Beneath the surface: Amino acid variation underlying two decades of dengue virus antigenic dynamics in Bangkok, Thailand.

Neutralizing antibodies are important correlates of protection against dengue. Yet, determinants of variation in neutralization across strains within the four dengue virus serotypes (DENV1-4) is imperfectly understood. Studies focus on structural DENV proteins, especially the envelope (E), the primary target of anti-DENV antibodies. Although changes in immune recognition (antigenicity) are often attributed to variation in epitope residues, viral processes influencing conformation and epitope accessibility also affect neutralizability, suggesting possible modulating roles of nonstructural proteins. We estimated effects of residue changes in all 10 DENV proteins on antigenic distances between 348 DENV collected from individuals living in Bangkok, Thailand (1994-2014). Antigenic distances were derived from response of each virus to a panel of twenty non-human primate antisera. Across 100 estimations, excluding 10% of virus pairs each time, 77 of 295 positions with residue variability in E consistently conferred antigenic effects; 52 were within ±3 sites of known binding sites of neutralizing human monoclonal antibodies, exceeding expectations from random assignments of effects to sites (p = 0.037). Effects were also identified for 16 sites on the stem/anchor of E which were only recently shown to become exposed under physiological conditions. For all proteins, except nonstructural protein 2A (NS2A), root-mean-squared-error (RMSE) in predicting distances between pairs held out in each estimation did not outperform sequences of equal length derived from all proteins or E, suggesting that antigenic signals present were likely through linkage with E. Adjusted for E, we identified 62/219 sites embedding the excess signals in NS2A. Concatenating these sites to E additionally explained 3.4% to 4.0% of observed variance in antigenic distances compared to E alone (50.5% to 50.8%); RMSE outperformed concatenating E with sites from any protein of the virus (ΔRMSE, 95%IQR: 0.01, 0.05). Our results support examining antigenic determinants beyond the DENV surface.

Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing.

Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P. aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP8), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P. aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

An Innovative, Prospective, Hybrid Cohort-Cluster Study Design to Characterize Dengue Virus Transmission in Multigenerational Households in Kamphaeng Phet, Thailand.

Difficulties inherent in the identification of immune correlates of protection or severe disease have challenged the development and evaluation of dengue vaccines. There persist substantial gaps in knowledge about the complex effects of age and sequential dengue virus (DENV) exposures on these correlations. To address these gaps, we were conducting a novel family-based cohort-cluster study for DENV transmission in Kamphaeng Phet, Thailand. The study began in 2015 and is funded until at least 2023. As of May 2019, 2,870 individuals in 485 families were actively enrolled. The families comprise at least 1 child born into the study as a newborn, 1 other child, a parent, and a grandparent. The median age of enrolled participants is 21 years (range 0-93 years). Active surveillance is performed to detect acute dengue illnesses, and annual blood testing identifies subclinical seroconversions. Extended follow-up of this cohort will detect sequential infections and correlate antibody kinetics and sequence of infections with disease outcomes. The central goal of this prospective study is to characterize how different DENV exposure histories within multigenerational family units, from DENV-naive infants to grandparents with multiple prior DENV exposures, affect transmission, disease, and protection at the level of the individual, household, and community.

Association between semi-quantitative microbial load and respiratory symptoms among Thai military recruits: a prospective cohort study.

BACKGROUND: Multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. However, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. We assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. METHODS: We obtained throat and nasal swab samples from military trainees at two Thai Army barracks. Specimens were collected at the start and end of 10-week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). We analysed the samples using a commercial multiplex respiratory panel comprising 33 bacterial, viral and fungal targets. We used random effects tobit models to compare cycle threshold (Ct) value distributions from non-acute and acute samples. RESULTS: We analysed 341 non-acute and 145 acute swab samples from 274 participants. Haemophilus influenzae type B was the most commonly detected microbe (77.4% of non-acute and 64.8% of acute samples). In acute samples, nine specific microbe pairs were detected more frequently than expected by chance. Regression models indicated significantly lower microbial load in non-acute relative to acute samples for H. influenzae non-type B, Streptococcus pneumoniae and rhinovirus, although it was not possible to identify a Ct-value threshold indicating causal etiology for any of these organisms. CONCLUSIONS: Semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections.

Comparative genome sequencing of Rickettsia rickettsii strains that differ in virulence.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. Strains of R. rickettsii differ dramatically in virulence. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, to Iowa, which causes no fever. To identify potential determinants of virulence in R. rickettsii, the genomes of two additional strains were sequenced for comparison to known sequences (comparative genome sequencing [CGS]). R. rickettsii Morgan and R strains were compared to the avirulent R. rickettsii Iowa and virulent R. rickettsii Sheila Smith strains. The Montana strains Sheila Smith and R were found to be highly similar while the eastern strains Iowa and Morgan were most similar to each other. A major surface antigen, rickettsial outer membrane protein A (rOmpA), is severely truncated in the Iowa strain. The region of ompA containing 13 tandem repeats was sequenced, revealing only seven shared SNPs (four nonsynonymous) for R and Morgan strains compared to Sheila Smith, with an additional 17 SNPs identified in Morgan. Another major surface antigen and autotransporter, rOmpB, exhibits a defect in processing in the Iowa strain such that the beta fragment is not cleaved. Sequence analysis of ompB reveals identical sequences between Iowa and Morgan strains and between the R and Sheila Smith strains. The number of SNPs and insertions/deletions between sequences of the two Montana strains and the two eastern strains is low, thus narrowing the field of possible virulence factors.

Genome sequence of the Klebsiella quasipneumoniae bacteriophage EKq1 with activity against Klebsiella pneumoniae.

We describe the genome of a lytic phage EKq1 isolated on Klebsiella quasipneumoniae, with activity against Klebsiella pneumoniae. EKq1 is an unclassified representative of the class Caudoviricetes, similar to Klebsiella phages VLCpiS8c, phiKp_7-2, and vB_KleS-HSE3. The 48,244-bp genome has a GC content of 56.43% and 63 predicted protein-coding genes.

Complete genome sequence of the broad host range Acinetobacter baumannii phage EAb13.

We describe the genome of a lytic phage EAb13 isolated from sewage, with broad activity against multidrug-resistant Acinetobacter baumannii. EAb13 is an unclassified siphovirus. Its genome consists of 82,411 bp, with 40.15% GC content, 126 protein-coding sequences, 1 tRNA, and 2,177 bp-long direct terminal repeats.

Genome Sequence of Staphylococcus aureus Phage ESa2.

We describe the genome of a lytic phage, ESa2, isolated from environmental water and specific for Staphylococcus aureus. ESa2 belongs to the family Herelleviridae and genus Kayvirus. Its genome consists of 141,828 bp, with 30.25% GC content, 253 predicted protein-coding sequences, 3 tRNAs, and 10,130-bp-long terminal repeats.

Identification and Characterization of vB_PreP_EPr2, a Lytic Bacteriophage of Pan-Drug Resistant Providencia rettgeri.

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections.

Antibody persistence and immune memory response following primary vaccination and boosting with live attenuated SA 14-14-2 Japanese encephalitis vaccine (CD-JEV) in Bangladesh: A phase 4 open-label clinical trial.

INTRODUCTION: Japanese encephalitis (JE) virus is one of the leading causes of viral encephalitis across temperate and tropical zones of Asia. The live attenuated SA 14-14-2 JE vaccine (CD-JEV) is one of three vaccines prequalified by the World Health Organization (WHO) to prevent JE. WHO currently recommends a single CD-JEV dose for infants in endemic settings. However, in the absence of long-term immunogenicity data, WHO has indicated a need for long-term immunogenicity studies to inform optimal dosing schedules and determine the need for booster doses. METHODS: This Phase 4, open-label clinical study measured neutralizing antibody (NAb) titers in Bangladeshi children three and four years after primary CD-JEV vaccination and 7 and 28 days after a booster CD-JEV vaccination given four years after primary vaccination. The study also assessed the tolerability and safety of the booster dose. A NAb titer of ≥1:10 was considered seroprotective. RESULTS: Of 560 children vaccinated between 10 and 12 months of age with CD-JEV three years earlier and enrolled in this study from 30 July 2015 through 03 January 2016, 52 (9.3%; 95% CI: 7.2-12.0) had a seroprotective titer at enrollment. One year later, of 533 children, 66 (12.4%; 95% CI: 9.9-15.5) had a seroprotective titer before receiving a booster dose. Of 524 children who received a booster CD-JEV dose, 479 (91.4%; 95% CI: 88.7-93.5) and 514 (98.1%; 95% CI: 96.5-99.0) were seroprotected 7 and 28 days later, respectively. The geometric mean titer (GMT) was 6 (95% CI: 6-6) at baseline, 105 (95% CI: 93-119) 7 days post-booster, and 167 (95% CI: 152-183) 28 days post-booster. No vaccine-associated neurologic adverse events or other serious adverse events were noted following the booster dose. CONCLUSIONS: Although most children did not have measurable antibody titers three and four years after a single primary CD-JEV dose, more than 90% of seronegative children had a strong anamnestic response within one week of a booster dose. This suggests that these children were immune despite the absence of measurable NAb prior to their booster.ClinicalTrials.gov Identifier: NCT02514746.

Independent Evaluation of Two Prototype Immunochromatographic Tests for Dengue Fever Developed by InBios.

INTRODUCTION: Dengue fever, caused by any of the four dengue viruses (DENV1-4), is endemic in more than 100 countries around the world. Each year, up to 400 million people get infected with dengue virus. It is one of the most important arthropod-borne viral diseases. Dengue's global presence poses a medical threat to deploying military personnel and their dependents. An accurate diagnosis followed by attentive supportive care can improve outcomes in patients with severe dengue disease. Dengue diagnostic tests based on PCR and ELISA platforms have been developed and cleared by the U.S. FDA. However, these diagnostic assays are laborious and usually require highly trained personnel and specialized equipment, which presents a significant challenge when conducting operations in austere and resource-constrained areas. InBios International, Inc. (Seattle, WA) has developed two rapid and instrument-free immunochromatographic test prototype devices (multiplex and traditional formats) for dengue diagnosis. MATERIALS AND METHODS: To determine the performance of the InBios immunochromatographic tests, 183 clinical samples were tested on both prototype devices. Both assays were performed without any instruments and the results were read in 20 minutes. RESULTS: The traditional format had better overall performance (sensitivity: 97.4%; specificity: 90%) than the multiplex format (sensitivity: 86.9%; specificity: 63.3%). The traditional format was superior in serotype-specific detection with 100% overall sensitivity for DENV1, DENV3, and DENV4 and 93.3% sensitivity for DENV2 compared to the multiplex format (91.7%, 78.3%, 83.3%, and 96.3% for DENV1, 2, 3, and 4, respectively). The traditional format was easier to read than the multiplex format. The multiplex format was simpler and faster to set up than the traditional format. CONCLUSIONS: The InBios traditional format had a better overall performance and readability profile than the multiplex format, while the multiplex format was easier to set up. Both formats were highly sensitive and specific, were easy to perform, and did not require sophisticated equipment. They are ideal for use in resource-limited settings where dengue is endemic. Based on our overall assessment, the traditional format should be considered for further development and used in the upcoming multicenter clinical trial toward FDA clearance.

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella.

Shigellosis is a leading global cause of diarrheal disease and travelers' diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Bacteriophages (phages) are a promising anti-infective option for human disease. Major gaps remain in understanding their potential utility. METHODS: This is a randomized, placebo-controlled, double-blind study of a single dose of intravenous phage in approximately 72 clinically stable adult cystic fibrosis volunteers recruited from up to 20 US sites with Pseudomonas aeruginosa airway colonization. The single dose of phage consists of a mixture of four anti-pseudomonal phages. Six sentinel participants will be sequentially enrolled with dose escalation of the phage mixture by one log10 beginning with 4 × 107 plaque-forming units in an unblinded stage 1. If no serious adverse events related to the study product are identified, the trial will proceed to a double-blinded stage 2. In stage 2a, 32 participants will be randomly assigned to one of three phage dosages or placebo in a 1:1:1:1 allocation. An interim analysis will be performed to determine the phage dosage with the most favorable safety and microbiological activity profile to inform phage dosing in stage 2b. During stage 2b, up to 32 additional volunteers will be randomized 1:1 to the phage or placebo arm. Primary outcomes include (1) the number of grade 2 or higher treatment-emergent adverse events, (2) change in log10 P. aeruginosa total colony counts in sputum, and (3) the probability of a randomly selected subject having a more favorable outcome ranking if assigned to receive phage therapy versus placebo. Exploratory outcomes include (1) sputum and serum phage pharmacokinetics, (2) the impact of phage on lung function, (3) the proportion of P. aeruginosa isolates susceptible to the phage mixture before and after study product administration, and (4) changes in quality of life. DISCUSSION: This trial will investigate the activity of phages in reducing P. aeruginosa colony counts and provide insights into the safety profile of phage therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT05453578. Registered on 12 July 2022.

Complementation of Rickettsia rickettsii RelA/SpoT restores a nonlytic plaque phenotype.

Spotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains of Rickettsia rickettsii, the virulent R strain and the avirulent Iowa strain. A nonlytic variant of R. rickettsii R, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated as relA/spoT-like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. In R. rickettsii R, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intact relA/spoT gene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncated relA/spoT gene in the Iowa lytic plaque variant restored the nonlytic phenotype. The relA/spoT mutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.

Genome Sequences of 17 Diverse Pseudomonas aeruginosa Phages.

Here, we describe genome sequences of 17 Pseudomonas aeruginosa phages, including therapeutic candidates. They belong to the families Myoviridae, Podoviridae, and Siphoviridae and six different genera. The genomes ranged in size from 42,788 to 88,805 bp, with G+C contents of 52.5% to 64.3% and numbers of coding sequences from 58 to 179.

Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens.

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several "in-house" components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility.

Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

Immunogenicity and safety of concurrent or sequential administration of live, attenuated SA 14-14-2 Japanese encephalitis vaccine (CD-JEV) and measles-mumps-rubella vaccine in infants 9-12 months of age in the Philippines: A non-inferiority Phase 4 randomized clinical trial.

INTRODUCTION: Japanese encephalitis (JE) virus is the leading cause of viral encephalitis across temperate and tropical zones of Asia. The live attenuated SA 14-14-2 JE vaccine (CD-JEV) is one of three vaccines prequalified by the World Health Organization (WHO) to prevent JE. When incorporating a new vaccine into a country's Expanded Program on Immunization (EPI), it is important to show that the new vaccine can be administered concurrently with other routine pediatric vaccines without impairing the immune responses or changing the safety profiles of the co-administered vaccines. This Phase 4 open-label study evaluated the safety and immunogenicity of measles-mumps-rubella (MMR) vaccine co-administered with CD-JEV. METHODS: The study randomized 628 healthy Filipino children aged between 9 and 10 months to receive MMR and CD-JEV concurrently or separately. MMR immunogenicity was measured 56 days after MMR vaccination using a measles plaque reduction neutralization test (PRNT), anti-mumps immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), and anti-rubella IgG ELISA, respectively. Neutralizing antibody against JE virus was measured 28 days after CD-JEV vaccination using PRNT. Safety was assessed through solicitation of immediate reactions, adverse events (AEs) within 14 days of vaccination, unsolicited AEs occurring within 28 days, and serious adverse events (SAEs) during participation in the study. RESULTS/CONCLUSIONS: During the study, no post-vaccinal encephalitis cases or related SAEs were reported in either group. Concurrent immunization with CD-JEV and MMR vaccines was not associated with any unusual safety signals when compared with sequential immunization. No significant differences between the regimens were seen in seropositivity or serology titer/concentration results for any of the antigens. Co-administration of CD-JEV and MMR was non-inferior to single administration of either vaccine.

Longitudinal Analysis of Memory B and T Cell Responses to Dengue Virus in a 5-Year Prospective Cohort Study in Thailand.

Prior exposure to dengue virus (DENV) has a profound impact on the outcome of infection, which varies according to the interval between infections. Antibodies secreted by B cells and cytokines secreted by T cells are thought to contribute both to protective immunity against DENV and the pathogenesis of dengue disease. We analyzed peripheral blood mononuclear cells (PBMC) collected from Thai children over a 5-year prospective cohort study to define the dynamics of DENV-specific memory B and T cell responses and the impact of symptomatic or subclinical DENV infections. To measure B cell responses, PBMC were stimulated with IL-2 plus R848 and culture supernatants were tested for DENV-binding antibodies by ELISA. To measure T cell responses, PBMC were stimulated in dual-color ELISPOT assays with overlapping peptide pools of structural and non-structural proteins from the four DENV types. B cell responses were low to one or more DENV types prior to symptomatic infection and increased with reactivity to all four types after infection. Subjects who had a subclinical infection or who did not experience a DENV infection during the study period showed strong memory B cell responses to all four DENV types. T cell responses to DENV peptides demonstrated a cytokine hierarchy of IFN-γ > IL-2 > IFN-γ/IL-2. T cell responses were low or absent prior to secondary infections. The trends in T cell responses to DENV peptides over 3 year post-infection were highly variable, but subjects who had experienced a secondary DENV1 infection showed higher cytokine responses compared to subjects who had experienced a secondary DENV2 or subclinical infection. The longitudinal nature of our study demonstrates persistent memory B cell responses over years and a lasting but variable impact of secondary DENV infection on DENV-specific T cell responses.

Genomic comparison of virulent Rickettsia rickettsii Sheila Smith and avirulent Rickettsia rickettsii Iowa.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. To identify genes involved in the virulence of R. rickettsii, the genome of an avirulent strain, R. rickettsii Iowa, was sequenced and compared to the genome of the virulent strain R. rickettsii Sheila Smith. R. rickettsii Iowa is avirulent in a guinea pig model of infection and displays altered plaque morphology with decreased lysis of infected host cells. Comparison of the two genomes revealed that R. rickettsii Iowa and R. rickettsii Sheila Smith share a high degree of sequence identity. A whole-genome alignment comparing R. rickettsii Iowa to R. rickettsii Sheila Smith revealed a total of 143 deletions for the two strains. A subsequent single-nucleotide polymorphism (SNP) analysis comparing Iowa to Sheila Smith revealed 492 SNPs for the two genomes. One of the deletions in R. rickettsii Iowa truncates rompA, encoding a major surface antigen (rickettsial outer membrane protein A [rOmpA]) and member of the autotransporter family, 660 bp from the start of translation. Immunoblotting and immunofluorescence confirmed the absence of rOmpA from R. rickettsii Iowa. In addition, R. rickettsii Iowa is defective in the processing of rOmpB, an autotransporter and also a major surface antigen of spotted fever group rickettsiae. Disruption of rompA and the defect in rOmpB processing are most likely factors that contribute to the avirulence of R. rickettsii Iowa. Genomic differences between the two strains do not significantly alter gene expression as analysis of microarrays revealed only four differences in gene expression between R. rickettsii Iowa and R. rickettsii strain R. Although R. rickettsii Iowa does not cause apparent disease, infection of guinea pigs with this strain confers protection against subsequent challenge with the virulent strain R. rickettsii Sheila Smith.