A new quaternary structure epitope on dengue virus serotype 2 is the target of durable type-specific neutralizing antibodies.

UNLABELLED: Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. IMPORTANCE: Dengue virus causes fever and dengue hemorrhagic fever. Dengue serotype 2 (DENV2) is widespread and frequently responsible for severe epidemics. Natural DENV2 infections stimulate serotype-specific neutralizing antibodies, but a leading DENV vaccine did not induce a similar protective response. While groups have identified epitopes of single monoclonal antibodies (MAbs), the molecular basis of DENV2 neutralization by polyclonal human immune sera is unknown. Using a recombinant DENV displaying serotype 2 epitopes, here we map the main target of DENV2 polyclonal neutralizing antibodies induced by natural infection and a live DENV2 vaccine candidate. Proper display of the epitope required the assembly of viral envelope proteins into higher-order structures present on intact virions. Despite the complexity of the epitope, it was possible to transplant the epitope between DENV serotypes. Our findings have immediate implications for evaluating dengue vaccines in the pipeline as well as designing next-generation vaccines.

Location and length distribution of somatic hypermutation-associated DNA insertions and deletions reveals regions of antibody structural plasticity.

Following the initial diversity generated by V(D)J recombination, somatic hypermutation is the principal mechanism for producing further antibody repertoire diversity in antigen-experienced B cells. While somatic hypermutation typically results in single-nucleotide substitutions, the infrequent incorporation of genetic insertions and deletions has also been associated with the somatic hypermutation process. We used high-throughput antibody sequencing to determine the sequence of thousands of antibody genes containing somatic hypermutation-associated insertions and deletions (SHA indels), which revealed significant differences between the location of SHA indels and somatic mutations. Further, we identified a cluster of insertions and deletions in the antibody framework 3 region, which corresponds to the hypervariable region 4 (HV4) in T-cell receptors. We propose that this HV4-like region, identified by SHA indel analysis, represents a region of under-appreciated affinity maturation potential. Finally, through the analysis of both location and length distribution of SHA indels, we have determined regions of structural plasticity within the antibody protein.

High-throughput antibody sequencing reveals genetic evidence of global regulation of the naïve and memory repertoires that extends across individuals.

Vast diversity in the antibody repertoire is a key component of the adaptive immune response. This diversity is generated centrally through the assembly of variable, diversity and joining gene segments, and peripherally by somatic hypermutation and class-switch recombination. The peripheral diversification process is thought to only occur in response to antigenic stimulus, producing antigen-selected memory B cells. Surprisingly, analyses of the variable, diversity and joining gene segments have revealed that the naïve and memory subsets are composed of similar proportions of these elements. Lacking, however, is a more detailed study, analyzing the repertoires of naïve and memory subsets at the level of the complete V(D)J recombinant. This report presents a thorough examination of V(D)J recombinants in the human peripheral blood repertoire, revealing surprisingly large repertoire differences between circulating B-cell subsets and providing genetic evidence for global control of repertoire diversity in naïve and memory circulating B-cell subsets.

Regulatory T cell expression of CLA or α(4)ß(7) and skin or gut acute GVHD outcomes.

Regulatory T cells (Tregs) are a suppressive subset of CD4(+) T lymphocytes implicated in the prevention of acute GVHD (aGVHD) after allo-SCT (ASCT). To determine whether increased frequency of Tregs with a skin-homing (cutaneous lymphocyte Ag, CLA(+)) or a gut-homing (α(4)ß(7)(+)) phenotype is associated with reduced risk of skin or gut aGVHD, respectively, we quantified circulating CLA(+) or α(4)ß(7)(+) on Tregs at the time of neutrophil engraftment in 43 patients undergoing ASCT. Increased CLA(+) Tregs at engraftment was associated with the prevention of skin aGVHD (2.6 vs 1.7%; P=0.038 (no skin aGVHD vs skin aGVHD)), and increased frequencies of CLA(+) and α(4)ß(7)(+) Tregs were negatively correlated with severity of skin aGVHD (odds ratio (OR), 0.67; 95% confidence interval (CI), 0.46-0.98; P=0.041) or gut aGVHD (OR, 0.93; 95% CI, 0.88-0.99; P=0.031), respectively. This initial report suggests that Treg tissue-homing subsets help to regulate organ-specific risk and severity of aGVHD after human ASCT. These results need to be validated in a larger, multicenter cohort.

Surfing a genetic association interaction network to identify modulators of antibody response to smallpox vaccine.

The variation in antibody response to vaccination likely involves small contributions of numerous genetic variants, such as single-nucleotide polymorphisms (SNPs), which interact in gene networks and pathways. To accumulate the bits of genetic information relevant to the phenotype that are distributed throughout the interaction network, we develop a network eigenvector centrality algorithm (SNPrank) that is sensitive to the weak main effects, gene-gene interactions and small higher-order interactions through hub effects. Analogous to Google PageRank, we interpret the algorithm as the simulation of a random SNP surfer (RSS) that accumulates bits of information in the network through a dynamic probabilistic Markov chain. The transition matrix for the RSS is based on a data-driven genetic association interaction network (GAIN), the nodes of which are SNPs weighted by the main-effect strength and edges weighted by the gene-gene interaction strength. We apply SNPrank to a GAIN analysis of a candidate-gene association study on human immune response to smallpox vaccine. SNPrank implicates a SNP in the retinoid X receptor α (RXRA) gene through a network interaction effect on antibody response. This vitamin A- and D-signaling mediator has been previously implicated in human immune responses, although it would be neglected in a standard analysis because its significance is unremarkable outside the context of its network centrality. This work suggests SNPrank to be a powerful method for identifying network effects in genetic association data and reveals a potential vitamin regulation network association with antibody response.

Integrated analysis of genetic and proteomic data identifies biomarkers associated with adverse events following smallpox vaccination.

Complex clinical outcomes, such as adverse reaction to vaccination, arise from the concerted interactions among the myriad components of a biological system. Therefore, comprehensive etiological models can be developed only through the integrated study of multiple types of experimental data. In this study, we apply this paradigm to high-dimensional genetic and proteomic data collected to elucidate the mechanisms underlying the development of adverse events (AEs) in patients after smallpox vaccination. As vaccination was successful in all of the patients under study, the AE outcomes reported likely represent the result of interactions among immune system components that result in excessive or prolonged immune stimulation. In this study, we examined 1442 genetic variables (single nucleotide polymorphisms) and 108 proteomic variables (serum cytokine concentrations) to model AE risk. To accomplish this daunting analytical task, we employed the Random Forests (RF) method to filter the most important attributes, then we used the selected attributes to build a final decision tree model. This strategy is well suited to integrated analysis, as relevant attributes may be selected from categorical or continuous data. Importantly, RF is a natural approach for studying the type of gene-gene, gene-protein and protein-protein interactions we hypothesize to be involved in the development of clinical AEs. RF importance scores for particular attributes take interactions into account, and there may be interactions across data types. Combining information from previous studies on AEs related to smallpox vaccination with the genetic and proteomic attributes identified by RF, we built a comprehensive model of AE development that includes the cytokines intercellular adhesion molecule-1 (ICAM-1 or CD54), interleukin-10 (IL-10), and colony stimulating factor-3 (CSF-3 or G-CSF) and a genetic polymorphism in the cytokine gene interleukin-4 (IL4). The biological factors included in the model support our hypothesized mechanism for the development of AEs involving prolonged stimulation of inflammatory pathways and an imbalance of normal tissue damage repair pathways. This study shows the utility of RF for such analytical tasks, while both enhancing and reinforcing our working model of AE development after smallpox vaccination.

Evaporative cooling feature selection for genotypic data involving interactions.

MOTIVATION: The development of genome-wide capabilities for genotyping has led to the practical problem of identifying the minimum subset of genetic variants relevant to the classification of a phenotype. This challenge is especially difficult in the presence of attribute interactions, noise and small sample size. METHODS: Analogous to the physical mechanism of evaporation, we introduce an evaporative cooling (EC) feature selection algorithm that seeks to obtain a subset of attributes with the optimum information temperature (i.e. the least noise). EC uses an attribute quality measure analogous to thermodynamic free energy that combines Relief-F and mutual information to evaporate (i.e. remove) noise features, leaving behind a subset of attributes that contain DNA sequence variations associated with a given phenotype. RESULTS: EC is able to identify functional sequence variations that involve interactions (epistasis) between other sequence variations that influence their association with the phenotype. This ability is demonstrated on simulated genotypic data with attribute interactions and on real genotypic data from individuals who experienced adverse events following smallpox vaccination. The EC formalism allows us to combine information entropy, energy and temperature into a single information free energy attribute quality measure that balances interaction and main effects. AVAILABILITY: Open source software, written in Java, is freely available upon request.

Automated image analysis of atomic force microscopy images of rotavirus particles.

A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.

Hybrid grammar-based approach to nonlinear dynamical system identification from biological time series.

We introduce a grammar-based hybrid approach to reverse engineering nonlinear ordinary differential equation models from observed time series. This hybrid approach combines a genetic algorithm to search the space of model architectures with a Kalman filter to estimate the model parameters. Domain-specific knowledge is used in a context-free grammar to restrict the search space for the functional form of the target model. We find that the hybrid approach outperforms a pure evolutionary algorithm method, and we observe features in the evolution of the dynamical models that correspond with the emergence of favorable model components. We apply the hybrid method to both artificially generated time series and experimentally observed protein levels from subjects who received the smallpox vaccine. From the observed data, we infer a cytokine protein interaction network for an individual's response to the smallpox vaccine.

Respiratory syncytial virus vaccine development.

Development of an RSV vaccine for infants has been hindered by the lack of an ideal animal model that exhibits disease, and the challenge of effectively immunizing very young infants who are immunologically immature. Nevertheless, significant progress has been made recently in developing live attenuated viruses and protein subunit vaccine candidates. Numerous vaccine candidates are currently in early clinical trials. This paper reviews the significant obstacles to development of RSV vaccines, and the progress made to date.

Influence of maternal antibodies on neonatal immunization against respiratory viruses.

Vaccines that successfully prevent severe infant respiratory virus diseases should induce protection at a very young age because of the low age of patients who are hospitalized owing to these viruses. Candidate respiratory virus vaccines are being tested in infants who are naïve to infection but seropositive to the viral agents because they possess maternal IgG antibodies (Abs). Transplacental maternal Abs may be partially protective against disease caused by respiratory virus infections. Carefully conducted studies have shown that these Abs can also profoundly suppress or enhance infant immune responses to immunization. The mechanisms underlying regulation of immune responses to viruses by maternal Abs are under investigation. This article explores the current knowledge regarding the effect of maternal Abs on respiratory virus and measles virus immunization, and it reviews the current approaches to overcoming Ab-mediated immunosuppression.

Passively acquired antibodies suppress humoral but not cell-mediated immunity in mice immunized with live attenuated respiratory syncytial virus vaccines.

A respiratory syncytial virus (RSV) vaccine will need to be administered by 1 mo of age to protect young infants; therefore, it will need to be effective in the presence of maternally acquired RSV Abs. In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates of different level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The replication of the RSV vaccines was suppressed in the lower, but not the upper, respiratory tract of the passively immunized mice. Immunization with either vaccine candidate was highly efficacious against challenge with wild-type RSV in both passively immunized and control mice. Nonetheless, a high level of immunity was seen even in passively/actively immunized animals that failed to develop a humoral immune response, suggesting that T cells mediated the immunity. Depletion of CD4+ and CD8+ T cells in passively/actively immunized and control animals at the time of challenge with wild-type RSV demonstrated that CD4+ and CD8+ T cells made significant independent contributions to the restriction of replication of RSV challenge virus in both the upper and lower respiratory tracts. Although passively acquired serum RSV Abs suppressed the primary systemic and mucosal Ab responses of IgM, IgG, and IgA isotypes, B lymphocytes were nevertheless primed for robust secondary Ab responses. Thus, immunity mediated by CD4+ and CD8+ T cells and Abs can be readily induced in mice by live RSV vaccine candidates in the presence of physiologic levels of RSV neutralizing Abs.

Genetic and structural determinants of virus neutralizing antibodies.

Neutralizing antibodies (Abs) are the principal protective mechanism against disease caused by reinfection with viruses. Ab-mediated neutralization of viruses is a complex process comprising multiple mechanisms. Every structural aspect of Abs is potentially capable of modulating the level of neutralizing activity or the mechanisms of neutralization. The focus of our laboratory is to understand the genetic and structural basis of Ab-mediated neutralization of human viral pathogens. We demonstrated the unexpected finding that virus antigen-binding fragments of Abs (Fabs) mediate potent virus neutralizing effects in vivo. This work has led to a broad investigation of the importance of the genetics, chemistry, and structure of the combining site to the neutralizing activity of antiviral Abs. Ongoing work in our laboratory reveals that effect or functions specified by the Ab isotype such as polymer formation, interactions with complement, interactions with Fc receptors, and the ability to transcytose mucosal epithelia, also modulate the mechanism and level of neutralizing effects mediated by antiviral Abs.

CD40-ligand in primate cardiac allograft and viral immunity.

Our laboratory has studied the role of CD40 ligand (CD40L, CD154) in the primate immune response to allogenic and infectious challenges. We find that intensive early blockade of CD40L reliably attenuates acute rejection of primate cardiac allografts. Monotherapy fails to prevent late graft loss, which often occurs in association with rising antidonor antibody titers and allograft vasculopathy, despite continuing anti-CD40L therapy. In contrast, the primary humoral response to T helper dependent influenza viral antigen is inhibited during anti-CD40L therapy, and responses to subsequent immunization are blunted after discontinuation of therapy. These results are encouraging with regard to the tolerogenic potential of costimulatory blockade for specific T helper dependent antigens. However, these findings also indicate that pathogenic allograft responses in primates are probably not entirely CD40L-dependent. As such, additional immunomodulatory strategies are needed to facilitate tolerance to a transplanted organ.

Recurrent Achromobacter xylosoxidans bacteremia associated with persistent lymph node infection in a patient with hyper-immunoglobulin M syndrome.

Achromobacter xylosoxidans (formerly Alcaligenes xylosoxidans) is a rare but important cause of bacteremia in immunocompromised patients, and strains are usually multiply resistant to antimicrobial therapy. We report an immunocompromised patient with hyper-immunoglobulin M syndrome who suffered from 14 documented episodes of A. xylosoxidans bacteremia. Each episode was treated and resulted in rapid clinical improvement, with blood cultures testing negative for bacteria. Between episodes, A. xylosoxidans was isolated from an excised right axillary lymph node, whereas the culture of the central venous catheter, removed at the same time, was negative. Multiple cultures from sputum, stool, and urine samples, as well as from gastrointestinal biopsies or environmental sources, were negative. Results from antibiotic sensitivity testing and pulsed-field gel electrophoresis suggested that a single strain of A. xylosoxidans caused the recurrent bacteremias in this patient; this strain originated from persistently infected lymph nodes. Lymphoid hyperplasia is a prominent characteristic of hyper-IgM syndrome and may serve as a source of bacteremia with low-pathogenicity organisms.

Recurrent pneumococcal arthritis as the presenting manifestation of X-linked agammaglobulinemia.

Pneumococcal arthritis in children older than 24 months is unusual and can suggest underlying immunodeficiency. We report a case of recurrent pneumococcal arthritis as the presenting manifestation of X-linked agammaglobulinemia.

Evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy.

A live-attenuated, intranasal respiratory syncytial virus (RSV) candidate vaccine, cpts-248/404, was tested in phase 1 trials in 114 children, including 37 1-2-month-old infants-a target age for RSV vaccines. The cpts-248/404 vaccine was infectious at 104 and 105 plaque-forming units in RSV-naive children and was broadly immunogenic in children >6 months old. Serum and nasal antibody responses in 1-2 month olds were restricted to IgA, had a dominant response to RSV G protein, and had no increase in neutralizing activity. Nevertheless, there was restricted virus shedding on challenge with a second vaccine dose and preliminary evidence for protection from symptomatic disease on natural reexposure. The cpts-248/404 vaccine candidate did not cause fever or lower respiratory tract illness. In the youngest infants, however, cpts-248/404 was unacceptable because of upper respiratory tract congestion associated with peak virus recovery. A live attenuated RSV vaccine for the youngest infant will use cpts-248/404 modified by additional attenuating mutations.

A RhoA-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3.

The fusion glycoproteins of human respiratory syncytial virus (RSV) and human parainfluenza virus type-3 (PIV-3) mediate virus entry and syncytium formation. Interaction between the fusion protein of RSV and RhoA, a small GTPase, facilitates virus-induced syncytium formation. We show here a RhoA-derived peptide inhibits RSV and syncytium formation induced by RSV and PIV-3, both in vitro by inhibition of cell-to-cell fusion and in vivo by reduction of peak titer by 2 log10 in RSV-infected mice. These findings indicate that the interaction between these two paramyxovirus fusion proteins and RhoA is an important target for new antiviral strategies.

Passive IgA monoclonal antibody is no more effective than IgG at protecting mice from mucosal challenge with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a mucosally restricted pathogen that can cause severe respiratory disease. Although parenteral administration of sufficient RSV-specific IgG can reduce severity of lower respiratory tract infection in high-risk infants, delivery of antibody by direct airway administration is an attractive alternative. Topical and parenteral administration of an IgA monoclonal antibody (MAb) specific for the RSV F glycoprotein was compared with an IgG MAb, specific for the same antigenic site, for ability to protect mice against RSV infection. Administration of RSV-specific IgG was more effective in reducing RSV titers in lung (4.6 log10 pfu/g) than IgA MAb (3.6 log10 pfu/g) when given intranasally immediately prior to infection (P=.005). RSV titers in the nose were reduced only by prophylactic administration of IgG parenterally. Therefore, topical administration of IgA is no more effective than topically administered IgG and is less effective than systemically administered IgG for protecting against RSV infection.