Biology subjects

Shifflett, K.

Publications and source records attributed to Shifflett, K..

6 recordsLinked to original sources

bioRxiv2021

Serum from COVID-19 patients early in the pandemic shows limited evidence of cross-neutralization against variants of concern

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in a variety of clinical symptoms ranging from no or mild to severe disease. Currently, there are multiple postulated mechanisms that may push a moderate to severe disease into a critical state. Human serum contains abundant evidence of the immune status following infection. Cytokines, chemokines, and antibodies can be assayed to determine the extent to which a patient responded to a pathogen. We examined serum and plasma from a cohort of patients infected with SARS-CoV-2 early in the pandemic and compared them to negative-control sera. Cytokine and chemokine concentrations varied depending on the severity of infection, and antibody responses were significantly increased in severe cases compared to mild to moderate infections. Neutralization data revealed that patients with high titers against an early 2020 isolate had detectable but limited neutralizing antibodies against newly circulating SARS-CoV-2 variants of concern. This study highlights the potential of re-infection for recovered COVID-19 patients.

microbiology↗

bioRxiv2021

Optimization of single dose VSV-based COVID-19 vaccination in hamsters

The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need the for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8+ T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4+ T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.

microbiology↗

bioRxiv2021

Pathogenic and transcriptomic differences of emerging SARS-CoV-2 variants in the Syrian golden hamster model

Following the discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid spread throughout the world, new viral variants of concern (VOC) have emerged. There is a critical need to understand the impact of the emerging variants on host response and disease dynamics to facilitate the development of vaccines and therapeutics. Syrian golden hamsters are the leading small animal model that recapitulates key aspects of severe coronavirus disease 2019 (COVID-19). In this study, we show that intranasal inoculation of SARS-CoV-2 into hamsters with the ancestral virus (nCoV-WA1-2020) or VOC first identified in the United Kingdom (B.1.1.7) and South Africa (B.1.351) led to similar gross and histopathologic pulmonary lesions. Although differences in viral genomic copy numbers were noted in the lungs and oral swabs of challenged animals, infectious titers in the lungs were comparable. Antibody neutralization capacities varied, dependent on the original challenge virus and cross-variant protective capacity. Transcriptional profiling indicated significant induction of antiviral pathways in response to all three challenges with a more robust inflammatory signature in response to B.1.1.7. Furthermore, no additional mutations in the spike protein were detected at peak disease. In conclusion, the emerging VOC showed distinct humoral responses and transcriptional profiles in the hamster model compared to the ancestral virus.

microbiology↗

bioRxiv2021

A human factor H-binding protein of Bartonella bacilliformis and potential role in serum resistance

Bartonella bacilliformis is a Gram-negative bacterium and etiologic agent of Carrions disease; a potentially life-threatening illness endemic to South America. B. bacilliformis is a facultative parasite that infects human erythrocytes (hemotrophism) and the circulatory system, culminating in a variety of symptoms, including a precipitous drop in hematocrit, angiomatous lesions of the skin (verruga peruana) and persistent bacteremia. Because of its specialized niche, serum complement imposes a continual selective pressure on the pathogen. In this study, we demonstrated the marked serum-resistance phenotype of B. bacilliformis, the role of factor H in serum complement resistance, and binding of host factor H to four membrane-associated polypeptides of [~]131, 119, 60 and 43 kDa by far-western (FW) blots. The [~]119-kDa protein was identified as ABM44634.1 by mass spectrometry; a protein annotated as a 116.5-kDa outer membrane autotransporter (encoded by the BARBAKC583_1133 locus). We designated the protein as factor H-binding protein A (FhbpA). FhbpA possesses three structural motifs common to all autotransporter proteins (i.e., a signal peptide, autotransporter {beta}-barrel domain and passenger domain). Recombinant FhbpA passenger domain, but not the recombinant autotransporter domain, was able to bind human factor H when analyzed by FW blots. Phylogenetic analyses of the passenger domain suggest that it is well-conserved among Bartonella autotransporters, with closest matches from Bartonella schoenbuchensis. Transcriptomic analyses of B. bacilliformis subjected to conditions mimicking the sand fly vector or human host, and infection of human blood or vascular endothelial cells showed maximal expression of fhbpA under human-like conditions and during infection of blood and endothelial cells. Expression during HUVEC infection was significantly higher compared to all other conditions by DESeq2. Surface binding of serum factor H by FhbpA is hypothesized to play a protective role against the alternative pathway of complement fixation during B. bacilliformis infection of the human host. Author SummaryB. bacilliformis is a bacterial pathogen that colonizes the circulatory system of humans, where it can cause a life-threatening illness unless treated. Serum complement is a major effector of innate humoral immunity and a significant obstacle that must be evaded for successful survival and colonization by pathogens, especially those residing in the vasculature. In this study, we examined the serum complement resistance phenotype of B. bacilliformis and identified four membrane-associated proteins that bind serum factor H; a protein used by the host to protect its own tissues from complement activation. One of the proteins was identified by mass spectrometry, characterized, and designated factor H-binding protein A (FhbpA). FhbpA is a predicted autotransporter, and we determined that the translocated " passenger" domain of the protein is responsible for binding factor H. We also determined that expression of the fhbpA gene was highest during infection of human blood and especially vascular endothelial cells or under conditions that simulate the human host. The results suggest that FhbpA binding of host serum factor H protects the bacterium against complement activation during infection.

microbiology↗

bioRxiv2021

Rapid protection from COVID-19 in nonhuman primates vaccinated intramuscularly but not intranasally with a single dose of a recombinant vaccine

The ongoing pandemic of Coronavirus disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single dose, fast-acting vesicular stomatitis virus-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (IM) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, intranasal (IN) vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to control animals. While IM and IN vaccination both induced neutralizing antibody titers, only IM vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of IM-vaccinated animals only. Overall, the data demonstrates that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study. One sentence summaryVSV vaccine protects NHPs from COVID-19 in 10 days

microbiology↗

bioRxiv2019

Coxiella burnetii small RNA 12 binds CsrA regulatory protein and transcripts for the CvpD type IV effector, regulates pyrimidine and methionine metabolism, and is necessary for optimal intracellular growth and vacuole formation during infection

Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small non-coding RNAs (sRNAs) of C. burnetii. One of them, named CbsR12 (Coxiella burnetii small RNA 12) is highly expressed during growth in axenic medium and becomes even more dominant during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in single-stranded segments of stem loops with consensus AGGA/ANGGA motifs. From this foundation, we determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro. Moreover, through a combination of in vitro and in vivo assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds to and upregulates translation of carA transcripts coding for carbamoyl phosphate synthetase A; an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of metK transcripts coding for S-adenosyl methionine (SAM) synthase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of cvpD transcripts, coding for a type IVB effector protein, in vitro and in vivo. Finally, we found that CbsR12 is necessary for full expansion of Coxiella-containing vacuoles (CCVs) and affects bacterial growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first detailed characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK expression. CbsR12 is also one of only a few identified trans-acting sRNAs that interacts with CsrA. Results illustrate the importance of sRNA-mediated regulation in establishment of the intracellular CCV niche.\n\nAuthor summaryC. burnetii is an obligate intracellular bacterial pathogen that is transmitted to humans from animal reservoirs. Upon inhalation of aerosolized C. burnetii, the agent is phagocytosed by macrophages in the lung. The pathogen subverts macrophage-mediated degradation and resides in a large, intracellular, acidic vacuole, termed the Coxiella-containing vacuole (CCV). Small RNAs (sRNAs) are not translated into proteins. Instead, they target mRNAs in order to up- or down-regulate their stability and translation. Alternatively, some sRNAs bind to regulatory proteins and serve as \"sponges\" that effectively sequester the proteins and inhibit their function. C. burnetiis CbsR12 sRNA is highly expressed during infection in order to expand the CCV, and it works by a variety of mechanisms, including: 1) directly regulating transcripts of several metabolic genes that aid in bacterial replication, 2) binding to and regulating transcripts of a type IV effector protein that aids in infection, and 3) indirectly regulating an unknown number of genes by binding to a homolog of the global regulatory protein, CsrA. CbsR12 represents one of only a few sRNAs known to bind and sequester CsrA while also directly regulating mRNAs.

microbiology↗