Patent Application
20160244510
The present invention relates, in general, to HIV-1-reactive antibodies and, in at least certain specific embodiments, to broadly neutralizing antibodies (bnAbs) (and fragments and derivatives thereof) and to compositions comprising same. The invention further relates to methods of using such bnAbs (and fragments and derivatives thereof) and compositions in immunotherapy regimens (e.g., passive immunotherapy regimens). The antibodies (and fragments and derivatives thereof) disclosed herein can also be used in methods of identifying candidate immunogens for use in inducing an immune response against HIV-1 in a mammal (e.g., a human). The invention also relates to such methods and to immunogens so identified.
Induction of antibodies with neutralization breadth is a primary goal of HIV-1 vaccine development (Karlsson Hedestam et al, Nat. Rev. Microbiol 6(2):143-155 (2008). Broadly neutralizing antibodies (bnAbs) have been demonstrated to protect against acute infection in animal models (Mascola et al, Nat. Med. 6(2):207-210 (2000), Hessell et al, J. Virol. 84(3):1302-1313 (2010)), and, since 2009, a large number of new monoclonal antibodies have been isolated (Burton et al, Science 337(6091):183-186 (2012), Bonsignori et al, Trends Microbiol. 20(11):532-539 (2012), Mascola and Haynes, Immunol. Rev. 254(1):225-244 (2013)), providing new strategies for vaccine design aimed at eliciting those antibodies (Haynes et al, Nat. Biotechnol. 30(5):423-433 (2012)). It has been shown that bnAbs only arise after several years of HIV-1 infection (Tomaras et al, J. Virol. 82(24):12449-12463 (2008), Gray et al, J. Virol. 85(10):4828-4840 (2011)). Studies of bnAbs given to chronically (Armbruster et al, AIDS 16(2):227-233 (2002)) or acutely (Mehandru et al, J. Virol. 81(20):11016-11031 (2007)) HIV-1 infected subjects showed little impact on the course of infection, suggesting that the presence of bnAbs alone in most individuals with established infection cannot prevent disease progression
All current HIV-1 envelope (Env) immunogens induce a narrow neutralizing antibody response that in standard TZM-bl pseudovirus neutralization assays predominantly inhibit tier 1 HIV-1 Env pseudoviruses (Montefiori et al, J. Infect. Dis. 206(3):431-441 (2012)). Thus, a critical question for vaccine development is whether easily induced, narrow neutralizing antibodies can mediate immune pressure sufficient to select for virus escape mutants.
The ALVAC/AIDSVAX B/E vaccine used in the RV144 vaccine efficacy trial in Thailand induced an estimated vaccine efficacy of 31.2%; however, antibodies induced by this vaccine were capable of neutralizing only tier 1 laboratory-adapted HIV-1 strains but not tier 2 strains across HIV-1 clades (Montefiori et al, J. Infect. Dis. 206(3):431-441 (2012), Liao et al, Immunity 38(1):176-186 (2013)). Interestingly, RV144-induced antibodies directed to the first and second variable (V1V2) region of Env gp120 correlated with decreased infection risk (Haynes et al, N. Engl. J. Med. 366(14):1275-1286 (2012)), and V2 antibodies isolated from vaccinees, while not capturing nor neutralizing HIV-1 primary strain virions, did bind to the surface of primary virus-infected cells and mediated antibody-dependent cellular cytotoxicity (ADCC) Liao et al, Immunity 38(1):176-186 (2013)). A study of virus sequences from breakthrough infections in RV144 participants showed vaccine-induced immune pressure at amino acid position 169 in V2 of Env gp120 (Rolland et al, Nature 490(7420):417-420 (2012)). These data are consistent with the hypothesis that the estimated 31.2% protection in RV144 was mediated by antibodies targeted at the V2 region but, at present, it is not known whether tier 1 neutralization or ADCC effector functions were responsible.
Determining whether an in vitro assay is a surrogate for in vivo protection against HIV-1 is difficult and requires passive protection studies of rhesus macaques challenged with simian-human immunodeficiency virus (SHIV) (Mascola et al, Nat. Med. 6(2):207-210 (2000), Moldt et al, Proc. Natl. Acad. Sci. USA 109(46):18921-18925 (2012), Girard and Plotkin, Curr. Opin. HIV AIDS 7(1):2012)) or demonstration that a particular immune response can select for virus escape mutants in vivo (Goonetilleke et al, J. Exp. Med. 206(6):1253-1272 (2009), Miura et al, J. Viro. 83(6):2743-2755 (2009)). In order to determine whether tier 1 neutralizing antibodies are capable of exerting immune pressure, a chronically HIV-1 clade C infected Tanzanian individual (CH0457) has been studied over two years and both tier 1 (narrow) and tier 2 (broad) neutralizing antibodies have been isolated from the same individual. In addition, a large panel of full-length env genes from multiple time points was isolated from CH0457 that were used to generate autologous pseudoviruses for testing for antibody-mediated virus neutralization and evidence of antibody-mediated immune pressure. The present invention results, at least in part, from studies demonstrating that bnAbs with the ability to broadly neutralize tier 2 viruses exerted profound immune pressure (94% escape mutants) on the autologous virus quasispecies. In contrast, a clonal lineage of CD4 binding site (CD4bs) narrow neutralizing antibodies with neutralizing activity only against tier 1 viruses exerted minimal autologous virus immune pressure (13% escape mutants) during the time studied. These data suggest that HIV-1 Env tier 1 neutralizing antibodies will not be able to prevent HIV-1 transmission by virion neutralization.
In general, the present invention relates to HIV-1-reactive antibodies. In at least certain specific embodiments, the invention relates to bnAbs (and fragments and derivatives thereof) and to compositions comprising same. The invention further relates to methods of using such bnAbs (and fragments and derivatives thereof) and compositions in immunotherapy regimens (e.g., passive immunotherapy regimens). The invention also relates to methods of using such bnABs (and fragments and derivatives thereof) to identify candidate immunogens that can induce an immune response against HIV-1 in a mammal (e.g., a human), and to immunogens so identified.
Certain objects and advantages of the present invention will be clear from the description that follows.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The global HIV/AIDS epidemic remains a global health threat. While there is not yet a cure, significant advances in the treatment of HIV-1 infection have occurred. A vaccine effective against the most common modes of transmission of the virus will likely need to induce antibodies that have the capacity to block infection by a wide array of possible viral targets and that can be present at mucosal surfaces (e.g., the lower gastrointestinal tract and genital tract).
One class of antibodies capable of blocking infection by a wide array of HIV-1 strains is bnAbs. Within the last five years, there have been a large number of new bnAbs isolated with a concomitant increase in the number of known epitope targets for bnAbs. These targets reflect relatively conserved epitopes on HIV-1 and have consisted of regions that mimic human antigens and are thereby under immune tolerance control (Yang et al, J. Exp. Med. 210(2):241-256 (2013)), post-translational modifications added by human cells (Pejchal et al, Science 334(6059)L1097-1103 (2011), Sanders et al, J. Virol. 76(14):7293-7305 (2002)), or epitopes that must be conserved to maintain functionality of the HIV-1 envelope protein (Sanders et al, J. Virol. 76(14):7293-7305 (2002)). A great deal of effort has been invested in developing vaccines that elicit such antibodies (Esparza, Vaccine 31(35):3502-3518 (2013)). The identification of additional targets for bnAbs remains a priority.
Antibody responses at mucosal surfaces consist of antibodies of the IgG and IgA classes. IgG antibodies are the predominant isotype found in plasma and can be actively or passively transported across anatomical barriers. IgA antibodies can also be found in plasma at lower concentrations but can also be locally produced and actively transported across mucosal barriers. IgA antibodies are particularly adapted for survival at mucosal surfaces; e.g., IgA2 antibodies are resistant to some bacterial proteases found in respiratory tract pathogens. It is expected that for protection against HIV-1 infection that would occur via mucosal surfaces (e.g., sexual transmission, breast milk transmission), IgA antibodies will be critically important.
A series of antibodies have been isolated from a chronically HIV-1-infected subject from Tanzania (CH0457). Using antigen-specific flow cytometry, B cells expressing HIV-1-reactive antibodies were isolated from this subject. Genes from these cells were isolated by overlapping PCR and antibodies expressed for screening. Based on the screening, a number of HIV-1-reactive antibodies were identified that were sent for neutralization assays; of these antibodies, three (CH27, CH28 and CH44) were found to be broadly neutralizing. Two of these three antibodies (CH27 and CH28) were of the IgA2 isotype. These are the first natural IgA bnAbs that have been isolated.
Mapping of this group of bnAbs revealed that they did not map to any known bnAb specificity. These data suggest that this group of bnAbs binds to a novel bnAb epitope. Combined with the fact that the isolated bnAbs were of the IgA isotype, this group of antibodies represent a target for vaccine development and represent a therapeutic for the prevention of mucosal HIV-1 transmission.
The present invention relates to the bnAbs disclosed herein (e.g., the IgA bnAbs), to antibodies having the specificity of the disclosed bnAbs, and to fragments (e.g., antigen-binding fragments) and derivatives thereof, and to methods of using same to inhibit HIV-1 infection in a subject (e.g., a human). The invention includes intact antibodies and fragments (e.g., Fab, Fab′, F(ab′)2, FV, CDR (see
Preferred antibodies of the invention for therapeutic use include those comprising variable heavy (VH) and light (VL) chain amino acid sequences selected from those shown in
The antibodies, and fragments/derivatives thereof, described above can be formulated as a composition (e.g., a pharmaceutical composition). Suitable compositions can comprise the bnAb or fragment (or derivative thereof) dissolved or dispersed in a pharmaceutically acceptable carrier (e.g., an aqueous medium). The compositions can be sterile and can be in an injectable form (e.g., a form suitable for intravenous injection). The antibodies or fragments (or derivatives thereof) can also be formulated as a composition appropriate for topical administration to the skin or mucosa (e.g., intrarectal or intravaginal administration). Such compositions can take the form of liquids, ointments, creams, gels and pastes. The antibodies or fragments (or derivatives thereof) can also be formulated as a composition appropriate for intranasal administration. The antibodies or fragments (or derivatives thereof) can be formulated so as to be administered as a post-coital douche or with a condom. Standard formulation techniques can be used in preparing suitable compositions.
The bnAbs and fragments thereof (and derivatives) described herein have utility, for example, in settings including the following:
i) in the setting of anticipated known exposure to HIV-1 infection, the antibodies described herein, or fragments thereof, (or derivatives thereof) and be administered prophylactically (e.g., IV, topically or intranasally) as a microbiocide,
ii) in the setting of known or suspected exposure, such as occurs in the setting of rape victims, or commercial sex workers, or in any homosexual or heterosexual transmission without condom protection, the antibodies described herein or fragments thereof (or derivatives thereof) can be administered as post-exposure prophylaxis, e.g., IV or topically, and
iii) in the setting of Acute HIV infection (AHI), the antibodies described herein, or fragments thereof, (or derivatives thereof) can be administered, alone or in combination with another anti-HIV-1 therapeutic, as a treatment for AHI to control the initial viral load or for the elimination of virus-infected CD4 T cells.
Suitable dose ranges can depend on the antibody or fragment (or derivative thereof—e.g., toxin- or radioisotope-bound derivative) and on the nature of the formulation and route of administration. Optimum doses can be determined by one skilled in the art without undue experimentation. Doses of antibodies in the range of 1-50 mg/kg can be used. If, for example, antibodies or fragments, with or without toxins, are used or antibodies are used that can be targeted to specific CD4 infected T cells, then less antibody or fragment can be used (e.g., from 5 mg/kg to 0.01 mg/kg).
In accordance with the invention, the bnAbs or antibody fragments (or derivatives) described herein can be administered prior to contact of the subject or the subject's immune system/cells with HIV-1 or, for example, within about 48 hours of such contact. Administration within this time frame can maximize inhibition of infection of vulnerable cells of the subject with HIV-1.
Antibodies of the invention and fragments thereof can be produced recombinantly using nucleic acids comprising nucleotide sequences encoding, for example, VH and VL chains (or CDRs) selected from those shown in
The antibodies of the present invention can be used as probes to identify their specificity and to identify candidate immunogens that can elicit this new class of antibodies. Candidate immunogens can be selected based on binding to the antibodies and their inferred intermediates. Binding can be assessed, for example, using surface plasmon resonance, ELISA, and multiplex binding (Luminex-based) assays. In addition, binding activity can be assessed by testing for the ability of these antibodies to block the binding of other molecules, such as other antibodies, soluble CD4, or other molecules. Binding can also be assessed using functional assays such as neutraliztion or ADCC. The invention includes methods of identifying such immunogens and immunogens so identified.
Certain aspects of the invention can be described in greater detail in the non-limiting Example that follows. (See also Moody et al, Retrovirology 9 (Suppl 2): 035 (2012).)
In certain embodiments the invention provides antibodies with dual targeting specificity. In certain aspects the invention provides bi-specific molecules that are capable of localizing an immune effector cell to an HIV-1 envelope expressing cell, so as facilitate the killing of the HIV-1 envelope expressing cell. In this regard, bispecific antibodies bind with one “arm” to a surface antigen on target cells, e.g. HIV-1 envelope, and with the second “arm” to an activating, invariant component of the T cell receptor (TCR) complex, eg. CD3. The simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Hence, the immune response is re-directed to the target cells and is independent of peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs. In this context it is crucial that CTLs are only activated when a target cell is presenting the bispecific antibody to them, i.e. the immunological synapse is mimicked. Particularly desirable are bispecific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells.
In certain embodiments, such bispecific molecules comprise one portion which targets HIV-1 envelope and a second portion which binds a second target. In certain embodiments, the first portion comprises VH and VL sequences, or CDRs from CH27, 28, or CH44 (
In certain aspects the invention provides use of the antibodies of the invention, including bispecific antibodies, in methods of treating and preventing HIV-1 infection in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the antibodies of the invention in a pharmaceutically acceptable form. In certain embodiment, the methods include a composition which includes more than one HIV-1 targeting antibody. In certain embodiments, the HIV-1 targeting antibodies in such combination bind different epitopes on the HIV-1 envelope. In certain embodiments, such combinations of bispecific antibodies targeting more than one HIV-1 epitope provide increased killing of HIV-1 infected cells. In other embodiments, such combinations of bispecific antibodies targeting more than one HIV-1 epitope provide increased breadth in recognition of different HIV-1 subtypes.
Broadly neutralizing antibodies (bnAbs) against HIV-1 have activity in vitro against difficult-to-neutralize (tier 2) viruses while antibodies that arise following vaccination or early in HIV-1 infection have activity only against easy-to-neutralize (tier 1) viruses. The capacity for antibodies that neutralize only heterologous tier 1 viruses to exert selection pressure on HIV-1 is not known. To study this question, we isolated tier 1 virus-nAbs that bind to the third variable loop (V3) or the CD4 binding site (CD4bs) from two HIV-1-infected individuals and determined the antibody sensitivity of autologous HIV-1 strains sampled over time. We found functional autologous viruses could be neutralized by these V3 and CD4bs antibodies, and found that resistant forms of HIV-1 accumulated over time, suggesting Ab-mediated viral selection pressure. One clinical setting where transfer of both autologous nAbs and virus can occur is that of mother-to-child transmission (MTCT). In this setting, high levels of maternal V3 and CD4bs autologous nAbs may may be able to reduce transmission, regardless of autologous nAb breadth and potency against heterologous viruses.
Induction of antibodies with neutralization breadth is a primary goal of HIV-1 vaccine development (1). All current HIV-1 envelope (Env) immunogens frequently induce neutralizing antibodies (nAbs) that inhibit only easy-to-neutralize (tier 1) HIV-1 strains (2). In contrast, broadly neutralizing antibodies (bnAbs) that can potently neutralize a variety of difficult-to-neutralize (tier 2) HIV-1 strains that have been associated with HIV-1 transmission (3) are not induced by current vaccines (1, 2, 4, 5).
The initial autologous nAb response in HIV-1-infected subjects is generally restricted to neutralizing the infecting transmitted/founder virus (6-13). Epitopes frequently targeted by the initial autologous nAbs are the third constant region-variable loop 4 (C3-V4) domain (8, 10, 13), the base of the third variable (V3) loop (11, 12, 14), the first and second variable loop (V1V2) regions (9, 10, 12, 15), and the CD4 binding site (CD4bs) (16, 17). In chronic HIV-1 infection, virus escape mutants are selected that repopulate the plasma virus pool, and neutralization breadth accrues to varying degrees in different individuals (18). In addition, antibodies to V3 and the CD4bs arise that can neutralize heterologous tier 1 but not tier 2 HIV-1 isolates (2, 19-24). However, the neutralization sensitivity of the autologous repopulated plasma virus pool to this type of V3 and CD4bs nAbs has not been studied. Here, we have isolated from two chronically HIV-1-infected individuals V3 and CD4bs nAbs with breadth only for tier 1 but not tier 2 heterologous viruses, and for comparison, CD4bs bnAbs with tier 2 neutralization breadth; and determined the ability of these Abs to neutralize a large pane of autologous viruses as well as to select virus escape mutants.
Isolation of nAbs with Restricted or Broad Neutralizing Activity from Chronically Infected Individuals.
From chronically HIV-1 infected individual CH0457, we isolated two clonal lineages as well as single monoclonal antibodies (mAbs) using antigen-specific memory B cell flow cytometry sorting (
The second clonal lineage of mAbs from CH0457, CH27 (
From a second individual, CH505, previously described to have a CD4bs bnAb lineage (represented by CH103 in
Virus Evolution in Chronically Infected Individual CH0457.
We amplified a total of 209 CH0457 env gene sequences by single genome amplification (SGA) from 10 time points over a two year period during chronic infection (weeks 0, 2, 4, 8, 12, 16, 24, 48, 72, and 96 post-enrollment). An average of 21 (range 12-35) SGA env sequences were analyzed for each time point. Phylogenetic analysis showed that the Env sequences continuously evolved over time (
Neutralization of Autologous Viruses by bnAbs and Tier 1 Virus-Neutralizing mAbs.
We made 84 pseudoviruses from these env sequences (
We next determined the neutralization activities of the CH27 CD4bs lineage bnAbs against the panel of 84 autologous pseudoviruses derived from viral RNA from plasma samples. Five autologous viruses were weakly neutralized by one of three lineage CH27 bnAbs (range 32-50 μg/mL), while the other 79 pseudoviruses (94%) were resistant to the CH27 lineage bnAbs (
Thus, to seek definitive evidence of evolutionary selection exerted by the CH27 bnAb lineage, we amplified proviral env genes archived in peripheral blood mononuclear cells (PBMC) from the earliest time point (termed week 0) in this study. Like plasma-derived Env pseudoviruses, the majority of the PBMC-derived Env pseudoviruses were resistant to the lineage CH27 bnAbs (
Next, we asked if 7 of the CH0457 tier 1 virus-nAbs (5 CH13 lineage CD4bs mAbs, and 2 V3 mAbs CH14 and CH48) could neutralize autologous HIV-1 pseudoviruses. We found that the V3 and CD4bs mAbs were able to neutralize autologous viruses throughout the 2-year study period, including PBMC-archived viruses (
Sensitivity to the CH13 lineage and to the two V3 mAbs peaked at week 24 after enrollment; by week 48 of follow-up, most viruses were resistant to the V3 mAbs (
The 3 CH0457 viruses sensitive to the CD4bs CH13 lineage (w48.20, w72.2, and w72.18) were all located within the persistent minor clade (3/10 in the minor clade vs. 0/17 in the dominant clade; Fisher's exact test p=0.04). The fact that both in the CDbs CH27 lineage and in the CH13 lineage the sensitive viruses persisted longest in the minor clade but not in the dominant clade raises the possibility that the viruses in the minor clade may be emerging from an immunologically protected site (eg, brain or the CD4 T cell latent pool) where antibody pressure would be limited (27). Across all time points, 32/84 (38%) of autologous pseudoviruses were resistant to the CD4bs nAbs while 17/84 (20%) were resistant to the V3 loop mAbs. Analysis of CH0457 Env sequences did not demonstrate an accumulation of Env mutations at the putative nAb contact sites suggested by epitope mapping (Tables S2, S3, S4).
To determine if autologous virus neutralization by autologous tier-1 virus nAbs was a phenomenon unique to individual CH0457, we studied two V3 nAbs (DH151 and DH228; Table S6) isolated from a second HIV-1-infected African individual, CH505, 41 weeks after transmission (16). CH505 also developed a CD4bs clonal lineage (termed CH103) at 136 weeks after transmission (16). CH505 was studied earlier during infection compared with CH0457, thus Env selection by bnAbs was ongoing in individual CH505 at the time of study and many autologous Env pseudoviruses were only partially resistant to the CH103 bnAb lineage (
Autologous Virus Neutralization Sensitivity.
To assess the susceptibility of autologous viruses to heterologous nAbs, we performed neutralization assays with a panel of tier 1 virus-neutralizing antibodies and bnAbs. Of 84 CH0457 autologous pseudoviruses, 73 (87%) were sensitive to the heterologous VRC01-like CD4bs bnAb CH31 (29) (
Next, we tested each of the 84 CH0457 Env pseudoviruses against the pooled serum product HIVIG-C and a subset of Env pseudoviruses against well-characterized HIV-1 patient serum samples (
The initial autologous neutralizing antibody response that arises in acute HIV-1 infection is specific for the autologous virus with little tier 1 autologous virus breadth (31-33). This response differs from the autologous nAb response in chronic infection where breadth for heterologous tier 1 viruses can develop. When autologous neutralizing antibodies begin to show heterologous tier 1 breath, it is possible that such antibodies may be enroute to developing some degree of bnAb activity as occurred in the CH103 CD4bs lineage (16).
The CD4bs and V3 antibody lineages studied here were able to neutralize tier 1B and select tier 2 autologous HIV-1 isolates. We speculate that this was possible because the mAbs and viruses isolated in the present study co-evolved in the same HIV-1-infected individuals. During HIV-1 infection, virus quasispecies evolve that have different degrees of Env reactivity; viruses with high intrinsic activity (ie, tier 1A viruses) (30) are more reactive with both soluble CD4 and neutralizing antibodies (34). Thus, in these individuals, autologous viruses with low Env reactivity (ie, tier 1B or tier 2 viruses) (34, 35) can act as templates for antibody evolution, giving rise to antibodies that bind and neutralize autologous virus Envs with low reactivity (
The ability of autologous neutralizing antibodies that arise in acute HIV-1 infection to exert immune pressure has been demonstrated by studies of the evolution of transmitted/founder viruses and plasma antibodies (31, 33, 36). In particular, the initial autologous-specific neutralizing antibody response to HIV-1 appears within the first year of infection and is associated with the development of resistant viruses in virtually all infected individuals (31, 33). A critical question is why neutralization of autologous viruses by tier 1 heterologous virus-neutralizing antibodies like the CH13 lineage from CH0457 and DH151 and DH228 V3 mAbs from CH505 has not been previously observed? The simplest answer is that testing of a large series of autologous Envs isolated in the setting of a chronically infected individual from whom multiple specificities of recombinantly-produced neutralizing mAbs have also been isolated has not been performed.
To date, HIV-1 vaccine efficacy trials have not convincingly demonstrated a protective effect of vaccine-elicited tier 1 virus-neutralizing antibodies (37, 38). In particular, the only vaccine study to date that demonstrated a degree of protection, the RV144 trial, did not elicit bnAbs (2, 39) and has been postulated to have as correlates of protection antibody dependent cellular cytotoxicity (ADCC)-mediating antibodies (37, 40-42) and V3 antibodies (43). The present study reaffirms that tier 1 virus-neutralizing antibodies would be of limited benefit in protection from infection against heterologous tier 2 viruses. However, in our study we show that such antibodies could neutralize autologous tier 1B and tier 2 HIV-1 Envs with which they co-evolved (
Materials and Methods:
The clinical material used for the present study was obtained as a part of the CHAVI 001 observational study. The participants studied here were identified during the screening of CHAVI 001 and CHAVI 008 subjects for the presence of neutralization breadth (47). The present work was performed under a protocol approved by the Duke University Health System Institutional Review Board for Clinical Investigations. These original studies with human subjects from which we obtained the clinical material herein studied were approved by the Kilimanjaro Christian Medical Centre Research Ethics Committee, the Tanzania National Institutes for Medical Research Ethics Coordinating Committee, and the Institutional Review Boards of the London School of Hygiene and Tropical Medicine and Duke University as well as by the NIH Human Subject Review Committee.
Clinical Material.
The participants in this study (CH0457 and CH505) were recruited in 2008 in Tanzania and Malawi, respectively. At the time of recruitment, CH0457 had been chronically infected with a subtype C virus for an unknown period. This participant did not receive antiretroviral drug therapy during the study period. Peripheral blood collections were performed at weeks 0, 2, 4, 8, 12, 16, 24, 48, 72, and 96 of observation. Blood was processed for peripheral blood mononuclear cells (PBMC), plasma, and serum, all of which were cryopreserved for transport to the research laboratories. Participant CH505 was recruited early following infection and has been described previously (16).
Flow Cytometry Panel Antibodies, Recombinant Proteins, and Assay Control Antibodies.
The gp120ConC core protein was produced as described (48) and labeled with Pacific Blue and Alexa Fluor (AF) 647 using fluorochrome labeling kits (Invitrogen, Carlsbad, Calif.). The protein batches were confirmed to bind to CD4 expressed on the surface of the H9 T cell line as a quality control after conjugation. Setup for flow cytometry was performed as described (49). Sorting was performed using antibodies reactive with surface IgM (FITC), surface IgD (phycoerythrin [PE]), CD3 (PE-Cy5), CD16 (PE-Cy5), CD235a (PE-Cy5), and CD19 (allophycocyanin [APC]-Cy7) (BD Biosciences, San Jose, Calif.); CD14 (PE-Cy5) (Invitrogen, Carlsbad, Calif.); CD27 (PE-Cy7) and CD38 (APC-Alexa Fluor 700) (Beckman Coulter, Brea, Calif.).
Hyperimmune HIV-1 globulin subtype C (HIVIG-C) is a mixture of purified IgG from 5 subtype C HIV-1-infected plasma donors in South Africa (Johannesburg blood bank). (50). Genetic subtype was confirmed by SGA sequencing of the plasma Envs. The 5 IgG samples included in HIVIG-C were selected among 35 IgG samples for having the greatest magnitude and breadth of neutralizing activity against a panel of 6 tier 2 viruses. Palivizumab, a humanized monoclonal antibody against the F protein of respiratory syncytial virus, was purchased from MedImmune, LLC (Gaithersburg, Md.). Negative control CH65 is a mAb directed against the sialic acid binding site of hemagglutinin (51, 52). Positive control CH31 is a bnAb directed against the CD4bs (29, 53), as is positive control CH106 (16). Positive control was CD4bs-directed BNAb HJ16 (25).
Antibody Reactivity by Binding Antibody Multiplex Assay and Enzyme-Linked Immunosorbant Assay (ELISA).
Expressed mAbs were studied for reactivity to HIV-1 antigens using a standardized custom binding antibody multiplex assay using Luminex (54). All assays were run under conditions compliant with Good Clinical Laboratory Practice, including tracking of positive controls by Levy-Jennings charts. FDA-compliant software, Bio-Plex Manager, version 5.0 (Bio-Rad, Hercules, Calif.), was utilized for the analysis of specimens. Screening by binding antibody multiplex assays was performed against a panel of HIV-1 antigens (gp140ConC, gp120ConC full length, gp140ConB, gp140ConG, gp140JR.FL); mAbs that had a blank-bead-subtracted value greater than 2000 units and greater than 1000 times the mAb IgG concentration in g/mL were evaluated further. Binding of all mAbs was confirmed by subsequent assays on mAbs prepared from transfected cells at large scales.
ELISA testing of mAbs was performed as described (55); testing was considered positive if the optical density reading at 405 nm was above 0.3 units and greater than 4-fold over background.
Flow Cytometric Analysis and Single-Cell Sorting.
We previously reported that CH0457 had broad neutralizing activity in plasma that could be absorbed by a subtype C consensus (ConC) gp120 protein that lacked V1V2 and V3 loops (gp120ConC core) (47). To isolate neutralizing antibody-producing memory B cells, we used antigen-specific sorting. Fluorescently-labeled gp120ConC core protein was used to isolate Env-reactive memory B cells using a dual-color technique (13, 56). We sorted samples from the week 8 and week 12 time points, and in both cases we isolated antigen-specific B cells from which immunoglobulin (Ig) genes were recovered (Fig. S1 online). In total, we isolated 19 heavy chains with paired light chains and found that when expressed as mAbs, 12/19 (63%) were reactive with one or more consensus Env proteins from clades A, B, C, G and CRF01_AE; 11 of these mAbs were carried forward for further study (Table S1).
Single-cell sorting was performed using a BD FACSAria II (BD Biosciences, San Jose, Calif.) and the flow cytometry data were analyzed using FlowJo (Treestar, Ashland, Oreg.). Antigen-specific memory B cells were identified by using gp120ConC core labeled with Alexa Fluor 647 and Pacific Blue; cells were gated on CD3− CD14− CD16− CD235a− CD19+ surface IgD-gp120ConC core+/+. Single cells were directly sorted into 96-well plates containing 20 μL per well of reverse transcription (RT) reaction buffer (5 μL of 5′ first-strand cDNA buffer, 0.5 μL of RNaseOUT [Invitrogen, Carlsbad, Calif.], 1.25 μL of dithiothreitol, 0.0625 μL Igepal CA-630 [Sigma, St. Louis, Mo.], 13.25 μL of distilled H2O [dH2O; Invitrogen, Carlsbad, Calif.]); plates were stored at −80° C. until use and after sorting were again stored at −80° C. until PCR was performed.
PCR Isolation and Analysis of Immunoglobulin (Ig) VH, Vκ, and Vλ Genes.
Single-cell PCR was performed as described (49, 57, 58). PCR amplicons were sequenced in forward and reverse directions using a BigDye sequencing kit on an ABI 3730XL (Applied Biosystems, Foster City, Calif.). Sequence base calling was performed using Phred (59, 60), forward and reverse strands were assembled using an algorithm based on the quality scores at each position (61). Local alignment with known sequences was used to determine Ig isotype (62); V, D, and J region genes, complementarity-determining region 3 (CDR3) lengths, and mutation frequencies were determined using SoDA (63). Clonal lineages of antibodies were determined as described (51, 56) and were confirmed by alignment of complete V(D)J sequences. Maximum-likelihood trees for clonal lineages were generated using V(D)J regions (excluding constant region sequences); trees were constructed (dnaml), reorganized (retree), and plotted (drawgram) with the PHYLIP package, version 3.69 (64).
Expression of VH and Vκ/λ as Full-Length IgG1 mAbs.
PCR was used to assemble isolated Ig VH and Vκ/λ gene pairs into linear full-length Ig heavy- and light-chain gene expression cassettes as described (57). Human embryonic kidney cell line 293T (ATCC, Manassas, Va.) was grown to near confluence in six-well tissue culture plates (Becton Dickinson, Franklin Lakes, N.J.) and transfected with 2 μg per well of both IgH and Igκ/λ purified PCR-produced cassettes using Effectene (Qiagen, Valencia, Calif.). Culture supernatants were harvested 3 days after transfection and concentrated 4-fold using centrifugal concentrators; expressed IgG was quantitated by ELISA (65); tested mAbs were expressed at 10 μg/mL up to 20 mg/mL. Larger-scale production of mAbs was performed using synthesized linear IgH and Igκ/λ gene constructs (GeneScript, Piscataway, N.J.).
Amplification of Full-Length Env Genes.
Viral RNA (vRNA) was prepared from plasma samples (400 μL) using the EZ1 Virus Mini Kit V2.0 on BIO ROBOT EZ1 (Qiagen; Valencia, Calif.). Reverse transcription was performed with 20 μL of vRNA and 80 pmol primer 1.R3.B3R (5′-ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3′) in 50 μL using Superscript III (Invitrogen; Carlsbad, Calif.). The 3′ half genomes were amplified by single genome amplication (SGA) as previous described (66, 67), using 07For7 (5′CAAATTAYAAAAATTCAAAATTTTCGGGTTTATTACAG-3′) and 2.R3.B6R (5′-TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′) as first round primers, and VIF1 (5′-GGGTTTATTACAGGGACAGCAGAG-3′) and Low2c (5′-TGAGGCTTAAGCAGTGGGTTCC-3′) as the second round primers. The PCR products were purified with the QiaQuick PCR Purification kit (Qiagen; Valencia, Calif.). The env gene sequences were obtained by cycle-sequencing and dye terminator methods with an ABI 3730XL genetic analyzer (Applied Biosystems; Foster City, Calif.). Individual sequence contigs from each env SGA were assembled and edited using the Sequencher program 4.7 (Gene Codes; Ann Arbor, Mich.).
Amplification of HIV-1 Env Genes from PBMCs by SGA.
Proviral DNA was extracted from 3×106 PBMCs at the enrollment (week 0) time point using the QIAamp DNA Blood and Tissue kit (Qiagen; Valencia, Calif.). The HIV-1 rev/env cassette was amplified from the genomic DNA using the single genome amplification (SGA) method. The PCR primers and conditions were the same as those used for viral RNA templates extracted from plasma.
Generation of Pseudoviruses.
The CMV promoter was added to the 5′ end of each env gene amplified by SGA using the promoter addition PCR (pPCR) method as described (68). The pPCR product was used for generation of pseudoviruses by cotransfecting with the env-deficient HIV-1 backbone pSG3Δenv into 293T cells in a 6-well tissue culture plate using FuGENE6 transfection reagent (Roche Diagnostics; Indianapolis, Ind.) according to manufacturer instructions. Transfected cells were maintained in DMEM with 10% FBS at 37° C. with 5% CO2. Forty-eight hours after transfection, supernatants were harvested and stored in 20% FBS medium at −80° C.
Neutralization Assay in TZM-bl Cells.
Neutralizing antibody assays in TZM-bl cells were performed as described (69). Antibodies were tested at concentrations up to 50 μg/mL using eight serial 3-fold dilutions. Control antibodies include HJ16 which was generously provided by D. Corti (Institute for Research in Biomedicine, Universita della Svizzera Italiana, Bellinzona, Switzerland). Env-pseudotyped viruses were added to the antibody dilutions at a predetermined titer to produce measurable infection and incubated for 1 h. TZM-bl cells were added and incubated for 48 h. Firefly luciferase (Luc) activity was measured as a function of relative luminescence units (RLU) using a Britelite Luminescence Reporter Gene Assay System as described by the supplier (Perkin-Elmer Life Sciences, Waltham, Mass.). Neutralization was calculated as the reduction in RLU in test wells compared with control wells after subtraction of background RLU in cell control wells and reported as mAb 50% inhibitory concentration (IC50) in μg/mL. Env-pseudotyped viruses were prepared in 293T cells and titrated in TZM-bl cells as described (69).
Mapping of mAb Specificities by Neutralization.
Single amino acid substitutions were introduced into the consensus C (ConC) or B.RHPA Env by oligonucleotide-directed PCR mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Alanine or conserved mutations were introduced in C1 (L125A), V1 (R132A/T), C2 (S256A, N289K), C3 (T372V, T373M, S375M), C5 (G471E), the β23 sheet of C4 (R456W), as well as the CD4bs (D-loop: N276A/Q, T278A, N279D and α5: D474A, M475A, R476A). The ability of antibodies to neutralize pseudoviruses containing Env point mutations was assessed and compared to the wild-type pseudovirus neutralization. A fifteen-fold or higher increase in IC50 titer from the wild-type to the mutant was considered positive.
Statistical Analysis.
Graphs of the data were created using GraphPad Prism (GraphPad Software, La Jolla, Calif.) with layout in Illustrator CS5 (Adobe, San Jose, Calif.). Statistical tests were performed in SAS, version 9.2 (SAS Institute, Cary, N.C.) or in R, version 2.15.2 (R Foundation for Statistical Computing, Vienna, Austria). The statistical test used is noted when p values are presented. Env sequence phylogenies were inferred using PhyML (70) with the HIVw substitution model (71).
Isolation of nAbs.
Antibodies from CH0457 were isolated by antigen-specific B cell sorting using a clade C consensus Env protein. Clonal lineage CH13 consisted of six monoclonal antibodies (mAbs) of IgG1 isotype (CH13, CH16, CH17, CH18, CH45, CH46) that used VH1˜69*01/JH3*02 and VK1-39*01/JK4*01 genes. Epitope mapping with binding and neutralization assays demonstrated that the CH13 lineage antibody bound to the CD4bs and were sensitive to mutations at D386, E370, I371, S375, and K421 (
The second group of mAbs, clonal lineage CH27 (
Plasma samples from CH0457 taken from weeks 8 and 96 were tested against the same panel of heterologous viruses (
We isolated restricted V3 neutralizing antibodies from a second HIV-1-infected African individual, CH505, 41 weeks after transmission (2). This individual eventually developed a CD4bs clonal lineage (termed CH103) at 136 weeks after transmission (2).
Validation of CH0457 Sequence Integrity.
To determine if there was any evidence for multiple infection or contamination, particularly given that there were two distinctive clades in the CH0457 sample, we did the following tests using the tools at the Los Alamos HIV database (www.hiv.lanl.gov). First we made a DNA consensus of the sequences from the persistent minor clade and the major lineage in CH0457. We then then used HIV-BLAST to these compare the two consensus sequences against the HIV database. Both consensus sequences are closest to natural sequences from CH0457 in already GenBank, supporting that they came from the same quasispecies, and same individual. At the DNA level, the consensus from the persistent minor clade shared between 94 and 97% identity in Blast searches with other CH0457 sequences from the cominant clade. In contrast, the next closest match shared only 87%; it was a C clade isolate from Malawi. We then extracted all full length Env sequences from Tanzania; there were 388 of them. We combined these with the HIV subtyping reference set, and the consensus sequences from CH0457, and made a neighbor joining phylogeny based on these 435 reference and Tanzanian sequences. The two consensus sequences from the 2 distinctive within-subject CH0457 lineages always clustered together, among natural sequences from CH0457, forming a monophyletic group with high bootstrap support in a neighbor joining tree (data not shown, as this was a quality control check). This again indicates that the unusual clade is not a recurrent contamination, or independent infecting strain, and that both lineages evolved from a single infecting strain within CH0457, and had diverged prior to the first sample in taken during chronic infection.
This view is was further supported by the addition of the PBMC proviral DNA sequences from the enrollment time point, that were considered to be biologically “archived” in the host representing virus that had been replicating prior to the time of enrollment. These sequences revealed intermediate steps between the two distinctive lineages found in the CH0457 SGA sequences (
†Additional mutants tested for which no binding change was observed: B.HXBc2 K429E, D474V, M475S; B.YU2 G473A, M475A, ΔV1/V2/V3.
‡NR = not reactive to B.HXB2c or B.YU2 gp120 proteins.
†Other mutants of clade C consensus tested that did not show changes >20 fold for any tested mAb: L125A, S256A, N289K, G471E, M475A, R476A.
‡NR = no neutralization of clade C consensus.
§— = not tested.
†ConB = clade B consensus; ConC = clade C consensus; ConS = group M consensus.
‡NB = no binding observed.
†ConB = clade B consensus; ConC = clade C consensus; ConS = group M consensus.
‡NB = no binding observed.
HIV-1 reporter viruses used in ADCC assays were replication-competent infectious molecular clones (IMC) designed to encode the viruses listed in the left column of the Table in
A luciferase-based ADCC assay was performed as previously described (H. X. Liao et al., Immunity 38, 176 (Jan. 24, 2013), Pollara J, Bonsignori M, Moody M A, et al. HIV-1 Vaccine-Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities. J Virol. 2014; 88(14):7715-7726.) Briefly, HIV-1 infected cells, HIV-1 IMCSF162 and IMCWITO infected CEM.NKRCCR5 cells were used as targets. Whole PBMC obtained from a HIV seronegative donor with the F/V Fc-gamma Receptor (FcRγ) IIIa phenotype were used as the source of NK effector cells. After overnight resting, the PBMC were used as effector cells at an effector to target ratio of 30:1. The target and effector cells were incubated in the presence of 5-fold serial concentrations of plasma/Ab starting at 1:50 dilution for 6 hours at 37° C. in 5% CO2.] The final read-out was the luminescence intensity generated by the presence of residual intact target cells that have not been lysed by the effector population in presence of ADCC-mediating mAb. The % of killing was calculated using the formula:
In this analysis, the RLU of the target plus effector wells represents spontaneous lysis in absence of any source of Ab. Plasma samples collected from a HIV-1 seronegative and seropositive donor were used as negative and positive control samples, respectively, in each assay.
Results are shown in
All documents and other information sources cited herein are hereby incorporated in their entirety by reference.
This application claims the benefit of U.S. Application Ser. No. 61/883,220 filed Sep. 27, 2013, the entire contents of which application are hereby incorporated by reference
This invention was made with government support under Grant Nos. U19 AI067854 and UM1 AI100645 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/057743 | 9/26/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61883220 | Sep 2013 | US |
