Anti-hantavirus antibodies in human sera in Czechoslovakia.

By indirect immunofluorescence using antigens of hantavirus Hantaan and CG 18-20 on Vero E6 cells were examined 5,827 samples of sera from 5,299 probands of Czechoslovakia. In 49 persons (0.94%) were found antibodies of titres 1:32 and higher. Two groups of elderly farm workers showed a cluster of positive individuals amounting to 9.9% and 29.4% respectively. The ratio of positivities in some other, randomly and specifically selected groups was deep below 1%. The partial results for the group of farmers were confirmed by RIA test. Occasionally antibodies only to one of the hantavirus serotypes, at other time, to both were found. The authors discuss the findings of antibodies of the two serotypes in humans as related to the evidenced existence of two hantavirus antigen serotypes in animals.     

Anti-O-phosphotyrosine antibodies in human sera

Antibodies reactive with O-phosphotyrosine (PTYR) were detected in 60 out of 621 inpatients, with high frequencies in hematologic and lung malignancies, hepatic diseases, cerebrovascular diseases, and autoimmune diseases. Affinity-purified antibodies proved capable of recognizing PTYR-containing proteins in a human carcinoma cell line, A431, both by immunofluorescent staining and by immunoaffinity chromatography, but had no detectable affinity for phosphorylated serine or threonine, or for the nucleotides tested. In these respects, the antibodies observed in human sera were indistinguishable from anti-PTYR antibodies raised experimentally in rabbits or mice.     

Anti-infective activity of anti-influenza antibodies from human colostrum]

Antibodies against influenza virus of A, B serological types and PC-virus were detected in the colostrum collected after an epidemic. These antibodies belonged to the secretory IgA form. The secretory antibodies preparation made of colostrum, and its IgA fraction instilled intransally to mice and rats prevented the development of infection caused by 100--10 ID50/0.1 ml of the influenza A virus. The protective action of the antibodies of IgA class was due to its capacity to become fixed on the surface of the cells of the mucosal epithelium of the respiratory tract.     

Prevalence of antibodies to human papillomavirus type 8 in human sera.

The epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV-8) poses a high risk for malignant conversion of skin lesions in patients with epidermodysplasia verruciformis. For seroepidemiological studies, the HPV-8 open reading frames for E1, E2, E4, E6, E7, and L1 were bacterially expressed as beta-galactosidase fusion proteins, which were purified by preparative gel electrophoresis. Cleavage with the protease FXa at the engineered recognition site separated the beta-galactosidase polypeptide part from the viral polypeptide. Western blot analysis of 445 serum samples from a randomly selected population with the entire L1 as antigen revealed HPV-8-specific immunoglobulin G antibodies in 20% of the samples. The percentage of positive sera did not significantly differ in different age groups. In some sera, we could also detect immunoglobulin M antibodies. The use of two shortened L1 polypeptides as antigen indicated that there are at least two reactive epitopes in the case of HPV-8 L1. Several sera contained antibodies to the early proteins E1, E2, E4, and E7. E1 and E7 were predominantly detected by sera which were negative for L1. In one case, we found antibodies to E6. Two of four sera of patients with epidermodysplasia verruciformis reacted with HPV-8 L1. The prevalence of anti-HPV-8-L1 antibodies in patients with malignant melanomas was comparable to that in the normal population (27.8%) but was significantly higher in patients with cervical cancer (37.5%), basaliomas (40%), and squamous cell skin carcinomas (72.7%) and in immunocompromised patients with Hodgkin's disease (47.7%).     

Isotype profiles of anti-influenza antibodies in mice bearing the xid defect.

The humoral response to influenza A/PR8 virus was examined in the CBA/N and C3J.xid strains of mice, both of which bear an X-linked genetic defect (xid), and in strains lacking this defect. Hemagglutination-inhibiting antibody titers and measurement of virus-specific antibodies by solid-phase radioimmunoassay indicated that the xid defect does not impair the production of an adequate anti-influenza antibody response. However, investigation of the isotypes of PR8 virus-specific antibodies disclosed a relative decrease in the levels of IgG3 and IgG1 in the xid-bearing strains. This was observed after both intraperitoneal immunization and aerosol infection. The isotype differences were not reflected in the susceptibility of these strains to influenza virus infection.     

Characterization of DNA used to assay sera for anti-DNA antibodies; determination of the specificities of anti-DNA antibodies in SLE and non-SLE rheumatic disease states.

Commercial 14C-labeled KB cell DNA, widely used to assay sera for anti-DNA antibodies, was chromatographed on benzoylated-naphthoylated-DEAE-cellulose (BNDC) and on hydroxyapatite (HAP). On BNDC, only 25% of the 14C label eluted with 1 M NaC1 (KB fraction I) characteristic of ds-DNA. Fifty-five percent of the label eluted with 50% formamide-1 M NaC1 (KB fraction II) characteristic of ss or denatured DNA. On HAP, however, none of the 14C label eluted with 0.2 M phosphate buffer as anticipated for ss-DNA, but, rather, all of the 14C label eluted with 0.4 M phosphate, characteristic of ds-DNA. after pretreatment with S1 endonuclease of Aspergillus oryzae, which selectively digests ss regions, however, 42% of the 14C label was lost from the 0.4 M phosphate peak. These results indicated that more than half of this 14C-KB-cell DNA preparation was ds-DNA with ss regions which was undetectable by HAP chromatography. 3H-ds-DNA and circular 3H-ss-DNA prepared from T7 and phiX174 bacteriophage, respectively, were found to be chromatographically pure on both BNDC and HAP. None of 10 non-SLE sera (rheumatoid arthritis 3, mixed connective tissue disease 4, scleroderma 1, ulcerative colitis 1, and pulmonary fibrosis with chronic active hepatitis 1), previously believed to contain anti-ds-DNA antibodies on the basis of KB cell DNA testing and detectable antibodies against KB fraction 1 or T7 DNA: all of 10 KB cell DNA positive SLE sera had antibodies against both. Additionally, none of the 10 non-SLE sera had antibodies against KB cell DNA when retested with DNA that had been pretreated with S1 endonuclease. Seven of these 10, however, as well as all 10 SLE sera, had antibodies against phiX174 DNA, KB fraction II DNA and alkali-denatured T7 DNA. The data support the conclusions that 1) false positive tests for anti-ds-DNA antibodies can result from contamination of ds-DNA with ds-DNA having ss regions, and 2) non-SLE sera do not contain antibodies specific for ds-DNA at levels comparable to those found in SLE sera but rather contain high levels of antibodies reacting with ss regions or mixed DNA.     

Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy

The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar–protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.     

Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D

Background

Surfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens.

Methods

We tested antiviral activities of recombinant human SP-D against a panel of IAV strains that vary in glycosylation sites on their hemagglutinin (HA). For these experiments a recombinant version of human SP-D of the Met11, Ala160 genotype was used after it was characterized biochemically and structurally.

Results

Oligosaccharides at amino acid 165 on the HA in the H3N2 subtype and 104 in the H1N1 subtype are absent in collectin-resistant strains developed in vitro and are important for mediating antiviral activity of SP-D; however, other glycans on the HA of these viral subtypes also are involved in inhibition by SP-D. H3N2 strains obtained shortly after introduction into the human population were largely resistant to SP-D, despite having the glycan at 165. H3N2 strains have become steadily more sensitive to SP-D over time in the human population, in association with addition of other glycans to the head region of the HA. In contrast, H1N1 strains were most sensitive in the 1970s–1980s and more recent strains have become less sensitive, despite retaining the glycan at 104. Two H5N1 strains were also resistant to inhibition by SP-D. By comparing sites of glycan attachment on sensitive vs. resistant strains, specific glycan sites on the head domain of the HA are implicated as important for inhibition by SP-D. Molecular modeling of the glycan attachment sites on HA and the carbohydrate recognition domain of SPD are consistent with these observations.

Conclusion

Inhibition by SP-D correlates with presence of several glycan attachment sites on the HA. Pandemic and avian strains appear to lack susceptibility to SP-D and this could be a contributory factor to their virulence.     

Comparative specificities of trehalases from various species.

1. Using derivatives or non-symmetrical analogs of alpha,alpha-trehalose, we studied the catalytic specificities of trehalases from various species: Pseudomonas fluorescens, Melolontha vulgaris, porcine and human kidneys. 2. alpha,Beta-trehalose, beta,beta-trehalose, 6,6'dideoxy alpha,alpha-trehalose, alpha-D-xylopyranosyl alpha-D-xylopyranoside were shown to be neither substrates nor inhibitors. 3. 6'deoxy alpha,alpha-trehalose, alpha-D-glucopyranosyl alpha-D-xylopyranoside, alpha-D-allopyranosyl alpha-D-glucopyranoside and alpha-D-galactosyl alpha-D-glucopyranoside, which all possess an intact alpha-D-glucopyranosyl residue, were split by all these trehalases. 4. alpha-D-glucopyranosyl alpha-D-mannopyranoside, alpha,alpha-trehalosamine are competitive inhibitors. 5. These results show the importance of the primary alcohol group at C-6, of the equatorial configuration of the OH groups at C-2, C-3 and C-4 and of the modification of the structure at C-2 of the substrate for the catalytic activity.     

Cytomegalovirus early and late membrane antigens detected by antibodies in human convalescent sera.

Using indirect immunofluorescence and a panel of human convalescent-phase sera, we identified cytomegalovirus (CMV) early and late membrane antigens (CMV-EMA and CMV-LMA, respectively) as separate entities on the surfaces of viable CMV-infected fibroblasts starting at 6 to 12 and 36 to 48 h postinoculation, respectively. For expression of CMV-EMA and CMV-LMA, infectious virus and active protein synthesis were required, whereas the expression of CMV-LMA, in addition, required viral DNA synthesis. Our data suggest that CMV-EMA and CMV-LMA form an individual set of CMV antigens that are different from intracellular CMV antigens and possibly (partly) different from the viral envelope.     

Protective antibodies in human sera against encapsulated strains of Staphylococcus epidermidis

Passive protective antibodies in 100 samples of normal human sera against challenge with three representative capsular type strains of Staphylococcus epidermidis in mice were examined. Six of them passively protected mice against capsular type I; 17 protected against capsular type II; one against capsular type III; and one against both capsular types I and II. The activities were sensitive to 2-mercaptoethanol and were absorbed out either with rabbit anti-human IgG serum, rabbit anti-human IgA serum or rabbit anti-human IgM serum. Also, the sera activities absorbed out with cell surface polysaccharide extracted from three representative capsular type strains. These results indicate that the protective activities were specifically related to three major immunoglobulins against the above cell surface polysaccharides.