Antibody-dependent cell-mediated cytotoxicity in simian immunodeficiency virus-infected rhesus monkeys

With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.     

Monoclonal antibodies to phosphatidylinositol phosphate neutralize human immunodeficiency virus type 1: role of phosphate-binding subsites

Both a murine monoclonal antibody to phosphatidylinositol phosphate (PIP) and a human monoclonal antibody (4E10) that is known to have broadly neutralizing capabilities against primary isolates of human immunodeficiency virus type 1 (HIV-1) bound to PIP, as determined by enzyme-linked immunosorbent assay. Each of the antibodies had antigen subsite binding specificities in aqueous medium for small phosphate-containing molecules and for inositol. The anti-PIP monoclonal antibody inhibited infection by two HIV-1 primary isolates in neutralization assays employing primary human peripheral blood mononuclear cells. The data suggest that PIP or related lipids having free phosphates could serve as targets for the neutralization of HIV-1.     

Broad and potent neutralizing antibody responses elicited in natural HIV-2 infection

Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-2(7312A), HIV-2(ST), and HIV-2(UC1). Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC(50)) NAb titers of 1.7 × 10(5), 2.8 × 10(4), and 3.3 × 10(4), respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-2(7312A) proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4(+) T cells and tested for neutralization sensitivity on human CD4(+) T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.     

Protection in macaques immunized with HIV-1 candidate vaccines can be predicted using the kinetics of their neutralizing antibodies

Background

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.

Methods and Findings

We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV_SF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.

Conclusions

Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses.     

Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.     

Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1

The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1.     

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.     

Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies

We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10(8) cells) plus 8 ml of whole blood from protected animals to na?ve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.     

Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.     

Examination of sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals for antibodies reactive with peptides corresponding to the principal neutralizing determinant of HIV-1 gp120 and for in vitro neutralizing activity.

Sera from human immunodeficiency virus type 1 (HIV-1)-infected individuals from the United States and Tanzania were examined for antibody reactivity to four synthetic peptides which corresponded to the principal neutralizing determinant from the V3 region of HIV-1 gp120. We observed that the majority of sera from both countries contained antibodies reactive with a V3 peptide whose sequence is based on that of the HIV-1 MN isolate. We were unable to establish a relationship between the presence of V3-reactive antibodies, as measured by enzyme-linked immunosorbent assay and neutralization of homologous HIV-1 isolates, in sera from either the United States or Tanzania. We observed that some sera which contained high antibody titers to the V3 peptides failed to neutralize HIV-1, while others with no antibody reactivity to the panel of V3 peptides exhibited in vitro neutralizing activity. These results suggest that neutralizing epitopes exist outside the V3 loop and that the presence of V3-reactive antibodies in sera does not imply in vitro neutralization of the homologous HIV-1 isolate. In addition, it appears that the V3 loop may consist of both neutralizing and nonneutralizing epitopes. The identification of neutralizing as well as nonneutralizing epitopes will be important for the design of potential HIV-1 vaccines.     

Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus.

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.     

Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection

The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.     

Antibody-dependent cellular cytotoxicity detects type- and strain-specific antigens among human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus SIVmac isolates.

Human cell lines were infected with different strains of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as with a simian immunodeficiency virus SIVmac isolate and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. Sera from HIV-1- or HIV-2-infected subjects provided the antibody, and lymphocytes from normal donors provided the effector cells. About 60% of HIV-1 antibody-positive sera mediated ADCC when tested against any given HIV-1 isolate-infected target cell (human T-cell lymphotropic virus type IIIB, B40, A2587), and about 75% of HIV-2 antibody-positive sera mediated ADCC when tested against target cells infected with HIV-2 isolates (lymphadenopathy-associated virus type 2 and SBL-6669) or simian immunodeficiency virus from macaques. Within each type, individual sera showed different reactivity patterns, and the probability that a serum was ADCC positive was higher when it was tested against several strains. When the ADCC reactivity of sera against different strains was compared, diversity as detected by ADCC appeared to be greater among HIV-1 strains than among HIV-2 strains. For HIV-1, 54 to 67% of the sera gave concordant ADCC reactions, whereas for HIV-2 and SIVmac, 91% of the sera gave concordant results. Almost no strain-specific differences were seen between SBL-6669 and lymphadenopathy-associated virus type 2. As we determined previously, HIV-1 and HIV-2 did not cross-react in ADCC. The results indicated that HIV-1 and HIV-2 antibody-positive sera mediate both strain- and type-specific ADCC. HIV-2 antibody-positive sera seem to mediate ADCC with broader reactivity and to a higher frequency compared with HIV-1 antibody-positive sera.     

Alterations in antibody-dependent cellular cytotoxicity during the course of HIV-1 infection. Humoral and cellular defects

HIV-1-specific cell-mediated cytotoxicity (CMC) is a form of antibody-dependent cellular cytotoxicity (ADCC) in which HIV-1-specific antibodies arm NK cells directly to become cytotoxic for targets bearing HIV-1 antigenic determinants. This non-MHC-restricted cytotoxic activity is present in early stages of disease and declines markedly with disease progression. To understand the cellular and humoral factors contributing to the reduction in this activity, the conditions under which maximal arming of cells occurs was examined in vitro. With the use of a large patient cohort, a strong positive correlation was found between the capacity of a serum to direct lysis in standard ADCC assays and its ability to arm NK cells. Patients with minimal HIV-1-specific ADCC-directing antibodies exhibited low levels of CMC and were unable to arm normal effector cells in vitro. The lack of sufficient ADCC-directing antibodies was found to be one cause of defective CMC in some patients. Unlike asymptomatics, only a weak positive correlation was found between arming and ADCC with sera from AIDS patients, indicating that a factor other than absolute HIV-1 specific antibody titer was responsible for decreased CMC in this patient population. Another group of patients was found to have diminished CMC despite the presence of antibodies in the serum that were fully capable of arming normal effector cells to become cytotoxic for gp120-expressing targets. When compared with those of normal individuals, lymphocytes from seropositive patients mediated significantly reduced levels of cytotoxicity in ADCC and arming assays with the use of a high titered HIV-1-specific serum. In both assay systems, the magnitude and frequency of dysfunction in antibody-dependent cytolysis was found to be greater among AIDS patients than among asymptomatic individuals. The demonstration of both cellular and humoral defects in the ability of seropositive individuals to manifest ADCC reactivities strongly suggests that HIV-1 infection may significantly compromise the effectiveness of this potentially important cytolytic reactivity in vivo.     

Effective ex vivo neutralization of human immunodeficiency virus type 1 in plasma by recombinant immunoglobulin molecules.

We tested the ability of human monoclonal antibodies (immunoglobulin G1b12 [IgG1b12] and 19b) and CD4-based molecules (CD4-IgG2 and soluble CD4 [sCD4]) to neutralize human immunodeficiency virus type 1 directly from the plasma of seropositive donors in an ex vivo neutralization assay. IgG1b12 and CD4-IgG2, at concentrations from 1 to 25 micrograms/ml, were found to be effective at reducing the HIV-1 titer in most plasma samples. When viruses recovered from plasma samples were expanded to produce virus stocks, no correlation between the neutralization sensitivities to IgG1b12 and CD4-IgG2 of the in vitro passaged stocks and those of the ex vivo neutralizations performed directly on the plasma was observed. These differences could be due to changes in neutralization sensitivity that occur after one passage of the virus in vitro, or they could be related to the presence of complement or antibodies in the plasma. Furthermore, differences in expression of adhesion molecules on plasma-derived and phytohemagglutinin-activated peripheral blood mononuclear cell-derived viruses could be involved. These studies suggest that IgG1b12 and CD4-IgG2 have broad and potent neutralizing activity in both in vitro and ex vivo neutralization assays and should be considered for use as potential immunoprophylactic or therapeutic agents.     

Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.     

Nonneutralizing antibodies are able to inhibit human immunodeficiency virus type 1 replication in macrophages and immature dendritic cells

Only five monoclonal antibodies (MAbs) neutralizing a broad range of primary isolates (PI) have been identified up to now. We have found that some MAbs with no neutralizing activities according to the "conventional" neutralization assay, involving phytohemagglutinin-stimulated peripheral blood mononuclear cells as targets, efficiently inhibit the replication of human immunodeficiency virus type 1 (HIV-1) PI in macrophages and immature dendritic cells (iDC). The mechanism of inhibition is distinct from the neutralization of infectivity occurring via Fab fragments and involves the interaction of the F portion with the FcgammaRs present on macrophages and iDC. We propose that, if such nonneutralizing inhibitory antibodies limit mucosal HIV transmission, they should be induced by vaccination.     

A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.     

Characterization of neutralizing profiles in HIV-1 infected patients from whom the HJ16, HGN194 and HK20 mAbs were obtained

Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an 'extended incubation phase' PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development.     

Human immunodeficiency virus type 1 neutralization measured by flow cytometric quantitation of single-round infection of primary human T cells

There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV-1 infection of peripheral blood mononuclear cells (PBMC). Initial experiments demonstrated that essentially all productively infected PBMC could be identified by flow cytometric detection of intracellular p24 antigen (p24-Ag). After infection of PBMC with HIV-1, p24(+) lymphocytes could be distinguished beginning 1 day postinfection, and the majority of CD8(-) T cells were p24-Ag positive by 3 to 4 days postinfection. To directly quantify first-round infection, we included a protease inhibitor in PBMC cultures. The resulting 2-day assay was highly sensitive and specific for the detection of HIV-1-infected PBMC. Serial dilutions of virus stocks demonstrated that the number of target cells infected was directly related to the amount of infectious virus input into the assay. In neutralization assays, the flow cytometric enumeration of first-round infection of PBMC provided quantitative data on the number of target cells infected and on the inactivation of infectious virus due to reaction with antibody. We also used this single-round assay to compare the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag per 100 PBMC. The precision and reproducibility of this assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines.