Margaret E. Ackerman

Polyfunctional Fc-Effector Profiles Mediated by IgG Subclass Selection Distinguish RV144 and VAX003 Vaccines

RV144 vaccination induced polyfunctional antibody Fc-effector responses, whereas VAX003 vaccination increased inhibitory IgG4 antibodies. More Is Better for Protection Against HIV Recently, results from the first protective HIV phase 2B RV144 vaccine trial pointed to an unexpected signature of protection, not associated with the traditional mechanisms of vaccine-induced immunity, namely, neutralizing antibodies and killer T cell immunity. Instead, protection was associated with specific subpopulations of antibodies that were able to direct killing of HIV-infected cells. However, little is known about the properties of these killer antibodies or their biophysical features. In a new study, Chung et al. functionally profiled antibodies raised by the protective RV144 vaccine trial and its nonprotective predecessor, the VAX003 vaccine trial, both conducted in Thailand. RV144 vaccination uniquely induced antibodies capable of directing several different antiviral functions in a coordinated manner. In contrast, VAX003 vaccination predominantly induced single or uncoordinated antiviral responses. Functional coordination was regulated by the selection of antibody responses directed at vulnerable regions on the HIV envelope that were specifically tuned to enhanced functionality through the selection of a specific antibody subclass, IgG3, known to harbor strong antiviral activity. Collectively, these data suggest that vaccines able to induce broader antibody functional profiles, through the selection of more potent antibody subclasses, which target vulnerable regions of the virus, may represent a new means by which to achieve protection from HIV infection in the absence of neutralization. The human phase 2B RV144 ALVAC-HIV vCP1521/AIDSVAX B/E vaccine trial, held in Thailand, resulted in an estimated 31.2% efficacy against HIV infection. By contrast, vaccination with VAX003 (consisting of only AIDSVAX B/E) was not protective. Because protection within RV144 was observed in the absence of neutralizing antibody activity or cytotoxic T cell responses, we speculated that the specificity or qualitative differences in Fc-effector profiles of nonneutralizing antibodies may have accounted for the efficacy differences observed between the two trials. We show that the RV144 regimen elicited nonneutralizing antibodies with highly coordinated Fc-mediated effector responses through the selective induction of highly functional immunoglobulin G3 (IgG3). By contrast, VAX003 elicited monofunctional antibody responses influenced by IgG4 selection, which was promoted by repeated AIDSVAX B/E protein boosts. Moreover, only RV144 induced IgG1 and IgG3 antibodies targeting the crown of the HIV envelope V2 loop, albeit with limited coverage of breakthrough viral sequences. These data suggest that subclass selection differences associated with coordinated humoral functional responses targeting strain-specific protective V2 loop epitopes may underlie differences in vaccine efficacy observed between these two vaccine trials.

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.

Protective Efficacy of Adenovirus/Protein Vaccines Against SIV Challenges in Rhesus Monkeys

To defeat SIV, add a protein boost Despite 30 years of effort, no HIV-1 vaccine exists. Barouch et al. evaluated one promising strategy in rhesus macaques, a preclinical model commonly used to test potential HIV-1 vaccine candidates. They immunized monkeys with adenovirus-36 vectors engineered to express SIV (simian immunodeficiency virus) genes and then boosted them with a recombinant gp120 envelope glycoprotein (Env) from SIV. This regimen afforded greater protection than a strategy that instead used a viral vector–based boost. A parallel trial using a SHIV (simian/human immunodeficiency virus)–based vaccine and challenge model produced similar results. Whether this particular approach will be equally successful in humans remains to be tested. Science, this issue p. 320 A viral vector–recombinant envelope glycoprotein–based HIV-1 vaccine strategy protected 50% of monkeys from infection. Preclinical studies of viral vector–based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys.

Protein-Protein Fusion Catalyzed by Sortase A

Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality — demonstrating the robust and facile nature of this reaction.

Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control

Elite controllers (ECs) represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC) that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune–recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

A modular IgG-scFv bispecific antibody topology

Here we present a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv is fused to the C-terminus of the light chain of an IgG to create an IgG-scFv bifunctional antibody. When expressed in mammalian cells and purified by one-step protein A chromatography, the bsAb retains parental affinities of each binding domain, exhibits IgG-like stability and demonstrates in vivo IgG-like tumor targeting and blood clearance. The extension of the C-terminus of the light chain of an IgG with an scFv or even a smaller peptide does appear to disrupt disulfide bond formation between the light and heavy chains; however, this does not appear to affect binding, stability or in vivo properties of the IgG. Thus, we demonstrate here that the light chain of an IgG can be extended with an scFv without affecting IgG function and stability. This format serves as a standardized platform for the construction of functional bsAbs.

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition.

A recombinant vaccine containing Aventis Pasteurs canarypox vector (ALVAC)–HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC–simian immunodeficiency virus (SIV) and gp120 alum (ALVAC–SIV + gp120) equivalent vaccine, but not an ALVAC–SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1.

Broadly HIV-1-neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1-infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1-infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patients plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells.

High-throughput, multiplexed IgG subclassing of antigen-specific antibodies from clinical samples.

In vivo, the activity of antibodies relies critically on properties of both the variable domain, responsible for antigen recognition, and the constant domain, responsible for innate immune recognition. Here, we describe a flexible, microsphere-based array format for capturing information about both functional ends of disease-specific antibodies from complex, polyclonal clinical serum samples. Using minimal serum, we demonstrate IgG subclass profiling of multiple antibody specificities. We further capture and determine the subclass of epitope-specific antibodies. The data generated in this array provides a profile of the humoral immune response with multi-dimensional metrics regarding properties of both variable and constant IgG domains. Significantly, these properties are assessed simultaneously, and therefore information about the relationship between variable and constant domain characteristics is captured, and can be used to predict functions such as antibody effector activity.

Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcγR2a and FcγR2b

ABSTRACT While development of an HIV vaccine that can induce neutralizing antibodies remains a priority, decades of research have proven that this is a daunting task. However, accumulating evidence suggests that antibodies with the capacity to harness innate immunity may provide some protection. While significant research has focused on the cytolytic properties of antibodies in acquisition and control, less is known about the role of additional effector functions. In this study, we investigated antibody-dependent phagocytosis of HIV immune complexes, and we observed significant differences in the ability of antibodies from infected subjects to mediate this critical effector function. We observed both quantitative differences in the capacity of antibodies to drive phagocytosis and qualitative differences in their FcγR usage profile. We demonstrate that antibodies from controllers and untreated progressors exhibit increased phagocytic activity, altered Fc domain glycosylation, and skewed interactions with FcγR2a and FcγR2b in both bulk plasma and HIV-specific IgG. While increased phagocytic activity may directly influence immune activation via clearance of inflammatory immune complexes, it is also plausible that Fc receptor usage patterns may regulate the immune response by modulating downstream signals following phagocytosis—driving passive degradation of internalized virus, release of immune modulating cytokines and chemokines, or priming of a more effective adaptive immune response.