A tool for evaluating heterogeneity in avidity of polyclonal antibodies.

Diversity in specificity of polyclonal antibody (pAb) responses is extensively investigated in vaccine efficacy or immunological evaluations, but the heterogeneity in antibody avidity is rarely probed as convenient tools are lacking. Here we have developed a polyclonal antibodies avidity resolution tool (PAART) for use with label-free techniques, such as surface plasmon resonance and biolayer interferometry, that can monitor pAb-antigen interactions in real time to measure dissociation rate constant (kd ) for defining avidity. PAART utilizes a sum of exponentials model to fit the dissociation time-courses of pAb-antigens interactions and resolve multiple kd contributing to the overall dissociation. Each kd value of pAb dissociation resolved by PAART corresponds to a group of antibodies with similar avidity. PAART is designed to identify the minimum number of exponentials required to explain the dissociation course and guards against overfitting of data by parsimony selection of best model using Akaike information criterion. Validation of PAART was performed using binary mixtures of monoclonal antibodies of same specificity but differing in kd of the interaction with their epitope. We applied PAART to examine the heterogeneity in avidities of pAb from malaria and typhoid vaccinees, and individuals living with HIV-1 that naturally control the viral load. In many cases, two to three kd were dissected indicating the heterogeneity of pAb avidities. We showcase examples of affinity maturation of vaccine induced pAb responses at component level and enhanced resolution of heterogeneity in avidity when antigen-binding fragments (Fab) are used instead of polyclonal IgG antibodies. The utility of PAART can be manifold in examining circulating pAb characteristics and could inform vaccine strategies aimed to guide the host humoral immune response.

Polyclonal Broadly Neutralizing Antibody Activity Characterized by CD4 Binding Site and V3-Glycan Antibodies in a Subset of HIV-1 Virus Controllers.

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection.

A MUC16 IgG Binding Activity Selects for a Restricted Subset of IgG Enriched for Certain Simian Immunodeficiency Virus Epitope Specificities.

We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.

HLA class II-Restricted CD8+ T cells in HIV-1 Virus Controllers.

A paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. Another recent study highlighted the presence of HIV-1-specific class II-restricted CD8+ T cells in HIV-1 patients who naturally control infection (virus controllers; VCs). However, questions regarding class II-restricted CD8+ T cells ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In this study, we investigated the distribution and anti-viral properties of HLA-DRB1*0701 and DQB1*0501 class II-restricted CD8+ T cells in different HIV-1 patient cohorts; and whether class II-restricted CD8+ T cells represent a unique T cell subset. We show that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronically infected patients, but not in healthy seronegative donors. We also demonstrate that VC CD8+ T cells inhibit virus replication in both a class I- and class II-dependent manner, and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection.

Antibody-Dependent Cellular Cytotoxicity (ADCC)-Mediating Antibodies Constrain Neutralizing Antibody Escape Pathway.

Both neutralization and antibody-dependent cellular cytotoxicity (ADCC) may be required for effective protection against HIV-1 infection. While there is extensive information on the targets of early neutralizing antibody (nAb) responses, much less is known about the targets of ADCC responses, which are more difficult to characterize. In four individuals recruited during acute HIV-infection, ADCC responses were detected 3-7 weeks prior to nAb responses. To determine the relative influence of ADCC and nAb responses on virus evolution, we performed an in-depth investigation of one individual (CAP63) who showed the highest nAb and ADCC responses. Both nAbs and ADCC antibodies targeted the V4 region of the Env, although there were some differences in epitope recognition. We identified accelerated viral evolution in this region concurrent with emergence of nAb activity, but not ADCC activity. Deep sequencing demonstrated that most nAb escape mutations were strongly selected for, however one nAb escape mutation that rendered the virus highly susceptible to autologous ADCC responses, was suppressed despite not affecting viral fitness. This escape mutation also rendered the virus more sensitive to autologous responses, as well as monoclonal antibodies targeting CD4-induced epitopes, compared to the wildtype virus. In conclusion, ADCC responses and nAbs in donor CAP63 recognized overlapping but unique epitopes in the V4 region, and while ADCC activity was present prior to nAbs, it did not drive viral evolution during this time. However, ADCC responses may select against nAb escape pathways that expose other common ADCC epitopes thereby restricting viral replication and expansion.

Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk.

Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5' UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Beyond Oncolytics: E1B55K-Deleted Adenovirus as a Vaccine Delivery Vector.

Type 5 human adenoviruses (Ad5) deleted of genes encoding the early region 1B 55-kDa (E1B55K) protein including Onyx-015 (dl1520) and H101 are best known for their oncolytic potential. As a vaccine vector the E1B55K deletion may allow for the insertion of a transgene nearly 1,000 base pairs larger than now possible. This has the potential of extending the application for which the vectors are clinically known. However, the immune priming ability of E1B55K-deleted vectors is unknown, undermining our ability to gauge their usefulness in vaccine applications. For this reason, we created an E1B55K-deleted Ad5 vector expressing full-length single chain HIVBaLgp120 attached to a flexible linker and the first two domains of rhesus CD4 (rhFLSC) in exchange for the E3 region. In cell-based experiments the E1B55K-deleted vector promoted higher levels of innate immune signals including chemokines, cytokines, and the NKG2D ligands MIC A/B compared to an E1B55K wild-type vector expressing the same immunogen. Based on these results we evaluated the immune priming ability of the E1B55K-deleted vector in mice. The E1B55K-deleted vector promoted similar levels of Ad5-, HIVgp120, and rhFLSC-specific cellular and humoral immune responses as the E1B55K wild-type vector. In pre-clinical HIV-vaccine studies the wild-type vector has been employed as part of a very effective prime-boost strategy. This study demonstrates that E1B55K-deleted adenoviruses may serve as effective vaccine delivery vectors.