Our results demonstrate marginally significant prevalence of C pneumoniae DNA in patients with CAD compared with healthy subjects (P=0.082). In contrast, the prevalence of IgG seropositivity among the 2 groups did not reach statistical significance (P=0.306). We also provide unequivocal evidence for the presence of C pneumoniae DNA predominantly among the circulating CD3+ T-cell population.

A Agtll recombinant library of Chlamydia trachomatis serovar L2 chromosomal DNA was screened with a 29-mer synthetic oligonucleotide specific to the N-terminal amino acids of a predominant 18-kDa chlamydial protein. (11,23), they are placed in a distinct order, Chlamydiales (18).The presence of a condensed, discrete, and electron-dense nucleoid in EB is a notable feature, unique among members of the family Eubacteriaceae (2). Wagar and Stephens reported three DNA-binding proteins with molecular masses of 58, 25, and 17 kDa specific to the EB form of chlamydiae (21). Nucleoproteins that are involved in DNA compaction and transcriptional regulation have been identified among prokaryotes (3, 13). These nucleoproteins share biochemical properties, such as DNA packaging, charge, and low molecular weights, with eukaryotic histones. All higher organisms contain small, basic DNA-binding proteins called histones which are conserved in their primary structure (10).We report here the cloning and sequencing of an 18-kDa developmentally regulated protein-encoding gene from C.

Recently we demonstrated that normal polyspecific immunoglobulin given intravenously (IVIG) and plasma samples from patients treated with IVIG neutralize the mitogenic and cytokine-inducing activities of group A streptococcal (GAS) superantigens. Here we investigated whether this neutralizing activity is mediated by antibodies to these superantigens. IVIG and plasma samples collected from a patient with GAS necrotizing fasciitis post-IVIG infusions markedly inhibited the mitogenic activity elicited by the streptococcal pyrogenic exotoxins SpeB and SpeC, as well as by GAS culture supernatant. Immunoblot analysis showed marked increases in the levels of antibodies to SpeC and proteins in the GAS culture supernatant in post-IVIG over those of pre-IVIG plasma samples. Removal of antisuperantigen antibodies in IVIG by adsorption to SpeC-and GAS culture supernatant-coupled Sepharose markedly reduced the neutralizing ability of IVIG against respective stimuli. The neutralizing activity was totally recovered in the eluted antibodies. By contrast, although pre-and post-IVIG plasma samples contained antibodies to SpeA, these antibodies did not block the activity of this superantigen. Nonspecific immunomodulatory activity of IVIG was ruled out because neither the IVIG nor the affinity-purified antibodies significantly inhibited the response to the polyclonal T-cell mitogen phytohemagglutinin A. These data provide direct evidence that the neutralizing activity in IVIG, and in patient plasma samples following IVIG treatment is mediated by antibodies to superantigens and indicate that the quality rather than the quantity of these antibodies may be more clinically relevant.

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies downregulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar L 2 with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37°C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase ␣-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.Chlamydia trachomatis is an obligate intracellular bacterium with a complex life cycle that involves two developmental forms, the extracellular, small (0.2-to 0.3-m) elementary bodies (EBs) and the intracellular, large (1-m) fragile reticulate bodies (RBs) (10, 14). The EB is metabolically inactive and resistant to osmotic lysis due to its rigid outer membrane. Proliferation of the organism is initiated by EB uptake and enlargement in the host cell, circumventing phagosomal fusion to form noninfectious, metabolically active RBs which divide within the cytoplasmic inclusions by binary fission (19). The life cycle is completed with the reorganization of RBs into EBs, which subsequently leave the disrupted host cell ready to infect new cells (29). However, little is known about the signals that trigger thes...

BACKGROUND The SARS-CoV-2 virus has mutated and evolved since the inception of the COVID-19 pandemic bringing into question the future effectiveness of current vaccines and antibody therapeutics. With evolution of the virus updated methods for the evaluation of the immune response in infected and vaccinated individuals are required to determine the durability of the immune response to SARS-CoV-2 variants. METHODS We developed a multiplexed surrogate virus neutralization test (plex-sVNT) that simultaneously measures the ability of antibodies in serum to inhibit binding between angiotensin converting enzyme-2 (ACE2) and 7 SARS-CoV-2 trimeric spike protein variants, including wild type, B.1.1.7(α), B.1.351(β), P.1(γ), B.1.617.2(δ), B.1.617.1(κ), and B.1.429(ε). The assay was validated against a plaque reduction neutralization test (PRNT). We evaluated 170 samples from 97 COVID-19 patients and 281 samples from 188 individuals that received the Pfizer-BioNTech or Moderna mRNA vaccines. RESULTS The plex-sVNT demonstrated >96% concordance with PRNT. Antibody neutralization activity was significantly reduced for all SARS-CoV-2 variants compared to wild type in both the infected and vaccinated cohorts. There was a decline in overall antibody neutralization activity, within both cohorts, out to 5 months post infection or vaccination, with the rate of decline being more significant for the vaccinated. CONCLUSIONS The plex-sVNT provides a correlative measure to PRNT and a convenient approach for evaluating antibody neutralization against SARS-CoV-2 variants. Neutralization of SARS-CoV-2 variants is reduced compared to wild type and declines over the ensuing months after exposure or vaccination within each cohort, however it is still unknown what degree of neutralizing capacity is protective.