Influenza virus ISCOMs: biochemical characterization

Immunostimulating complexes (ISCOMs) have been prepared from influenza A virus envelope glycoproteins, i.e. haemagglutinin (HA) and neuraminidase (NA). An ISCOM consists of a matrix, which is the micellar form of the glycoside, Quil A, in hydrophobic interaction with both the envelope glycoproteins (HA/NA). The Quil A bound to the ISCOM amounted to 50 micrograms mg-1 (5%) of ISCOM protein. ISCOMs were morphologically identified as symmetrical cage-like structures of approximately equal to 40 nm in diameter with hexagonal or pentagonal subunits of approximately equal to 12 nm. The sedimentation coefficient was approximately equal to 19 S as compared to 30 S for the glycoprotein micelles. The biological activities of the HA and NA are preserved in both ISCOMs and micelles.     

Influenza B in households: virus shedding without symptoms or antibody response

In the fall of 1983, 53 households were enrolled in a double-blind trial of alpha 2-interferon as an intranasal spray to prevent common colds. During the winter/spring of 1984, 26 households were infected with influenza type B, as shown by isolation of the virus (19 households) and/or significant antibody titer rises (seven households). Interferon did not prevent influenza B infection or modify resulting illness. Of 37 persons shedding virus, 12 were asymptomatic. All were older than age 12 years, and 10 did not respond with antibody by any of the five test methods employed (complement fixation, hemagglutination inhibition, enzyme-linked immunosorbent assay (ELISA), neutralization, and Western blot). In contrast, of 13 symptomatic persons shedding virus from whom sera were available, 11 had significant antibody titer rises. Infection rates were highest among teenagers, but also surprisingly high among the 11 persons observed who were aged 50 years or older, four of whom were infected. The case-to-case interval in household transmission varied between one and nine days. Longer intervals of one, two, and four months between infections among family members were also observed, suggesting repeated introductions. Neither virus isolation alone nor serologic tests was sufficient to estimate infection rates.     

Bovine leukaemia virus ISCOMs: biochemical characterization

The glycoprotein gp51 of bovine leukaemia virus (BLV) has been included in an immunostimulating complex (ISCOM). The ISCOM was characterized biochemically in SDS-polyacrylamide gel electrophoresis showing the presence of proteins of estimated molecular weights of 50 and 30 kDa. Immunoblotting showed that gp51 was present in the ISCOM. The BLV-ISCOM had a S-value of 19 S and the electronmicrograph showed the cage-like structure as previously reported for other ISCOMs. About 17% of the total amount of gp51 in the cell culture fluid was recovered in the ISCOMs. The largest loss of gp51 was encountered during the sedimentation of the virus. An ELISA, utilizing monoclonal antibodies to defined epitopes for capture was developed to control the antigenicity of epitopes, e.g. those known to induce neutralizing antibodies. Using this device as a quality control for epitopes the following could be stated. First, ISCOMs prepared from virus solubilized with the non-ionic detergents Triton X-100 or MEGA did not react with neutralizing monoclonal antibodies. In contrast, ISCOMs prepared from virus solubilized with the non-ionic detergents Tween-20, Tween-80 or octyl glucoside did react with the neutralizing antibodies. Second, the neutralizing epitopes were better exposed in ISCOMs than the other epitopes of gp51. In a preliminary experiment it was shown that gp51 in ISCOMs was highly immunogenic.     

Incorporation of soluble antigens into ISCOMs: HIV gp120 ISCOMs induce virus neutralizing antibodies.

Through a process of covalent attachment of palmitic acid, we have incorporated recombinant gp120 of HIV strain IIIB into ISCOMs. Rabbits immunized with ISCOMs incorporating 10 micrograms gp120 produced high levels of gp120-specific antibody, comparable to the response to ten times as much antigen in complete Freund's adjuvant. The ISCOM-induced antisera showed virus neutralizing activity against the homologous strain, but failed to neutralize two heterologous strains of HIV-1. The antisera recognized non-conformationally determined epitopes on gp120, and antibody binding to gp120 was affected by glycosylation of the viral glycoprotein.     

High antibody responses in rabbits immunized with influenza virus ISCOMs containing a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA

Immunostimulating complexes (ISCOMs) are spherical structures where immunogens are presented as multimers in a matrix of the adjuvant Quil A. ISCOMs have been shown to enhance the immunogenicity of several antigens important to both human and veterinary vaccine development. We have coupled a fusion protein, designated ZZ-M2, comprising eight copies of the C-terminal repeat subunit EENV of the Plasmodium falciparum blood-stage antigen Pf155/RESA and two IgG-binding domains of staphylococcal protein A (SpA), to preformed influenza virus envelope protein ISCOMs. Rabbits immunized with the conjugated ISCOMs produced high titres of antibodies even after the first injection. These antibodies reacted with the EENV repeat sequence in ELISA and with Pf155/RESA in immunofluorescence on infected erythrocytes. The antibody response, which was sustained for more than 20 weeks, was efficiently boosted and superior or equal to that obtained after immunization with ZZ-M2 in Freund's complete adjuvant. In contrast, the antibody response induced in rabbits immunized with ZZ-M2 in Syntex Adjuvant Formulation-MF (SAF-MF) was weak and of short duration. The antibodies produced after immunization with ZZ-M2 coupled to influenza virus ISCOMs mainly recognized epitopes formed by two or more EENV subunits and were highly specific for Pf155/RESA. Furthermore, the antibodies efficiently inhibited merozoite reinvasion of erythrocytes in vitro, indicating that they recognized epitopes exposed on the native antigen. In addition, the ZZ-M2-conjugated ISCOMs also induced high titres of antibodies reacting with SpA or the influenza virus envelope protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent antibody staining of rabies virus antigens in the salivary glands of rabid animals

Staining with fluorescent antibody of street rabies antigens in smears made from the salivary glands of rabid animals is described.     

Rift Valley fever virus vaccine trial: study of neutralizing antibody response in humans

The serological response to immunization with a formalin inactivated Rift Valley fever (RVF) vaccine was studied in 963 Swedish UN soldiers serving in the Sinai peninsula. Antibody titres were determined with a plaque reduction neutralization test (PRNT). Attempts were made to give all soldiers three injections (1 ml s.c. days 0, 7-10 and 28-30) but 128 soldiers received only two injections. In a group of 51 fully vaccinated individuals, repeated blood samples were collected. Fifty of the vaccinees seroconverted. Serum collected six weeks after the first vaccination revealed the highest antibody titres. The geometric mean titre then decreased rapidly during the following two weeks. Six months after vaccination sera were collected from 433 vaccinees who had received three injections and 379 had antibodies detectable by PRNT (88% PRNT greater than or equal to 10). The corresponding figures one and two years after vaccination were 223 seropositives out of 255 (91% PRNT greater than or equal to 10) and 91 out of 123 (74% PRNT greater than or equal to 10), respectively. Multiple stepwise regression showed that three injections gave a better antibody response than two injections. This analysis also showed that the magnitude of the antibody response was reduced with increasing age. Slight, local and general side effects were reported in 6% of the vaccinees and these reactions occurred in individuals with relatively higher antibody response.     

Humoral immune response elicited in rats by measles viral membrane antigens presented in liposomes and ISCOMs

The immunogenicity of measles virus glycoproteins presented associated to liposomes or ISCOMs was compared with that of whole virus and solubilized membrane proteins in W/Fu rats. The rats were immunized three times at ten-day intervals with syngeneic peritoneal exudate cells fed in vitro with the various antigen preparations. A strong and persisting antibody response with haemagglutinin inhibitory and neutralization activity was observed in rats immunized with liposomes, ISCOMs, or virus. The responses were very similar despite the lower dose of protein received by rats immunized with ISCOMs (1 microgram protein) or with liposomes (20 micrograms protein). By contrast, injection of peritoneal exudate cells previously fed in vitro with soluble H and F glycoproteins resulted in only a poor and transient response. The sera from rats immunized with virus, liposomes or ISCOMs contained antibodies immunoprecipitating mainly H and F glycoproteins. Despite a strong enrichment in F polypeptides during the preparation of ISCOMs, they induced an equal anti-H and anti-F antibody response.     

Influenza B virus reinfection

Four outbreaks of influenza B infection occurred in Houston, Texas in the years 1976-1984. In the Houston Family Study, age-related infection and illness rates in the recent two epidemics resembled those reported previously. A total of 118 persons, including 35 children followed from birth, were followed longitudinally through this entire period and 331 persons were studied through at least two outbreaks. Fifty-nine (88%) of 67 children studied for four outbreaks were infected and 25% had a second infection; about half of the adults had one infection but only one of 51 was reinfected. Infection rates were proportionally lower for those followed through 2-3 outbreaks. Those with documented infection were protected decreasingly over time against reinfection and associated illness in subsequent epidemics. Such protection decreased in efficacy from 65% after 2-3 years, to 46% after 4-5 years, and to no protection after seven years.     

Influenza A and B antibody status in Tanzania

Sera from 200 babies and young children and from 205 mother-newborn pairs were tested for haemagglutination inhibiting antibody against three A-H1N1, one A-H2N2, four A-H3N2, and two influenza B viruses. The results indicated that a higher concentration of antibody against all influenza A and B viruses tested was found more frequently in maternal sera than in neonatal sera. High prevalences of antibody and high geometric mean titres against the A-H2N2-1957 and A-H3N2-1968 viruses from the eras 1957-1968 and 1968-now, respectively, were found in the age groups above 15 years. The antibodies against former and recent epidemic influenza viruses of the A-H3N2 subtype, found among different adult age groups in Mwanza, Tanzania, showed a pattern similar to that in the population of The Netherlands.     

Influenza A virus recycling revisited

Current textbooks link influenza pandemics to influenza A virus subtypes H2 (1889-91), H3 (1990), H1 (1918-20), H2 (1957-58) and H3 (1968), a pattern suggesting subtype recycling in humans. Since H1 reappeared in 1977, whatever its origin, some workers feel that H2 is the next pandemic candidate. This report reviews the publications on which the concept of influenza A virus subtype recycling is based and concludes that the data are inconsistent with the purported sequence of events. The three influenza pandemics prior to 1957-58 were linked with subtypes through retrospective studies of sera from the elderly, or through seroarchaeology. The pandemic seroarchaeological model for subtype H1 has been validated by the recent recovery of swine virus RNA fragments from persons who died from influenza in 1918. Application of the model to pre-existing H3 antibody among the elderly links the H3 subtype to the pandemic of 1889-91, not that of 1900 as popularly quoted. Application of the model to pre-existing H2 antibody among the elderly fails to confirm that this subtype caused a pandemic in the late 1800's, a finding which is consistent with age-related excess mortality patterns during the pandemics of 1957 (H2) and 1968 (H3). H2 variants should be included in pandemic planning for a number of reasons, but not because of evidence of recycling. It is not known when the next pandemic will occur or which of the 15 (or more) haemagglutinin subtypes will be involved. Effective global surveillance remains the key to influenza preparedness.     

Mucosal antibody response to vaginal infection with herpes simplex virus in pre-vaccinated guinea-pigs

The mucosal antibody response, in female guinea-pigs vaccinated with the Skinner herpes simplex virus vaccine, has been investigated. The HSV-specific secretory IgA response was assessed using the cross-reactivity of an antiserum raised against human secretory component. Animals vaccinated subcutaneously at a distant site were shown to respond to subsequent infection with HSV by the production of HSV-specific vaginal IgG and secretory IgA. No vaginal HSV-specific antibodies were found in infected, non-vaccinated animals.