Gene Editing of Primary Rhesus Macaque B Cells.

B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.

Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent d-peptide HIV entry inhibitor.

Cholesterol-PIE12-trimer (CPT31) is a potent d-peptide HIV entry inhibitor that targets the highly conserved gp41 N-peptide pocket region. CPT31 exhibited strong inhibitory breadth against diverse panels of primary virus isolates. In a simian-HIV chimeric virus AD8 (SHIVAD8) macaque model, CPT31 prevented infection from a single high-dose rectal challenge. In chronically infected animals, CPT31 monotherapy rapidly reduced viral load by ∼2 logs before rebound occurred due to the emergence of drug resistance. In chronically infected animals with viremia initially controlled by combination antiretroviral therapy (cART), CPT31 monotherapy prevented viral rebound after discontinuation of cART. These data establish CPT31 as a promising candidate for HIV prevention and treatment.

Of Mice, Macaques, and Men: Broadly Neutralizing Antibody Immunotherapy for HIV-1.

The neutralizing antibodies targeting the HIV-1 envelope protein have been a major focus for HIV therapy. Early studies with anti-HIV-1 neutralizing monoclonal antibodies (mAbs) administered to infected individuals showed some promise, as they resulted in transient reductions in plasma viremia in some recipients. However, resistant viral variants rapidly emerged. A major development during the past 6 to 7 years has been the isolation and characterization of highly potent and broadly neutralizing mAbs (bNAbs) from infected individuals known as "elite neutralizers." These "next-generation" bNAbs have been tested in animal model systems and shown to effectively control virus replication, particularly following combination immunotherapy. The success of these preclinical animal studies has led to human clinical trials using an individual bNAb for therapy. This review examines recent findings from animal models and human clinical trials and discusses the future use of bNAbs for HIV-1 treatment.

Early antibody therapy can induce long-lasting immunity to SHIV.

Highly potent and broadly neutralizing anti-HIV-1 antibodies (bNAbs) have been used to prevent and treat lentivirus infections in humanized mice, macaques, and humans. In immunotherapy experiments, administration of bNAbs to chronically infected animals transiently suppresses virus replication, which invariably returns to pre-treatment levels and results in progression to clinical disease. Here we show that early administration of bNAbs in a macaque simian/human immunodeficiency virus (SHIV) model is associated with very low levels of persistent viraemia, which leads to the establishment of T-cell immunity and resultant long-term infection control. Animals challenged with SHIVAD8-EO by mucosal or intravenous routes received a single 2-week course of two potent passively transferred bNAbs (3BNC117 and 10-1074 (refs 13, 14)). Viraemia remained undetectable for 56-177 days, depending on bNAb half-life in vivo. Moreover, in the 13 treated monkeys, plasma virus loads subsequently declined to undetectable levels in 6 controller macaques. Four additional animals maintained their counts of T cells carrying the CD4 antigen (CD4+) and very low levels of viraemia persisted for over 2 years. The frequency of cells carrying replication-competent virus was less than 1 per 106 circulating CD4+ T cells in the six controller macaques. Infusion of a T-cell-depleting anti-CD8ß monoclonal antibody to the controller animals led to a specific decline in levels of CD8+ T cells and the rapid reappearance of plasma viraemia. In contrast, macaques treated for 15 weeks with combination anti-retroviral therapy, beginning on day 3 after infection, experienced sustained rebound plasma viraemia when treatment was interrupted. Our results show that passive immunotherapy during acute SHIV infection differs from combination anti-retroviral therapy in that it facilitates the emergence of potent CD8+ T-cell immunity able to durably suppress virus replication.

Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants.

Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian-human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants.

Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques.

It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti-HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (∼1:100) and potentially achievable by vaccination.

Rapid development of glycan-specific, broad, and potent anti-HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque.

It is widely believed that the induction of a broadly neutralizing antibody (bNAb) response will be a critical component of a successful vaccine against HIV. A significant fraction of HIV-infected individuals mount bNAb responses, providing support for the notion that such responses could be elicited through vaccination. Infection of macaques with simian immunodeficiency virus (SIV) or SIV/HIV chimeric virus (SHIV) has been widely used to model aspects of HIV infection, but to date, only limited bNAb responses have been described. Here, we screened plasma from 14 R5-tropic SHIV-infected macaques for broadly neutralizing activity and identified a macaque with highly potent cross-clade plasma NAb response. Longitudinal studies showed that the development of broad and autologous NAb responses occurred coincidentally in this animal. Serum-mapping studies, using pseudovirus point mutants and antigen adsorption assays, indicated that the plasma bNAbs are specific for epitopes that include carbohydrates and are critically dependent on the glycan at position 332 of Env gp120. The results described herein provide insight into the development and evolution of a broad response, suggest that certain bNAb specificities may be more rapidly induced by immunization than others, and provide a potential model for the facile study of the development of bNAb responses.

Neutralizing antibody titers conferring protection to macaques from a simian/human immunodeficiency virus challenge using the TZM-bl assay.

We previously reported that passive transfer of polyclonal neutralizing antibodies (NAbs) sufficient to generate a titer of 1:38 in the plasma would confer sterilizing protection to 99% of macaques challenged intravenously with 75 TCID(50) of SHIV(DH12). Neutralizing activity in that study was measured in an MT4 cell assay in which infection was completely blocked (EC(100)). In the current study, the TZM-bl system was used to measure EC(50) neutralizing titers in several of the same macaque plasma samples and the relationship between these titers and in vivo protection was determined. The antiviral EC(50) NAb titers measured in individual plasma samples were higher than those previously obtained in the MT4 system. Furthermore, the geometric mean EC(50) NAb titers against pseudotyped SHIV(DH12) were 33-fold greater than the EC(100) titers measured in the MT4 cell assay against the replication-competent SHIV(DH12) inoculated into animals. An augmented probit regression model was used to generate curves relating TZM-bl EC(50) NAb titers and protection from a virus challenge; estimated titers conferring various levels of protection were then determined. In TZM-bl assays using pseudotyped SHIV(DH12), representative percent in vivo protection/estimated EC(50) titers were 99%/1:4467, 90%/1:1175, 80%/1:676, 50%/1:234, and 33%/1:141. Because it is likely that contributions from other arms of the immune system will contribute to vaccine-induced control, the range of EC(50) NAb titers we have derived may be more informative for evaluating the protective value of NAb activity from TZM-bl assays.

Dissecting the factors involved in the locomotion mode of neuronal migration in the developing cerebral cortex.

Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cdelta (PKCdelta), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCdelta hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCdelta in the early and final phases of migration, respectively.

Noninvasive in vivo imaging of CD4 cells in simian-human immunodeficiency virus (SHIV)-infected nonhuman primates.

Since the earliest days of the HIV epidemic, the number of CD4(+) T cells per unit volume of blood has been recognized as a major prognostic factor for the development of AIDS in persons with HIV infection. It has also been generally accepted that approximately 2% of total body lymphocytes circulate in the blood. In the present study, we have used a nondepleting humanized anti-CD4 monoclonal antibody labeled with the gamma emitter indium-111 to visualize the CD4(+) T-cell pool in vivo in nonhuman primates with simian HIV infection. A strong correlation was noted between radiotracer uptake in spleen, tonsil, axillary lymph nodes, and peripheral blood CD4 T-cell counts (rho = 0.75, 0.93, and 0.85, respectively, P < .005). The relationship between radiotracer retention in lymphoid tissues and CD4(+) T-cell counts in the circulation was governed by an exponential law. These data provide an estimate for the total number of lymphocytes in the body as being between 1.9 and 2.9 x 10(12) and suggest that the partition between peripheral blood and lymphoid tissue is between 0.3% and 0.5%.

Although macrophage-tropic simian/human immunodeficiency viruses can exhibit a range of pathogenic phenotypes, a majority of isolates induce no clinical disease in immunocompetent macaques.

Unlike prototypical lentiviruses like visna and caprine arthritis-encephalitis viruses, which are mainly macrophage tropic (M-tropic), primate lentiviruses primarily target CD4+ T lymphocytes. We previously reported that during the late phase of highly pathogenic chimeric simian/human immunodeficiency virus (SHIV) infections of rhesus macaques, when CD4+ T cells have been systemically eliminated, high levels of viremia are maintained from productively infected macrophages. The availability of several different M-tropic SHIVs from such late-stage immunocompromised animals provided the opportunity to assess whether they might contribute to the immune deficiency induced by their T-cell-tropic parental viruses or possibly cause a distinct disease based on their capacity to infect macrophages. Pairs of rhesus monkeys were therefore inoculated intravenously with six different M-tropic SHIV preparations, and their plasma viral RNA loads, circulating lymphocyte subset numbers, and eventual disease outcomes were monitored. Only one of these six M-tropic SHIVs induced any disease; the disease phenotype observed was the typical rapid, complete, and irreversible depletion of CD4+ T cells induced by pathogenic SHIVs. An analysis of two asymptomatic monkeys, previously inoculated with an M-tropic SHIV recovered directly from alveolar macrophages, revealed that this inoculum targeted alveolar macrophages in vivo, compared to a T-cell-tropic virus, yet no clinical disease occurred. Although one isolate did, in fact, induce the prototypical rapid, irreversible, and complete loss of CD4+ T cells, indicating that M-tropism and pathogenicity may not be inversely related, the majority of M-tropic SHIVs induced no clinical disease in immunocompetent macaques.

Involvement of a Rac activator,P-Rex1, in neurotrophin-derived signaling and neuronal migration.

Rho-family GTPases play key roles in regulating cytoskeletal reorganization, contributing to many aspects of nervous system development. Their activities are known to be regulated by guanine nucleotide exchange factors (GEFs), in response to various extracellular cues. P-Rex1, a GEF for Rac, has been mainly investigated in neutrophils, in which this molecule contributes to reactive oxygen species formation. However, its role in the nervous system is essentially unknown. Here we describe the expression profile and a physiological function of P-Rex1 in nervous system development. In situ hybridization revealed that P-Rex1 is dynamically expressed in a variety of cells in the developing mouse brain, including some cortical and DRG neurons. In migrating neurons in the intermediate zone, P-Rex1 protein was found to localize in the leading process and adjacent cytoplasmic region. When transfected in pheochromocytoma PC12 cells, P-Rex1 can be activated by NGF, causing an increase in GTP-bound Rac1 and cell motility. Deletion analyses suggested roles for distinct domains of this molecule. Experiments using a P-Rex1 mutant lacking the Dbl-homology domain, a dominant-negative-like form, and small interfering RNA showed that endogenous P-Rex1 was involved in cell migration of PC12 cells and primary cultured neurons from the embryonic day 14 cerebral cortices, induced by extracellular stimuli (NGF, BDNF, and epidermal growth factor). Furthermore, in utero electroporation of the mutant protein into the embryonic cerebral cortex perturbed radial neuronal migration. These findings suggest that P-Rex1, which is expressed in a variety of cell types, is activated by extracellular cues such as neurotrophins and contributes to neuronal migration in the developing nervous system.

Transfer of neutralizing IgG to macaques 6 h but not 24 h after SHIV infection confers sterilizing protection: implications for HIV-1 vaccine development.

Passive transfer of high-titered antiviral neutralizing IgG, known to confer sterilizing immunity in pig-tailed monkeys, has been used to determine how soon after virus exposure neutralizing antibodies (NAbs) must be present to block a simian immunodeficiency virus (SIV)/HIV chimeric virus infection. Sterilizing protection was achieved in three of four macaques receiving neutralizing IgG 6 h after intravenous SIV/HIV chimeric virus inoculation as monitored by PCR analyses of and attempted virus isolations from plasma, peripheral blood mononuclear cell, and lymph node specimens. In the fourth animal, the production of progeny virus was suppressed for >4 weeks. A delay in transferring NAbs until 24 h after virus challenge resulted in infection in two of two monkeys. These results suggest that even if a vaccine capable of eliciting broadly reactive NAbs against primary HIV-1 were at hand, the Abs generated must remain at, or rapidly achieve, high levels within a relatively short period after exposure to virus to prevent the establishment of a primate lentivirus infection.

Amino acid deletions are introduced into the V2 region of gp120 during independent pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV) infections of rhesus monkeys generating variants that are macrophage tropic.

Highly pathogenic simian immunodeficiency virus/HIV chimeric viruses (SHIVs) cause extremely rapid, irreversible, and systemic depletions of CD4(+) T lymphocytes in inoculated rhesus monkeys. In the absence of this T cell subset, virus production can be sustained for several months by tissue macrophage. During independent infections of seven animals with uncloned virus stocks, SHIV variants emerged bearing amino acid deletions that affected specific residues of the gp120 V2 loop. Some of these macrophage-phase SHIVs replicated to high levels in alveolar macrophage.