Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.     

Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C

Identifying the viral epitopes targeted by broad neutralizing antibodies (NAbs) that sometimes develop in human immunodeficiency virus type 1 (HIV-1)-infected subjects should assist in the design of vaccines to elicit similar responses. Here, we investigated the activities of a panel of 24 broadly neutralizing plasmas from subtype B- and C-infected donors using a series of complementary mapping methods, focusing mostly on JR-FL as a prototype subtype B primary isolate. Adsorption with gp120 immobilized on beads revealed that an often large but variable fraction of plasma neutralization was directed to gp120 and that in some cases, neutralization was largely mediated by CD4 binding site (CD4bs) Abs. The results of a native polyacrylamide gel electrophoresis assay using JR-FL trimers further suggested that half of the subtype B and a smaller fraction of subtype C plasmas contained a significant proportion of NAbs directed to the CD4bs. Anti-gp41 neutralizing activity was detected in several plasmas of both subtypes, but in all but one case, constituted only a minor fraction of the overall neutralization activity. Assessment of the activities of the subtype B plasmas against chimeric HIV-2 viruses bearing various fragments of the membrane proximal external region (MPER) of HIV-1 gp41 revealed mixed patterns, implying that MPER neutralization was not dominated by any single specificity akin to known MPER-specific monoclonal Abs. V3 and 2G12-like NAbs appeared to make little or no contribution to JR-FL neutralization titers. Overall, we observed significant titers of anti-CD4bs NAbs in several plasmas, but approximately two-thirds of the neutralizing activity remained undefined, suggesting the existence of NAbs with specificities unlike any characterized to date.     

An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing human immunodeficiency virus type 1 antibody 2G12 and elicits mannose-specific gp120-binding antibodies

The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man(8)GlcNAc(2), and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Delta och1 Delta mnn1 Delta mnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man alpha1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man alpha1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.     

Improved induction of antibodies against key neutralizing epitopes by human immunodeficiency virus type 1 gp120 DNA prime-protein boost vaccination compared to gp120 protein-only vaccination

A major challenge in human immunodeficiency virus type 1 (HIV-1) vaccine development is to elicit potent and broadly neutralizing antibodies that are effective against primary viral isolates. Previously, we showed that DNA prime-protein boost vaccination using HIV-1 gp120 antigens was more effective in eliciting neutralizing antibodies against primary HIV-1 isolates than was a recombinant gp120 protein-only vaccination approach. In the current study, we analyzed the difference in antibody specificities in rabbit sera elicited by these two immunization regimens using peptide enzyme-linked immunosorbent assay and a competitive virus capture assay. Our results indicate that a DNA prime-protein boost regimen is more effective than a protein-alone vaccination approach in inducing antibodies that target two key neutralizing domains: the V3 loop and the CD4 binding site. In particular, positive antibodies targeting several peptides that overlap with the known CD4 binding area were detected only in DNA-primed sera. Different profiles of antibody specificities provide insight into the mechanisms behind the elicitation of better neutralizing antibodies with the DNA prime-protein boost approach, and our results support the use of this approach to further optimize Env formulations for HIV vaccine development.     

Expression, Purification, and Inhibitory Properties of Human Proteinase Inhibitor 8

In a previous report, the cDNA for human proteinase inhibitor 8 (PI8) was first identified, isolated, and subcloned into a mammalian expression vector and expressed in baby hamster kidney cells. Initial studies indicated that PI8 was able to inhibit the amidolytic activity of trypsin and form an SDS-stable approximately 67-kDa complex with human thrombin [Sprecher, C. A., et al. (1995) J. Biol Chem. 270, 29854-29861]. In the present study, we have expressed recombinant PI8 in the methylotropic yeast Pichia pastoris, purified the inhibitor to homogeneity, and investigated its ability to inhibit a variety of proteinases. PI8 inhibited the amidolytic activities of porcine trypsin, human thrombin, human coagulation factor Xa, and the Bacillus subtilis dibasic endoproteinase subtilisin A through different mechanisms but failed to inhibit the Staphylococcus aureus endoproteinase Glu-C. PI8 inhibited trypsin in a purely competitive manner, with an equilibrium inhibition constant (Ki) of less than 3.8 nM. The interaction between PI8 and thrombin occurred with a second-order association rate constant (kassoc) of 1.0 x 10(5) M-1 s-1 and a Ki of 350 pM. A slow-binding kinetics approach was used to determine the kinetic constants for the interactions of PI8 with factor Xa and subtilisin A. PI8 inhibited factor Xa via a two-step mechanism with a kassoc of 7.5 x 10(4) M-1 s-1 and an overall Ki of 272 pM. PI8 was a potent inhibitor of subtilisin A via a single-step mechanism with a kassoc of 1.16 x 10(6) M-1 s-1 and an overall Ki of 8.4 pM. The interaction between PI8 and subtilisin A may be of physiological significance, since subtilisin A is an evolutionary precursor to the intracellular mammalian dibasic processing endoproteinases.     

Neutralizing Antibodies in Sera from Macaques Immunized with Attenuated Simian Immunodeficiency Virus

Infection with attenuated simian immunodeficiency virus (SIV) in rhesus macaques has been shown to raise antibodies capable of neutralizing an animal challenge stock of primary SIVmac251 in CEMx174 cells that correlate with resistance to infection after experimental challenge with this virulent virus (M. S. Wyand, K. H. Manson, M. Garcia-Moll, D. C. Montefiori, and R. C. Desrosiers, J. Virol. 70:3724–3733, 1996). Here we show that these neutralizing antibodies are not detected in human and rhesus peripheral blood mononuclear cells (PBMC). In addition, neutralization of primary SIVmac251 in human and rhesus PBMC was rarely detected with plasma samples from a similar group of animals that had been infected either with SIVmac239Δnef for 1.5 years or with SIVmac239Δ3 for 3.2 years, although low-level neutralization was detected in CEMx174 cells. Potent neutralization was detected in CEMx174 cells when the latter plasma samples were assessed with laboratory-adapted SIVmac251. In contrast to primary SIVmac251, laboratory-adapted SIVmac251 did not replicate in human and rhesus PBMC despite its ability to utilize CCR5, Bonzo/STRL33, and BOB/gpr15 as coreceptors for virus entry. These results illustrate the importance of virus passage history and the choice of indicator cells for making assessments of neutralizing antibodies to lentiviruses such as SIV. They also demonstrate that primary SIVmac251 is less sensitive to neutralization in human and rhesus PBMC than it is in established cell lines. Results obtained in PBMC did not support a role for neutralizing antibodies as a mechanism of protection in animals immunized with attenuated SIV and challenged with primary SIVmac251.     

Recombinant Vaccine-Induced Protection against the Highly Pathogenic Simian Immunodeficiency Virus SIVmac251: Dependence on Route of Challenge Exposure

Vaccine protection from infection and/or disease induced by highly pathogenic simian immunodeficiency virus (SIV) strain SIV_mac251 in the rhesus macaque model is a challenging task. Thus far, the only approach that has been reported to protect a fraction of macaques from infection following intravenous challenge with SIV_mac251 was the use of a live attenuated SIV vaccine. In the present study, the gag, pol, and env genes of SIV_K6W were expressed in the NYVAC vector, a genetically engineered derivative of the vaccinia virus Copenhagen strain that displays a highly attenuated phenotype in humans. In addition, the genes for the α and β chains of interleukin-12 (IL-12), as well as the IL-2 gene, were expressed in separate NYVAC vectors and inoculated intramuscularly, in conjunction with or separate from the NYVAC-SIV vaccine, in 40 macaques. The overall cytotoxic T-lymphocyte (CTL) response was greater, at the expense of proliferative and humoral responses, in animals immunized with NYVAC-SIV and NYVAC–IL-12 than in animals immunized with the NYVAC-SIV vaccine alone. At the end of the immunization regimen, half of the animals were challenged with SIV_mac251 by the intravenous route and the other half were exposed to SIV_mac251 intrarectally. Significantly, five of the eleven vaccinees exposed mucosally to SIV_mac251 showed a transient peak of viremia 1 week after viral challenge and subsequently appeared to clear viral infection. In contrast, all 12 animals inoculated intravenously became infected, but 5 to 6 months after viral challenge, 4 animals were able to control viral expression and appeared to progress to disease more slowly than control animals. Protection did not appear to be associated with any of the measured immunological parameters. Further modulation of immune responses by coadministration of NYVAC-cytokine recombinants did not appear to influence the outcome of viral challenge. The fact that the NYVAC-SIV recombinant vaccine appears to be effective per se in the animal model that best mirrors human AIDS supports the idea that the development of a highly attenuated poxvirus-based vaccine candidate can be a valuable approach to significantly decrease the spread of human immunodeficiency virus (HIV) infection by the mucosal route.     

Derivation and Characterization of a Simian Immunodeficiency Virus SIVmac239 Variant with Tropism for CXCR4

Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4⁺ CXCR4⁺ CCR5⁻ SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4⁻ SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4⁻ SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.Simian immunodeficiency viruses (SIVs) share many structural and biological features with human immunodeficiency virus (HIV), including target cell entry via interactions of the viral envelope glycoprotein (Env) with CD4 and a chemokine coreceptor. For HIV, the most important coreceptors in vivo are CCR5 (2, 13, 19, 21, 22) and CXCR4 (30). HIV type 1 (HIV-1) strains that use only CCR5 (R5 viruses) predominate during the early stages of infection and are critical for transmission (84, 90), as evidenced by the finding that individuals lacking a functional CCR5 protein due to a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (16, 54, 82). Although R5 viruses generally persist in late-stage disease, viruses that can use CXCR4, either exclusively (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of subtype B-infected individuals (15, 43). This coreceptor switch is associated with a more rapid decline in peripheral blood CD4⁺ T cells and a faster progression to AIDS (15, 43, 77), although it is unclear if CXCR4-using viruses are a cause or a consequence of progressing immunodeficiency. Like HIV, the vast majority of SIVs use CCR5 to establish infection (11, 12, 45). However, although CXCR4-using SIVs have been reported (47, 52, 65, 68, 69), their occurrence is rare, especially in models of pathogenic infection, where only one CXCR4-using SIV has been identified (17, 60, 71).This paucity of CXCR4-using SIVs is surprising for several reasons. First, SIV Envs tend to be more promiscuous than HIV-1 Envs and frequently use alternative coreceptors in addition to CCR5, including GPR1, GPR15, CXCR6, and CCR8 (20, 27, 29, 80, 81, 92) but not CXCR4. Second, HIV-2, which is more closely related to SIVmac than to HIV-1 (56, 57), commonly uses CXCR4 in vitro and in vivo (3, 28, 33, 58, 59, 67). Third, rhesus CXCR4 is ∼98% identical to human CXCR4 in amino acid sequence and can function as a coreceptor for HIV-1 in vitro (12). Finally, chimeric simian-human immunodeficiency viruses (SHIVs) that contain X4 HIV Envs on an SIV core can replicate to high levels in vivo and cause disease in rhesus macaques (39, 86). Moreover, it was recently shown that coreceptor switching can occur in rhesus macaques infected with an R5 SHIV (35). Thus, there does not appear to be any block per se against the use of rhesus CXCR4 as an entry coreceptor either in vitro or in vivo, suggesting that SIV is less capable of adapting to use CXCR4 and/or that mutations required for CXCR4 utilization may lead to a virus that is less fit and/or more susceptible to immune control in this host.For HIV-1, the Env determinants for CXCR4 use have been well documented and often involve the acquisition of positively charged amino acids in the V3 loop (18, 32, 87), particularly at positions 11, 24, and 25 (6, 18, 31, 32, 38, 75). Although the SIVmac239 V3 loop is a critical determinant for Env-coreceptor interactions (44, 63, 72), attempts to create an X4 SIVmac239 by introducing positively charged residues into the V3 loop (63) or by inserting a V3 loop from X4 HIV-1 (44) have been unsuccessful. SIVmac155T3, the only CXCR4-using variant of SIVmac that has been identified to date, was isolated from a rhesus macaque with advanced disease and contains additional positively charged residues in V3, although the determinants for CXCR4 use have not been determined (60, 71).Given questions concerning the possible determinants for and/or barriers to coreceptor switching in SIV, we sought to derive a CXCR4-using variant of the well-characterized pathogenic R5 SIV clone SIVmac239. Here we show that SIVmac239 could indeed acquire CXCR4 utilization when it was adapted in vitro for high-efficiency replication in the CXCR4⁺ CCR5⁻ human SupT1 cell line. An env clone from this virus could use CXCR4 in cell-cell fusion and reporter virus infection assays and conferred CXCR4 tropism to a replication-competent SIV. Although V3 mutations were important for CXCR4 use, an L328W change at the V3 crown rather than the acquisition of positively charged residues was required, as was an unusual K47E mutation in the conserved C1 domain of gp120. These changes also caused the highly neutralization-resistant SIVmac239 strain to become more neutralization sensitive to sera and plasmas from SIVmac239-infected animals, and particularly to soluble CD4. These results indicate that mutations distinct from those typically seen for HIV-1 may be required for SIVmac to gain CXCR4 utilization and suggest that these changes render this virus more susceptible to humoral immune control. Collectively, our findings indicate that there are likely to be strong viral and host selection pressures against CXCR4 use that may contribute to the paucity of X4 coreceptor switching for SIVmac in vivo.     

Evidence that Ecotropic Murine Leukemia Virus Contamination in TZM-bl Cells Does Not Affect the Outcome of Neutralizing Antibody Assays with Human Immunodeficiency Virus Type 1

The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.It was recently reported that TZM-bl cells, which are commonly used to assess neutralizing antibodies (Abs) against human immunodeficiency virus type 1 (HIV-1), are contaminated with an ecotropic murine leukemia virus (MLV) (22). TZM-bl (also called JC.53bl-13) is a HeLa cell derivative that was engineered by amphotropic retroviral transduction to express CD4 and CCR5 (17) and was further engineered with an HIV-1-based vector to contain Tat-responsive reporter genes for firefly luciferase (Luc) and Escherichia coli β-galactosidase (24). These engineered features made TZM-bl cells highly susceptible to HIV-1 infection in a readily quantifiable assay for neutralizing Abs. Many published studies used this cell line for assessments of HIV-1 neutralization; these include several recent reports describing the magnitude, breadth, and epitope specificity of the neutralizing Ab response in infected individuals (14, 18-20), neutralization escape (25), and the neutralization phenotype of transmitted/founder viruses (10). TZM-bl cells are also gaining popularity for assessments of vaccine-elicited neutralizing Ab responses (13). The validity of these and other published results, together with a rationale for the continued use of TZM-bl cells in assessing neutralizing Abs against HIV-1, are very dependent on establishing to what extent, if any, MLV contamination affects the outcome of the assay.It was suggested that ecotropic MLV entered TZM-bl cells via the progenitor JC.53 cell line as an amphotropic MLV pseudotype (22). In this regard, JC.53 cells were constructed from HeLa cells in two stages by using ping-pong technology to amplify the pSFF vector derived from the replication-defective and highly truncated Friend spleen focus-forming virus (3). When used with this vector, this procedure has previously resulted in stable vector expression (17) without formation of replication-competent MLV recombinants (8, 11). A panel of HeLa-CD4 clones was made that express different amounts of CD4 and where the high-expression HI-J clone was used to make a derivative panel of clones (termed JC), including JC.53, that expressed diverse levels of CCR5 (9, 16, 17). In addition, the HeLa-CD4 clone HI-R that expressed low levels of CD4 was used to make another panel of CCR5-expressing clones (termed RC). To investigate this newly reported issue, cell extracts from these clonal panels and from TZM-bl cells were analyzed for MLV Gag antigens by Western immunoblotting. Representative data, as shown in Fig. ​Fig.1A,1A, confirm that JC.53 and TZM-bl cells express MLV Gag antigens, whereas the progenitor HI-J clone of HeLa-CD4 cells and many but not all of the other HeLa-CD4/CCR5 clones in the JC panel lack MLV antigens.Open in a separate windowFIG. 1.Characterization of HeLa clones for MLV Gag expression, HIV-1 susceptibility, and cell surface expression of HIV-1 fusion receptors. (A) MLV Gag antigen expression in HeLa cells and derivative clones expressing CD4 or CD4 and CCR5. Cell lysates were prepared from the cell clones and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with Abs to MLV Gag antigens (upper blot). The lysates were also probed with anti-tubulin antibodies (lower blot). Lane 1, HeLa cells; lanes 2 and 3, HeLa CD4 clones HI-R and HI-J, respectively; lanes 4, 5, and 6, HeLa-CD4/CCR5 clones JC.10, JC.48, and JC.53, respectively; lane 7, TZM-bl cells; lane 8, psi-2 packaging cells positive for MLV Gag. (B) HIV-1 infectivity on the HeLa-CD4/CCR5 JC panel. Target cells were infected with HIV-1 isolate JRCSF that had been produced from clone JC.53 cells (black) or with JRCSF produced from transfected HEK293T cells (red). The target cells were also infected with the JR-FL isolate produced from peripheral blood mononuclear cells (PBMC; green). The HeLa-CD4/CCR5 target cells had a CCR5 expression range of 2 × 10³ (clone JC.10) to 1.3 × 10⁵ (clones JC.53 and TZM-bl) CCR5 molecules/cell. Each set of three data points at a given CCR5 expression level represents a single HeLa-CD4/CCR5 JC clone. None of the HIV-1 isolates was able to infect HeLa-CD4 cells lacking CCR5. The blue asterisks indicate clones that are negative for MLV Gag proteins. Clones JC.48 (used for subsequent infection and neutralization assays) and JC.53 (progenitor of TZM-bl cells) are specifically labeled. (C) Surface expression of CD4, CCR5, and CXCR4 on TZM-bl and JC.48 cells was assessed by flow cytometry using the same stocks of cells that were used in infection and neutralization assays in Fig. ​Fig.2.2. Surface staining was performed with phycoerythrin-conjugated mouse monoclonal Abs to CD4, CCR5 (CD195), and CXCR4 (CD184). Background staining was performed with isotype-matched control Abs. All Abs for flow cytometry were purchased from BD Biosciences Pharmingen (San Diego, CA). Results are shown as the mean fluorescence intensity (MFI) of positive cells. Most cells (>90%) stained positive in each case.Initial studies of HI-R cells and other clonal panels that were made using these methods also suggested a lack of MLV antigens (data not shown). We then determined the titers of replication-competent HIV-1_JRCSF preparations using JC.53 and TZM-bl cells as well as other representative HeLa-CD4/CCR5 clones in the JC panel. The results are plotted in Fig. ​Fig.1B1B as a function of cellular CCR5 content. Clones having more than a low threshold level of ∼8,000 CCR5/cell were equally susceptible to infection regardless of whether they contained MLV antigens, clearly demonstrating that HIV-1_JRCSF titers were not significantly affected by MLV. As expected, titers obtained with JC.53 and TZM-bl cells were also equivalent. In addition, these results demonstrate that HIV-1_JRCSF preparations made in JC.53 cells and in cells lacking MLV antigens (i.e., HEK293T cells and human peripheral blood mononuclear cells) were unable to infect HeLa cells lacking CCR5. The results in Fig. ​Fig.1B1B were expected because previous studies demonstrated that ecotropic MLVs cannot infect human cells or even bind to the human CAT-1 receptor paralog (1, 6, 21, 23). Moreover, it has been shown that ecotropic host range MLVs do not interfere with superinfection by any retrovirus capable of infecting human cells, including gibbon ape leukemia virus, amphotropic MLV, baboon endogenous virus, and feline leukemia virus subgroup C (21). In view of the report by Takeuchi et al. (22), we were surprised to find that JC.53 and TZM-bl cells express very small amounts of ecotropic MLV Env glycoproteins, as indicated by immunofluorescence microscopy and by their resistance to complement-dependent killing by a cytotoxic antiserum specific for MLV envelope glycoproteins (6). Nevertheless, the cell clones that contained MLV Gag all released ecotropic host range virions that replicated in murine NIH 3T3 cells but not in human cells (data not shown).To determine whether MLV affects the measurement of neutralizing Abs in TZM-bl cells, parallel assays were performed in TZM-bl and JC.48 cells; these latter cells were determined to be MLV free by Western blot analysis (Fig. ​(Fig.1)1) and by an inability to transfer MLV infection to NIH 3T3 cells (data not shown). Because JC.48 cells express CCR5 at somewhat lower levels than JC.53 cells (∼2-fold lower; Fig. ​Fig.1B),1B), it may be expected that they would be less susceptible to HIV-1 infection than are TZM-bl cells. Differences in susceptibility to HIV-1 infection may require the use of adjusted virus doses to achieve equivalent assay performance when measuring neutralizing Abs. Indeed, levels of CD4 and CCR5 were approximately twofold lower on JC.48 cells than on TZM-bl cells, whereas levels of CXCR4 were approximately equal (Fig. ​(Fig.1C).1C). We therefore measured the susceptibility of both cell lines to infection by three molecularly cloned Env-pseudotyped viruses, each bearing an Env from a different CCR5-tropic HIV-1 subtype B virus (SF162.LS, Bal.26, and QH0692.42). Infection was quantified by Luc activity expressed as relative luminescence units (RLU). Because JC.48 cells do not contain a reporter gene, the Env-pseudotyped viruses were prepared by cotransfection with the NL4-3.Luc.R-E- reporter backbone plasmid (7). Identical Luc-containing, Env-pseudotyped virus stocks were used in both cell lines. As shown in Fig. ​Fig.2A,2A, the infectivity of each pseudotyped virus was somewhat diminished in JC.48 cells compared to the infectivity in TZM-bl cells. Nonetheless, the levels of infectivity in JC.48 cells remained acceptable for neutralization assays.Open in a separate windowFIG. 2.HIV-1 infectivity and neutralization in TZM-bl and JC.48.CD4.CCR5 cells. (A) TZM-bl and JC.48 cells were incubated with serial fourfold dilutions (11 dilutions total) of three HIV-1 Env-pseudotyped viruses in quadruplicate in 96-well culture plates. Luc activity was measured after 48 h of incubation and is expressed as RLU after subtraction of background luminescence from cell control wells. Squares, TZM-bl cells; triangles, JC.48 cells. (B) Neutralization assays were performed with three HIV-1 Env-pseudotyped viruses in either TZM-bl or JC.48 cells. Input virus doses were adjusted to yield equivalent infectivity in both cell lines. Black bars, TZM-bl; gray bars, JC.48. Top panel: sCD4, monoclonal Abs, and HIVIG (purified immunoglobulin G from pooled HIV-1-positive plasmas). Bottom panel: individual HIV-1-positive plasma samples. The same three stocks of virus were used in both experiments. All three Env-pseudotyped viruses were prepared with the NL4-3.Luc.R-E- reporter backbone plasmid.With this information in hand, neutralization assays were performed in JC.48 and TZM-bl cells using adjusted virus doses that yielded equivalent infectivity levels in both cell lines. These neutralization assays were performed in a 96-well format as described previously (12), where the 50% inhibitory dose (ID₅₀) was reported as either the concentration or sample dilution at which RLU were reduced by 50% compared to RLU in virus control wells (cells plus virus without test sample) after subtraction of background RLU from cell control wells (cells only). A wide variety of serologic reagents was tested, including sCD4, a monoclonal Ab to the CD4 binding site of gp120 (immunoglobulin G1b12) (15); a monoclonal Ab that recognizes a glycan-specific epitope on gp120 (2G12) (5); two monoclonal Abs that recognize adjacent epitopes in the membrane proximal external region of gp41 (2F5 and 4E10) (2, 4); and serum samples from seven antiretroviral-naive HIV-1-infected individuals. As shown in Fig. ​Fig.2B,2B, results in the two cell lines were similar for all three viruses and all serologic reagents tested. Indeed, ID₅₀ values in the two cell types agreed within twofold, which is within the normal range of variability of the assay. These results indicate that equivalent neutralization results were obtained in both cell lines.In summary, we found no evidence that ecotropic MLV contamination in TZM-bl cells has a measurable effect on HIV-1 neutralization when these cells are used as targets for infection. This outcome indicates that the presence of ecotropic MLV in TZM-bl cells does not alter the ability of Ab to neutralize HIV-1, nor does it interfere with the detection of neutralization by using HIV-1 Tat-regulated reporter gene expression in a single-cycle infection assay. However, we discourage the use of TZM-bl cells to generate HIV-1 stocks, because the latter would likely be contaminated with ecotropic MLV and contain pseudovirions with mixtures of HIV-1 and ecotropic MLV Env glycoproteins. For this reason, we have begun efforts to produce an uncontaminated, second-generation panel of HeLa-CD4/CCR5 cell clones that express diverse amounts of CCR5 and to isolate a TZM-bl variant lacking MLV antigens.