Improving peptide-MHC class I binding prediction for unbalanced datasets

<p>Abstract</p> <p>Background</p> <p>Establishment of peptide binding to Major Histocompatibility Complex class I (MHCI) is a crucial step in the development of subunit vaccines and prediction of such binding could greatly reduce costs and accelerate the experimental process of identifying immunogenic peptides. Many methods have been applied to the prediction of peptide-MHCI binding, with some achieving outstanding performance. Because of the experimental methods used to measure binding or affinity between peptides and MHCI molecules, however, available datasets are enriched for nonbinders, and thus highly unbalanced. Although there is no consensus on the ideal class distribution for training sets, extremely unbalanced datasets can be detrimental to the performance of prediction algorithms.</p> <p>Results</p> <p>We have developed a decision-theoretic framework to construct cost-sensitive trees to predict peptide-MHCI binding and have used them to 1) Assess the impact of the training data's class distribution on classifier accuracy, and 2) Compare resampling and cost-sensitive methods as approaches to compensate for training data imbalance. Our results confirm that highly unbalanced training sets can reduce the accuracy of classifier predictions and show that, in the peptide-MHCI binding context, resampling methods do not improve the classifier performance. In contrast, cost-sensitive methods significantly improve accuracy of decision trees. Finally, we propose the use of a training scheme that, when the training set is enriched for nonbinders, consistently improves the overall classifier accuracy compared to cost-insensitive classifiers and, in particular, increases the sensitivity of the classifiers. This method minimizes the expected classification cost for large datasets.</p> <p>Conclusion</p> <p>Our method consistently improves the performance of decision trees in predicting peptide-MHC class I binding by using cost-balancing techniques to compensate for the imbalance in the training dataset.</p

Tat-SF1 Is Not Required for Tat Transactivation but Does Regulate the Relative Levels of Unspliced and Spliced HIV-1 RNAs

.To directly address the involvement of Tat-SF1 in HIV-1 gene expression, we depleted Tat-SF1 in HeLa cells by conventional expression of shRNAs and in T- Rex -293 cells containing tetracycline-inducible shRNAs targeting Tat-SF1. We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability. HIV-1 infectivity decreased in Tat-SF1-depleted cells, but only when multiple rounds of infection occurred. Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle. Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.Tat-SF1 is not required for regulating HIV-1 transcription, but is required for maintaining the ratios of different classes of HIV-1 transcripts. These new findings highlight a novel, post-transcriptional role for Tat-SF1 in the HIV-1 life cycle

Envelope-specific antibodies and antibody-derived molecules for treating and curing HIV infection

HIV-1 is a retrovirus that integrates into host chromatin and can remain transcriptionally quiescent in a pool of immune cells. This characteristic enables HIV-1 to evade both host immune responses and antiretroviral drugs, leading to persistent infection. Upon reactivation of proviral gene expression, HIV-1 envelope (HIV-1 Env) glycoproteins are expressed on the cell surface, transforming latently infected cells into targets for HIV-1 Env-specific monoclonal antibodies (mAbs), which can engage immune effector cells to kill productively infected CD4+ T cells and thus limit the spread of progeny virus. Recent innovations in antibody engineering have resulted in novel immunotherapeutics such as bispecific dual-affinity re-targeting (DART) molecules and other bi- and trispecific antibody designs that can recognize HIV-1 Env and recruit cytotoxic effector cells to kill CD4+ T cells latently infected with HIV‑1. Here, we review these immunotherapies, which are designed with the goal of curing HIV-1 infection

Initiation of Human Immunodeficiency Virus Type 1 (HIV-1) Transcription is Inhibited by Noncytolytic CD8+ Suppression

The replication of human immunodeficiency virus type 1 (HIV-1) can be inhibited by noncytolytic CD8+ T cell mediated suppression, an immune response that specifically targets HIV-1 gene expression. Clinical studies demonstrate that this immune response may play an important role in the host defense against HIV infection. In this study, we examined the distinct steps in viral gene expression for inhibition by noncytolytic CD8+ T cells. A primary HIV-1 infection system of CD4+ enriched peripheral blood mononuclear cells was utilized to examine the HIV-1 life cycle as a relevant ex vivo system. Established CD8+ T cell lines from two HIV+ long-term nonprogressors were used to examine differences at the level of transcriptional initiation and elongation of the HIV genome. This infection system coupled with the results from real-time measurement of newly transcribed RNA transcripts determined that there was a significant decrease (5-8 fold) in short intracellular viral RNA transcripts. These data strongly favor a role for the initiation of virus transcription in noncytolytic CD8+ T cell mediated suppression

Superior efficacy of a human immunodeficiency virus vaccine combined with antiretroviral prevention in simian-human immunodeficiency virus-challenged nonhuman primates

International audienc

TitrationAnalysis: a tool for high throughput binding kinetics data analysis for multiple label-free platforms [version 1; peer review: 3 approved]

Label-free techniques including Surface Plasmon Resonance (SPR) and Biolayer Interferometry (BLI) are biophysical tools widely used to collect binding kinetics data of bimolecular interactions. To efficiently analyze SPR and BLI binding kinetics data, we have built a new high throughput analysis tool named the TitrationAnalysis. It can be used as a package in the Mathematica scripting environment and ultilize the non-linear curve-fitting module of Mathematica for its core function. This tool can fit the binding time course data and estimate association and dissociation rate constants (ka and kd respectively) for determining apparent dissociation constant (KD) values. The high throughput fitting process is automatic, requires minimal knowledge on Mathematica scripting and can be applied to data from multiple label-free platforms. We demonstrate that the TitrationAnalysis is optimal to analyze antibody-antigen binding data acquired on Biacore T200 (SPR), Carterra LSA (SPR imaging) and ForteBio Octet Red384 (BLI) platforms. The ka, kd and KD values derived using TitrationAnalysis very closely matched the results from the commercial analysis software provided specifically for these instruments. Additionally, the TitrationAnalysis tool generates user-directed customizable results output that can be readily used in downstream Data Quality Control associated with Good Clinical Laboratory Practice operations. With the versatility in source of data input source and options of analysis result output, the TitrationAnalysis high throughput analysis tool offers investigators a powerful alternative in biomolecular interaction characterization

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&lt;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

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine

H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials

IntroductionHemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research.MethodsAs a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination.ResultsThis qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (&lt;25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria.DiscussionThese results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials