Alpaca single B cell interrogation and heavy-chain-only antibody discovery on an optofluidic platform.

In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Advancing HIV Broadly Neutralizing Antibodies: From Discovery to the Clinic.

Despite substantial progress in confronting the global HIV-1 epidemic since its inception in the 1980s, better approaches for both treatment and prevention will be necessary to end the epidemic and remain a top public health priority. Antiretroviral therapy (ART) has been effective in extending lives, but at a cost of lifelong adherence to treatment. Broadly neutralizing antibodies (bNAbs) are directed to conserved regions of the HIV-1 envelope glycoprotein trimer (Env) and can block infection if present at the time of viral exposure. The therapeutic application of bNAbs holds great promise, and progress is being made toward their development for widespread clinical use. Compared to the current standard of care of small molecule-based ART, bNAbs offer: (1) reduced toxicity; (2) the advantages of extended half-lives that would bypass daily dosing requirements; and (3) the potential to incorporate a wider immune response through Fc signaling. Recent advances in discovery technology can enable system-wide mining of the immunoglobulin repertoire and will continue to accelerate isolation of next generation potent bNAbs. Passive transfer studies in pre-clinical models and clinical trials have demonstrated the utility of bNAbs in blocking or limiting transmission and achieving viral suppression. These studies have helped to define the window of opportunity for optimal intervention to achieve viral clearance, either using bNAbs alone or in combination with ART. None of these advances with bNAbs would be possible without technological advancements and expanding the cohorts of donor participation. Together these elements fueled the remarkable growth in bNAb development. Here, we review the development of bNAbs as therapies for HIV-1, exploring advances in discovery, insights from animal models and early clinical trials, and innovations to optimize their clinical potential through efforts to extend half-life, maximize the contribution of Fc effector functions, preclude escape through multiepitope targeting, and the potential for sustained delivery.

Single-dose bNAb cocktail or abbreviated ART post-exposure regimens achieve tight SHIV control without adaptive immunity.

Vertical transmission accounts for most human immunodeficiency virus (HIV) infection in children, and treatments for newborns are needed to abrogate infection or limit disease progression. We showed previously that short-term broadly neutralizing antibody (bNAb) therapy given 24 h after oral exposure cleared simian-human immunodeficiency virus (SHIV) in a macaque model of perinatal infection. Here, we report that all infants given either a single dose of bNAbs at 30 h, or a 21-day triple-drug ART regimen at 48 h, are aviremic with almost no virus in tissues. In contrast, bNAb treatment beginning at 48 h leads to tight control without adaptive immune responses in half of animals. We conclude that both bNAbs and ART mediate effective post-exposure prophylaxis in infant macaques within 30-48 h of oral SHIV exposure. Our findings suggest that optimizing the treatment regimen may extend the window of opportunity for preventing perinatal HIV infection when treatment is delayed.

Multimeric Epitope-Scaffold HIV Vaccines Target V1V2 and Differentially Tune Polyfunctional Antibody Responses.

The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing polyfunctional antibodies with characteristics associated with protection.

Reduced Cell-Associated DNA and Improved Viral Control in Macaques following Passive Transfer of a Single Anti-V2 Monoclonal Antibody and Repeated Simian/Human Immunodeficiency Virus Challenges.

A high level of V1V2-specific IgG antibodies (Abs) in vaccinees' sera was the only independent variable that correlated with a reduced risk of human immunodeficiency virus (HIV) acquisition in the RV144 clinical trial. In contrast, IgG avidity, antibody neutralization, and antibody-dependent cellular cytotoxicity each failed as independent correlates of infection. Extended analyses of RV144 samples demonstrated the antiviral activities of V1V2-specific vaccine-induced antibodies. V2-specific antibodies have also been associated with protection from simian immunodeficiency virus (SIV), and the V2i-specific subset of human monoclonal antibodies (MAbs), while poor neutralizers, mediates Fc-dependent antiviral functions in vitro The objective of this study was to determine the protective efficacy of a V2i-specific human MAb, 830A, against mucosal simian/human immunodeficiency virus (SHIV) challenge. V2i MAb binding sites overlap the integrin binding site in the V2 region and are similar to the epitopes bound by antibodies associated with reduced HIV infection rates in RV144. Because the IgG3 subclass was a correlate of reduced infection rates in RV144, we compared passive protection by both IgG1 and IgG3 subclasses of V2i MAb 830A. This experiment represents the first in vivo test of the hypothesis emanating from RV144 and SIV studies that V2i Abs can reduce the risk of infection. The results show that passive transfer with a single V2i MAb, IgG1 830A, reduced plasma and peripheral blood mononuclear cell (PBMC) virus levels and decreased viral DNA in lymphoid tissues compared to controls, but too few animals remained uninfected to achieve significance in reducing the risk of infection. Based on these findings, we conclude that V2i antibodies can impede virus seeding following mucosal challenge, resulting in improved virus control.IMPORTANCE Since the results of the HIV RV144 clinical trial were reported, there has been significant interest in understanding how protection was mediated. Antibodies directed to a subregion of the envelope protein called V1V2 were directly correlated with a reduced risk, and surprisingly low virus neutralization was observed. To determine whether these antibodies alone could mediate protection, we used a human monoclonal antibody directed to V2 with properties similar to those elicited in the vaccine trial for passive infusions in rhesus macaques and challenge with SHIV. The single V2 antibody at the dose given did not significantly reduce the number of infections, but there was a significant reduction in the seeding of virus to the lymph nodes and a decrease in plasma viremia in the HIV antibody-infused macaques compared with the control antibody-infused animals. This finding shows that V2 antibodies mediate antiviral activities in vivo that could contribute to a protective HIV vaccine.

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 ß-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

Cell extrinsic alterations in splenic B cell maturation in Flt3-ligand knockout mice.

B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3-ligand (FL-/-) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL-/- mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL-/- mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF-R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL-/- mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL-/- mice. A Bcl2 transgene did not rescue TS cells in FL-/- mice, uncoupling FL-deficiency to Bcl2-dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL-/- mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

Flt3 signaling regulates the proliferation, survival, and maintenance of multipotent hematopoietic progenitors that generate B cell precursors.

Flt3 signaling plays a crucial role in regulating the survival and differentiation of lymphoid progenitors into B cell precursors (BCPs) in bone marrow. To define further the role of Flt3 signaling in lymphoid progenitor survival, mice deficient in Flt3 ligand that also expressed a Bcl2 transgene (Eµ-bcl2tg flt3l(-/-)) were generated. Intracellular flow cytometry established transgene expression in primitive hematopoietic progenitors, including lineage-negative Sca-1(+) c-kit(+) (LSK(+)) CD27(-) cells enriched for functional hematopoietic stem cells. Compared with flt3l(-/-) mice, Eµ-bcl2tg flt3l(-/-) mice had significantly increased multipotential progenitors (MPPs), IL-7R(+) common lymphoid progenitors, and B cell precursors. To determine whether forced expression of Bcl2 was sufficient to restore lymphoid priming in the absence of Flt3 signaling Eµ-bcl2tg flt3l(-/-)rag1-gfp(+) mice were generated. Analysis of Eµ-bcl2tg flt3l(-/-)rag1-gfp(+) mice revealed that the Bcl2 transgene had no effect on lymphoid priming before CD19 expression. Thus, forced expression of a survival gene can bypass the requirement for threshold levels of Flt3 signaling requisite for lymphoid priming. Temporal Flt3 ligand (FL) replacement therapy in flt3l(-/-) mice revealed specific requirements for Flt3 signaling in the expansion and maintenance of Flt3(+hi) MPP and Flt3(+) all lymphoid progenitors, but not Flt3(+) B lymphoid progenitors (BLPs), the immediate precursors of BCPs. BCPs were restored after temporal in vivo FL treatment, albeit with delayed kinetics. Together, these results show that Flt3 regulates the proliferation, survival, and maintenance of developmental stage-specific hematopoietic progenitors that give rise to BCPs.

Hoxa9 and Flt3 signaling synergistically regulate an early checkpoint in lymphopoiesis.

Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9(-/-)Flt3l(-/-) mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3(+) MPP in bone marrow than the single knockouts. Although LMPP and Flt3(+) CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l(-/-) mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l(-/-) mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150(+hi)CD34(-)CD41(+) myeloid-biased stem cells in Hoxa9(-/-)Flt3l(-/-) mice. In contrast, CD150(-) MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150(-)Flt3(+) cells from wild-type control, Hoxa9(-/-), and Flt3l(-/-) single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.

Differential requirement for Hoxa9 in the development and differentiation of B, NK, and DC-lineage cells from Flt3+ multipotential progenitors.

Hoxa9 is a homeodomain transcription factor important for the generation of Flt3+hiIL-7R- lymphoid biased-multipotential progenitors, Flt3+IL-7R+ common lymphoid progenitors (CLPs), and B cell precursors (BCP) in bone marrow (BM). In addition to B-cell, Flt3+IL-7R+ CLPs possess NK and DC developmental potentials, although DCs arise from Flt3+IL-7R- myeloid progenitors as well. In this study, we investigated the requirement for Hoxa9, from Flt3+ or Flt3- progenitor subsets, in the development of NK and DC lineage cells in BM. Flt3+IL-7R+Ly6D- CLPs and their Flt3+IL-7R+Ly6D+ B lineage-restricted progeny (BLP) were significantly reduced in hoxa9-/- mice. Interestingly, the reduction in Flt3+IL-7R+ CLPs in hoxa9-/- mice had no impact on the generation of NK precursor (NKP) subsets, the differentiation of NKP into mature NK cells, or NK homeostasis. Similarly, percentages and numbers of common dendritic progenitors (CDP), as well as their plasmacytoid or conventional dendritic cell progeny in hoxa9-/- mice were comparable to wildtype. These findings reveal distinct requirements for Hoxa9 or Hoxa9/Flt3 molecular circuits in regulation of B versus NK and DC development in BM.

Changes at the 3'-untranslated region stabilize Rubisco activase transcript levels during heat stress in Arabidopsis.

Inhibition of photosynthesis by heat stress is accompanied by functional impairment of Rubisco's chaperone, activase (RCA), resulting in deactivation of Rubisco. Since activase is extremely sensitive to thermal denaturation, changes in expression of RCA at the transcript or protein level could provide a mechanism for acclimation of photosynthesis to prolonged heat stress. Using quantitative real-time PCR (qPCR) we show steady-state RCA transcript levels in Arabidopsis thaliana are stabilized during prolonged exposure to moderate heat (35  °C). A survey of RCA transcripts indicates heat stress did not alter the relative abundance of transcripts encoding α and ß-isoforms of activase that are produced by alternative splicing of the pre-mRNA. Instead, mRNA stabilization in heat-stressed plants coincided with a significant reduction in the average length of activase 3'-untranslated regions, and was associated with enrichment of an uncharacterized activase mRNA splice variant, AtRCAß2. Transcript-specific qPCR revealed AtRCAß2 mRNA was more stable than AtRCAα and AtRCAß mRNA in heat-stressed plants. Using an inducible transgenic system, we found that RCA transcripts lacking their native 3'-untranslated region were significantly more stable than their full-length counterparts in vivo. Using this system, stability of the RCA protein was examined over 24 h in vivo, in the absence of RCA transcription. At both optimal and elevated temperatures, RCA protein levels remained stable in plants lacking RCA mRNA, but increased when RCA mRNA was present, particularly in heat-stressed plants. This study reveals a possible mechanism, involving post-transcriptional regulation of an important photosynthesis regulatory gene, for acclimation of photosynthesis to heat stress.