Boosting of HIV Envelope Cd4 Binding Site Antibodies with Long Variable Heavy Third Complementarity Determining Region in the Randomized Double Blind Rv305 Hiv-1 Vaccine Trial

The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1- uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports

Figure 4. Apoptotic sperm cells are efficiently phagocytosed in the presence of fibrils.

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens

Multivariate analysis of FcR-mediated NK cell functions identifies unique clustering among humans and rhesus macaques

Rhesus macaques (RMs) are a common pre-clinical model used to test HIV vaccine efficacy and passive immunization strategies. Yet, it remains unclear to what extent the Fc-Fc receptor (FcR) interactions impacting antiviral activities of antibodies in RMs recapitulate those in humans. Here, we evaluated the FcR-related functionality of natural killer cells (NKs) from peripheral blood of uninfected humans and RMs to identify intra- and inter-species variation. NKs were screened for FcγRIIIa (human) and FcγRIII (RM) genotypes (FcγRIII(a)), receptor signaling, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by a cocktail of monoclonal IgG1 antibodies with human or RM Fc. FcγRIII(a) genetic polymorphisms alone did not explain differences in NK effector functionality in either species cohort. Using the same parameters, hierarchical clustering separated each species into two clusters. Importantly, in principal components analyses, ADCC magnitude, NK contribution to ADCC, FcγRIII(a) cell-surface expression, and frequency of phosphorylated CD3ζ NK cells all contributed similarly to the first principal component within each species, demonstrating the importance of measuring multiple facets of NK cell function. Although ADCC potency was similar between species, we detected significant differences in frequencies of NK cells and pCD3ζ+ cells, level of cell-surface FcγRIII(a) expression, and NK-mediated ADCC (P<0.001), indicating that a combination of Fc-FcR parameters contribute to overall inter-species functional differences. These data strongly support the importance of multi-parameter analyses of Fc-FcR NK-mediated functions when evaluating efficacy of passive and active immunizations in pre- and clinical trials and identifying correlates of protection. The results also suggest that pre-screening animals for multiple FcR-mediated NK function would ensure even distribution of animals among treatment groups in future preclinical trials

Characterization of Amyloid Fibrils in Seminal Fluid and Their Interaction With Pathogens

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2013.Several proteolytic cleavage products of prostatic acid phosphatase and semenogelin have been identified in seminal fluid that are cationic in nature and have amyloidogenic propensity. These cationic amyloid fibrils, the best described of which is the semen derived enhancer of viral infection (SEVI), have been shown to electrostatically interact with human immunodeficiency virus type 1 (HIV-1) and enhance infection in vitro by allowing virus to associate with the target cells more efficiently. Here, we used biophysical characterization of short self-assembling amyloid fibrils to determine the fibril phenotypes capable of enhancing HIV-1 infection. We also developed new tools to study fibril function. To do this, we screened a targeted library of small molecules in order to identify compounds that efficiently bound to SEVI. Our lead compound bound SEVI and reduced SEVI-mediated enhancement of HIV-1 infection. Additionally, there was enhanced fluorescence from the small molecule when bound to fibrils, suggesting that it may have utility as a novel detection reagent for the identification of SEVI and related fibrils in biological samples. The biological function of semen cationic amyloid fibrils is currently unknown. Since they share characteristics of other antimicrobial peptides, we hypothesized that they may also possess antimicrobial properties. We found that SEVI did not directly inhibit the growth of bacteria, but exerted indirect antimicrobial effects, consistent with a role in promoting bacterial clearance. Specifically, it bound both Gram-positive and Gram-negative bacteria in a charge-dependent manner, promoted their aggregation, facilitated phagocytic uptake by macrophages and neutrophils, enhanced bacterially induced pro-inflammatory cytokine product from macrophage, and protected against vaginal infection in a N. gonorrhoeae murine model. Collectively the data reported in this thesis shed light on the fundamental mechanisms by which cationic amyloid fibrils enhance HIV-1 infection, and suggest that these fibrils act principally by neutralizing charge repulsion between the virus and the host cell, and not by accelerating the sedimentation rate of bound virus particles. Additionally, we identified a novel small molecule that binds efficiently to SEVI and other fibrils, and is also capable of inhibiting SEVImediated enhancement of HIV infection. Finally, we demonstrated that SEVI has indirect antimicrobial activity, suggesting that the natural biologic function(s) of semen derived cationic amyloid fibrils may include a role in the prevention of bacterial infections in the human reproductive tract

Roles of MHC class Ia and class Ib in hsp70 mediated anti-tumor responses in the frog <i>Xenopus</i> (43.11)

Abstract The heat shock proteins (hsps) gp96 and hsp70 mediate potent antigen-dependent anti-tumor responses in both mammals and Xenopus. We have developed an adoptive cell transfer assay using peritoneal leukocytes as antigen presenting cells (APCs), and shown that hsp72, but not hsc73, is as potent as gp96 to prime T cell responses in vivo against the Xenopus thymic tumor 15/0 that is tumorigenic when transplanted in MHC-compatible Xenopus clones. The 15/0 tumor expresses several nonclassical MHC class Ib genes and β2-m, however it does not express classical MHC class Ia. Despite the lack of class Ia expression by 15/0, Xenopus adults generate potent cytotoxic CD8 T cell effectors (CCU-CTLs) that specifically recognize and kill the 15/0 tumor. We postulate that hsp72, but not hsc73, can prime class Ib-mediated anti-tumor CCU-CTL responses by interacting with APCs. To reveal the roles of class Ia and class Ib in this process we silenced surface expression of both molecules on APCs and assessed the effects in priming hsp72- or hsc73-mediated tumor immunity by adoptive transfer. To further elucidate the involvement of class Ia and class Ib in hsp70-mediated T cell function in vivo, we developed reliable transgenic techniques for our Xenopus clones and obtained F0 animals with silenced β2-m expression in vivo. This study allows us to gain better perspective into the intricate relationship between the two hsps, class Ia and class Ib molecules in T cell function as well as tumor immunity.</jats:p

Enhancement of HIV-1 Infectivity by Simple, Self-Assembling Modular Peptides

AbstractSemen-derived enhancer of viral infection (SEVI), an amyloid fibril formed from a cationic peptide fragment of prostatic acidic phosphatase (PAP), dramatically enhances the infectivity of human immunodeficiency virus type 1 (HIV-1). Insoluble, sedimentable fibrils contribute to SEVI-mediated enhancement of virus infection. However, the SEVI-forming PAP(248–286) peptide is able to produce infection-enhancing structures much more quickly than it forms amyloid fibrils. This suggests that soluble supramolecular assemblies may enhance HIV-1 infection. To address this question, non-SEVI amyloid-like fibrils were derived from general amphipathic peptides of sequence Ac-Kn(XKXE)2-NH2. These cationic peptides efficiently self-assembled to form soluble, fibril-like structures that were, in some cases, able to enhance HIV-1 infection even more efficiently than SEVI. Experiments were also performed to determine whether agents that efficiently shield the charged surface of SEVI fibrils block SEVI-mediated infection-enhancement. To do this, we generated self-assembling anionic peptides of sequence Ac-En(XKXE)2-NH2. One of these peptides completely abrogated SEVI-mediated enhancement of HIV-1 infection, without altering HIV-1 infectivity in the absence of SEVI. Collectively, these data suggest that soluble SEVI assemblies may mediate infection-enhancement, and that anionic peptide supramolecular assemblies have the potential to act as anti-SEVI microbicides