Austin W. Boesch

Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity

While the induction of a neutralizing antibody response against HIV remains a daunting goal, data from both natural infection and vaccine-induced immune responses suggest that it may be possible to induce antibodies with enhanced Fc effector activity and improved antiviral control via vaccination. However, the specific features of naturally induced HIV-specific antibodies that allow for the potent recruitment of antiviral activity and the means by which these functions are regulated are poorly defined. Because antibody effector functions are critically dependent on antibody Fc domain glycosylation, we aimed to define the natural glycoforms associated with robust Fc-mediated antiviral activity. We demonstrate that spontaneous control of HIV and improved antiviral activity are associated with a dramatic shift in the global antibody-glycosylation profile toward agalactosylated glycoforms. HIV-specific antibodies exhibited an even greater frequency of agalactosylated, afucosylated, and asialylated glycans. These glycoforms were associated with enhanced Fc-mediated reduction of viral replication and enhanced Fc receptor binding and were consistent with transcriptional profiling of glycosyltransferases in peripheral B cells. These data suggest that B cell programs tune antibody glycosylation actively in an antigen-specific manner, potentially contributing to antiviral control during HIV infection.

Fc receptor-mediated phagocytosis in tissues as a potent mechanism for preventive and therapeutic HIV vaccine strategies

Although the development of a fully protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a weakly protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of nonneutralizing antibodies, able to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), suggestive of an important role of Fc-mediated functions in protection. Similarly, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, but the Fc effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc receptors present within vulnerable tissues. We found negligible Fc receptor-expressing natural killer cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc receptor-expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC-mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches.

Highly parallel characterization of IgG Fc binding interactions

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.

Multiplexed Fc array for evaluation of antigen-specific antibody effector profiles

Antibodies are widely considered to be a frequent primary and often mechanistic correlate of protection of approved vaccines; thus evaluating the antibody response is of critical importance in attempting to understand and predict the efficacy of novel vaccine candidates. Historically, antibody responses have been analyzed by determining the titer of the humoral response using measurements such as an ELISA, neutralization, or agglutination assays. In the simplest case, sufficiently high titers of antibody against vaccine antigen(s) are sufficient to predict protection. However, antibody titer provides only a partial measure of antibody function, which is dependent on both the variable region (Fv) to bind the antigen target, and the constant region (Fc) to elicit an effector response from the innate arm of the immune system. In the case of some diseases, such as HIV, for which an effective vaccine has proven elusive, antibody effector function has been shown to be an important driver of monoclonal antibody therapy outcomes, of viral control in infected patients, and of vaccine-mediated protection in preclinical and clinical studies. We sought to establish a platform for the evaluation of the Fc domain characteristics of antigen-specific antibodies present in polyclonal samples in order to better develop insights into Fc receptor-mediated antibody effector activity, more fully understand how antibody responses may differ in association with disease progression and between subject groups, and differentiate protective from non-protective responses. To this end we have developed a high throughput biophysical platform capable of simultaneously evaluating many dimensions of the antibody effector response.

Prospects for engineering HIV-specific antibodies for enhanced effector function and half-life

Purpose of reviewA wealth of recent animal model data suggests that as exciting possibilities for the use of antibodies in passive immunotherapy strategies continue to develop, it will be important to broadly consider how antibodies achieve anti-HIV-1 effect in vivo. Recent findingsBeyond neutralization breadth and potency, substantial evidence from natural infection, vaccination, and studies in animal models points to a critical role for antibody Fc receptor (FcR) engagement in reducing risk of infection, decreasing postinfection viremia, and delaying viral rebound. Supporting these findings in the setting of HIV, the clinical maturation of recombinant antibody therapeutics has reinforced the importance of Fc-driven activity in vivo across many disease settings, as well as opportunely resulted in the development and exploration of a number of engineered Fc sequence and glycosylation variants that possess differential binding to FcRs. Exploiting these variants as tools, the individual and concerted effects of antibody effector functions such as antibody-dependent cellular cytotoxicity, antibody-dependent cell-mediated virus inhibition, phagocytosis, complement-dependent cytotoxicity, antibody half-life, and compartmentalization are now being explored. As exciting molecular therapies are advanced, these studies promise to provide insight into optimal in-vivo antibody activity profiles. SummaryCareful consideration of recent progress in understanding protective antibody activities in vivo can point toward how tailoring antibody activity via Fc domain modification may enable optimization of HIV prevention and eradication strategies.

Intestinal Immunity Is a Determinant of Clearance of Poliovirus After Oral Vaccination

BACKGROUND Response to challenge with live, attenuated, oral polio vaccine (OPV) is a measure of immunity induced by prior immunization. METHODS Using stool samples from a study from Oman in which an initial schedule of inactivated polio vaccine (IPV) was followed by an OPV type 1 challenge, we quantitated virus shed, sequenced capsid proteins of recovered virus, and developed assays for neutralization of poliovirus and mucosal immunoglobulin A (IgA) detection. RESULTS Neutralizing activity correlated with detection of polio-specific IgA in stool suspensions collected 7 days after OPV type 1 challenge. Both neutralization and IgA in stool were associated with cessation of virus shedding by day 7. Rapid development of an IgA response with cessation of shedding suggests that IPV primed for the early response to challenge. Correlation of neutralization activity and IgA detection provides evidence that polio-specific IgA intestinal antibody is a determinant of mucosal shedding/transmission and that IgA functions through neutralization of virus. In contrast, neither presence nor quantity of serum or intestinal antibody induced by IPV prior to challenge correlated with cessation of shedding. CONCLUSIONS These assays provide an opportunity to study other immunization schedules to gain a broader understanding of the appearance and duration of a protective mucosal response to polio vaccination.

The role of Fc receptors in HIV prevention and therapy.

Over the past decade, a wealth of experimental evidence has accumulated supporting the importance of Fc receptor (FcR) ligation in antibody‐mediated pathology and protection in many disease states. Here we present the diverse evidence base that has accumulated as to the importance of antibody effector functions in the setting of HIV prevention and therapy, including clinical correlates, genetic associations, viral evasion strategies, and a rapidly growing number of compelling animal model experiments. Collectively, this work identifies antibody interactions with FcR as important to both therapeutic and prophylactic strategies involving both passive and active immunity. These findings mirror those in other fields as investigators continue to work toward identifying the right antibodies and the right effectors to be present at the right sites at the right time.

A Multiplexed Assay to Detect Antimicrobial Peptides in Biological Fluids and Cell Secretions

Mucosal tissues represent the front line in defense against potential pathogens, and one means by which mucosa provide protection is via the secretion of antimicrobials which can interfere with potential pathogens as well as recruit and modify the responses of immune cells. Here we describe adaptation of ELISA assays to microsphere format, facilitating simultaneous quantification of antimicrobial peptides including elafin, MIP3α, HBD2, HBD3, SLPI, RANTES, SDF1, lactoferrin, LL-37, and HNP1-3. The multiplexed assay exhibits excellent reproducibility, shows linearity over a two order of magnitude concentration range for most analytes, is compatible with biological fluids such as cervicovaginal lavage fluid, and presents significant cost and sample savings relative to traditional ELISA assays.

Vaccine-induced mucosal immunity to poliovirus: analysis of cohorts from an open-label, randomised controlled trial in Latin American infants

Summary Background Identification of mechanisms that limit poliovirus replication is crucial for informing decisions aimed at global polio eradication. Studies of mucosal immunity induced by oral poliovirus (OPV) or inactivated poliovirus (IPV) vaccines and mixed schedules thereof will determine the effectiveness of different vaccine strategies to block virus shedding. We used samples from a clinical trial of different vaccination schedules to measure intestinal immunity as judged by neutralisation of virus and virus-specific IgA in stools. Methods In the FIDEC trial, Latin American infants were randomly assigned to nine groups to assess the efficacy of two schedules of bivalent OPV (bOPV) and IPV and challenge with monovalent type 2 OPV, and stools samples were collected. We selected three groups of particular interest—the bOPV control group (serotypes 1 and 3 at 6, 10, and 14 weeks), the trivalent attenuated OPV (tOPV) control group (tOPV at 6, 10, and 14 weeks), and the bOPV–IPV group (bOPV at 6, 10, and 14 weeks plus IPV at 14 weeks). Neutralising activity and poliovirus type-specific IgA were measured in stool after a monovalent OPV type 2 challenge at 18 weeks of age. Mucosal immunity was measured by in-vitro neutralisation of a type 2 polio pseudovirus (PV2). Neutralisation titres and total and poliovirus-type-specific IgG and IgA concentrations in stools were assessed in samples collected before challenge and 2 weeks after challenge from all participants. Findings 210 infants from Guatemala and Dominican Republic were included in this analysis. Of 38 infants tested for mucosal antibody in the tOPV group, two were shedding virus 1 week after challenge, compared with 59 of 85 infants receiving bOPV (p<0·0001) and 53 of 87 infants receiving bOPV–IPV (p<0·0001). Mucosal type 2 neutralisation and type-specific IgA were noted primarily in response to tOPV. An inverse correlation was noted between virus shedding and both serum type 2 neutralisation at challenge (p<0·0001) and mucosal type 2 neutralisation at challenge (p<0·0001). Interpretation Mucosal type-2-specific antibodies can be measured in stool and develop in response to receipt of OPV type 2 either in the primary vaccine series or at challenge. These mucosal antibodies influence the amount of virus that is shed in an established infection. Funding Bill & Melinda Gates Foundation.

IgG Binding Characteristics of Rhesus Macaque FcγR.

Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.