Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcgammaR) and their ability to elicit the effector functions of human FcgammaR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies

Methane observations from the Greenhouse Gases Observing SATellite: Comparison to ground‐based TCCON data and model calculations

We report new short-wave infrared (SWIR) column retrievals of atmospheric methane (X_(CH4)) from the Japanese Greenhouse Gases Observing SATellite (GOSAT) and compare observed spatial and temporal variations with correlative ground-based measurements from the Total Carbon Column Observing Network (TCCON) and with the global 3-D GEOS-Chem chemistry transport model. GOSAT X_(CH4) retrievals are compared with daily TCCON observations at six sites between April 2009 and July 2010 (Bialystok, Park Falls, Lamont, Orleans, Darwin and Wollongong). GOSAT reproduces the site-dependent seasonal cycles as observed by TCCON with correlations typically between 0.5 and 0.7 with an estimated single-sounding precision between 0.4–0.8%. We find a latitudinal-dependent difference between the X_(CH4) retrievals from GOSAT and TCCON which ranges from 17.9 ppb at the most northerly site (Bialystok) to −14.6 ppb at the site with the lowest latitude (Darwin). We estimate that the mean smoothing error difference included in the GOSAT to TCCON comparisons can account for 15.7 to 17.4 ppb for the northerly sites and for 1.1 ppb at the lowest latitude site. The GOSAT X_(CH4) retrievals agree well with the GEOS-Chem model on annual (August 2009 – July 2010) and monthly timescales, capturing over 80% of the zonal variability. Differences between model and observed X_(CH4) are found over key source regions such as Southeast Asia and central Africa which will be further investigated using a formal inverse model analysis

Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography

INTRODUCTION: Chikungunya virus (CHIKV) is a re-emerging mosquito transmitted alphavirus of global concern. Neutralizing antibodies and antibody Fc-effector functions have been shown to reduce CHIKV disease and infection in animals. However, the ability to improve the therapeutic activity of CHIKV-specific polyclonal IgG by enhancing Fc-effector functions through modulation of IgG subclass and glycoforms remains unknown. Here, we evaluated the protective efficacy of CHIKV-immune IgG enriched for binding to Fc-gamma receptor IIIa (FcγRIIIa) to select for IgG with enhanced Fc effector functions. METHODS: Total IgG was isolated from CHIKV-immune convalescent donors with and without additional purification by FcγRIIIa affinity chromatography. The enriched IgG was characterized in biophysical and biological assays and assessed for therapeutic efficacy during CHIKV infection in mice. RESULTS: FcγRIIIa-column purification enriched for afucosylated IgG glycoforms. In vitro characterization showed the enriched CHIKV-immune IgG had enhanced human FcγRIIIa and mouse FcγRIV affinity and FcγR-mediated effector function without reducing virus neutralization in cellular assays. When administered as post-exposure therapy in mice, CHIKV-immune IgG enriched in afucosylated glycoforms promoted reduction in viral load. DISCUSSION: Our study provides evidence that, in mice, increasing Fc engagement of FcγRs on effector cells, by leveraging FcγRIIIa-affinity chromatography, enhanced the antiviral activity of CHIKV-immune IgG and reveals a path to produce more effective therapeutics against these and potentially other emerging viruses

Intestinal Immunity to Poliovirus Following Sequential Trivalent Inactivated Polio Vaccine/Bivalent Oral Polio Vaccine and Trivalent Inactivated Polio Vaccine-only Immunization Schedules: Analysis of an Open-label, Randomized, Controlled Trial in Chilean Infants.

Background: Identifying polio vaccine regimens that can elicit robust intestinal mucosal immunity and interrupt viral transmission is a key priority of the polio endgame. Methods: In a 2013 Chilean clinical trial (NCT01841671) of trivalent inactivated polio vaccine (IPV) and bivalent oral polio vaccine (bOPV; targeting types 1 and 3), infants were randomized to receive IPV-bOPV-bOPV, IPV-IPV-bOPV, or IPV-IPV-IPV at 8, 16, and 24 weeks of age and challenged with monovalent oral polio vaccine type 2 (mOPV2) at 28 weeks. Using fecal samples collected from 152 participants, we investigated the extent to which IPV-bOPV and IPV-only immunization schedules induced intestinal neutralizing activity and immunoglobulin A against polio types 1 and 2. Results: Overall, 37% of infants in the IPV-bOPV groups and 26% in the IPV-only arm had detectable type 2-specific stool neutralization after the primary vaccine series. In contrast, 1 challenge dose of mOPV2 induced brisk intestinal immune responses in all vaccine groups, and significant rises in type 2-specific stool neutralization titers (P < .0001) and immunoglobulin A concentrations (P < 0.0001) were measured 2 weeks after the challenge. In subsidiary analyses, duration of breastfeeding also appeared to be associated with the magnitude of polio-specific mucosal immune parameters measured in infant fecal samples. Conclusions: Taken together, these results underscore the concept that mucosal and systemic immune responses to polio are separate in their induction, functionality, and potential impacts on transmission and, specifically, provide evidence that primary vaccine regimens lacking homologous live vaccine components are likely to induce only modest, type-specific intestinal immunity

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

Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neu- tralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the proper- ties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies

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

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

Intestinal antibody responses to a live oral poliovirus vaccine challenge among adults previously immunized with inactivated polio vaccine in Sweden.

BACKGROUND: Our understanding of the acquisition of intestinal mucosal immunity and the control of poliovirus replication and transmission in later life is still emerging. METHODS: As part of a 2011 randomised, blinded, placebo-controlled clinical trial of the experimental antiviral agent pocapavir (EudraCT 2011-004804-38), Swedish adults, aged 18-50 years, who had previously received four doses of inactivated polio vaccine (IPV) in childhood were challenged with a single dose of monovalent oral polio vaccine type 1 (mOPV1). Using faecal samples collected before and serially, over the course of 45 days, after mOPV1 challenge from a subset of placebo-arm participants who did not receive pocapavir (N=12), we investigated the kinetics of the intestinal antibody response to challenge virus by measuring poliovirus type 1-specific neutralising activity and IgA concentrations. RESULTS: In faecal samples collected prior to mOPV1 challenge, we found no evidence of pre-existing intestinal neutralising antibodies to any of the three poliovirus serotypes. Despite persistent high-titered vaccine virus shedding and rising serum neutralisation responses after mOPV1 challenge, intestinal poliovirus type 1-specific neutralisation remained low with a titer of ≤18.4 across all time points and individuals. Poliovirus types 1-specific, 2-specific and 3-specific IgA remained below the limit of detection for all specimens collected postchallenge. INTERPRETATION: In contrast to recent studies demonstrating brisk intestinal antibody responses to oral polio vaccine challenge in young children previously vaccinated with IPV, this investigation finds that adults previously vaccinated with IPV have only modest intestinal poliovirus type 1-specific neutralisation and no IgA responses that are measurable in stool samples following documented mOPV1 infection