Human Anti–HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1

Human immunodeficiency virus-1 continues to be a worldwide health issue, with an estimated 35 million lives lost to date (1). In 2016, 1 million people died of HIV-1–related causes. Despite the length of time HIV-1 has been wreaking havoc on its victims, further improvements in the prevention and treatment of HIV-1 are still critically needed.

Humanized mice offer invaluable animal models to study the treatment and prevention of HIV-1 infection, because human tissues engrafted in these mice can be infected with HIV-1. Generally, there are three different humanized models for engraftment of human immune systems in immunodeficient mice: engraftment with human PBMCs (Hu-PBL-SCID), engraftment with human CD34⁺ hematopoietic stem cells (HSC) (Hu-SRC-SCID), and engraftment with human fetal tissues (bone marrow, liver, thymus) (BLT and SCID-hu). Hu-PBL-SCID mice are generated by injection of human peripheral blood leukocytes and support examination of human T cell function (2). However, due to the rapid onset of T cell–mediated xenogeneic graft-versus-host disease, there is a limited window of opportunity for experiments with Hu-PBL-SCID mice. In the second model, Hu-SRC-SCID mice, HSC derived from fetal liver, cord blood, bone marrow, or G-CSF–mobilized peripheral blood are injected (2, 3). Hu-SRC-SCID mice support engraftment of a functional human immune system, including B cells, T cells, myeloid cells, and APCs. However, human innate immune cell populations developing in Hu-SRC-SCID mice are present at very low numbers in the blood, and human T cells develop primarily within the murine thymus, which lacks HLA expression needed for development of HLA-restricted T cells (2). Finally, the BLT model involves the transplantation of human fetal liver and thymus, and i.v. injections of autologous fetal liver HSC. This model enables robust development of a functional immune system, provides much higher percentages of human T cells, supports efficient development of HLA-restricted conventional and regulatory T cells, and is the only model that leads to the generation of a robust mucosal human immune system (3). This combination of features is ideal for studying HIV-1 infection, as it predominantly occurs at the mucosal surfaces. Of course, there are caveats to BLT mice as well, including a limited supply of fetal tissue for engraftment, the requirement for skilled technicians to perform engraftment protocols, development of a wasting syndrome that limits the life span of the mice, and difficulty in generating class-switched, affinity-matured B cell responses following antigenic challenge.

For our studies on preventing and treating HIV-1 infection with mAbs, we selected the BLT model. Studies using SCID-hu and immunodeficient mice engrafted with human CD34⁺ HSC (hu-HSC) mice revealed the characteristics of latency during the early stages of infection (4). However, as improvements to the engraftment of BLTs have been made, they have become a powerful model for studying HIV-1 for their unique characteristics allowing for the mimicry of a full human immune system. We describe in this article the generation of human mAbs to HIV-1 from infected NOD-scid IL2rγ^null (NSG)-BLT mice. Despite the BLT mouse model having previously been shown difficult to illicit a robust Ab response (5, 6), there are unique characteristics of HIV-1 infection, such as the chronic production of viral Ags, with inflammation helping to drive the response (7). The mAbs isolated in this study were incredibly diverse in variable repertoire, isotype, and subclass and displayed neutralization activity. Thus, the engraftment of immunodeficient mice with human immune cells in combination with infection of HIV-1 enables the generation and isolation of fully human mAbs to specific targets and Ags for which immunized individuals are either not available or fail to generate a humoral immune response.

Stock NOD.Cg-Prkdc^scidIl2rγ^tm1Wjl/SzJ (NSG) mice were obtained from colonies maintained at The Jackson Laboratory (Bar Harbor, ME) by L.D.S. All procedures with animals were done in accordance with the guidelines of the Animal Care and Use Committee of the University of Massachusetts Medical School and The Jackson Laboratory and conformed to the recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, Eighth Edition, 2011). NSG-BLT mice were generated using standard protocols previously described (8, 9). Mice were infected with virus isolate BaL or with virus generated by cloned pNL43 bearing the JR-FL env, via i.p. injection. Plasma samples were collected over time via submandibular or tail nick bleeds and used in ELISAs to monitor Ab development, as well as to measure HIV-1 genomic RNA equivalents by RT-PCR. A total of four animals were chosen to perform fusions.

Splenocytes were fused with human myeloma cells HMMA 2.5 using polyethylene glycol (PEG solution, P7181; Sigma-Aldrich) and selection with 1% hypoxanthine-aminopterin-thymidine (Mediatech), and 0.2% ouabain, as previously described (10, 11). Fusions were screened for HIV-1 Ab by capture ELISA using gp41 (Meridian Life Science), gp120, or gp120-CD4 full-length single chain construct [FLSC; gp120 plus CD4 D1 and D2 domains (12)]. gp120 and gp120-CD4 FLSC were made by transient transfection of 293T with expression plasmids and purified using lectin from Galanthus nivalis (snowdrop) agarose (L8275; Sigma-Aldrich) as previously described (13). Positive-producing hybrids were scaled up and Ab purified from supernatants using a Protein G Sepharose (IgG; GE Healthcare) or CaptureSelect Affinity Matrix (IgA; Thermo Fisher Scientific).

RNA extraction from hybridomas was performed using QIAGEN RNEasy Plus Mini Kit. cDNA was synthesized using SuperScript III First-Strand Synthesis System (Invitrogen) and then amplified using either H, L, or alternate λ chain primer sets (Table I). PCRs were run on an agarose gel; bands were extracted and purified using QIAGEN Gel Extraction Mini Kit. Ten microliters of gel-extracted product was sent to Genewiz, with separate reactions for each sequencing primer (Table II). Sequences were then analyzed using ImMunoGeneTics.

The neutralization activity of the Abs against four isolates of HIV-1 was determined by a luciferase assay using TZM-bl cells. Abs were titered in serial dilutions and incubated with virus stock diluted to 100 median tissue culture infectious dose for 1 h at 37°C. TZM-bl cells were then added at a concentration of 1 × 10⁴ cells per well with DEAE-Dextran (Sigma-Aldrich). After 48 h of incubation at 37°C, 100 μl of supernatant was removed from the wells and 100 μl of Britelite Plus substrate was added and incubated for 5 min. Plates were then read on a luminometer to determine relative light units. Percent neutralization was determined based on control wells of virus and media, and IC₅₀ and IC₉₀ values were calculated by regression analysis.

Three months post-tissue engraftment, NSG-BLT mice were screened for the level of human cell chimerism in the blood by flow cytometry. Peripheral blood samples were evaluated for human CD45⁺ cells, CD3⁺ T cells, and CD20⁺ B cells. Successfully engrafted NSG-BLT mice with >20% human CD45⁺ cells and >20% human CD3⁺ T cells (as a percentage of CD45) in blood were used in experiments. Within each cohort, >75% of animals were engrafted with at least 20% human CD45⁺ cells. For those animals engrafted with >20% of human CD45⁺ cells, among the CD45⁺ cells, the range of either CD3⁺ T cells or CD20⁺ B cells was 20–70%. Successfully engrafted mice were infected with HIV-1 BaL or NL43-JRFL via i.p. injection. Blood samples were tested by ELISA for anti–HIV-1 Ab using gp41, gp120, or gp120-CD4 FLSC Ag ELISA and viral load. The inclusion of gp120-CD4 FLSC, which is a single chain construct of CD4 and gp120, allows reactivity with epitopes only exposed upon CD4 binding (gp120-CD4 FLSC), in contrast to gp120 without CD4 (12). Fig. 1 shows a representative animal, in which there was an increase in serum Ab reactive against gp41 or gp120-CD4 FLSC, as a function of time post-HIV-1-infection. Fig. 2 demonstrates two other animals of the same cohort and their Ab response to gp120-CD4 FLSC over time, comparing IgG versus IgA. It should be noted that there was no correlation between the number of B cells (as indicated by CD20⁺ cells) and Ab titers, albeit this is a small dataset.

All but one animal were euthanized within 3 mo of infection (one animal was euthanized 8 mo postinfection), spleens were harvested, and single cell suspensions were collected for fusion. Out of 20 total mice between three different cohorts, four mice were selected for fusion based on Ab titer (and survival for at least 3 mo postinfection). As shown in Table III, recovery of splenocytes varied, which in turn led to variation in the number of hybrids to be screened. Altogether, between three different fusions, more than 70 different Abs to either gp41 or gp120-CD4 FLSC were isolated during the initial screening process, with a random selection undergoing further characterization (Table IV). Perhaps due to splenocytes being frozen previously, the yield of hybridomas to be screened from fusion F878 was less than for fusions in which fresh splenocytes were used. It should be noted that >80% of the screened wells with hybridomas secreted either IgG or IgA Ig, demonstrating the ability to capture Ab-producing B cells using these methods.

The immunoreactivity of purified Ab with HIV-1 env-encoded Ags was determined using gp41 and gp120 or gp120-CD4 FLSC (Fig. 3). Human mAbs F240, reactive with gp41, F425-A1g8, reactive with CD4i epitope (expressed by gp120-CD4 FLSC), and b12, reactive with gp120/CD4 binding site, were included for comparison. End point concentrations were determined by linear regression based on the best-fit curve for the control. For each assay, it was determined that the immunoreactivity for each mAb isolated from the humanized mice was very similar, if not more immunoreactive than that observed for a known positive control Ab. It is interesting to note that the Ab isolated as an IgA showed higher affinity for gp120-CD4 FLSC when compared with its control IgG.

Neutralization of HIV-1 was tested in vitro using TZM-bl cells as targets and a panel of four HIV-1 isolates (67970, BaL, JR-CSF, and SF-162) grown in PBMCs. Serial dilutions of all mAbs were tested, and IC₅₀ and IC₉₀ values were calculated (Table V). The Ab F861 A7f10 had the most neutralizing activity across all HIV-1 isolates, with IC₅₀ values ranging from <0.02 to 25 μg/ml, whereas IC₉₀ values ranged from 0.02 to 39 μg/ml, depending on the virus. Tested Abs from fusions F860, F862, and F878 displayed neutralizing activity against isolate SF-162, with IC₅₀ values 2.64, 12.14, 4.65, and 1.3 μg/ml, respectively (Table V). Thus, Abs can be isolated from HIV-1–infected humanized mice that vary in immunoreactivity and neutralization across more than one clade of virus and target Ag.

To explore the variety of Abs generated from fusions, a random sampling of isolated human mAbs were chosen for sequencing, and gene family usage was explored (Table IV). The H and L chains of several human mAbs were sequenced and analyzed using ImMunoGeneTics V-Quest software to determine variable gene family usage, somatic hypermutation, and affinity maturation. Based on these analyses, it is clear that Ab isotype and subclass were not restricted, but there was a much higher representation of λ light chains (78%). In looking at gene family usage for the selected human mAbs, our data display comparable results to previous studies that explored Ig repertoires in NSG mice in comparison with humans (14). Although no single gene family was dominant, VH1 and VH3 families were most common, which is comparable to gene family usage in humans (15, 16). It is of interest that mAbs F860 A2e10 and F861 A7f10, despite being different isotypes, share the same gene family, IgHV1-69*01, and yield more neutralizing ability and wider breadth than members of other families, respectively. In preceding studies comparing gene family usage in healthy versus HIV-1–infected individuals, an increase of VH1 gene families could be seen (15). Most of the L chain genes were relatively unchanged from germline sequences (>95% homology); there was more divergence from germline for H chain genes. Two Abs were 85–90% homologous to germline; one was 94% homologous, with the remaining five >95% homologous, to germline. Broadly neutralizing Abs to HIV tend to have high somatic mutation rates (17). However, in this study, the most effective Ab, F861 A7f10 was not highly mutated. Classically, somatic mutation occurs predominantly in the CDR regions, whereas for HIV, framework regions can also be found significantly mutated (18). Such extensive somatic mutation, especially of the framework regions, may not be necessarily critical for neutralizing activity (19, 20), but perhaps it is specific mutations that may only occur in the presence of high mutation activity that are critical (21). It can also be possible that further maturation of this Ab response would increase potency and breadth of neutralization with this particular (or other) Ab. While the sample size in this study was limited, these data would suggest that although it has been observed that most B cells in BLT mice do not undergo selection processes in the germinal center (22), some B cells can be stimulated to undergo affinity maturation and somatic hypermutation.

Although much has been learned in the field of HIV-1 research in regard to disease pathogenesis, there is still a need for a more effective prophylactic that will not only impede the spread of HIV-1 but will prevent infection from occurring. With infected individuals being capable of producing broadly neutralizing Abs, treating HIV-1 using a prophylactic Ab is under active development (23–26). As we have shown in this article, Ag-specific, affinity-matured, and somatically mutated Abs can be generated in the BLT mouse model, making it ideal for producing broadly neutralizing Abs. Furthermore, given that the animals have human immune systems, the potential for countless immunization strategies that will ensure the production of millions of splenocytes against the exact target being examined is possible.

HIV-1 infection of the mice was monitored by viral load and human IgG and IgA Ab response using Ag-specific ELISA with gp120, gp41, or gp120/CD4 complex as Ags. Approximately 8–12 wk postinfection, spleens were harvested and splenocytes were fused with human fusion partner HMMA 2.5 to isolate Ab-expressing hybridomas. Interestingly, mouse 13.41, which was infected with JRFL, had survived up to 8 mo at time of sacrifice. This is rare in that, given the high level of augmentation the mice undergo, in combination with potential onset of graft-versus-host disease, they do not normally survive for such extended periods of time (2, 3, 27). Lead clones were scaled and purified for testing in TZM-bl neutralization functional assays to determine neutralization and cytotoxic ability of the Abs.

Previous studies using humanized mice for immunization with virus or Ag have generally demonstrated lower serum Ab titers, characterized by mostly IgM Ag-specific responses with lower IgG (14). In addition to serum reactivity, IgM mAb have been isolated from humanized mice (28). When the human IL-6 gene was knocked into the murine locus, there was an increase in serum Ag-specific IgG with improved diversity (29). As shown in Fig. 1, even without insertion of the human IL-6 gene, we observed a dramatic rise in Ag-specific Ab following infection. In addition, we were not limited to detecting an IgG response but detected an IgA response as well. This finding is intriguing, as IgA Abs may be the first encountered at the mucosal surface upon infection. However, intriguingly, there is a limited IgA response to HIV-1, especially as compared with other mucosal infections (30–32). It should be noted that neutralizing activity of the mAbs was not necessarily restricted to the infected isolate. As shown in Table V, Ab F861 A7f10 neutralized Tier 1 and Tier 2 strains, with an IC₅₀ of 25.01 when being tested against BaL. The same Ab had an IC₅₀ of <0.01 and an IC₉₀ of <0.02 against JR-CSF and SF-162, respectively. Out of the sampling of Abs tested that were reactive with gp120-CD4 FLSC, F861 A7f10 was the only IgG3 that had neutralizing ability across multiple clades. It has been shown that IgG3 Abs typically appear before other IgG subclasses throughout the course of viral infection (33).

Ab sequences were also determined. A robust, specific Ab response of both IgG and IgA isotypes occurred in response to HIV-1 infection. Over 70 hybridomas were created that were not only immunoreactive with env Ags, but some of which also had neutralization activity. Moreover, variable family usage was not limited and somatic mutation was evident, indicating ongoing stimulation and proliferation of B cells in response to infection. Ultimately, by using this mouse model, not only were we able to generate large amounts of Ag-specific Abs but it offered astounding variation of isotypes and gene family usage. Thus, in contrast to some other mouse models, such as the HuMab mouse, there are no limitations to the gene families that are available for use (14, 34).

In summary, we have demonstrated that human mAbs can be isolated from HIV-infected humanized mice. The Abs were highly immunoreactive, functional, and somatically mutated and represented diverse gene family usage, as well as isotype selection. Further use of these animals, in HIV-1 as well as other disease areas, may lead to the isolation of additional diverse, functional human mAbs for active or passive immunotherapy.

The authors have no financial conflicts of interest.