A recombinant virus-like particle vaccine against adenovirus-7 induces a potent humoral response.

Adenoviruses (AdVs) cause infections in humans that range from mild to severe, and can cause outbreaks particularly in close contact settings. Several human AdV types have been identified, which can cause a wide array of clinical manifestations. AdV types 4 and 7 (AdV-4 and AdV-7), which are among the most commonly circulating types in the United States, are known to cause acute respiratory disease that can result in hospitalization and rarely, death. Currently, the only vaccines approved for use in humans are live virus vaccines against AdV-4 and AdV-7, though these vaccines are only authorized for use in U.S. military personnel. While they are efficacious, use of these live virus vaccines carries considerable risks of vaccine-associated viral shedding and recombination. Here, we present an alternative vaccination strategy against AdV-7 using the virus-like particle platform (AdVLP-7). We describe the production of stable recombinant AdVLP-7, and demonstrate that AdVLP-7 is structurally analogous to wild-type AdV-7 virions (WT AdV-7). Preclinical immunogenicity studies in mice show that AdVLP-7 elicits a potent humoral immune response, comparable to that observed in mice immunized with WT AdV-7. Specifically, AdVLP-7 induces high titers of antibodies against AdV-7-specific antigens that can effectively neutralize AdV-7.

Differential Genome Replication of a Unique Single-Amino-Acid Mutation in the Adenovirus-4 Component of the Live Oral Adenovirus Type 4 and Type 7 Vaccine.

The FDA-approved Adenovirus Type 4 and Type 7 Vaccine, Live, Oral is highly effective and essential in preventing acute respiratory diseases (ARDs) in U.S. military recruits. Our study revealed the presence of a previously undetected mutation, not found in the wild-type human adenovirus type 4 (HAdV-4) component of the licensed vaccine, which contains an amino acid substitution (P388T) in the pre-terminal protein (pTP). This study demonstrated that replication of the T388 HAdV-4 vaccine mutant virus is favored over the wild type in WI-38 cells, the cell type utilized in vaccine manufacturing. However, results from serial human stool specimens of vaccine recipients support differential genome replication in the gastrointestinal tract (GI), demonstrated by the steady decline of the percentage of mutant T388 vaccine virus. Since vaccine efficacy depends upon GI replication and the subsequent immune response, the mutation can potentially impact vaccine efficacy.

A Phase 1 Two-Arm, Randomized, Double-Blind, Active-Controlled Study of Live, Oral Plasmid-Derived Adenovirus Type 4 and Type 7 Vaccines in Seronegative Adults.

PXVX0047 is an investigational vaccine developed for active immunization to prevent febrile acute respiratory disease (ARD) caused by adenovirus serotypes 4 (Ad4) and 7 (Ad7). PXVX0047 consists of a modernized, plasmid-derived vaccine that was generated using a virus isolated from Wyeth Ad4 and Ad7 vaccine tablets. A phase 1 two-arm, randomized, double-blind, active-controlled study was conducted to evaluate the safety profile and immunogenicity of the investigational adenovirus vaccines. The two components of PXVX0047 were administered orally together in a single dose to 11 subjects. For comparison, three additional subjects received the Ad4/Ad7 vaccine that is currently in use by the US military. The results of this study show that the tolerability and immunogenicity of the PXVX0047 Ad7 component are comparable with that of the control Ad4/Ad7 vaccine; however, the immunogenicity of the PXVX0047 Ad4 component was lower than expected. Clinical trial number NCT03160339.

Clustered cases of human adenovirus types 4, 7, and 14 infections in US Department of Defense Beneficiaries during the 2018-2019 season.

Human adenoviruses (HAdV) are genetically diverse and can infect a number of tissues with severities varied from mild to fatal. HAdV types 3, 4, 7, 11, 14, 21, and 55 were associated with acute respiratory illnesses outbreaks in the United States and in other countries. The risk of outbreaks can be effectively controlled by HAdV vaccination or mitigated by screening and preventive measures. During the influenza season 2018-2019, the DoD Global Respiratory Pathogen Surveillance Program (DoDGRS) received 24 300 respiratory specimens. HAdV samples that produced positive cytopathic effects in viral cultivation were subjected to next-generation sequencing for genome sequence assembly, genome typing, whole genome phylogeny, and sequence comparative analyses. A variety of HAdV types were identified in this study, including HAdV types 1-7, 14, 55, and 56. HAdV types 4, 7, and 14 were found in clustered cases in Colorado, Florida, New York, and South Carolina. Comparative sequence analyses of these isolates revealed the emergence of novel genetic mutations despite the stability of adenovirus genomes. Genomic surveillance of HAdV suggested possible undetected outbreaks and shed light on prevalence, genetic divergence, and viral evolution of HAdV. Continued surveillance will inform risk assessment and countermeasures.

Live Oral Adenovirus Type 4 and Type 7 Vaccine Induces Durable Antibody Response.

Human adenoviruses (AdV) are mostly associated with minimal pathology. However, more severe respiratory tract infections and acute respiratory diseases, most often caused by AdV-4 and AdV-7, have been reported. The only licensed vaccine in the United States, live oral AdV-4 and AdV-7 vaccine, is indicated for use in the military, nearly exclusively in recruit populations. The excellent safety profile and prominent antibody response of the vaccine is well established by placebo-controlled clinical trials, while, long-term immunity of vaccination has not been studied. Serum samples collected over 6 years from subjects co-administered live oral AdV-4 and AdV-7 vaccine in 2011 were evaluated to determine the duration of the antibody response. Group geometric mean titers (GMT) at 6 years post vaccination compared to previous years evaluated were not significantly different for either AdV-4 or AdV-7 vaccine components. There were no subjects that demonstrated waning neutralization antibody (NAb) titers against AdV-4 and less than 5% of subjects against AdV-7. Interestingly, there were subjects that had a four-fold increase in NAb titers against either AdV-4 or AdV-7, at various time points post vaccination, suggesting either homotypic or heterotypic re-exposure. This investigation provided strong evidence that the live oral AdV-4 and AdV-7 vaccine induced long-term immunity to protect from AdV-4 and AdV-7 infections.

Human Adenovirus Type 55 Distribution, Regional Persistence, and Genetic Variability.

Human adenovirus type 55 (HAdV-55) causes acute respiratory disease of variable severity and has become an emergent threat in both civilian and military populations. HAdV-55 infection is endemic to China and South Korea, but data from other regions and time periods are needed for comprehensive assessment of HAdV-55 prevalence from a global perspective. In this study, we subjected HAdV-55 isolates from various countries collected during 1969-2018 to whole-genome sequencing, genomic and proteomic comparison, and phylogenetic analyses. The results show worldwide distribution of HAdV-55; recent strains share a high degree of genomic homogeneity. Distinct strains circulated regionally for several years, suggesting persistent local transmission. Several cases of sporadic introduction of certain strains to other countries were documented. Among the identified amino acid mutations distinguishing HAdV-55 strains, some have potential impact on essential viral functions and may affect infectivity and transmission.

Composition and variation of respiratory microbiota in healthy military personnel.

Certain occupational and geographical exposures have been associated with an increased risk of lung disease. As a baseline for future studies, we sought to characterize the upper respiratory microbiomes of healthy military personnel in a garrison environment. Nasal, oropharyngeal, and nasopharyngeal swabs were collected from 50 healthy active duty volunteers eight times over the course of one year (1107 swabs, completion rate = 92.25%) and subjected to pyrosequencing of the V1-V3 region of 16S rDNA. Respiratory bacterial taxa were characterized at the genus level, using QIIME 1.8 and the Ribosomal Database Project classifier. High levels of Staphylococcus, Corynebacterium, and Propionibacterium were observed among both nasal and nasopharyngeal microbiota, comprising more than 75% of all operational taxonomical units (OTUs). In contrast, Streptococcus was the sole dominant bacterial genus (approximately 50% of all OTUs) in the oropharynx. The average bacterial diversity was greater in the oropharynx than in the nasal or nasopharyngeal region at all time points. Diversity analysis indicated a significant overlap between nasal and nasopharyngeal samples, whereas oropharyngeal samples formed a cluster distinct from these two regions. The study produced a large set of pyrosequencing data on the V1-V3 region of bacterial 16S rDNA for the respiratory microbiomes of healthy active duty Service Members. Pre-processing of sequencing reads showed good data quality. The derived microbiome profiles were consistent both internally and with previous reports, suggesting their utility for further analyses and association studies based on sequence and demographic data.

Adenovirus type 4 respiratory infections with a concurrent outbreak of coxsackievirus A21 among United States Army Basic Trainees, a retrospective viral etiology study using next-generation sequencing.

Human adenoviruses (HAdV), in particular types 4 and 7, frequently cause acute respiratory disease (ARD) during basic military training. HAdV4 and HAdV7 vaccines reduced the ARD risk in U.S. military. It is important to identify other respiratory pathogens and assess their potential impact on military readiness. In 2002, during a period when the HAdV vaccines were not available, throat swabs were taken from trainees (n = 184) with respiratory infections at Fort Jackson, South Carolina. Viral etiology was investigated initially with viral culture and neutralization assay and recently in this study by sequencing the viral isolates. Viral culture and neutralization assays identified 90 HAdV4 isolates and 27 additional cultures that showed viral cytopathic effects (CPE), including some with picornavirus-like CPE. Next-generation sequencing confirmed these results and determined viral genotypes, including 77 HAdV4, 4 HAdV3, 1 HAdV2, 17 coxsackievirus A21 (CAV21), and 1 enterovirus D68. Two samples were positive for both HAdV4 and CAV21. The identified genotypes are phylogenetically close to but distinct from those found during other years or in other military/non-military sites. HAdV4 is the predominant respiratory pathogen in unvaccinated military trainee. HAdV4 has temporal and demographic variability. CAV21 is a significant respiratory pathogen and needs to be evaluated for its current significance in military basic trainees.

Frequency of influenza H3N2 intra-subtype reassortment: attributes and implications of reassortant spread.

BACKGROUND: Increasing evidence suggests that influenza reassortment not only contributes to the emergence of new human pandemics but also plays an important role in seasonal influenza epidemics, disease severity, evolution, and vaccine efficacy. We studied this process within 2091 H3N2 full genomes utilizing a combination of the latest reassortment detection tools and more conventional phylogenetic analyses. RESULTS: We found that the amount of H3N2 intra-subtype reassortment depended on the number of sampled genomes, occurred with a steady frequency of 3.35%, and was not affected by the geographical origins, evolutionary patterns, or previous reassortment history of the virus. We identified both single reassortant genomes and reassortant clades, each clade representing one reassortment event followed by successful spread of the reassorted variant in the human population. It was this spread that was mainly responsible for the observed high presence of H3N2 intra-subtype reassortant genomes. The successfully spread variants were generally sampled within one year of their formation, highlighting the risk of their rapid spread but also presenting an opportunity for their rapid detection. Simultaneous spread of several different reassortant lineages was observed, and despite their limited average lifetime, second and third generation reassortment was detected, as well as reassortment between viruses belonging to different vaccine-associated clades, likely displaying differing antigenic properties. Some of the spreading reassortants remained confined to certain geographical regions, while others, sharing common properties in amino acid positions of the HA, NA, and PB2 segments, were found throughout the world. CONCLUSIONS: Detailed surveillance of seasonal influenza reassortment patterns and variant properties may provide unique information needed for prediction of spread and construction of future influenza vaccines.

Genome Sequence of Bellavista Virus, a Novel Orthobunyavirus Isolated from a Pool of Mosquitoes Captured near Iquitos, Peru.

A novel orthobunyavirus, Bellavista virus, was isolated from Culex (Melanoconion) portesi mosquitoes in the Bellavista neighborhood of Iquitos, Peru, in 2009. The assembled segment L, M, and S sequences of strain PRD0552 are 6,950, 4,469, and 1,256 bases in length, respectively, comprising complete protein-coding sequences and partial terminal untranslated sequences.

Molecular Epidemiology of Adenovirus Type 21 Respiratory Strains Isolated From US Military Trainees (1996-2014).

BACKGROUND: The circulation of human adenovirus type 21 (HAdV21) in the United States has been documented since the 1960s in association with outbreaks of febrile respiratory illness (FRI) in military boot camps and civilian cases of respiratory disease. METHODS: To describe the molecular epidemiology of HAdV21 respiratory infections across the country, 150 clinical respiratory isolates obtained from continuous surveillance of military recruit FRI, and 23 respiratory isolates recovered from pediatric and adult civilian cases of acute respiratory infection were characterized to compile molecular typing data spanning 37 years (1978-2014). RESULTS: Restriction enzyme analysis and genomic sequencing identified 2 clusters of closely related genomic variants readily distinguishable from the prototype and designated 21a-like and 21b-like. A-like variants predominated until 1999. A shift to b-like variants was noticeable by 2007 after a 7-year period (2000-2006) of cocirculation of the 2 genome types. US strains are phylogenetically more closely related to European and Asian strains isolated over the last 4 decades than to the Saudi Arabian prototype strain AV-1645 isolated in 1956. CONCLUSIONS: Knowledge of circulating HAdV21 variants and their epidemic behavior will be of significant value to local and global FRI surveillance efforts.

16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles.

BACKGROUND: Sample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability. RESULTS: The bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment. CONCLUSIONS: Mock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples.

Adenovirus type 4 and 7 vaccination or adenovirus type 4 respiratory infection elicits minimal cross-reactive antibody responses to nonhuman adenovirus vaccine vectors.

Antivector immunity may limit the immunogenicity of adenovirus vector vaccines. We tested sera from individuals immunized with adenovirus type 4 and 7 (Ad4 and Ad7, respectively) vaccine or naturally infected with Ad4 for their ability to neutralize a panel of E1-deleted human and chimpanzee adenoviruses (ChAd). Small statistically significant increases in titers to ChAd63, ChAd3, human Ad35, and human Ad5 were observed. Neutralizing antibodies elicited by Ad4 infection or immunization results in a small amount of adenovirus cross-reactivity.

Genomic characterization of group C Orthobunyavirus reference strains and recent South American clinical isolates.

Group C orthobunyaviruses (family Bunyaviridae, genus Orthobunyavirus), discovered in the 1950s, are vector-borne human pathogens in the Americas. Currently there is a gap in genomic information for group C viruses. In this study, we obtained complete coding region sequences of reference strains of Caraparu (CARV), Oriboca (ORIV), Marituba (MTBV) and Madrid (MADV) viruses, and five clinical isolates from Peru and Bolivia, using an unbiased de novo approach consisting of random reverse transcription, random anchored PCR amplification, and high throughput pyrosequencing. The small, medium, and large segments encode for a 235 amino acid nucleocapsid protein, an approximately 1430 amino acid surface glycoprotein polyprotein precursor, and a 2248 amino acid RNA-dependent RNA polymerase, respectively. Additionally, the S segment encodes for an 83 amino acid non-structural protein, although this protein is truncated or silenced in some isolates. Phylogenetically, three clinical isolates clustered with CARV, one clustered with MTBV, and one isolate appeared to be a reassortant or a genetic drift resulted from the high variability of the medium segment which was also seen in a few other orthobunyaviruses. These data represent the first complete coding region sequences for this serocomplex of pathogenic orthobunyaviruses. The genome-wide phylogeny of reference strains is consistent with the antigenic properties of the viruses reported in the original serological studies conducted in the 1960s. Comparative analysis of conserved protein regions across group C virus strains and the other orthobunyavirus groups revealed that these group C viruses contain characteristic domains of potential structural and functional significance. Our results provide the basis for the developments of diagnostics, further genetic analyses, and future epidemiologic studies of group C viruses.

Enhanced de novo assembly of high throughput pyrosequencing data using whole genome mapping.

Despite major advances in next-generation sequencing, assembly of sequencing data, especially data from novel microorganisms or re-emerging pathogens, remains constrained by the lack of suitable reference sequences. De novo assembly is the best approach to achieve an accurate finished sequence, but multiple sequencing platforms or paired-end libraries are often required to achieve full genome coverage. In this study, we demonstrated a method to assemble complete bacterial genome sequences by integrating shotgun Roche 454 pyrosequencing with optical whole genome mapping (WGM). The whole genome restriction map (WGRM) was used as the reference to scaffold de novo assembled sequence contigs through a stepwise process. Large de novo contigs were placed in the correct order and orientation through alignment to the WGRM. De novo contigs that were not aligned to WGRM were merged into scaffolds using contig branching structure information. These extended scaffolds were then aligned to the WGRM to identify the overlaps to be eliminated and the gaps and mismatches to be resolved with unused contigs. The process was repeated until a sequence with full coverage and alignment with the whole genome map was achieved. Using this method we were able to achieved 100% WGRM coverage without a paired-end library. We assembled complete sequences for three distinct genetic components of a clinical isolate of Providencia stuartii: a bacterial chromosome, a novel bla NDM-1 plasmid, and a novel bacteriophage, without separately purifying them to homogeneity.

A phase 3, randomized, double-blind, placebo-controlled study of the safety and efficacy of the live, oral adenovirus type 4 and type 7 vaccine, in U.S. military recruits.

Adenovirus (ADV) types 4 (ADV-4) and 7 (ADV-7) are presently the major cause of febrile acute respiratory disease (ARD) in U.S. military recruits. We conducted a multi-center, randomized, double-blind, placebo-controlled phase 3 study of the new vaccine to assess its safety and efficacy. Healthy adults at two basic training sites were randomly assigned to receive either vaccine (two enteric-coated tablets consisting of no less than 4.5 log10 TCID50 of live ADV-4 or ADV-7) or placebo in a 3:1 ratio. Volunteers were observed throughout the approximate eight weeks of their basic training and also returned for four scheduled visits. The primary endpoints were prevention of febrile ARD due to ADV-4 and seroconversion of neutralizing serum antibodies to ADV-7, which was not expected to circulate in the study population during the course of the trial. A total of 4151 volunteers were enrolled and 4040 (97%) were randomized and included in the primary analysis (110 were removed prior to randomization and one was removed after randomization due to inability to swallow tablets). A total of 49 ADV-4 febrile ARD cases were identified with 48 in the placebo group and 1 in the vaccine group (attack rates of 4.76% and 0.03%, respectively). Vaccine efficacy was 99.3% (95% CI, 96.0-99.9; P<0.001). Seroconversion rates for vaccine recipients for ADV-4 and ADV-7 were 94.5% (95% CI, 93.4-95.5%) and 93.8% (95% CI: 93.4-95.2%), respectively. The vaccine was well tolerated as compared to placebo. We conclude that the new live, oral ADV-4 and ADV-7 vaccine is safe and effective for use in groups represented by the study population.

Quantitative predictions of binding free energy changes in drug-resistant influenza neuraminidase.

Quantitatively predicting changes in drug sensitivity associated with residue mutations is a major challenge in structural biology. By expanding the limits of free energy calculations, we successfully identified mutations in influenza neuraminidase (NA) that confer drug resistance to two antiviral drugs, zanamivir and oseltamivir. We augmented molecular dynamics (MD) with Hamiltonian Replica Exchange and calculated binding free energy changes for H274Y, N294S, and Y252H mutants. Based on experimental data, our calculations achieved high accuracy and precision compared with results from established computational methods. Analysis of 15 micros of aggregated MD trajectories provided insights into the molecular mechanisms underlying drug resistance that are at odds with current interpretations of the crystallographic data. Contrary to the notion that resistance is caused by mutant-induced changes in hydrophobicity of the binding pocket, our simulations showed that drug resistance mutations in NA led to subtle rearrangements in the protein structure and its dynamics that together alter the active-site electrostatic environment and modulate inhibitor binding. Importantly, different mutations confer resistance through different conformational changes, suggesting that a generalized mechanism for NA drug resistance is unlikely.