HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients.

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.

In vivo CD8+ T-cell suppression of siv viremia is not mediated by CTL clearance of productively infected cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef.

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.

Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.

BACKGROUND: Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco. RESULTS: We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. CONCLUSION: We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of clade B.

In this investigation we have generated and defined the immunogenicity of two novel HIV/AIDS vaccine candidates based on the highly attenuated vaccinia virus strains, MVA and NYVAC, efficiently expressing in the same locus (TK) and under the same viral promoter the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef antigens of clade B (referred as MVA-B and NYVAC-B). In infected human HeLa cells, gp120 is released from cells and GPN is produced as a polyprotein; NYVAC-B induces severe apoptosis but not MVA-B. The two poxvirus vectors showed genetic stability of the inserts. In BALB/c and in transgenic HHD mice for human HLA-A2 class I, both vectors are efficient immunogens and induced broad cellular immune responses against peptides represented in the four HIV-1 antigens. Some differences were observed in the magnitude and breadth of the immune response in the mouse models. In DNA prime/poxvirus boost protocols, the strongest immune response, as measured by fresh IFN-gamma and IL-2 ELISPOT, was obtained in BALB/c mice boosted with NYVAC-B, while in HHD mice there were no differences between the poxvirus vectors. When the prime/boost was performed with homologous or with combination of poxvirus vectors, the protocols MVA-B/MVA-B and NYVAC-B/NYVAC-B, or the combination NYVAC-B/MVA-B gave the most consistent broader immune response in both mouse models, although the magnitude of the overall response was higher for the DNA-B/poxvirus-B regime. All of the immunization protocols induced some humoral response against the gp160 protein from HIV-1 clone LAV. Our findings indicate that MVA-B and NYVAC-B meet the criteria to be potentially useful vaccine candidates against HIV/AIDS.

High and inducible expression of human immunodeficiency virus type 1 (HIV-1) Nef by adenovirus vector does not disturb potent antigen presentation by monocyte-derived dendritic cells.

Numerous studies indicated that Nef is a pleiotropic factor. Although it has been shown that Nef impairs the antigen-presenting activity of dendritic cells, more recent studies have shown no such impairment. This issue is critical for designing a vaccine expressing Nef. To refine our knowledge regarding the effect of Nef on dendritic cells, we developed constitutive and inducible adenovirus vector systems that express high levels of Nef in monocyte-derived dendritic cells (MDDCs). We showed here that Nef expression clearly downregulated CD4 expression of MDDCs but had little or no effect on other surface molecules, including MHC class I. Nef also did not affect the functional maturation of MDDCs. Use of the inducible Nef-expression system clearly revealed that adenovirus infection per se modulates cytokine secretion and the expression of apoptosis-related molecules in MDDCs, whereas Nef had no effect on these functions. Moreover, the antigen-presenting activity of MDDCs was not disturbed by the presence of Nef. On the contrary, we found that Nef-expressing MDDCs generated from HIV-1-infected individuals efficiently activated Nef-reactive T cells. Therefore, although adenovirus vector may modulate some aspects of MDDC function, Nef-expressing adenovirus would be served as one of HIV vaccine candidates.

Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy.

In the present paper we describe the cloning and extracellular expression of the HIV-1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low-temperature fed-batch strategy for decreasing end-product degradation by proteases. The nef gene in a pPICZalphaA vector was integrated into the genome of three different P. pastoris strains, namely X-33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild-type strain (X-33) was found to be the best choice. The decreased end-product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44-fold utilizing the low-temperature strategy compared with the standard fermentation. Purification of histidine-tagged Nef was performed in one step using a Ni(2+)-nitrilotriacetate-Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix-assisted laser-desorption ionization-time-of-flight MS, reversed-phase HPLC and N-terminal-sequence analysis.

Evasion of cytotoxic T lymphocytes is a functional constraint maintaining HIV-1 Nef expression.

Nef expression is not required for HIV-1 replication and is highly targeted by CD8+ CTL, raising the question of why Nef expression is not lost in order to evade immunity in vivo. We explore whether MHC class I (MHC-I) down-regulation to evade CTL in general is a selective pressure maintaining Nef. HIV-1 with functional Nef (wild type, WT) is compared to virus containing a Nef point mutation (M20A) that selectively ablates MHC-I down-regulation. WT-infected cells are relatively resistant to cytolysis and less suppressed for viral replication by Gag- and RT-specific CTL compared to M20A. These viruses grow similarly in vitro in the absence of CTL, but the presence of Gag- or RT-specific CTL strongly favors WT overgrowth of M20A. Finally, while in vitro selection by Nef-specific CTL readily drives disruption of the nef reading frame, the addition of Gag- or RT-specific CTL markedly limits such escape. These data indicate that MHC-I down-regulation is an important function favoring Nef maintenance due to a net selective advantage in the setting of the general CTL response.

Codon-optimized reading frames facilitate high-level expression of the HIV-1 minor proteins.

We have constructed reading frames for the HIV-1 YU-2 minor proteins Vpr, Vpu, Vif and Nef that are codon-optimized for high-level expression in mammalian cells. We show that, in the absence of the Rev/Rev-response element system, these codon-optimized reading frames result in greatly increased levels of expression of the corresponding proteins in cell culture systems when compared with the native reading frame. Northern blot analysis shows that the increase in expression found with the codon-optimized reading frames is largely owing to increased steady-state mRNA levels.

Nef expressed from human immunodeficiency virus type 1 extrachromosomal DNA downregulates CD4 on primary CD4+ T lymphocytes: implications for integrase inhibitors.

Recently developed integrase inhibitors targeting the HIV-1 integrase (IN) protein block integration of HIV DNA in the target cell, preventing subsequent virus replication. In the absence of integration, viral DNA is shunted towards the formation of extrachromosomal DNA (E-DNA). Although HIV-1 E-DNA does not support productive replication, it is transcriptionally active and produces viral proteins. However, the significance of E-DNA in virus replication and pathogenesis is poorly understood. In this study, the functional activity of the HIV-1 Nef protein expressed in the absence of viral integration was analysed. Using both a recombinant HIV-1 IN defective virus and a diketo acid IN inhibitor, evidence was provided showing that Nef expressed from E-DNA downregulates CD4 surface expression on primary CD4(+) T lymphocytes. These results suggest that proteins expressed in the absence of integration may have potential clinical consequences, an issue that should be further explored with the introduction of IN inhibitors.

Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef.

To characterize newly arising replication of human immunodeficiency virus (HIV) type 1 in vivo at the cellular level, distinct viral RNA species in peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients were monitored during 2 weeks of structured treatment interruption (STI). HIV-1 RNA encoding tat/rev and PBMC-associated virions were almost completely depleted during antiretroviral therapy and emerged simultaneously after 2 weeks of STI, thus specifically reflecting productive viral infection at the cellular level. The magnitude of these correlates of reappearing cellular viral replication was predicted by during-therapy levels of nef transcripts in PBMCs. Significant rebound of plasma viremia, representing the progeny of a broader range of anatomical compartments, preceded and predicted productive infection in PBMCs. Thus, cellular viral rebound in PBMCs likely was primed before STI by the expression of nef in HIV-1-infected PBMCs that lacked virion production and was subsequently triggered by the plasma viremia that preceded the recurrence of productively infected PBMCs.

Human immunodeficiency virus type 1 Nef protein mediates neural cell death: a neurotoxic role for IP-10.

HIV-1 Nef is expressed in astrocytes, but a contribution to neuropathogenesis and the development of HIV-associated dementia (HAD) remains uncertain. To determine the neuropathogenic actions of the HIV-1 Nef protein, the brain-derived (YU-2) and blood-derived (NL4-3) Nef proteins were expressed in neural cells using an alphavirus vector, which resulted in astrocyte death (P < 0.001). Supernatants from Nef-expressing astrocytes also caused neuronal death, suggesting the release of neurotoxic molecules by astrocytes. Analysis of pro-inflammatory gene induction in astrocytes expressing Nef revealed increased IP-10 mRNA expression (4000-fold) that was Nef sequence dependent. Recombinant IP-10 caused selective cell death in neurons (P < 0.001) but not astrocytes, and the cytotoxicity of supernatant from astrocytes expressing Nef YU-2 was blocked by an antibody directed against the chemokine receptor CXCR3 (P < 0.001). SCID/NOD mice implanted with a Nef YU-2-expressing vector displayed abnormal motor behavior (P < 0.05), neuroinflammation, and neuronal loss relative to controls. Analysis of mRNA levels in brains from patients with HAD also revealed increased expression of IP-10 (P < 0.05), which was confirmed by immunoreactivity detected principally in astrocytes. Phylogenetic and protein structure analyses of Nef sequences derived from HIV/AIDS patients with and without HAD suggested viral evolution toward a neurotropic Nef protein. These results indicate that HIV-1 Nef contributes to neuropathogenesis by directly causing astrocyte death together with indirect neuronal death through the cytotoxic actions of IP-10 on neurons. Furthermore, Nef molecular diversity was evident in brain tissue among patients with neurological disease and which may influence IP-10 production by astrocytes.

CD4 phosphorylation partially reverses Nef down-regulation of CD4.

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (approximately 95%), whereas that induced by PMA was partial (approximately 40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.

Endogenously expressed HIV-1 nef down-regulates antigen-presenting molecules, not only class I MHC but also CD1a, in immature dendritic cells.

The effects of Nef molecules on immature dendritic cells (iDCs) were analyzed using recombinant human immunodeficiency virus type 1 (HIV-1) with intact nef gene, pseudotyped with vesicular stomatitis virus glycoprotein, HIV/VSV-G/+Nef. When iDCs were infected with HIV/VSV-G/+Nef, the surface expression of CD1a, a molecule for presenting glycolipid/lipid antigens, was selectively down-regulated among CD1 molecules (CD1a, -b, -c, and -d) as well as class I MHC. Moreover, the CD1a molecules were also down-modulated and co-localized with DsRed2-tagged-Nef in CD1a-transfected cells. Their co-localization was dependent upon CD1a cytoplasmic tail and the CD1a was redistributed from cell surface to LAMP-1+ late endosomal/lysosomal compartment. These findings reveal that the HIV-1-Nef interferes with the intracellular trafficking of CD1a, and suggest the involvement of CD1a-restricted immune effectors in the protective immunity against HIV-1 infection, which implicates the feasibility of virus-derived glycolipid/lipid antigens together with epitope peptides for the vaccine development.

Antibody-dependent cellular cytotoxicity via humoral immune epitope of Nef protein expressed on cell surface.

Antibodies against various proteins of HIV type 1 (HIV-1) can be detected in HIV-1-infected individuals. We previously reported that the level of Ab response against one Nef epitope is correlated with HIV-1 disease progression. To elucidate the mechanism for this correlation, we examined Ab-dependent cellular cytotoxicity (ADCC) against target cells expressing Nef. We observed efficient cytotoxicity against Nef-expressing target cells in the presence of patient plasma and PBMCs. This ADCC activity was correlated with the dilution of plasma from HIV-1-infected patients. Addition of a specific synthetic peptide (peptide 31:FLKEKGGLE) corresponding to the Nef epitope reduced cell lysis to approximately 50%. These results suggest that PBMCs of HIV-1-infected patients may exert ADCC via anti-Nef Abs in the patients' own plasma and serve as a mechanism used by the immune system to regulate HIV-1 replication.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Coxiella burnetii avoids macrophage phagocytosis by interfering with spatial distribution of complement receptor 3.

Phagocytosis is a highly localized event requiring the formation of spatially and temporally restricted signals. Numerous microorganisms have taken advantage of this property to invade host cells. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that has developed a survival strategy in macrophages based on subversion of receptor-mediated phagocytosis. The uptake of C. burnetii is mediated by alpha(v)beta(3) integrin and is restricted by impaired cross-talk of alpha(v)beta(3) integrin and complement receptor 3 (CR3) (CD11b/CD18). In this study, we showed that CR3 molecules remained outside the pseudopodal extensions induced by C. burnetii in THP-1 monocytes, although alpha(v)beta(3) integrin was present in the pseudopods. Chemoattractants such as RANTES restored CR3 localization to the front of pseudopodal extensions and increased C. burnetii phagocytosis, demonstrating that the localization of CR3 is critical for bacterial uptake. In addition, monocyte activation due to the expression of HIV-1 Nef protein also restored CR3-mediated phagocytosis of C. burnetii by allowing CR3 redistribution toward bacterial-induced pseudopods. The redistribution of CR3 and increased C. burnetii phagocytosis in THP-1 cells stimulated by RANTES or expressing Nef were associated with the inhibition of intracellular replication of C. burnetii. Hence, the localization of CR3 is critical for bacterial phagocytosis and also for the control of bacterial replication. This study describes a nonpreviously reported strategy of phagocytosis subversion by intracellular pathogens based on altered localization of monocyte receptors.

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells.

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes.

Recent studies indicate that the time required for virus-infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV-1-specific CTL, we measured virus production in cultures containing CD4(+) T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev-specific CTL prevented at least 2 log(10) more HIV-1 production, in 10 days, than similar numbers of RT-specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL-target cell interactions during successive infection cycles. The results show that substantially higher CTL-mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT-specific CTL were at least as effective as the Rev-specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

Development of a novel allo-independent HIV-1 virus preparation for use in immunoassays.

The study of the immunologic response to whole human immunodeficiency virus type 1 (HIV-1) antigen is limited by the presence of highly immunogenic human leukocyte antigen (HLA) alloantigens on the envelope of wild type virus. This paper outlines the production of HIV-1 infectious virions free of HLA for use as whole viral antigens in immunoassays. An infectious molecular clone of HIV-1 was transfected into the K-562 cell line, which does not express HLA on the cell surface. After a 30-day selection period, to ensure stable transfection, cells and culture supernatants were analyzed for productive HIV-1 infection and virion infectivity. An enzyme-linked immunosorbent assay (ELISA) confirmed the presence of p24 in the culture supernatants. Molecular confirmation of HIV-1 transfection was achieved by gene amplification. Flow cytometric analysis was used to identify gp120 on the surface of the infected cells. Viral supernatants were tested for HIV infectivity in peripheral blood mononuclear cells (PBMCs). The usefulness of this viral preparation as whole virus antigens was validated using PBMCs from HIV-infected individuals. These results indicate the successful production of HIV-1 infectious virions, which do not have HLA molecules on their viral envelope, and demonstrate their utility for immunoassays.