Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Improved molecular typing assay for rhinovirus species A, B, and C.

Human rhinoviruses (RVs), comprising three species (A, B, and C) of the genus Enterovirus, are responsible for the majority of upper respiratory tract infections and are associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types necessitate regular updating of the diagnostic assays for the accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5' untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which are observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes for seminested PCR assays provided higher specificity and sensitivity for virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignments for RV-C isolates, based on phylogenetic analysis of partial VP4/VP2 coding sequences. The improved assay was evaluated by typing RVs in >3,800 clinical samples. RVs were successfully detected and typed in 99% of the samples that were RV positive in multiplex diagnostic assays.

Human rhinovirus species and season of infection determine illness severity.

RATIONALE: Human rhinoviruses (HRVs) consist of approximately 160 types that cause a wide range of clinical outcomes, including asymptomatic infections, common colds, and severe lower respiratory illnesses. OBJECTIVES: To identify factors that influence the severity of HRV illnesses. METHODS: HRV species and types were determined in 1,445 nasal lavages that were prospectively collected from 209 infants participating in a birth cohort who had at least one HRV infection. Questionnaires were used during each illness to identify moderate to severe illnesses (MSI). MEASUREMENTS AND MAIN RESULTS: Altogether, 670 HRV infections were identified, and 519 of them were solitary infections (only one HRV type). These 519 viruses belonged to 93 different types of three species: 49 A, 9 B, and 35 C types. HRV-A (odds ratio, 8.2) and HRV-C (odds ratio, 7.6) were more likely to cause MSI compared with HRV-B. In addition, HRV infections were 5- to 10-fold more likely to cause MSI in the winter months (P < 0.0001) compared with summer, in contrast to peak seasonal prevalence in spring and fall. When significant differences in host susceptibility to MSI (P = 0.004) were considered, strain-specific rates of HRV MSI ranged from less than 1% to more than 20%. CONCLUSIONS: Factors related to HRV species and type, season, and host susceptibility determine the risk of more severe HRV illness in infancy. These findings suggest that anti-HRV strategies should focus on HRV-A and -C species and identify the need for additional studies to determine mechanisms for seasonal increases of HRV severity, independent of viral prevalence, in cold weather months.

TAK - 021, an inactivated Enterovirus 71 vaccine candidate, provides cross-protection against heterologous sub-genogroups in human scavenger receptor B2 transgenic mice.

BACKGROUND: Enterovirus 71 (EV71) is a major cause of outbreaks of hand, foot and mouth disease, most frequently in children, and is a public health concern in the Asia-Pacific region. Takeda is developing TAK-021, an inactivated EV71 vaccine candidate based on sub-genogroup B2 strain MS87. In a phase I clinical trial, TAK-021 was safe, well tolerated, and immunogenic in healthy adults and elicited cross-neutralizing antibodies against heterologous EV71 sub-genogroup viruses. TAK-021 confers protection from lethal challenge with a mouse-adapted homologous strain in AG129 mice. However, it has not been determined whether TAK-021 can provide cross-protection against heterologous EV71 sub-genogroups. METHODS: We examined the efficacy of TAK-021 against challenge with EV71 sub-genogroups B4, B5, C1, C2, and C4 on day 42 (short-term) and sub-genogroups B5 and C4 on day 120 (long-term) after immunization of human scavenger receptor B2 transgenic (hSCARB2-tg) mice with TAK-021 on days 0 and 28. Antibody titers were monitored over 120 days using plaque reduction neutralization test of the homologous vaccine virus. RESULTS: TAK-021 elicited neutralizing antibody (nAb) in greater than 90% of the mice and nAb persisted through day 120. Challenge of control animals led to weight loss and death, as well as virus detection in various organs and histopathological lesions in the brain. All mice that received two doses of TAK-021 developed nAb and survived a short-term challenge given on day 42, while more than 80% survived a long-term challenge given on day 120. EV71 was detected less frequently and at lower levels in organs of immunized mice compared to non-immunized control mice. CONCLUSIONS: The results show that TAK-021 can confer protection in mice against the EV71 sub-genogroups tested.

Weekly monitoring of children with asthma for infections and illness during common cold seasons.

BACKGROUND: Exacerbations of childhood asthma and rhinovirus infections both peak during the spring and fall, suggesting that viral infections are major contributors to seasonal asthma morbidity. OBJECTIVES: We sought to evaluate rhinovirus infections during peak seasons in children with asthma and to analyze relationships between viral infection and illness severity. METHODS: Fifty-eight children aged 6 to 8 years with asthma provided 5 consecutive weekly nasal lavage samples during September and April; symptoms, medication use, and peak flow were recorded. Rhinoviruses were identified by using multiplex PCR and partial sequencing of viral genomes. RESULTS: Viruses were detected in 36% to 50% of the specimens, and 72% to 99% of the viruses were rhinoviruses. There were 52 different strains (including 16 human rhinovirus C) among the 169 rhinovirus isolates; no strains were found in more than 2 collection periods, and all but 2 children had a respiratory tract infection. Virus-positive weeks were associated with greater cold and asthma symptom severity (P < .0001 and P = .0002, respectively). Furthermore, virus-positive illnesses had increased duration and severity of cold and asthma symptoms and more frequent loss of asthma control (47% vs 22%, P = .008). Although allergen-sensitized versus nonsensitized children had the same number of viral infections, the former had 47% more symptomatic viral illnesses (1.19 vs 0.81 per month, P = .03). CONCLUSIONS: Rhinovirus infections are nearly universal in children with asthma during common cold seasons, likely because of a plethora of new strains appearing each season. Illnesses associated with viruses have greater duration and severity. Finally, atopic asthmatic children experienced more frequent and severe virus-induced illnesses.

An inactivated enterovirus 71 vaccine is safe and immunogenic in healthy adults: A phase I, double blind, randomized, placebo-controlled, study of two dosages.

BACKGROUND: Hand, foot and mouth disease (HFMD), especially that caused by enterovirus 71 (EV71) infection, is a public health concern in the Asia-Pacific region. We report a phase I clinical trial of an EV71 candidate vaccine (INV21) based on a binary ethylenimine inactivated B2 sub-genotype formulated with aluminum hydroxide. METHODS: In this double-blind, placebo-controlled, randomized, dose escalation study adult volunteers received two vaccinations 28 days apart of low or high dose formulations of the candidate vaccine and were then monitored for safety and reactogenicity for four weeks after each dose, and for their immune responses up to 28 weeks. RESULTS: Of 36 adults enrolled, 35 completed the study as planned. Either no or mild adverse events were observed, mainly injection site pain and tiredness. Seroconversion was 100% after two vaccinations. High geometric mean neutralizing antibody titers (GMT) were observed 14 days post first dose, peaking 14 days post second dose (at Day 42) in both high and low dose groups; GMTs on days 14, 28, 42, and 56 were 128, 81, 323, 203 and 144, 100, 451, 351 in low- and high-dose groups, respectively. Titers for both doses declined gradually to Day 196 but remained higher than baseline and the placebo groups, which had low GMTs throughout the duration of the study. Cross-neutralizing antibody activity against heterologous sub-genotypes was demonstrated. CONCLUSION: These data show that the EV71 candidate vaccine is safe and immunogenic in adults and supports further clinical development as a potential pediatric vaccine by initiating a dose-escalation study for determining the dose-dependent safety and immunogenicity of the vaccine in young naïve children.

MtDNA point mutations are associated with deletion mutations in aged rat.

The age-dependent accumulation of point mutations in the control region of human mtDNA has been suggested to contribute to aging processes. We investigated whether mtDNA point mutations accumulate to detectable levels in this region of mtDNA from aged Fischer 344 X Brown Norway F(1) hybrid rats. The control region and a portion of the major arc region (nucleotides 4386-7707) of the mtDNA were PCR-amplified and directly sequenced from microdissected single cardiomyocytes and single skeletal muscle fibers of 36-month old rats. Point mutations were not observed in these regions of the full-length mtDNA. Point mutations were, however, associated with deletion mutations, especially in cardiac cells. Approximately 40% of the deletion mutations identified in heart contained a point mutation, whereas only 1.9% of deletion mutations in skeletal muscle contained a point mutation. Point mutations were located adjacent to the deletion breakpoints and each point mutation was unique. In aged rats, point mutations are clonally expanded only when associated with deletion events suggesting that there are important differences between rats and humans in the mechanisms that cause mtDNA abnormalities.

Molecular identification and quantification of human rhinoviruses in respiratory samples.

PCR-based molecular assays have become standard diagnostic procedures for the identification and quantification of human rhinoviruses (HRVs) and other respiratory pathogens in most, if not all, clinical microbiology laboratories. Molecular assays are significantly more sensitive than traditional culture-based and serological methods. This advantage has led to the recognition that HRV infections are common causes for not only upper airway symptoms but also more severe lower respiratory illnesses. In addition, molecular assays improve turnaround time, can be performed by technicians with ordinary skills, and can easily be automated. This chapter describes two highly sensitive and specific PCR-based methods for identifying and quantifying HRVs. The first is a two-step PCR method for the detection and typing of HRV. The second is a pan-HRV real-time quantitative (q) PCR method for measuring viral loads in respiratory samples.