that soluble chemokine-GAG complexes represent seven-transmembrane ligands that do not activate receptors yet suppress HIV infection. Such complexes may be considered as therapeutic formulations for the treatment of HIV-1 infection.

Intracellular delivery of a cytolytic T-lymphocyte epitope peptide by pertussis toxin to major histocompatibility complex class I without involvement of the cytosolic class I antigen processing pathway.

A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.

A new monoclonal antibody, mAb 4A12, identifies a role for the glycosaminoglycan (GAG) binding domain of RANTES in the antiviral effect against HIV-1 and intracellular Ca2+ signaling.

The beta-chemokine RANTES (regulated on activation, normal T cell expressed and secreted) suppresses the infection of susceptible host cells by macrophage tropic strains of HIV-1. This effect is attributed to interactions of this chemokine with a 7-transmembrane domain receptor, CCR5, that is required for virus-cell fusion and entry. Here we identify domains of RANTES that contribute to its biological activities through structure-function studies using a new monoclonal antibody, mAb 4A12, isolated from mice immunized with recombinant human RANTES. This monoclonal antibody (mAb) blocked the antiviral activity of RANTES in infectivity assays with HIV-1Bal, and inhibited the mobilization of intracellular Ca2+ elicited by RANTES, yet recognized this chemokine bound to cell surfaces. Epitope mapping using limited proteolysis, reversed phase high-performance liquid chromatography, and mass spectrometry suggest that residues 55-66 of RANTES, which include the COOH-terminal alpha-helical region implicated as the glycosaminoglycan (GAG) binding domain, overlap the determinant recognized by mAb 4A12. This is supported by affinity chromatography studies, which showed that RANTES could be eluted specifically by heparin from a mAb 4A12 immunoaffinity matrix. Removal of cell surface GAGs by enzymatic digestion greatly reduced the ability of mAb 4A12 to detect RANTES passively bound on cell surfaces and abrogated the ability of RANTES to elicit an intracellular Ca2+ signal. Taken together, these studies demonstrate that the COOH-terminal alpha-helical region of RANTES plays a key role in GAG-binding, antiviral activity, and intracellular Ca2+ signaling and support a model in which GAGs play a key role in the biological activities of this chemokine.

Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta.

OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines. METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay. RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS. CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.

Induction of mucosal and systemic responses against human immunodeficiency virus type 1 glycoprotein 120 in mice after oral immunization with a single dose of a Salmonella-HIV vector.

Previous studies from our group showed that a Salmonella-HIV vector vaccine that expressed recombinant HIV-1 envelope protein gp120 stably in the vector cytoplasm elicited type 1 helper T cell (Th1) responses to gp120. Despite the promise of such vaccines, a major limitation in their use was that multiple immunizations were required to elicit even small responses. For this reason, we sought a modified vector configuration that would induce more potent gp120-specific T cell responses exhibiting a broader spectrum of effector functions after a single inoculation. In this article we describe the construction and immunogenicity of a Salmonella-HIV vector that displays a truncated derivative of HIV-1(IIIB) envelope in the periplasm of the vector. A single oral dose of this Salmonella vector, called H683(pW58-asd+), generated a gp120-specific proliferation response in the spleen 14 days after immunization. In agreement with our previous findings, the gp120-specific splenic CD4+ T cells elicited by H683(pW58-asd+) displayed a Th1 phenotype; however, gp120-specific splenic CD4+ Th2 cells were also evident. In addition, this strain induced strong gp120-specific IgA antibody-secreting cell (ASC) responses in the intestinal lamina propria and mesenteric lymph nodes. As many as 2% of the total lamina propria and mesenteric lymph node IgA ASCs were found to be specific for gp120 28 days after a single oral dose of H683(pW57-asd+). Because the proliferative response following a single dose of H683(pW58-asd+) was comparable to that seen previously after three doses of an analogous construct expressing recombinant gp120 in the cytoplasm, these observations suggest that Salmonella-vectored secreted HIV-1 antigens elicit higher T cell responses than their cytoplasmically bound analogs.

Oral bacterial vaccine vectors for the delivery of subunit and nucleic acid vaccines to the organized lymphoid tissue of the intestine.

Bacterial vaccine vectors have the potential to deliver a number of antigens from bacterial, protozoan and viral pathogens. To further develop the utility of bacterial vaccine vectors we are currently evaluating three model systems: 1. A Salmonella-ETEC Vaccine Vector; 2. A Salmonella-HIV Vaccine Vector, and 3. Novel Live Bacterial Nucleic Acid Vaccine Vectors. Through our studies, and those of others, significant progress has been made toward bacterial vaccine vector systems that effectively deliver subunit and nucleic acid vaccines to the organized lymphoid tissue of the intestine. The practical reality of these findings is discussed.

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.

In vitro effects of anti-HIV immunotoxins directed against multiple epitopes on HIV type 1 envelope glycoprotein 160.

We have used a panel of anti-gp160 MAbs to construct anti-HIV immunotoxins by coupling antibodies to ricin A chain (RAC). The ability of the immunotoxins to kill HIV-1-infected cells and halt the spread of infection was tested in tissue culture on persistently and acutely infected cell lines and primary lymphocyte cultures stimulated with phytohemagglutinin (PHA blasts). Laboratory strains and clinical isolates of HIV both were tested. The constitution and antigen-binding capacity of the immunotoxins were confirmed by ELISA and indirect immunofluorescence. Immunotoxins that bind epitopes exposed on the cell surface effectively killed persistently infected cells, although killing was not directly proportional to binding of immunotoxin to cell. The activity of anti-gp41, but not anti-gp120, immunotoxins was markedly enhanced in the presence of soluble CD4 or peptides corresponding to the CDR3 region of CD4. CD4-mediated enhancement of anti-gp41 immunotoxin activity was observed for laboratory strains neutralized by sCD4 and for clinical isolates that were resistant to neutralization by sCD4. Immunotoxin action was potentiated by brefeldin A, bafilomycin A1, cortisone, and an amphipathic fusion peptide, but not by cytochalasin D, nocodazol, monodansyl cadaverine, or trans-retinoic acid. Anti-HIV immunotoxins are useful tool with which to study the functional expression of gp120/gp41 antigens on the surface of HIV-infected cells, as well as potential AIDS therapeutics. Because these studies relate to the accessibility of viral antigens to antibody-mediated attack, these studies also have relevance for vaccine development.