Concerted synergy between viral-specific IgG and CD8 + T cells is critical for clearance of an HCV-related rodent hepacivirus.

BACKGROUND AND AIMS: Evidence assessing the role of B cells and their antibodies, or lack thereof, in the spontaneous resolution of acute HCV infection is conflicting. Utilization of a strictly hepatotropic, HCV-related rodent hepacivirus (RHV) model circumvents many of the challenges facing the field in characterizing the immunological correlates of dichotomous infection outcomes. This study seeks to elucidate the importance of B cells in the clearance of acute RHV infection. APPROACH AND RESULTS: µMT mice were infected i.v. with RHV and found to develop chronic infection for over a year. Wild-type (WT) mice depleted of B cells also exhibited persistent viremia that resolved only upon B cell resurgence. The persistent infection developed by B1-8i and AID cre/cre mice revealed that antigen-specific, class-switched B cells or their antibodies were crucial for viral resolution. Virus-specific CD8 + and CD4 + T cells were characterized in these mice using newly developed major histocompatibility complex class I and II tetramers and ex vivo peptide stimulation. Immunoglobulin G (IgG) was purified from the serum of RHV- or lymphocytic choriomeningitis virus Armstrong-infected mice after viral clearance and passively transferred to AID cre/cre recipients, revealing viral clearance only in αRHV IgG recipients. Further, the transfer of αRHV IgG into B cell-depleted recipients also induced viral resolution. This ability of RHV-specific IgG to induce viral clearance was found to require the concomitant presence of CD8 + T cells. CONCLUSIONS: Our findings demonstrate a cooperative interdependence between immunoglobulins and the T cell compartment that is required for RHV resolution. Thus, HCV vaccine regimens should aim to simultaneously elicit robust HCV-specific antibody and T cell responses for optimal protective efficacy.

Phenotype and fate of liver-resident CD8 T cells during acute and chronic hepacivirus infection.

Immune correlates of hepatitis C virus (HCV) clearance and control remain poorly defined due to the lack of an informative animal model. We recently described acute and chronic rodent HCV-like virus (RHV) infections in lab mice. Here, we developed MHC class I and class II tetramers to characterize the serial changes in RHV-specific CD8 and CD4 T cells during acute and chronic infection in C57BL/6J mice. RHV infection induced rapid expansion of T cells targeting viral structural and nonstructural proteins. After virus clearance, the virus-specific T cells transitioned from effectors to long-lived liver-resident memory T cells (TRM). The effector and memory CD8 and CD4 T cells primarily produced Th1 cytokines, IFN-γ, TNF-α, and IL-2, upon ex vivo antigen stimulation, and their phenotype and transcriptome differed significantly between the liver and spleen. Rapid clearance of RHV reinfection coincided with the proliferation of virus-specific CD8 TRM cells in the liver. Chronic RHV infection was associated with the exhaustion of CD8 T cells (Tex) and the development of severe liver diseases. Interestingly, the virus-specific CD8 Tex cells continued proliferation in the liver despite the persistent high-titer viremia and retained partial antiviral functions, as evident from their ability to degranulate and produce IFN-γ upon ex vivo antigen stimulation. Thus, RHV infection in mice provides a unique model to study the function and fate of liver-resident T cells during acute and chronic hepatotropic infection.

Recombinant measles virus expressing prefusion spike protein stabilized by six rather than two prolines is more efficacious against SARS-CoV-2 infection.

Measles virus (MeV) has been an excellent vector platform for delivering vaccines against many pathogens because of its high safety and efficacy, and induction of long-lived immunity. Early in the COVID-19 pandemic, a recombinant MeV (rMeV) expressing the prefusion full-length spike protein stabilized by two prolines (TMV-083) was developed and tested in phase 1 and 1/2 clinical trials but was discontinued because of insufficient immunogenicity and a low seroconversion rate in adults. Here, we compared the immunogenicity of rMeV expressing a soluble prefusion spike (preS) protein stabilized by two prolines (rMeV-preS-2P) with a rMeV expressing a soluble preS protein stabilized by six prolines (rMeV-preS-6P). We found that rMeV-preS-6P expressed approximately five times more preS than rMeV-preS-2P in cell culture. Importantly, rMeV-preS-6P induced 30-60 and six times more serum immunoglobulin G and neutralizing antibody than rMeV-preS-2P, respectively, in IFNAR-/- mice. IFNAR-/- mice immunized with rMeV-preS-6P were completely protected from challenge with a mouse-adapted SARS-CoV-2, whereas those immunized with rMeV-preS-2P were partially protected. In addition, hamsters immunized with rMeV-preS-6P were completely protected from the challenge with a Delta variant of SARS-CoV-2. Our results demonstrate that rMeV-preS-6P is significantly more efficacious than rMeV-preS-2P, highlighting the value of using preS-6P as the antigen for developing vaccines against SARS-CoV-2.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

A highly efficacious live attenuated mumps virus-based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV.

BACKGROUND AND AIMS: Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. APPROACH AND RESULTS: We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. CONCLUSIONS: The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

BACKGROUND AND AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH AND RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

A safe and highly efficacious measles virus-based vaccine expressing SARS-CoV-2 stabilized prefusion spike.

The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR-/-mice, IFNAR-/--hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2-specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

Vaccination to prevent T cell subversion can protect against persistent hepacivirus infection.

Efforts to develop an effective vaccine against the hepatitis C virus (HCV; human hepacivirus) have been stymied by a lack of small animal models. Here, we describe an experimental rat model of chronic HCV-related hepacivirus infection and its response to T cell immunization. Immune-competent rats challenged with a rodent hepacivirus (RHV) develop chronic viremia characterized by expansion of non-functional CD8+ T cells. Single-dose vaccination with a recombinant adenovirus vector expressing hepacivirus non-structural proteins induces effective immunity in majority of rats. Resolution of infection coincides with a vigorous recall of intrahepatic cellular responses. Host selection of viral CD8 escape variants can subvert vaccine-conferred immunity. Transient depletion of CD8+ cells from vaccinated rats prolongs infection, while CD4+ cell depletion results in chronic viremia. These results provide direct evidence that co-operation between CD4+ and CD8+ T cells is important for hepacivirus immunity, and that subversion of responses can be prevented by prophylactic vaccination.

Viral testing of 18 consecutive cases of equine serum hepatitis: A prospective study (2014-2018).

BACKGROUND: Three flaviviruses (equine pegivirus [EPgV]; Theiler's disease-associated virus [TDAV]; non-primate hepacivirus [NPHV]) and equine parvovirus (EqPV-H) are present in equine blood products; the TDAV, NPHV, and EqPV-H have been suggested as potential causes of serum hepatitis. OBJECTIVE: To determine the prevalence of these viruses in horses with equine serum hepatitis. ANIMALS: Eighteen horses diagnosed with serum hepatitis, enrolled from US referral hospitals. METHODS: In the prospective case study, liver, serum, or both samples were tested for EPgV, TDAV, NPHV, and EqPV-H by PCR. RESULTS: Both liver tissue and serum were tested for 6 cases, serum only for 8 cases, and liver only for 4 cases. Twelve horses received tetanus antitoxin (TAT) 4-12.7 weeks (median = 8 weeks), 3 horses received commercial equine plasma 6-8.6 weeks, and 3 horses received allogenic stem cells 6.4-7.6 weeks before the onset of hepatic failure. All samples were TDAV negative. Two of 14 serum samples were NPHV-positive. Six of 14 serum samples were EPgV-positive. All liver samples were NPHV-negative and EPgV-negative. EqPV-H was detected in the serum (N = 8), liver (N = 4), or both samples (N = 6) of all 18 cases. The TAT of the same lot number was available for virologic testing in 10 of 12 TAT-associated cases, and all 10 samples were EqPV-H positive. CONCLUSIONS AND CLINICAL IMPORTANCE: We demonstrated EqPV-H in 18 consecutive cases of serum hepatitis. EPgV, TDAV, and NPHV were not consistently present. This information should encourage blood product manufacturers to test for EqPV-H and eliminate EqPV-H-infected horses from their donor herds.

New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product.

Equine serum hepatitis (i.e., Theiler's disease) is a serious and often life-threatening disease of unknown etiology that affects horses. A horse in Nebraska, USA, with serum hepatitis died 65 days after treatment with equine-origin tetanus antitoxin. We identified an unknown parvovirus in serum and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares <50% protein identity with its phylogenetic relatives of the genus Copiparvovirus. Next, we experimentally infected 2 horses using a tetanus antitoxin contaminated with EqPV-H. Viremia developed, the horses seroconverted, and acute hepatitis developed that was confirmed by clinical, biochemical, and histopathologic testing. We also determined that EqPV-H is an endemic infection because, in a cohort of 100 clinically normal adult horses, 13 were viremic and 15 were seropositive. We identified a new virus associated with equine serum hepatitis and confirmed its pathogenicity and transmissibility through contaminated biological products.

Viral persistence, liver disease, and host response in a hepatitis C-like virus rat model.

The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity, and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus from wild rats (Rattus norvegicus), RHV-rn1; acquired the complete viral genome sequence; and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' untranslated regions. We used site-directed and random mutagenesis to determine that only the first of the two microRNA-122 seed sites in the viral 5' untranslated region is required for viral replication and persistence in rats. Next, we used the clone-derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability to induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation, and macrovesicular and microvesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct-acting antiviral therapy, sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats. CONCLUSION: We developed RHV-rn1-infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity, and pathogenesis. (Hepatology 2018).

The Surface Protein SdrF Mediates Staphylococcus epidermidis Adherence to Keratin.

Background: Staphylococcus epidermidis, a major component of skin flora, is an opportunist, often causing prosthetic device infections. A family of structurally related proteins mediates staphylococcal attachment to host tissues, contributing to the success of S. epidermidis as a pathogen. We examined the ability of the surface protein SdrF to adhere to keratin, a major molecule expressed on the skin surface. Methods: A heterologous Lactococcus lactis expression system was used to express SdrF and its ligand-binding domains. Adherence to keratin types 1 and 10, human foreskin keratinocytes, and nasal epithelial cells was examined. Results: SdrF bound human keratins 1 and 10 and adhered to keratinocytes and epithelial cells. Binding involved both the A and B domains. Anti-SdrF antibodies reduced adherence of S. epidermidis to keratin and keratinocytes. RNA interference reduced keratin synthesis in keratinocytes and, as a result, SdrF adherence. Direct force measurements using atomic force microscopy showed that SdrF mediates bacterial adhesion to keratin 10 through strong and weak bonds involving the A and B regions; strong adhesion was primarily mediated by the A region. Conclusions: These studies demonstrate that SdrF mediates adherence to human keratin and suggest that SdrF may facilitate S. epidermidis colonization of the skin.

In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.

207-nm UV light - a promising tool for safe low-cost reduction of surgical site infections. I: in vitro studies.

BACKGROUND: 0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ~200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure. AIMS: The aim of this work was to test the biophysically-based hypothesis that ~200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues. METHODS: A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue. RESULTS: We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp. CONCLUSIONS: As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.

The role of ionic interactions in the adherence of the Staphylococcus epidermidis adhesin SdrF to prosthetic material.

Staphylococcus epidermidis infections are common complications of prosthetic device implantation. SdrF, a surface protein, appears to play a critical role in the initial colonization step by adhering to type I collagen and Dacron™. The role of ionic interactions in S. epidermidis adherence to prosthetic material was examined. SdrF was cloned and expressed in Lactococcus lactis. The effect of pH, cation concentration, and detergents on adherence to different types of plastic surfaces was assessed by crystal violet staining and bacterial cell counting. SdrF, in contrast with controls and other S. epidermidis surface proteins, bound to hydrophobic materials such as polystyrene. Binding was an ionic interaction and was affected by surface charge of the plastic, pH, and cation concentration. Adherence of the SdrF construct was increased to positively charged plastics and was reduced by increasing concentrations of Ca(2+) and Na(+). Binding was optimal at pH 7.4. Kinetic studies demonstrated that the SdrF B domain as well as one of the B subdomains was sufficient to mediate binding. The SdrF construct also bound more avidly to Goretex™ than the lacotococcal control. SdrF is a multifunctional protein that contributes to prosthetic devices infections by ionic, as well as specific receptor-ligand interactions.

Identification of a highly transmissible animal-independent Staphylococcus aureus ST398 clone with distinct genomic and cell adhesion properties.

UNLABELLED: A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage ϕ3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans. IMPORTANCE: Staphylococcus aureus strains have generally been considered to be species specific. However, cross-species transfers of S. aureus clones, such as ST398 methicillin-resistant S. aureus (MRSA), from swine to humans have been reported. Recently, we observed the emergence of ST398 methicillin-susceptible S. aureus (MSSA) as a colonizing strain of humans in northern Manhattan. Here we report that ST398 is a frequent cause of MSSA infections in this urban setting. The ST398 MSSA clone was readily transmitted within households, independent of animal contact. We discovered that human ST398 MSSA genomes were smaller than that of the LA-ST398 strain S0385 due to fewer mobile genetic elements. Human and LA-ST398 strains also differed in their composition of adhesion genes and their ability to bind to human skin keratinocytes, providing a potential mechanism of S. aureus host adaptation. Our findings illustrate the importance of implementing molecular surveillance of MSSA given the evidence for the rapid and clinically undetected spread of ST398 MSSA.

Similar effects of selective laser trabeculoplasty and prostaglandin analogs on the permeability of cultured Schlemm canal cells.

PURPOSE: To evaluate whether selective laser trabeculoplasty and prostaglandin analogs regulate the permeability of cultured human Schlemm canal cells by inducing intercellular junction disassembly. DESIGN: Laboratory investigation. METHODS: Intercellular junctions were made visible in living cells by making them fluoresce after transfection with a plasmid expressing the zonula occludens 1 protein tagged with green fluorescent protein. Schlemm canal cells were treated by direct laser irradiation; by exposure to media conditioned by either lasered Schlemm canal cells or trabecular meshwork cells; by exposure to the prostaglandin analogs latanoprost, bimatoprost, and travoprost; or by the addition of the nonprostaglandin agents brimonidine, timolol, and dorzolamide. Junction disassembly was monitored using fluorescence microscopy, and permeability alterations were measured as changes in conductivity using flow meters. RESULTS: The direct laser irradiation of Schlemm canal cells caused a 3-fold increase in conductivity. Exposure of the cells to media conditioned by lasered Schlemm canal cells or trabecular meshwork cells induced junction disassembly and a 2- to 4-fold increase in conductivity. Exposure to prostaglandin analogs also induced junction disassembly and a 4- to 16-fold increase in conductivity, whereas the 3 nonprostaglandin agents tested were ineffective in both regards. CONCLUSIONS: Exposure to factors secreted by lasered Schlemm canal cells and lasered trabecular meshwork cells and the application of prostaglandin analogs induced junction disassembly while increasing the permeability of Schlemm canal cells. These findings support our hypothesis that selective laser trabeculoplasty and prostaglandin analogs share a common mechanism that likely mediates their pressure-lowering effects.

Monocyte modulation of aqueous outflow and recruitment to the trabecular meshwork following selective laser trabeculoplasty.

OBJECTIVES: To determine whether selective laser trabeculoplasty (SLT) induces monocyte recruitment to the trabecular meshwork (TM) in human and monkey eyes and whether monocytes increase both aqueous outflow in vivo and the conductivity of human Schlemm canal endothelial cells (SCEs) in vitro. METHODS: Monocyte recruitment was examined morphometrically in control human and monkey eyes and compared with that following SLT applied 1 to 3 days earlier. Outflow facility was measured for up to 4 days after the intracameral infusion of autologous macrophages in rabbits. Schlemm canal endothelial cell conductivity was measured using flow meters after exposing cultured SCEs to monocytes and monocyte-secreted factors for 24 hours. RESULTS: Our estimates show that the TM in the human eye normally had an average of 15 003 monocytes, while in the monkey eye there were 3181 monocytes, and this number increased 4- to 5-fold following SLT. The intracameral infusion of autologous macrophages in rabbits increased outflow facility 2-fold in a rapid and sustained manner. Human monocytes and monocyte-secreted factors increased SCE conductivity 2-fold in vitro. CONCLUSIONS: The number of monocytes/macrophages in the TM increases substantially after SLT and monocytes augment both outflow facility and SCE conductivity. Clinical Relevance These findings indicate that the innate immune system in general and monocytes in particular play an important role in aqueous outflow homeostasis. The recruitment of monocytes in increased numbers after SLT likely plays a role in lowering the intraocular pressure after this procedure. The intracameral introduction of autologous monocytes harvested from a vein could have therapeutic potential as a cell-based individualized treatment of glaucoma.