Complement contributes to antibody-mediated protection against repeated SHIV challenge.

The first clinical efficacy trials of a broadly neutralizing antibody (bNAb) resulted in less benefit than expected and suggested that improvements are needed to prevent HIV infection. While considerable effort has focused on optimizing neutralization breadth and potency, it remains unclear whether augmenting the effector functions elicited by broadly neutralizing antibodies (bNAbs) may also improve their clinical potential. Among these effector functions, complement-mediated activities, which can culminate in the lysis of virions or infected cells, have been the least well studied. Here, functionally modified variants of the second-generation bNAb 10-1074 with ablated and enhanced complement activation profiles were used to examine the role of complement-associated effector functions. When administered prophylactically against simian-HIV challenge in rhesus macaques, more bNAb was required to prevent plasma viremia when complement activity was eliminated. Conversely, less bNAb was required to protect animals from plasma viremia when complement activity was enhanced. These results suggest that complement-mediated effector functions contribute to in vivo antiviral activity, and that their engineering may contribute to the further improvements in the efficacy of antibody-mediated prevention strategies.

Loose Coupling between the Voltage Sensor and the Activation Gate in Mammalian HCN Channels Suggests a Gating Mechanism.

Voltage-gated potassium (Kv) channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels share similar structures but have opposite gating polarity. Kv channels have a strong coupling (>109) between the voltage sensor (S4) and the activation gate: when S4s are activated, the gate is open to >80% but, when S4s are deactivated, the gate is open <10-9 of the time. Using noise analysis, we show that the coupling between S4 and the gate is <200 in HCN channels. In addition, using voltage clamp fluorometry, locking the gate open in a Kv channel drastically altered the energetics of S4 movement. In contrast, locking the gate open or decreasing the coupling between S4 and the gate in HCN channels had only minor effects on the energetics of S4 movement, consistent with a weak coupling between S4 and the gate. We propose that this loose coupling is a prerequisite for the reversed voltage gating in HCN channels.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 µg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

Protection of Newborn Macaques by Plant-Derived HIV Broadly Neutralizing Antibodies: a Model for Passive Immunotherapy during Breastfeeding.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

An HIV Vaccine Targeting the V2 Region of the HIV Envelope Induces a Highly Durable Polyfunctional Fc-Mediated Antibody Response in Rhesus Macaques.

The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of nonneutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen was highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only an ∼5× reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted.IMPORTANCE The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively nonneutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

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.

Clogging-free continuous operation with whole blood in a radial pillar device (RAPID).

Pillar-based passive microfluidic devices combine the advantages of simple designs, small device footprint, and high selectivity for size-based separation of blood cells. Most of these device designs have been validated with dilute blood samples. Handling whole blood in pillar-based devices is extremely challenging due to clogging. The high proportion of cells (particularly red blood cells) in blood, the varying sizes and stiffness of the different blood cells, and the tendency of the cells to aggregate lead to clogging of the pillars within a short period. We recently reported a ra dial pi llar d evice (RAPID) design for continuous and high throughput separation of multi-sized rigid polystyrene particles in a single experiment. In the current manuscript, we have given detailed guidelines to modify the design of RAPID for any application with deformable objects (e.g. cells). We have adapted RAPID to work with whole blood without any pre-processing steps. We were successful in operating the device with whole blood for almost 6 h, which is difficult to achieve with most pillar-based devices. The availability of multiple parallel paths for the cells and the provision for a self-generating cross flow in the device design were the main reasons behind the minimal clogging in our device. We also observed that a vibrator motor attached to the inlet tubing occasionally disturbed the cell clumps. As an illustration of the improved device design, we demonstrated up to ∼ 60-fold enrichment of platelets.

Achieving Potent Autologous Neutralizing Antibody Responses against Tier 2 HIV-1 Viruses by Strategic Selection of Envelope Immunogens.

Advancement in immunogen selection and vaccine design that will rapidly elicit a protective Ab response is considered critical for HIV vaccine protective efficacy. Vaccine-elicited Ab responses must therefore have the capacity to prevent infection by neutralization-resistant phenotypes of transmitted/founder (T/F) viruses that establish infection in humans. Most vaccine candidates to date have been ineffective at generating Abs that neutralize T/F or early variants. In this study, we report that coimmunizing rhesus macaques with HIV-1 gp160 DNA and gp140 trimeric protein selected from native envelope gene sequences (envs) induced neutralizing Abs against Tier 2 autologous viruses expressing cognate envelope (Env). The Env immunogens were selected from envs emerging during the earliest stages of neutralization breadth developing within the first 2 years of infection in two clade B-infected human subjects. Moreover, the IgG responses in macaques emulated the targeting to specific regions of Env known to be associated with autologous and heterologous neutralizing Abs developed within the human subjects. Furthermore, we measured increasing affinity of macaque polyclonal IgG responses over the course of the immunization regimen that correlated with Tier 1 neutralization. In addition, we report firm correlations between Tier 2 autologous neutralization and Tier 1 heterologous neutralization, as well as overall TZM-bl breadth scores. Additionally, the activation of Env-specific follicular helper CD4 T cells in lymphocytes isolated from inguinal lymph nodes of vaccinated macaques correlated with Tier 2 autologous neutralization. These results demonstrate the potential for native Env derived from subjects at the time of neutralization broadening as effective HIV vaccine elements.

Envelope variants circulating as initial neutralization breadth developed in two HIV-infected subjects stimulate multiclade neutralizing antibodies in rabbits.

UNLABELLED: Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE: Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable of generating potent neutralization are likely to be critical constituents in an effective HIV vaccine. However, vaccines tested thus far have elicited only weak antibody responses and very modest, waning protection. We hypothesized that B cells develop broad antibodies by exposure to the evolving viral envelope population and tested this concept using multiple envelopes from two subjects who developed neutralization breadth within a few years of infection. We compared different combinations of envelopes from each subject to identify the most effective immunogens and regimens. In each subject, use of HIV envelopes circulating during the early development and maturation of breadth generated more-potent antibodies that were modestly cross neutralizing. These data suggest a new approach to identifying envelope immunogens that may be more effective in generating protective antibodies in humans.

What women want from women's reproductive health research: a qualitative study.

BACKGROUND: Researchers are being urged to involve patients in the design and conduct of studies in health care with limited insight at present into their needs, abilities or interests. This is particularly true in the field of reproductive health care where many conditions such as pregnancy, menopause and fertility problems involve women who are otherwise healthy. OBJECTIVE: To ascertain the feasibility of involving patients and members of the public in research on women's reproductive health care (WRH). SETTING: University and tertiary care hospital in north-east Scotland; 37 women aged 18-57. METHOD: Four focus groups and one individual interview were audio-recorded and verbatim transcripts analysed thematically by two researchers using a grounded theory approach. RESULTS AND DISCUSSION: Most participants were interested in WRH, but some participated to promote a health issue of special concern to them. Priorities for research reflected women's personal concerns: endometriosis, polycystic ovary syndrome, menopause, fertility risks of delaying parenthood and early post-natal discharge from hospital. Women were initially enthusiastic about getting involved in research on WRH at the design or delivery stage, but after discussion in focus groups, some questioned their ability to do so or the time available to commit to research. None of the respondents expected payment for any involvement, believing that the experience would be rewarding enough in itself. CONCLUSIONS: Involving patients and public in research would include different perspectives and priorities; however, recruiting for this purpose would be challenging.

Cost of fertility treatment and live birth outcome in women of different ages and BMI.

STUDY QUESTION: What is the impact of different age and BMI groups on total investigation and treatment costs in women attending a secondary/tertiary care fertility clinic? SUMMARY ANSWER: Women in their early to mid-30s and women with normal BMI had higher cumulative investigation and treatment costs, but also higher probability of live birth. WHAT IS KNOWN ALREADY: Female age and BMI have been used as criteria for rationing publically funded fertility treatments. Population-based data on the costs of investigating and treating infertility are lacking. STUDY DESIGN, SIZE AND DURATION: A retrospective cohort study of 2463 women was conducted in a single secondary/tertiary care fertility clinic in Aberdeen, Scotland from 1998 to 2008. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants included all women living in a defined geographical area referred from primary care to a specialized fertility clinic over an 11-year period. Women were followed up for 5 years or until live birth if this occurred sooner. Mean discounted cumulative National Health Service costs (expressed in 2010/2011 GBP) of fertility investigations, treatments (including all types of assisted reproduction), and pregnancy (including delivery episode) and neonatal admissions were calculated and summarized by age (≤ 30, 31-35, 36-40, >40 years) and BMI groupings (<18.50, 18.50-24.99 (normal BMI), 25.00-29.99, 30.00-34.99, ≥ 35.00 kg/m(2)). Further multivariate modelling was carried out to estimate the impact of age and BMI on investigation and treatment costs and live birth outcome, adjusting for covariates predictive of the treatment pathway and live birth. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 2463 women referred, 1258 (51.1%) had a live birth within 5 years, with 694 (55.1%) of these being natural conceptions. The live birth rate was highest among women in the youngest age group (64.3%), and lowest in those aged >40 years (13.4%). Overall live birth rates were generally lower in women with BMI >30 kg/m(2). The total costs of investigations were generally highest among women younger than 30 years (£491 in those with normal BMI), whilst treatment costs tended to be higher in 31-35 year olds (£1,840 in those with normal BMI). Multivariate modelling predicted a cost increase associated with treatment which was highest among women in the lowest BMI group (across all ages), and also highest among women aged 31-35 years. The increase in the predicted probability of live birth with exposure to treatment was consistent across age and BMI categories (∼ 10%), except in the oldest age group where a slightly smaller increase in the probability of live birth was observed. The ratio of increased costs to the increased probability of live birth in women who were treated increased markedly in women over the age of 40 years, but tended to fall as BMI increased within all age groups. LIMITATIONS AND REASON FOR CAUTION: Our results, based on retrospective observational data from a single centre, have limited generalizability and are not free from clinician and clinic selection bias which can influence the choice of treatments as well as their costs. WIDER IMPLICATIONS OF THE FINDINGS: Spontaneous live birth rates were particularly high in younger women with unexplained infertility, suggesting that expectant management is a reasonable option in this group. The policy of not over-investigating older women and offering early treatment where appropriate still incurred the highest costs per additional live birth associated with treatment, owing to the lower probability of treatment success. The increased additional cost for each live birth associated with treatment for women with decreasing BMI across all age groups, suggests that it may be possible to identify a more targeted approach to treatment. STUDY FUNDING/COMPETING INTERESTS: This study was partly funded by an NHS endowment grant (Grant Number 12/48) and D.J.M. by a Chief Scientist Office Postdoctoral Fellowship (Ref PDF/12/06). There are no conflicts of interest to declare.

Continuous Perfusion Experiments on 3D Cell Proliferation in Acoustic Levitation.

An acoustofluidic trap is used for accurate 3D cell proliferation and cell function analysis in levitation. The prototype trap can be integrated with any microscope setup, allowing continuous perfusion experiments with temperature and flow control under optical inspection. To describe the trap function, we present a mathematical and FEM-based COMSOL model for the acoustic mode that defines the nodal position of trapped objects in the spherical cavity aligned with the microscope field of view and depth of field. Continuous perfusion experiments were conducted in sterile conditions over 55 h with a K562 cell line, allowing for deterministic monitoring. The acoustofluidic platform allows for rational in vitro cell testing imitating in vivo conditions such as cell function tests or cell-cell interactions.

Immune perturbation following SHIV infection is greater in newborn macaques than in infants.

Transmission of HIV-1 to newborns and infants remains high, with 130,000 new infections in 2022 in resource poor settings. Half of HIV-infected newborns, if untreated, progress to disease and death within 2 years. While immunologic immaturity likely promotes pathogenesis and poor viral control, little is known about immune damage in newborns and infants. Here we examined pathologic, virologic, and immunologic outcomes in rhesus macaques exposed to pathogenic SHIV at 1-2 weeks, defined as newborns, or at 4 months of age, considered infants. Kinetics of plasma viremia and lymph node seeding DNA were indistinguishable in newborns and infants, but levels of viral DNA in gut and lymphoid tissues 6-10 weeks post-infection were significantly higher in newborns versus either infant or adult macques. Two of six newborns with the highest viral seeding required euthanasia at 25 days. We observed age-dependent alterations in leukocyte subsets and gene expression. Compared with infants, newborns had stronger skewing of monocytes and CD8+ T cells toward differentiated subsets and little evidence of type I interferon responses by transcriptomic analyses. Thus, SHIV infection reveals distinct immunological alterations in newborn and infant macaques. These studies lay the groundwork for understanding how immune maturation affects pathogenesis in pediatric HIV-1 infection.

HIV-1 interaction with an O-glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralization by antibodies.

Bacteria dysbiosis has been associated with an increased risk of HIV-1 transmission and acquisition. The prevalent idea is that bacteria dysbiosis compromises mucosal integrity and promotes inflammatory conditions to cause recruitment and activation of immune cells that harbor or are targeted by HIV-1. However, it is also possible that HIV-1 directly binds bacteria or bacterial products to impact virus infectivity and transmissibility. This study evaluated HIV-1 interactions with bacteria through glycan-binding lectins. The Streptococcal Siglec-like lectin SLBR-N, which is part of the fimbriae shrouding the bacteria surface and recognizes α2,3 sialyated O-linked glycans, was noted for its ability to enhance HIV-1 infectivity in the context of cell-free infection and cell-to-cell transfer. Enhancing effects were recapitulated with O-glycan-binding plant lectins, signifying the importance of O-glycans. Conversely, N-glycan-binding bacterial lectins FimH and Msl had no effect. SLBR-N was demonstrated to capture and transfer infectious HIV-1 virions, bind to O-glycans on HIV-1 Env, and increase HIV-1 resistance to broadly neutralizing antibodies targeting different regions of Env. Hence, this study highlights the potential contribution of O-glycans in promoting HIV-1 infection through the exploitation of O-glycan-binding lectins from commensal bacteria at the mucosa.

HIV-1 interaction with an O-glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies.

Bacteria dysbiosis and its accompanying inflammation or compromised mucosal integrity is associated with an increased risk of HIV-1 transmission. However, HIV-1 may also bind bacteria or bacterial products to impact infectivity and transmissibility. This study evaluated HIV-1 interactions with bacteria through glycan-binding lectins. The Streptococcal Siglec-like lectin SLBR-N, a part of the fimbriae shrouding the bacteria surface that recognizes α2,3 sialyated O-linked glycans, was noted for its ability to enhance HIV-1 infectivity in the context of cell-free infection and cell-to-cell transfer. Enhancing effects were recapitulated with O-glycan-binding plant lectins, signifying the importance of O-glycans. N-glycan-binding bacterial lectins FimH and Msl had no effect. SLBR-N was demonstrated to capture and transfer infectious HIV-1 virions, bind to O-glycans on HIV-1 Env, and increase HIV-1 resistance to neutralizing antibodies targeting different regions of Env. This study highlights the potential contribution of O-glycan-binding lectins from commensal bacteria at the mucosa in promoting HIV-1 infection.

Engineered deletions of HIV replicate conditionally to reduce disease in nonhuman primates.

Antiviral therapies with reduced frequencies of administration and high barriers to resistance remain a major goal. For HIV, theories have proposed that viral-deletion variants, which conditionally replicate with a basic reproductive ratio [R0] > 1 (termed "therapeutic interfering particles" or "TIPs"), could parasitize wild-type virus to constitute single-administration, escape-resistant antiviral therapies. We report the engineering of a TIP that, in rhesus macaques, reduces viremia of a highly pathogenic model of HIV by >3log10 following a single intravenous injection. Animal lifespan was significantly extended, TIPs conditionally replicated and were continually detected for >6 months, and sequencing data showed no evidence of viral escape. A single TIP injection also suppressed virus replication in humanized mice and cells from persons living with HIV. These data provide proof of concept for a potential new class of single-administration antiviral therapies.

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.

Vaccination with immune complexes modulates the elicitation of functional antibodies against HIV-1.

Introduction: Neutralizing antibodies (Abs) are one of the immune components required to protect against viral infections. However, developing vaccines capable of eliciting neutralizing Abs effective against a broad array of HIV-1 isolates has been an arduous challenge. Objective: This study sought to test vaccines aimed to induce Abs against neutralizing epitopes at the V1V2 apex of HIV-1 envelope (Env). Methods: Four groups of rabbits received a DNA vaccine expressing the V1V2 domain of the CRF01_AE A244 strain on a trimeric 2J9C scaffold (V1V2-2J9C) along with a protein vaccine consisting of an uncleaved prefusion-optimized A244 Env trimer with V3 truncation (UFO-BG.ΔV3) or a V1V2-2J9C protein and their respective immune complexes (ICs). These IC vaccines were made using 2158, a V1V2-specific monoclonal Ab (mAb), which binds the V2i epitope in the underbelly region of V1V2 while allosterically promoting the binding of broadly neutralizing mAb PG9 to its V2 apex epitope in vitro. Results: Rabbit groups immunized with the DNA vaccine and uncomplexed or complexed UFO-BG.ΔV3 proteins (DNA/UFO-UC or IC) displayed similar profiles of Env- and V1V2-binding Abs but differed from the rabbits receiving the DNA vaccine and uncomplexed or complexed V1V2-2J9C proteins (DNA/V1V2-UC or IC), which generated more cross-reactive V1V2 Abs without detectable binding to gp120 or gp140 Env. Notably, the DNA/UFO-UC vaccine elicited neutralizing Abs against some heterologous tier 1 and tier 2 viruses from different clades, albeit at low titers and only in a fraction of animals, whereas the DNA/V1V2-UC or IC vaccines did not. In comparison with the DNA/UFO-UC group, the DNA/UFO-IC group showed a trend of higher neutralization against TH023.6 and a greater potency of V1V2-specific Ab-dependent cellular phagocytosis (ADCP) but failed to neutralize heterologous viruses. Conclusion: These data demonstrate the capacity of V1V2-2J9C-encoding DNA vaccine in combination with UFO-BG.ΔV3, but not V1V2-2J9C, protein vaccines, to elicit homologous and heterologous neutralizing activities in rabbits. The elicitation of neutralizing and ADCP activities was modulated by delivery of UFO-BG.ΔV3 complexed with V2i mAb 2158.

Corrigendum: Vaccination with immune complexes modulates the elicitation of functional antibodies against HIV-1.

[This corrects the article DOI: 10.3389/fimmu.2023.1271686.].