Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization.

UNLABELLED: The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 µg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE: To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different antibody binding arms could potentially display neutralization characteristics better than those of any single parental antibody. Here we show that bispecific antibodies contain the binding specificities of the two parental antibodies and that a single bispecific antibody can neutralize 97% of viral strains with a high overall potency. These findings support the use of bispecific antibodies for the prevention or treatment of HIV-1 infection.

Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 µg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 µg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

Human dendritic cells as targets of dengue virus infection.

Dengue virus infections are an emerging global threat. Severe dengue infection is manifested as dengue hemorrhagic fever and dengue shock syndrome, both of which can be fatal complications. Factors predisposing to complicated disease and pathogenesis of severe infections are discussed. Using immunohistochemistry, immunofluorescence, flow cytometry, and ELISA techniques, we studied the cellular targets of dengue virus infection, at both the clinical (in vivo) and the laboratory (in vitro) level. Resident skin dendritic cells are targets of dengue virus infection as demonstrated in a skin biopsy from a dengue vaccine recipient. We show that factors influencing infection of monocytes/macrophages and dendritic cells are different. Immature dendritic cells were found to be the cells most permissive for dengue infection and maybe early targets for infection. Immature dendritic cells exposed to dengue virus produce TNF-alpha protein. Some of these immature dendritic cells undergo TNF-alpha mediated maturation as a consequence of exposure to the dengue virus.

Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRF01_AE) generated by the polymerase chain reaction.

We previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend these findings by describing a novel vector system to recover infectious molecular clones from long PCR amplicons. Directional ligation of 9.2-kb proviral amplicons into a recovery vector reconstitutes missing LTR sequences, providing candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional infectious molecular clones generated, from primary isolates of subtype B (HIV-1(WR27)) and circulating recombinant form 01_AE (subtype E) (HIV-1(CM235)) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first report of infectious molecular clones from circulating recombinant form 01_AE (subtype E).

Human skin Langerhans cells are targets of dengue virus infection.

Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukocyte antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60-80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.

Drug resistance patterns, genetic subtypes, clinical features, and risk factors in military personnel with HIV-1 seroconversion.

BACKGROUND: Regular testing of military personnel identifies early HIV infection; this identification provides a sentinel cohort in which to describe the evolving molecular epidemiology of HIV-1 transmission. OBJECTIVE: To describe the prevalence and epidemiologic correlates associated with the acquisition of non-subtype B and drug-resistant HIV infections. DESIGN: Cross-sectional study. SETTING: Military referral hospital. PATIENTS: 95 military personnel with HIV-1 seroconversion. MEASUREMENTS: Self-reported questionnaire, CD4 cell counts, plasma HIV-1 RNA levels, and nucleic acid sequence analysis for drug-resistant mutations and HIV-1 genetic subtype. RESULTS: 95 patients were enrolled between February 1997 and February 1998. The likely geographic location of HIV-1 acquisition was overseas in 8% of patients, the United States in 68%, and either overseas or the United States in 24%. Seven patients (7.4%) had subtype E infection; the remainder had subtype B infection. Eight of 31 (26%) treatment-naive patients had mutations in the reverse transcriptase or protease gene associated with drug resistance. CONCLUSIONS: The percentage of HIV-1 non-subtype B infection and antiretroviral drug-resistant mutations was relatively high in U.S. military personnel with recently acquired HIV-1 infection.

Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies.

The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4(+)-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4(+)-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4(+) T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease.

Determination of Syncytium-Inducing Phenotype of Primary HIV-1 Isolates Using MT-2 cells.

HIV-1 is routinely isolated by cocultivation of patient PBMC with mitogen-stimulated HIV-uninfected donor PBMC (see Chapter 1 ). In this culture system, HIV-1 primarily replicates in CD4(+) T-lymphocytes, and such viruses are termed clinical or primary isolates. As early as 1986, the in vitro replicative capacity and cell tropism of primary HIV-1 isolates were shown to be important in the pathophysiology of HIV-1 infection (1). High replication capacity in PBMC and virus growth and syncytium formation in neoplastic T-cell lines were found to correlate with severity of HIV-1-disease (2-5). Compared to syncytium-inducing (SI) isolates, nonsyncytium-inducing (NSI) strains did not replicate in neoplastic T-cell lines and showed preferential replication in cells of the monocyte-macrophage lineage (6,7). Thus, NSI viruses have often been termed macrophage tropic, whereas SI strains are termed T-cell line tropic.

Determination of HIV-1 Chemokine Coreceptor Tropism Using Transduced Human Osteosarcoma (HOS) Cells.

CD4 was identified in 1984 as the receptor for HIV-1 (1,2). However, it was soon apparent that a second receptor was necessary for HIV-1 infection of CD4(+) cells. This coreceptor was first identified by Berger and colleagues who showed that fusion and entry of T-cell line-adapted strains of HIV-1 into CD4(+) cells were mediated by a member of the seven transmembrane chemokine receptor family (3). This protein was initially termed "fusin" and later found to be the α-chemokine receptor CXCR4. Subsequent reports by several laboratories rapidly identified a ß-chemokine receptor, CCR5, that mediated entry of macrophage tropic HIV-1 isolates into CD4(+) cells (4-8). Since these initial reports, our understanding of HIV-1 cell entry has continued to evolve. It now seems clear that primary HIV-1 strains with a nonsyncytium-inducing pheno-type (in MT2 cells) utilize the CCR5 coreceptor and are designated "R5," whereas syncytium-inducing viruses preferentially utilize CXCR4, but may be dual tropic and are designated either "X4" or X4R5, respectively (9,10). Some viruses also appear to be able to utilize chemokine receptors CCR2B and CCR3 (7-10). In addition, the role of chemokine coreceptors in the pathophysiology of HIV-1 infection and disease progression is just beginning to be understood. Individuals who are homozygous for a 32-bp deletion in the CCR5 gene are only rarely found to be HIV-1-infected, and the heterozygous CCR5/Δccr5 genotype has been associated with a survival advantage against HIV-1 disease progression (11-14).

Neutralizing antibodies from the sera of human immunodeficiency virus type 1-infected individuals bind to monomeric gp120 and oligomeric gp140.

Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation.

Neutralizing monoclonal antibodies block human immunodeficiency virus type 1 infection of dendritic cells and transmission to T cells.

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14(+) blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3(+) T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.

Exclusive and persistent use of the entry coreceptor CXCR4 by human immunodeficiency virus type 1 from a subject homozygous for CCR5 delta32.

Individuals who are homozygous for the 32-bp deletion in the gene coding for the chemokine receptor and major human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 (CCR5 -/-) lack functional cell surface CCR5 molecules and are relatively resistant to HIV-1 infection. HIV-1 infection in CCR5 -/- individuals, although rare, has been increasingly documented. We now report that the viral quasispecies from one such individual throughout disease is homogenous, T cell line tropic, and phenotypically syncytium inducing (SI); exclusively uses CXCR4; and replicates well in CCR5 -/- primary T cells. The recently discovered coreceptors BOB and Bonzo are not used. Although early and persistent SI variants have been described in longitudinal studies, this is the first demonstration of exclusive and persistent CXCR4 usage. With the caveat that the earliest viruses available from this subject were from approximately 4 years following primary infection, these data suggest that HIV-1 infection can be mediated and persistently maintained by viruses which exclusively utilize CXCR4. The lack of evolution toward the available minor coreceptors in this subject underscores the dominant biological roles of the major coreceptors CCR5 and CXCR4. This and two similar subjects (R. Biti, R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang, Nat. Med. 3:252-253, 1997; I. Theodoreu, L. Meyer, M. Magierowska, C. Katlama, and C. Rouzioux, Lancet 349:1219-1220, 1997) showed relatively rapid CD4+ T-cell declines despite average or low initial viral RNA load. Since viruses which use CXCR4 exclusively cannot infect macrophages, these data have implications for the relative infection of the T-cell compartment versus the macrophage compartment in vivo and for the development of CCR5-based therapeutics.

Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.

Rapid disease progression without seroconversion following primary human immunodeficiency virus type 1 infection--evidence for highly susceptible human hosts.

A patient is described who rapidly progressed from primary human immunodeficiency virus (HIV) type 1 infection to death without seroconversion but with consistently high plasma viremia. His asymptomatic sex partner had been HIV-1 seropositive for >8 years prior to transmission. Analysis of viral sequences from these subjects and controls confirmed the transmission event. Although the biologic properties of the patient's virus were unremarkable, he had poor functional immune responses to HIV and an HLA haplotype associated with rapid disease progression. The disparity between immune responses and clinical course in this transmission pair, coupled with infection with an unremarkable HIV-1 isolate, underscores the crucial importance of host factors in HIV-1 pathogenesis.

Human immunodeficiency virus type 1 neutralizing antibody serotyping using serum pools and an infectivity reduction assay.

Classification of human immunodeficiency virus type 1 (HIV-1) by neutralization serotype may be important for the design of active and passive immunization strategies. Neutralizing antibody serotyping is hindered by the lack of standard reagents and assay format, and by the weak activity of many individual sera. To facilitate cross-clade neutralization analysis, we used an infectivity reduction assay (IRA) and selected clade-specific serum (or plasma) pools from subjects infected with clade B and E HIV-1, respectively. Several serum pools were utilized; some were selected for strong neutralizing activity against intraclade viruses and others were derived from conveniently available samples. Against a panel of 51 clade B and E viruses, serum pools displayed strong neutralization of most intraclade viruses and significantly diminished cross-clade neutralization. Results were confirmed against a blinded panel of 20 viruses. The data indicate that the phylogenetic classification of virus subtypes B and E corresponds to two distinct neutralization serotypes. This approach to neutralizing antibody serotyping may be useful in defining the antigenic relationship among viruses from other clades.

Overexpression of nef as a marker for restricted HIV-1 infection of astrocytes in postmortem pediatric central nervous tissues.

In previous studies, using polymerase chain reaction amplification of HIV-1 genes directly from pathologic tissues of children who died with AIDS encephalopathy, we showed that the reading frame of the HIV-1 regulatory nef gene is open, suggesting that the nef protein was expressed. We now show, using immunocytochemistry and in situ hybridization with nef-specific probes in postmortem pediatric CNS tissues, that nef mRNA and protein are present in up to 20% of astrocytes in tissue sections selected for extensive histopathology. By contrast, HIV-1 structural proteins such as gag and their coding mRNAs are present in multinucleated giant cells that harbor productive infection and are the hallmark of HIV-1 infection in the CNS. These findings are consistent with the nonproductive infection of glial cells observed in vitro, and imply that HIV-1 infection of astrocytes is restricted to early regulatory gene products, of which nef is the best target as it is expressed at high levels and is membrane-anchored. In developing central nervous tissues of children, restricted and latent HIV-1 infection of astrocytes may be extensive and contribute significantly to HIV-1 neuropathogenesis.