Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody.

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

Evaluation of activity of Graves' orbitopathy with multiparameter orbital magnetic resonance imaging (MRI).

Background: When quantitative magnetic resonance imaging (MRI) is used to assess the activity of Graves' orbitopathy (GO), the examination is generally focused on a specific orbital tissue, especially the extraocular muscles (EOMs). However, GO usually involves the entire intraorbital soft tissue. The aim of this study was to use multiparameter MRI on multiple orbital tissues to distinguish the active and inactive GO. Methods: From May 2021 to March 2022, consecutive patients with GO were prospectively enrolled at Peking University People's Hospital (Beijing, China) and divided into those with active disease and those with inactive disease based on a clinical activity score. Patients then underwent MRI, including sequences of conventional imaging, T1 mapping, T2 mapping, and mDIXON Quant. Width, T2 signal intensity ratio (SIR), T1 values, T2 values, and fat fraction of EOMs, as well as water fraction (WF) of orbital fat (OF), were measured. Parameters were compared between the 2 groups, and a combined diagnostic model was constructed using logistic regression analysis. Receiver operating characteristic analysis was used to test the diagnostic performance of the model. Results: Sixty-eight patients with GO (27 with active GO, 41 with inactive GO) were included in the study. The active GO group had higher values of EOM thickness, T2 SIR, and T2 values, as well as higher WF of OF. The diagnostic model, which included EOM T2 value and WF of OF, demonstrated a good ability to distinguish between active and inactive GO (area under the curve, 0.878; 95% CI: 0.776-0.945; sensitivity, 88.89%; specificity, 75.61%). Conclusions: A combined model incorporating the T2 value of EOMs and the WF of OF was able to identify cases of active GO, potentially offering an effective and noninvasive method to assess pathological changes in this disease.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY-GUIDED PHOTODYNAMIC THERAPY FOR ACUTE CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To compare the anatomic results of optical coherence tomography angiography (OCTA)-guided half-dose photodynamic therapy (PDT) versus indocyanine green angiography (ICGA)-guided PDT in eyes with acute central serous chorioretinopathy. METHODS: This study is a prospective, single-center, noninferiority, double-masked, randomized, controlled clinical trial. Fifty-one eyes of 45 patients with acute central serous chorioretinopathy were recruited, and randomized to an ICGA-guided group and an OCTA-guided group. The primary outcome measures were the rates of complete subretinal fluid (SRF) resolution at 1 month and 3 months. RESULTS: Forty-six eyes of 40 patients finished the follow-up and were analyzed. In the OCTA-guided group, the SRF was completely resolved in 13 (56.5%) eyes within 1 month and in 21 (91.3%) eyes within 3 months. In the ICGA-guided group, the SRF was resolved in 16 (69.6%) of the eyes within 1 month and in 22 (95.7%) of the eyes by 3 months. Optical coherence tomography angiography-guided PDT was demonstrated noninferior to ICGA-guided PDT for SRF resolution rate at 3 months (P = 0.016), but not at 1 month (P = 0.311) for acute central serous chorioretinopathy patients. Subretinal fluid did not recur in any of the eyes in the OCTA-guided group, but did recur in 2 eyes (8.7%) of the ICGA-guided group during the 3-month follow-up period. CONCLUSION: Optical coherence tomography angiography-guided PDT seemed to be noninferior to ICGA-guided PDT for resolution of SRF at 3 months in eyes with acute central serous chorioretinopathy.

Short-Term Results of Switch from Conbercept to Bevacizumab or Ranibizumab in Eyes with Persistent Neovascular Age-Related Macular Degeneration.

PURPOSE: To study the short-term anatomical and functional outcomes in patients with neovascular age-related macular degeneration (nAMD) who were previously treated with conbercept and switched to ranibizumab or bevacizumab due to persistent activity. METHODS: This retrospective single-arm study included nAMD patients who were followed up for at least three months after switching from at least 3 monthly intravitreal conbercept injections to bevacizumab or ranibizumab for persistent choroidal neovascularization (CNV) activity. The demographic data, treatments, best-corrected visual acuity (BCVA), central macular thickness (CMT), and the height of pigmented epithelial detachment (PED) before and after switching were recorded and analyzed. RESULTS: A total of 64 eyes of 64 patients were included with a mean follow-up of 9.6 ± 3.0 months. The average number of injections of conbercept was 3.6 ± 0.8 (range, 3-5) before switching. 18 eyes were switched to bevacizumab, and the other 46 eyes were switched to ranibizumab. After switching, mean BCVA slowly improved from 0.73 ± 0.48 to 0.64 ± 0.41 (p=0.0132) at one month after the last intravitreal injection of ranibizumab or bevacizumab during the mean follow-up of 4.4 ± 2.0 months. One month after switching, the mean CMT decreased significantly from 294.9 ± 121.8 µm to 230.9 ± 107.0 µm (p < 0.0001) and kept stable during the follow-up. There was a significant reduction of maximum PED height (mPEDH) at the first month after switching (from 384.3 ± 340.3 µm to 287.2 ± 245.2 µm, p=0.0018) and kept stable during the follow-up. The mean PED height at foveal center (cPEDH) showed a regression over time after switching (from 169.3 ± 230.6 µm to 130.5 ± 180.2 µm, p=0.0227) and also kept stable during the follow-up. The proportion of patients with IRF was slightly increased but not statistically significant before switching. After switching, this proportion decreased significantly from 96.9% to 81.3% at one month after the first intravitreal injection of ranibizumab or bevacizumab (p=0.0086). The proportion of patients with SRF did not change significantly before and after switching. The mean decrease of mPEDH and cPEDH at the last follow-up after switching was significantly larger in the IVR subgroup than in the IVB subgroup (p=0.023 and 0.010). CONCLUSION: Our results indicate that switching from intravitreal conbercept injections to bevacizumab or ranibizumab can lead to significant improvement of CMT, PED, and IRF and slight improvement of BCVA in a short period of time for persistent nAMD patients.

Optical Coherence Tomography Angiography Compared with Indocyanine Green Angiography in Central Serous Chorioretinopathy.

The purpose of this article is to compare optical coherence tomography angiography (OCTA) and indocyanine green angiography (ICGA) in patients with central serous chorioretinopathy (CSC). OCTA, ICGA and fluorescein angiography (FA) images of all enrolled patients were collected and compared. Abnormal areas were annotated on en face choriocapillaris OCTA and ICGA images and compared with each other. We found three main types of anomalies in choriocapillaris OCTA images: type A, coarse granulated high reflective area (61 eyes [92.4%]); type B, roundish dark halo around Type A (54 eyes [81.8%]); and type C, coarse granulated low reflective area (66 eyes [100%]). There were 54 eyes (81.8%) that exhibited all three types abnormalities, 7 (10.6%) had only type A and C abnormalities, and 5 (7.6%) had only type C abnormalities. The Mean JI of type A on OCTA and hyperfluorescence area on ICGA was 0.84 ± 0.15 and 0.82 ± 0.23 for grader 1 and 2, respectively. Type A area on OCTA had a statistically larger area than hyperfluorescence on ICGA (P = 0.01 [paired t-test]). In summary, abnormalities were found on OCTA images of CSC. Coarse granulated high reflective area in OCTA corresponded well with the hyper-permeability area in ICGA in most of the eyes.

A functional polymorphism in the promoter of αA-crystallin increases the risk of nAMD.

OBJECTIVE: To analyze the association between the promoter of αA-crystallin (CRYAA) variants with neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in a northern Chinese population. METHODS: We performed a case-control study in a group of Chinese patients with nAMD (n = 345) or PCV (n = 371) and contrasted the results against an independent control group comprising 514 mild cataract patients without any evidence of age-related maculopathy. An association analysis of allele frequencies was performed for 6 single-nucleotide polymorphisms (SNPs) at the CRYAA locus (rs3761381, rs3761382, rs79545821, rs13053109, rs7278468, and rs117396767). Differences in the observed genotypic distributions between the cases and controls were tested using chi-square tests, and logistic regression models were used to calculate the odds ratio (OR) and 95% confidence interval (CI) of nAMD or PCV. RESULTS: The CRYAA rs7278468 variant was significantly associated with neovascular age-related macular degeneration (OR = 1.253, 95% CI 1.018-1.542, P = 0.033). No association was detected between the other five SNPs and nAMD (P > 0.05). No association was detected between these six SNPs and PCV (P > 0.05). CONCLUSIONS: Our data suggest CRYAA rs7278468 increases the risk of nAMD. The data might provide crucial information for future clinical studies on the mechanisms of nAMD and may require larger studies to accurately dissect.

A Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice From Secondary Pneumococcal Pneumonia.

BACKGROUND: Secondary bacterial infections after influenza can be a serious problem, especially in young children and the elderly, yet the efficacy of current vaccines is limited. Earlier work demonstrated that a replication-incompetent PB2-knockout (PB2-KO) influenza virus possessing a foreign gene in the coding region of its PB2 segment can serve as a platform for a bivalent vaccine. METHODS: In the current study, we generated the PB2-KO virus expressing pneumococcal surface protein A (PspA), PB2-KO-PspA virus, the replication of which is restricted to PB2-expressing cells. We then examined the protective efficacy of intranasal immunization with this virus as a bivalent vaccine in a mouse model. RESULTS: High levels of influenza virus-specific and PspA-specific antibodies were induced in the serum and airways of immunized mice. The intranasally immunized mice were protected from lethal doses of influenza virus or Streptococcus pneumoniae. These mice were also completely protected from secondary pneumococcal pneumonia after influenza virus infection. CONCLUSIONS: These findings indicate that our recombinant influenza virus serves as a novel and powerful bivalent vaccine against primary and secondary pneumococcal pneumonia as well as influenza.

A bivalent vaccine based on a replication-incompetent influenza virus protects against Streptococcus pneumoniae and influenza virus infection.

UNLABELLED: Streptococcus pneumoniae is a major causative pathogen in community-acquired pneumonia; together with influenza virus, it represents an important public health burden. Although vaccination is the most effective prophylaxis against these infectious agents, no single vaccine simultaneously provides protective immunity against both S. pneumoniae and influenza virus. Previously, we demonstrated that several replication-incompetent influenza viruses efficiently elicit IgG in serum and IgA in the upper and lower respiratory tracts. Here, we generated a replication-incompetent hemagglutinin knockout (HA-KO) influenza virus possessing the sequence for the antigenic region of pneumococcal surface protein A (PspA). Although this virus (HA-KO/PspA virus) could replicate only in an HA-expressing cell line, it infected wild-type cells and expressed both viral proteins and PspA. PspA- and influenza virus-specific antibodies were detected in nasal wash and bronchoalveolar lavage fluids and in sera from mice intranasally inoculated with HA-KO/PspA virus, and mice inoculated with HA-KO/PspA virus were completely protected from lethal challenge with either S. pneumoniae or influenza virus. Further, bacterial colonization of the nasopharynx was prevented in mice immunized with HA-KO/PspA virus. These results indicate that HA-KO/PspA virus is a promising bivalent vaccine candidate that simultaneously confers protective immunity against both S. pneumoniae and influenza virus. We believe that this strategy offers a platform for the development of bivalent vaccines, based on replication-incompetent influenza virus, against pathogens that cause respiratory infectious diseases. IMPORTANCE: Streptococcus pneumoniae and influenza viruses cause contagious diseases, but no single vaccine can simultaneously provide protective immunity against both pathogens. Here, we used reverse genetics to generate a replication-incompetent influenza virus carrying the sequence for the antigenic region of pneumococcal surface protein A and demonstrated that mice immunized with this virus were completely protected from lethal doses of infection with either influenza virus or Streptococcus pneumoniae. We believe that this strategy, which is based on a replication-incompetent influenza virus possessing the antigenic region of other respiratory pathogens, offers a platform for the development of bivalent vaccines.

Protective properties of a fusion pneumococcal surface protein A (PspA) vaccine against pneumococcal challenge by five different PspA clades in mice.

An increase in the appearance of nonvaccine serotypes in both children and adults with invasive pneumococcal disease (IPD) after introduction of pneumococcal conjugate vaccine represents a limitation of this vaccine. In this study, we generated three recombinant pneumococcal surface protein A (PspA) proteins comprising PspA families 1 and 2, and we examined the reactivity of antisera raised in mice immunized with a PspA fusion protein in combination with CpG oligonucleotides plus aluminum hydroxide gel. The protective effects of immunization with PspA fusion proteins against pneumococcal challenge by strains with five different PspA clades were also examined in mice. Flow cytometry demonstrated that PspA3+2-induced antiserum showed the greatest binding of PspA-specific IgG to all five challenge strains with different clades. PspA2+4- or PspA2+5-induced antiserum showed the lowest binding of PspA-specific IgG to clade 3. Immunization with PspA3+2 afforded significant protection against pneumococcal challenge by five strains with different clades in mice, but immunization with PspA2+4 or PspA2+5 failed to protect mice from pneumococcal challenge by strains with clades 1 and 3. The binding of PspA-specific IgG in antisera raised by three PspA fusion proteins was examined in 68 clinical isolates from adult patients with IPD. Immunization of mice with PspA3+2-induced antiserum with a high binding capacity for clinical isolates expressing clades 1-4, but not clade 5. Our results suggest that the PspA3+2 vaccine has an advantage over the PspA2+4 or PspA2+5 vaccine in terms of a broad range of cross-reactivity with clinical isolates and cross-protection against pneumococcal challenge in mice.

Importance of the icmN gene in the growth of Legionella pneumophila in amoebic cells at low temperature.

Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37 °C. In particular, intracellular growth in amoebae was completely impaired at 25 °C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37 °C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37 °C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.

Intranasal vaccination with pneumococcal surface protein A plus poly(I:C) protects against secondary pneumococcal pneumonia in mice.

Effective pneumococcal vaccines are required for preventing secondary bacterial pneumonia, a life-threatening condition, during epidemics of influenza. We examined whether nasal administration of a low dose of pneumococcal surface protein A (PspA) plus polyinosinic-polycytidylic acid (poly(I:C)) could protect against a fatal secondary pneumococcal pneumonia after influenza A virus infection in mice. PspA-specific IgG but not IgA level was higher in the airways and blood of mice nasally administered a low dose of PspA plus poly(I:C) than in mice nasally administered PspA alone or poly(I:C) alone. Binding of PspA-specific IgG increased C3 deposition on the bacterial surface. The survival rate during secondary infection was higher in mice immunized with PspA plus poly(I:C) than in mice immunized with poly(I:C) alone. The significant reduction in bacterial density in the lung and blood was associated with increased survival of immunized mice with secondary pneumonia. Passive transfer of sera from mice immunized with PspA plus poly(I:C) increased the survival of mice infected with secondary pneumonia. Our data suggest that an intranasal PspA vaccine has promising protective effects against secondary pneumonia after influenza and that PspA-specific IgG plays a critical role in this protection.

A novel insertion sequence, IS1642, of Mycobacterium avium, which forms long direct repeats of variable length.

A new insertion sequence (IS), IS1642, was identified in a Mycobacterium avium strain isolated from a human patient. IS1642 had a size of 1642 bp and contained a single ORF encoding a probable transposase of 503 amino acid residues homologous (79% identity) to that of IS1549 found in Mycobacterium smegmatis. The IS1642 included imperfect inverted repeats (5'-cctgacttttatca-3', 5'-tgataaaagtcggg-3') on its ends, and was flanked by direct repeats of variable length ranging from 5 to 161 bp. It was suggested that the IS1642 was widely distributed in many M. avium strains of human patients, and the Southern blot profile of IS1642 was very diverse among the strains examined. The transposition event of IS1642 was observed by in vitro repeated passages, showing that the IS1642 is actually a transposable element. In light of these characteristics, IS1642 could be a new useful marker when genotyping with high discrimination is required.

Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila.

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.