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Abstract Background Understanding the level of exposure to Lassa virus (LASV) in at-risk communities allows for the administration of effective preventive interventions to mitigate epidemics of Lassa fever. We assessed the seroprevalence of LASV antibodies in rural and semiurban communities of two cosmopolitan cities in Nigeria with poorly understood Lassa epidemiology. Methods A cross-sectional study was conducted in ten communities located in the Abuja Municipal Area Council (AMAC), Abuja, and Ikorodu Local Government Area (LGA), Lagos, from February 2nd to July 5th, 2022. Serum samples collected from participants were analyzed for IgG and IgM antibodies using a ReLASV® Pan-Lassa NP IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit. A questionnaire administered to participants collected self-reported sociodemographic and LASV exposure information. Seroprevalence of LASV IgG/IgM was estimated overall, and by study site. Univariate and multivariate log-binomial models estimated unadjusted and adjusted prevalence ratios (aPRs) and 95% confidence intervals (CI) for site-specific risk factors for LASV seropositivity. Grouped Least Absolute Shrinkage and Selection Operator (LASSO) was used for variable selection for multivariate analysis. Results A total of 628 participants with serum samples were included in the study. Most participants were female (434, 69%), married (459, 73%), and had a median age of 38 years (interquartile range 28–50). The overall seroprevalence was 27% (171/628), with a prevalence of 33% (126/376) in Abuja and 18% (45/252) in Lagos. Based on site-specific grouped LASSO selection, enrollment in the dry season (vs. wet; aPR, 95% CI: 1.73, 1.33–2.24), reported inconsistent washing of fruits and vegetables (aPR, 95% CI: 1.45, 1.10–1.92), and a positive malaria rapid test (aPR, 95% CI: 1.48, 1.09-2.00) were independently associated with LASV seropositivity in Abuja, whereas, only a self-reported history of rhinorrhea (PR, 95% CI: 2.21, 1.31–3.72) was independently associated with Lassa seropositivity in Lagos. Conclusions The LASV seroprevalence was comparable to that in other areas in Nigeria. Our findings corroborate those from other studies on the importance of limiting human exposure to rodents and focusing on behavioral factors such as poor hygiene practices to reduce exposure to LASV.

Summary: The rapid emergence of SARS-CoV-2 variants of concern (VoC) and the threat of future zoonotic sarbecovirus spillover emphasizes the need for broadly protective next-generation vaccines and therapeutics. We utilized SARS-CoV-2 spike ferritin nanoparticle (SpFN), and SARS-CoV-2 receptor binding domain ferritin nanoparticle (RFN) immunogens, in an equine model to elicit hyperimmune sera and evaluated its sarbecovirus neutralization and protection capacity. Immunized animals rapidly elicited sera with the potent neutralization of SARS-CoV-2 VoC, and SARS-CoV-1 pseudoviruses, and potent binding against receptor binding domains from sarbecovirus clades 1b, 1a, 2, 3, and 4. Purified equine polyclonal IgG provided protection against Omicron XBB.1.5 virus in the K18-hACE2 transgenic mouse model. These results suggest that SARS-CoV-2-based nanoparticle vaccines can rapidly produce a broad and protective sarbecovirus response in the equine model and that equine serum has therapeutic potential against emerging SARS-CoV-2 VoC and diverse sarbecoviruses, presenting a possible alternative or supplement to monoclonal antibody immunotherapies.

ABSTRACTIdentification of the diverse animal hosts responsible for spill-over events from animals to humans is crucial for comprehending the transmission patterns of emerging infectious diseases, which pose significant public health risks. To better characterize potential animal hosts of Lassa virus (LASV), we assessed domestic and non-domestic animals from 2021–2022 in four locations in southern Nigeria with reported cases of Lassa fever (LF). Birds, lizards, and domestic mammals (dogs, pigs, cattle and goats) were screened using RT-qPCR, and whole genome sequencing was performed for lineage identification on selected LASV positive samples. Animals were also screened for exposure to LASV by enzyme-linked immunosorbent assay (ELISA). Among these animals, lizards had the highest positivity rate by PCR. Genomic sequencing of samples in most infected animals showed sub-lineage 2 g of LASV. Seropositivity was highest among cattle and lowest in pigs. Though the specific impact these additional hosts may have in the broader virus-host context are still unknown – specifically relating to pathogen diversity, evolution, and transmission – the detection of LASV in non-rodent hosts living in proximity to confirmed human LF cases suggests their involvement during transmission as potential reservoirs. Additional epidemiological data comparing viral genomes from humans and animals, as well as those circulating within the environment will be critical in understanding LASV transmission dynamics and will ultimately guide the development of countermeasures for this zoonotic health threat.

Abstract The repeat emergence of SARS-CoV-2 variants of concern (VoC) with decreased susceptibility to vaccine-elicited antibodies highlights the need to develop next-generation vaccine candidates that confer broad protection. Here we describe the antibody response induced by the SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine candidate adjuvanted with the Army Liposomal Formulation including QS21 (ALFQ) in non-human primates. By isolating and characterizing several monoclonal antibodies directed against the Spike Receptor Binding Domain (RBD), N-Terminal Domain (NTD), or the S2 Domain, we define the molecular recognition of vaccine-elicited cross-reactive monoclonal antibodies (mAbs) elicited by SpFN. We identify six neutralizing antibodies with broad sarbecovirus cross-reactivity that recapitulate serum polyclonal antibody responses. In particular, RBD mAb WRAIR-5001 binds to the conserved cryptic region with high affinity to sarbecovirus clades 1 and 2, including Omicron variants, while mAb WRAIR-5021 offers complete protection from B.1.617.2 (Delta) in a murine challenge study. Our data further highlight the ability of SpFN vaccination to stimulate cross-reactive B cells targeting conserved regions of the Spike with activity against SARS CoV-1 and SARS-CoV-2 variants.

Abstract 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.

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.

ABSTRACTZika virus (ZIKV) strains belong to the East African, West African, and Asian/American phylogenetic lineages. RNA viruses, like ZIKV, exist as populations of genetically-related sequences whose heterogeneity may impact viral fitness, evolution, and virulence. Genetic diversity of representative ZIKVs from each lineage was examined using next generation sequencing (NGS) paired with downstream entropy and single nucleotide variant (SNV) analysis. Comparisons showed that inter-lineage diversity was statistically supported, while intra-lineage diversity varied. Intra-lineage diversity was significant for East but not West Africa strains. Furthermore, intra-lineage diversity for the Asian/American lineage was not supported for human serum isolates; however, a placenta isolate differed significantly. Relative entropy values were higher in the pre-membrane/membrane (prM/M) gene of several ZIKV strains. Additionally, the East African lineage contained a greater number of synonymous SNVs, while a greater number of non-synonymous SNVs were identified for American strains. Further, inter-lineage SNVs were dispersed throughout the genome, whereas intra-lineage non-synonymous SNVs for Asian/American strains clustered within prM/M and NS1 gene. This comprehensive analysis of ZIKV genetic diversity provides a repository of SNV positions across lineages. We posit that increased non-synonymous SNV populations and increased relative genetic diversity of the prM/M and NS1 proteins provides more evidence for their role in ZIKV virulence and fitness.

ABSTRACT One paradigm to explain the complexity of viral RNA populations is that the low fidelity of the RNA-dependent RNA polymerase (RdRp) drives high mutation rates and consequently genetic diversity. Like most RNA viruses, wild-type yellow fever virus (YFV) replication is error-prone due to the lack of proofreading by the virus-encoded RdRp. However, there is evidence that replication of the live attenuated YF vaccine virus 17D, derived from wild-type strain Asibi, is less error-prone than wild-type RNA viruses. Recent studies comparing the genetic diversity of wild-type Asibi and 17D vaccine virus found that wild-type Asibi has the typical heterogeneous population of an RNA virus, while there is limited intra- and interpopulation variability of 17D vaccine virus. Utilizing chimeric and mutant infectious clone-derived viruses, we show that high and low genetic diversity profiles of wild-type Asibi virus and vaccine virus 17D, respectively, are multigenic. Introduction of either structural (pre-membrane and envelope) genes or NS2B or NS4B substitutions into the Asibi and 17D backbone resulted in altered variant population, nucleotide diversity, and mutation frequency compared to the parental viruses. Additionally, changes in genetic diversity of the chimeric and mutant viruses correlated with the phenotype of multiplication kinetics in human alveolar A549 cells. Overall, the paradigm that only the error-prone RdRp controls genetic diversity needs to be expanded to address the role of other genes in genetic diversity, and we hypothesize that it is the replication complex as a whole and not the RdRp alone that controls genetic diversity. IMPORTANCE With the advent of advanced sequencing technology, studies of RNA viruses have shown that genetic diversity can contribute to both attenuation and virulence and the paradigm is that this is controlled by the error-prone RNA-dependent RNA polymerase (RdRp). Since wild-type yellow fever virus (YFV) strain Asibi has genetic diversity typical of a wild-type RNA virus, while 17D virus vaccine has limited diversity, it provides a unique opportunity to investigate RNA population theory in the context of a well-characterized live attenuated vaccine. Utilizing infectious clone-derived viruses, we show that genetic diversity of RNA viruses is complex and that multiple genes, including structural genes and NS2B and NS4B genes also contribute to genetic diversity. We suggest that the replication complex as a whole, rather than only RdRp, drives genetic diversity, at least for YFV.

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.