Search

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.

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.

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.

Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. Candidate live-attenuated vaccine (LAV) viruses with engineered deletions in the 3’ untranslated region (UTR) provide immunity and protection in animal models of ZIKV infection, and phenotypic studies show that LAVs retain protective abilities following in vitro passage. The present study investigated the genetic diversity of wild-type (WT) parent ZIKV and its candidate LAVs using next generation sequencing analysis of five sequential in vitro passages. The results show that genomic entropy of WT ZIKV steadily increases during in vitro passage, whereas that of LAVs also increased by passage number five but was variable throughout passaging. Additionally, clusters of single nucleotide variants (SNVs) were found to be present in the pre-membrane/membrane (prM), envelope (E), nonstructural protein NS1 (NS1), and other nonstructural protein genes, depending on the specific deletion, whereas in the parent WT ZIKV, they are more abundant in prM and NS1. Ultimately, both the parental WT and LAV derivatives increase in genetic diversity, with evidence of adaptation following passage.

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 103 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92-2.41) and 10:1 (CT increase of 3.03-3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.