HIV Antibody Fc N-Linked Glycosylation Is Associated with Viral Rebound.

Changes in antibody glycosylation are linked to inflammation across several diseases. Alterations in bulk antibody galactosylation can predict rheumatic flares, act as a sensor for immune activation, predict gastric cancer relapse, track with biological age, shift with vaccination, change with HIV reservoir size on therapy, and decrease in HIV and HCV infections. However, whether changes in antibody Fc biology also track with reservoir rebound time remains unclear. The identification of a biomarker that could forecast viral rebound time could significantly accelerate the downselection and iterative improvement of promising HIV viral eradication strategies. Using a comprehensive antibody Fc-profiling approach, the level of HIV-specific antibody Fc N-galactosylation is significantly associated with time to rebound after treatment discontinuation across three independent cohorts. Thus virus-specific antibody glycosylation may represent a promising, simply measured marker to track reservoir reactivation.

Mining for humoral correlates of HIV control and latent reservoir size.

While antiretroviral therapy (ART) has effectively revolutionized HIV care, the virus is never fully eliminated. Instead, immune dysfunction, driven by persistent non-specific immune activation, ensues and progressively leads to premature immunologic aging. Current biomarkers monitoring immunologic changes encompass generic inflammatory biomarkers, that may also change with other infections or disease states, precluding the antigen-specific monitoring of HIV-infection associated changes in disease. Given our growing appreciation of the significant changes in qualitative and quantitative properties of disease-specific antibodies in HIV infection, we used a systems approach to explore humoral profiles associated with HIV control. We found that HIV-specific antibody profiles diverge by spontaneous control of HIV, treatment status, viral load and reservoir size. Specifically, HIV-specific antibody profiles representative of changes in viral load were largely quantitative, reflected by differential HIV-specific antibody levels and Fc-receptor binding. Conversely, HIV-specific antibody features that tracked with reservoir size exhibited a combination of quantitative and qualitative changes marked by more distinct subclass selection profiles and unique HIV-specific Fc-glycans. Our analyses suggest that HIV-specific antibody Fc-profiles provide antigen-specific resolution on both cell free and cell-associated viral loads, pointing to potentially novel biomarkers to monitor reservoir activity.

Route of immunization defines multiple mechanisms of vaccine-mediated protection against SIV.

Antibodies are the primary correlate of protection for most licensed vaccines; however, their mechanisms of protection may vary, ranging from physical blockade to clearance via the recruitment of innate immunity. Here, we uncover striking functional diversity in vaccine-induced antibodies that is driven by immunization site and is associated with reduced risk of SIV infection in nonhuman primates. While equivalent levels of protection were observed following intramuscular (IM) and aerosol (AE) immunization with an otherwise identical DNA prime-Ad5 boost regimen, reduced risk of infection was associated with IgG-driven antibody-dependent monocyte-mediated phagocytosis in the IM vaccinees, but with vaccine-elicited IgA-driven neutrophil-mediated phagocytosis in AE-immunized animals. Thus, although route-independent correlates indicate a critical role for phagocytic Fc-effector activity in protection from SIV, the site of immunization may drive this Fc activity via distinct innate effector cells and antibody isotypes. Moreover, the same correlates predicted protection from SHIV infection in a second nonhuman primate vaccine trial using a disparate IM canarypox prime-protein boost strategy, analogous to that used in the first moderately protective human HIV vaccine trial. These data identify orthogonal functional humoral mechanisms, initiated by distinct vaccination routes and immunization strategies, pointing to multiple, potentially complementary correlates of immunity that may support the rational design of a protective vaccine against HIV.

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans.

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K(+) channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K(+) channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K(+) channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.

External pH modulates EAG superfamily K+ channels through EAG-specific acidic residues in the voltage sensor.

The Ether-a-go-go (EAG) superfamily of voltage-gated K(+) channels consists of three functionally distinct gene families (Eag, Elk, and Erg) encoding a diverse set of low-threshold K(+) currents that regulate excitability in neurons and muscle. Previous studies indicate that external acidification inhibits activation of three EAG superfamily K(+) channels, Kv10.1 (Eag1), Kv11.1 (Erg1), and Kv12.1 (Elk1). We show here that Kv10.2, Kv12.2, and Kv12.3 are similarly inhibited by external protons, suggesting that high sensitivity to physiological pH changes is a general property of EAG superfamily channels. External acidification depolarizes the conductance-voltage (GV) curves of these channels, reducing low threshold activation. We explored the mechanism of this high pH sensitivity in Kv12.1, Kv10.2, and Kv11.1. We first examined the role of acidic voltage sensor residues that mediate divalent cation block of voltage activation in EAG superfamily channels because protons reduce the sensitivity of Kv12.1 to Zn(2+). Low pH similarly reduces Mg(2+) sensitivity of Kv10.1, and we found that the pH sensitivity of Kv11.1 was greatly attenuated at 1 mM Ca(2+). Individual neutralizations of a pair of EAG-specific acidic residues that have previously been implicated in divalent block of diverse EAG superfamily channels greatly reduced the pH response in Kv12.1, Kv10.2, and Kv11.1. Our results therefore suggest a common mechanism for pH-sensitive voltage activation in EAG superfamily channels. The EAG-specific acidic residues may form the proton-binding site or alternatively are required to hold the voltage sensor in a pH-sensitive conformation. The high pH sensitivity of EAG superfamily channels suggests that they could contribute to pH-sensitive K(+) currents observed in vivo.