Kari Johansen

Mucosal and Plasma IgA from HIV-1-Exposed Uninfected Individuals Inhibit HIV-1 Transcytosis Across Human Epithelial Cells

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

Evolution of human calicivirus RNA in vivo: accumulation of mutations in the protruding P2 domain of the capsid leads to structural changes and possibly a new phenotype.

ABSTRACT In the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype. FUT2 gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor+, a finding in accordance with the hypothesis of FUT2 gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with the FUT2 gene characterization, may lead to a better understanding of calicivirus pathogenesis.

Lactobacilli Expressing Variable Domain of Llama Heavy-Chain Antibody Fragments (Lactobodies) Confer Protection against Rotavirus-Induced Diarrhea

BACKGROUND Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable domain of llama heavy-chain (VHH) antibody fragments against rotavirus. METHODS VHH were expressed in Lactobacillus paracasei, in both secreted and cell surface-anchored forms. Electron microscopy was used to investigate the binding efficacy of VHH-expressing lactobacilli. To investigate the in vivo function of VHH-expressing lactobacilli, a mouse pup model of rotavirus infection was used. RESULTS Efficient binding of the VHH antibody fragments to rotavirus was shown by enzyme-linked immunosorbent assay and scanning electron microscopy. VHH fragments expressed by lactobacilli conferred a significant reduction in infection in cell cultures. When administered orally, lactobacilli-producing surface-expressed VHH markedly shortened disease duration, severity, and viral load in a mouse model of rotavirus-induced diarrhea when administered both fresh and in a freeze-dried form. CONCLUSIONS Transformed lactobacilli may form the basis of a novel form of prophylactic treatment against rotavirus infections and other diarrheal diseases.

VIRAL DIARRHEA IN CHILDREN IN BEIJING, CHINA

A study was undertaken from November 1994 to August 1996 to determine the role of viruses in children (⩽5 years of age) hospitalized at Beijing Children Hospital, Beijing China, for acute diarrhea. Stool samples from diarrheal patients were investigated by ELISA, electron microscopy, and RT‐PCR for the presence of rotavirus, calicivirus, astrovirus, and adenovirus. Group A rotavirus was detected in 55.9% of all diarrheal patients and comprised 82.5% of all viruses detected. Group A rotavirus samples were further characterized for their G‐type specificity by RT‐PCR. Four major G types (1–4) were identified. G1 to G4 accounted for 58.9%, 15.7%, 16.8%, and 6.3%, respectively, of the serotyped samples. Almost all rotavirus infections occurred in children less than 1 year of age, with a significant clustering during the winter months. Group C rotavirus was detected in one 18‐month‐old child. Astroviruses, caliciviruses, and adenoviruses were detected in 8.5%, 7.6%, and 2.5% of the hospitalized children, respectively. This, the first viral etiological study of childhood diarrhea in China, concludes that rotavirus G1–4 strains play an important role in severe diarrhea in Beijing children. J. Med. Virol. 57:390–396, 1999.

Comparison of rubella seroepidemiology in 17 countries: progress towards international disease control targets

OBJECTIVE To standardize serological surveillance to compare rubella susceptibility in Australia and 16 European countries, and measure progress towards international disease-control targets. METHODS Between 1996 and 2004, representative serum banks were established in 17 countries by collecting residual sera or community sampling. Serum banks were tested in each country and assay results were standardized. With a questionnaire, we collected information on current and past rubella vaccination programmes in each country. The percentage of seronegative (< 4 IU/ml) children (2-14 years of age) was used to evaluate rubella susceptibility, and countries were classified by seronegativity as group I (< 5%), group II (5-10%) or group III (> 10%). The proportion of women of childbearing age without rubella protection (< or = 10 IU/ml) was calculated and compared with WHO targets of < 5%. FINDINGS Only Romania had no rubella immunization programme at the time of the survey; the remaining countries had a two-dose childhood schedule using the measles, mumps and rubella (MMR) vaccine. The percentage of susceptible children defined five countries as group I, seven as group II and four as group III. Women of childbearing age without rubella protection were < 5% in only five countries. CONCLUSION Despite the low reported incidence in many countries, strengthening the coverage of the routine two-dose of MMR vaccine among children is needed, especially in group III countries. Catch-up campaigns in older age groups and selective targeting of older females are needed in many countries to ensure necessary levels of protective immunity among women of childbearing age.

Incidence and estimates of the diease burden of rotavirus in Sweden

Laboratory and hospitalization data from two childrens hospitals with large primary catchment areas and national laboratory and hospitalization data for children under 4 y of age with acute diarrhoea were compiled to estimate the number of hospitalizations and the cost burden associated with rotavirus diarrhoea in Sweden. According to our estimates 1500‐1700 rotavirus‐associated hospitalizations occur annually in Sweden in children under 4 y of age (3.7 hospitalizations/lOOO children/y). This number represents 2.3% of admissions for all diagnoses in children of this age group. The cost of these hospitalizations is 13.5–15 million Swedish crowns (US

Identification of viral agents associated with diarrhea in young children during a winter season in Beijing, China

1.8–2 million). Serotyping by PCR for two years revealed that serotype 1 (GI) was the most common (49% and 58%, respectively) identified. Serotypes 2‐4 were identified in the following proportions G2 (23% and 5%), G3 (21% and 0%) and G4 (7% and 16%). The national laboratory report data for 1993‐96 show that as much as 7‐13% of rotavirus infections occur in elderly people. □G‐typing, incidence, rotavirus

Towards elimination : measles susceptibility in Australia and 17 European countries

Abstract Background Viral diarrhea remains a major cause of childhood morbidity and mortality worldwide. Although rotavirus was extensively studied in China, few comprehensive studies of all viral agents related to diarrhea in children have been conducted. Objectives Our study was performed to investigate the role of enteric viruses in acute diarrhea in our country and to evaluate methods that could be used in routine diagnostics. Study design One hundred stool samples were collected from children under 5 years of age seeking medical care for acute diarrhea during the winter season 2000/2001 in Beijing Childrens Hospital. All specimens were initially screened microscopically for leucocytes/red blood cells. Samples with negative results were analyzed for virus presence using commercial EIAs and/or in-house RT-PCRs. Results At least one viral agent was found in 67% of the specimens. The frequency of rotavirus, astrovirus, norovirus and enteric adenovirus was 59%, 8%, 6% and 2%, respectively. Dual infections were found in 9.0% (6/67) of the positive samples. The results from rotavirus and astrovirus EIAs were concordant with those of rotavirus and astrovirus RT-PCRs. Conclusions Enteric viruses play an important role in pediatric diarrhea during the winter season in China. A combination of microscopic examination of stool samples with specific EIA assays to detect virus antigen in stool specimens may be suitable for routine diagnostics.

Nasal Immunization of Mice with Virus-Like Particles Protects Offspring against Rotavirus Diarrhea

OBJECTIVE To evaluate age-specific measles susceptibility in Australia and 17 European countries. METHODS As part of the European Sero-Epidemiology Network 2 (ESEN2), 18 countries collected large national serum banks between 1996 and 2004. These banks were tested for measles IgG and the results converted to a common unitage to enable valid intercountry comparisons. Historical vaccination and disease incidence data were also collected. Age-stratified population susceptibility levels were compared to WHO European Region targets for measles elimination of < 15% in those aged 2-4 years, < 10% in 5-9-year-olds and < 5% in older age groups. FINDINGS Seven countries (Czech Republic, Hungary, Luxembourg, Spain, Slovakia, Slovenia and Sweden) met or came very close to the elimination targets. Four countries (Australia, Israel, Lithuania and Malta) had susceptibility levels above WHO targets in some older age groups indicating possible gaps in protection. Seven countries (Belgium, Bulgaria, Cyprus, England and Wales, Ireland, Latvia and Romania) were deemed to be at risk of epidemics as a result of high susceptibility in children and also, in some cases, adults. CONCLUSION Although all countries now implement a two-dose measles vaccination schedule, if the WHO European Region target of measles elimination by 2010 is to be achieved higher routine coverage as well as vaccination campaigns in some older age cohorts are needed in some countries. Without these improvements, continued measles transmission and outbreaks are expected in Europe.

Humoral and cell‐mediated immune responses in humans to the NSP4 enterotoxin of rotavirus

ABSTRACT Rotavirus is the major cause of diarrhea among young infants in both humans and animals. Immune protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. We have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and/or lactation. Adult female mice were nasally immunized with virus-like particles (VLPs) made of viral proteins VP2 and 6 (VLP2/6) or VP 2, 6, and 7 (VLP2/6/7) derived from the RF rotavirus strain in the presence or absence of cholera toxin. Both vaccines elicited serum and milk antibodies against the respective VPs. Four days after parturition, suckling pups were challenged orally with RF rotavirus. Pups from mothers immunized with VLP2/6/7 but not VLP2/6 were protected against rotavirus diarrhea, indicating that VP7 plays a key role in protection. Protection was mediated by milk rather than serum antibodies, and mucosal adjuvants were not required. In conclusion, VLPs containing VP7 administered nasally to mothers represent a promising vaccine candidate for the protection of suckling newborns against rotavirus-induced diarrhea, even in the absence of a mucosal adjuvant.